Richard A.

Kaslow
Lawrence R. Stanberry
James W. Le Duc
Editors

Viral Infections
of Humans
Epidemiology and Control
Fifth Edition
Viral Infections of Humans
Richard A. Kaslow • Lawrence R. Stanberry
James W. Le Duc
Editors

Viral Infections of Humans

Epidemiology and Control

Fifth Edition
Editors
Richard A. Kaslow James W. Le Duc
Department of Epidemiology Department of Microbiology and Immunology
University of Alabama at Birmingham University of Texas Medical Branch
School of Public Health Galveston, TX
Birmingham, AL USA
USA

Lawrence R. Stanberry
Departmant of Pediatrics
Columbia University College of Physicians
and Surgeons
New York, NY
USA

ISBN 978-1-4899-7447-1 ISBN 978-1-4899-7448-8 (eBook)

DOI 10.1007/978-1-4899-7448-8
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Preface to the Fifth Edition

It has been 17 years since the release of the last edition—an inordinately long interval in view
of the dizzying pace of discovery in the biomedical sciences. That lengthy interval presented
certain challenges in deciding whether and how to embark on the production of a new edition.
An immediate reality was that a new volume had to be comprehensive enough to retain the
durable parts of the increasingly obsolete earlier edition while capturing the myriad new
advances. That significantly greater breadth and depth would require an expanded editorial
team. We were fortunate that the three of us together were sufficiently familiar with current
experts across our exciting and rapidly evolving field to recruit a stellar group of authorities in
their respective specialties. Most auspiciously, although we had not worked so closely together
before, we all brought our experience with the previous edition and a commitment to the high
standards that Al Evans imparted to everyone involved in the four earlier editions of the text.
The time elapsed since the earlier edition also meant that many chapters and the roster of
authors had to be entirely replaced or substantially modified by a largely new group of con-
tributors since relatively few of those involved in the earlier chapters remained available. In the
end, dozens of new authors were engaged.
Perhaps the most difficult decision had to be made primarily by Springer. Despite the fact
that the publishing world was undergoing a revolutionary transformation to digital media, a
determination had to be made on whether a printed book could still be both relevant to the
discipline and financially viable. In that regard, during the several years since the first conver-
sations about the new edition with William Tucker, Khristine Queja, and others at Springer,
they have steadfastly supported the project even in such an uncertain environment surrounding
print publications. To vindicate their judgment as well as our own leadership on the venture,
we aspired to a product that would greatly surpass a typical new edition; we hope readers con-
clude that it meets that test by any reasonable measure.
The text now contains four sections. The first covers principles and methodology, including
new chapters on methods of detection and modeling and a largely new perspective on surveil-
lance and biomarker epidemiology. Astonishing technologic progress is reflected in the chap-
ters discussing the proliferation of virologic and immunologic assay platforms, routine
applications of RT-PCR, and more recent incorporation of nucleic acid sequencing into
research and even clinical diagnostic identification. Some chapters also cite early dividends
from the expanding disciplines of bioinformatics and computational biology. The chapter on
disease surveillance systems and techniques highlights sophisticated approaches to data gath-
ering, with ever faster reporting aided not only by conventional electronic transmission capa-
bility but also increasingly by the hardware and software on portable devices that power social
media. Although surveillance still depends heavily on seroepidemiology, biomarker studies of
viral infections in large populations now increasingly involve collection and storage of sam-
ples other than serum, opening further opportunities to study host-agent interactions at the
cellular and molecular level. A new chapter on modeling focuses on the more theoretical
aspects of transmission, particularly person-to-person transmission; on the other hand, refer-
ence to decision models on projected future numbers of cases, clinical utility of a diagnostic
assay, or cost-effectiveness of interventions to combat particular infections appear in the
chapters addressing those agents.

v
vi Preface to the Fifth Edition

The remaining sections categorize viruses and the infections they cause into three groups:
those involved primarily in acute disease, those involved primarily in chronic disease including
malignancy, and those involved in both. Of the 47 chapters—13 more than in the previous edi-
tion—each has been substantially revised or written entirely anew. Where an earlier single
chapter may have covered multiple viruses related to each other either clinically (e.g., hepati-
tis, gastroenteritis) or epidemiologically (e.g., vector-borne diseases), they are now treated
more expansively. Four separate chapters cover hepatic diseases (hepatitis A, hepatitis B and
D, hepatitis C, and hepatocellular carcinoma); three cover gastroenteritis due to a number of
enteric viruses (noroviruses, sapoviruses, astroviruses, rotaviruses, enteroviruses, and hepatitis
E virus); and six separate chapters cover countless viruses transmitted by arthropods or small
mammal vectors. Although prion-associated spongiform encephalopathies are not viral dis-
eases, the chapter on these conditions reflects recognition that many of their clinical and epi-
demiologic features resemble those of chronic viral infections closely enough to be instructive
in this context.
Viral Infections in Humans has now been in print for more than 40 years. Many dozens of
basic, clinical, and population scientists have contributed to one or more editions. And each
successive edition has built on the solid foundations laid by colleagues who have preceded us.
Of the 90-odd authors connected with this fifth edition, we three editors are among only 9 who
also contributed to the fourth. Many who participated earlier are no longer alive, and we
thought it was fitting at least to honor those listed below who have passed away during the
years since the previous edition, including Caroline Breese Hall, who shared in preparing the
chapter on HHV-6 included in this volume.

Abram S. Benenson 2003

Francis L. Black 2007
Floyd W. Denny 2001
Alfred S. Evans 1996
Clarence J. Gibbs Jr. 2001
Eli Gold 2011
Caroline B. Hall 2012
Joseph L. Melnick 2001
Robert E. Shope 2004
Thomas H. Weller 2008

We remember all of them here in gratitude and admiration. Each made noteworthy—some
even monumental—contributions to our field, and each unquestionably enhanced the value of
this latest edition.
Finally, we owe special thanks to our wives—Leanne, Elizabeth, and Maryellen—who have
patiently endured the many annoying diversions of our attention.

Birmingham, AL, USA Richard A. Kaslow

New York, NY, USA Lawrence R. Stanberry
Galveston, TX, USA James W. Le Duc
Contents

Part I Concepts and Methods

1 Epidemiology and Control: Principles, Practice and Programs . . . . . . . . . . . . . 3

Richard A. Kaslow
2 Diagnosis, Discovery and Dissection of Viral Diseases . . . . . . . . . . . . . . . . . . . . . 39
W. Ian Lipkin and Thomas Briese
3 Immunological Detection and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Robert L. Atmar
4 Surveillance and Seroepidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Ruth Jiles, Monina Klevens, and Elizabeth Hughes
5 Viral Dynamics and Mathematical Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Nimalan Arinaminpathy, Charlotte Jessica E. Metcalf, and Bryan T. Grenfell

Part II Viruses Causing Acute Syndromes

6 Adenoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Xiaoyan Lu, Amita Joshi, and Phyllis Flomenberg
7 Alphaviruses: Equine Encephalitis and Others . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Scott C. Weaver and Ann M. Powers
8 Arenaviruses: Lassa Fever, Lujo Hemorrhagic Fever, Lymphocytic
Choriomeningitis, and the South American Hemorrhagic Fevers . . . . . . . . . . . 147
Daniel G. Bausch and James N. Mills
9 Bunyaviruses: Hantavirus and Others. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Alexander N. Freiberg, Dennis A. Bente, and James W. Le Duc
10 Coronaviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Arnold S. Monto, Benjamin J. Cowling, and J.S. Malik Peiris
11 Enteroviruses and Parechoviruses: Echoviruses,
Coxsackieviruses, and Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
M. Steven Oberste and Susan I. Gerber
12 Enteroviruses: Enterovirus 71 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Mong How Ooi and Tom Solomon
13 Enteroviruses: Polio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Olen M. Kew
14 Filoviruses: Marburg and Ebola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Thomas G. Ksiazek

vii
viii Contents

15 Flaviviruses: Dengue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

Stephen J. Thomas, Timothy P. Endy, and Alan L. Rothman
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others. . . . . . . . . . . . . . 383
Stephen J. Thomas, Luis J. Martinez, and Timothy P. Endy
17 Hepatitis A Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Daniel Shouval
18 Hepatitis E Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Xiang-Jin Meng
19 Influenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
John J. Treanor
20 Noroviruses, Sapoviruses, and Astroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Ben A. Lopman, Jan Vinjé, and Roger I. Glass
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox . . . . . . . . . . . . . 501
Brett W. Petersen, Kevin L. Karem, and Inger K. Damon
22 Paramyxoviruses: Henipaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Stephen P. Luby and Christopher C. Broder
23 Paramyxoviruses: Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
William J. Moss and Diane E. Griffin
24 Paramyxoviruses: Mumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
Steven A. Rubin
25 Paramyxoviruses: Parainfluenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Janet A. Englund and Anne Moscona
26 Paramyxoviruses: Respiratory Syncytial Virus
and Human Metapneumovirus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
James E. Crowe Jr. and John V. Williams
27 Parvoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
Kevin E. Brown
28 Rhabdovirus: Rabies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
Kira A. Christian and Charles E. Rupprecht
29 Rhinoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
Ian M. Mackay and Katherine E. Arden
30 Rotaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
Monica Malone McNeal and David I. Bernstein
31 Rubella Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
Walter Orenstein and Susan E. Reef

Part III Viruses Causing Acute and Chronic Syndromes and/or Malignancy

32 Hepatitis Viruses: Hepatitis B and Hepatitis D. . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Alison A. Evans, Chari Cohen, and Timothy M. Block
33 Hepatitis Viruses: Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Ponni V. Perumalswami and Robert S. Klein
34 Hepatitis Viruses: Hepatocellular Carcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
Ju Dong Yang, Roongruedee Chaiteerakij, and Lewis R. Roberts
Contents ix

35 Human Herpesviruses: Cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805

Robert F. Pass
36 Human Herpesviruses: Herpes Simplex Virus Types 1 and 2 . . . . . . . . . . . . . . . 829
Christine Johnston, Rhoda Ashley Morrow, and Lawrence R. Stanberry
37 Human Herpesviruses: Human Herpesvirus 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . 855
Mary T. Caserta and Caroline Breese Hall
38 Epstein-Barr Virus (EBV): Infectious Mononucleosis
and Other Nonmalignant EBV-Associated Diseases. . . . . . . . . . . . . . . . . . . . . . . 867
Karen F. Macsween and Ingólfur Johannessen
39 Kaposi’s Sarcoma-Associated Herpesvirus: Epidemiology,
Biological Characteristics and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897
Ronit Sarid and Maria Luisa Calabrò
40 Human Herpesviruses: Malignant Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . 933
Jennifer A. Kanakry and Richard F. Ambinder
41 Epstein-Barr Virus: Nasopharyngeal Carcinoma
and Other Epithelial Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953
Lawrence S. Young, Christopher W. Dawson, and Ciaran B.J. Woodman
42 Human Herpesviruses: Varicella and Herpes Zoster . . . . . . . . . . . . . . . . . . . . . . 971
John W. Gnann Jr.
43 Human Immunodeficiency Viruses Types 1 and 2 . . . . . . . . . . . . . . . . . . . . . . . . 1001
Richard A. Kaslow, Emily J. Erbelding, and Paul A. Goepfert
44 Human Papillomaviruses: Cervical Cancer and Warts . . . . . . . . . . . . . . . . . . . . 1063
Georgios Deftereos and Nancy B. Kiviat
45 Human T-Cell Leukemia Viruses Types 1 and 2. . . . . . . . . . . . . . . . . . . . . . . . . . 1105
Edward L. Murphy and Roberta L. Bruhn
46 Polyomaviruses: Progressive Multifocal Leukoencephalopathy
and Other Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135
Raphael P. Viscidi, Loubna Tazi, and Keerti V. Shah

Part IV Other Transmissible Agents

47 Prion Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165

Ermias D. Belay and Jason C. Bartz
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187
Contributors

Richard F. Ambinder, MD, PhD Division of Hematologic Malignancies,

Johns Hopkins University, Cancer Research Building (CRB I), Baltimore, MD, USA
Katherine E. Arden, PhD Queensland Paediatric Infectious Diseases Laboratory,
Queensland Children’s Medical Research Institute, Sir Albert Sakzewski Virus Research
Centre, Children’s Health Queensland Hospital and Health Service,
The University of Queensland, Herston, QLD, Australia
Nimalan Arinaminpathy, DPhil Department of Infectious Disease Epidemiology,
Imperial College London, London, UK
Robert L. Atmar, MD Departments of Medicine and Molecular Virology
and Microbiology, Baylor College of Medicine, Houston, TX, USA
Jason C. Bartz, PhD Department of Medical Microbiology and Immunology,
Creighton University, Omaha, NE, USA
Daniel G. Bausch, MD, MPH&TM Department of Tropical Medicine,
Tulane School of Public Health and Tropical Medicine, New Orleans, LA, USA
Ermias D. Belay, MD Division of High-Consequence Pathogens and Pathology,
NCEZID, Centers for Disease Control and Prevention, Atlanta, GA, USA
Dennis A. Bente, DVM, PhD Galveston National Laboratory,
Department of Microbiology and Immunology, University of Texas Medical Branch,
Galveston, TX, USA
David I. Bernstein, MD, MA Division of Infectious Diseases, Department of Pediatrics,
Cincinnati Children’s Hospital Medical Center, University of Cincinnati,
Cincinnati, OH, USA
Timothy M. Block, PhD Drexel Institute for Biotechnology and Virology Research,
Drexel University, College of Medicine and Hepatitis B Foundation, Doylestown, PA, USA
Thomas Briese, PhD Department of Epidemiology, Columbia University
Mailman School of Public Health, New York, NY, USA
Christopher C. Broder, PhD Emerging Infectious Diseases Graduate Program,
Department of Microbiology and Immunology, Uniformed Services University
of the Health Sciences, Bethesda, MD, USA
Kevin E. Brown, MD Virus Reference Department, Center for Infection,
Health Protection Agency, London, UK
Roberta L. Bruhn, MS, PhD Blood Systems Research Institute, San Francisco, CA, USA
Maria Luisa Calabrò, BScD Immunology and Molecular Oncology, Veneto Institute of
Oncology, IOV-IRCCS, Padova, Italy

xi
xii Contributors

Mary T. Caserta, MD Department of Medicine, University of Rochester

School of Medicine and Dentistry, Rochester, NY, USA
Roongruedee Chaiteerakij, MD, MSc Division of Gastroenterology and Hepatology,
Mayo Clinic College of Medicine, Rochester, MN, USA
Department of Medicine, Faculty of Medicine, King Chulalongkorn Memorial Hospital,
Bangkok, Thailand
Kira A. Christian, DVM, MPH Division of Global Disease Detection and Emergency
Response, Center for Global Health, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Chari Cohen, MPH, DrPH(c) Hepatitis B Foundation, Doylestown, PA, USA
Benjamin J. Cowling, PhD, FFPH Division of Epidemiology and Biostatistics, School of
Public Health, The University of Hong Kong, Pokfulam, Hong Kong SAR
James E. Crowe Jr., MD Vanderbilt Vaccine Center, Departments of Pediatrics,
Pathology, Microbiology and Immunology, Vanderbilt Vaccine Center, Vanderbilt University
Medical Center, Nashville, TN, USA
Inger K. Damon, MD, PhD Poxvirus and Rabies Branch, Division of High-Consequence
Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, GA, USA
Christopher W. Dawson, PhD School of Cancer Sciences, University of Birmingham,
Birmingham, UK
Georgios Deftereos, MD Department of Pathology, University of Washington,
Harborview Medical Center, Seattle, WA, USA
Timothy P. Endy, MD, MPH Infectious Disease Division, Department of Medicine,
State University of New York, Upstate Medical University, Syracuse, NY, USA
Janet A. Englund, MD Department of Pediatric Infectious Diseases,
Seattle Children’s Hospital, University of Washington, Seattle, WA, USA
Emily J. Erbelding, MD Division of AIDS, National Institute of Allergy
and Infectious Diseases, Bethesda, MD, USA
Alison A. Evans, ScD Department of Epidemiology and Biostatistics,
Drexel University School of Public Health, Philadelphia, PA, USA
Phyllis Flomenberg, MD Division of Infectious Diseases, Department of Medicine,
Thomas Jefferson University, Philadelphia, PA, USA
Alexander N. Freiberg, PhD Robert E. Shope BSL-4 Laboratory, Department of Pathology,
University of Texas Medical Branch, Galveston, TX, USA
Susan I. Gerber, MD Epidemiology Branch, Division of Viral Diseases, National Center
for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Roger I. Glass, MD Fogarty International Center, National Institute of Health,
Bethesda, MD, USA
John W. Gnann Jr., MD Division of Infectious Diseases, Department of Medicine, Medical
University of South Carolina, Charleston, SC, USA
Paul A. Goepfert, MD Division of Infectious Diseases, Department of Medicine,
University of Alabama at Birmingham, Birmingham, AL, USA
Contributors xiii

Bryan T. Grenfell, PhD Department of Ecology and Evolutionary Biology and Woodrow
Wilson School of Public and International Affairs, Princeton University, Princeton, NJ, USA
Diane E. Griffin, MD, PhD W. Harry Feinstone Department of Molecular
Microbiology and Immunology, Bloomberg School of Public Health,
Johns Hopkins University, Baltimore, MD, USA
Caroline Breese Hall, MD Department of Medicine, University of Rochester
School of Medicine and Dentistry, Rochester, NY, USA
Department of Pediatrics, University of Rochester School of Medicine and Dentistry,
University of Rochester Medical Center, Rochester, NY, USA
Elizabeth Hughes, MS, DrPh Division of Viral Hepatitis, National Center for HIV/AIDS,
Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Ruth Jiles, MS, MPH, PhD Division of Viral Hepatitis, Department of Health and Human
Services Centers for Disease Control and Prevention, Office of Infectious Diseases, National
Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Atlanta, GA, USA
Ingólfur Johannessen, MD, PhD Department of Laboratory Medicine,
Royal Infirmary of Edinburgh, Edinburgh, UK
Christine Johnston, MD, MPH Division of Allergy and Infectious Disease,
University of Washington, Virology Research Clinic, Seattle, WA, USA
Amita Joshi, PhD Division of Vaccines and Biologics Research, Merck & Co,
West Point, PA, USA
Jennifer A. Kanakry, MD Division of Hematology, School of Medicine,
Johns Hopkins University, Baltimore, MD, USA
Kevin L. Karem, PhD Laboratory Reference and Research Branch,
Division of Sexually Transmitted Disease and Prevention, National Center for HIV/AIDS,
Viral Hepatitis, STD, and TB Prevention, Atlanta, GA, USA
Richard A. Kaslow, MD, MPH Department of Epidemiology, University of Alabama
at Birmingham School of Public Health, Birmingham, AL, USA
Olen M. Kew, PhD Polio and Picornavirus Laboratory Branch, Division of Viral Diseases,
National Center for Immunization and Respiratory Diseases, Centers for Disease
Control and Prevention, Atlanta, GA, USA
Nancy B. Kiviat, MD Department of Pathology, Harborview Medical Center,
University of Washington, Seattle, WA, USA
Robert S. Klein, MD Division of Infectious Diseases, Department of Medicine,
St. Luke’s and Roosevelt Hospitals, New York, NY, USA
Monina Klevens, DDS, MPH Division of Viral Hepatitis, National Center for HIV/AIDS,
Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Thomas G. Ksiazek, DVM, PhD Department of Pathology, Sealy Center for Vaccine
Development, University of Texas Medical Branch, Galveston, TX, USA
W. Ian Lipkin, MD Department of Epidemiology, Columbia University Mailman
School of Public Health, New York, NY, USA
Department of Neurology and Pathology, Columbia University College of Physicians
and Surgeons, Center for Infection and Immunity, New York, NY, USA
xiv Contributors

James W. Le Duc, PhD Department of Microbiology and Immunology, University of Texas

Medical Branch, Galveston, TX, USA
Ben A. Lopman, PhD Division of Viral Diseases, National Center for Immunization
and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Xiaoyan Lu, MS Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of
Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Stephen P. Luby, MD Center for Innovation in Global Health, Woods Institute of the
Environment, Stanford University, Stanford, CA, USA
Ian M. Mackay, PhD Queensland Paediatric Infectious Diseases Laboratory,
Queensland Children’s Medical Research Institute, Sir Albert Sakzewski
Virus Research Centre, Children’s Health Queensland Hospital and Health Service,
The University of Queensland, Herston, QLD, Australia
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular
Biosciences, The University of Queensland, St Lucia, QLD, Australia
Karen F. Macsween, MSc, MD Department of Laboratory Medicine, Royal Infirmary of
Edinburgh, Edinburgh, UK
Luis J. Martinez, MD Viral Diseases Branch, Walter Reed Army Institute of Research,
Silver Spring, MD, USA
Monica Malone McNeal, MS Laboratory of Specialized Clinical Studies,
Division of Infectious Diseases, Department of Pediatrics,
Cincinnati Children’s Hospital Medical Center, University of Cincinnati,
Cincinnati, OH, USA
Xiang-Jin Meng, MD, PhD Department of Biomedical Sciences and Pathobiology,
Center for Molecular Medicine and Infectious Diseases, Virginia Polytechnic Institute
and State University (Virginia Tech), Blacksburg, VA, USA
Charlotte Jessica E. Metcalf, PhD Department of Ecology and Evolutionary Biology and
the Woodrow Wilson School, Princeton University, Princeton, NJ, USA
James N. Mills, PhD Population Biology, Ecology and Evolution Program,
Emory University, Atlanta, GA, USA
Arnold S. Monto, MD Department of Epidemiology, School of Public Health,
University of Michigan, Ann Arbor, MI, USA
Rhoda Ashley Morrow, PhD Faculty Affairs Office, Vaccine and Infectious
Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Anne Moscona, MD Departments of Pediatrics and of Microbiology and Immunology,
Weill Cornell Medical Centers, New York, NY, USA
William J. Moss, MD, MPH Departments of Epidemiology, International Health and the
W. Harry Feinstone Department of Molecular Microbiology and Immunology,
Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
Edward L. Murphy, MPH, MD Departments of Laboratory Medicine
and Epidemiology/Biostatistics, University of California San Francisco,
San Francisco, CA, USA
Blood Systems Research Institute, Baltimore, MD, USA
M. Steven Oberste, PhD Division of Viral Diseases, National Center for
Immunization and Respiratory Diseases, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Contributors xv

Mong How Ooi, MD Institute of Health and Community Medicine,

Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
Walter Orenstein, MD Department of Medicine, Emory University School of Medicine,
Atlanta, GA, USA
Robert F. Pass, MD Pediatric Hospital Medicine, University of Alabama at Birmingham,
Birmingham, AL, USA
J.S. Malik Peiris, DPhil Division of Public Health Laboratory Sciences, School of Public
Health, The University of Hong Kong, Pokfulam, Hong Kong SAR
Ponni V. Perumalswami, MD, MCR Division of Liver Diseases,
Mount Sinai Medical Center, New York, NY, USA
Brett W. Petersen, MD, MPH Poxvirus and Rabies Branch, Division of High-Consequence
Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, GA, USA
Ann M. Powers, PhD Arbovirus Diseases Branch, Division of Vector Borne Diseases,
National Center for Emerging and Zoonotic Diseases, Centers for Disease Control and
Prevention, Fort Collins, CO, USA
Susan E. Reef, MD Vaccine Preventable Disease Eradication and Elimination Branch,
Global Immunization Division, Center for Global Health, Centers for Disease Control and
Prevention, Atlanta, GA, USA
Lewis R. Roberts, MBChB, PhD Division of Gastroenterology and Hepatology,
Mayo Clinic College of Medicine, Rochester, MN, USA
Alan L. Rothman, MD Laboratory of Viral Immunity and Pathogenesis,
Institute for Immunology and Informatics, College of the Environment and Life Sciences,
University of Rhode Island, Providence, RI, USA
Steven A. Rubin, PhD Division of Viral Products, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, MD, USA
Charles E. Rupprecht, PhD Global Alliance for Rabies Control, Manhattan, KS, USA
LYSSA, LLC, Lawrenceville, GA, USA
Ronit Sarid, PhD The Mina and Everard Goodman Faculty of Life Sciences,
Bar Ilan University, Ramat Gan, Israel
Keerti V. Shah, MD, MPH Department of Molecular Microbiology and Immunology,
Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, MD, USA
Daniel Shouval, MD Division of Medicine, Institute of Gastroenterology and Liver
Diseases, Hadassah-Hebrew University Hospital, Jerusalem, Israel
Tom Solomon, BA, BM, BCh Brain Infections Group, Institute of Infection and Global
Health, University of Liverpool, Liverpool, UK
Department of Neurological Science, Walton Centre NHS Foundation Trust, Liverpool, UK
NIHR Health Protection Research Unit in Emerging and Zoonotic Infection, Liverpool, UK
Lawrence R. Stanberry, MD, PhD Department of Pediatrics, Columbia University
College of Physicians and Surgeons, New York, NY, USA
Loubna Tazi, PhD Division of Biology, Kansas State University, Manhattan, KS, USA
Stephen J. Thomas, MD Viral Diseases Branch, Walter Reed Army Institute
of Research, Silver Spring, MD, USA
xvi Contributors

John J. Treanor, MD Department of Medicine, University of Rochester Medical Center,

Rochester, NY, USA
Jan Vinjé, PhD Division of Viral Diseases, National Center for Immunization and
Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Raphael P. Viscidi, MD Department of Pediatrics, Johns Hopkins School of Medicine,
Baltimore, MD, USA
Scott C. Weaver, PhD Institute for Human Infections and Immunity, University of Texas
Medical Branch, Galveston, TX, USA
John V. Williams, MD Division of Infectious Diseases, Department of Pediatrics,
Vanderbilt University Medical Center, Nashville, TN, USA
Ciaran B.J. Woodman, MD School of Cancer Sciences,
University of Birmingham, Birmingham, UK
Ju Dong Yang, MD, MSc Division of Gastroenterology and Hepatology,
Mayo Clinic College of Medicine, Rochester, MN, USA
Lawrence S. Young, PhD, DSc Warwick Medical School, The University of Warwick,
Coventry, UK
College of Medical and Dental Sciences, Medical School Building,
University of Birmingham, Birmingham, UK
 Part I
Concepts and Methods
 Epidemiology and Control: Principles,
Practice and Programs 1
Richard A. Kaslow

1 Introduction: Infection and Disease 2 The Agent

The epidemiology of infectious diseases is concerned with This section addresses general properties of viruses that are
the circumstances under which both infection and disease important to an understanding of their epidemiology but
occur in a population and the factors that influence their fre- not their basic genetic, chemical, or structural composition
quency, spread, and distribution. It is critical to distinguish or details of their physiologic or multiplicative properties.
between infection and disease because the factors that gov- Chapters of this book on specific viral infections provide
ern their occurrence may be different and because infection some information on these topics. Other sources to be con-
without disease is common with many viruses. Infection sulted include textbooks on basic virology, such as Fields
indicates the introduction and multiplication of a biological Virology [2] and others, and books on the more general topic
agent within a host, leading to an interaction often manifest of microbiology and infectious diseases [3, 4].
as an immune response. It is determined largely by environ- Several intrinsic properties of all pathogens, including
mental factors that govern exposure to the agent and by both viruses, are of importance in the production of infection and
intrinsic susceptibility and personal behavior of the host. disease; these properties can be quantified and permit com-
Disease represents the host response to infection when it parisons among viruses. One property, infectivity, is ability
is severe enough to evoke a recognizable pattern of clini- to infect the preferred target cell in a standard assay, usually
cal manifestations. The factors that influence the occurrence measured as the minimum number of infectious units or par-
and severity of this response vary with the characteristics of ticles required. It may involve the capacity for attachment to,
virus involved, its portal of entry, and the dose or inoculum entry into, and multiplication within a variety of host cells.
size; but the most important determinants for many common A second property, pathogenicity, is defined as the ability of
infections lie within the host. Of these, the age and general an infectious agent to produce clinically significant disease.
health at the time of infection, genetic background, and A third property, virulence, has been used synonymously
immune status of the host are the most crucial. with the previous two terms but more formally refers to the
This chapter deals with the principles, observational proportion of infections that produce serious disease, classi-
methods, and control techniques applicable to viral infection cally measured as a ratio of fatal cases to the total number of
and disease in general; these concepts and approaches are infections. A fourth property is what might be described as
explored in greater detail in individual chapters concerned adaptability, a reflection of genetic responsiveness to external
with specific viruses or groups of viruses. For broader and perturbations often determined by the type and organization
fuller presentations of the epidemiologic principles, see texts of its nucleic acid. Over their long evolutionary history, most
in Suggested Reading, and for widely accepted definitions, viruses have either gradually accumulated key mutations
see Ref. [1]. or even integrated entire host genes that alter their capac-
ity to cause disease or permit them to evade or circumvent
specific host mechanisms of resistance. The proper series
of mutations may have enabled a virus to utilize an alter-
R.A. Kaslow, MD, MPH native biochemical pathway or conferred variable degrees
Department of Epidemiology, University of Alabama of resistance to antiviral agents. Rapidly replicating RNA
at Birmingham School of Public Health,
viruses like influenza, human immunodeficiency virus, and
2230 California St NW Apt 2BE,
Birmingham, AL 20008-3950, USA hepatitis C virus are more error (mutation) prone than many
e-mail: [email protected] DNA viruses. More sophisticated mechanisms of evasive

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 3

DOI 10.1007/978-1-4899-7448-8_1, © Springer Science+Business Media New York 2014
4 R.A. Kaslow

adaptation have been adopted by larger DNA viruses like the biologic origins versus those most likely due to other factors
herpesviruses. One such mechanism utilized by human her- that correlate closely with race. Genetic factors are discussed
pesvirus 8 is exemplified by the incorporation of several viral below. For the nongenetic explanations, race is usually a sur-
homologues of human genes (e.g., IL6, IRF3), which may rogate marker for some other often readily apparent factor.
mimic their human counterparts and/or usurp their function Geographic distribution is the most obvious reason for large
[5, 6]. All of these properties are factors that may influence racial differences. Viruses that have reservoirs or life cycles
not only the capacity to initiate infection and produce disease that involve nonhumans would be expected to occur only in
but also the transmissibility of the agent. regions of the world where the animal populations may coin-
cide with humans of one particular racial group. Exposure
may differ by race because of behavioral differences. New
3 The Host HIV-1 infections in young gay men in the United States have
surged more in blacks because of their relatively exclusive
Characteristics of the host that influence the occurrence of exposure network (see Chap. 43). HIV-2 is heavily concen-
both infection and disease are numerous and highly variable. trated in individuals of West African and Portuguese ances-
Some host factors that alter resistance and susceptibility to try because the virus probably originated and mainly spread
infection are identical to those that modulate the develop- in the former Portuguese colonies on the west coast of sub-
ment and natural history of disease, but many others affect Saharan Africa.
only one or the other. Age, sex, and race are the most long- Other host biological and behavioral factors are known
standing and obvious factors for both. or likely to influence acquisition of viral infection: general
Most of the effect of age on resistance to infection either nutritional status and specific micronutrient or vitamin defi-
is attributable to the development of immunity over time or ciency, cigarette smoking, medication, use of alcohol and
reflects the mode of transmission. Infants and young chil- other non-medicinal substances, occupation, marital status,
dren are susceptible to a whole range of infections that and sexual practices.
confer lifelong immunity once acquired. Many viral infec- Many of the above-mentioned factors may not only pre-
tions that are now largely prevented wherever vaccines are dispose to or protect from infection but also modulate the
available (e.g., measles, mumps, rubella, varicella, and rota- nature, severity, or course of disease. Table 1.1 contains a list
virus) and others like respiratory syncytial virus have his- of the host factors implicated with varying degrees of cer-
torically caused major childhood morbidity and mortality. tainty as modulators of occurrence, severity, pace, or dura-
Acquisition of certain herpesviruses (herpes simplex virus, tion of some viral diseases.
cytomegalovirus, and Epstein–Barr virus) begins in the peri- It is biologically based immunity that is the fundamental
natal period via congenital transmission followed by slow host characteristic governing not only acquisition but also,
and steady increases until adolescence, most likely through probably more profoundly than the other factors, the patho-
relatively casual direct contact. Then occurrence accelerates genesis and evolution of infection. Indeed, the influences of
somewhat for the next several decades during the period of age, sex, race, and some of those others reflect in part the
more intimate oral or sexual contact before leveling off after accompanying biologic functions that vary with those char-
middle age. acteristics. There are many examples in which these host fac-
The severity of an acute infection and the course of chronic tors are almost certainly operating as surrogates for immune
one infection may also differ by the age at acquisition. For
reasons not well understood, hepatitis A in older children and Table 1.1 Host factors that influence the occurrence, course, or
severity of disease
adults can be much more clinically apparent than in younger
children who are often asymptomatic. Similarly, poliovirus 1. Age at onset of infection
is much more likely to produce paralytic disease in older per- 2. Sex
sons than in young children. The age at which an individual 3. Race
becomes infected with HIV-1 is a strong determinant of the 4. Genetic factors controlling an enormous array of immune
response elements
natural history of that infection (see Ref. [7] and Chap. 43).
5. Preexisting level of specific or nonspecific immunity
For most viral infections, especially those occurring prin- 6. Iatrogenic immunosuppression
cipally in childhood, the two sexes appear approximately 7. Behavior and lifestyle: sexual practices, smoking, alcohol, and
equally susceptible. In adolescents and adults, differential recreational drugs
rates of acquisition by sex may be due to obvious differences 8. Dual infection or superinfection with other agents
in anatomy or in the mode of transmission, as in the case of 9. Preexisting chronic conditions and pregnancy
human papilloma virus and HIV-1, respectively. 10. Nutritional status
Differences in infection rate by race or ethnicity can be 11. Psychological status (e.g., motivation, emotional crises, attitudes
divided into those known or likely to have genetic or other toward illness) [8]
1 Epidemiology and Control: Principles, Practice and Programs 5

status. Age is prime example. Childhood respiratory and or additionally, these and other specialized cells may secrete
enteric viruses produce life-threatening disease in infants cytokines, chemokines, complement, peptides, or other
but little or no illness in adults who have developed natural immunoactive substances, for which their target cells carry
immunity. Conversely, infections with hepatitis A and polio surface or intracellular receptors. Thus, countless different
viruses are often asymptomatic in young children but may cell types in elaborate interlocking pathways execute any of
produce significant clinical hepatitis and paralytic poliomy- a variety of assigned functions—signaling, activating, inhib-
elitis in older individuals. Aging adults develop increasingly iting, attracting, killing, etc.
severe infections as their immunity in general and specifically Humoral immunity is generated by a cascade of inter-
to previously encountered viruses wanes. Sex differences in actions among dendritic cells, T and B lymphocytes, and
disease expression are generally less obvious. Disease due to plasma cells culminating in the production of immuno-
influenza virus is more or less equally common and severe in globulins that serve as antibodies to the virus that initi-
men and women in general, but pregnant women experience ated the response. Once activated by a virus, the humoral
more serious illness [9]. immune system engages in sequential production of immu-
Apparent racial disparities exist in rates of infection, dis- noglobulins of the IgM, IgG, and IgA classes in different
ease frequency, or expression. Various sources of surveillance proportions that bind to surface or intracellular antigenic
data suggest that persons of African ancestry in the United components of the virus. This binding improves with avid-
States have higher rates of influenza, HCV, HPV, and other ity (tightness) and affinity (antigen specificity) that increase
viral infections, while those of Asian ancestry have higher during the days or weeks following onset of infection. The
rates of HBV infection. However, in each case these differ- antibodies, often in conjunction with complement or other
ences are readily attributable to geographic, socioeconomic, proteins, may neutralize or kill the virus directly or recruit
or behavioral influences on acquisition, as noted earlier, and cells with cytotoxic capabilities into the vicinity to accom-
race is simply a marker of some combination of those factors plish that task.
rather than signifying an underlying immunologic or other Both cellular and humoral systems demonstrate what are
biologic basis for the difference. On the other hand, some known as innate and adaptive responses. Certain effector
racial or ethnic differences in the disease manifestations cells and molecules preexist in the host before any pathogen
(severity or natural history) will undoubtedly prove to be due is encountered. These relatively unvarying elements have
to underlying immunogenetic heterogeneity. been recognized as the “first responders” because they medi-
Immunity exists in a continuum ranging from lowest ate intrinsic or innate functions that are automatically and
(i.e., complete susceptibility) to highest (i.e., complete resis- uniformly invoked in the first hours and days after a viral
tance). Resistance is either intrinsic or induced, and it can be infection or other foreign intrusion. Soon after the innate
induced either naturally or artificially. The basis for natural response is underway, the adaptive parts of the systems are
resistance or immunity to viral infection is briefly discussed activated. Dendritic and other cells use molecules on their
just below; the strategy and resources employed for induc- surface to present antigenic fragments to effector T lym-
ing resistance by immunization are addressed later in this phocytes individually in a process exquisitely specific to the
chapter. offending virus. This specificity is generated by the huge
Mediators of natural resistance include components of variation in the human leukocyte antigen (HLA) genes that
primary defense systems such as cilia, mucus, the integu- encode those surface proteins used for microbial antigen
ment, and other physical and chemical inhibitors. However, presentation. These molecules not only activate helper and
resistance is even more dependent on the principal organs cytotoxic T lymphocytes but also interact with another set of
involved in generating the immune response—spleen, bone cognate receptors on the surface of natural killer (NK) cells;
marrow, lymph nodes, and other lymphoid tissue—the struc- these NK cell receptors are also encoded by highly complex
tures on which the highly complex cellular and humoral multigenic systems such as the leukocyte receptor complex
immune systems are founded. Multiple cellular arms of (LCR), the natural cytotoxicity receptor (NCR) family, and
the immune system dedicated to defending against viral the killer lectin-like receptor (KLR) family. The antibody-
infection. They operate through dozens of known and as- mediated humoral response is likewise largely adaptive. As
yet-unknown interlocking pathways in which monocytes; with cell-mediated antigen presentation and the cytotoxic
macrophages; dendritic cells; a wide array of T-helper, sup- systems, the organization of the genes encoding immuno-
pressor, and regulatory cells along with B lymphocytes and globulins are likewise programmed for genetically governed
plasma cells are all orchestrated to stimulate or respond to rearrangements to provide for maximum adaptability and
each other in a coherent and effective manner. They may do specificity. Moreover, not only do most antibodies bind with
so by a variety of mechanisms. Communication and func- specificity for a particular class of viruses or a single member
tion may occur through direct cell–cell contact or binding of the class, they also undergo structural maturation over time
between receptor–ligand pairs on cell surfaces. Alternatively to conform even more closely to the specific target antigen.
6 R.A. Kaslow

All of these processes depend primarily on differential poor sanitation and fecal contamination clearly increases
expression of hundreds to thousands of genetic loci rela- exposure and transmission efficiency.
tively directly involved in the immune response. Many of Climate also alters the social behavior of the host. In set-
the simplest, often monogenic immunodeficiencies with tings where high temperatures promote not only contact of
Mendelian patterns of inheritance and the infectious diseases hosts with water through swimming or drinking but also viral
that complicate them have been well catalogued, although replication, transmission of waterborne gastrointestinal dis-
new examples continue to surface [10]. On the other hand, eases is increased in polluted areas. Warm weather also brings
very little of the overall genetic contribution to more com- closer contact with insect vectors of arboviruses and with dogs
plex multigenic infectious traits have yet been elucidated, but and other animal sources of rabies. Conversely, in the cooler
research in this field is charging ahead. An example of very seasons, people collect indoors, where crowding promotes
direct genetic mediation of resistance to viral infection is the transmission of airborne and droplet-borne infections. Spread
remarkable protection against HIV-1 infection afforded by in indoor environments is amplified by relative concentration
the deletion of a portion of the gene for the viral co-receptor of military personnel in barracks, residents of assisted living
(CCR5) in individuals of European ancestry, described fur- facilities, and assembly and dispersal of students coincid-
ther in Chap. 43. Other clear instances of genetic mediation ing with the periodic openings and closings of educational
are the increased susceptibility to norovirus infection among institutions. Furthermore, the relatively hot and dry environ-
carriers of the O blood group antigen compared with carri- ment in many homes and commercial buildings may impair
ers of A or B antigen [11] and the predisposition of children the protective mechanisms on mucous surfaces and promote
with polymorphisms in the Toll-like receptor 3 gene (TLR3) entry and attachment of certain respiratory viruses.
to herpes simplex viral encephalitis [12]. In the past two In tropical settings, heavy monsoon rains bring groups
decades, the explosion of knowledge in immunology and indoors and closer together much as they do during winter
genetics has paralleled the rapid advances in molecular and in colder climates. The incidence of common upper respira-
computational technology. A more detailed summary of the tory diseases in college students was as high in the warm cli-
rapidly expanding knowledge of immune system function is mate where the University of the Philippines is situated as in
beyond the scope of this overview. The reader is referred to the intemperate winters at the University of Wisconsin [16].
textbooks and the most current reviews of specific subjects Community studies in India [17], Trinidad [18], and Panama
for more complete coverage [13–15]. [19] have documented high morbidity from influenza and
other respiratory diseases in tropical settings where people
aggregate inside, as with heavy rainfall or school attendance.
4 The Environment Cultural as well as physical environment can contribute
to the spread of infection, as exemplified by the patterns
The external environment can exert its influences on the virus of spread of HIV infection among gay bathhouse patrons,
itself or its mode of spread or on the biological or behavioral injectable substance abusers in “shooting” galleries, women
aspects of the host response. Although viruses survive or offering sexual favors in exchange for crack cocaine [20],
die within defined ranges of such physical factors as tem- and long-distance truck drivers patronizing commercial sex
perature, humidity, and air currents, variability between viral workers [21].
groups is high. Chemicals may be used as antiseptics or
microbicides to kill viruses prophylactically, and the list of
pharmaceuticals now available for antiviral therapy has been 5 Agent, Host, and Environment
growing rapidly (see Sect. 10). These environmental factors Interaction
may enhance or diminish survival of viruses, but they prob-
ably have a greater impact on routes of transmission and on It is obviously the interplay of those three cardinal ele-
patterns of host behavior. ments that dictates where, when, and in whom viral infec-
In addition to physical and chemical factors, biological tion occurs. Their interaction further determines whether the
variables play a role as well. For insect-borne agents like the infection produces disease, how soon it begins, how it mani-
equine encephalitis viruses, whose survival may depend on fests itself, how severe it becomes, and how long it lasts.
migration patterns of an avian host or on overwintering in To initiate infection a virus must be in the appropri-
a mammalian reservoir, the environment exerts an obvious ate structural and functional state, reflected in the intrin-
role in restricting transmission of infection and occurrence sic properties described above. It must also be present
of disease to those areas that have the proper temperature, in a quantity sufficient to penetrate the target host. That
humidity, vegetation, and other features necessary for the quantity, the inoculum, is the amount of virus available
insects. For viruses potentially transmitted by water, such as to be transmitted; and the minimum size of a viral inoc-
hepatitis A virus and noroviruses, a warm environment with ulum needed to initiate infection varies with infectivity,
1 Epidemiology and Control: Principles, Practice and Programs 7

a property discussed earlier, as well as with other features 6 Incubation and Latency Periods
of those three cardinal elements. Upon introduction of the
minimum inoculum and successful attachment to and pen- The interval of time between the sufficient exposure and the
etration of its target cells, the infection is established. Then, appearance of the first symptoms in the human host is the
again depending on various features of the agent, host, and intrinsic incubation period. Viruses transmitted by arthro-
environment, the virus elicits a response that may or may pods or other animals have their own (extrinsic) incubation
not manifest clinically. periods that vary with both the response within the animal
The degree of response an agent can evoke in the host and the external environmental factors that affect the ani-
immune system is known as its immunogenic potential or mal populations. However, here the reference is only to the
immunogenicity. It commonly refers to the ability of the intrinsic incubation period. The variation in this interval in
natural infection to confer protection against reinfection or different diseases is considerable (Fig. 1.1). Viruses that do
significant disease upon reexposure. This protective ability not require distant spread but are able to produce disease
has typically been measured as the quantity of antibody that through multiplication at the site of implantation, such as
neutralizes or incapacitates the virus, but viruses may gen- the respiratory tract, tend to have short incubation periods
erate antibodies that do not neutralize or protect. Many of on the order of 2–5 days. Arthropod-borne viral infections
the classic acute childhood infections (e.g., measles, mumps, like dengue and yellow fever may have onsets as early as
rubella, chicken pox, and other exanthematous conditions) 2–3 days after a mosquito bite [22], whereas inoculation by
are not merely strongly immunogenic but also usually con- small mammals may lead to longer incubation periods [23].
fer lifelong immunity. In contrast, other viruses can either Viruses that require hematogenous spread and involve dis-
readily alter their antigenic makeup like influenza and some tant target organs such as the skin or CNS have incubation
flaviviruses or produce persistent chronic infection like periods of 2–3 weeks. Rabies virus, dependent on spread
herpesviruses; by a variety of mechanisms, they have inge- along nerves, has a long and variable incubation period rang-
niously avoided generating highly neutralizing antibody. ing from 8 days to a year or more. Of course, transmission
HIV-1 is the master of evasive and adaptive mechanisms that of the prion-associated kuru is exceptional with onset docu-
allow it both to evolve rapidly and to persist for years with- mented as long 34–56 years after exposure [24]. In some dis-
out stimulating antibodies that neutralize. eases (e.g., poliomyelitis, dengue, hepatitis, and infectious
In the context of immunization, immunogenicity repre- mononucleosis), early symptoms or even a rash may accom-
sents the extent to which a vaccine can provide the same or pany the period of initial invasion or viremia. With these
similar degree of protection achieved in the course of natu- infections an early phase may not be clinically recognized or
ral infection. Many of the regimens for vaccination against occurs before the patient seeks medical care.
those highly immunogenic infections have succeeded in Knowledge of incubation periods has many practi-
mimicking very high levels of neutralizing antibody if not cal uses. Epidemiologically, the former helps define the
lifelong protection. The more basic biologic characteristics period of infectiousness: a patient is not usually infectious
of the agent and the host that determine immunogenicity are until close to the time of the appearance of clinical symp-
addressed in texts on virology and immunology (see Sect. 1). toms. In epidemics, knowledge of the mean, minimum, and

DAYS
DISEASE
10 20 30 40 50 100

Norwalk
Influenze Range
Commin Cold
Adenovirus Most Common
Dengue
Herpes Simplex
Exanthem Subiitum (HHV-6)
Enterovirus
Polimyelitis
Mcasles
Smallpox
Chickenpox
Erythema Infectiosum
Mumps
Rubella
Hepatitis A
Hepatitis E
Inf. Mononucleosis 360
Fig. 1.1 Incubation periods in
Rabies
viral diseases (Based on data Hepatitis C
from Heymann [25]; Evans Hepatitis B
and Kaslow [210])
8 R.A. Kaslow

maximum intrinsic incubation periods can be used to iden- As with HIV infection, the latency period for different
tify the probable time of exposure to the index case or other outcomes of HTLV-I varies. The interval between pre-
source of infection. The duration of infectivity depends sumed infection via breast milk and onset of adult T-cell
on the persistence of the virus and its exit into the envi- leukemia/lymphoma differs from the interval between pre-
ronment. Clinically, the duration of the incubation period sumed sexual transmission and onset of tropical spastic
helps to identify the likelihood of viral exanthem after a paraparesis or myelopathy. The concept of a latency period
known exposure or to differentiate hepatitis A from hepati- is also applicable to long-delayed virus-induced cancer
tis B infections. For prophylaxis, it determines the feasibil- as seen with an EBV-induced nasopharyngeal carcinoma
ity of prevention of the clinical illness by immunoglobulin, KSHV-induced Kaposi sarcoma, HBV- and HCV-induced
as in hepatitis A and varicella zoster infections, rubella, hepatocellular carcinoma, and HPV-induced cervical car-
and rabies, as well as the potential success of postexposure cinoma (see Chaps. 34, 39, 40, 41, 44, and 45). In kuru,
rabies vaccination. the latency period from exposure by ingestion of infected
In addition to the viruses that produce acute infections, brain or other tissues or by absorption via abraded skin at
there are delayed effects of certain common viruses in which a cannibalistic feast to the onset of disease can actually be
the “incubation period” represents a true or apparent inter- as long as several decades (see Chap. 47) [24].
val of “latency” lasting several to many years, during which
there is little if any viral replication. Examples include the
relationship of measles virus to subacute sclerosing panen- 7 Patterns of Response
cephalitis, in which infection in infancy may be associated
with involvement of the CNS some 5–10 years later [26]. The response to viral infection varies along a biological
Certain papovaviruses cause widespread inapparent infec- gradient in terms of both the nature and the severity of the
tions in childhood. Rarely, reactivation occurs later in life clinical syndrome produced. That variation is the product of
in the form of progressive multifocal leukoencephalopathy. the interaction of agent, host, and environment. The empha-
This phenomenon is seen in patients with Hodgkin’s disease sis here is on the biological gradient as manifested in the
in association with depression of cell-mediated immunity human as a whole; qualitative and quantitative differences
and more recently in AIDS patients [27]. in responses that occur at the cellular and molecular levels
With HIV infection, a primary clinical response may are more properly the subject of basic virology and immu-
occur within the first 2 months or so after infection in a sub- nology texts.
stantial proportion of newly infected persons (see Sect. 7.1
and Chap. 43). During the subsequent clinically quiescent
period, CD4 lymphocytes are destroyed at highly vari- 7.1 The Biological Gradient
able rates. Accordingly, clinical latency continues until
immunosuppression is significant enough to permit new Response to a virus by an individual may range from com-
opportunistic pathogens to superinfect or long-latent agents pletely asymptomatic to severe or fatal. The ratio of inap-
to reactivate and produce clinical disease. The average time parent (or subclinical) to apparent (or clinical) responses
from initial HIV infection to that clinical event may be varies from one virus to another; representative examples
shorter or longer depending not only on the degree of viral are shown in Table 1.2. The clinical response may be abrupt
control and immunosuppression under host genetic influence or more gradual, sometimes appearing long after the initial
but also on the other cofactors required for any specific clini- infection. It may be self-limited or arise from viral persis-
cal AIDS outcome. For example, Kaposi’s sarcoma tends tence or reactivation or both.
to occur at a somewhat earlier stage of immunosuppression GBV-C infection increases with age but produces no
than cerebral atrophy with dementia or lymphoma for rea- known clinical illness [28]. Initial infection with BK and JC
sons presumably related to the unknown determinants of strains of polyomavirus, which show high prevalence and
these conditions. Besides differences in the properties of the rates of acquisition of antibody in school children and adults
virus itself, such as the capacity to penetrate cells or induce [29, 30], is not known to affect them clinically. However, in
syncytium formation in vitro, the route of transmission and immunocompromised patients (e.g., with AIDS, Hodgkin’s
host factors determine the rate of progression of HIV infec- disease, or renal transplantation), previously asymptomatic
tion. The average AIDS-free interval is shorter for infants JC virus infection may evolve into progressive multifocal
and children and for recipients of large volumes of blood leukoencephalopathy[31]. Other primary or often reactivated
products. The interval is longest among the youngest adults infections are common in immunodeficient states; regardless
and then gradually shorter with increasing age [7]. There is of their clinical expression in normal hosts, most of the her-
also conclusive evidence for differential predisposition due pesviruses are more likely to cause mild to life-threatening
to immunogenetic factors. complications.
1 Epidemiology and Control: Principles, Practice and Programs 9

Table 1.2 The gradient of subclinical/clinical ratio in selected viral infections (inapparent/apparent)
Estimated subclinical/ Percentage of infection
Virus Clinical feature Age at infection clinical ratio with clinical features
GBV-C None known Child to adult ∞ 0
Poliomyelitis Paralysis Child ±1,000:1 0.1–1
Epstein–Barr Heterophile-positive 1–5 >100:1 1
infectious mononucleosis 6–15 10–100:1 1–10
16–25 2–3:1 35–50
Hepatitis A Jaundice <5 20:1 5
5–9 11:1 10
10–15 7:1 14
Adult 2–3:1 35–50
Rubella Rash 5–20 2:1 50
Influenza Fever, cough Young adult 1.5:1 60
Norovirus Gastroenteritis <2 1:5 60–90
HIV-1 Multiple Any age 1:99 99
Measles Rash, fever 5–20 1:99 99+
Rabies CNS symptoms Any age 0:100 100

A second group of viruses cause predominantly mild or The biological gradient of many viral infections can be
asymptomatic infection when acquired in early childhood viewed as analogous to an iceberg in which clinically apparent
but symptomatic and sometimes severe clinical disease when illness represents only a small proportion of the response pat-
infection is delayed. Examples of this are hepatitis A, polio- tern and the usually larger amount represents unrecognized
virus, and EBV. and inapparent infection. A similar analogy may exist at the
At the other end of the spectrum are acute infections cellular level. Figure 1.2 portrays these concepts, but Table 1.2
caused by agents like measles and rabies viruses or chronic demonstrates that the precise shape of the iceberg for any
infection with a virus like HIV-1 or the agents of spongiform given infection can vary considerably at the clinical level.
encephalopathies, in which clinically recognized illness usu-
ally accompanies the infection but the course of infection
may vary from days to decades. Rabies infection of man is 7.2 Clinical Syndromes: Manifestations,
the epitome of an infection from which death is virtually Etiologic Agents, and Frequencies
inevitable after characteristic symptoms develop.
The subclinical/clinical ratio for HIV-1 infection defies The tissue and organs targeted by human viruses can respond
the more straightforward categorization possible for many to infection in a limited number of ways; any of several
other viral infections. A syndrome resembling mononucleo- viruses (or other causative agents) may trigger the same
sis with fever, fatigue, headache, lymph node swelling, joint general response pattern. The clinician must often rely on
and muscle aching, rash, sore throat, and other features epidemiologic and clinical features and simple laboratory
occurs frequently in newly infected individuals. However, tests in making a tentative etiologic diagnosis. This diagnos-
the proportion reported to have experienced this syndrome tic reasoning may be based heavily on the known frequency
is higher or lower depending on the method of ascertainment of potential causative agents in the age group of the patient,
(e.g., presentation at a sexually transmitted disease clinic, the specific geographic or physical setting, the season, and
follow-up of cohort of initially uninfected homosexual men) their epidemic behavior. This section summarizes the major
and the clinical definition [32–34]. Likewise, the vast major- clinical syndromes produced by some of the more common
ity of HIV-1 infections follow a highly variable course—for human viruses. It includes (1) a very brief description of the
a median of about 9 years in most studies of untreated indi- clinical manifestations; (2) identification of major etiologic
viduals—free of the serious (AIDS-defining) illness; and on agents; (3) general frequency distributions by factors such as
the average, HIV-2 infection progresses even more slowly. age and geography, not necessarily applicable at special times
However, laboratory evidence usually indicates that immu- such as an epidemic or off-season or in settings like hospitals,
nologic deterioration is continuing at some rate even when nursing homes, and day-care facilities; and (4) selected other
the infection is clinically silent. The natural history of HIV features relevant to the understanding of their occurrence in
infection and the factors that modulate it are addressed in populations. For more detail, consult the chapter correspond-
detail in Chap. 43. ing to the agent of interest or more specialized texts.
10 R.A. Kaslow

Fig. 1.2 “Iceberg” concept of CELL RESPONSE* HOST RESPONSE

infectious diseases at level of the
cell and at level of the host. LYSIS OF CELL DEATH OF ORGANISM
Within any cell population,

DISCERNABLE EFFECT

CLINICAL DISEASE
varying patterns of cell response CLASSICAL AND
also occur. *Generic response INCLUSION BODY FORMATION
SEVERE DISEASE
[208] (Reproduced in Evans OR
and Kaslow [210]) CELL TRANSFORMATION
OR
MODERATE SEVERITY
CELL DYSFUNCTION
MILD ILLNESS

BELOW VISUAL CHANGE VIRAL MULTIPLICATION INFECTION WITHOUT

SUBCLINICAL DISEASE
WITHOUT VISIBLE CHANGE CLINICAL ILLNESS
OR INCOMPLETE (ASYMPTOMATIC
VIRAL MATURATION INFECTION)

EXPOSURE WITHOUT EXPOSURE

ATTACHMENT AND/ WITHOUT
OR CELL ENTRY INFECTION

ICEBERG CONCEPT OF INFECTION

It is important to note that in recent years newer sam- Approximately nine of ten children under 2 years old
ple collection approaches, rapid immunologic assays, and have experienced an episode of otitis [37]. The usual viral
highly sensitive molecular techniques have gradually supple- etiologies are respiratory syncytial virus, parainfluenza
mented and, in certain cases, supplanted the more traditional (types 1–3), influenza (A and B), enterovirus, and rhinovirus
virologic and serologic tests, but they have come into use [38]; however, the distribution of pathogens differs by age
unevenly in different places (Chap. 2). Therefore, general- and location. Exposure to day care or a family member with
izations made in this discussion must be tempered by con- a URI may be a predisposing factor.
sideration of the methodology for sampling, detection, and Influenza virus is the leading cause of both upper and
identification that may have been used to generate the find- lower respiratory infections in middle-aged and older adults.
ings at different times in different settings. Up to one-fifth of the entire US population may develop
influenza in any particular season. Seasonal influenza typi-
7.2.1 Common Respiratory Tract Syndromes cally lasts from November until March. However, in an
Many viruses and viral groups can evoke symptoms and atypical year, influenza activity may be present in summer
signs in the upper respiratory tract (e.g., eye, ear, nose, and and early autumn, as was true of H1N1 in 2009, when it
throat symptoms; Viral infections of the lower respiratory overlapped with seasonal influenza, and of swine-associated
tract trigger cough with little or no sputum production, short- H3N2v infection that occurred in 2012 in the setting of late
ness of breath, and changes in breath sounds on examina- summer county fairs [39].
tion). Fever is less prominent than with bacterial or fungal Several viruses causing respiratory tract infection are
infection. relatively recent discoveries if not new to humans, and they
Viral upper respiratory infection (URI) is extremely com- are responsible for an appreciable proportion of viral respi-
mon in most temperate climates; it is the most frequent cause ratory tract infection (see Chaps. 10, 26, and 27). Human
of absence from work or school in the United States [35]. metapneumovirus can cause upper and lower respiratory
A combination of rhinitis and pharyngitis (common cold) tract infections (i.e., a cold, cough, bronchitis, pneumonia,
occurs most frequently in children under 5 years old, who and exacerbation of asthma) in people of all ages but more
may have multiple episodes in a single year. URIs are still severe forms at the extremes of age and complicating immu-
frequent in older children, but incidence declines steadily nosuppression. Coronaviruses, although known for years
through adulthood to greater than 60 years of age. Other in animals and as the cause of more benign URI, came to
manifestations of viral infection include sinusitis, which may attention dramatically during the large epidemic of severe
be clinically or radiographically apparent in a very large pro- acute respiratory syndrome (SARS) in 2003, and resurfaced
portion of those with viral URI [36]. again in the variant form producing Middle East Respiratory
1 Epidemiology and Control: Principles, Practice and Programs 11

Syndrome (MERS). Since then a succession of new human Viral gastroenteritis caused by adenoviruses and astrovi-
coronavirus strains has been associated with outbreaks of ruses has been studied more selectively, mostly in the United
both upper and lower infections in different parts of the States. Specific serotypes of adenoviruses tend to cause dis-
world. An even newer member of the parvovirus family, a ease in a year-round, endemic form, primarily in very young
bocavirus, has been at least presumptively linked with respi- children. Up to 15 % of gastroenteritis in some settings may
ratory symptoms and otitis media [40]. be due to astrovirus infection. Older children and adults are
Over the years, numerous investigators have tried to esti- less affected. The infection is probably transmitted person to
mate the relative frequency of the different viral etiologies person, and it has caused infection in hospitalized and immu-
of clinical syndromes of acute respiratory diseases [40–48]. nodeficient patients [11]. As many as another 10 % of cases
However, not surprisingly, the advent of newer more sensi- in children may be caused by astroviruses, often in insti-
tive diagnostic techniques has made it increasingly clear that tutions like day-care centers and hospitals, where an even
the distributions of viral pathogens vary greatly not only by higher proportion of the transmitted enteric infections may
age and season but also by geography and proximity to ani- be due to this agent [53, 54]. Elderly and immunocompro-
mal populations, previous human population experience, and mised patients appear to be at elevated risk [55, 56].
other factors.
7.2.3 Common Central Nervous System
7.2.2 Gastroenteritis Syndromes
This syndrome consists of nausea, vomiting, and/or diar- Multiple viral agents are involved as causes of the syndromes
rhea of varying severity, with relatively little fever or other of inflammation of the spinal cord (aseptic meningitis) and
features (e.g., significant abdominal pain or gross fecal the brain (encephalitis) or a combination (meningoencephali-
blood) more typical of bacterial gastroenteritis. Worldwide, tis). Comprehensive reviews of these infections can be found
gastroenteritis causes serious morbidity and mortality, with in Refs. [57–59]. The clinical features of these syndromes
the greatest toll in children under 5 years old, due to severe are quite variable, from mild headache and/or stiff neck
dehydration. In a study from the mid-1980s in the United with or without fever and subtle changes in cortical function
States, diarrhea occurred in children under age 3 years in (i.e., orientation, wakefulness, concentration, etc.) to more
childcare at a rate of approximately three episodes per year profound manifestations of spinal cord or brain dysfunc-
[49]. Members of four families, rotaviruses, noroviruses, tion (including cranial nerve abnormalities; compromise of
enteric adenoviruses, and astroviruses account for most of critical brain stem function; confusion, irritability, and poor
the disease caused by viruses, and each has epidemiologic feeding in very young children; and seizures). In younger
characteristics that depend heavily on age and geography. children, long-term complications may include hydrocepha-
Infections with these viruses tend to occur continually in lus, vision and hearing loss, weakness or paralysis, cranial
children but only sporadically in adults. In the developed nerve deficits, and learning and behavior problems.
world, mortality is quite low although illness and hospital- The annual incidence of aseptic meningitis in the United
ization are more common. States from all causes is approximately 11 in 100,000 per-
Prior to the introduction of an effective vaccine, rota- sons, leading to 25,000–50,000 hospitalizations, mostly in
virus infections were the leading cause of diarrheal illness children. However, many more mild and unconfirmed cases
in children under 2 years of age in the United States and must go unreported. Again, the distribution of etiologic
many well-developed countries. The toll is still great in chil- agents varies strongly with age, geography, and availability
dren under 5 years old in a number of places throughout the of vaccines.
world, where it may account for as much as 30 % of mor- The incidence of viral meningitis in infants may be as
tality due to diarrheal disease [50]. As coverage with the much as ten times that in older children. Worldwide, over-
vaccine expands, that picture is expected to change dramati- all incidence is less easily estimated. Enteroviruses (echo-
cally [51]. Caliciviruses and particularly the most notorious viruses, coxsackieviruses, and others) are among the major
member of that family, norovirus, are now the leading cause causes in infants, and they may account for at least 80 % of
of gastroenteritis among adults in the United States; these cases in countries where vaccines against mumps, polio, and
infections often occur in epidemic form as “winter vomiting measles have significantly lowered the incidence of men-
disease” and are responsible for more than 90 % of outbreaks ingitis accompanying those conditions. On the other hand,
[52]. Facile interpersonal spread accounts for high second- in Asia, Japanese B encephalitis virus accounts for many
ary attack rates in family members and health-care personnel thousands of meningitis cases each year and probably hun-
attending ill patients. dreds of thousands more that remain clinically inapparent.
12 R.A. Kaslow

Additional, less common causes include other flaviviruses Table 1.3 Viral causes of common exanthems
(West Nile virus; see below), herpesviruses (VZV and oth- Type of rash Examplesa
ers), lymphocytic choriomeningitis virus, other arboviruses Macular/papular CMV, HHV-6, HBV, HIV
(Eastern and Western equine encephalitis), and HIV-1. Measles, atypical measles (vaccine)
Because both enteroviral and arboviral infections show Rubella
strong peaks in occurrence during the spring, summer, and Echovirus
early fall, the overall incidence in aseptic meningitis exhibits Enterovirus 71
clear seasonality. Coxsackievirus
Encephalitis is less common than meningitis due to viral Adenovirus
infection in the United States, with an annual incidence of Parvovirus B19 (erythema infectiosum)
about 20,000 cases or 3–8 in 100,000 persons. However, Vesicular/pustular Varicella zoster virus
Smallpox
because many cases are not seen as part of an outbreak, an
Eczema herpeticum
etiologic diagnosis requires a brain biopsy; therefore, only
Eczema vaccinatum
more severe and hospitalized cases are likely to receive such a
Coxsackievirus (esp. A16)
specific diagnosis. HSV-1, the most frequent cause, is respon- Enterovirus 71
sible for about 10 % of those cases. Arboviruses account for Adenovirus [63]
another 150 to several thousand cases, with variability accord- EBV [63]
ing to the intensity of seasonal occurrence and striking peaks CMV [63]
during sporadic epidemics. These have included Venezuelan Petechial or purpuric Coxsackievirus (esp. A9)
equine encephalitis (1969–1971, several hundred cases), St. Echovirus, esp. 9
Louis encephalitis virus infection (1975, 3,000 cases), West EBV
Nile virus (encephalitis or meningitis, 2,800 cases in 2003 Atypical measles (vaccine)
and 2,700 cases in 2012), and smaller numbers of La Crosse Parvovirus B19 [64]
encephalitis and Western equine encephalitis cases [59]. In Erythema multiforme HSV-1 [65]
centers where concerted diagnostic effort is made, additional Coxsackievirus A
agents of sporadic cases of encephalitis and rarer central ner- Echovirus
vous system syndromes include other herpesviruses (EBV Adenovirus
Others Coxsackievirus A
and VZV) and influenza A virus.
Echovirus
As with meningitis, accurate international estimates of
HHV-8
the incidence of encephalitis are unreliable, but epidemic
a
Examples of agents for each type of rash in table adapted from Cherry
disease patterns similar to those in the United States occur
[66], except as noted by other citations [63–65]
throughout the world. For example, in a 1995 outbreak of
Venezuelan equine encephalitis in Colombia and Venezuela,
of the 75,000 humans estimated to have developed infection, 7.2.5 Common Cutaneous Syndromes
neuroinvasive disease occurred in about 3,000, and 300 peo- Many viruses can produce one or more forms of eruption of
ple died. Recently, henipaviruses in the paramyxovirus fam- the skin (exanthem). The most common acute viral infec-
ily have accounted for clusters of encephalitis and related tions involving the skin are listed in Table 1.3; many are
neurologic disease in Australia along with Malaysia and classic exanthems of childhood including measles, rubella,
Singapore (see Chap. 22). varicella, erythema infectiosum or fifth disease caused by
parvovirus B19, and roseola infantum (exanthem subitum)
7.2.4 Ocular Syndromes caused primarily by human herpesvirus type 6 (HHV-6) [61].
Redness, watering, photophobia, feeling of irritation, and Rashes also occur in children or adults with coxsackieviruses
swelling of the membrane covering the eye (conjunctivitis) and echoviruses [62], with certain adenoviruses (such as type
and the cornea (keratitis) are often severe symptoms epi- 7), occasionally with EBV mononucleosis (often brought
demic keratoconjunctivitis (EKC) [60]. Outbreaks are on by a reaction to ampicillin), and with other herpesvi-
commonly caused by very contagious human adenovirus ruses. HIV also causes a rash in a small proportion of newly
infections. They are often nosocomial and have frequently infected patients. The distribution of these agents as causes
occurred in such settings as the neonatal intensive care unit of cutaneous conditions clearly varies by age, geography,
and ophthalmologic clinics, where contaminated contact vaccination status, and other host factors. The circumstances
lenses, eye drops, or instruments used for tonometry or slit- in which these many combinations of types of rash and
lamp examinations have been the modes of transmission agents are observed are not easily generalizable; the chapters
(see Chap. 6). on the individual agents should be consulted.
1 Epidemiology and Control: Principles, Practice and Programs 13

7.2.6 Hepatitis translated to high rates of chronic childhood infection and

Five principal agents are currently recognized as widespread perpetuated the cycle. Throughout the world and in propor-
causes of viral hepatitis: hepatitis A virus (HAV), hepatitis B tion to their representation in each population, hepatitis B
virus (HBV), hepatitis C virus (HCV), hepatitis delta virus has affected injection drug users (IDUs), recipients of con-
(HDV), and hepatitis E virus (HEV). However, viruses in a taminated blood products, those exposed to contaminated
number of other families can produce hepatitis. In the parts medical equipment, and men who have sex with men.
of the world where yellow fever virus is prevalent, the syn- Because HCV is transmitted more by the parenteral and less
drome is common. Less common agents are several herpes- by the sexual route, in the developed world, hepatitis C has
viruses (HSV-1, CMV, and EBV). All are associated with been more confined to IDUs and others who have been exposed
inflammation of the liver but with different severity, ranging to contaminated blood and needles. Central Asia and East Asia
from asymptomatic to acute mild liver tenderness and jaun- along with North Africa and the Middle East have the highest
dice, often accompanied by malaise and anorexia, to fulmi- prevalences of the HCV infection (>3.5 %) [69] as well as its
nant hepatic failure in a fortunately small fraction of cases. incident sequelae hepatitis, cirrhosis, and cancer. Because of
Two of the agents (HAV and HEV) produce more acute, the highly variable absolute and relative prevalences of HBV
usually mild to moderate self-limited illness, with milder and HCV infection and the other risk factors for those compli-
illness in younger individuals. As enteric pathogens, they cations, it is difficult to estimate the incidence of HCV-induced
occur in geographic distributions that reflect socioeconomic cirrhosis or cancer or the risk attributable to HCV.
conditions in those regions. Hepatitis A tends to occur spo- The delta virus (HDV) is an unusual partially defective
radically in older individuals where immunity is high, often RNA virus, closely dependent on the presence of HBV for its
because poor sanitation permitted the virus to saturate the pathogenic expression if not for its multiplication. When HDV
population at younger age. Conversely, epidemic HAV dis- coinfects simultaneously with HBV, it may trigger an acute
ease is more often seen in populations left susceptible as hepatitis syndrome but generally does not appreciably alter
a result of better hygiene. In the United States and other the course of disease. However, when HDV superinfects in
countries where the vaccine against HAV is widely used, a preexisting case of HBV infection, progression to cirrhosis
the decline in cases of hepatitis A has been striking. About is considerably more likely and often more rapid. The defec-
1.5 million cases occur annually worldwide. Hepatitis E is tive virus was first described in Europe, but it has been found
found worldwide, with different viral genotypes determining at high prevalence in countries of sub-Saharan Africa, South
the disease distribution. Globally, genotypes 1 and 2 account America, and Asia where HBV is highly endemic, more often
for 70,000 deaths and 3.4 million cases often in outbreaks in parenterally infected blood product recipients or injection
particularly in South and East Asia but in other developing drug users and occasionally in men who have sex with men.
countries as well, whereas genotype 3 occurs more sporadi-
cally in countries with better sanitation [67]. 7.2.7 Perinatal Conditions
The three others (HBV, HCV, and HDV) have strikingly Infections of the fetus, neonate, and infant may be acquired
different clinical patterns. Both HBV and HCV may produce from the mother in utero via placental transfer or during pas-
a range of symptoms including an acute syndrome much like sage through the birth canal or from other individuals post-
that of HAV and HEV. However, a small proportion of HBV partum via nosocomial and other similar close contact. The
and the majority of HCV infections become chronic and major syndromes and their etiologic agents are tabulated
often lifelong. In their chronic forms both infections may below (Table 1.4). The specific clinical manifestations vary by
remain essentially quiescent for a lifetime or progress at quite agent and timing of infection relative to gestation. Vertically
variable rates to chronic inflammation and cirrhosis, with or transmitted rubella, HSV-1/HSV-2, and cytomegalovirus
without symptoms along the way, and both are responsible in infections have long been implicated as causes of especially
some proportion for virus-induced hepatocellular carcinoma severe congenital multiorgan anomalies [70]. Estimates of
(see Chaps. 32, 33, and 34). Of the approximately two billion occurrence of perinatal infection vary according to location,
persons with HBV infection worldwide, 240 million have personal hygienic and sexual activities, obstetric practices,
some degree of persistent infection of the liver, and 600,000 utilization of vaccines, and other factors. Table 1.4 cata-
succumb to the sequelae [68]. The patterns of occurrence of logues the major clinical consequences of the large number
these diseases are driven primarily by geographic and behav- of agents responsible for perinatal infections.
ioral factors. Highly effective transmission by the parenteral, The adverse outcomes of rubella during pregnancy, once
sexual, and perinatal routes accounts for the ubiquitous dis- numerically and clinically very important, have thankfully
tribution of hepatitis B with particularly high prevalence in been virtually eliminated in the United States and other
Asia. Until the introduction of vaccine, high prevalence in countries with effective vaccination programs. The fetal
childbearing adults who transmitted the infection perinatally complications of the other vaccine-preventable diseases
14 R.A. Kaslow

Table 1.4 Effects of transplacental fetal infection

Effect of infection on the fetus and newborn infanta
Intrauterine growth Persistent
retardation and low Developmental Congenital postnatal
Organism or disease Prematurity birth weight anomalies disease infection
Viruses
Rubella − + + + +
Cytomegalovirus + + + + +
Herpes simplex + − − + +
Varicella zoster − (+) + + +
Mumps − − − (+) −
Rubeola + − − + −
Vaccinia − − − + −
Smallpox + − − + −
Coxsackie B − − (+) + −
Echoviruses − − − − −
Poliovirus − − − + −
Influenza − − − − −
Hepatitis B + − − + +
Human immunodeficiency virus (+) (+) (+) (+) +
Lymphocytic choriomeningitis virus − − − + −
Parvovirus − − − (+) −
Modified from Ref. [70]
a
+, Evidence for effect; –, no evidence for effect; (+), association of effect with infection has been suggested and is under consideration

have also been drastically reduced. That has left CMV as The risk of perinatal HBV infection is primarily that of
the most common serious congenital disease due to viral transmission of the virus from mothers who are chronically
infection in the United States [71]. It may infect more than infected (i.e., HBsAg carriers). Although infection is not ter-
2 % of all pregnant women. About one-third of women with atogenic and the reported impact on gestational age or birth
primary infection will transmit the virus to their newborn weight must be quite infrequent [76], there are adverse con-
infants, but acquisition by mother and infant varies greatly sequences for the infected neonate who receives no immu-
with age, location, and socioeconomic conditions [72]. Both noprophylaxis. About 5–8 % become hepatitis B surface
infection and serious sequelae are more common with pri- antigen carriers during the first 6 months of life. Moreover,
mary than with recurrent infection. Congenital infection may young adults infected in infancy are far more likely than
be symptomatic in as many as 15 % of infected neonates those not infected until later to manifest not only persistent
[73]. Although these infections are usually benign, the virus antigenemia but cirrhosis and, after an even longer latency
can produce anomalies such as microencephalopathy, cho- period, hepatocellular carcinoma. Rates of infection in preg-
rioretinitis, deafness, and mental retardation in a small pro- nant women show great geographic variation. Congenital
portion of those infected. Irreversible anomalies due to CMV and intrapartum HBV infection transmitted by maternal car-
infection occur in more than 5,000 children (0.1–0.2 %) each riers has long been considerably more common in Asia and
year. Africa than in Europe and the Western Hemisphere and has
Herpes simplex virus infections occur quite variably remained so. The pattern is changing in some places and will
among pregnant women in different geographic, ethnic, continue to do so wherever infant vaccination has been used
and socioeconomic subpopulations. Infection in infants is routinely for more than two decades, and the earliest univer-
almost always complicated by mucocutaneous (skin, eye, sally vaccinated birth cohorts are well into their childbearing
and mouth) lesions (75 %), encephalitis (57 %), pneumonia years.
(18 %), or disseminated infection with combinations of Untreated HIV-1 infection is transmitted from about
the three (30 %). Prompt recognition is important because 25–30 % infected mothers to their child/children. Adverse
antiviral therapy is often effective in reducing morbidity. outcomes of pregnancy similar to those of perinatal herpes-
Estimates of incidence come from multiple sources. An virus and certain other infections have been reported with
early study from a single location found a frequency of 1 per fetal and neonatal HIV-1 infection in some settings, but more
1,500 live births [74]. A more weighted population-based recent systematic studies have not revealed clear causal
extrapolation from 2006 US hospitalization data yielded an associations [77, 78]. An accelerated course of infection
incidence of 9.6 per 100,000 births [75]. and certain distinctive features (e.g., lymphocytic interstitial
1 Epidemiology and Control: Principles, Practice and Programs 15

pneumonitis) have been observed, potentially attributable to cause a spectrum of cervical and vaginal cytological abnor-
the unique attack on the immune system. Several regimens malities that are largely benign but in diminishing propor-
of antiretroviral agents alone or in combination have proved tions may progress to higher-grade neoplasia and culminate
increasingly effective in interrupting perinatal transmission. in invasive cervical carcinoma in a minority of women car-
Parvovirus B19 has been repeatedly linked to anemia, rying those specific types. Annually worldwide, HPV causes
neurological anomalies, hydrops fetalis, and fetal death (see about 500,000 cases of cervical cancer and ten million addi-
Chap. 27). In an earlier prospective study of 156 mothers tional cases of premalignant neoplasia [87]. About 80 % of
infected with this virus, 12 % delivered babies with some the cases and deaths occur in the developing world. The inva-
abnormality [79]. The investigators estimated the overall sive cancer has a 50 % case fatality ratio. Analogous patho-
fetal risk to be 9 %, although higher in the second trimester, logic processes lead to anal and penile carcinoma, largely in
and the transplacental transmission rate to be 33 %. Adverse men who have sex with men. The incidence rates of these
outcomes, particularly fetal loss and hydrops (a syndrome cancers appear to be considerably lower than rates of cervi-
associated with destruction of red blood cells in utero), fol- cal cancer in the same population [88].
low a proportion of the infections with this virus [79]. With
a predilection for myocytes, it has also been implicated in 7.2.9 Urinary Tract Syndromes
myocarditis [80, 81]. Acute hemorrhagic cystitis is the most prominent abnormal-
ity of the urinary tract caused by viruses. When the blad-
7.2.8 Genital Tract Syndromes der inflammation is caused by the principal etiologic agents
Two families of viruses, HSV-1 and HSV-2, and several in children (adenoviruses, particularly serotypes 11 and 21
subtypes of HPV are common causes of conditions of the of subgroup B), hematuria may be recognized along with
genital tract. Both HSV types can produce a spectrum of fea- pain or burning, but the condition is often less symptomatic.
tures ranging from no or minimal symptoms to an episode Another possible viral etiology of a urinary tract syndrome
of fever; headache; myalgia; regional adenopathy; painful, (manifested as renal tubular damage) is BK virus, which,
tender blisters and ulceration on the epithelial surfaces of like other polyomavirus-related infections, tends to be patho-
the genital organs and adjacent areas, including the anus and genic primarily in immunosuppressed patients.
rectum; and dysuria. Small proportions of infected persons
develop complications such as aseptic meningitis and proc- 7.2.10 Febrile Illness with Hemorrhage
titis (in men). Symptoms are more frequent and prominent Viral hemorrhagic fever may begin with a nonspecific pro-
with primary than with recurrent infection. Genital HSV drome of fever and chills, malaise, myalgia, headache, rash,
infections are acquired through sexual contact, beginning flushing, and in some cases vomiting and diarrhea. These
with sexual debut and increasing through the earlier years may rapidly progress to a multiorgan syndrome with pete-
of highest sexual activity in most areas of the world, where chial and more extensive bleeding but which is often less
they are often the leading cause of genital ulcer disease. In life threatening than vascular collapse with varying degrees
recent years type 1 has matched or surpassed type 2 as a of pulmonary edema, hypotension, shock, and renal failure.
frequent cause of primary genital infection, particularly in Variations of this syndrome are caused by a numerous and
younger persons. Infection with one of the two HSV types is growing list of mostly vector-borne viruses in four fami-
extremely common; tens of millions of people in the United lies: arenaviruses, filoviruses, bunyaviruses, and flaviviruses
States have genital HSV infection [82]. Estimates from CDC (Table 1.5). Collectively, the viruses that produce this syn-
indicated that in 2008, approximately 776,000 new HSV-2 drome are found throughout the world although each indi-
infections occurred, with half being in persons under 25 vidual agent is confined to the region where the principal
years of age [83]. arthropod vector or other animal reservoir lives. Certain of
The two major manifestations of HPV infection are geni- the diseases occur sporadically, for example, when a single
tal warts and squamous cell neoplasia. HPV types 6 and 11 individual has incidental contact with an infected animal. The
and less frequently other types are responsible for genital occurrence of a cluster of cases is more ominous; because
warts (condyloma acuminata) on epithelial surfaces in the of the often fulminant illness and the unpredictable human-
genital and anal area. The annual incidence of new cases of to-human propagation of cases, including nosocomial trans-
genital warts in the United States is about 360,000 persons mission, publicity about the event can have disproportional
[84]. Within the past decade, the cumulative incidence of community impact. These phenomena have typified the
genital warts in Scandinavian women up to age 45 years was ominously large outbreak of Ebola virus infection in West
about 10 % [85]. Among the unvaccinated group of women Africa. From time to time, one of these illnesses may present
followed in a multinational trial of quadrivalent vaccine, an immediate, major public health problem with hundreds
approximately 1 % of the women developed warts each year of cases; there is, for example, concern about the potential
[86]. HPV types 16 and 18 and less frequently other types for hemorrhagic dengue on a large scale in Southeast Asia.
16 R.A. Kaslow

Table 1.5 Families of viruses that may cause hemorrhagic fever

Arenaviridae Bunyaviridae Filoviridae Flaviviridae
Argentine hemorrhagic fever Crimean–Congo hemorrhagic fever (CCHF) Ebola hemorrhagic fever Denguea
Bolivian hemorrhagic fever Hantavirus pulmonary syndrome (HPS) Marburg hemorrhagic fever Kyasanur forest disease
Sabia-associated hemorrhagic fever Hemorrhagic fever with renal syndrome Omsk hemorrhagic fever
(HFRS)
Venezuelan hemorrhagic fever Rift Valley fever Tick-borne encephalitis
Lassa fever
Lymphocytic choriomeningitis (LCM)
Adapted from [89]
a
Dengue may occur in hemorrhagic form in certain circumstances (see Chap. 15)

Overall, the patterns of occurrence of these diseases are too survival on the temporal accumulation of new susceptibles.
variable to allow generalizations about their geographic dis- However, as noted earlier HIV has the powerful dual advan-
tribution or the magnitude of the problem; they are addressed tage of a retrovirus—capacity not only to vary its antigenic
more individually in Chaps. 8, 9, 14, and 15. structure but also to establish latency for years.

8 Occurrence and Spread 8.1 Routes of Transmission

in Populations
The major routes of transmission of selected viral infections
In the broadest terms, viral propagation within an individual are listed in Table 1.6. Viruses that have several alternate
depends on properties such as the efficiency of spread from routes have an increased chance of infection. The sequence of
cell to cell, either by direct involvement of contiguous cells events in transmission involves release of the virus from the
or by transport via body fluids to other susceptible cells; the cell, exit from the body, transport through the environment in
number of cells infected; and the consequences of viral mul- a viable form, and appropriate entry into a susceptible host.
tiplication on the cell itself and on the organism as a whole. Some viruses are released from cells at the end of the cycle
Further biochemical and physiologic details of these cellular of multiplication. Others do not complete this cycle (incom-
processes are the subjects of more basic science texts. The plete viruses), and some are less effective at escaping (e.g.,
next several sections are concerned with the occurrence and vaccinia). Many viruses are released from cells by budding,
propagation of infection in populations, along with factors acquiring a lipoprotein coat or envelope as they go through
that influence those events. the cell membrane; these include herpesviruses, togavi-
Survival of human viruses depends on their long-term ruses, myxoviruses, paramyxoviruses, and coronaviruses.
viability in human populations. Sustained viability, in turn, Nonenveloped viruses not released by budding are the ade-
depends on the patterns of occurrence and propagation— noviruses, parvoviruses, poxviruses, picornaviruses, and reo-
within a host and between hosts. A virus must be capable viruses. Some of these latter are released by cell lysis. Once
of (1) infecting a host chronically without killing its cells; released, viruses find their way to new hosts via one or more
(2) infecting a host acutely and severely but escaping from portals such as the respiratory tract (influenza, adenoviruses,
the dying host cells as an intact, replicable entity in a man- RSV), skin (VZV and smallpox virus), blood (HIV, HTLV-I
ner that ensures its transport to a new susceptible host; or and HTLV-II, HBV, HCV, and arboviruses), gastrointestinal
(3) infecting chronically or acutely but rapidly adapting to tract (enteroviruses, noroviruses, caliciviruses), genital tract
biological adversity such as exhaustion of susceptible hosts. (HIV, HTLV-I, HSV-2, and HPV), and placenta (rubella,
Herpesviruses and other persistent viruses have evolved to HIV, CMV, HSV-1, and HSV-2). A more detailed presenta-
establish durable relationships with their immunologically tion of these major routes of spread follows.
competent hosts. Arboviruses can destroy their hosts as long
as the former can survive and replenish themselves in their 8.1.1 Respiratory
nonhuman reservoirs and vectors. It is the viruses like influ- The respiratory route is probably the most important method
enza A and HIV, endowed with the greatest adaptability in of spread for most common viral diseases of man and is the
the form of antigenic variation, that pose the greatest threat least subject to effective environmental control.
because they are not self-limited in their pathogenicity or Viruses transmitted principally by the airborne route
in their dependence on favorable environmental conditions. include the agents of many classic childhood infections (e.g.,
Without its capacity for antigenic variation, influenza virus rhinoviruses, measles, rubella, mumps, varicella, influenza,
would, like measles or rubella virus, probably depend for parainfluenza, respiratory syncytial virus). Of course, these
1 Epidemiology and Control: Principles, Practice and Programs 17

Table 1.6 Transmission of viral infections

Routes of exit Mode of transmission Examplea Factors Routes of entryb
Respiratory tract Bite Rabies Animal Skin
Saliva EBV Kissing Mouth
Prechewed food, infants
HBV Dental work
HIVc Sexual
Aerosol Influenza, measles Cough, sneeze Respiratory
Oropharynx to hands, surfaces HSV, RSV, rhinovirus Fomites Oropharynx
Gastrointestinal tract Stool to hands Enteroviruses Poor hygiene Oropharynx
Stool to water, milk food HAV, rhinoviruses Seafood, water, etc. Mouth
HAV, HEV
Thermometer HAV Nurses Rectal
Skin Air Pox viruses Vesicles Respiratory
Skin to skin Molluscum contagiosum warts Abrasions Abraded skin
Blood Mosquitos Alphaviruses, flaviviruses Extrinsic incubation period Skin
Ticks Group B togaviruses Transovarial transmission Skin
Transfusions of blood and its HIV, HBV, HCV, HTLV-I/HTLV-II, Carrier in plasma or lymphs Skin
products CMV, EBV
Needles for injection HIV, HBV, HDV Drug addicts, tattooing Skin
Urine Rarely transmitted CMV, measles, mumps, rubella Unknown Unknown
Genital Cervix HSV, CMV, HBV, HIV, HPV, rubella Sexual, perinatal Genital
Semen CMV, HBV, HIV Heterosexual, homosexual Genital, rectal
Placenta Vertical to fetus CMV, HBV, HIV, rubella Infection in pregnancy Blood
Eye Tonometer Adenovirus Glaucoma test Eye
Breast Breastfeeding CMV, HIV, HTLV-I Maternal viremia Mouth
Multiple organs Transplant Rabies, Creutzfeldt–Jakob disease Surgery
Cornea, kidney
a
CMV cytomegalovirus, EBV Epstein–Barr virus, HAV hepatitis A virus, HBV hepatitis B virus, HCV hepatitis C virus, HDV hepatitis delta virus,
HEV hepatitis E virus, HIV human immunodeficiency virus, HPV human papilloma virus, HTLV-I/HTLV-II human T-lymphotropic virus type I/
human T-lymphotropic virus type II, RSV respiratory syncytial virus
b
Transmission does not always follow standard routes (Modified table from Evans and Kaslow [210] (Table 3), Springer has copyright)
c
Likelihood of transmission by this route is controversial

are also transmitted among adults by this route as well. Others 4 min, and 10-μm particles 17 min. This means that parti-
are transmitted by more direct contact with the nose or mouth cles under 10 μm have a relatively long circulation time in
or their mucosal secretions (e.g., EBV, HSV, and rabies virus). the ordinary room. Particles 6–10 μm or larger in diameter
Various other factors that affect airborne transmission of are more readily trapped upon direct impact in the nose and
respiratory viruses include the intensity and method of pro- nasopharynx. Further into the airway, flow diminishes to the
pulsion of discharges from the mouth and nose, the size of point where smaller particles 0.5–5 μm in diameter settle on
the aerosol droplets created, and the resistance to desicca- the tracheal and bronchial walls by sedimentation, and par-
tion. Much of the early work on the transmission of respira- ticles of 0.5 μm in diameter or smaller can enter and deposit
tory viruses was done by Knight and his group [90]. Direct in the alveoli. As infectious secretions are discharged in
transmission of infection occurs via personal contact such as large numbers by coughing or sneezing, the initially hygro-
kissing, touching of contaminated objects (hands, handker- scopic particles of 1.5-μm diameter lose moisture and shrink
chiefs, soft drink bottles), and direct impingement of large in ambient air and then regain their original dimensions from
droplets produced by coughing or sneezing. These last two the saturated air as they are again deposited in the respira-
behaviors are regarded as personal contact because of the tory tract. Of course, virus particles in an aerosol may not
short range of the heavy droplets formed. necessarily settle at a level in the respiratory tree with the
They also create aerosols varying in size up to >20 μm that optimally susceptible cells for that agent.
permit transmission of infection at a distance. Dispersion of High concentrations of particles of rhinovirus (and other
an aerosol depends on air currents and on particle size. In viruses, including influenza) on fingers, hands, and hard
still air, a spherical particle with a unit density of 100-μm surfaces indicate that infection via hands may be at least as
diameter requires 10 s to fall the height of the average room important a route of spread as aerosols containing lower con-
(3 m), and 40-μm particles require 1 min, 20-μm particles centrations. This is supported by the continual inadvertent
18 R.A. Kaslow

contact of hands with the nose or eyes [91]. Therefore, Hepatitis viruses and the enteroviruses also flourish in cer-
frequent hand washing, using antiseptic-containing fluids, tain institutional settings (mental hospitals, institutions for
sprays, or gels, is recommended for controlling the spread of retarded children, some prisons) and in countries where per-
rhinoviruses, respiratory syncytial virus, and influenza and sonal hygiene is lacking or difficult to practice or where poor
other viruses [92–95]. environmental control is present.
Aerosolization of certain viral agents may occur from For viruses spread by enteric routes, environmental con-
suction devices and from catheters in intensive care units trol is much more effective than for those transmitted by the
and from blood products in dialysis units. These include not respiratory route. Thus, good personal hygiene, especially
only respiratory and intestinal agents but also agents such as washing of hands after defecation, proper cleanliness and
HBV that circulate cell-free in the blood and cell-associated cooking of food, pasteurization of milk, good waste disposal,
viruses such as CMV, EBV, HIV, and HTLV-1. Viruses aero- and purification of drinking water supplies have all proved to
solized from urine or fecal material can be inhaled; hanta- be effective preventive measures. On the other hand, some
virus and arenaviruses are examples of agents that may be enteroviruses may also multiply in the respiratory tract and
spread by aerosols created from soil containing the rodent be transmitted by the respiratory route; therefore, this alter-
urine or feces in which those viruses are excreted. nate pathway is of epidemiologic importance even in the face
of good personal and environmental hygiene.
8.1.2 Enteric (Oral–Fecal)
Transmission by the oral–fecal route via the gastrointestinal 8.1.3 Direct Cutaneous Contact
tract as the portal of entry is probably the second most fre- Direct penetration of intact skin is a less common route of
quent means of spread of common viral infections. viral entry, and when it occurs, the viruses are usually car-
The major enteric viruses are enteroviruses, rotaviruses, ried in fluid or some other vehicle. Human papillomaviruses,
and calici-, polio-, echo-, and coxsackieviruses, along with the agents causing warts, enter through abraded skin and
HAV and HEV. Adenoviruses and reoviruses also multiply anogenital epithelium; prions, the agents of kuru, may also
in and shed from the intestinal tract, but this route of trans- gain access that way. With rabies, viral penetration of the
mission is not usually of epidemiologic importance. Despite skin invariably involves an animal bite or some other source
their name, although enteroviruses multiply in cells lining of contaminated biological fluid.
the gastrointestinal tract, they rarely produce local disease The skin serves as a portal of exit for those few viruses
there; they rather target the central nervous system and skin that produce skin vesicles or pox lesions that release infec-
and produce their major symptoms and pathology in those tious particles on rupture. These include herpes simplex,
organs. Likewise, hepatitis A and E viruses attack the liver, smallpox, varicella zoster, and vaccinia viruses. The viruses
not enteric mucosal cells. of certain maculopapular exanthems may also be present in
Viruses can directly infect susceptible cells of the oro- the skin, as in rubella, but this does not seem to be an impor-
pharynx, but to induce intestinal infection, virus-containing tant avenue of escape, since vesicles are not formed and skin
material must be swallowed, successfully resist the hydro- involvement occurs late in the disease, when the virus may
chloric acid in the stomach and the bile acids in the duode- be bound by antibody; indeed, the antigen–antibody com-
num, and access susceptible cells in the intestine. Viruses plex may be responsible for the rash itself.
with envelopes do not normally survive exposure to these
acids, salts, and enzymes in the gut. 8.1.4 Sexual
Agents excreted via the gastrointestinal tract must suc- The genital tract serves as a portal of both entry and exit for
cessfully infect other susceptible persons via fecally con- viruses that infect the genital tissues themselves and more
taminated hands, food, water, milk, thermometers, insects, remote target organs. It is also the source of intrapartum
or other vehicles. Both HAV and HEV are stable viruses in infection as the fetus passes through the birth canal. Herpes
water and, when present in sufficient dosage, may not be simplex types 1 and 2 cause ulcerative lesions of the anal and
inactivated by ordinary levels of chlorine. Outbreaks of viral genital mucosa and the surrounding skin. Sexual transmis-
hepatitis have occurred from sewage-contaminated water, as sion of these viruses can produce oropharyngeal lesions as
in the huge outbreaks in New Delhi, India, in 1955 [96], and well. These and other viruses (most frequently, CMV, HIV,
subsequently often due to HEV and in less dramatic attacks HBV, and HPV) can also be transmitted to their target cells
elsewhere. Furthermore, HAV, at least, can persist over long in the anogenital epithelial layer (HPV), in nearby subepithe-
periods in oysters and clams obtained from fecally contami- lial lymphoid system (HIV and CMV), or in remote hepatic
nated waters. Milk and water have also served as vehicles tissues (HBV). Receptive anal intercourse is a particularly
of transmission of other viral agents: caliciviruses and important method of spread of these infections. Three of
poliomyelitis viruses. This is especially hazardous because these four viruses (CMV, HIV, and HBV) along with rubella
these foods are so often eaten without having been cooked. virus are present in cervicovaginal secretions and can infect
1 Epidemiology and Control: Principles, Practice and Programs 19

infants perinatally (see Sect. 8.1.5). Finally certain types of 8.1.7 Vector-Borne: Insects and Mammals
HPV, principally 16 and 18, cause a substantial proportion of Humans may be primary or incidental hosts for numer-
cervical infection leading to a spectrum of mucosal abnor- ous vector-borne viral infections. They may be transmitted
malities including cervical cancer. through direct injection by mosquitos, ticks, flies, and other
CMV, HBV, and HIV are present in the semen and/ arthropods; they may also be spread directly through saliva
or female genital secretions and can be transmitted during during the bite of larger mammal; or they may be spread more
either heterosexual or homosexual intercourse [97–101]. indirectly through aerosolization of urine from a rodent. For
Long-term asymptomatic cervical or semen carrier states some viruses, their life cycle involves multiplication in their
exist and make recognition and control difficult. vector. In many arboviral infections, virus acquired from the
The presence of other genital infections has been shown human or animal host during viremia requires a period of
in repeated studies to predispose to transmission of HIV-1 multiplication in the vector before it is infectious. Examples
by the HIV-1-infected partner and acquisition of HIV-1 by of this include the transmission of yellow fever virus by
the susceptible partner. Most epidemiologic data indicate Aedes aegypti mosquitos and of the seasonally epidemic St.
that the ulcerative lesions of syphilis, chancroid, and HSV-2 Louis, California, and equine encephalitis viruses. The vec-
infection mechanically facilitate penetration of the epithe- tor life cycles can be complex, including overwinter survival
lial barriers of the genital tract by HIV [102, 103]. However, with transovarian or sexual transmission within mosquito
suggestions of predisposition by non-ulcerative infections populations.
like gonorrhea, chlamydiasis, trichomoniasis, and bacte- A range of mammals (dogs, bats, raccoons, skunks, foxes,
rial vaginosis are consistent with an alternative to enhanced and others) carry rabies virus in their tissues and bodily flu-
epithelial penetration, namely, recruitment and activation of ids including saliva. They can transmit to a human during a
macrophages and other inflammatory cells responsive to the bite, with the likelihood of transmission varying according to
mucosal breach (see Chap. 43). several factors: the mammalian species, the level of viremia,
the duration of its infection, the nature and location of the
8.1.5 Intrauterine and Intrapartum bite, and others. Bats may also transmit henipaviruses and
Viruses may infect the fetus either by direct contact via the coronaviruses. Rodent species can also carry and transmit
birth canal or by hematogenous spread via the placenta to viruses in their urine or feces. Examples of rodent transmis-
the fetus within the uterus. Herpes simplex virus and CMV sion include hantaviruses and Lassa fever virus. The viruses
infections can initiate intrauterine infection by more direct transmitted by vectors can vary greatly in their host range
local contact, whereas CMV, hepatitis B, rubella, varicella, from a fairly high degree of species specificity (as with den-
and HIV all infect the placenta by the hematogenous route. gue in Aedes aegypti or Lassa fever virus in one or possibly
CMV is the most common congenital infection, whereas more closely related rat species) to a much broader range (as
congenital rubella has sharply declined wherever vaccination with rabies in a number of different mammalian families).
has become routine. Acquisition of EBV or HBV in the early Another kind of transmission by a vector is simply
postnatal period is associated with persistence of infection mechanical, involving adherence of material containing
and substantially increased risk of subsequent cancer (see virus to an insect and transportation from one host to another.
Chaps. 32, 34, 40 and 41). This type requires neither incubation time in the insect vector
nor any specificity for either the arthropod host or the virus.
8.1.6 Blood-Borne Enteric viruses like polio and possibly hepatitis viruses may
Direct inoculation of viruses into the blood requires some be carried in this way.
object sharp enough to penetrate the skin and the wall of
a blood vessel. That can happen naturally through the bite 8.1.8 Urinary
of an arthropod vector (see Sect. 8.1.7) or iatrogenically Interestingly, although viruses such as CMV and measles are
through an injection, either with an inadvertently contami- excreted in the urine, this portal of exit has not been estab-
nated needle (e.g., by an injection drug user or in medical lished as being of epidemiologic or clinical importance.
practice) or for administration of medicinal blood products Considering the wide variety of viruses that can multiply in
(e.g., a transfusion). The agents best known for transmission human kidney tissue cultures in vitro and the likely role they
by this route include hepatitis viruses (HBV, HCV, and play in immune complex nephritis, it seems surprising that
HDV), retroviruses (HIV and HTLV), and herpesviruses renal infections with these viruses are not observed more fre-
(EBV, CMV). The mechanism of transmission for each of quently in humans.
these viruses is similarly straightforward, with the likelihood
of infection dependent on a combination of the intrinsic viral 8.1.9 Nosocomial
properties described above (Sect. 2), the delivery mechanics, The unique populations and physical circumstances found
and the dose or the size of the inoculum delivered. in hospitals and other health-care facilities lead to the
20 R.A. Kaslow

transmission of many viral infections by several of the fore- consequences [122]. Corneal transplant has also been associ-
going routes. The more than two-dozen viruses that have ated with transmission of the slow (Creutzfeldt–Jakob) virus
been documented as being nosocomially transmitted include [123]. These incidents have prompted the transplantation
many viruses that affect the respiratory tract as well as those community to adopt exacting protocols for excluding organs
transmitted through the respiratory tract to other organs and and tissue that may be infected with potentially harmful
cells (e.g., CMV, HSV-1, HSV-6, and VZV) [104], hepati- viruses. Lassa fever, Ebola, and Marburg viruses have often
tis viruses (including HAV, HBV, and HCV) [105], enteric been transmitted as nosocomial infections [124]. Lassa fever,
viruses (mainly rotavirus [106] and caliciviruses [107]), the in particular, has infected patients and staff, especially in the
viruses of several exanthems (rubella and measles) [108], obstetrical wards via infected placentas.
and picornaviruses [106, 109–111].
Blood-Borne Route
Respiratory Route Blood transfusion has long been a potential source of viral
Respiratory tract infections spread by droplets or droplet infection, and with each significant pathogen discovered to
nuclei like influenza may disseminate naturally through both be transmissible by transfusion (HBV, HIV-1/HIV-2, HCV,
the relatively vulnerable patients and the personnel in close HTLV-I/HTLV-II, and West Nile virus), the US Food and
proximity to each other. Nosocomial respiratory syncytial Drug Administration has orchestrated the development and
virus (RSV) infection in patients and staff has been a major implementation of universal donor screening [125]. Other
concern in the pediatric nursery, intensive care wards, or viruses that are capable of transmission by blood products
other population groups [92] at high risk due to crowding (e.g., CMV, EBV, parvovirus B19) appear to be relatively
and often an immature immune system. Outbreaks among harmless and/or ubiquitous enough that screening is not
adults and elderly patients in health-care facilities have also deemed necessary.
been reported, with influenza being a major concern [112– Of major concern for patients is accidental exposure
115]. In these settings, infections, diseases, and outbreaks during procedures where there is direct contact with a poten-
of CMV, HSV, VZV, enteroviruses, myxoviruses, metapneu- tially contaminated tissue (e.g., transplantation) or instrument
moviruses, and parainfluenza viruses have also occurred (e.g., needle, dental or dialysis equipment, or endoscope). In
[116–118]. regions of the world with adequate resources and training,
Although viruses may also theoretically be transferred universal blood precautions and the use of safety measures
from one patient to another by contaminated ventilating for disposing or auto-inactivating contaminated needles have
equipment, with the exception of occasional reports about largely but not entirely [126] eliminated percutaneous trans-
herpesviruses (CMV and HSV-1) [119, 120], this type of mission of HBV, HCV, HIV, and other viruses by needlestick
nosocomial transmission has not been recognized as an injuries. During the first 20 years of the HIV-1 epidemic in
important one. Lack of concerted effort to detect viruses the United States, 57 cases of infection were due to occupa-
rather than their actual absence may explain the dearth of tional injury in health-care personnel [127]. From 1999 to
information about this phenomenon. 2012, no further cases had been documented. The Centers
for Disease Control (CDC) continues to collect data and
Enteric Route inform personnel about those risks [128, 129].
As noted above, enteric viral infections are very common
in acute and long-term health-care settings, and outbreaks
(mainly due to rotavirus [106] and caliciviruses [121]) 8.2 Measures of Occurrence
can involve large numbers of patients. The circumstances
in which they are transmitted within health-care facilities Incidence is the number of new events (e.g., instances of
do not differ greatly from those operating outside them infection or cases of disease) occurring in some time inter-
(Sect. 8.1.2), although they appear to have a tendency to val. Generally, the incidence rate is the number of new events
attack immunocompromised patients. divided by the number of people at risk. The incidence rate
may be expressed more specifically as the number of events
Direct Contact with Biologic Tissue and Fluids per unit of population per unit of time or as the number of
Transmission of cutaneous and mucosal viral infections in events in a fixed total population during a fixed total time
health-care settings by direct contact is not especially com- period. The latter is considered a cumulative incidence but
mon (VZV, HPV, HSV). Although viruses naturally trans- is often called an “attack rate” in an epidemic setting, where
mitted through the skin following bites (rabies, in particular) the total time period under consideration is established by
rarely propagate that way in hospital settings, contact by per- the circumstances.
sonnel with tissue, saliva, and other secretions from trans- In the public health context, incidence may refer to new
plant patients infected with rabies virus has led to serious infections or new cases of disease. In the past, incident
1 Epidemiology and Control: Principles, Practice and Programs 21

infection was usually documented by isolation or pathologic the cumulative rate of infection with the agent over recent
visualization of newly acquired virus or by an assay for sero- and some years past depending on the persistence of the
conversion—the appearance of new specific antibody or a antibody. For neutralizing or other long-lasting antibody,
defined increase in the titer or concentration of preexisting it reflects the cumulative or lifetime experience with that
antibody; occasionally, new infection would be recognized agent. If the antibody measured is present only transiently,
by a change in the concentration of a specific biomarker as is often true of immunoglobulin M (IgM) antibody, then
like an enzyme. In the current molecular era, a variety of its prevalence indicates infection acquired within a recent
techniques for detecting viral nucleic acid are being used to period and may even approximate incidence. As with anti-
establish newly acquired infection (see Chap. 2). In calculat- body, the detection of nucleic acid could signify either recent
ing an infection incidence rate, the denominator would ide- or more persistent infection, depending on the natural history
ally consist of all individuals in a population considered to be of the infection. In calculating a viral disease prevalence, the
both exposed to the agent and susceptible (i.e., lack antibody denominator is usually all individuals in the total population.
or other evidence of prior infection). It should be clear that the number of new (incident) cases
Incident disease has always meant the presence of clini- and the number of existing (prevalent) cases are related
cal manifestations—some combination of self-reported to each other. More specifically, the prevalence of infec-
symptoms, objective physical signs, and positive results of tion or disease is determined by the duration of incident
specific diagnostic tests; and the disease incidence rate, is cases: the longer the duration of an incident condition, the
based on all individuals in a population whether or not they higher the number of prevalent cases of that condition. That
are infected. proportionality can be represented as prevalence = inci-
Mortality can be thought of as the incidence of death. In dence × duration. More strictly speaking, the term in the
calculating mortality, because the number with infection is equation should be average duration, which may vary con-
seldom known, the total population is customarily used as siderably for more chronic infections. Two corollaries of
the denominator, even though that rate generally underesti- this relationship are that the prevalence of acute self-lim-
mates the actual rate of death due to a specific viral infection. iting illnesses of short duration would closely mirror the
The case fatality ratio, another measure of the deadliness of incidence, and the more variable the duration of the condi-
an infection, is the proportion of all persons with cases who tion, the less reliable the relationship in a particular popula-
have died. tion may be.
In public health practice, the populations at risk or infected
can only be estimated with variable degrees of accuracy
depending on the circumstances. Evidence of prior infection 8.3 Patterns of Occurrence
is often not available. Commonly, the best estimate possible
is simply the total population present during a particular time Infections at the population level do not happen randomly;
interval within the geographic area or physical space (e.g., they occur in patterns, with different characteristics and in
hospital) encompassed by the detection or reporting system. different magnitudes.
Public health agencies generally tabulate statistics for infec-
tion or disease in the form of annual rates. 8.3.1 Secular, Periodic, and Seasonal Trends
Prevalence is the number of persons with infection or Secular trends in occurrence with no obvious periodicity
cases of disease existing at a designated time or in a time have been observed over longer intervals than a single year.
interval. The prevalence rate is the number of such cases Numbers may rise or fall depending on natural factors. Short-
divided by the population at risk. The time period involved or long-lived climate changes can alter the balance of vec-
may be a given instant of time (point prevalence) or a fixed tor and host populations, leading to significant increases or
period such as a year (period prevalence). The term period decreases in incident arboviral infections. Local populations
prevalence thereby incorporates both the number of new experience gradually declining rates of a particular strain of
(incident) cases and the duration of illness (number of old respiratory virus as population-level immunity develops fol-
cases persisting from the previous reporting period). It is lowing repeated exposures.
used most commonly for chronic diseases. Other factors, natural or human in origin, may produce
Prevalence of infection is usually measured by a test for periodic fluctuations that are not strictly seasonal because
the presence of a substance (e.g., antibody, antigen, fragment they do not recur each year at the same time. In the absence
of nucleic acid, or other component) in biological fluid or tis- of high levels of vaccination, herd immunity to a disease
sue samples from a given population at the time of their col- like measles may produce periodic but not strictly seasonal
lection. In calculating a prevalence, the denominator consists waves of new cases every 2–4 years depending on how
of persons whose samples were tested. For viral infections, fast new susceptibles are introduced into a population (see
the prevalence of antibody (i.e., seroprevalence) represents Sect. 8.3.2). Human migration may account for periodicity
22 R.A. Kaslow

Fig. 1.3 Seasonal occurrence of a

selected viral diseases. Panel
80
(a) Arboviral infections (of the
central nervous system)—cases
due to California serogroup
viruses, by month, United States,
1975–1993 (Reproduced from
Koo et al. [130]). Panel 60
(b) Pneumonia and influenza
mortality for 122 US cities, week

Reported Cobes
ending April 6, 2013 (Reproduced
from CDC [131])
40

20

0
1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994
Year (months)

b
12 Pnemonia and Influenza Mortality
fog 122 U.S. Citied
Week Ending April 6 2013

10
% of All Deaths Due to P&I

Epidemic Threshold

6
Seasonal Baseline

2008 2009 2010 2011 2012 2013

4
20 30 40 50 10 20 30 40 50 10 20 30 40 50 10 20 30 40 60 10 20 30 40 50 10
Weeks

that is predictable but might not occur every year in the viruses, Fig. 1.3, Panel a) or the return of favorable condi-
same season. tions for replication (e.g., for enteric viruses) during warmer
Seasonality is one pattern that has been characteristic of weather. For others (many respiratory infections), the con-
many viral infections in the absence of widespread vaccine- ventional explanation is that increases in the winter reflect
induced immunity. Many of the childhood viral diseases a combination of environmental factors (temperature and
(e.g., measles, rubella, mumps, chicken pox, polio, influenza humidity) that both directly enhance the viral replication
gastroenteritis, etc.) and certain viral diseases of adults (e.g., and transmissibility and indirectly lead to more conducive
vector-borne meningoencephalitis and influenza) show strik- host behavior (more time spent indoors under artificial con-
ing natural seasonal fluctuation. ditions of heat and ventilation). For a number of the diseases
For some of these illnesses, the explanation is obvious— where vaccines have been effective at radically decreas-
rising incidence in summer parallels the resurgence of the ing the incidence, the seasonality is barely or no longer
vector population (e.g., for mosquito-borne encephalitis discernible.
1 Epidemiology and Control: Principles, Practice and Programs 23

8.3.2 Endemic and Epidemic Occurrence population and despite the existence of a large tourist trade
Infection and infectious diseases occur in populations in dif- [132]. The introduction of more susceptibles or of more
ferent patterns. Incidence rates may be constant or may show infected persons may tip this balance. On the other hand,
seasonal or secular trends. When the pattern of a given condi- antibodies to viruses characterized by persistent or recurrent
tion is stable or constant over a period of years, it is usually viral excretion, such as herpesviruses and adenoviruses, have
considered endemic. A number of chronic viral infections been present in every population thus far tested, no matter
have affected certain populations in more or less the same how remote or isolated [133].
pattern for decades; although their patterns may be distinc- Of recent, an increasing concern is a possible deliberate
tive in different subgroups, infections with herpesviruses, introduction of an agent capable of spreading mass illness and
like cytomegalovirus, Epstein–Barr virus, and HHV-6, have death. The leading viral candidates for use in an attack of bio-
largely remained endemic. Some could even be considered terrorism (i.e., those causing smallpox, hemorrhagic fevers,
holoendemic—affecting entire populations, nearly univer- arboviral encephalitis, or a severe pulmonary syndrome) are
sally and more severely in childhood and less so in older those for which the target population has little or no immunity.
individuals. In contrast, an epidemic or outbreak of disease is To be effective as an agent of bioterrorism, the proportion of
recognized as the occurrence of cases in excess of the number susceptibles to infection with the candidate would need to be
expected for a population based on its past experience. The high, and the pattern and magnitude of the ensuing epidemic
definition of “excess” is an arbitrary one and depends on the would therefore be particularly difficult to predict.
relative concentration of recent and previous cases in the place, A critical characteristic of populations that strongly
time period, and population group of interest. In a country or influences its experience with infectious diseases in gen-
region where a naturally occurring disease has not been seen eral and the occurrence of an epidemic in particular is
for years, a few instances (e.g., encephalitis cases in summer) herd immunity. The herd immunity level is the cumulative
or even a single case (e.g., of poliomyelitis anywhere in the proportion of persons immune to a given disease within a
Western Hemisphere or Japanese B encephalitis in a country community. This proportion is, in turn, highly dependent on
where none has been documented for years) could be deemed such variables as the probability of contact between a source
an epidemic. On the other hand, a large and rising number of of infection and the susceptible person, the portal of entry
cases of winter respiratory illness (e.g., due to the introduc- accessible, the contagiousness of the agent, and the degree
tion of a new influenza strain) may signify normal seasonal of individual host immunity (all of which are often quan-
fluctuation. For that pattern to be declared an epidemic usu- titatively summarized as the basic reproduction number
ally requires application of more sophisticated criteria (i.e., (or ratio)). The term refers to the average number of new
deaths from influenza and pneumonia in 122 cities exceeding cases of infection transmitted by an index case during its
established threshold based on a 5-year average), for example, interval of infectivity (see Chap. 5) [134, 135]. High preva-
with the 2012–2013 season (Fig. 1.3 Panel b). When several lence of protective antibody to a virus among persons in a
continents are involved, as is the case with the global distribu- given community makes an outbreak with that virus most
tion of HIV/AIDS, the disease is said to be pandemic. unlikely. Herd immunity to highly communicable infections
Three essential requirements for an outbreak of viral dis- such as measles, mumps, rubella, and influenza appear to
ease are the presence of an infected host or contaminated require levels of antibody at least 75 % and even as high as
reservoir, an adequate number of susceptibles, and an effec- 94 % to be effective (Table 1.7).
tive method of contact and transmission between them. If
the agent is not endemic within the community, then the
introduction of an infected person, animal reservoir, or vec-
Table 1.7 Basic reproduction numbers and herd immunity thresholds
tor of transmission is needed to initiate a naturally occurring for selected vaccine-preventable viral diseases
outbreak. While the infection may have long been endemic
Disease R0 Herd immunity threshold (%)
in some remote place, an accident of nature (e.g., importa-
HIV/AIDS 2–5 –
tion of an animal carrying the agent) or change in human
Influenza (1918 A/H1N1) 2–3 –
behavior (e.g., a lapse in hygienic practice in a hospital)
Measles 12–18 83–94
triggers its emergence in a new location. This is particularly Mumps 4–7 75–86
important in an island or isolated population group, where a Polio 5–7 80–86
virus disappears after no more persons remain susceptible, Rubella 6–7 83–85
if persistent viral excretion does not occur to permit infec- Severe acute respiratory 2–5 –
tion of newborns. Rubella, for example, disappeared from syndrome (SARS)
Barbados for 10 years despite an accumulation in the num- Smallpox 5–7 80–85
ber of susceptibles to a level representing about 60 % of the Adapted from [136–140]
24 R.A. Kaslow

In an open college community, a preexisting level of immu- tion and illness, and recognition of patterns of occurrence all
nity to rubella of 75 % failed to prevent an outbreak of this involve rigorous comparisons of data from exposed, infected,
disease [141]. In a rubella outbreak among military recruits and/or diseased individuals with similar data from unexposed
with a 95 % level of immunity, 100 % of the susceptibles were or unaffected individuals. That form of inquiry has come to
infected [142]. Close and prolonged contact is apparently be called analytic epidemiology, the basic methods of which
extraordinarily efficient at spreading infection. Other princi- are summarized below. Finally, in conjunction with these
ples are also worth emphasizing: (1) the concept of herd immu- numerical and graphical methods, advances in the theory
nity is even less valid where several strains of virus exist and and methods of mathematical statistics have refined empiri-
cross protection is not complete; (2) even the identical strain of cal and predictive models of virus–host adaptation, transmis-
virus that does not naturally confer complete immunity (e.g., sion and vector dynamics, incidence, epidemic trajectories,
certain herpesviruses) may reinfect; (3) reactivation of latent and other phenomena of infectious disease. Chapter 5 is a
infection may produce disease, especially in immunocompro- concise view of current approaches to modeling of viral
mised hosts; and (4) the presence of antibody in the forms usu- transmission dynamics.
ally measured for such persistent viruses as HIV or hepatitis C The analysis of the results assembled and integrated
indicates that humoral immunity is incomplete at best. during the data gathering and descriptive phase primarily
involves evaluating relationships or associations with
standard measures. These measures and the investigative
9 Investigative Approaches approaches to estimating them are summarized very briefly
here; more elaborate treatment of the epidemiologic and sta-
9.1 Descriptive Epidemiology tistical principles underlying them can be found in any of
several textbooks on epidemiology as noted in Sect. 1.
The first steps in understanding infection and disease in
populations constitute so-called descriptive epidemiology. 9.2.1 Cohort Studies
It generally involves integrating the data that best characterize The search for associations in epidemiologic data often con-
the phenomenon under investigation in the terms discussed sists of comparing rates of infection in cohorts of individu-
in the preceding sections: the agent, environment, and host; als exposed and unexposed to a specific risk factor or rates
the distinctive pathogenetic features; the patterns of occur- of disease in cohorts of infected and uninfected individuals.
rence in time and place; and the mode(s) of transmission. Cohort studies may be cross-sectional or longitudinal in
The technologies for detecting and defining the agent, quan- design. In the former, the exposure and the outcome are mea-
tifying environmental conditions, and profiling the immune sured in the population at a single time point. There are many
status of the host have all been advancing at an exceedingly relationships for which the timing of the exposure relative
rapid pace. So have the noninvasive and imaging procedures to the disease cannot be established through cross-sectional
for characterizing the clinical and pathophysiologic features observation; however, there are many others for which the
of the disease. They have facilitated the pinpointing in time pathogenesis is understood or the exposure is unambiguously
and location of cases of infection or illness, often reveal- antecedent to the outcome (e.g., genetically mediated rapid
ing early key information about the origins and the mode of progression of HIV infection or shingles in an individual
transmission of infection. with serologic evidence of VZV infection). In contrast, lon-
The next steps after cases are recognized involve system- gitudinal cohort studies are often more powerful because the
atically defining, counting, and reporting their occurrences; temporal relationship between the exposure and the outcome
tabulating their frequencies; comparing their rates; graphing can be explicitly established. Although ulcerative inflam-
their trends; and evaluating and communicating the results of mation due to HSV-2 is a risk factor for acquisition of HIV
these efforts. The usual approaches for capturing and inter- infection, it could also be a more prominent consequence of
preting these events in the context of public health and dis- HIV-induced immunosuppression. Only longitudinal obser-
ease control come under the broad heading of surveillance, vation of precise temporal relationships between the onset
which is covered in detail in Chap. 4. of these two infections and the timing of their clinical mani-
festations would allow definitive inferences about causality.
The most informative measure of association in a cohort
9.2 Analytic Epidemiology study is usually the relative risk, used synonymously with
risk ratio or rate ratio. Relative risk is estimated as the ratio
Beyond the collection of data for descriptive and surveillance of the rate of the event (infected or ill) among exposed or
purposes, insight into infectious disease at the population infected persons, respectively, to the rate of such an event
level also emerges from more sophisticated computational in the unexposed or uninfected persons. In cohort studies in
and pictorial representation of the salient events and rela- which all or representative samples of individuals exposed
tionships. Hypothesis testing and other formal evaluation of and unexposed to a virus are observed for a period of time
causal relationships, exploration of co-determinants of infec- until some proportion of each sample develops disease, a
1 Epidemiology and Control: Principles, Practice and Programs 25

Table 1.8 Standard analytic table for estimating relative risk and relative odds
Infected/ill Uninfected/well
Exposed/infected a b (a + b)
Unexposed/uninfected c d (c + d)
Affected (a + c) Unaffected (b + d) Total (a + b + c + d)

Fig. 1.4 Predicted survival

1.0
curves (time from first HBeAg-
positive result to clearance) at Age at diagnosis 10y
ages 10, 20, and 50 years. Age at diagnosis 20y
Significant covariates included in
0.8 Age at diagnosis 50y
Proportion of Patients with HBeAg

the model are age at diagnosis of

HBsAg (including a nonlinear
effect), an interaction of initial
recorded status of HBeAg
(positive or negative) and age, and 0.6
the number of HBeAg
measurements recorded. The
overall probability of clearing
HBeAg within 10 years was 0.4
72.5 %. In those who cleared
HBeAg, the median time to
clearance was 5.6 years (range, <1
to 29.4 years). Older carriers were 0.2
significantly more likely than
younger ones to clear HBeAg
(P < 0.001) (Adapted from
McMahon et al. [143]) 0.0

0 5 10 15 20 25 30
Years from First HBeAg-Positive Test Result

“true” relative risk is calculated as the ratio of the eventual are compared, the putative causal relationship is measured
cumulative incidences in the groups with and without expo- as the relative odds, a term synonymous and interchange-
sure or [a/(a + b)]/[c/(c + d)] (Table 1.8). able with the odds ratio. Based on mathematical principles,
For conditions that develop over variable lengths of time, for diseases that are uncommon in the population, the relative
if the actual time between exposure/infection and disease is odds, calculated as [a × d/b × c], represent a close approxima-
known, it is often preferable to incorporate that time element tion to the relative risk (Table 1.8). The case–control study
into the estimate relative risk. That can be done either graphi- design is especially productive in epidemic situations where
cally in survival (Kaplan–Meier) plots (Fig. 1.4) or numeri- the investigation needs to be structured around cases that are
cally by application of statistical techniques of which a very reported or sought in the absence of a clear causal exposure.
useful and popular one is known as a (Cox) proportional The analysis of case–control studies is usually performed with
hazards regression, yielding a relative hazard. Several use- the technique of logistic regression, which can accommodate
ful pieces of information about the timing of events can be independent variables other than the putative causal factor.
extracted from the survival curves. As in other regression The major limitation is the many forms of bias that can dis-
models, a proportional hazards model can include multiple tort the estimate of the strength of the key relationship, due to
factors to be assessed for their independent contribution to difficulties in selecting and assessing study subjects with the
the outcome (disease). tight comparability necessary for proper analysis.

9.2.2 Case–Control Studies

When an investigation can only be done feasibly on infection 9.3 Investigation of an Epidemic
or disease that has already occurred following presumed expo-
sure to some etiologic agent in the past, properly designed This brief introduction to the strategy and tactics of epi-
comparison of those exposed and affected (cases of infection or demic investigations is best supplemented by more exten-
disease) with those exposed and unaffected (controls) can pro- sive guidance found elsewhere [144–146]. From the
vide a valid measure of association. When the available cases earlier discussion of the origin and nature of epidemics,
and, for efficiency, only a small subset of all the non-cases it should be clear that no two are exactly alike, but just
26 R.A. Kaslow

Table 1.9 Investigation of an epidemic world (see Chaps. 7, 8, 9, 14, 15, and 16,). Common source
1. Determine whether an outbreak is occurring outbreaks of viral infections from water, food, milk, or other
2. Verify the diagnosis environmental sources have not historically been as common
3. Establish a case definition as those due to bacterial infections. However, such outbreaks
4. Enumerate cases have been recognized increasingly in recent years. Some
5. Conduct descriptive analyses of the preliminary data examples include spread of adenoviruses by tonometers in
6. Develop hypotheses about the cause of illness and the source eye clinics or in swimming pools, hepatitis A by public water
of infection
supplies or by seafood, or enteroviruses by fecally contami-
7. Evaluate the hypotheses with analytic methods
nated water, food, or milk (see Chap. 17).
8. Conduct additional epidemiologic, environmental,
or laboratory studies
As the nature, magnitude, mode of transmission, and
9. Develop and implement prevention and control measures likely etiologic agent of the outbreak become clearer, the
10. Communicate the findings analyses can be refined and focused on more conclusive
Adapted from Dicker et al. [145] (CDC publication, no copyright) tests of hypotheses about individuals or other sources and
the modulating factors such as age, sex, occupation, recre-
ation, and other geographic or social characteristics of cases.
as there are patterns to endemic occurrence of disease, The results of these analyses may assist in excluding or add-
there are patterns to epidemics. A systematic approach to ing cases and focus control measures on individuals most
their investigation should take account of those patterns by directly affected as well as those at continued risk. The epi-
incorporating the sequence of steps outlined in Table 1.9, demiologic discovery of the cause and other features of the
although this list is neither exhaustive nor in strict priority outbreak along with the implementation of control measures
order of execution. will further oblige and enable the investigative team to com-
Outbreaks may come to attention in a variety of ways. municate effectively with the numerous stakeholders in a
An insightful clinician may suddenly see one or more unex- typically quite visible public health event.
pected cases of a syndrome or atypical presentations of infec-
tion, just as happened at the onset of the HIV/AIDS epidemic
[147]; or a laboratory may identify an unusual viral strain in 10 Control and Prevention
the course of routine testing (e.g., a novel influenza variant)
[148]; or a health department officer may recognize a cluster The basic strategy for controlling a disease is to break one
of cases of, say, norovirus gastroenteritis as multiple entries or more links in the chain of causation or pathogenetic con-
in computerized syndromic surveillance system [149]. With tinuum. Any such a strategy must rest on the fundamen-
any such initial event, it may not be possible to establish a tal knowledge of the agent, host, and environment for the
diagnosis only at the level of a clinical syndrome and not particular infection; the control may be physical, chemi-
with etiologic specificity. At this initial stage, a somewhat cal, or biological in nature. In the simplest terms, control
specific but simple working definition should include key at the level of the agent usually means incapacitating or
epidemiologic and clinical features for the purposes of case destroying it with a physical force as with ultraviolet or
finding. This definition can be altered in its sensitivity or light or freezing. A variety of chemicals can also be used
specificity later, when the spectrum of clinical features has externally, particularly on inanimate surfaces (i.e., viru-
been clarified and laboratory studies have been completed. cidal disinfectants such as detergents, methylene blue, and
The definition is primarily for the purposes of beginning to chlorine or other compounds that depend on the activity of
enumerate cases. This enumeration is accompanied simul- hypochlorite). At the level of the environment, the strategy
taneously with the organization and analyses of the clinical might involve an engineering solution such as improving
data and construction of a plot (epidemic curve) of cases water supplies or sewage disposal or installing negative-
according to onset or recognition and/or a plot of the location pressure ventilation systems, or it might involve avoiding,
of cases. Categorization by occurrences by infection, case, or suppressing, or eradicating the vector population using a
death may provide information about the seriousness of this widely sprayed insecticide or some other intervention that
outbreak relative to previous ones. alters the vector habitat. Effective prevention can also be
Even at this early point, if the pattern of cases or other achieved at the personal level with behavioral intervention
information reveals a likely mode of transmission, control to prevent contact (e.g., isolating or cohorting infected indi-
measures should be instituted. Not surprisingly many epi- viduals in separate rooms, wearing protective clothing such
demics of viral infection result from person-to-person spread, as masks or gowns, applying a topical insect repellant, or
particularly by the respiratory route, in open communities washing hands with any of a variety of antiseptic agents).
or in relatively closed populations like health or extended Because of space limitation, for more information on the
care facilities. Vector-borne spread also accounts for a sig- principles and practices of isolation and barrier, sanitation,
nificant portion of epidemic viral infection throughout the disinfection and antisepsis, environmental engineering,
1 Epidemiology and Control: Principles, Practice and Programs 27

and vector control, the reader is referred to more general Each chapter on individual viral infections provides more
texts and references on selected topics here; many others on information about the antiviral pharmaceuticals in use or in
these numerous topics are available [95, 150–153]. trial for those viruses. For another review of antivirals used in
The remainder of this section will concentrate on strat- specific infections, refer to Ref. [154]. Here simply for rapid
egies that depend on biological activity of pharmaceutical reference and comparison is a summary tabulation of the dis-
agents that can be injected, ingested, or topically applied eases and viruses (other than HIV/AIDS, which is covered
in humans. These are antiviral products that target specific in greater depth in Chap. 43), the corresponding antiviral
strains or broad classes of viruses and vaccines and immu- drugs available at the time of publication, their mechanism
nobiologics that capitalize on specific host responses to par- of action and/or stage of replication affected, and their use
ticular viruses. for treatment and/or prophylaxis (Table 1.10).
In addition to the antiviral agents covered here, several new
candidates in familiar classes as well as new classes of drugs
10.1 Therapy and Chemoprophylaxis are under experimental and in some cases human investigation.
They include numerous new drugs of several classes for HCV;
Strategies for designing antiviral agents focus on the cyclopropavir for CMV infection; a methanocarbathymidine
numerous vulnerable points in the replicative cycle of a compound active against several herpesviruses; a drug similar
virus that are, ideally, distinct from any point along the to cidofovir with activity against polyoma-, adeno-, pox-, and
synthetic pathways of the human host. There is a growing herpesviruses; favipiravir, which inhibits replication of influ-
list of candidate drugs with established clinical antiviral enza and probably arena-, bunya-, and hantaviruses; pleconaril
efficacy, and new compounds can now be designed in silico and other drugs that inhibit picornaviruses; drugs targeting
with computer programs that can produce exquisitely pre- kinase pathways; and other agents with a broader antiviral spec-
cise models of protein–protein interactions that help pre- trum, such as those that bind to the uniquely viral intermediary
dict their likely biologic effects. Of course, as promising as double-stranded RNA while initiating apoptosis [155–157].
these advances are, the ideal is rarely achieved. Limitations
still include the poor correlation of effects predicted by in
silico, in vitro, or animal studies with those observed in 10.2 Immunization
humans, strain differences in response, easy emergence of
resistance, and unforeseen acute toxicity. Delayed adverse Each chapter on a viral infection for which an effective or
consequences like oncogenicity and teratogenicity are also promising immunizing agent is available includes details
a concern. of that virus-specific immunization. This section presents

Table 1.10 Antiviral drugs licensed/generally available for human use

Treatment (Rx),
Condition Virus Agent Mechanism of action prophylaxis (Px)
Cold sores, fever blisters, HSV-1 Acyclovir, ganciclovir, famciclovir, Nucleos(t)ide analogue, inhibits Rx, Px
labial herpes zanamivir, valacyclovir, vidarabinea deoxypyrimidine kinase as a
Genital herpes HSV-2 foscarnet, and others competitive substrate
Chicken pox, zoster VZV Rx
Cytomegalovirus CMV Cidofovir, fomivirsen, foscarnet
Influenza A Influenza A virus Amantadine, rimantadine Interferes with ion channel protein Rx, Px
Influenza A Influenza A virus (M2) and uncoating
Respiratory syncytial virus RSV Rimantadine Rx
Influenza A and B Influenza Oseltamivir, zanamivir Blocks release by inhibiting Rx, Px
neuraminidase
Chronic hepatitis B HBV Adefovir, emtricitabine, lamivudine, Nucleos(t)ide analogue Rx
tenofovir
Chronic hepatitis C HCV 1. Interferon-α 1. Cytokine initiating intracellular Rx
immune response cascade
2. Ribavirin 2. Nucleoside analogue
3. Boceprevir, telaprevir, simprevir 3. Inhibits replication by binding to the
nonstructural protein, serine protease
4. Sofosbuvir 4. RNA polymerase
Respiratory syncytial virus RSV Ribavirin Nucleoside analogue Rx
infection
Reproduced and adapted from [155]
a
Now only for ophthalmic use
28 R.A. Kaslow

general perspectives and more concrete information, includ- panying illness. The full set of objectives for creating a good
ing the objectives of vaccination, types of immunization for vaccine for active immunization is listed in Table 1.11. Both
viral infections, schedules for immunizing target populations live and killed vaccines are used, and Table 1.12 compares
with agents licensed in the United States, prospects for eradi- the two types. Active viral vaccines containing live attenu-
cation of viral diseases, and national and international pro- ated virus (measles, mumps, rubella, and smallpox viruses,
grams concerned with immunization. For deeper treatment of poliovirus, adenovirus, and VZV) have generally been
the principles and practice of vaccinology, consult Ref. [158]. more immediately, broadly, and durably protective than
killed vaccines or other constructs, especially when they
10.2.1 Active Immunization are administered by the natural portal of entry to produce
Active vaccines are administered with the goal of stimulating local immunity. However, there are a growing number of
antibody production by the host to provide a high degree and exceptions to that rule—live virus preparations that are less
long duration of protection but with no or minimal accom- fully successful (e.g., VZV vaccine against herpes zoster,
nasally administered influenza vaccine) while certain non-
replicating types formulated as recombinants that appear to
Table 1.11 Objectives for a vaccine provide unexpectedly high protection (e.g., HBV and HPV
1. Elicit a good humoral, cellular, and/or mucosal immune response vaccines). In the case of hepatitis A, both inactivated and
that correlates with protection against clinical disease and live attenuated formulations are highly effective, although
reinfection
immunity generated by the latter may be somewhat longer
2. Provide protection lasting at least several years and preferably a
lifetime, similar to that induced by natural infection that leads to lasting [159].
lifetime immunity Limitations to the wide applicability of live vaccines
3. Result in minimal immediate side effects or adverse reactions or include the difficulty of successfully attenuating the candi-
mild disease and result in no delayed effects (e.g., late date strain without reversion to virulence, the avoidance of
reactivation, CNS involvement, or cancer)
viral persistence and the risk of reactivation, and the elimi-
4. Require a simple regimen for administration in a form and a
schedule acceptable to the target population and the general public nation of possible oncogenicity. These have been major
5. Pass a favorable cost/benefit analysis, where the health and hurdles for vaccines against herpesviruses, and it is diffi-
economic benefit of administration clearly outweighs the cost and cult to measure some of these attributes in the laboratory.
risk of natural disease and the adverse consequences of Efforts to produce live vaccines with temperature-sensitive
administration mutants that replicate in the upper respiratory but not in

Table 1.12 Comparison of live Live Killed

and killed vaccines
Immune response
Humoral antibody (IgG) +++ +++
Local antibody (IgA) +++ +
Cell-mediated immunity +++ +
Duration of response Longer Shorter
Epidemiologic response
Prevents reinfection by natural route +++ ++
Stops spread of “wild” virus to others ++ +
Some vaccine viruses (polio) spread to others +++ 0
Creates herd immunity if enough persons are vaccinated +++ 0
Characteristics of the vaccine
Usually heat stable, may need to keep below freezing point
until just prior to administration ++ 0
Vaccine virus may mutate or increase in virulence + +
Antigenic site limited or lost during preparation
(e.g., formalin treatment) 0 +
Contraindicated in immunosuppressed persons +++ 0
Side reactions:
Systemic (viremia) + 0
Local 0 ++
Number of doses for successful take 1 2–3
The table is a simplification and may not apply to all vaccines. Some live vaccines, e.g., polio, are relatively
heat stable. Knowledge of the presence of and degree of protection by cell-mediated immunity is inadequate
for many vaccines
1 Epidemiology and Control: Principles, Practice and Programs 29

19–23 13–15 16–18

Vaccine Birth 1 mo 2 mos 4 mos 6 mos 9 mos 12 mos 15 mos 18 mos 2-3 yrs 4-6 yrs 7-10 yrs 11-12 yrs
mos yrs yrs
nd
Hepatitis B1 (HepB) 1st dose 2 dose 3rddose

Rotavirus2 (RV) RV1 (2-dose 1st dose 2

nd
dose See
series); RV5 (3-dose series) footnote 2
Diphtheria, tetanus, & acel- nd rd
1st dose 2 dose 3 dose 4th dose 5th dose
lular pertussis3 (DTaP: <7 yrs)
Tetanus, diphtheria, & acel-
(Tdap)
lular pertussis4 (Tdap: ≥7 yrs)
Haemophilus influenzae type 1st dose
nd
2 dose
See 3rd or 4th dose,
b5 (Hib) footnote 5 See footnote 5
Pneumococcal conjugate6 nd
1st dose 2 dose 3rd dose 4th dose
(PCV13)
Pneumococcal polysaccha-
ride6 (PPSV23)
Inactivated poliovirus7 (IPV) nd
3rd dose
1st dose 2 dose 4th dose
(<18 yrs)
Influenza8 (IIV; LAIV) 2 doses Annual vaccination (IIV only) Annual vaccination (IIV or LAIV)
for some: See footnote 8
Measles, mumps, rubella9 nd
1st dose 2 dose
(MMR)
nd
Varicella1 0 (VAR) 1st dose 2 dose

Hepatitis A11 (HepA) 2-dose series, See footnote 11

Human papillomavirus1 2 (3-dose

(HPV2: females only; HPV4: series)
males and females)
Meningococcal1 3 (Hib-Men-
CY ≥6 weeks; MenACWY-D
≥9 mos; MenACWY-CRM See footnote 13 1st dose Booster

≥2 mos)

Range of Range of recommended Range of recommended Range of recommended ages Not routinely
recommended ages for ages for catch-up ages for certain high-risk during which catch-up is recommended
all children immunization groups encouraged and for certain
high-risk groups
This schedule includes recommendations in effect as of January 1, 2014. Any dose not administered at the recommended age should be administered at a subsequent visit, when indicated and feasible. The use of a
combinationvaccine generally is preferred over separate injections of its equivalent component vaccines. Vaccination providers should consult the relevant Advisory Committee on Immunization Practices (ACIP) statement
for detailed recommendations, available online at http://www.cdc.gov/vaccines/hcp/acip-recs/index.html. Clinically significant adverse events that follow vaccination should be reported to the Vaccine Adverse Event Reporting
System (VAERS) online (http://www.vaers.hhs.gov) or by telephone (800-822-7967).Suspected cases of vaccine-preventable diseases should be reported to the state or local health department. Additional information, i
ncluding precautions and contraindications for vaccination, is available from CDC online (http://www.cdc.gov/vaccines/recs/vac-admin/contraindications.htm) or by telephone (800-CDC-INFO [800-232-4636]).

This schedule is approved by the Advisory Committee on Immunization Practices (http//www.cdc.gov/vaccines/acip), the American Academy of Pediatrics (http://www.aap.org), the American Academy of Family Physicians
(http://www.aafp.org), and the American College of Obstetricians and Gynecologists (http://www.acog.org).

Fig. 1.5 Recommended immunization schedule for persons aged 0 through 18 years—United States 2013. These recommendations must be read
with the footnotes that follow [161]

the lung have culminated in the licensure of a cold-adapted administered soon (preferably within hours) after exposure
nasal influenza vaccine recommended for use in healthy and when it contains a sufficiently high titer of antibody that
2–49-year-olds. will be effective against the agent.
A comprehensive overview of general immunization Some preparations are derived from persons known to
principles and recommendations can be found in Ref. [160]. be convalescent from the disease and from persons hyper-
The schedule for immunization of immunocompetent infants, immunized against it or by selecting only donors shown to
children, and adolescents is shown in Fig. 1.5 [161], and have high antibody titers. Passive immunization is generally
for immunocompetent adults in Fig. 1.6 [211]. They reflect limited to well-defined exposures to rabies virus or in immu-
recommendations that took effect in 2013 and will obviously be nocompromised patients who are susceptible to HAV, HBV,
modified as new agents and regimens are found to be effective. VZV, CMV, and vaccinia (unlikely in the absence of small-
The above pages of the CDC website are the definitive pox immunization but potentially useful if vaccinia virus
sources of information about the requirements for immuni- gains acceptance as a carrier for other antigens).
zation and details of administration, precautions, contraindi-
cations, and other aspects in the United States. References to
CDC recommendations are also available for immunization 10.3 Disease Eradication and Elimination
in other specific situations: health-care personnel [162], spe-
cial health conditions [163], pregnancy [164], and interna- Smallpox was the first and only disease to have been officially
tional travel [165]. declared eradicated from the earth. From the moment of that
historic accomplishment in 1977, this consummate success
10.2.2 Passive Immunization of the WHO eradication program has inspired initiatives to
Passive immunization with an Ig preparation is an expedi- replicate it with other diseases. The definition of eradication,
ent useful in short-term prevention primarily when it can be telegraphed in Table 1.13, can be more fully understood in
30 R.A. Kaslow

VACCINE AGE GROUP 19-21 years 22-26 years 27-49 years 50-59 years 60-64 years ≥ 65 years

Influenza 2,* 1 dose annually

Tetanus, diphtheria, pertussis (Td/Tdap) 3,* Substitute 1-time dose of Tdap for Td booster; then boost with Td every 10 yrs

4,* 2 doses
Varicella

Human papillomavirus (HPV) Female 5,* 3 doses

Human papillomavirus (HPV) Male 5,* 3 doses

6 1 dose
Zoster

7,* 1 or 2 doses
Measles, mumps, rubella (MMR)

Pneumococcal 13-valent conjugate (PCV13) 8,* 1 dose

9,10 1 or 2 doses 1 dose

Pneumococcal polysaccharide (PPSV23)

Meningococcal 11,* 1 or more doses

Hepatitis A 12,* 2 doses

13,* 3 doses
Hepatitis B

Haemophilus influenza type b (Hib) 14,* 1 or 3 doses

*Covered by the Vaccine Injury Compensation Program

For all persons in this category who Report all clincially significant postvaccination reactions to the Vaccine Adverse Event Reporting System (VAERS). Reporting forms and instructions on filling
meet the age requirements and who a VAERS report are available at www.vaers.hhs.gov or by telephone, 800-822-72967.
lack documentation of vaccination or
have no evidence of previous infection; Information on how to file a Vaccine Injury Compensation Program claim is available at www.hrsa.gov/vaccinecompensation or by telephone 800-338-2382.
zoster vaccine recommended regardless
To file a claim for vaccine injury, contact the U.S. Court of Federal Claims, 717 Madison Place, N.W., Washington, D.C. 20005; telephone, 202-357-6400.
of prior episode of zoster
Recommended if some other risk Additional information about the vaccines in this schedule, extent of available data, and contraindications for vaccination is also available at
factor is present (e.g., on the basis of
medical, occupational, lifestyle, or other
www.cdc.gov/vaccines or from the CDC-INFO Contact Center at 800-CDC-INFO (800-232-4636) in English and Spanish, 8:00 a.m. - 8:00 p.m. Eastern
indication) Time, Monday - Friday, excluding holidays.
No recommendation
Use of trade names and commercial sources is for identificationo nly and does not imply endorsement by the U.S. Department of Health and Human Services.

The recommendations in this schedule were approved by the Centers for Disease Control and Prevention’s (CDC) Advisory Committee on Immunization
Practices (ACIP), the American Academy of Family Physicians (AAFP), the American College of Physicians (ACP), American College of Obstetricians and
Gynecologists (ACOG) and American College of Nurse-Midwives (ACNM).

Fig. 1.6 Recommended adult immunization schedule by vaccine and age group [211]

the context of viral infection as “…disappear[ance] from all Table 1.13 Disease eradication
countries of the world because transmission of the causative
Definitions
organism has ceased in an irreversible manner” [166]. A less Eradication
ambitious goal, elimination, denotes disappearance of the Zero disease globally as a result of deliberate efforts
causal agent within a large geographic area; this goal has Control measures no longer needed
often been viewed as an intermediate step toward eradica- Elimination
tion. The critical distinction made in Table 1.13 is that once a Zero disease in a defined geographic area as a result
disease is eradicated, it is no longer necessary to maintain the of deliberate efforts
elaborate public health apparatus required for ongoing sur- Control measures needed to prevent reestablishment
veillance and control. The implications for achieving either of transmission
Criteria for assessing the eradicability of a disease
result in practice are that (1) every real or potential occur-
Scientific feasibility
rence in every location within the target area must be taken
Epidemiologic susceptibility (e.g., no nonhuman reservoir, ease
seriously, (2) the infection and the interventions to control of spread, naturally induced immunity, ease of diagnosis)
it must be carefully monitored, (3) program structure and Effective, practical intervention available (e.g., vaccine, curative
function must be nimble and thorough in response to any treatment)
suggestion of difficulty or failure, and (4) the rising cost of Demonstrated feasibility of elimination (e.g., documented
preventing each successive case cannot be allowed to justify elimination from island or other geographic unit)
diverting resources away from the goal [167]. Political will and popular support
Only a few other viral diseases have come close enough to Perceived burden of the disease (e.g., extent, deaths, other
effects; relevance to rich and poor countries)
meeting the criteria for potential eradication or elimination to
Expected cost of eradication
receive serious consideration. In 1993 the International Task Synergy of eradication efforts with other interventions (e.g.,
Force for Disease Eradication promulgated its list of recom- potential for added benefits or savings)
mended target diseases for eradication or elimination, and the Need for eradication rather than control
Task Force recommendations and assessment were updated Reproduced from Ref. [167]
1 Epidemiology and Control: Principles, Practice and Programs 31

Table 1.14 Diseases considered as candidates for global eradication by the International Task Force for Disease Eradication
Disease Current annual toll Chief obstacles for eradication Conclusion
Measles 780,000 deaths, mostly Lack of suitably effective vaccine for young infants Potentially eradicable
among children
Mumps Unknown Lack of data on impact in developing countries; Potentially eradicable
difficult diagnosis
Poliomyelitis 2,000 cases of paralytic Insecurity; low vaccine coverage; increased national Eradicable
disease; 200 deaths commitment needed WHA 41.28 (1988)
Rubella Unknown Lack of data on impact in developing countries; Potentially eradicable
difficult diagnosis
Diseases/conditions of which some aspects could be eliminated
Hepatitis B 250,000 deaths Carrier state, infections in utero not preventable; Not now eradicable, but could eliminate
need routine infant vaccination transmission over several decades
Rabies 52,000 deaths No effective way to deliver vaccine to wild animals Could eliminate urban rabies
that carry the disease
Reproduced from Ref. [169]

in 2008 (Table 1.14) [168, 169]. Global efforts against both diseases is at least imaginable, there have been very recent
poliomyelitis and measles have been the most concerted. The resurgences of measles [175], mumps [176], and chicken
successes and setbacks are covered in the respective chapters pox [177], most likely for a combination of reasons: wan-
on the two conditions (Chaps. 13 and 23). ing immunity despite what had been deemed adequate vac-
Although the original Task Force and those who have cination recommendations and coverage, gaps in coverage
updated their work acknowledged certain key obstacles, in among minority and other populations, and more general
reality, even when the commitment is strong, funding is sel- resistance to vaccination stimulated by negative publicity
dom entirely sufficient for the tasks at hand, motivation of all in social media along with pockets of objection on religious
key stakeholders is difficult to sustain, and political and social grounds.
insecurities remain an unpredictable threat. The intensive effort
to eradicate polio has been a test case. In 1988 WHO formally
initiated its campaign to eradicate poliomyelitis. Under the 10.4 Programmatic Approaches
overall leadership of WHO, it and numerous public and private to Immunization
organizations (e.g., CDC, Rotary International, UNICEF, and
the Bill and Melinda Gates Foundation) have persevered in 10.4.1 United States
this mission for more than 20 years [170]. It is noteworthy that Creating, testing, producing, licensing, recommending, and
even before the inception of this more coordinated effort, in monitoring vaccines share many aspects with analogous
1979 Rotary International began its remarkable commitment steps involved in pharmaceutical development and market-
to polio eradication, and for three decades, through its world- ing. Descriptions of those elaborate processes are beyond the
wide chapters, it performed tireless vaccine campaign work scope of this chapter. On the other hand, because vaccines
that has recently captured the attention of major news organi- are administered almost entirely to basically healthy indi-
zations [171, 172, 176]. By 2006 polio remained endemic in viduals, often on a global population scale, various distinc-
only four countries, whereupon gains continued but began to tive programmatic features of their use are worth considering
be punctuated by periodic setbacks. Then new blows began to here briefly. The following summary of the major domestic
be struck in late 2012 as a result of lethal attacks on health- and international programs involved in vaccine development
care workers conducting polio immunization campaigns in and delivery may help the reader to appreciate the enormous
Pakistan and Nigeria and even on their police escorts [173], as commitment to immunization for prevention and control of
well as introductions into countries where war had degraded viral infections in general and to understand how a vaccine
the public health infrastructure. Continual political and social for any specific infection covered in this text fits into this
unrest has fueled the spread of lies about motives of the health larger context.
workers and the consequences of immunization [174]. All of In the United States, besides certain private sector phar-
this turmoil has seriously threatened the eradication program. maceutical manufacturers, the National Institute of Allergy
Unfortunately, even in the United States, where wide- and Infectious Diseases [a component of the National
spread if not universal elimination of other childhood viral Institutes of Health within the Department of Health and
32 R.A. Kaslow

Human Services (DHHS)], the Department of Defense, and tis, rotavirus, rubella, and varicella zoster. There is general
other agencies conduct and/or support a broad variety of agreement that VFC had been a vital force in ensuring rela-
basic microbiological and immunological research fostering tively high levels of immunization in vulnerable populations,
the creation of new and better vaccines. The Food and Drug thereby contributing to the major successes in reducing vac-
Administration oversees the development and licensure of cine-preventable diseases in the past two decades.
all vaccines for human use; through many years of legisla-
tion, regulation, and policy-making, this agency has built Advisory Committee on Immunization
an elaborate system for ensuring that the products of vac- Practices (ACIP)
cine manufacturers are safe and effective. Such pre-market This committee is chartered by Federal law [181] to advise
design, development, and production activities are beyond CDC and, in effect, all government agencies and the pub-
the scope of this chapter. Although these Federal agencies lic at large about the appropriate use of vaccines to control
remain closely involved with monitoring and reviewing the those diseases for which immunizing agents are available.
safety and effectiveness of vaccines once they are licensed The committee meets regularly to review statistics on vaccine-
and distributed, the National Vaccine Program and CDC are preventable diseases; new experimental and other research
the DHHS components in the US Federal government that findings relevant to vaccine safety and efficacy; informa-
have primary authority for implementing and monitoring tion about current vaccines, including labeling and package
their use in populations. The DHHS and particularly the CDC inserts; newly licensed products; policies and guidelines of
are engaged in other activities related to vaccine use, but the other organizations; cost considerations; and other aspects
following paragraphs highlight its principal responsibilities. of immunization policies and programs. Recommendations
may be forthcoming on any of those topics; in addition, rec-
National Vaccine Program Office ommendations may also cover for modification of schedules,
This group oversees and coordinates all of the DHHS agen- for administration of vaccines in the Vaccines for Children
cies and offices involved with vaccine development and uti- Program, or for introduction of new vaccines into the pro-
lization. In 2010, in part with a view toward achieving the gram. This advice carries heavy weight throughout the public
immunization-related objectives established by the Federal health, health-care provider, public and private health insur-
government in Healthy People 2020, the NVP/DHHS pub- ance, and legal communities. The primary legal authority for
lished the 2010 National Vaccine Plan, a carefully articulated matters of public health and disease control is vested in the
set of goals and objectives and recommendations on how to states, and most of them follow ACIP guidelines closely.
reach them [178]. The five overarching goals of the plan
are to (1) develop new and improved vaccines; (2) enhance Vaccine Adverse Event Reporting System (VAERS)
the vaccine safety system; (3) support communications to In 1986, congressional legislation required CDC and FDA to
enhance informed vaccine decision-making; (4) ensure a sta- develop this system for receiving, monitoring, and respond-
ble supply of, access to, and better use of recommended vac- ing to reports of potential side effects and complications
cines in the United States; and (5) increase global prevention of immunization [182, 183]. Every effort is made to docu-
of death and disease through safe and effective vaccination. ment any such untoward event following administration of a
There also a separate implementation plan, which details the licensed vaccine, regardless of the degree of certainty about
tactics to be pursued in meeting the goals [179]. the causal relationship to the vaccination. Each year, the sys-
tem receives some 30,000 reports of events, the vast majority
Surveillance of Vaccine-Preventable Diseases of which are mild.
Through its long-standing relationships with state and local
health departments, CDC has implemented increasingly 10.4.2 International
elaborate systems and methods for reporting of selected WHO
infectious diseases, a number of which are viral and a sub- In 1974 the World Health Assembly, encouraged by the
set of which are vaccine preventable. These systems are success of the smallpox eradication campaign, created the
reviewed in detail in Chap. 4. Expanded Program on Immunization (EPI) [184] to ensure
that children everywhere would receive routine immuniza-
Vaccines for Children Program (VFC) tions. The early goals of the program were to assist in devel-
In 1994 the Federal government initiated and began to fund oping the appropriate immunization policies and systems. In
this program to provide free vaccines to children for whom its evolving role, it has promoted the key objectives of ser-
they would otherwise be unaffordable [180]. As administered vice delivery, vaccine storage with temperature control (cold
by CDC, the program currently provides vaccines against the chain maintenance), timely vaccine distribution, surveillance
following viral infections: hepatitis A, hepatitis B, human of disease and vaccination rates, health-care personnel train-
papillomavirus, influenza, measles, mumps, poliomyeli- ing, and efficient program management.
1 Epidemiology and Control: Principles, Practice and Programs 33

More recently (2006), WHO and UNICEF produced the [187]. The strategy incorporates five themes: making routine
Global Immunization Vision and Strategy (GIVS) [185] vaccines available, introducing new vaccines as they become
aimed at reducing morbidity and mortality from vaccine- available, using innovative and market-based approaches to
preventable diseases during the decade ahead, not just in financing and implementing immunization programs, pro-
children but in all segments of the population. The strategy moting decisions about the deployment of vaccines based
includes immunizing more broadly, introducing a range of on scientifically sound evidence, and advocating for the
new vaccines and technologies, integrating vaccination and support for vaccine programs by other stakeholders. The
other preventive health care, and coordinating programs on Supporting Independent Immunization and Vaccine Advisory
a global level. The overall strategy contains numerous goals Committees Initiative is one of those funded by the Gates
from which countries can select to tailor their own specific Foundation to promote National Immunization Technical
programs. Within only a few years after its adoption, GIVS Advisory Groups (NITAGs) [188]. These groups provide rec-
has been successful in stimulating the establishment of a ommendations on immunization policies and programs (e.g.,
number of national immunization plans. schedules for administration of existing vaccines, improved
coverage, and introduction of new vaccines) [189].
GAVI
As a culmination of the World Economic Forum in Davos,
Switzerland, at the beginning of the new millennium, major 10.5 An Emerging Challenge
stakeholders in the global immunization (UN agencies, donor to Immunization
governments, vaccine industry leaders, aid organizations,
and others) formed a consortium called the Global Alliance Government and nongovernment organizations alike are
for Vaccines and Immunization (GAVI). The mission of this engaged in an enormous multipronged worldwide immu-
entity is to bring new vaccines to all children of the devel- nization enterprise. There are many natural and legitimate
oping world. The specific goals include intense focus on concerns about the prospects for continuing success with
the more than 20 million children worldwide in poor areas each vaccine-preventable disease. The obstacles in every
who would otherwise remain unvaccinated against vaccine- domain—scientific, political, economic, cultural, and oth-
preventable diseases, acceleration of delivery of new vac- ers— are formidable. It was especially distressing to learn
cines to the poorer countries as soon as possible after they that one of those obstacles, opposition on religious/ethnic
are available in the wealthier ones, and channeling support grounds, had motivated the murder of innocent Pakistani and
for academic and industrial research on new vaccines tar- Nigerian health workers in 2012–2013 [190, 191]. In retro-
geted to the developing world. spect, such extreme but, hopefully, isolated acts should not
Early GAVI efforts in the realm of viral diseases con- be all that surprising as part of the spectrum of social or reli-
centrated on vaccines against hepatitis B and yellow fever. gious opposition to vaccination campaigns in countries with
Lately, attention has turned to delivery of rotavirus, the poorly educated populations. However, on a more ominous
second dose of measles, human papillomavirus, Japanese note, beginning in the 1990s, the United States and other
encephalitis, and rubella vaccines. Two examples of current developed countries have witnessed a gradual increase in
initiatives include the intention to immunize 700 million the numbers of parents who are refusing to permit their chil-
children against measles and rubella by 2020 and plans for dren to receive required routine immunizations. Because the
administering HPV vaccine to 180,000 preadolescent girls in refusals have tended to concentrate among certain subsets of
the first phase of a campaign to protect girls in many devel- the population who may be clustered geographically [192,
oping countries against cervical cancer. The alliance has 193], the relatively higher proportions of children whose par-
depended heavily on grants and donor government pledges ents have denied them vaccination have led to outbreaks of
along with private philanthropic contributions, but it has sub- vaccine-preventable disease [194]. The resulting increased
stantially capitalized on more innovative financing for pur- risk of such diseases as measles engenders particular con-
chases of existing vaccines and for mutual assurances about cern because children unvaccinated against them not only are
the future availability of vaccines and the funds needed to vulnerable in their own right but also pose significant risk
purchase and deliver them. More details about the goals and to their contacts who may remain unprotected—because of
accomplishments of GAVI to date can be found at Ref. [186]. exemption from vaccine requirements, a medical precaution
or contraindication for live vaccine, vaccination exclusion
Bill and Melinda Gates Foundation for young age, or inadequate response to vaccine [195].
As a powerful force in the quest to control vaccine- Religious beliefs have often been cited reasons for request-
preventable diseases, for years this foundation has supported ing exemption, but other anti-vaccine forces are at work too
both traditional and innovative approaches to the develop- [196]. Although incidents with contaminated products had
ment and delivery of vaccines for the places in greatest need raised largely transient concern in the more distant past [197],
34 R.A. Kaslow

it was the reported but now thoroughly discredited research 6. Shin YC, Joo CH, Gack MU, Lee HR, Jung JU. Kaposi’s sarcoma-
on the role of vaccination as a cause of autism that probably associated herpesvirus viral IFN regulatory factor 3 stabilizes
hypoxia-inducible factor-1 alpha to induce vascular endothelial
accounted for the first more serious and lasting rupture in growth factor expression. Cancer Res. 2008;68:1751–9.
public confidence in the benefits of immunization in general 7. Rosenberg PS, Goedert JJ, Biggar RJ. Effect of age at serocon-
[198, 199]. The news media, in their predilection for con- version on the natural AIDS incubation distribution. Multicenter
troversy, have not always presented a properly iunbalanced hemophilia Cohort Study and the International Registry of
Seroconverters. AIDS. 1994;8:803–10.
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N, Levine MM. Enteric adenovirus infection and childhood diar-
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2007;31:25–31. Jones & Bartlett; 2012.
 Diagnosis, Discovery and Dissection
of Viral Diseases 2
W. Ian Lipkin and Thomas Briese

Only a few years ago, viral diagnosis was largely an exercise 1 Emerging Infectious Diseases
for academic researchers and public health practitioners with and Biodefense
focus on epidemiologic analyses and outbreak prevention,
detection, and control. Opportunities for therapeutic inter- In an era when travel and trade are increasingly global,
vention were limited to only a few applications such as her- patients with what were once considered exotic infectious
pesvirus infections, influenza, and HIV/AIDS; hence, once a diseases restricted to the developing world, like dengue
bacterial or fungal infection was excluded, clinicians were fever, Ebola, or chikungunya, now present in clinics and
limited to providing supportive care for what was presumed emergency rooms in North America and Europe. Nonstop
to be a viral syndrome. Public health organizations tracked flights of less than 24 h connect the world’s major airports;
the incidence of viral infections and the development of hence, physicians must be prepared to expect the unexpected.
resistance to the few antiviral drugs in use and provided input In New York City, for example, more than 12 million pas-
to governments and the pharmaceutical industry regarding sengers annually pass through John F. Kennedy (JFK) airport
selection of vaccine targets. More recently, interest in viral from more than 100 international destinations. With this
diagnostics has burgeoned with the advent of new tools for traffic volume in one metropolitan airport alone, it is not sur-
detection and discovery, global recognition of pandemic risk, prising that human and stowaway passengers like mosqui-
high-throughput drug screening, rational drug design, and toes have been implicated in the transmission of West Nile
immunotherapeutics. An additional impetus has been the virus, HIV, influenza virus, Mycobacterium tuberculosis,
implication of viruses in chronic illnesses not previously SARS coronavirus, and chikungunya virus. Exotic agents
attributed to infection. The objective of this chapter is to can also transit internationally in legal and illegal (bushmeat)
review the factors responsible for the rise in awareness of food products and companion animals. The annual traffic in
viral infections, methods for diagnosis and monitoring viral bushmeat through Charles de Gaulle airport in Paris is esti-
infections, and future prospects for improvements in discov- mated at 273 tonnes [1]. In work with nonhuman primate,
ery, detection, and response to the challenges of clinical rodent, and bat bushmeat seized at JFK by the Wildlife
virology. Conservation Society, EcoHealth Alliance, and the Centers
for Disease Control and Prevention, we have found evidence
of infection with retroviruses, herpesviruses, and pathogenic
bacteria [2]. Illegal importation of companion animals such
as birds, primates, and rodents has been linked to outbreaks
of poxviruses and Salmonella [3, 4].
Approximately 70 % of emerging infectious diseases are
zoonoses—infections that are transmitted to humans from
W.I. Lipkin, MD (*)
Departments of Epidemiology, wildlife or domestic animals [5, 6]. The majority of zoonotic
Neurology and Pathology, Center for Infection and Immunity, diseases can be attributed to anthropogenic change. Loss of
Columbia University College of Physicians and Surgeons, wildlife habitat to development and consumption of bush-
722 West 168th Street, Room 1703a, New York, NY 10032, USA
meat necessitated by poverty or due to cultural preference
e-mail: [email protected]
increases opportunities for cross-species jumps. Global
T. Briese, PhD
warming may also increase the geographic range of phlebo-
Department of Epidemiology,
Columbia University Mailman School of Public Health, tomus insects like mosquitoes and ticks that serve as reser-
722 West 168th Street, Room 1703a, New York, NY 10032, USA voirs and vectors for infectious agents [7]. Given that there

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 39

DOI 10.1007/978-1-4899-7448-8_2, © Springer Science+Business Media New York 2014
40 W.I. Lipkin and T. Briese

are more than 50,000 vertebrate species, if we assume an of access to the appropriate sample. Infectious agents that do
average of 20 endemic viruses per vertebrate species, the not shed into saliva, nasopharyngeal secretions, blood, urine,
potential reservoir of vertebrate viruses can be estimated at CSF, or feces may be detected in tissue biopsies. Alternatively,
one million. Although it is unlikely that all of them can be pathogenetic mechanisms may be indirect, or consequences
transmitted to humans and cause disease, it is sobering to of infection may be delayed obscuring the relationship
consider the challenge of detecting and responding even to between the causative agent and the disease.
1 % of them (10,000 novel viruses). The most straightforward mechanisms for viral pathogen-
In the aftermath of the fall of the Twin Towers on esis are cellular damage due to replication and lysis, apopto-
September 11, 2001, in New York City, and the anthrax sis, autophagy, or immune responses to proteins expressed
attacks that followed, many western governments became on infected cells. However, viruses can also induce systemic
concerned about bioterrorism. Early investments in surveil- damage through cytokine storm resulting in shock, acute
lance for biological weapons gave way to surveillance for respiratory distress, and/or organ failure, cause immunosup-
emerging infections when sober reflection led to recogni- pression resulting in opportunistic infection, or break toler-
tion that the latter were more likely threats to public health. ance for self, resulting in autoimmune disease. Infection can
However, advances in synthetic biology over the past be cryptic, impairing differentiated cell functions like hor-
decade have been so dramatic that clinicians and public mone secretion, inducing neoplasia, or impairing develop-
health practitioners must again consider the possibility that mental programs that may not become apparent for months
high-threat known and novel pathogens may arise through or years. In summary, the challenge in viral diagnostics is to
deliberate genomic manipulation either in the form of bio- develop strategies for not only detecting footprints of the
weaponeering, inadvertent release of high-threat human agent itself in target tissues but also enduring shadows of
pathogens, or legitimate gain-of-function research, whereby infection in accessible compartments.
low-risk agents become high risk. The scientific and larger
communities are currently grappling with the implications
of gain-of-function research in the context of experiments 3 Culture
designed to understand virulence and transmission of H5N1
(avian) influenza viruses [8–13]. Once the mainstay of viral diagnostics, culture now receives
less emphasis in clinical microbiology, chiefly because
assays require days rather than hours; thus, information
2 Impact of Mechanisms obtained is unlikely to directly impact patient management.
of Pathogenesis on Viral Diagnostics Culture nonetheless continues to play an important role in
public health as well as basic and clinical research because it
Establishing a causative link between a virus and disease can enables insights into pathogenesis and the efficacy of drugs,
be straightforward or complex. In some instances, the virus antibodies, and vaccines. The presence of virus can be
responsible for the induction of disease is present at a site of detected by changes in cell morphology at the level of light
organ pathology, and there is precedent for the same or a microscopy including lysis, rounding, and syncytia forma-
related virus causing similar disease. A classic example is tion—fusion of cells as revealed by an increase in size and
herpes encephalitis where the detection of herpesvirus the presence of more than one nucleus—visualization of
sequences by polymerase chain reaction (PCR) in the cere- pathognomonic structures by electron microscopy; or viral
brospinal fluid (CSF) of a patient with encephalitis provides proteins that bind antibodies as revealed through immuno-
a clear diagnosis and suggests a specific therapeutic inter- histochemistry or immunofluorescent microscopy. A wide
vention [14]. PCR alone can be inadequate. In West Nile range of cell lines has been established for culturing viruses.
encephalitis, PCR of CSF is less reliable than assays of CSF Some are immortalized; others are primary cultures that can
for IgM antibodies to the virus [15, 16]. In some instances, only be propagated for a few generations. Although some
the footprints of an agent cannot be found in or adjacent to viruses can grow in many cell types, others have fastidious
the affected organ but can be detected in other compartments. requirements. Some viruses have never been cultured despite
PCR detection of enterovirus, for example, in the feces of a implication in disease. In some instances, propagation fail-
patient with aseptic meningitis, provides strong evidence of ure may be overcome by adaptation with serial passaging in
enteroviral meningitis [17]. Despite these examples of suc- the presence of a second, permissive type of cell (cocultiva-
cess, an etiological agent is not identified by any test in up to tion), the use of antibodies or RNAi to suppress innate
70 % of what is presumed to be viral encephalitis. Similar immune responses, or cells obtained from genetically modi-
figures pertain in viral pneumonias. fied animals. However, serial passaging can lead to adapta-
There are several explanations for surveillance and diag- tion, including changes in virulence (the capacity of the virus
nostic failure. In some instances, the problem is simply lack to cause disease) or tropism (the cells and organs the virus
2 Diagnosis, Discovery and Dissection of Viral Diseases 41

can infect). Indeed, serial passage may be utilized to develop for detecting and quantitating a specific known agent [22,
less virulent strains that can be used as vaccines. A potential 23], these assays may nonetheless fail with templates of vari-
confound in characterizing samples that may contain more able sequence composition, especially if this affects the
than one virus is that the culture environment can select for region of reporter molecule binding. This can be particularly
the agent that is more fit to replicate—that may or may not be challenging in the diagnosis of RNA virus infections as RNA
the agent of interest. In an attempt to address this potential viruses are characterized by high mutation rates and include
confound as well as to propagate viruses that fail to grow in species with high genetic strain variability. In comparison,
simple cultures, investigators have developed cultures that consensus PCR assays are less likely to be confounded by
include more than one cell type. In some instances these sequence divergence but are also less sensitive than the spe-
complex cultures are designed to replicate the architecture of cific PCR assays. Nested PCR tests that can employ consen-
an organ like the respiratory tract. An alternative to culture in sus or specific primers in two sequential amplification
cells is animal inoculation. An advantage of animal inocula- reactions with either one (hemi-nested) or two (fully nested)
tion is that the presence of a wide range of cell types is asso- primers located 3′ with respect to the first primer set may
ciated with expression of a wide range of receptors that may both accommodate sequence variation and be more sensitive
allow virus entry. Most investigators use suckling mice than fluorescent or beacon-based singleplex assays. However,
because their innate immune responses are immature. Others whereas in quantitative fluorescence- or beacon-based real-
use mice genetically modified to abrogate immune responses. time assays reporter readings are taken indirectly without
opening the reaction vessels (“closed system”), nested PCR
systems bear a high risk of contamination because of the
4 Molecular Assays transfer of (amplified) material from the first to the second,
nested reaction [24, 25], even if scrupulous experimental
Nucleic acid tests (NATs) have largely replaced culture in hygiene is observed. Recently, automated (closed) systems
viral diagnostics due to advantages in cost, speed, and ease have been developed that allow contamination-free transfer
of use. Common NAT platforms include polymerase chain between separate reaction compartments of single-use car-
reaction (PCR), in situ hybridization, microarray, and high- tridges that may present new opportunities for nested assay
throughput sequencing. design.

4.1 Singleplex Assays 4.2 Multiplex Assays

These assays, designed to detect individual viruses, are the As signs and symptoms of disease are rarely pathognomonic
most common NATs employed in clinical microbiology. of a single agent, particularly early in the course of an illness,
They take several forms, but quantitative real-time PCR, many microbial candidates must be entertained simultane-
wherein nucleic acid replication results in either cleavage or ously. Multiplex NATs provide such an opportunity. The
release of a fluorescence-labeled probe oligonucleotide that number of candidates considered may range from 10 to 50
binds to a sequence region between the regular forward and with multiplex PCR systems to thousands with microarray
reverse primers, is the most popular. The continuous (“real platforms to the entire tree of life with unbiased high-
time”) reading of the reporter fluorescence signal affords throughput sequencing approaches. However, genetic targets
these systems with unprecedented dynamic range and low compete for assay components in multiplex assays, and thus
false-negative rate. The required equipment, thermal cycler they may be less sensitive than a singleplex assay. In com-
with fluorescence detector and (laptop) computer for data pensation, multiplex assays provide the advantage of consis-
analysis, is cost competitive, and rugged battery-powered tently interrogating each sample for a wide range of agents
instruments are available for field use. Loop-mediated iso- without the selection bias introduced by singleplex testing.
thermal amplification (LAMP) tests do not require program- This comprehensive coverage is particularly important for
mable thermal cyclers [18–20]. In the laboratory, LAMP surveillance and applications.
products are detected in conventional dye-stained agarose
gels, but in field applications the estimation of product accu- 4.2.1 Multiplex PCR
mulation through turbidity or dye reading by the naked eye is Multiplex PCR assays are more difficult to establish than sin-
also possible [21]. The sensitivity of all such assays is high- gleplex assays because primer sets may differ in optimal reac-
est when primers and/or probe sequences perfectly match the tion conditions (e.g., annealing temperature or magnesium
selected single genetic target. Fluorescence-based TaqMan concentration). Furthermore, complex primer mixtures are
or molecular beacon assays, for example, typically have more likely to result in primer-primer interactions that reduce
detection limits of <10 molecules per assay. Although ideal assay sensitivity and/or specificity. To advance multiplex
42 W.I. Lipkin and T. Briese

primer design, we developed Greene SCPrimer, a software via a divergent PCR product weight, but like MassTag PCR,
program that automates consensus primer design over a mul- it too requires subsequent sequencing of the product for
tiple sequence alignment with customizable primer length, detailed characterization. A wide variety of syndrome-spe-
melting temperature, and degree of degeneracy [26]. cific MassTag PCR panels have been developed and applied
Gel-based multiplex PCR assays are limited by size dif- to the detection of viruses, bacteria, fungi, and parasites asso-
ferentiation of the amplification products in agarose gels and ciated with acute respiratory diseases, diarrheas, tick-borne
the concomitant requirement for short product sizes (approx. diseases, encephalitides/meningitides, and hemorrhagic
90–250 base pairs) to ensure high sensitivity and fidelity [24, fevers [41–50].
25]. Multiplexing can be achieved in fluorescence- or Although multiplex PCR methods are designed to detect
beacon-based real-time assays to the degree by which differ- known agents, they can nonetheless facilitate pathogen dis-
ent fluorescent reporter emission peaks can be unequivocally covery. MassTag PCR requires only two differently tagged
separated. At present up to five fluorescent reporter dyes are primers per target that may include degenerate positions to
detected simultaneously, although multiplexing may be address genetic variation of larger taxonomic groups such as
increased to some extent by double-labeling strategies and/or a whole species or genus, and its use to investigate influenza-
melting curve analyses. “Sloppy Molecular Beacons” like illness in New York State revealed the presence of a
address this limitation in part by binding to related targets at novel rhinovirus clade by the employed conserved enterovi-
different melting temperatures [27]; however, they are not rus/rhinovirus primer set [42]. This discovery enabled fol-
suited to detect targets that differ by more than a few low-up studies across the globe wherein this third species of
nucleotides. rhinovirus, rhinovirus C, was implicated not only in
The Bio-Plex (or Luminex) platform employs flow cytom- influenza-like illnesses but also in asthma, pediatric pneumo-
etry to detect multiple PCR amplification products bound to nia, and otitis media [44, 51–63].
matching oligonucleotides that are attached to differently
colored fluorescent beads [28, 29]. By combining multiplex 4.2.2 Microarray Assays
PCR amplification systems with various protocols for direct Whereas multiplex PCR systems support rapid high-
or indirect (tag-mediated) bead hybridization of the prod- throughput diagnosis with highest sensitivity for a limited
ucts, assay panels have been developed that permit detection number of agents, microarray-based systems provide detec-
of up to approx. 20 genetic targets simultaneously [30–32]; tion of all known pathogens for which sequence information
the most commonly used respiratory panels range from 9 to is available, but at the expense of some degree of sensitivity.
20 plex [33–37]. Like real-time PCR, these assays rely for Modern printing technologies can generate high-quality
assay specificity on a three-oligonucleotide interaction with arrays with several million features, a printing density that
the target sequence. They are thereby limited in their toler- enables not only detection of a wide range of infectious
ance for mutated or variant templates when compared to agents but also discrimination of medically important types
mass spectroscopy (MS)-coupled platforms that require only or subtypes. Examples of the latter application include respi-
two oligonucleotide-binding sites, such as MassTag PCR or ratory virus resequencing arrays that identify the different
the Ibis T5000 system. influenza virus HA and NA subtypes [64–68].
Two platforms are established that combine PCR with MS The discovery array platforms currently in use are the
for sensitive, simultaneous detection of large numbers of tar- GreeneChip and the Virochip [69, 70]. The panmicrobial
gets. The Ibis T5000 system uses matrix-assisted laser version of the GreeneChip, addressing viruses and in addi-
desorption/ionization (MALDI) MS to directly determine the tion pathogenic bacteria, fungi, and parasites, led to the rec-
molecular weights of the generated PCR products and to ognition of Plasmodium falciparum infection in a case of
compare them for identification with a database of known or unexplained fatal hemorrhagic fever during the 2004–2005
predicted product weights [38–40]. MassTag PCR uses atmo- Marburg virus outbreak in Angola [70]. A variant of the
spheric pressure chemical ionization (APCI) MS to detect GreeneChip facilitated recently the implication of Reston
molecular weight reporter tags attached via a photo-cleavable Ebola virus in a respiratory disease outbreak on pig farms in
linkage to PCR primers [41]. Whereas the Ibis system or the the Philippines [71]. In 2003, the Virochip supported the
subsequent electrospray ionization (ESI)-based Plex-ID sys- characterization of the SARS coronavirus and was also used
tem requires analytical MS to determine the exact weight of subsequently to diagnose parainfluenza virus 4 and infection
the PCR products and thus depends on advanced mass spec- with a human metapneumovirus variant in cases of acute
troscopic data analysis, MassTag PCR can be performed respiratory disease [69, 72, 73].
using smaller instruments and does not require sophisticated Both platforms rely on random PCR strategies to amplify
analyses because it only records the known masses of the and label nucleic acids for detection. In comparison to mul-
40–80 reporter tags used in a given multiplex test. The Ibis tiplex consensus PCR methods employed with some tar-
system may be able to alert of variants of known organisms geted array applications or resequencing arrays, this limits
2 Diagnosis, Discovery and Dissection of Viral Diseases 43

sensitivity especially with complex sample types. In tissue of known and novel agents, as well as detection of genetic
specimens, for example, the sensitivity may not exceed 106– features that may be associated with drug or vaccine resis-
107 copies per assay because host and pathogen nucleic acids tance, or provide insight into provenance and evolution.
compete for PCR reagents. Thus, these platforms have been
more successful with samples containing comparatively low
levels of competing nucleic acid, such as virus culture super- 6 Proof of Causation
natant, serum, respiratory specimens, spinal fluid, or urine.
Improvements in sensitivity to a range of 103–104 copies per Finding the nucleic acid footprint of a virus is frequently only
assay have been achieved with methods for host DNA diges- the first stage in implicating it in disease. There is no func-
tion and/or the depletion of host ribosomal RNA (rRNA) tional equivalent in viruses to the pathogenicity islands found
prior to amplification through subtraction or use of random in bacteria, wherein specific sequences acquired through hori-
primers selected for lack of complementarity to rRNA [74]. zontal gene transfer confer specific pathogenic properties. The
In current array platforms, virus detection is achieved via best established criteria for proof of causation in infectious
fluorescent reporter systems—either through direct incorpo- disease were developed in the late 1800s by Koch and Loeffler
ration of fluorescent nucleotides into the PCR product that is [80]. Known as Koch’s postulates they stipulate that an agent
bound to the array or with a “sandwich approach” whereby be present in every case of the disease, be specific for the dis-
fluorescent-branched chains of DNA are added to the prod- ease, and be sufficient to reproduce the disease after culture
uct after it is bound to the array [75, 76]. However, new and inoculation into a naive host. In the 1930s, Rivers sug-
arrays are in development that will detect viral sequences gested that the development of specific immunity to an agent
through changes in electrical conductance. Such platforms following the appearance of disease could be used in demon-
would enhance portability by eliminating the need for fluo- strating causation [81]. Adapting the original postulates to the
rescent scanners. They may also increase sensitivity and molecular era, Fredricks and Relman later established that
reduce costs by eliminating the need for PCR amplification. microbial sequences may be used as surrogates for culturing
the actual organism [82]. Lipkin and colleagues recently
established levels of confidence in the strength of association
5 High-Throughput Sequencing between an agent and a disease that considers viral burden and
distribution, specific immunity, and prevention or ameliora-
High-throughput sequencing has transformed microbiology tion of disease with use of specific drugs or vaccines [12].
by enabling discovery as well as diagnostics. Unlike PCR or Given the sensitivity of molecular methods, it is imperative
array methods where investigators must choose the patho- that physicians and researchers consider the biological plausi-
gens to be considered or are limited by known sequence bility of an assay result and, where feasible, pursue confirma-
information, high-throughput sequencing has the potential to tion with an independent assay, particularly when engaged in
simultaneously detect not only all viruses but also bacteria, pathogen discovery.
fungi, and parasites. Although the technology is presently
limited to specialized laboratories, sequencing is becoming
increasingly accessible as instruments become smaller, 7 Future Perspectives
methods become more user friendly, and costs decrease.
Over the past 10 years, the cost has decreased 10,000-fold NATs are rapidly replacing classical culture methods in clin-
from $5,000 per 1,000 nucleotides in 2001 to $0.5 per 1,000 ical microbiology laboratories. Although some NATs, such
nucleotides in 2012 [77]. Even more impressive perhaps is as microarrays and high-throughput sequencing, still require
the time required to generate sequence data. Projects that substantial investment in equipment and personnel, diagnos-
required weeks only a decade ago are now completed in tic platforms are becoming more accessible and less expen-
hours [78]. sive through miniaturization and improvements in methods
Current sequencing platforms analyze libraries of ampli- for bioinformatic analysis. Systems using handheld microar-
fied nucleic acids. However, some platforms in development rays, for example, are in development that will ultimately
will have the capacity to directly sequence nucleic acid. enable diagnosis at the bedside or in the field. Benchtop
Irrespective of the platform, raw sequence reads are filtered sequencers are also in production. It is inevitable that as
for quality and redundance before assembly into contiguous sequencing costs continue to decrease, clinicians will seek
sequence streams. These streams, known as contigs, as well information concerning not only the presence of a single
as reads that cannot be assembled, are aligned to databases candidate organism but also the predisposition of the host to
using bioinformatic algorithms that examine homology at disease based on genetic factors and coinfections with other
the nucleotide and deduced amino acid levels in all six poten- microflora. These improvements will bring dramatic benefits
tial reading frames [79]. The alignments allow identification to medicine and public health.
44 W.I. Lipkin and T. Briese

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 Immunological Detection
and Characterization 3
Robert L. Atmar

1 Introduction the disease process very difficult. Viruses such as cytomega-

lovirus (CMV), adenoviruses, and enteroviruses are shed in
Immunological methods are valuable strategies for the disease states but also may be shed asymptomatically for
diagnosis and characterization of viral infections. These
­ prolonged periods of apparent good health. An additional
methods rely on antigen–antibody interactions, and they can complicating factor is the differentiation of primary infec-
be adapted to allow direct detection of the virus (antigen tion from reactivation of disease, a problem common to
detection) or to identify the host’s immune response to the the study of infections with viruses such as herpes simplex
virus infection (antibody detection). The methods are also (HSV) or CMV, especially in immunocompromised hosts
used to identify and characterize virus isolates following in such as transplant recipients or patients infected with HIV.
vitro or in vivo propagation. Although molecular detection is Interpretation of laboratory results in such situations requires
often more sensitive than antigen detection, immunological a thorough understanding of the pathogenesis and epidemi-
methods still have an important role in the study of the epi- ology of the virus.
demiology, pathology, and assessment of clinical disease Failure to detect a virus or viral antigen does not necessar-
associated with viral infection. ily mean that the virus was not present previously or did not
The development of simple, rapid, and often relatively cause disease. Although failure to detect a virus may be a
inexpensive antigen detection test kits has revolutionized result of inappropriate or inadequate specimen collection
both clinical care and laboratory practice. An understanding and handling, it may also be a function of the time course of
of various detection methods is increasingly important in the the disease, the age or antibody status of the patient, and the
design and interpretation of epidemiologic studies. The vast technical resources available to detect or cultivate the virus.
array of laboratory tests now permits enhanced detection of The investigator today has many options to diagnose the
viral antigens, although the clarification of the classic issue presence or past presence of a viral infection, but careful epi-
of “causation” of disease remains blurred. demiologic and laboratory studies are still required to ulti-
The significance of detection or lack of detection of a mately link the viral agent to a specific disease process.
virus or viral antigen remains difficult to interpret. Isolation
of a virus from a normally sterile site, such as tissue, cere-
brospinal fluid (CSF), or blood, is generally highly signifi- 2 Historical Background
cant and usually establishes the etiology of the infection.
The identification of certain viruses, such as influenza or The recognition of immune-mediated virus neutraliza-
respiratory syncytial virus (RSV) in respiratory specimens, tion dates back to the late 1800s when Sternberg extended
also is diagnostic because an asymptomatic carrier state has observations of other scientists of the time and described
not been shown to exist. However, prolonged and generally the neutralization of vaccinia infectivity using serum from a
asymptomatic excretion or shedding of other viruses can recently vaccinated calf [1]. It was another several decades
make the determination of the effect of a particular virus on before diagnostic viral serological methods were developed,
including complement fixation in the 1930s and hemaggluti-
nation inhibition in the 1940s [2]. Immunofluorescent meth-
R.L. Atmar, MD ods for detecting virus antigen were developed in the 1950s
Departments of Medicine and Molecular Virology and Microbiology,
Baylor College of Medicine, 1 Baylor Plaza, MS BCM280,
by labeling virus-specific antibodies with a fluorescent
Houston, TX 77030, USA reporter such as fluorescein isothiocyanate [3]. A little more
e-mail: [email protected] than one decade after the initial description in 1959 of the

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 47

DOI 10.1007/978-1-4899-7448-8_3, © Springer Science+Business Media New York 2014
48 R.L. Atmar

­radioimmunoassay [4] for detecting human plasma insulin, A related concept is antibody avidity, which is a measure of
the method was adapted for virus antigen detection [5]. The the overall binding strength of an antibody to the correspond-
radioisotope used as a reporter was replaced later during the ing antigen. Antibody avidity is correlated to its affinity, but
1970s with enzymes such as alkaline phosphatase and horse- it is also affected by the number (or valency) of interactions
radish peroxidase that allowed colorimetric detection [6]. between the antibody and antigen. For IgG antibody, the
The method was also readily modified (see below) to allow valency is up to two. Since the antigen–antibody interaction is
detection of antibody. A further modification of immunoas- in equilibrium, the presence of multiple binding interactions
say detection methods followed with the use of substrates can maintain the antigen–antibody complex once formed dur-
that allowed detection of the antigen–antibody interaction ing periods of dissociation between an epitope and paratope.
via measurement of a chemiluminescent reaction. There are many factors that affect the interaction between
The diagnostic procedures used for evaluation of specific an antigen and antibody and the ability to one or the other in
viral infections are presented in individual chapters of this a diagnostic assay. These include the temperature of the reac-
book. However, certain common aspects of immunological tion conditions and the ionic strength of the solutions used in
methods used for virus diagnosis are important for the design the assay. As noted above, antibody affinity and concentra-
and implementation of epidemiologic studies. These general tion are particularly important when developing an antigen
issues are addressed below. detection assay. The relative concentration of antigen-­
specific antibody is lowest in postinfection sera. It can be
increased through hyperimmunization with the antigen to
3  rinciples of Antigen–Antibody
P generate polyclonal antisera, and the relative concentration
Interactions can be increased further by affinity purification of the anti-
body. The highest relative concentrations are reached through
An antigen is defined as a substance capable of stimulating the generation of monoclonal antibodies, and selection of
an immune response when introduced into the body (e.g., of high-affinity monoclonal antibodies for use in diagnostic
an immunized animal). When the immune response is the assays is now widely used in the development of antigen
production of antibody, the generated antigen-specific anti- detection assays [7].
body can bind directly to the antigen. The portion of the anti- Another factor that can influence the assay development
gen recognized by and specifically interacting with the is nonspecific interactions between the antibody and other
antibody molecule is called an epitope. An antigen may have substances in the reaction mix (e.g., attachment to the reac-
more than one epitope. When this occurs the epitopes either tion vessel, other microbial antigens). Although these are
may be distinct molecular structures (and thus distinct epi­ generally low-affinity interactions, the concentrations of the
topes) or they may be the same molecular structure (the same competing substances can be high enough to affect the read-
epitope) repeated many times. The portion of the antibody out of the assay. Many of the low-affinity interactions can be
interacting with the antigen is called a paratope. The ability removed by washing steps, use of blocking reagents and use
of the epitope and paratope to bind to each other is the basis of lower concentrations of antibody, but each antibody used
of the specificity of an antigen–antibody interaction. in a diagnostic assay should be evaluated for the presence of
The binding of antibody to antigen is thought to be the these nonspecific reactions. In addition, the use of appropriate
result of electrostatic and van der Waals bonding over short controls can identify problems with assay performance [8].
distances, with the kinetics of the reaction following the law
of mass action. The law can be represented mathematically
as follows: 4 Specimen Collection
K d = [ Ag ⋅ Ag ] / [ Ag ]
The appropriate collection of specimens is of the utmost
importance for the successful identification of viruses in
where Kd is the dissociation constant, [Ag ⋅ Ag] is the con- clinical samples. The source of the specimen, the timing of
centration of the antigen–antibody complex, [Ag] is the anti- collection in relation to onset of symptoms, the rapidity and
gen concentration, and [Ab] is the antibody concentration. method of delivery to the laboratory, and the clinical and epi-
From this law, the formation of antigen–antibody com- demiologic data provided to the laboratory all are important
plexes reaches equilibrium, and the amount of complex variables that relate to the likelihood of successful identifi-
formed is proportional to the concentration of the antibody cation of a viral pathogen. Knowledge of the restrictions of
and antigen present. The binding constant is a measure of the the diagnostic assay to be used is also important; many anti-
strength of interaction between one epitope and one paratope gen detection assays are only approved for use when applied
and is also referred to as the affinity of the antibody. The to a limited number of clinical sample types. For example,
higher the affinity of a paratope for a corresponding epitope, some influenza antigen assays should only be used with
the greater the strength of binding and the lower the Kd value. nasal swabs while other assays are approved for detection
3  Immunological Detection and Characterization 49

of ­influenza in a broad array of respiratory samples types, sample should be obtained 2–4 weeks later to look for a rise
including nasal aspirate, nasopharyngeal swab, throat swab, in virus-specific antibody titer.
and bronchoalveolar lavage [9].

4.3 Clinical Data

4.1 Source
Clinical information may be useful in helping one choose the
The clinical syndrome caused by a virus and its pathogenesis types of diagnostic assays that should be performed on a
of infection determine the specimen(s) that is most appro- clinical specimen. The time of year and age of the patient are
priate for virus identification. Viruses that primarily cause examples of epidemiologic information that will influence
disease at a mucosal surface or cause vesicular skin lesions the likelihood of identifying certain viral infections. For
generally can be identified in specimens taken from those example, rotavirus infections occur seasonally and are more
sites. However, viruses causing generalized or congenital dis- common in young children, while norovirus infections often
ease or causing symptoms in an internal organ (e.g., central occur in outbreaks and are more common in older individu-
nervous system) often can be identified in specimens taken als. Enteroviruses are the most common cause of viral men-
from multiple different sites. Viruses that cause respiratory ingitis and tend to occur seasonally in epidemics, whereas
tract disease such as influenza viruses, RSV, and rhinoviruses HSV type 1 is the most common cause of sporadic viral
are most frequently identified in samples of respiratory secre- meningoencephalitis. Knowledge of the pertinent epidemio-
tions; viruses that cause gastroenteritis, such as rotaviruses, logic information will permit the use of appropriate enzyme
caliciviruses, and astroviruses, are identified in fecal speci- immunoassays (EIAs), immunofluorescence assays, and
mens; and viruses that cause generalized or congenital dis- other diagnostic methods to identify a potential viral patho-
eases, such as measles, CMV, and mumps, are identified from gen in the clinical specimen.
respiratory secretions, urine, and blood. However, antigen
detection methods may not be available for some of these dif-
ferent sample types. Furthermore, some samples may be more 5 Detection of Viral Antigens
likely to yield a positive sample than others (e.g., fecal speci-
men vs. rectal swab for rotavirus and bronchoalveolar lavage The detection of viruses or viral components is the foun-
for respiratory viruses in immunocompromised patients) dation of diagnostic virology. Although the detection of
[10, 11]. The reader is referred to specific chapters for infor- antibodies to specific viral proteins remains an important
mation on the ideal specimen type for specific viruses. element of viral epidemiology, the ability to isolate and/or
characterize viral pathogens initially is critically important.
The performance of any diagnostic test in a reproducible,
4.2 Timing of Collection sensitive, and specific manner is crucial in the study of viral
diseases. Combinations of various techniques, including
Specimens to be used for virus identification should be centrifugation-­enhanced tissue culture, antibody–antigen
obtained early in the course of the illness. For many viral detection, and detection of viral nucleic acid, can be used to
infections, viral shedding begins before the onset of symp- supplement classic tissue culture methods.
toms, peaks during the illness, and disappears around the Methods for the detection of virus-specific antigens have
time that symptoms resolve. There are notable exceptions; allowed rapid identification of a wide variety of viruses
enteroviruses and adenoviruses may be shed in the feces for (Table  3.1). Specific monoclonal antibodies conjugated to
weeks to months, and congenitally acquired CMV is shed in biochemical markers may provide high levels of sensitivity
the urine for prolonged periods. Some factors that influence and specificity [13]. The key to the success of the assays out-
the likelihood of successful virus identification include the lined below is the use of reliable virus-specific antibody.
type of virus, the site from which the sample was obtained, Molecular biological techniques that permit the production
the test being used, the age of the patient being sampled (e.g., of relatively large quantities of avid monoclonal antibodies
younger children shed influenza viruses longer than adults), have facilitated viral antigen detection.
and the immune competence of the patient (e.g., immuno-
compromised hosts shed HSV from genital lesions longer
than do immunocompetent adults) [12]. 5.1 Latex Agglutination Techniques
A serum sample should be collected early in the course of
illness for potential use in identification of a viral infection. Viral-specific antibodies linked to latex beads can be used to
For some viral infections, the identification of IgM antibody detect viral antigens in a clinical sample. The presence of
or the presence of high titers of antibody is sufficient to con- viral antigen in the sample results in cross-linking of
firm a virus infection. A second, or convalescent, serum the beads that can be identified by visual inspection. This
50 R.L. Atmar

Table 3.1  Antigen detection methods used for diagnosis by virus group
Virus group Immunofluorescence Enzyme immunoassay Immunohistochemistry
Enteric
Adenovirus ++
Astrovirus +
Norovirus +
Rotavirus ++
Respiratory
Adenovirus ++ ++ +
Coronavirus + +
Human metapneumovirus ++ ++ +
Influenza ++ ++ +
Parainfluenza ++ ++ +
Respiratory syncytial virus ++ ++ +
Herpes viruses
Cytomegalovirus ++ +
Epstein–Barr virus +
Herpes simplex ++ +
Human herpesvirus 8 +
Varicella–zoster virus ++
Others
Arenavirus ++
Filovirus ++ +
Hantavirus +
Hepatitis B virus ++
Hepatitis D virus +
Human papillomavirus +
Rabies ++ +
Rubeola +
+ occasionally used, ++ commonly used

strategy has most commonly been applied to detection of and examined microscopically for direct visualization of the
viral enteric pathogens like rotavirus and adenovirus, and it fluorescence of the infected cells in the specimen. In the
has the advantage of low complexity and providing rapid indirect IF test, two different antisera are used: an unlabeled
results (in less than 15 min). However, a major disadvantage virus-specific antibody capable of binding to a specific viral
is its relatively lower sensitivity (<70 %) compared to other antigen is used first and is followed by a fluorescein-labeled,
antigen detection methods (>80–90 %) or culture [14, 15]. species-­specific antibody directed against the species in
which the first antibody was raised. If a reaction occurs
between the first antiserum and the clinical specimen, the
5.2 Immunofluorescence Techniques second antibody will bind to the antigen–antibody complex
and fluorescence of the virus-infected cells can be detected.
Viral-specific antibodies conjugated to a fluorescein-labeled The two IF methods each have advantages and disadvan-
moiety have been used to identify viral pathogens since the tages. Both tests allow an assessment of the quality of the
late 1950s [16]. Immunofluorescence (IF) assays are widely sample in that samples that do not contain cells are poor
used for the rapid detection of viruses in clinical samples quality and cannot be interpreted. The indirect IF is usually
and for definitive identification of a virus in tissue culture more sensitive because several fluorescein-conjugated mol-
that may allow viral antigens to be sought before or after ecules potentially are able to bind to each virus-specific anti-
cytopathic effect (CPE) is evident. In the direct IF test body molecule attached to the viral antigen, resulting in
(Fig. 3.1), the virus-specific antibody labeled with a fluores- amplification of the fluorescence. The direct IF test may
cent dye such as fluorescein isothiocyanate or, less com- offer enhanced specificity due to lower background fluores-
monly, rhodamine isothiocyanate is allowed to react for a cence. The indirect IF method requires more reagents and
short time with cells obtained from a clinical specimen or more time to perform. Whether monoclonal or polyclonal
from an inoculated cell culture. After allowing time for an antisera are optimal for use in either test method is still
antigen–antibody reaction to occur, the slides are washed debated. The use of monoclonal antibodies generally
3  Immunological Detection and Characterization 51

Immunofluorescence visualized under routine cell culture conditions [26, 27].

Disadvantages of IF techniques include the need for fluores-
cent microscopes, difficulty in the interpretation of clinical
2°Ab specimens that have a high level of nonspecific fluorescence,
and the fact that prepared slides are not generally stable over
periods longer than 1 month [20].

1°Ab
1°Ab
5.3 Immunocytochemical Staining

Immunocytochemical staining is a sensitive and specific

Ag Ag method for detecting viral antigens with labeled antibodies.
This technique, pioneered by Coons [28] in 1942, has been
used to study the structure and function of a variety of viral
Direct Indirect
proteins and continues to be utilized in both the research and
Fig. 3.1  Schematic of immunofluorescence. Ag antigen, Ab antibody clinical laboratories. It has been used both for detection of
viral infection of a monolayer prior to the appearance of
cytopathic effect and in rapid screening assays for drug resis-
­provides the lowest background but may be limited by the tance [29, 30]. This method utilizes reagents similar to those
high specificity of these reactions. This problem can usually used in the IF assay except that the fluorescent marker is
be overcome by using a pool of monoclonal antibodies. replaced by an enzyme. When enzyme-specific substrates
The use of IF for the direct detection of viral antigens are provided, a colored precipitate forms at the site of reac-
in clinical samples and the confirmation of viral growth in tion. Typical enzymes used to detect viral antigens include
cell cultures has increased with the widespread commercial alkaline phosphatase and horseradish peroxidase. A major
availability of relatively inexpensive antibodies specific for drawback of alkaline phosphatase-based reagents is their
many of the herpesviruses and respiratory viruses. The IF lack of stability; a major drawback of peroxidase as a marker
method has the advantage of allowing rapid viral diagno- is the fact that this enzyme is endogenous to some mamma-
sis in properly obtained specimens [17–20]. When working lian tissue, thus requiring either elimination of the endoge-
with large numbers of clinical specimens, the time required nous enzyme or use of a nonmammalian enzyme, glucose
for sample collection, processing, and interpretation of the oxidase [31].
stained slide becomes substantial. The enthusiasm for this Advantages of immunoenzymatic staining compared to
technique in clinical specimens has varied due to the time IF staining include the virtual permanence of stained prepa-
and degree of technical competence required to read such rations and the ability to view slides using an ordinary light
samples, the availability of other, the less labor-intensive microscope. Both direct and indirect staining with immuno-
antigen detection methods, and the frequency of false-­ peroxidase and other enzymes have been utilized to detect
negative results obtained because of the dependence of the many viruses. Refinements have been developed that allow
assay on having a high degree of viral antigen expression even greater sensitivity than that seen with indirect staining
in the clinical sample. Nevertheless, the appropriate use of without the need to conjugate enzyme to an antibody. For
this test can result in reliable and sensitive rapid diagnosis example, a modification of these techniques has been a four-­
from clinical samples. The use of IF for the detection of RSV layer sandwich technique involving (1) virus-specific anti-
in pediatric patients by an experienced laboratory can detect body raised in species X, (2) an excess amount of a second
up to 90–95 % of the samples positive by culture [21, 22], antibody raised against the species X antibody, (3) a complex
although many laboratories report rates of 60–80 % [23–25]. of peroxidase and antiperoxidase antibody (raised in species
The combination of IF techniques with cell culture has X), and (4) reducing substrate for peroxidase [32]. The sec-
increased the sensitivity of cell culture while providing a ond antibody acts as a bridge, binding to both the virus and
positive result in a shorter time period. With the use of cen- the antiperoxidase antibody. Similar unlabeled assay meth-
trifugation or other methods of enhancement of viral replica- ods have been described for alkaline phosphatase–antialka-
tion and pools of varying antibodies, cell cultures can be line phosphatase and glucose oxidase–antiglucose oxidase
incubated between 1 and 3 days and then stained for a variety [33, 34]. The sensitivity of antigen detection has been further
of virus antigens using indirect or direct IF methods. For improved by more recently developed signal amplification
some viruses, such as CMV or VZV, specific antibodies methods, including avidin–biotin complexes (binding of 4
directed toward early or nonstructural antigens permit biotins per streptavidin), chain polymer-conjugated technol-
the rapid diagnosis within 48 h, well before CPE would be ogy where multiple enzyme and antibody molecules are
52 R.L. Atmar

attached to an inert molecule such as dextran, and the use of Enzyme Immunoassay
tyramine conjugates as substrates for horseradish peroxidase
that allow signal amplication as much as 100-fold [35].
Sub Signal

5.4 Radioimmunoassay
Enz
Radioimmunoassay (RIA) techniques have been valuable for
the detection of many compounds in laboratories and clinical
medicine. Initially, the technique was developed for the
determination of endogenous human plasma insulin levels Detecting Ab
[4]. The first important use of RIA in diagnostic virology
was for the detection of hepatitis B surface antigen [5]. The
original RIA described by Yalow and Berson [36] was a
Ag
competitive binding assay in which the competition between
an unlabeled antigen and a radiolabeled antigen reacting
with a limited amount of antibody over a short period of time
was monitored. Variations in RIA methods have been devel-
Capture Ab
oped, with the most common being the direct and indirect
solid-phase RIA. In the direct solid-phase RIA, antigen or
antibody are captured on a solid support and detected by
radiolabeled (usually 125I) antibody or antigen, respectively.
The amount of signal increases proportionally to the amount Fig. 3.2  Schematic of sandwich enzyme immunoassay. Ag antigen, Ab
of antigen or antibody present in the sample. In indirect antibody, Enz enzyme, Sub substrate
assays, the capture of antigen or antibody to the solid phase
prevents the binding of labeled antibody or antigen, respec- s­ pecific for the enzyme is added and a color reaction occurs
tively, so that the amount of signal detected is inversely pro- that can be monitored by spectrophotometry or by direct
portional to the amount of antigen or antibody present. RIA visualization. The test is quite simple to run, requiring only
methods currently are utilized mainly for the detection of standardization of reagents and techniques such as dilution,
antigens and antibodies of viral hepatitis [37]. The use of incubation, and washing. The principles involved in EIA are
RIA for the detection of various hepatitis markers has dem- similar to those involved in immunofluorescence and RIA,
onstrated the assay’s high degree of sensitivity. For the most but the EIA test has the distinct advantages of being simple
part, RIA methods have been replaced by EIA for routine to perform, utilizing reagents that have long shelf lives, are
diagnostic purposes due to the complexity of the assay, the inexpensive, and do not require sophisticated technical eval-
use of radioisotopes, lack of standardized commercially uation to determine results. Advantages of the EIA technique
available reagents, and high equipment costs. also include sensitivity (less than 1 ng/ml), specificity, rapid-
ity, safety, automation potential, and low cost, particularly
when many specimens require evaluation.
5.5 EIA Variations in the methodology for EIA testing include the
materials used, the procedures for incubation and detection,
Enzyme immunoassays, or EIAs, have gained widespread and the interpretation of results. Many different test kits are
acceptance in virology laboratories for the detection of a vari- commercially available and in widespread clinical use for
ety of viral antigens and antibodies. The assays used in this the detection of common viral pathogens such as RSV, influ-
method rely on antibodies directed against a specific virus or enza, adenovirus, HIV, norovirus, and rotavirus; EIA tests
viral antigen that are adsorbed or directly linked to polysty- have been devised and reported for nearly all virus groups
rene wells in microtiter plates, plastic beads, or membrane-­ and continue to be used widely for clinical and research
bound material. When viral antigen is present in a specimen, purposes.
it binds to the immobilized “capture” antibody and a sec-
ond “detecting” antibody conjugated to an enzyme such as
horseradish peroxidase or alkaline phosphatase then attaches 5.6 Optical Immunoassay (OIA)
to the antigen, forming a three-layer “sandwich” consisting
of the immobilized antibody, the antigen, and the detect- The OIA utilizes a virus-specific antibody coated onto a thin
ing antibody with enzyme attached (Fig. 3.2). A ­substrate molecular film on a silicon wafer surface. The clinical ­sample
3  Immunological Detection and Characterization 53

Fig. 3.3  Schematic of lateral Lateral Flow Immunoassay

flow immunoassay. V viral
antigen, Ab antibody Sample Ab-Gold Test Control Wick
Pad Conjugate Line Line Pad

V
V V
V
V V V

Capillary Flow

is treated to extract and expose any viral antigens present and 5.8 Time-Resolved Fluoroimmunoassay
is then placed on the surface of the chip. Viral antigen is (TR-FIA)
captured and the resulting antigen–antibody complex
changes the optical thickness of the film on the chip. The The TR-FIA is an immunoassay that replaces the reporter
change in the surface thickness is magnified through addition molecule with a lanthanide metal. When exposed to the
of a second virus-specific antibody conjugated to horserad- appropriate wavelength of light, the lanthanide will fluoresce
ish peroxidase followed by addition of a substrate such as [44]. Compared to fluorescein and background autofluores-
tetramethylbenzidine (TMB). The presence of virus antigen cence, which have fluorescence decay times of less than five
is then detected by a change in the color of reflected light nanoseconds, the lanthanides have much longer decay times
from gold to purple. Kits have been developed for influenza of 1,000 to 1 million nanoseconds [8]. The format of the anti-
and respiratory syncytial virus detection [38, 39]. gen detection TR-FIA is similar to that of a sandwich EIA,
where a microtiter plate is coated with a virus-specific cap-
ture antibody and is then blocked. The clinical sample and
5.7 Lateral Flow Immunoassay antibody conjugated to the lanthanide is added next, and
after a suitable incubation period, the unbound components
The lateral flow immunoassay, also called the immunochro- are removed by washing. An enhancement solution is added
matographic assay, is an immunoassay that is performed on and the well is exposed to the appropriate wavelength of
chromatographic paper along a single axis (Fig. 3.3). The light. A fluorometer is used to measure fluorescence for 1 s,
clinical sample is applied to an absorbent pad and then is and the pattern of fluorescence allows the separation of
drawn by capillary action through a conjugate pad. If viral antigen-­specific signal from background fluorescence.
antigen is present in the clinical sample, it will interact with Several different lanthanides are available for use, but euro-
a virus-specific antibody conjugated to a colored particle pium is frequently used because of its long fluorescence
(often colloidal gold). The fluid in the sample carries the decay time and the difference between its excitation
antigen–antibody complex to a reaction membrane to which wavelength (~340–360 nm) and emission wavelength
­
another virus-specific antibody has been immobilized in a (~615 nm) [8]. TR-FIA has been developed for detection of
line perpendicular to the capillary flow direction. The anti- a variety of viral pathogens [45].
gen–antibody–conjugate complex is captured and can be
observed as a colored line on the membrane. The sample is
carried further across the reaction membrane to a control 6  aboratory Methods for Virus
L
line. Antibody specific for the antibody–conjugate is immo- Characterization
bilized along the control line, and visualization of the control
line indicates that the sample migrated across the membrane Further characterization of a virus obtained from a clinical
and picked up the antibody–conjugate as designed. An specimen is frequently desirable once an agent has been iso-
absorbent pad is beyond the reaction pad and acts as a waste lated. This can be done in a variety of ways, depending on
reservoir, drawing the clinical sample across the other pads what is known about the virus and what additional information
by capillary action. is being sought. For example, if a previously unrecognized
The simplicity of the lateral flow immunoassay design virus is recovered, characterization of its physicochemical as
allows the use of these assays as point-of-care tests. Results well as biological, antigenic, and genomic properties would
can usually be obtained within 15 min of sample collection. be useful. Various immunological methods can be used for
Tests have been developed for detection of respiratory and this purpose because of the general availability of immune
enteric virus as well as dengue viruses [40–43]. reagents for most human viruses. Immunofluorescence,
54 R.L. Atmar

radioimmunoassay, and enzyme immunoassay formats may inhibition) and permit the identification of influenza A and B
be used in a fashion similar to that described for the virus viruses, parainfluenza viruses, and adenoviruses [51].
detection in clinical specimens (Sect. 5). Other methods for
virus identification and characterization include virus neu-
tralization assays, hemagglutination-­inhibition assays, and 6.3 Agar Gel Immunodiffusion
epitope-blocking enzyme immunoassays using monoclonal
antibodies. Agar gel immunodiffusion, or agar gel precipitation, has
been used for the characterization of a variety of viral anti-
gens using standard, or reference, antisera. A thin layer of
6.1 Neutralization Assays agarose is made in a plate or on a slide, and small wells
are cut into the agarose. The unknown antigen and known
Virus neutralization assays detect the loss of virus infectiv- antiserum are placed in separate wells, and the proteins in the
ity that results from the interaction of virus with specific wells diffuse through the agarose. If the antiserum reacts
antibody. Unknown viruses may be identified using virus-­ with the virus antigen, a precipitation band appears. Though
specific antisera, and antibody to a specific virus present in a less sensitive than other methods and largely replaced by
serum sample can be detected or quantitated (see Sect. 7.1). molecular assays, this method offers high specificity and is
The loss of infectivity can be measured in a number of ways, simple to perform. It has been used for the identification of
depending on the biological systems capable of supporting orthopoxviruses, typing of influenza viruses, and subtyping
virus growth, the types of viruses being sought, and the capa- of hepatitis B viruses [51–53]. It also has been used to char-
bilities of the laboratory performing the studies. The principal acterize unknown sera with known virus antigens [54].
biological systems used for neutralization assays are tissue
culture, embryonated chicken eggs, and adult and suckling
mice [46]. Cell culture systems are used most commonly 6.4 Antigenic Characterization
because they support the growth of a large number of viruses,
are widely available, are easier to work with than the other The antigenic differences or similarities between vaccine
two systems, and lack an immune system (that may influence and wild-type strains or among virus strains that have been
test results). Embryonated hen’s eggs and mice are used as isolated from different geographic locations or at different
for primary isolation. Neutralization cannot be measured for times may be examined in a number of ways. The availabil-
some viruses (e.g., norovirus) because their infectivity cannot ity of monoclonal antibodies permits the examination of
be measured in currently available culture systems. these relationships and may detect differences or similarities
Pools of virus-specific antisera have been used to decrease that cannot be detected by polyclonal antisera [55, 56]. These
the number of neutralization assays needed to serotype assays examine the ability of a given monoclonal antibody to
enteroviruses [47]. Each serum pool contains antisera to a interact with a particular virus strain and are performed using
discrete number of enteroviruses, and antiserum to a given the same formats used for polyclonal antisera: RIA or EIA,
enterovirus is present in one to three pools. Thus, the pattern neutralization (if antibody is neutralizing), immunoprecipi-
of neutralization obtained from the use of only eight inter- tation, hemagglutination inhibition (if the virus has hemag-
secting serum pools allows the identification of 42 different glutination activity), and so forth.
enteroviruses [48]. Methods for the production of intersect- Monoclonal antibodies also have been used to map anti-
ing serum pools have been published [49]. genic sites on virus proteins. When a virus is grown in the
presence of a monoclonal antibody that normally neutralizes
it, the only progeny virus will be escape mutants, or viruses
6.2 Hemagglutination and that are no longer neutralized by the antibody. Frequently,
Hemagglutination-­Inhibition Assays escape mutants arise after substitution of a single nucleotide,
resulting in a single amino acid change, and the location of
The ability to agglutinate erythrocytes, a property shared by the change can be determined by sequencing the virus gene(s)
many viruses, can be used for the identification of some of encoding the viral protein(s) important in neutralization (e.g.,
these viruses. The differential hemagglutination of rat, human rotavirus) [57, 58]. A less precise map of antigenic sites can
group O, and monkey erythrocytes by different adenovirus be obtained through the use of a panel of monoclonal anti-
serotypes allows their separation into groups so that fewer bodies by determining whether an individual monoclonal
type-specific sera need to be used in neutralization or hem- antibody competes with other monoclones for an antigenic
agglutination-inhibition assays [50]. Type-specific antisera site and whether the antibody has activity against the escape
can be used to prevent hemagglutination (­hemagglutination mutants raised by a different monoclonal antibody [59].
3  Immunological Detection and Characterization 55

7 Serological Diagnosis competition by IgG antibody for binding sites on the antigen;
(3) IgM antibody may persist for months to a year or more
The detection of newly developed, virus-specific antibody or after an infection occurred; and (4) heterotypic reactivation
the detection of an increase in titer of preexisting antibody is of IgM may be found with some infections (such as CMV or
important in viral diagnosis and is one of the most commonly EBV). For example, removal of Coombs antibody from sera
used methods in epidemiologic studies of viruses. Most pri- is necessary for the EBV–VCA–IgM test; otherwise, false-­
mary infections or reinfections result in the production of positive results may arise. Methods useful for the detection
specific antibodies. In addition, viruses such as EBV, HIV, of viral-specific IgM will be described below, but, in general,
rubella, hepatitis A and B viruses, and arboviruses are diffi- diagnosis using a single IgM sample needs to be carefully
cult to detect directly and the serological diagnosis may be controlled to exclude the detection of IgG or other interfer-
the only practical means of identifying the particular agent. ing substances.
The detection of specific IgM antibody may be used to Many different serological techniques have been used in
suggest a recent infection in a single serum specimen. the diagnosis of viral infections (Table 3.2). Factors involved
Detection of specific IgM antibody in the neonate is useful to in the selection of a specific antibody assay include specific-
diagnose congenital infections, because maternal IgM anti- ity, sensitivity, speed, technical complexity, cost, and avail-
body does not cross the placenta. IgM antibody also is useful ability of reagents (Table 3.3). All antibody assays rely on
to detect acute disease in a variety of other clinical situations, the proper collection and storage of sera and, ideally, the
including infection with CMV, rubella, hepatitis A and B, comparison of acute and convalescent specimens collected at
and EBV. Limitations to the use of IgM detection include the an interval of at least 2 weeks. The development of newer
following: (1) IgM-specific antibody is not restricted to pri- techniques, such as EIA, for antibody determination is
mary infection and may be seen with reactivated disease, replacing some of the older methods, such as complement
particularly with herpesviruses such as HSV or varicella– fixation, but an understanding of the available methods is
zoster; (2) false-positive responses may occur in the pres- important prior to choosing a laboratory test to evaluate a
ence of rheumatoid factor or false-negative results from specific question.

Table 3.2  Serological diagnosis of detected viruses

Serological methoda
Virus Neutralization Complement fixation Hemagglutination inhibition Immunoassay (EIA, IF)
Adenoviruses + + +
Arboviruses + + + ++
Coronaviruses + + +
Cytomegalovirus + +b ++
Enteroviruses + +
EBVc ++
Hepatitis B and C ++
HSV + +b ++
Influenza + +b ++ +
Measles + +b + ++
Mumps + ++
Norovirus/rotavirus +d ++
Parainfluenza + +b ++ +
Parvovirus +
Rabies + +
RSV + +b ++
Retroviruses + ++e
Rhinoviruses + +
Rubella + ++
VZV +b ++
a
+ Method used in research setting, ++ method in use and readily available in virology laboratories
b
Complement fixation method may lack sensitivity for these viruses
c
The absorbed heterophile test is commonly used for infectious mononucleosis, with the EBV–VCA–IgM needed if that test is negative
d
Selected strains only
e
Western blot commonly used as confirmatory test
56 R.L. Atmar

Table 3.3  Comparison of serological methods used to detect viral antibodies

Method Sensitivitya Specificity Cost Time to Dx Availability
Neutralization +++ ++ Expensive >1 week Research
Complement fixation + +/++ Inexpensive <1 day Widely available
Hemagglutination inhibition ++ ++ Inexpensive <1 day Research/reference labs
Enzyme immunoassay +++ ++ Inexpensive <1 day Widely available
Immunofluorescence ++/+++ ++ Moderate <1 day Research/reference labs
Radioimmunoassay +++ ++ Expensive 1–3 days Research
Immunoblot (Western blot) ++/+++ +++ Expensive <1–3 days Research/reference labs
+ relatively low, ++ moderate, +++ high
a

Problems specific to serodiagnosis of a viral infection assays. Colorimetric assays often are more sensitive than
include the broad cross-reactivity among some virus groups, assays relying on inhibition of CPE [60].
such as the coxsackie A viruses that cross-react with anti- The type of assay used to assess antibody is very impor-
bodies to coxsackie B and echoviruses. Another serious tant, particularly in the evaluation of susceptibility to
­limitation of this approach to diagnosis is the failure of some vaccine-­preventable or epidemic viruses such as measles or
individuals, particularly young children or immunocompro- rubella, where low levels of antibody may be predictive of
mised patients, to mount a detectable antibody response to a protection. Direct comparison of various laboratory methods
specific infection. However, serological methods remain used for the detection of virus-specific antibody may be
extremely important in epidemiologic studies because results important in designing studies or evaluating study results.
are not dependent on obtaining a specimen at the peak of ill- For example, analysis of CMV antibody using neutralization
ness, tests can be performed retrospectively for a variety of by plaque reduction has shown poor correlation with CMV
agents simultaneously, and large-scale studies can be con- antibody using EIA [61]. Different neutralization methods
ducted in a timely and cost-effective manner. also may give differing results, as has been shown in the
analysis of antibody to RSV, where microneutralization
assays appear to be more indicative of biological protection
7.1 Neutralization than either direct or competitive ELISA methods or
complement-­enhanced plaque reduction [62].
Serum specimens may be assayed for neutralizing antibody In general, measurement of neutralizing antibody is the
against a given virus by testing serial dilutions of the serum most specific method that reflects immunity, although other
against a standard dose of the virus. The antibody titer is tests for some viruses may be surrogate markers for this.
expressed as the highest serum dilution that neutralizes the However, neutralization tests are rather expensive since a
test dose of virus. As a bioassay, neutralization assays are demonstration of inhibition of viral replication in cell cul-
highly specific and quite sensitive. For many viral agents, ture, embryonated egg, or laboratory animal (such as the
the neutralizing antibody level is directly correlated with suckling mouse) is required.
immunity, an important clinical and epidemiologic end-
point. Disadvantages of the assay include the time required
to obtain a result and the relatively high cost, due to the labor 7.2 Complement Fixation
intensity and requirement for cell culture and titered viral
stocks. Neutralization assays may be carried out in a variety The complement fixation (CF) test is a relatively simple
of systems and the endpoint measured by a number of dif- technique that may be used successfully with a large variety
ferent procedures. Different neutralization systems include of viral antigens. First developed in 1909 by Wasserman and
plaque reduction neutralization, sometimes using comple- coworkers [63, 64] for the detection of syphilis antibodies,
ment enhancement, where the number of virus plaques in CF has been adapted to test for antibodies to many bacterial
control wells are compared with the number seen in cultures and fungal pathogens of animals and man. The CF test relies
inoculated with the virus–serum mixture; microneutraliza- on competition between two antigen–antibody systems for a
tion, an assay performed in microtiter plates requiring small fixed amount of complement, with the result ultimately dem-
amounts of sera; and colorimetric assays. Colorimetric onstrated by the lysis of erythrocytes. The serum is heated
assays rely on markers indicating metabolic inhibition of the at 50 °C for 30 min to inactivate any complement that may
virus in cell cultures or on antigen–antibody reactions with be present. Antigen and a known amount of complement are
antibody tagged or reacted with enzyme-linked antibodies. added to dilutions of serum. The complexes formed between
Colorimetric assays are generally analyzed by measure- the initial antigen and antibody bind the available free
ment of optical density and have the advantage of being less ­complement, thus preventing further reaction of the comple-
­time-­consuming and costly to set up and analyze than other ment in the second step of the assay. In the second step, a
3  Immunological Detection and Characterization 57

hemolytic indicator system using red blood cells (RBCs), evaluate antibody titers of influenza viruses, parainfluenza
which have been reacted with hemolysin to sensitize them viruses, adenoviruses, rubella, arboviruses, and some strains
to complement, is used to detect the free complement. The of picornaviruses and noroviruses [68, 69]. In this assay,
RBCs are reacted with hemolysin, or antibody to the RBC, serial dilutions of sera are allowed to react with a defined
and added to the assay. Lysis of the RBCs occurs if free amount (4 HA units) of viral hemagglutinin. Subsequently,
complement is present. Thus, the presence of hemolysis at agglutinable RBCs are added and the ability of the virus to
the conclusion of the assay is indicative of the absence of agglutinate the RBCs is measured. Properly treated sera con-
specific antibody, whereas the formation of clumped RBC taining antibody specific to the virus will prevent aggluti-
(often referred to as a “RBC button”) indicates a positive nation of the RBCs, resulting in formation of RBC buttons
test reaction. Antibody titers can be calculated using stan- in the test wells; sera lacking specific antibody will result
dard endpoint determinations. Antibodies detected by CF are in RBC agglutination. Nonspecific viral inhibitors can give
primarily of the IgG class and develop during the convales- rise to false-positive results in some systems, requiring that
cent stages of illness. The greatest application of the CF test the sera be properly prepared prior to use. The specificity
lies in the demonstration of a rise in antibody in convales- of the assay varies somewhat with the particular virus, with
cent compared with acute sera. The CF test has been widely influenza and parainfluenza virus systems being more spe-
used for the serodiagnosis of many human viral pathogens cific than the arbovirus system [69]. For example, the HAI
because of its broad reactivity and effectiveness in detecting test can identify specific strains of influenza viruses, whereas
changes in antibody titers and it is often the standard against it identifies only the group-specific antigens of the arbovi-
which new methods are compared. The CF test has largely ruses (with neutralization tests required for strain identifi-
been replaced in many laboratories by enzyme immunoas- cation). Advantages of the HAI assay are its simplicity, the
says, but it continues to maintain its usefulness in some cir- low cost for reagents and equipment, and speed of the assay.
cumstances because reagents are relatively inexpensive and Disadvantages of this assay include the fact that the system
readily available and the test is rapid, reliable, and relatively only works with those viruses that cause hemagglutination
easy to perform [65]. Another advantage of this assay is that and that nonviral-specific serum components may also inhibit
many antigens can be easily tested in the same sera sam- hemagglutination, thereby invalidating the test results.
ples simply by changing the antigen but keeping all other The immune adherence hemagglutination method is
reagents and conditions the same. The CF test is also adapt- another method that has been used in the clinical laboratory.
able to microtiter and automated methods. After an initial reaction between viral antigens and specific
Despite the widespread use of the CF assay over time and antibodies is allowed to occur, complement is added and
in many epidemiologic studies, the assay has some unique binds to the antigen–antibody complex, if present. Human
problems: (1) the test relies on a cascade of interactions erythrocytes then are added and reaction to the antigen–anti-
involving multiple biological reagents that must be carefully body–complement complex with the C3b receptor causes
monitored; (2) it is relatively insensitive because high con- hemagglutination. This method, commonly used as a
centrations of antigen are required to produce CF complexes microtiter procedure, has a well-defined endpoint and is
and the assay is unable to detect small changes in antibody rapid, inexpensive, and more sensitive than CF [70].
concentrations or low levels of antibody that may be predic- Disadvantages of this method include the inability to differ-
tive of protection in other assay systems (such as VZV or entiate between different immunoglobulin subclasses and its
measles) [66, 67]; and (3) sera containing antibodies to host difficulties with viruses that themselves agglutinate RBCs.
cell components or anti-complementary sera will not give a The direct agglutination of sheep or horse RBCs by serum
valid result. Newer laboratory methods, such as EIA methods, is a diagnostic test for the heterophile antibody of infectious
are now available commercially and have largely replaced mononucleosis. Removal of an inhibitory (Forssman) anti-
CF tests for many viral pathogens because of their ability to body by adsorption of the sera with guinea pig kidney
discriminate between IgG and IgM antibody, increased sen- extracts is required before testing. The hemolysis of beef
sitivity, and enhanced specificity at a similar cost. cells by sera from patients with acute infectious mononucle-
osis due to EBV is another diagnostic test that does not
require adsorption but is less sensitive (see Chap. 37).
7.3 Hemagglutination Inhibition Red blood cells or latex particles can be coated with viral
antigens and used to determine the presence or absence of viral
The hemagglutination-inhibition test (HAI) is based on the antibodies in reactions called passive hemagglutination and
ability of some viruses to attach to receptors on certain spe- passive latex agglutination, respectively. If viral ­antibodies
cies of erythrocytes and cause hemagglutination (Sect. 6.2). are present, the antigen-coated red blood cells or latex par-
While HAI may be used to identify an unknown virus with ticles are agglutinated. This assay is subject to a prozone
virus-specific antisera, it also is useful for the detection of effect, in which an excess of antibody reduces or eliminates
virus-specific antibodies in the serum. HAI may be used to agglutination leading to a false-negative result. To circumvent
58 R.L. Atmar

this ­problem, negative samples can be diluted and the assay labeled conjugate can interact with virus-specific antibodies
repeated. Commercial kits are available for the qualitative in the clinical sample. Recombinant viral antigen is used to
identification of antibodies to VZV and rubella virus [71]. capture the virus-specific antibody–antibody–conjugate
complex in a “test” line, and a “control” line containing anti-
human immunoglobulin is present to indicate that the assay
7.4 Immunoassay Techniques performed as intended. The test format provides a rapid qual-
itative answer with sensitivity and specificity similar to that
7.4.1 Enzyme Immunoassay obtained by more complex laboratory-based enzyme immu-
Enzyme-based immunoassays (EIAs), sometimes called noassays [71, 72]. These assays can be applied to serum as
enzyme-linked immunosorbent assays or ELISA, are well as to saliva, and they have been developed for several
widely used for many purposes, including the detection of different viruses including HSV, HIV, hepatitis C virus, and
antigen-­specific antibody. This methodology has replaced chikungunya virus [72–75].
other methods in many laboratories in part due to the com- Epitope-blocking assays using monoclonal antibodies
mercial availability of test materials and complete test kits. have been used to examine serological responses to a number
The most commonly used immunoassays employ a four- of viruses [76–81]. These assays measure the ability of a test
layer approach: antigen is bound directly to a surface, the serum to block the binding of a monoclonal antibody to a
unknown serum sample is then added, an enzyme-conjugated virus antigen. They have been particularly useful in deter-
antihuman IgG or IgM is added next, and an indicator sys- mining serotype-specific immune responses to multivalent
tem is used to determine the amount of reaction between the vaccines and in determining which of a number of cross-­
enzyme-linked antibody and the antigen–serum reaction. As reactive virus strains is responsible for a natural infection in
discussed in Sect. 5.5, this method has gained widespread use a given host.
due to its sensitivity, specificity, safety, simplicity, low cost,
and ability to be automated. The system lends itself to auto- 7.4.2 Other Immunoassays
mation because of readily available microtiter diagnostic sys- Other variations of the immunoassay include isoelectric
tems and because multiple tests for different antigens may be focusing and affinity immunoblotting. These techniques are
run by varying only the initial antigen in the system. Clinical useful because of enhanced sensitivity, particularly for diag-
specimens from various sources besides serum or plasma, nosis in the congenitally infected infant or for the differentia-
such as respiratory secretions, cerebrospinal fluid, and breast tion of passively acquired antibody from endogenous
milk, also may be tested in this system. The EIA test may antibody. Isoelectric focusing relies on the separation of
also be read visually and so is useful in developing countries serum antibody in thin-layer gels, with subsequent antibody
unable to afford the photometric reader used in developed detection by a reaction with antigen-coated membranes [82].
countries to accurately quantitate the antibody content. Clonal-specific antibodies can be detected by this method,
Difficulties inherent in immunoassay techniques include which may discriminate, for example, between unique
those associated with obtaining and standardizing purified, maternal and fetal antibodies.
sensitive, and specific IgG and IgM reagents. Although many The TR-FIA assay can be modified for detection of virus-­
of these reagents are commercially available, there can be specific antibodies by coating the well of the microtiter plate
variability in the specificity of the reagents so that during with viral antigen and labeling antihuman immunoglobulin
assay development the reagent specificity should be assessed. with the lanthanide reporter. The availability of lanthanides
In general, results from different laboratories using different with distinct emission spectra allows multiplexing of the
reagents are not directly comparable. Specific analysis for assay such that different antibody isotypes (e.g., IgG and
subclasses, particularly IgM, requires careful ­standardization IgA) can be measured in the same well [83, 84].
and quality control to assure reliability and specificity of the Radioimmunoassay techniques for the sensitive detection
assay. Attention to such detail is critically important in assays of antibody to viral antigens, pioneered in the 1970s for the
with life-threatening implications, such as the EIA assays serodiagnosis of hepatitis B, are used by research l­ aboratories
currently used in blood banks to detect the presence of anti- but otherwise are not widely utilized [5]. Immunoassays that
body to HIV or hepatitis B and C. Evaluation and standard- do not require radioisotopes and require less technical exper-
ization of tests, including commercially available kits, and tise, such as the EIA and IF tests, are more commonly
comparison among different products prior to use in research available.
and clinical settings remain an important part of EIA.
The lateral flow immunoassay described in Sect. 5.7 can 7.4.3 Immunohistochemical
be modified to detect viral antibody. Antihuman immuno- The most commonly used immunohistochemical technique
globulin or protein A labeled with conjugate is used in the to detect antibody is the immunofluorescence technique (IF).
reagent pad in place of viral antibody conjugates, and the Whereas direct techniques are used commonly to detect
3  Immunological Detection and Characterization 59

a­ntigen in infected tissue or cells, indirect immunohisto- preparing reagents such as purified virus and labeled antihu-
chemical techniques are used to detect antibody in sera or man IgG. The time interval from virus acquisition and sero-
other bodily fluids. Variations on the indirect IF test, such as conversion by immunoblot may vary for different patients, as
amplification immunoassay systems utilizing various sand- well as different viruses, indicating that diagnosis of infec-
wich techniques (double indirect IF or anticomplement IF) tion by immunoblot analysis, while extremely sensitive, is not
or chemical amplification systems utilizing biotin–avidin always definitive [87]. Furthermore, analysis by immunoblot
complexes, are used in research settings [65]. Indirect IF will not differentiate maternal from fetal HIV infection in
methods are available in many laboratories for a variety of many cases. Despite these problems, the immunoblot assay
assays, although more automated techniques such as EIA are is widely used today in laboratories around the world as the
replacing IF techniques in many clinical settings. “definitive” confirmatory test for HIV, and it is also used as
In the indirect IF test, tissue or cells containing viral anti- a confirmatory test in some circumstances for the serologi-
gen are fixed on a glass slide, a serum dilution is added, and cal diagnosis of hepatitis C virus infection [88]. It also has
a fluorochrome-conjugated antibody indicator system is used been used to differentiate type-specific serological responses
to detect the resulting reaction. The conjugated detector anti- to herpes simplex virus type 1 (HSV-1) and HSV-2 [89].
body can be varied to specifically measure the presence of
antibody classes, such as IgG, IgM, and IgA. IF techniques
provide sensitive methods to detect antibody that can be 8 Interpretation of Laboratory Tests
related to immunity to viruses, such as VZV, to detect con-
genital infections in newborns based on IgM-specific anti- Virus infection is usually identified by either detection of the
body, and to detect epitope-specific antibody [65, 85]. In virus or identification of a serological response to the virus.
particular, the anticomplement-amplified indirect IF tech- For some viral infections, both are required. However, the
nique, utilizing a four-layer reaction including antigen, interpretation of the viral diagnostic assays must be made in
patient serum, complement, and fluorochrome-conjugated the context of the assay sensitivity and specificity along with
anti-C3 antibody, has been useful for the detection of the seasonal, clinical, and other epidemiologic factors.
nuclear antigen of EBV, or EBNA [65]. The inclusion of Difficulties arise in interpretation of serological tests
standard positive and negative sera is needed in each test and when only a single convalescent serum sample is obtained
independent reading by two observers is recommended to and a high antibody titer is found or if high titers are present
minimize errors in interpretations. in both acute and convalescent sera without a fourfold rise.
Advantages of indirect immunohistochemical methods These results can reflect either current infection or persis-
include (1) the ability to use the system to detect antibody to tently high antibody levels from a previous infection.
many diverse viruses by varying only the initial step in the Significance may be attached to these findings if the disease
reaction, (2) the higher sensitivity and specificity compared is a rare one in which the presence of antibody is unique, if
with CF, (3) the simplicity and relative speed of an individual the test reflects a short-lasting antibody, or if specific immu-
test, and (4) the reproducibility of the test by experienced noglobulin M (IgM) antibody can be demonstrated. A rapid
personnel. Disadvantages of the test include the technical drop in antibody titer in a subsequent specimen is also sug-
complexity, the lack of automation, the requirement for spe- gestive of a recent infection. Sequential testing of other fam-
cialized cells and reagents, and the need for special equip- ily members also may be useful, since they may be in
ment, such as a fluorescence microscope and darkroom. different stages of apparent or inapparent infection with the
same virus. In an epidemic setting, comparison of the geo-
7.4.4 Immunoblot metric mean antibody titer of sera collected early in illness
An immunoblot, also referred as a Western blot, is another from one group of patients with that from another group of
widely used method for the detection of antibody to specific patients convalescing from the same illness may permit rapid
viral antigens. This technique relies on the incubation of identification of the outbreak.
patient serum with partially purified whole virus or recombi- At times, a virus may be identified or an antibody rise
nant viral proteins that have been separated by electrophoresis demonstrated that is not, in fact, causally related to the ill-
in a polyacrylamide gel and transferred onto nitrocellulose ness. Sometimes dual infections with two viruses, or with a
paper [86]. The assay is based on the same principal as EIA but virus and a bacterium, occur, and the interpretation of the
has the advantage of identifying antibodies specific for several role of an individual viral pathogen in the disease process
antigens of the same virus simultaneously. Quantitation of may be very difficult. On other occasions, no virus is isolated
the specific reactions can be determined by a densitometer. or the serological rise is not sufficient to demonstrate whether
Difficulties with immunoblots include the expense and time a specific virus is the real cause of the illness. A list of com-
required for the test, the technical requirements for performing mon causes for false-positive and false-negative results is
and interpreting the test, and the problems encountered with given in Table 3.4.
60 R.L. Atmar

Table 3.4  Viral diagnosis: some causes of false-positive and false-negative tests
False positive
  Antigen detection
   1. Persistent or reactivated virus from prior and unrelated infection
   2. Two microbial agents are present, and the one isolated is not the cause of the disease
   3. Mislabeled specimens
   4. Cross-contamination within the laboratory
  Serological rise
   1. Cross-reacting antigens
   2. Nonspecific inhibitors
   3. Double infection with only one agent producing the illness
   4. Rise to vaccination rather than natural infection
False negative
  Antigen detection
   1. Viral specimen taken too late or too early in illness
   2. Wrong site of multiplication sampled, e.g., throat rather than rectal swab
   3. Improper transport or storage of specimen
   4. Low assay sensitivity
  Serological rise
   1. Specimens not taken at proper time, i.e., too late in illness or too close together to show antibody rise
   2. Poor antibody response–low antigenicity of the virus or removal of antibody by immune-complex formation
   3. Wrong virus or wrong virus strain used in the test
   4. Nonspecific inhibitor obscures true antibody rise
   5. Wrong test used for the timing of the serum specimens

5. Hollinger FB, Vorndam V, Dreesman GR. Assay of Australia anti-

9 Unresolved Problems gen and antibody employing double-antibody and solid-phase
radioimmunoassay techniques and comparison with the passive
hemagglutination methods. J Immunol. 1971;107:1099–111.
Many problems still must be resolved in the area of viral 6. O’Beirne AJ, Sever JL. Enzyme immunoassay. In: Specter S, Lancz
diagnosis. The development of newer molecular assays with G, editors. Clinical virology manual. New York: Elsevier; 1992.
improved sensitivity have supplanted many antigen detection p. 153–88.
methods with lower sensitivity, but the ease of performance 7. Yolken RH, Viscidi R. Enzyme immunoassay and radioimmunoas-
say. In: Schmidt NJ, Emmons RW, editors. Diagnostic procedures
of the antigen detection methods still makes these assays for viral, rickettsial and chlamydial infections. 6th ed. Washington,
attractive to many laboratories. While molecular methodolo- D.C: American Public Health Association; 1989. p. 157–78.
gies offer the potential for great sensitivity and specificity, 8. Forghani B. Diagnosis by viral antigen detection. In: Jerome KR,
they often require specialized equipment for their perfor- editor. Lennette’s laboratory diagnosis of viral infection. 4th ed.
New York: Informa Healthcare; 2010. p. 113–32.
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10. Hertz MI, Englund JA, Snover D, Bitterman PB, McGlave PB.
virus to the observed disease process will remain in the hands Respiratory syncytial virus-induced acute lung injury in adult
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review of the literature. Medicine (Baltimore). 1989;68:269–81.
11. Stockman LJ, Staat MA, Holloway M, et al. Optimum diagnostic
assay and clinical specimen for routine rotavirus surveillance.
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 Surveillance and Seroepidemiology
4
Ruth Jiles, Monina Klevens, and Elizabeth Hughes

1 Introduction The techniques of surveillance and uses of surveillance

data were applied first to infectious diseases and subse-
The term surveillance was employed for years in the restric- quently to occupational, environmental, and chronic dis-
tive sense to imply follow-up of exposed persons to deter- eases, as well as to injuries and emergency preparedness [6,
mine whether disease developed within the limits of the 14]. Healthy People, the principal document of the US
incubation period. The dictionary definition of surveillance Department of Health and Human Services that outlines the
is “close observation, especially over a spy or criminal” [67]. health objectives for the United States, relies heavily on sur-
Surveillance, in the context of health, has been defined as the veillance data to establish goals, baseline measures, and
systematic collection of data pertaining to the occurrence of monitor progress towards achieving those goals [94]. The
specific diseases, the analysis and interpretation of these importance of surveillance and the need for improving sur-
data, and the dissemination of consolidated and processed veillance techniques, both in developed and developing
information to contributors, programs, and other interested countries, have been well documented [68]. To improve data
persons. The Centers for Disease Control and Prevention quality and timeliness of reporting, electronic transmission
(CDC) has described surveillance as the collection of “health of surveillance data, mainly laboratory results, was initially
related data essential to the planning, implementation, and implemented in the United States and France [39, 95]. Early
evaluation of public health practice, closely integrated with challenges included standardization of case definitions and
the timely dissemination of these data to those responsible reporting methods among official sources. These issues were
for preventing and controlling disease and injury” [89]. addressed in the US National Notifiable Disease Surveillance
Surveillance systems are developed and implemented System through consensus and development of standard case
for the ultimate purpose of preventing or controlling dis- definitions and reporting protocols.
eases. Historically, the principles of surveillance were well This chapter discusses the background and elements of
described and documented by Langmuir [57] and other traditional surveillance, the concept and uses of serological
officials of the US Public Health Service, CDC [6, 66], and and molecular epidemiology, and their application to the
by Raška [76, 77] for the World Health Organization control of viral infections.
(WHO) [108].

2 Surveillance
R. Jiles, MS, MPH, PhD (*)
Division of Viral Hepatitis, Department of Health and Human Traditionally, surveillance was based on the occurrence and
Services Centers for Disease Control and Prevention reporting of a case of clinical disease or death. Currently,
Office of Infectious Diseases, National Center for HIV/AIDS,
other life and infection-related events, such as births, hospi-
Viral Hepatitis, STD, and TB Prevention, 1600 Clifton Road,
NE Mail Stop G-37, Atlanta, GA 30333, USA talization, risk behaviors and exposures, treatment, and
e-mail: [email protected] healthcare system encounters, are also under surveillance.
M. Klevens, DDS, MPH • E. Hughes, MS, DrPh
Division of Viral Hepatitis, National Center for HIV/AIDS, Viral
Hepatitis, STD, and TB Prevention, Centers for Disease Control 2.1 Historical Background
and Prevention, 1600 Clifton Road, NE Mail Stop G-37,
Atlanta, GA 30333, USA
e-mail: [email protected]; A more detailed history of public health surveillance was
[email protected] published by Declich and Carter in 1994 [24] and summarized

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 63

DOI 10.1007/978-1-4899-7448-8_4, © Springer Science+Business Media New York 2014
64 R. Jiles et al.

in an earlier version of this chapter [33]. Based on these developed in response to occurrence of other epidemics.
accounts, various governmental actions to prevent spread of WHO established regional, national, and international con-
infections and diseases occurred during the seventeenth cen- trol programs with surveillance as an essential component of
tury. However, it is widely accepted that public health sur- each type of control program. While European unification
veillance dates back to 1662, when John Graunt published was anticipated, individual countries employed different
the Natural and Political Observations Made Upon the Bills approaches to surveillance; thus, there were concerns about
of Mortality. Graunt analyzed London’s mortality data col- comparability of data and coordination of activities [27]. The
lected for the Bills of Mortality and was the first to count the AIDS epidemic which began in the late 1970s and early
number of deaths from specific causes (numerator), estimate 1980s forced many countries to establish new surveillance
the population (denominator), and use the information to systems and improve existing ones.
determine death rates [41]. While a set of legal and ethical principles of surveillance
During the eighteenth century, some colonies in Rhode had been evolving gradually prior to the early 1980s, the
Island implemented various specific components of public pressing need for accurate information about HIV infection
health surveillance, requiring tavern keepers to report cases led to a broad array of new surveillance practices that have
of “contagious” diseases such as smallpox, yellow fever, and continued to test the balance between individual and com-
cholera among their customers. munity interests and responsibilities.
During the nineteenth century, several significant contribu-
tions were made in public health surveillance. Sir Edwin
Chadwick, Secretary of the Poor Law Commission in England, 2.2 Types of Surveillance
used surveillance data to demonstrate that poverty and disease
were associated and suggested improvement in the living con- Surveillance systems may be classified in a variety of ways:
ditions of the poor. In the United States, Lemuel Shattuck based on the method of reporting (passive or active), purpose
related living conditions to infant and maternal morbidity, of the system (Behavioral Risk Factor Surveillance System
mortality, and rates of death. He recommended collection of or BRFSS) [11], location (clinic based), or target population
health data by such factors as age, gender, and occupation. (Youth Risk Behavior Survey YRBS) [21].
William Farr, founder of a modern concept of surveillance, as The most common type of surveillance is passive. In pas-
the first Compiler of Abstracts for England and Wales, set up sive surveillance the reporter, usually a physician or labora-
a system that not only tracked births and deaths, but also rou- tory, regularly transmits a summary of all the cases of the
tinely recorded cause of death by occupation. Farr developed specified disease or diseases observed to health authorities
a nosology that is the basis for the International Classification during the reporting period.
of Diseases (ICD) (See http://www.cdc.gov/nchs/data/misc/ The US National Notifiable Disease Surveillance System
classification_diseases2011.pdf). (NNDSS) is an example of passive reporting [17]. States send
Surveillance concepts and methodologies were developed case reports of nationally notifiable diseases to the CDC on a
in response to national needs for disease surveillance or in weekly basis. States determine which diseases are reportable
response to epidemics. Surveillance data were needed to within their jurisdiction and who (laboratory, physician,
define the magnitude of the problem and to inform policy/ clinic, or some combination) initiates the report. State epide-
decision makers and others who were responsible for devel- miologists and CDC subject matter experts collaborate,
opment, implementation, and/or evaluation of prevention through the Council of State and Territorial Epidemiologists
and intervention programs and policies. Use of the term “sur- (CSTE), to develop case definitions and define reportable
veillance” in the United States began in 1949 with the devel- data elements for reportable diseases. Generally, laboratories
opment of a modified program at the CDC called and/or physicians report cases to the local health departments,
“Surveillance and Appraisal of Malaria.” In 1951, the con- local health departments report cases to state health depart-
cept was applied to the residual smallpox cases in the United ments, and state health departments notify CDC of confirmed
States. cases. Most laboratory reports and some physician reports are
On April 28, 1955, the Surgeon General of the United submitted electronically. However, reporting can be accom-
States directed the establishment of a “National Poliomyelitis plished in print, by telephone, even using toll-free numbers or
Surveillance Program” in response to paralytic polio cases automatic recording devices available at all hours. Time and
following the use of Salk vaccine (the “Cutter Incident”). lack of resources greatly limit such a system to a small per-
Based on this program, established at the CDC, surveillance centage of most reportable diseases, but as long as the system
methods became an effective tool in following trends in the and requirements remain unchanged, the changes in inci-
disease, in measuring the effectiveness of polio immuniza- dence may reflect meaningful patterns of disease.
tion programs, and in detecting suspected vaccine-associated Active surveillance requires contacting the reporters at
cases. Likewise, other surveillance systems and units were regular intervals and request for specific data on cases of
4 Surveillance and Seroepidemiology 65

specified diseases. It permits more extensive data collection Table 4.1 Selected sources of data for surveillance
on epidemiologic features of certain diseases. CDC’s 1. Viral statistics
Behavioral Risk Factor Surveillance System (BRFSS) is an 2. Reportable disease systems
active surveillance system [11]. States contact residents by 3. Surveys
telephone and administer a standardized questionnaire to eli- 4. Sentinel surveillance
gible consenting adults aged 18 years or older to obtain 5. Syndromic surveillance
information about a variety of chronic diseases and related 6. Registries
risk behaviors. Relevant to viral infections, the BRFSS 7. Laboratory records
includes questions about receipt of influenza, shingles, and 8. Pharmacy records
9. Administrative data
human papilloma virus vaccination. The BRFSS has also
been used by CDC to provide state-specific estimates of
HIV-related behaviors such as ever tested for HIV, where
tested, and why tested. Some states conduct active surveil- 2.3.1 Vital Statistics
lance for HIV/AIDS using other data sources. The oldest form of surveillance is mortality registration. In
For a few diseases, some special clinical feature of the most countries, death registration is legally required. As a
disease may be used to estimate prevalence data. For exam- result, almost all deaths are included in the registries. Causes
ple sudden severe acute lower respiratory illness may indi- of death listed on the death certificate is dependent on the
cate hantavirus infection or jaundice may be evidence of presence/absence of a physician or family member who is
hepatitis. In some instances, surveillance is based on labora- knowledgeable about the health of the deceased; severity of
tory data, as with the isolation of the agent or results of a disease, complexity of the disease, associated illnesses, and
serological test. In the case of viral hepatitis, the CDC/CSTE whether or not an autopsy or diagnostic laboratory testing
case definition includes both clinical features and laboratory was performed. For many viral diseases, such as hantavirus,
test results. Most surveillance systems cover political juris- rotavirus, and influenza, only a small percentage of cases are
dictions (e.g., national, state, and county). However, some fatal. However, the case fatality rate is high for other viral
surveillance systems cover high-risk or specific population diseases, such as AIDS, rabies, Lassa fever, and certain hem-
groups, requiring special reporting methods. Examples are orrhagic fevers. Thus, occurrence of viral infections is likely
military populations, hospitals, day-care centers, and homes underestimated from the death certificate data. However, vital
for the elderly. Most surveillance systems incorporate a list records are important to document severe complications of
of reportable diseases, but the focus of the reporting may be viral infections. In addition, surveillance from vital records
on specific disease entities, such as congenital defects as a can be useful for comparing viruses. For example, by 2007 in
reflection of the impact of rubella and cytomegalovirus the United States, the number of deaths associated with HIV
infections [28]. was lower than the number associated with hepatitis C [59].
An example of surveillance in a special population and Birth certificates are often used to monitor conditions such
location is CDC’s school-based Youth Risk Behavior Survey as congenital defects (due to rubella and cytomegalovirus
(YRBS) [21]. In this survey, questions related to viral dis- infection), diseases transmitted from mother to child (e.g.,
eases, for example, HIV, are self-reported by participating hepatitis B virus infections), and other conditions of new-
high school students. borns that may impact immediate or long-term health status.

2.3.2 Reportable Disease Systems

2.3 Sources of Surveillance Data The reporting of cases of specified infectious diseases also is
legally required in most countries. The US National
A wide variety of data sources are used for surveillance pur- Notifiable Diseases Surveillance System includes over 70
poses. Some data sources were designed for the purpose of diseases, of which approximately 30 are viral diseases
surveillance and are ipso facto “surveillance systems.” Other including 6 arboviruses, 6 hepatitides, and 6 types of hemor-
data sources are used secondarily for surveillance. Sources rhagic fevers. Reportable disease systems form the backbone
of surveillance data vary from country to country, state to of surveillance for most state and local health departments
state, and across local jurisdictions. Availability of surveil- and for many CDC programs. The advantages are that (1)
lance data is dependent on resources available to support the reports are usually made by physicians and/or laboratories,
system, such as appropriate laboratory facilities and trained (2) laboratory confirmation is generally available, and (3)
personnel. Table 4.1 lists some data sources that are com- there is typically an organized system of regional or national
monly used for surveillance purposes. These sources are tabulation and reporting. The disadvantages are as follows:
consistent with elements of surveillance summarized by (1) the absence of some viral diseases from the required list;
WHO [108]. (2) the notorious underreporting of diseases despite legal
66 R. Jiles et al.

requirements, primarily because of lack of resources to sup- or condition. For example, cancer registries collect informa-
port both reporting and education of physicians about the tion about type of cancer, anatomic location, stage of disease
need to report; and (3) the variability of reporting efficiency at diagnosis, treatment, and outcomes. Childhood immuniza-
from one jurisdiction to another. Lack of rapid, reliable, tion registries maintain a record of vaccinations received by
inexpensive diagnostic techniques also represents a discour- children within the jurisdiction of the registry [19].
aging obstacle to accurate identification and reporting of
many viral diseases [17]. 2.3.7 Laboratory Records/Investigations
Laboratory identification of the causative agent of many
2.3.3 Surveys viral infections has become a routine part of clinical care.
Many types of surveys of infectious disease have been used Laboratory records are especially useful for public health
in public health. Formal surveys support the collection of surveillance. Appropriate laboratory facilities and experi-
standardized information using standardized methods. enced personnel are needed for the isolation and/or serologi-
Although survey data can be collected quickly over a wide cal identification of the majority of viral infections. These
geographic area, the cost of data collection may be prohibi- may exist in national or regional public health laboratories,
tive. The National Health and Nutrition Examination Survey in specialized virus diagnostic institutes, or in university
(NHANES), conducted by the National Center for Health settings.
Statistics, a component of CDC, uses a complex sampling
design to collect nationally representative data on the health 2.3.8 Pharmacy Records/Investigations
and nutritional status of the US noninstitutionalized civilian Pharmacy data may be used to identify those who are treated
population [16]. NHANES is a source of data for a number for specific diseases, monitor uptake of new medications,
of infectious disease programs, mainly because the labora- and evaluate effectiveness of specific treatments. For exam-
tory and physical examination components allow both con- ple, when treatment for HIV/AIDS first became available,
firmation of case status and collection of health information. prescriptions for zidovudine were used to identify potentially
For example, the Division of Viral Hepatitis at CDC uses unreported cases [54].
NHANES data to estimate the prevalence of chronic hepati-
tis B and C for the US noninstitutionalized adult populations 2.3.9 Administrative Data
(see Sect. 3.3.2). These data consist of electronic records prepared usually for
billing or other administrative accounting and are sometimes
2.3.4 Sentinel Surveillance available in a de-identified format and at no cost for public
Sentinel surveillance is used as a less costly alternative to health use. Data from health plans such as Kaiser Permanente,
population-based surveillance. Sentinel surveillance systems Medicare, and Medicaid have proven to be useful to supple-
collect data from a sample of reporting sites. Generally a ment routine surveillance data. Hospital discharge data, like
select group of reporting sources—hospitals, healthcare pro- insurance/health plan data, provide useful information on
viders, agencies—are recruited to report all cases of one or diagnosis, surgical procedures, other billed treatments, com-
more notifiable conditions. In the United States, sentinel plications, length of stay, laboratory data, and associated
sites report all cases of influenza-like illness to their state costs. In addition to the wealth of data available in these
health department on a weekly basis. A network of sentinel sources, another attraction is that these data are already col-
providers in British Columbia, Canada, demonstrated the lected in electronic form, requiring fewer resources to ana-
usefulness of sentinel surveillance in documenting the effec- lyze and summarize.
tiveness of influenza vaccine [49].

2.3.5 Syndromic Surveillance 2.4 Surveillance Networks and Health

Syndromic surveillance uses clinical information about signs Information Exchanges
and symptoms of disease, before a diagnosis is made, as an
early warning signal of a potential outbreak. Many syn- 2.4.1 Surveillance Networks
dromic surveillance systems use electronic data from hospi- Surveillance networks grew out of the need for a more
tal emergency room visits. The value of such systems relies global approach to surveillance of infectious diseases.
on accurate assessment and coding of symptoms, as well as Surveillance of infectious diseases was often inadequate in
accurate data entry. the developing world due to a lack of resources and public
health infrastructure. The need for early warning of out-
2.3.6 Registries breaks of emerging and reemerging diseases led the
Registries are often established, usually at the state level, to Federation of American Scientists to support the establish-
collect information about persons diagnosed with a disease ment of the first infectious disease network, the Program for
4 Surveillance and Seroepidemiology 67

Monitoring Emerging Diseases (ProMED-mail) in 1993 WHO’s Global Influenza Program provides technical
[74]. ProMED-mail is an Internet-based system for report- support and guidance to Member States and maintains global
ing and disseminating information on infectious diseases surveillance for influenza. Virologic and epidemiologic data
outbreaks and acute exposures to toxins that impact human are collected from countries, areas, and territories through
health. This open-source network receives reports from cli- the influenza surveillance and monitoring system. Two plat-
nicians, public health officials and epidemiologists, labora- forms are provided for data collection and sharing: FluNet
tory scientists, medical missionaries, journalists, and for virologic data and FluID for epidemiologic. These sys-
laypersons. The editors also search the Web and press tems allow tracking of global trends, spread, and impact of
reports for information. A panel of experts screens, reviews, influenza [106].
and investigates reports before they are posted to the web-
site. ProMED-mail allows comparison of reports by geo- 2.4.2 Health Information Exchanges
graphic location. Thus, users of the system can identify During the late 1900s and early 2000s, there were concerns
similar outbreaks in both space and time. about the ability of the United States to respond to possible
Other surveillance networks include Canada’s Global acts of bioterrorism. Beginning with the anthrax attacks
Public Health Intelligence Network (GPHIN) [75] and the shortly after the September 11, 2001 destruction of the World
World Health Organization’s Global Outbreak Alert and Trade Center, epidemics of such emerging viral infections as
Response Network (GOARN) [103, 104]. West Nile virus, avian influenza, and SARS became a major
GPHIN, started in 1999, monitors internet websites, news issue in protecting the health of all Americans [58]. In
wires, and other internet media to gather and provide infor- response, the President of the United States signed Executive
mation on disease outbreaks and other public health events. Order 12225 on April 27, 2004, which created the Office of
This network collects information about infectious diseases, the National Coordinator of Health Information Technology
outbreaks, contaminated food and water, natural disasters, (ONCHIT) [58]. The primary objective of this order was to
bioterrorism events, and safety of products, drugs, and medi- formalize the administration of the office and to advance the
cal devices. GPHIN is part of GOARN [75]. development and growth of health information technology as
GOARN, started in 2000 by WHO, links existing net- vital to improving the quality of healthcare while reducing
works of government and academic centers of excellence, its cost. The first step in the process to achieve this critical
networks of laboratories, medical centers, scientific institu- objective was to use medical records maintained by health-
tions, and other international organizations. The goal of care providers to build a national network of electronic
GOARN is to provide countries with resources and expertise health records on the majority of Americans, by the year
necessary to respond to infectious disease outbreaks. 2014 [13].
GOARN provides and coordinates technical support, investi- Health information exchange (HIE) can be described as
gates the event, assesses risk, streamlines processes to rap- the “electronic movement of health-related information
idly deploy field teams, and supports national preparedness among organizations according to nationally recognized
[103, 104]. standards” developed by the Health Resources and Services
CDC surveillance systems may be viewed as networks Administration (HRSA) [92]. HIE encompasses two major
connecting the Federal agency with local, state, and territo- concepts: the electronic sharing of health-related informa-
rial health departments and international partners. A number tion among organizations and an organization that provides
of these surveillance networks focus on viral diseases. An services to enable the electronic sharing of health-related
example is the Influenza Surveillance Program [18]. The information [15, 46]. The primary goal of HIE is to facilitate
program which started in 1972 collaborates with local, state, access to and retrieval of patient clinical data to provide
and territorial health departments, clinical laboratories, more efficient, timely, effective, equitable, and safe health-
healthcare providers, and emergency departments to collects care. HIE secondary goals are to improve bidirectional com-
and analyzes information on influenza in the United States. munication, to enhance case reporting, and to focus on
Information is collected from five categories of influenza technology, interoperability, utilization standards, and har-
surveillance: viral surveillance, including surveillance for monious collaboration between all patient-centered health-
novel influenza A viruses; outpatient illness surveillance; care providers [13, 97]. The potential of HIEs to integrate
mortality surveillance; hospitalization surveillance; and geo- the electronic transfer of vital health information among
graphic distribution. Information from these five categories providers and public health agencies is critical in the effort
of surveillance is used to track influenza-related illnesses and to improve healthcare quality and increase safety for patients
deaths and to provide a comprehensive overview of activi- [46].
ties. CDC also collaborates closely with WHO in global sur- In 2007, CDC funded Health Information Exchanges to
veillance, including surveillance for novel influenza viruses support situational awareness project [12]. The objectives of this
and in influenza vaccine strain selection. project were to connect public health providers and organizations
68 R. Jiles et al.

with HIEs in order to improve public health with real-time may be collected for other purposes (e.g., blood donor
awareness of the health and status of healthcare facilities screening), but can be useful for public health purposes. In
within communities, bidirectional communication between the context of viral infections, seroepidemiology serves at
healthcare entities, and enhanced case reporting [93]. An least three objectives:
example of a successful merge of a HIE with a state partner 1. To supplement surveillance data and inform immuniza-
is the Indiana Health Information Exchange (IHIE) [40, 48]. tion and public health planning programs
The IHIE displayed the interoperability of health informa- 2. To generate hypotheses about the potential association
tion exchange by incorporating clinical messaging service between risk and occurrence of viral infectious diseases
(DOCS4DOCS®) to improve communication between 3. To assess old and newly recognized viruses in different
healthcare providers and public health agencies. With this population groups
system, clinical result messages can be forwarded to all phy- Just as epidemiology is concerned with the occurrence
sicians or targeted to clinical practices or specific geographi- and distribution of clinical cases in different populations,
cal areas [90]. In 2009 and 2010, the DOCS4DOCS® public serological epidemiology is concerned with the occurrence
health messaging system was utilized to alert nearly all phy- and distribution of various components of the blood that
sician practices and public health agencies about H1N1 indicate past or current infection, that are biochemical
influenza, a syphilis outbreak, an update on rabies treatment, markers for certain chronic infections, or that reveal the
and new vaccination requirements for school children [48]. genetic attributes of strains in various population groups.
Another example of a successful HIE is the Northwest The epidemiologic characteristics are detected in the labora-
Public Health Information Exchange (NW-PHIE) in tory rather than at the bedside; thus, laboratory support is
Washington and Idaho states [90]. This HIE evaluated new fundamentally necessary to conduct seroepidemiologic sur-
influenza surveillance efforts and compared these with exist- veys. Fortunately, laboratory testing is a part of routine clini-
ing influenza surveillance data feeds through the Influenza- cal care and is readily available in most countries.
Like-Illness Network (ILINET). Results indicated that the Largely based on the availability of resources to monitor
NW-PHIE data were timelier, more stable, more extensive, any given viral infection, we might consider that seroepide-
and more broadly representative of the community. The miologic data can be classified as:
NW-PHIE data accurately reflected trends in ILINET activ- 1. The primary objective of the survey (i.e., a planned and
ity at the state and community levels [13]. designed survey or study)
HIEs benefit public health by improving the safety of 2. A secondary use of specimens or data collected for other
patients and ensuring quality of healthcare. HIEs augment purposes (i.e., screening programs including blood
patient safety by serving as the connecting point for a stan- donors, clinics for high-risk individuals, and other rem-
dardized, organized process of data exchange across regional, nant sera)
state, and local jurisdictions; reducing duplication of ser- As a primary objective, serological surveys are frequently
vices which may result in lower healthcare costs; reducing integrated with other public health efforts. This section con-
operating costs by automating many day-to-day organiza- siders the history, methods, and uses of seroepidemiology as
tional tasks; providing management of the data exchange either a primary or secondary objective of data collection. As
process; and, most importantly, improving communication in the surveillance section, we draw heavily on the experi-
between providers and patients [46, 93]. ence in the United States, but recognize excellent seroepide-
miologic studies conducted in other countries.

3 Seroepidemiology
3.2 Historical Background
3.1 Introduction
The introduction of serological tests for the diagnosis of dis-
Seroepidemiology is the systematic collection and testing of ease provided the basis for later serological surveys. As early
blood samples from a target population, or a sample of a as 1916, the Wassermann test was applied routinely to
population, to identify current and past experiences with patients attending a prenatal clinic at Johns Hopkins Hospital
infectious diseases by means of biological markers (i.e., anti- by Williams [102] but this was more of a case-finding proce-
body, antigen, or other tests). Findings from the tests are the dure than an attempt to delineate disease patterns. In 1930,
outcomes that, when analyzed, are used to describe patterns the development of a neutralization test for poliomyelitis led
and potentially identify factors associated with the outcomes. Aycock and Kramer [4] to use the procedure to define the
Seroepidemiologic studies are used worldwide to measure immunity pattern of a given population; this is a landmark in
and characterize infectious diseases in the population. the history of serum surveys. In 1932, Soper et al. [85]
Sources of seroepidemiologic data are widely available and mapped out the occurrence of yellow fever in Brazil by
4 Surveillance and Seroepidemiology 69

antibody surveys under the auspices of the Rockefeller 3.3 Methodology

Foundation, and this technique has been widely used subse-
quently in studying arbovirus infections. Antibody surveys 3.3.1 Ethical Issues
for influenza also date back to the mid-1930s. The discovery Seroepidemiologic studies were used early in the HIV epidemic
of swine influenza virus by Shope [83] in 1931 and of human because there was a need to determine the unbiased frequency
influenza virus by Smith et al. [84] in 1933 was rapidly fol- of infection in different populations. A series of serosurveys
lowed by population studies to measure antibody to these were conducted to cover different populations including per-
viruses among persons of different age groups [2, 9, 37]. sons attending STD clinics [100], childbearing women [43],
The Yale Poliomyelitis Study Unit under Dr. John R. Paul and youth training programs [23]. To protect the identity of
employed serological survey techniques as early as 1935, infected persons, these surveys were conducted anonymously
and his analysis with Riordan of the poliomyelitis pattern in and data were unlinked, such that notifying the person whose
Alaskan Eskimos is a classic study [71]. He became one of blood was tested was not feasible. The ethical issues discussed
the foremost users and promoters of the concept of serologi- over time are described by Fairchild and Bayer [36]. The issues
cal epidemiology, and through his work and writing [69, 72], included participant consent, the participant’s right to know
the utilization of this technique in public health practice and and to control their information, and the responsibility of the
research studies has become a reality. The World Health health officials to inform the individuals of their test results.
Organization also took note of this development in 1960 and When treatment became available, serosurveys continued as
established three WHO Serum Reference Banks to practice long as voluntary counseling and testing was available to
and promote seroepidemiology in New Haven, Connecticut; survey participants. As clinical data became a more complete
Prague, Czechoslovakia; and Johannesburg, South Africa. source of similar surveillance information, the use of serosur-
An additional bank was established in 1971 in Tokyo, Japan. veys became ethically indefensible [36].
The activities and principles of these banks have been Currently, in the United States, Federal Regulations (45
reviewed in two WHO Technical Reports [105, 107], in a CFR 46.111) require that studies involving human research
book [72], and in several other publications [70, 73, 81]. subjects satisfy the following rules: (a) risks to participants
Although WHO no longer formally supports these banks, the are minimized; (b) risks to participants are reasonable rela-
rationale for proper collection, cataloging, and storage of tive to anticipated benefits, if any, to participants, and the
specimens for use by both primary and collaborating investi- importance of the knowledge that may reasonably be
gators has gained wide application in public health and aca- expected to result; (c) selection of participants is equitable
demic research institutions. For example, WHO collected (i.e., could any special problems arise when research involves
sera from household surveys in rural areas for the evaluation vulnerable populations, such as children, pregnant women,
of the effectiveness of penicillin in mass eradication pro- fetuses, prisoners, mentally disabled persons, economically
grams for yaws [42]. or educationally disadvantaged persons); (d) informed con-
Seroepidemiologic techniques were critical to the discov- sent will be sought from each prospective participant or the
ery by Blumberg et al. [7] in 1965 of a particular antigen in participant’s legally authorized representative; (e) informed
the serum of an Australian aborigine; it was uncovered in the consent will be appropriately documented; (f) the research
course of genetic studies of β-lipoprotein. Since the agent plan makes adequate provisions for ensuring the safety of
from which the antigen was derived could not be isolated or participants; and (g) there are adequate provisions to protect
cultivated in the laboratory, serological surveys using immu- the privacy of participants and to maintain the confidentiality
nodiffusion tests were carried out to detect its presence in the of data. Institutions conducting epidemiologic studies main-
sera of different population groups and different disease enti- tain “Institutional Review Boards” to ensure that investiga-
ties. The results provided the sole initial evidence that this tors address the above ethical requirements.
“Australia antigen” was associated causally with hepatitis B
or “long-incubation hepatitis” [8]. Herpes viruses, HHV-6 3.3.2 Statistical Considerations
and HHV-7, were not immediately recognized in association When serosurveys are designed as primary data collection,
with previously defined disease entities, although the former representativeness of the population is usually desirable. In
has been unequivocally shown to be the major, if not the the United States, NHANES combines interviews, physical
only, causal agent of exanthema subitum. examinations, and laboratory specimens from a nationally
Many countries recognize the value of such biological representative sample of about 5,000 persons each year.
resources and have created sizable repositories of serum and, These persons are located in different geographic areas
increasingly, tissue or other cellular material containing called primary sampling units, of which 15 are visited each
nucleic acid suitable for molecular and genetic analysis. year. Health interviews are conducted in respondents’ homes,
Seroprevalence studies of many different infections and from whereas health measurements are collected in specially
many countries have been published. designed and equipped mobile centers.
70 R. Jiles et al.

NHANES uses a complex sample survey design [16]. Table 4.2 Sources of human biological materials for surveillance
Primary sampling units are generally single counties, where where planned serum surveys from targeted populations are the primary
objective
the sampling frame is all counties in the United States. The
Objective Sources of biologic materials
additional stages of selection in the probability design con-
Primary Planned serum surveys from target populations
sist of clusters of households, where each person in a selected
Secondary Entrance and periodic examinations of different groups
household is screened for demographic characteristics, and (military, industry, health clinics)
one or more persons per household are selected for the sam- Blood donors in Red Cross and similar programs
ple. As with any complex probability sample, the sample (e.g., transplantation donors)
design information must be used when undertaking statisti- Public health laboratories:
cal analysis of the NHANES data. In particular, sample Serological tests for HIV (e.g., premarital)
weights and the first stage of the cluster design need to be Other immunologic and diagnostic tests
considered. Participants are compensated for participation Healthcare facilities:
and receive a report with their health results. Entry tests for blood chemistries or syphilis
For rapid surveys or for surveys in developing countries, Diagnostic tests for infectious diseases
Blood banks and transplantation programs
WHO has developed methods for cluster sampling that are
Prenatal; clinics (e.g., hemodialysis)
more easily implemented and analyzed than multistage proba-
Employee health services
bility sample surveys [5]. Using this method, and assuming the
Counseling and testing sites, clinics for sexually
survey is conducted to meet several health information needs, transmitted diseases
the first step is to plan carefully to ensure best use of resources. Drug treatment clinics
Sampling is conducted in several stages, starting with regions
of a country, then districts within regions, then communities
(e.g., villages, blocks, etc.) within the districts and households interest. To achieve efficiencies from this type of study, sero-
within the communities. Ideally, the community sampling logical surveys should be multipurpose in nature and can
framework should be a list of all the communities in the region, include measurement of antibodies, genetic material, and
and there should be a measure of the population size of the other clinical laboratory markers of health or illness. In the
community in order to sample with probability proportionate United States, the NHANES has produced a nationwide
to size. This assessment is helpful because weighting at the population-based health profile for more than two decades.
time of analysis then becomes unnecessary. The total popula- As one of its many components, it has provided valuable
tion divided by the number of communities to be selected seroprevalence data on a variety of viral infections, for
determines the sampling interval, which will be used to select example, measles, poliomyelitis, herpes simplex virus
the communities after a random start. Also ideally, there is enu- (HSV), hepatitis A virus (HAV) [53], and HBV [98].
meration of the households within the selected communities A list of several sources of material for survey analysis is
such that a simple random sample can be selected. If that shown in Table 4.2. Examples of sources of specimens for
framework is not available, methods from the 30 × 7 Expanded secondary use include blood collected for routine tests dur-
Program on Immunization can be used since the selection of ing physical examinations for the armed forces or industry or
households is conducted in the field using a central start; inter- during an outpatient visit or admission to a hospital and from
views are conducted until information on seven children aged neonates screened for specific heritable disorders. Sera sent
12–23 months are completed [22, 45]. A slight modification to to a public health laboratory for serological tests for syphilis,
the above rapid method is “compact segment sampling,” pro- viral diagnosis, or other diagnostic tests have also been
posed by Milligan et al. [62]. In this method, the communities employed. These collections of sera may not be representa-
to be sampled are divided into segments of equal population tive of the age, sex, and geographic distribution of the entire
size; then, one segment is randomly selected and all house- population; the nature of the biases introduced must be rec-
holds in the segment are included in the sample. ognized and evaluated. However, they are economical to
An important analysis issue for sample surveys of any obtain and sometimes may reveal important information
methodology is whether data have been collected consistently about the presence or absence of a certain virus in the com-
over time. When the same items have been collected using munity or about the occurrence of a recent outbreak. An
consistent methods, comparisons can be made over time, example of this type of serostudy was the description of the
adjusting for the distribution of the age of the population. frequency of HIV and hepatitis B and C infection among
injection drug users in several US cities [63].
3.3.3 Sources of Biological Material Specimen Management. The collection and management
When serostudies are designed for primary data collection, of specimens depends on the type of specimen (e.g., viruses
the collection of blood specimens can be targeted to a care- might be identified in stool samples, oral swabs). For blood,
fully and statistically selected sample of the population of the specimen must be collected and separated under sterile
4 Surveillance and Seroepidemiology 71

conditions. Aliquots of 0.5 ml each have been used by the virus can be identified in dried blood specimens, which can
WHO and CDC and are very useful for microtiter tests; sev- be stored at ambient temperature indefinitely. In general,
eral replicates of the entire collection may be prepared at the PCR requires specialized kits that differ for qualitative or
time of aliquoting so they can be shipped to other laborato- quantitative detection. Sequencing of select regions of ampli-
ries for testing. Sera are usually stored at −20 °C, often in a fied genetic material can be used to describe strains of virus
commercial warehouse. Temperatures of −70 °C are best but circulating in a region or country; however, because of cost,
are more expensive to maintain. Lymphocytes can also be these methods are frequently restricted to determine trans-
separated from anticoagulated blood, frozen at low tempera- mission during investigations of acute clusters of disease [1].
tures in fetal calf serum and dimethyl sulfoxide (DMSO), A full list of laboratory tests conducted using specimens
and later thawed for examination of stable cell surface mark- collected as part of the NHANES 2009–2010 can be found
ers and other cell-associated products. Cellular material, here: http://www.cdc.gov/nchs/nhanes/nhanes2009-2010/
even in extremely low concentration and a nonviable state, lab_methods_09_10.htm.
may be quite suitable for amplification and identification of
fragments of nucleic acid. These techniques offer powerful
tools for detecting genes characteristic of specific infectious 3.4 Advantages and Limitations
agents and other biological material of interest.
The use of serological surveys is an important means of sup-
3.3.4 Laboratory Tests plementing morbidity information, such as that obtained
The general principles and techniques of laboratory testing from routine case surveillance. Advantages are listed in
for viral infections are presented comprehensively in Chaps. Table 4.3. Because many viral infections may be clinically
2 and 3 of this text. The antibody tests most suitable to sero- mild or inapparent, laboratory confirmation is necessary for
logical surveys of specific viruses are detailed in each cor- accurate diagnosis of even overt cases. Data from serological
responding chapter of this book. The criteria for a satisfactory surveys can reveal total burden of infection (apparent and
test include simplicity, sensitivity, specificity, reliability, inapparent), both currently and in the past. Selection of tests
ability to detect long-lasting antibody, minimal interference that reflect antibody of long duration permits measurement
from nonspecific inhibitors, the availability of satisfactory of the cumulative experience of the population tested with
reagents, and the safety of the test for the laboratory techni- the disease in question; selection of a test based on short-
cian [29, 107]. The microtiter procedure developed by lived antibody such as immunoglobulin M (IgM) allows
Takatsy in 1950 in Hungary and popularized in this country identification of a recent infection or epidemic. Testing of
by Sever [82] in 1962 has become the standard method in two sera spaced in time permits measurement of the inci-
serological survey laboratories. It is adaptable to a wide vari- dence of infection during the interval period.
ety of antibody determinations, it requires a minimal amount An important advantage of carefully planned prospective
of serum (usually 0.1 ml) and other ingredients, and large serosurveys is that participants can become a cohort for
numbers of sera can be efficiently tested. Several automated other studies. For example, to determine the frequency with
methods of dilution and of adding various reagents have which hepatitis C-infected persons in the United States were
been introduced to speed the testing even more [107]. aware of their infection, the National Center for Health
The development of simple tests such as the enzyme- Statistics conducted a follow-up survey of positive cases. A
linked immunosorbent assays (ELISA) for antibody mea- full interview with these participants yielded not only
surement in microtiter plates has provided a sensitive method awareness, but also whether they sought medical care for
for identification of antibody in serum samples from survey their infection and their knowledge, attitudes, and practices
populations and can indicate both past and current infections related to hepatitis C [25].
[109]. The use of monoclonal antibodies in this and other
antibody tests permits highly specific identification of indi- Table 4.3 Advantages and limitations of seroepidemiologic studies by
data collection objective
vidual strains of the virus, a special advantage in determining
whether a new strain has been introduced in a community or Primary Secondary
if reinfection or reactivation has occurred in the individual. Advantages Sample is representative of Less expensive
the target population
Commercial kits for many antibodies are now available, and
Many possibilities of lab Saves time
new formulations are continually being devised for a variety markers and behaviors No participation bias
of clinical, public health, and research applications. Detection from volunteers
of virus and genetic characterization of viruses can be Limitations Expensive and Convenience sample
accomplished using polymerase chain reaction (PCR) meth- labor-intensive might not be
ods. Handling of specimens for PCR depends on two compo- Time consuming to design representative of the
and implement population of interest
nents: the source and virus, for example, varicella zoster
72 R. Jiles et al.

An advantage of serostudies in providing information to complement-fixing antibodies to cytomegalovirus, herpes

guide vaccination programs is that serostudies measure viruses, or dengue virus have been found to persist for
immunity and are likely more reliable than self-reported vac- years following infection.
cination coverage [26]. Another advantage is that aliquots of Measurement of IgG and IgM antibody by tests such as
sera from a collection can be shipped, frozen, over long dis- the ELISA or immunofluorescent antibody tests provides a
tances to a number of specialized reference laboratories for simple way in a single serum sample of reflecting current
testing; therefore, the work can be divided among participat- and past infection, respectively. Although IgM antibody usu-
ing laboratories. ally denotes a primary infection, certain viruses such as her-
The disadvantages of seroepidemiology are the cost, time, pes viruses may induce IgM on reactivation. It should also be
and effort involved in the selection of the target population, reemphasized that unlike prevalence data for clinical infec-
the collection and analysis of data and blood, and the need tious disease, serological prevalence data reflect total infec-
for and cost of laboratory facilities equipped to carry out the tion rates, representing both clinical and subclinical (or
tests. There must also be a satisfactory means of measuring asymptomatic) infections.
antibody for the particular virus to be studied, and the method Multipurpose antibody surveys have been carried out in
of carrying it out must be simple enough to allow perfor- a number of countries. In the United States, the successive
mance on a large-scale basis. Finally, because serostudies NHANES surveys have provided large numbers of serum
designed for primary data collection are labor-intensive and specimens for estimating cumulative exposure to various
prospective, there are significant delays in the availability of infections in representative samples of the population. For
data. For example, the NHANES data require about 2 years example, NHANES was critical in an understanding of
between the end of data collection and the availability of hepatitis A immunity in the United States because a vac-
datasets for public use. cination program was initiated in 1996 targeting select
higher incidence areas. Stratifying seroprevalence by
country of birth and race/ethnicity (Fig. 4.1) showed the
3.5 Uses of Serological and Molecular higher levels of immunity among foreign-born and
Techniques US-born Mexican Americans of any age. The impact of
the initial vaccination strategy was evident in an analysis
3.5.1 Prevalence of seroprevalence stratified by geographic region and age
Prevalence from serosurveys, or seroprevalence, is defined group (Fig. 4.2) [53].
as the number of persons whose sera contain a particular bio- In a Barbados seroprevalence study [32], a 10 % house-
marker among the total number of persons examined at that hold sample was randomly selected from a middle- and
point in time (frequently, 1 year). Unlike “case prevalence,” lower-socioeconomic-level community of 10,000 persons in
which indicates the existence of disease at the time of the Bridgetown. The results illustrate the type of information
survey, the presence of antibody reflects the cumulative that can be derived from this type of study. Of 100 sera from
experience, past and present, with an infectious agent. The children under age 10 tested, 30 % lacked protective levels
prevalence is a function both of prior and current infection of antitoxin against both diphtheria and tetanus, indicating
and of the duration of the antibody tested. In contrast, anti- the need for intensifying the immunization program against
gen indicates presence of the virus at that time; in cases of these diseases. The prevalence of protective levels against
latent or asymptomatic infection, it indicates prevalence of tetanus is a good indicator of the level of public health prac-
chronic infection. tice, since this antitoxin is acquired almost exclusively by
Many antibodies such as the neutralization antibody immunization procedures and not through natural infection.
for poliomyelitis or yellow fever virus; antibodies to The age distribution of antibodies to various viruses may
the HIV core, polymerase, and envelope; and the provide useful information on the behavior of these infec-
hemagglutination-inhibition antibody for influenza, para- tions in the community and of the need for immunization
influenza, rubella, measles, or arboviruses last for years, programs. On this basis, an active rubella immunization
perhaps a lifetime. Thus, the cumulative experience of a program was initiated in girls aged 12 years or younger. In
population can be measured and infection acquired in subsequent years through 1978, a few sporadic cases were
childhood can be detected in persons of middle or perhaps reported yearly, but no epidemic occurred [34]. Even spo-
even old age. Some waning of antibody titers (sometimes radic serosurveys have demonstrated utility, for example,
below the lowest detectable levels) may occur in older the researchers from the Barbados effort conducted a similar
age groups after a childhood infection or vaccination. survey in St. Lucia [31]. They found important and unex-
Similarly, the viral capsid antigen (VCA)-IgG antibody to pected differences compared to Barbados, including a
Epstein–Barr virus measured by the indirect immunofluo- higher prevalence of antibodies to dengue and rubella
rescence test has been found to be of long duration; even among young persons.
4 Surveillance and Seroepidemiology 73

Fig. 4.1 Age-specific 100

seroprevalence of antibodies Foreign-born
to hepatitis A virus by country 90 Mexican
of birth and race/ethnicity, Americans
NHANES, 1999–2006
80

70 US-born Mexican
Americans
60

Percentage
50
US-born non-
40 Hispanic Blacks

30

20 US-born non-
Hispanic Whites
10

0
6−11 12−19 20−29 30−39 40−49 50−59 60+
Age group (in years)

Fig. 4.2 Age-specific seroprevalence 80

of antibodies to hepatitis A virus
among US-born people by residence
70
in vaccinating or non-vaccinating
states based on 1999 99-06 Vaccinating
recommendations, NHANES, 60
1988–1994 and 1999–2006
50 99-06 Non-
Percentage

Vaccinating
40
88-94 Vaccinating
30

88-94 Non-
20
Vaccinating

10

0
0–12 12–20 20–22 30–32 40–42 50–52 60+
Age group (in years)

Initial tests for poliomyelitis antibody employing conven- after the 1972 survey, one in 1974–1975 and another in
tional microtiter neutralization procedures indicated that 27, 1977–1978. There have been no reported cases of poliomy-
42, and 54 % of those tested at 1:5 or 1:8 serum dilution elitis since 1972.
lacked antibody to poliomyelitis types 1, 2, and 3, respec- The 1972 Barbados serum collection was tested for
tively [32]. Subsequent tests on 304 sera using a 1:2 serum human retroviruses after the agents were discovered. The
dilution and longer serum–virus incubation periods indicated HTLV-1 antibody was found in 4.25 % overall, rising in age
that only 13.1 % lacked type 1 antibody, 6.5 % type 2 anti- from a 2.7 % prevalence for persons aged ≤10 years to 9.0 %
body, and 14.3 % type 3 antibody [34]. This emphasizes the among those aged 61–70 years [78]. Females had a higher
need for sensitive methods for detecting low levels of anti- prevalence rate (5.8 %) than males (2.3 %). The adult pattern
body. Two mass poliomyelitis programs were carried out raised the possibility of sexual transmission, and this was
74 R. Jiles et al.

strengthened by the finding of a prevalence rate of 14.1 % the natural history of HIV infections and the development of
among persons with a positive VDRL test for syphilis as AIDS. Further details of serological studies of HIV infection
compared with 3.5 % of those who were VDRL negative. can be found in Chap. 43.
There was evidence of household clustering and of vertical The calculation of incidence is possible from such cohort
transmission. studies because clinical, laboratory, and self-reported data
Advances in laboratory detection of viruses in oral fluids are collected on participants at regular intervals. Therefore,
allow surveillance in high-risk groups with less-invasive negative results are available, such that a time period can be
techniques. For example, unlinked anonymous surveillance calculated between the first positive and the most recent pre-
among injection drug users seeking services in England and vious negative result. Taken together, these and other cohort
Wales has been ongoing since 1990 [88]. The authors found studies have yielded critical data for the empirically based
that the prevalence of hepatitis C infection decreased from projections about burden of infection and disease in select
70 % in 1992 to 47 % in 1998 before rising again to 53 % in populations.
2006. Prevalence among women injecting drug users was
higher than among men, and that prevalence was highest in 3.5.3 Outbreak Detection
certain geographic areas that could be targeted for The advances in genetic characterization and communica-
prevention. tion online have combined to expand the scope and breadth
of serosurveillance into early detection of clusters of infec-
3.5.2 Incidence tious diseases. In the United States, norovirus is the most
Incidence is best calculated from cohort studies such that the common cause of foodborne disease outbreaks. Surveillance
appearance of the viral infection can be captured with a to detect early clusters of disease is conducted through rou-
known denominator of a well-defined population of persons tine notifiable diseases methods and by a laboratory network
at risk. However, cohort studies are costly and subject to called CaliciNet [96]. It is a national surveillance network
attrition over time. that includes a database for public health laboratories to sub-
Many prospective serological and clinical studies were mit gene sequences from human caliciviruses (noroviruses
used to investigate HIV infections and the development of and sapoviruses) identified from outbreaks. The information
AIDS and related clinical syndromes among high-risk popu- is used to link norovirus outbreaks that may be caused by
lations such as gay men, injection drug users, persons with common sources (such as food), monitor trends, and identify
hemophilia, and their contacts. Some of the earliest studies emerging norovirus strains.
were based on cohort methods using sera originally collected Measles in the United States provides an excellent exam-
for evaluation of HBV vaccine, and others started afresh with ple of the use of technology in outbreak detection. In 2000,
a new cohort [50]. The findings in the New York [86] and San the United States achieved measles elimination (defined as
Francisco cohorts [10] were as follows: (1) the prevalence interruption of year-round endemic measles transmission)
level of HIV antibody was 6.6 % in New York at entry into ( http://www.cdc.gov/measles/global-elimination.html ).
the study in 1978, and rose to 10.6 % by 1984; (2) in San However, importations of measles into the United States con-
Francisco the entering prevalence was 4.5 % in 1978, but tinue to occur, posing risks for measles outbreaks and sus-
rose dramatically to 73.1 % by 1985; and (3) the rate of viral tained measles transmission. During 2011, a total of 222
infection (seroconversion) among those lacking antibody on measles cases (incidence rate: 0.7 per one million population)
entry was 5.5–10.6 % per year in New York and 11.2 % in and 17 measles outbreaks (defined as three or more cases
San Francisco. Among gay and bisexual men entering the linked in time or place) were reported to CDC, compared
Multicenter AIDS Cohort Study (MACS) in Baltimore, with a median of 60 (range: 37–140) cases and four (range:
Chicago, Los Angeles, and Pittsburgh in late 1984 and early 2–10) outbreaks reported annually during 2001–2010.
1985 [50], seroprevalence ranged from 23 to 49 %. In a pat- Measles has also reappeared in other developed countries,
tern similar to those in other cohorts, the incidence rate of and a web-based, quality-controlled database with epidemio-
new infection in the MACS, documented at 3–5 % per year logic and nucleotide data for measles infection in the WHO/
in early 1985, declined rapidly during the next 3 years to Europe region was developed: the Measles Nucleotide
about 1 % [52]. In Africa and Thailand, measurements of Surveillance (MeaNS) [60, 79]. The major objectives of the
seroprevalence and incidence among prostitutes and other MeaNS initiative are to function as an epidemiologic surveil-
high-risk groups have repeatedly demonstrated the alarming lance tool and to monitor progress in measles control.
increases that have taken place over a very few years. Dynamic reports and graphical charts can be created on any
Transfusion-associated HIV infection systematically docu- user-selected fields in the MeaNS database (e.g., genotype or
mented through serosurveillance of hemophiliacs and other sequence variation in a geographical location or time period).
recipients [3, 54, 55], soon after known parenteral exposure, Information about MeaNS is available at http://www.hpa-
provided an important resource for comparative research on bioinformatics.org.uk/Measles/Public/Web_Front/main.php.
4 Surveillance and Seroepidemiology 75

3.5.4 Diagnostic Serology behavior. A program is regarded as effective when cases

Sera sent to large hospitals or public health laboratories for decrease or epidemics do not occur. This information may be
various tests can be frozen and stored for later antibody test- biased by the possibility that the decrease in clinical cases is
ing against other antigens. The specimens must be adequate related to poor reporting or that insufficient time has elapsed
in amount and free of bacterial contamination. Specimens for another epidemic to have occurred. Currently, our knowl-
sent for viral antibody tests usually fulfill these criteria and edge of the utilization of vaccines depends on such sources
are accompanied by minimal demographic and clinical data as sales records of manufactures, public clinic and physi-
concerning the patient. There are many uses for this type of cians’ data, direct interviews, and school entry surveys [20].
collection. All sera or exanthems coming from patients with Because of the inadequacy of these traditional surveillance
central nervous system, gastrointestinal, or respiratory infec- techniques in determining the need for and the effectiveness
tions can be tested at the time of receipt or, later, against a of a given vaccine, serological surveys could play an even
battery of viral antigens in order to reveal the profile of larger role in evaluation of immunization programs. Although
agents likely to have caused the syndromes. An example of the necessity for a venipuncture reduces the ease and accept-
this was the evaluation of the importance of a new virus, ability of this method, public health professionals and physi-
called “the California encephalitis virus” in the causation of cians can help surmount such barriers by conveying the
infections of the central nervous system by testing of all sera importance of seroepidemiology for such purposes. Newer
received in a state public health laboratory for this syndrome techniques that obviate the requirement for blood sampling
[44]. In Wisconsin, 5.7 % of 351 sera received in the state may further encourage the application of biological tools to
laboratory over the period 1961–1964 revealed evidence of the evaluation of vaccination effectiveness.
the new California viral infection [91]; in Minnesota, 4.1 % The uses of serological epidemiology in immunization
of 1,617 retrospectively tested sera contained this antibody. programs are summarized in Table 4.4. Much of this infor-
A second and related application is the determination of the mation could be obtained in no other way. Serological sur-
clinical spectrum associated with a newly discovered virus; veillance has been of particular importance for the new
this is accomplished by testing stored sera from patients with epidemiologic settings created by substituting vaccine
a variety of clinical syndromes and looking for evidence of immunity for natural immunity as for poliomyelitis, mea-
infection with the new agent. sles, rubella, and to a lesser extent mumps and influenza
A third application for sera stored over time is the mea- [30]. With vaccines against HBV universally recom-
surement of secular trends or antigenic shifts in viruses over mended for infants in the United States and a vaccine
time. This is especially useful in relation to influenza viruses. against varicella zoster virus, the patterns of susceptibility
A fourth use, employing freshly received sera from high-risk to and the distributions of these infections have changed
populations, is the search for influenza antibody patterns that substantially.
may reveal the beginning of an outbreak or a change in the
antigen composition of currently circulating strains; this was
used by Widelock et al. [101] at New York City public health Table 4.4 Uses of seroepidemiology in immunization programs
laboratories. Comparison of the geometric mean antibody
1. Cross-sectional surveys to determine the need for immunization
titer to influenza sera from persons in the acute phase of an programs in:
unidentified respiratory illness with the titer in others conva- (a) Different age groups
lescing from a similar illness may permit early identification 6–20 months—measure duration of maternal antibody
of an outbreak without waiting for serial samples from the School/college entry—identify omissions, failures, loss of
same persons. protective antibody
Finally, investigators in South Australia made efficient (b) Different geographic areas
use of samples taken in conjunction with blood donation. (c) Different socioeconomic groups
They searched for evidence of Ross River virus activity in (d) High-risk occupational groups
different locations during the arbovirus season by measuring 2. Follow-up measurements in immunized persons to determine:
(a) Proportion developing local, humoral, cell-mediated immune
IgA-, IgG-, and IgM-specific antibody in samples from Red
responses
Cross blood donors. Differences in antibody prevalence by (b) Quality and extent of response
region indicated prior activity and helped identify endemic (c) Nature and degree of interaction between vaccine components
areas but revealed that acute infection had not occurred in (d) Duration of response
that year at frequencies high enough to be detected [99]. (e) Level of protection against disease and asymptomatic infection
(f) Degree of spread of live vaccine strains to exposed and
3.5.5 Evaluation of Immunization Programs susceptible contacts
The effectiveness of an immunization program is tradition- 3. Periodic serological surveillance to identify groups who are not
ally judged on the basis of clinical cases or epidemic receiving vaccines or who have inadequate responses
76 R. Jiles et al.

Patterns of susceptibility and immunity to all of these 3.5.6 Biomarkers

viruses may now vary from place to place, from age group to Revolutionary advances in immunology and molecular biol-
age group, and in various socioeconomic settings, depending ogy have opened the possibilities for intensive analysis of
on the immunization program instituted by health depart- humoral and cellular material—what some are calling
ments and the activities of physicians and clinics rather than broadly “molecular epidemiology”—analysis that will clar-
on the inherent epidemiologic characteristics of the natural ify the role of viral infection in the pathogenesis of autoim-
infection and disease. The methods of immunization prac- mune, degenerative, neoplastic, and even “genetic” diseases
tice, the frequency of repeated immunization programs, and and will facilitate targeted drug and vaccine development.
the quality and duration of vaccine immunity will increas- An example of one such possibility for molecular study is a
ingly constitute the major determinants of the patterns of European network for hepatitis B virus. The HepSEQ (http://
these diseases. www.hepseqresearch.org) is a pioneering online resource for
Over the years, serological surveys in American cities, the management, characterization, and tracking of hepatitis
such as Syracuse [56], Cleveland [38], and Houston [61], B infection in the area. An important use will be to detect
have uncovered serious deficiencies in the antibody patterns mutations of the hepatitis B virus such as those consistent
for viral diseases for which vaccines are available. The US with antiviral resistance [64]. This information will be help-
military has used this approach to similar advantage. A ful in identifying transmission patterns that will guide pre-
national serosurvey of 1,547 Army recruits entering in 1989 vention efforts.
at ages 15–24 years documented 15–21 % seronegativity for
measles, mumps, and rubella; 7 % for varicella zoster virus;
and 2.3, 0.6, and 14.6 % for poliovirus types 1, 2, and 3, 4 Summary
respectively [51]. Likewise, a pre-vaccination survey of
1,568 US Navy and Marine entrants identified important dif- Systematic collection of cases of infection or disease and of
ferences in susceptibility to measles; susceptibility was great- biomarkers of infection or immunity is an essential tool in the
est among males, younger recruits and whites [87]. prevention and control of viral diseases. Surveillance and
Serosurveillance has also been used effectively in the devel- seroepidemiology have provided critical epidemiologic infor-
oping world for such purposes as a general assessment of mation to support public health policy at the local, national,
success in the WHO Expanded Program on Immunization and international levels. Continued advances in laboratory
[35] and a specific comparison of HIV-infected and unin- methods and communication systems will expand the applica-
fected Zairian infants for responses to polio vaccine [80]. The tions of surveillance and refine their precision and efficiency.
importance of surveillance programs and serological surveys
to evaluate immunization programs has long been recognized
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 Viral Dynamics and Mathematical
Models 5
Nimalan Arinaminpathy, Charlotte Jessica E. Metcalf,
and Bryan T. Grenfell

1 Introduction more complex life histories. After a brief discussion of mod-

els for the within-host dynamics of viruses, we conclude with
The application of mathematics to explore the dynamics and suggestions for gaps in knowledge and future research needs.
control of viral infections has a long history, which predates
its application in many other fields of biology, as well as the
germ theory itself [1]. A particularly fruitful area for devel- 2 Dynamics of Acute Immunizing
opment has been the dynamics of acute immunizing infec- Infections
tions, with their relatively simple natural history and rich
notification time series [2–4]. The last three decades have 2.1 Observed Epidemic Patterns
seen a considerable upsurge in the use of mathematical and
computational models to explore the dynamics and control Acute immunizing infections typically generate recurrent
of a wide range of viral infections. This phase arose initially epidemics in large communities. We illustrate these patterns
from developments in ecological population dynamics [4]; with the best documented case – the dynamics of measles in
it was then greatly accelerated, both by the explosion in large cities in England and Wales [2, 3]. Figure 5.1a shows
computational power and by the emergence of human the time series of raw weekly notifications for measles in
immunodeficiency virus (HIV) infection, severe acute respi- London from 1944 (shortly after measles cases became noti-
ratory syndrome (SARS), and other potential pandemic fiable) to 1994.
threats [5]. These data indicate three fairly distinctive dynamical eras:
In this chapter, we review basic concepts in infection • 1944–1950: Principally annual epidemics.
dynamics and control, via a synthesis of epidemic models and • 1950s–1960s: Regular, mainly biennial epidemic cycles,
data. We begin with acute immunizing infections, focusing with intervening small annual peaks – incidence rates are
on measles as a case study of the impact of herd immunity markedly seasonal (Sect. 2.4).
and other determinants of epidemic dynamics. We then • 1970s onwards: The vaccine era brought declining inci-
extend the discussion to dynamical modeling applications to dence, with increasing irregular, lower-amplitude epi-
a range of other acute and chronic viruses, with a variety of demic cycles. By the end of the series shown in this figure,
cases became very sporadic, with increasing levels of
mis-notification of clinically identified cases [7].
As well as these fluctuations in individual large cities,
N. Arinaminpathy, DPhil (*)
there are also rich dynamical patterns in the spatial spread of
Department of Infectious Disease Epidemiology,
Imperial College London, London, UK epidemics among large and small communities (Sect. 4).
e-mail: [email protected] Regional and temporal demographic variations, especially in
C.J.E. Metcalf, PhD birth rate, can also markedly influence dynamics (Sect. 2.4).
Department of Ecology and Evolutionary Biology and the Woodrow
Wilson School, Princeton University, Princeton, NJ, USA
e-mail: [email protected]
2.2 Epidemic Dynamics: The SEIR Model
B.T. Grenfell, PhD
Department of Ecology and Evolutionary Biology and
The striking epidemic patterns of acute immunizing infec-
Woodrow Wilson School of Public and International Affairs,
Princeton University, 211 Eno Hall, Princeton, NJ, 08544, USA tions are particularly well documented for widely notifiable
e-mail: [email protected] infections such as measles. The process of explaining these

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 81

DOI 10.1007/978-1-4899-7448-8_5, © Springer Science+Business Media New York 2014
82 N. Arinaminpathy et al.

a
10000
Weekly cases

5000
Vaccine era

0
45 50 55 60 65 70 75 80 85 90
b Year
Births
R=R0
c

Susceptible

R=1

Exposed

S
Infectious

Recovered
Time

Vaccination

Fig. 5.1 (a) Observed spatiotemporal dynamics of measles in England one infectious individual into a wholly susceptible population; the
and Wales, showing weekly aggregate time series of notifications for effect of birth rate on susceptibility is ignored. (d) Corresponding
London. (b) Basic flows of individuals captured by the SEIR model. (c) dynamics for the proportion of the population susceptible (line marked
Schematic time series of numbers of susceptible individuals, arising “S”) and the proportion infected (line marked “I”) (b, d: Taken from
from flows in panel b, and in particular following the introduction of Fig. 2, Grenfell [6]. Figure found on p. 38)

cycles, as well as that of addressing associated public health yet uninfected) susceptible individuals, infected, recovered,
issues, has spawned an extensive analytical literature, span- and other classes. An initial taxonomic split here is between the
ning public health epidemiology, theoretical biology, and SIR model (which crudely assumes that all infected individuals
population dynamics [4, 5, 8–10]. The key conceptual tool can pass on infection) and the SEIR (susceptible, exposed,
has been a family of compartmental dynamical models, infected, recovered) model, which adds an “exposed” (infected
based on the SIR (susceptible-infected-recovered) paradigm but not yet infectious) class [4]. SIR and SEIR models have
[2–5, 11, 12]. As described below, the SIR family success- qualitatively very similar dynamics [13]; however, we describe
fully captures many key features of the epidemiological the latter with its more realistic depiction of viral incubation.
dynamics and control of viral infections. The basic SEIR model, illustrated in Fig. 5.1b, reflects the fol-
The basic SIR formulation embodies the dynamics of lowing set of biological assumptions; we use measles as the
immunizing infections. Because viruses reproduce rapidly in classic acute exemplar here [14]. After a few months of mater-
the host, we ignore within-host kinetics in the simplest SIR nally derived passive immunity, infants enter the virus-naïve
models (but see Sect. 8); depending on model details, this leads “susceptible” (S) class; susceptibles can then become infected
to a compartmentalization of the host population between (as by close contact with infectious individuals (generally via
5 Viral Dynamics and Mathematical Models 83

respiratory aerosol for measles). Infection moves individuals 1

into the “exposed” (E) class, where they incubate but do not
transmit the infection for around a week; this leads to the infec- 0.8

Vaccination threshold, pc
tious (I) class, where virus is shed, again for approximately a
week, after which individuals enter the recovered state (R). In
0.6
the basic SEIR model, recovered individuals are assumed to be
immune for life, both to clinical disease and to retransmitting
the infection. However, subclinical infection (and hence boost- 0.4
ing of immunity [15, 16]) is in principle possible. The lifelong
sterilizing immunity induced by such an infection also makes 0.2
this an excellent potential vaccine candidate [4]. For the SEIR
model, vaccination is at its simplest assumed to be delivered to
0
a proportion p of infants at the end of the maternal immunity 0 2 4 6 8 10
period (i.e., near the effective “birth” of susceptibles). For indi- Basic reproduction number, R0
viduals who seroconvert, immunity is then often assumed to be
lifelong, moving susceptibles into the recovered class Fig. 5.2 The critical vaccination threshold (pc) and its dependence on
the basic reproduction number, R0. In the region above the blue line,
(Fig. 5.1c). There is also a considerable literature exploring
vaccination succeeds in local elimination of infection. Even in the
more operationally realistic age distributions and immunogenic region below the line, however, vaccination can substantially reduce
characteristics of vaccines in specific cases [5, 17]. overall transmission

2.3 Herd Immunity and the Impact herd immunity threshold (R = 1) becomes sc = 1/R0 (Fig. 5.1c);
of Vaccination thus, immunizing at a level above pc = 1 − sc = 1 − 1/R0 will elim-
inate local transmission. As described below, these calculations
We can lay bare much of the dynamical behavior of immunizing have been refined considerably to allow for heterogeneities in
epidemics by considering the case of the “simple” epidemic transmission with age, space, and other characteristics (Sects. 3,
(Fig. 5.1c, d), in which the outbreak occurs sufficiently rapidly 4, and 5). Nonetheless, our simple expression for pc is an
to ignore processes of host birth and death. The talismanic quan- extremely useful metaphor for epidemic control: (1) because of
tity here is the basic reproduction number (or ratio) of infection, indirect protection, not everyone needs to be vaccinated to
R0 [4, 5]. At its simplest, R0 is defined as the total number of eliminate transmission (corresponding to pc < 1) and (2) the
secondary cases caused by an infectious individual when intro- effort required to increase this level of immunization increases
duced into a well-mixed fully susceptible population. To illus- with transmission rates. The latter point is made clear by a
trate the ensuing dynamics, consider an epidemic of infection simple plot of pc against R0 (Fig. 5.2); more transmissible
with R0 > 0. Since each case initially causes R0 secondary infec- immunizing infections such as measles are harder to control
tions, the epidemic increases more or less exponentially over the than less transmissible agents such as smallpox. However, this
first few infectious generations (Fig. 5.1c). However, these refers to “random” mass vaccination; more targeted strategies
dynamics rapidly deplete the susceptible pool. This brings us to such as ring vaccination coupled to active surveillance can pro-
the other key parameter of epidemic spread: the effective repro- mote elimination even below pc, given (as with smallpox elimi-
duction number, defined as R = [S/N]R0. Here, S/N is the propor- nation [19]) the right biological characteristics and logistics.
tion of susceptibles in the population and R is the realized value Partial immunity and other characteristics of the population can
of R0 as the epidemic develops [4]. Since S declines over the complicate the picture still further (see Sect. 5.2 below).
course of the epidemic (Fig. 5.1c), so does R (Fig. 5.1d). Nonetheless, the metaphor that more transmissibility necessi-
This progressive decline in secondary infection rates tates a stronger vaccination effort remains.
through the epidemic is a manifestation of the key epidemio-
logical process of herd immunity [18] – increasing natural
immunity of the population that indirectly protects the remain- 2.4 Seasonal Transmission
ing susceptibles from infection. Eventually, the effective repro- and Recurrent Epidemic Dynamics
duction ratio declines through unity (Fig. 5.1c) as population
immunity increases; this corresponds to the herd immunity 2.4.1 Observed Epidemic Patterns
threshold, above which the epidemic will always decline, even in Developed Countries
if reintroduced to a closed population. This threshold is thus In the simplest analysis of an immunizing infection (Sect. 2.2),
also a key aim of vaccination campaigns [4, 5]. Remembering sustained cycles of infection will disappear, and the infected
that R = [S/N]R0, the associated susceptible proportion at the proportion in the population will settle to a constant level
84 N. Arinaminpathy et al.

0.03
Prop. infected

0.02 (a) Weakly damped epidemic without

seasonal forcing
0.01

0
5 10 15 20 25
Time
(b) Seasonal forcing drives
recurrent epidemics
x 10-3

Seasonal variation in
1.5 infection rate
Prop. Infected

0.5

0
0.07
4
0.06 3
2
0.05 1
Prop. Susceptible 0
Time (years)

Fig. 5.3 (a) Simulated SEIR dynamics for measles with a birth rate, amplitude set to 0.2; Grenfell and Bolker [20]). The green trajectory
but in the absence of seasonal forcing of transmission rate; for full shows the joint dynamics of susceptible and infectious densities through
model specification and parameters, see Grenfell and Bolker [20]. (b) time; area plots show the dynamics of infectious and susceptible indi-
Same model, with sinusoidal forcing of the infection as shown (forcing viduals (Taken from Fig. 3 in above book chapter, found on p.39)

(Fig. 5.3a). The question of what maintains the recurrent epi- with the biennial epidemic tendency of measles dynamics.
demics [9] generally observed for immunizing childhood However, during the postwar baby boom, births achieved suf-
infections (e.g., Fig. 5.1) drove researchers to seek the key ficient levels to shift measles dynamics into annual cycles
aspect of biological realism missing from the simplest SEIR (Fig. 5.4).
model. For measles in England and Wales, seasonal variation
in transmission driven by increased contact rates of children 2.4.2 Epidemic Dynamics in Developing
when schools were in session rapidly emerged as a possible Countries
candidate [12, 21] and has since received considerable empir- It was realized early that seasonality could also result in
ical support [2, 3]. Stochastic fluctuations in incidence could more complex dynamics, including chaotic fluctuations,
in principle also contribute to the maintenance of cycles [22, that is, very irregular dynamics with little long-term pre-
23]; however, for measles, the predominant driver is seasonal- dictability. Contexts with both high and low birth rates
ity in transmission [2, 3]. Birth rate may modulate the period- (and both high and low transmission since birth rates
icity of recurrent cycles driven by seasonality through its role and transmission act dynamically in very similar ways
in determining the rate of susceptible replenishment [24]. For for immunizing infections [24]) can promote complex
example, for most of the postwar pre-vaccination era in dynamics via coexisting attractors. Until recently, it was
London, seasonality generated sustained cycles by resonating thought that chaotic measles dynamics were not likely to
5 Viral Dynamics and Mathematical Models 85

be observed [3, 25], since observed seasonality in trans- with expectations of chaos (Fig. 5.5). Erratic boom and
mission was not strong enough to drive the associated vio- bust outbreaks are expected to continue even as routine
lent epidemics. However, recent analyses of measles in vaccination improves; and this suggests that high invest-
Niger revealed very strong seasonality (driven by move- ment in reactive vaccination and surveillance is important,
ment in and out of cities linked to rainfall); this, com- and pulsed vaccination approaches such as supplementary
bined with high transmission rates and the highest birth immunization activities could also play a helpful role in
rate in the world, results in irregular outbreaks consistent synchronizing dynamics [26].

Fig. 5.4 (a) Observed a

pre-vaccinations measles 400
dynamics for London (red
circles), corrected for under- 300
reporting, along with predictions
of an autoregressive time series
version of the SEIR model, 200
starting at the observed initial
density of cases and susceptibles 100
(for more details, see [126]) (b)
Cases (x100)

Birth rates accompanying the

0
dynamics in panel (a) (Taken
from Fig. 4 in above book
chapter, found on p. 41) 100

200

300

400
′44 ′49 ′54 ′59 ′64
Year
b
25
Births

15

a 25

25 290
0
1995 1999 2003

18.75 217.5
Cases per 1x105

Fig. 5.5 Time series dynamics of measles outbreaks from Niger. Niger
Rainfall (mm)

(a) Mean number of reported measles cases per 10,000 nationwide Niamey
in Niger from 1995 to 2004, and the mean monthly rainfall over the Rainfall
same time period (blue). Shaded regions give ±2 standard 12.5 145
deviations. Black curve, mean monthly cases of measles in Niamey
from 1986 to 2005. Inset monthly measles time series from 1995 to
2004. (b) Weekly measles case reports from seven departments of
6.25 72.5
Niger, 2001–2005. Red asterisk Niamey. Each department is an
aggregate of 3–8 arrondissements. (c) Case reports per month for the
city of Niamey from 1986 to 2005. The box indicates the time frame
shown in (b). Black dots months with 0 reported cases (Taken from 0 0
Fig. 1, Ferrari et al. [26]; http://www.nature.com/nature/journal/
v451/n7179/images/nature06509-f1.2.jpg) Sept Nov Jan Mar May July
86 N. Arinaminpathy et al.

Fig. 5.5 (continued)

b

12
8
12
Tahoua Agadez

4
8

0
4
2001 2002 2003 2004 2005

12
2001 2002 2003 2004 2005
Diffa

12
Cases per 1x105

8
Tillaberi

4
4

0
2001 2002 2003 2004 2005
0

2001 2002 2003 2004 2005

12
Zinder

8
12

Dosso

4
8

12

0
4

Maradi 2001 2002 2003 2004 2005

8
0

2001 2002 2003 2004 2005

4
0

c 2001 2002 2003 2004 2005

Niamey cases (per month)

6,000

4,000

2,000

0
1986 1988 1990 1992 1994 1996 1998 2000 2002 2004

3 Age Structure, Demography, infection [4]. Beyond these broad descriptors, however, there
and Serological Profiles is the added complication of possible relationships between
age and probability of exposure to infection. Such relation-
The mean age at which individuals become infected by ships may arise for a variety of reasons. For example, only
immunizing viral infection is generally lower for infections individuals beyond a certain age may work in areas where
with higher rates of transmission or R0 (defined above). the disease is transmitted, or the disease may be only sexu-
Intuitively, this occurs because the faster an immunizing ally transmitted. As a result, the force of infection, or prob-
virus is spreading through a population, the younger the age ability that a susceptible individual will be infected, will
at which individuals are likely to be exposed to it. Host show a distinct relationship with age.
demography modulates this relationship, and higher birth For directly transmitted infections, like measles, mumps,
rate countries with an R0 equivalent to that in lower birth rate rubella, and influenza, the age profile of the force of infec-
countries will tend to have a lower average age of infection tion is determined by the rate at which individuals of differ-
[27]. Maternal immunity, or protection of children after birth ent ages interact. There are two approaches to estimating this
by transfer of maternal antibodies, will also have an impact, variation. The first is to use age profiles of seropositivity to
increasing the average age of infection for children whose infer the pattern of the force of infection and, from this,
mothers have been exposed to infection. Since maternal anti- extrapolate to the pattern of contacts over age [28–31]. The
bodies rarely persist for much more than a year, this effect link between these age profiles and the force of infection
will be greatest for infections with very low average ages of over age can be made since once an individual has been
5 Viral Dynamics and Mathematical Models 87

a Estimated contact matrix (annual) b Polymod contact matrix (daily)

70

70
4000
10
60

60
5

2000
50

50
Age (Yrs)

Age (Yrs)
1
40

40
1000
30

30
20

20
500
10

10
0
0

0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Age (yrs) Age (yrs)

Fig. 5.6 (a) Contact matrix (annual) estimated from serology profiles and incidence data using maximum likelihood approaches (Ref. [30]), here show-
ing a semi-assortative mixing matrix; (b) POLYMOD contact matrix (daily) obtained from diary studies showing records across all of Europe (Ref. [34])

infected, she/he can never be infected again. Therefore, to be where the burden of disease shows an age profile. Rubella is
immune at any age, the individual must have contracted the a classic example. Infection during early childhood tends to
infection prior to that age. On a population level, cross- be mild, but infection during pregnancy may result in birth of
sectional seropositivity thus provides an indication of the a child with congenital rubella syndrome, consisting of a
total risk of infection for individuals up to a given age. range of birth defects (see Ref. [38]). A realistically complex
(Longitudinal age-serological profiles can be even more age structure of mixing, as detailed in empirical studies, may
powerful in quantifying epidemic risk [32]). thus be of crucial importance in establishing the burden of
The age profile of infection can be framed mathematically disease [39] but also how the burden of disease is likely to
[33], and parameters describing contacts between individuals change as a result of vaccination.
of different ages inferred [31]. The resulting estimated matrix
of contacts over age is known as the Who-Acquires-Infection-
from-Whom or WAIFW matrix. Alternatively, patterns of
contacts between individuals can be directly measured [34], 4 Spatial Dynamics
for example, using diary studies (Fig. 5.6); and by combining
this with the age profile of infection within the population, the So far, we have assumed that deterministic, spatially homo-
force of infection over age can be inferred [35]. geneous dynamics govern infectious disease outbreaks. In
The infection dynamics themselves may also influence the fact, epidemics often spread across a heterogeneous land-
relationship between age and force of infection. If outbreaks scape of human cities, towns, or rural communities, and this
are separated by long intervals during which little exposure spread depends partly on the links between those locations.
occurs, individuals may remain susceptible for many years This leads us to move from the deterministic SIR model
and thus contract the infection at a later age than might be described above to stochastic models, which account for the
predicted from the age profile of contacts alone [36]. More random nature of individual infection dynamics and
subtly, detailed network analyses show that even within a sin- demography – for instance, individuals may or may not
gle influenza outbreak, the burden of disease can cascade from become infected with a given average probability, so that by
children (where contacts are highest) to the less connected chance, particularly in small populations, no new infections
adults as immunity accumulates within the children, with may occur, and the chain of transmission may be broken.
implications for optimal vaccination distribution [37]. During the troughs between epidemic outbreaks in smaller
Understanding the processes underlying the average age communities, incidence may fall to such low numbers that
of infection has a practical importance for any infection local extinction is likely.
88 N. Arinaminpathy et al.

Based on this observation, Bartlett [9, 40] used analyses the SEIR model and vary the infectious period, from the
of epidemiological data and stochastic models to develop the roughly 1 week of measles to 10 years, corresponding to the
notion of critical community size (CCS), or population size approximate average pre-HAART treatment infectious
below which stochastic extinction is expected, which was period of HIV [4].
further developed by Black [41] in studies of measles persis- Figure 5.7 explores this comparison, simulating an infec-
tence in insular populations. Bartlett demonstrated the exis- tion which invades a partly susceptible population (full
tence of a CCS of around 300,000–500,000 for measles in details are given in Ref. [6]; note that for clarity, the epi-
England and Wales. For measles to persist in locations with demic curves for 1- and 10-year infectious periods are raised
a population size smaller than this CCS, immigration of above the curves for the more acute infections). Each simula-
infected immigrants from elsewhere in the metapopulation is tion refers to a virus with a different infectious period. We
necessary. The result is that the spread of measles across assume that R0 is identical for each of these infections. R0 is
England and Wales in the pre-vaccine era resembled travel- roughly given by the product of mean per capita infection
ing waves spreading out from London [6], with a substantial rate and infectious period; thus, to keep R0 constant:
epidemic lag in locations further away. The duration of the • A short infectious period implies a relatively high infec-
lag was also shaped by the size of the local populations. The tion rate.
duration of fade-outs following local extinction contains • A longer infectious period requires a lower infection rate.
information on the degree to which a particular location is This assumption imposes a simple evolutionary constraint
connected to the metapopulation as a whole [42]. Generally, on our set of model pathogens in Fig. 5.7, in that fitness
this points to larger places being more connected. More (roughly equating to R0) is kept constant as we increase the
detailed parametric analyses tend to confirm this (e.g., the infectious period. Figure 5.7 indicates, first, that increasing
gravity model [43]). the infectious period reduces, and eventually eliminates, the
Similar processes may operate for immunizing or lethal tendency for cyclical epidemics [48]; essentially, a longer
infections in animal populations, with, for example, the infectious period “fills in the troughs” following major epi-
spread of rabies through raccoon populations acting as an demics. As infectious period increases, we therefore see a
invasion wave structured by large landscape features such transition from seasonally driven biennial epidemics (at the
as rivers [44]. For more complex human infections, fea- measles extreme of a 1-week infectious period) to low-
tures such as imperfect immunity (see Sect. 5.2 below) will amplitude annual epidemics (at 1 month). For longer infec-
tend to shift the age class of hosts who disperse the infec- tious periods (1 year), we see slowly evolving epidemics
tion. That shift may impact on the main mechanism of dis- with little seasonal activity and a modest post-epidemic
persal [45] and move the scenario away from invasion into overshoot. Finally, for a 10-year infectious period, we see a
a locally coupled landscape to a more demographically smooth slow epidemic, with an essentially “logistic” rise to a
driven dynamic [46]. stable endemic plateau of incidence. Though crude, this
exercise captures the essential dynamical transmission from
the violent epidemics of acute infections to the much
5 Comparative Dynamics smoother and slower epidemic invasion of HIV [4]. Note that
the seasonal variation in infection rate is assumed similar for
So far, we have explored the epidemiological dynamics the “measles” and “HIV” cases; however, the latter com-
of acute, immunizing viral infections. Though the result- pletely eliminates associated seasonal fluctuations in inci-
ing dynamics are both important and fascinating from a dence due to the smoothing effect of prolonged infectious
dynamical perspective, the natural (life) history of most viral carriage.
infections departs, in one way or another, from this simple Figure 5.7 also illustrates a second major dynamical
case. We review these complexities, and their epidemiologi- impact arising from the trade-off of increased infectious
cal and control implications, in succeeding Sections. period against lowered infection rate. “Fast” infections are
much more prey to local stochastic extinction in the deep
troughs between epidemics (Sect. 2.4) than the much more
5.1 From Acute to Chronic endemic incidence promoted by longer infectious period. On
the other hand, acute immunizing infections can invade pop-
A dramatically different life history from the transient infec- ulations much more quickly than chronic infections for the
tion paradigm represented by measles is observed with infec- same R0 (Note that very imperfect immunity (corresponding
tions that are much more persistent (even lifelong). This to ‘SIS’ dynamics) could generate ‘fast’ invasion even for
difference may be expressed in terms of infectious period in relatively chronic infections (Figure 5.7, inset), because of
individual hosts [4]. To illustrate how increasing infectious the increased supply of susceptible individuals (Sect. 5.2)).
period alone modifies the violent epidemics of childhood Note that, despite these great variations in dynamics, the
infections, we retain the assumption of lifelong immunity of assumption of a common R0 means that the herd immunity
5 Viral Dynamics and Mathematical Models 89

Fig. 5.7 Numerical solutions of 1 week

seasonally forced SEIR models 105 105
2 weeks
(see Fig. 5.3b), showing changes
4 weeks
in the dynamics of infection SIS
caused by increasing the 1 year
infectious period (see color key) SIR 10 years
while maintaining the basic 104
reproduction number of infection
at a constant level. To simulate 0 5 10 15 20
crudely the initial dynamics of a
“novel” infection, the system
starts by introducing 6 % 103
infectives into a 20 % Cases
susceptible population (a real
novel epidemic might be much
more violent if everyone is
susceptible). Inset: Comparing 102
the “slow” dynamics generated
by a 10-year infectious period
with a (sketched) solution of an
SIS model with much faster
101
dynamics (Taken from Figure
in Box 2, Grenfell and
Harwood [47])

100
0 5 10 15 20
Time (years)

threshold for elimination of infection is the same across this ously immune individuals, the critical vaccination threshold
range of behaviors Sect. (2.3). (Sect. 2.3) is raised, making it more difficult to control the
spread of infection. For this reason, vaccination against
many imperfectly immunizing infections is aimed at direct
5.2 Departures from the SEIR Paradigm protection of those receiving vaccine, rather than at raising
indirect protection.
We have seen that the SEIR framework (Fig. 5.1b) can be a
powerful tool for capturing the dynamics of perfectly immu- 5.2.2 Partial Immunity
nizing infections such as measles. With its various refine- So far, the models we have considered assume that immune
ments to incorporate structure in the host population (such as individuals have solid, transmission-blocking immunity,
age and spatial distribution), it yields a rich variety of even if this should wane through time. In reality, however, it
dynamical behavior (Fig. 5.7). Given the range of immuno- is common for immunity to be clinically protective (i.e.,
logical complexities and different viral life histories, how- against symptomatic disease) but only partially protective
ever, it is clear that SEIR dynamics are only part of the story. from infection. This applies, for example, in the case of rota-
In such cases, it is often possible to adapt Fig. 5.1b to more virus, the leading cause of severe diarrhea in infants world-
faithfully reflect such important biological complexities. wide [53, 54]. Figure 5.8b illustrates a model structure
developed to capture these dynamics [46].
5.2.1 Waning Immunity
Many viruses are capable of reinfecting a host. This may be 5.2.3 Host Heterogeneity in Transmission
due to the waning of host immunity over time, as with respi- Recalling the qualitative differences between the dynamics
ratory syncytial virus (RSV) [49], or due to viral evolution of acute and chronic infections (Fig. 5.7), some viruses show
for immune escape (e.g., with norovirus [50] and influenza) a “mixed” character on the host population level: while some
[51, 52]: see Sect. 6 for a more in-depth treatment of the lat- individuals clear infection relatively quickly, others may
ter. In either case, the overall dynamical effect is for recov- continue to harbor the virus as “carriers,” either in a latent
ered, immune individuals to eventually reenter the susceptible phase or in a chronic infectious state, for a longer period. An
pool, as illustrated by Fig. 5.8a. example is hepatitis B, in which 5–10 % of adult patients
Where such waning of immunity is appreciable, the show chronic infection. As illustrated in Fig. 5.8c, this can be
implications for vaccination can be profound. As the suscep- represented mathematically by identifying two infectious
tible pool is replenished not only by births but also by previ- classes: one acute and the other chronic.
90 N. Arinaminpathy et al.

a
c
IA

S E I R

S E R

b
IC

S1 E1 I1 R1

S2 E2 I2 R2

Fig. 5.8 Examples for extensions of the basic SEIR model (Fig. 5.1b). tection, then additional SEIR stages may be incorporated to capture the
(a) Waning immunity can be represented by allowing individuals to return corresponding effects on transmission dynamics. (c) An example of pop-
from recovered (R) to susceptible (S) status at a given rate. (b) If acquired ulation heterogeneity, distinguishing “acute” cases (IA) from “carriers”
immunity affords clinical but not sterilizing (transmission-blocking) pro- (IC), the latter recovering at a slower rate than the former

Such factors can have implications for the dynamics However, there are also indications of a short period of
and persistence of infections in a population [55]. In par- cross-protective immunity (i.e., against all serotypes) for
ticular, where a disease shows epidemic cycles, facing the 2–9 months following infection [68], potentially mediat-
possibility of local extinction in the epidemic troughs, the ing some degree of competition between serotypes.
presence of a small number of carrier hosts can facilitate Several studies have addressed the dynamical implica-
viral persistence in the population, through these troughs. tions of interactions between ADE and such immunity
For example, in some cases infection with varicella zoster [69, 70]. Dengue dynamics are therefore complex and
virus can be followed decades later by infectious shingles, multifactorial: an understanding of these effects is impor-
an occurrence which could maintain the virus in small tant in understanding the potential for unexpected effects
populations [56]. from transmission-reducing interventions [71].

5.2.4 Complex Viral Strain Interactions

Strain structure, vector dynamics, and seasonality in trans-
mission offer yet more complexities, as in the example of 6 Dynamics and Evolution
dengue [57]. Spread by Aedes mosquitoes, dengue has shown of Immune Escape
a significant emergence worldwide in the last 50 years.
Dengue dynamics are notable for the global circulation of Had viruses been discovered by the time that Darwin pro-
four distinct serotypes (see Fig. 5.9a). Notably, prior immu- posed “descent with modification” in the mid-nineteenth cen-
nity to a given serotype can elevate the risk of severe disease tury, they may quickly have been recognized as fine examples
from subsequent infection by a different serotype [60–62]. It of evolution in action. Indeed, today there is wide acknowl-
has been proposed that this arises from antibody-dependent edgment of the inextricable role that evolution plays in viral
enhancement (ADE) between different serotypes [63, 64], dynamics [58] and in the control of many different viral infec-
and modeling studies suggest that ADE could play an impor- tions. RNA viruses in particular, lacking the replication fidel-
tant role in shaping the epidemiological and strain dynamics ity of a DNA genome, are capable of considerable mutation
of dengue, on the host population level [65–67]. rates [72]. In the presence of host immunity, natural selection
5 Viral Dynamics and Mathematical Models 91

a HIV-1, subtype B Dengue Influenza A/H3N2

(envelope (E) gene) (envelope gene) (HA gene)

DENV-1

DENV-3

DENV-2

DENV-4
Time

b INNOVATION SELECTIVE SWEEP EXPLORATION INNOVATION

Viral population (black dots) Population moves to new neutral Drift through neutral network; Mutation onto a new neutral
with mutation (yellow dot) onto network; phenotype changes genotype changes but no network
a new neutral network phenotype changes
Peak viral infection

Fig. 5.9 (a) A diversity of viral diversity: examples of major human (b) Schematic illustration of the “epochal evolution” model, which seeks
infections showing different patterns of evolution (Taken from (Ref. to explain notable features in the phylogeny of human influenza A (panel
[58]), and references therein; Adapted from Fig. 1, Grenfell et al. [58]) a, rightmost phylogeny) (Taken from figure in van Nimwegen [59])

will favor those mutants most capable of evading immunity In what follows, we explore both within-host and
while still being capable of spreading between hosts. population-level aspects of influenza evolution, with an
As a result of these high mutation rates, population-level emphasis on seasonal (interpandemic) influenza. We then set
evolutionary patterns that can arise are quite diverse. out the prevailing paradigms that seek to explain how these
Figure 5.9a illustrates how some infections, such as HIV, patterns arise, through complex interactions between viral
show significant population-level diversity [73], while oth- evolution and epidemiology.
ers, notably seasonal influenza A, exhibit a markedly differ-
ent pattern, with a “trunk-like” phylogeny on the population
level [74]. 6.1 Immune Escape and Herd Immunity
The case study of influenza demonstrates not only inter-
esting evolutionary patterns but also the following important Classic theoretical principles [4] provide a useful framework
features: in which to think about viral evolution, where “evolutionary
(i) The challenges that evolution can pose for infection fitness” is determined ultimately by the capacity for trans-
control mission in a given host population. Recall from Sect. 2.3 that
(ii) The complex interplay, across a range of physical in the presence of prior immunity in the population, the
scales, between the evolution of a pathogen and its suc- effective reproduction number, R, serves as a compact mea-
cess in spreading in a host population sure of transmission potential. Vaccination aims to lower R
(iii) The many outstanding questions that remain, in under- through reducing the number susceptible in the population;
standing how viruses adapt to continue reinfecting their however, evolving pathogens introduce the complexity of
hosts countering this effect, by evading immunity and thus acting
92 N. Arinaminpathy et al.

to restore R. Note the combination of immune escape and Two prevailing paradigms explaining these important
transmissibility encapsulated in R. In particular, on the scale phenomena, on both the genetic and antigenic levels, are the
of the host population and with a given level of existing epochal evolution model [82] and that of strain-transcending
immunity, the escape mutant with the greatest evolutionary immunity [83]. Both may be considered “phylodynamic”
fitness is that which maximizes R [5]. models [58], in the sense of aiming to capture the complex
interactions between viral immune escape, viral population
genetics, and epidemiology.
6.2 Molecular Aspects of Viral Evolution The picture of “strain-transcending immunity” [83]
invokes a temporary mode of immunity, which immediately
As discussed in Chap. 21, the influenza surface protein hemag- follows recovery from infection and protects the individual
glutinin (HA) is a key immune target, with anti-HA antibodies against infection by all influenza strains. Such broad immu-
capable of offering sterilizing immunity, that is, the potential to nity is postulated to arise, for example, either from T cells or
block host infection altogether. Indeed, raising such immunity from innate immunity [83]. Over the course of months, this
is a central function of current influenza vaccines. However, broad immunity gives way to more long-lived, more nar-
HA is also the most variable viral component, continually rowly acting immunity that is specific to the immunizing
under selective pressure to escape antibody binding [51]. For strain. On the population level, a key dynamical effect of
these reasons, while influenza evolution is by no means limited such immunity structure is to impose population-level con-
to HA, this viral component has attracted the most attention. straints on viral diversity, sufficiently strong to maintain a
An important mechanism of immune escape is through single dominant lineage through time.
conformational changes of HA epitopes to abrogate antibody The epochal evolution model [82] provides an alternative
binding, without compromising the ability of the virus to view. It builds on the proposal by Kimura in 1968 [84] that many
attach to host cells [75]. More recently, however, results amino acid substitutions in nature do not alter evolutionary fit-
from mouse experiments suggest that escape mutants can ness and are thus phenotypically neutral. If influenza HA evolu-
also acquire increased viral avidity for host cells [76]. tion can be thought of as tracing a series of paths through a space
Another potential immune escape strategy [77] is glycosyl- of genotypes, the epochal evolution model proposes that, in phe-
ation, or the attachment of oligosaccharides to the HA mol- notypic terms, such a space has a modular structure (Fig. 5.9b) in
ecule, to occlude epitopes from antibody binding. Such which each “module” is a group of genotypes sharing the same
strategies are not without potential functional costs for repli- antigenic phenotype and a transition between modules corre-
cation [78], and yet there are indications that human influ- sponds to the observed antigenic “jumps.” In particular, HA evo-
enza A/H3N2 has been accumulating glycosylation since its lution diffuses through the local genotype space, accumulating
introduction into the human population in 1968 [79, 80]. neutral substitutions and thus genotypic diversity, over several
years. Ultimately, a single strain accumulates the substitutions
required to transition to a new antigenic phenotype. At this point,
6.3 Population-Level Manifestations the emergent strain – owing to its antigenic novelty – causes a
of HA Evolution peak in infection and undergoes a selective sweep in the host
population, to the exclusion of other strains. Such an event thus
On a genetic level, the evolutionary pattern for influenza HA acts to periodically control antigenic diversity, maintaining the
(Fig. 5.9a), showing serial replacement of strains through “trunk-like” shape of the influenza phylogeny.
time, is often characterized as “drift.” The limited standing Alongside these two prevailing paradigms of influenza evo-
diversity, or “trunk-like” phylogeny, is especially paradoxi- lution, yet another model [85] proposes that observed strains
cal in light of the relatively high mutation rate of influenza A, are drawn from a limited set of antigenic types, with their selec-
where instead several lineages might have been expected to tion dependent on niches in host population immunity. While
emerge and coexist in the population. each of these models succeeds in capturing important features
Moreover, how do such genetic patterns translate to antigenic of influenza dynamics, they also highlight important gaps in
properties, that is, viral interaction with anti-HA antibodies? In our understanding of viral evolution and how to manage it.
2004, new techniques allowed a characterization of the antigenic
evolution of the influenza subtype H3N2 [74] during the years
since its introduction into humans in 1968. The resulting pattern 7 Coevolution and the Evolution
is characterized by “punctuations” in antigenic evolution, occur- of Virulence
ring roughly every 2–8 years, in contrast with the more gradual
pattern of genetic change shown in Fig. 5.9a. These punctuations The degree to which viruses harm hosts varies considerably.
have great importance for public health, often necessitating Both within and between virus species, some variants may
major reformulation of current, HA-based vaccines [81]. barely affect hosts at all, while others have significant health
5 Viral Dynamics and Mathematical Models 93

impacts, up to and including host mortality [86]. An evolu- interferon-alpha, ribavirin, and protease inhibitors, with
tionary perspective indicates that variation in virulence upwards of 50 % of treated patients being responders; how-
across clones should be shaped by the costs and benefits of ever, there is a need for a more rationally optimized approach.
virulence for parasite transmission [87]. The spread and per- Models capturing viral dynamics in treated and untreated
sistence of virulence mutations may be favored if they covary patients have contributed to an understanding of the action of
with transmission. However, since host mortality can inter- these therapies [100–102], estimates of parameters such as
rupt transmission, a range of values of virulence may lead to rates of viral growth and of viral RNA clearance [102, 103],
equivalent levels of overall fitness for the parasite [88]. and correlates of long-term response to therapy [104].
While it is broadly agreed that the theoretical framework and Modeling approaches have been used to study the kinetics
empirical evidence on the existence of virulence-transmission of acute infections too, notably in the context of influenza
trade-offs need to be further developed and extended [87], infection [105]. A key interest, for example, has been the
there are some systems where the general predictions appear relative importance of target cell depletion, innate immunity,
to be borne out (e.g., [89, 90]). Perhaps most famously, the and adaptive immunity in shaping the dynamics of viral
introduction of the myxoma virus into Australia was at first infection [106–108].
devastating to rabbit populations, causing near 100 % mor- Overall, while there remain significant gaps in our under-
tality. However, over subsequent years, the virulence of the standing of these and other major viral infections [103, 109],
infection decreased. Various lines of evidence suggest that this work demonstrates the clinically relevant insights that
this occurred in response to selection for increased transmis- can be derived from a careful study of within-host dynamics
sion via decreased virulence – swift rabbit mortality was not [91, 110].
an efficient mode of transmission for the virus.

9 Summary and Future Directions

8 Within-Host Dynamics
This chapter has broadly outlined the multitude of factors shap-
While we have so far largely discussed virus dynamics on the ing viral dynamics, as well as the role of mathematical
level of the host population, there are equally important pro- approaches in elucidating these factors and their interactions.
cesses to be considered on the within-host level. The “kinet- This growing body of work sets the stage for future directions.
ics” of viral infection arise from a complex array of factors The link between epidemiological modeling and policy is
including viral replication and variability, the dynamics of the long-standing. Bernoulli’s work provides perhaps the earliest
immune response, and pathogenicity to the host [91]. Over case study [1]; recent high-profile examples include the use
the past two decades, mathematical models have played an of models to guide responses to foot and mouth disease in the
important role in studying these dynamics, often motivated UK [111], smallpox [112], and influenza [113]. The key cri-
by the need to understand the actual and potential impact of terion of the effective deployment of models for policy is that
interventions such as treatment or immunization [91–93]. they are embedded in testable hypotheses, embodying the
A major example is HIV infection (see Chap. 43); its clin- best possible science. Continuing progress in this area thus
ical course is characterized by an initial acute phase of viral calls for better understanding of the basic principles.
replication, lasting on the order of weeks, followed by an Especially in the context of viral evolution, whether in
asymptomatic latent phase that can last decades before ulti- relation to immune escape or virulence, a complete biologi-
mately progressing to AIDS [94]. Major advances in the cal framework calls for a linkage of processes across dispa-
1990s showed that despite the long clinical timescale rate scales, from the macroscopic structure of the host
involved, in vivo HIV replication is in fact a rapid process, population to the molecular basis of viral replication and
with the viral life cycle being on the order of days [95, 96]. transmission. A key task at the heart of this challenge is argu-
Subsequent studies modeling the dynamics of drug resis- ably to quantify transmission potential in terms of immune
tance underlined the need for early and aggressive drug ther- escape, that is, to measure empirically how changes at the
apy [97]. Conversely, more recent work capturing the molecular level impact R (see Sect. 6.1). Indeed, recent
dynamics of the immune response has illustrated how immu- equine influenza experiments provide some important steps
nity raised by CD8+ T-cell vaccination elicits a response that in this direction [114].
is too weak and too late to achieve sterilizing immunity [98]. New and emerging ways of tracking disease may also
Hepatitis C virus (HCV) represents another major public help to shed new light on the global spread of viruses and
health challenge and, like HIV, is a rapidly mutating virus on how they may be better controlled. To name three exam-
capable of continually evading immunity to establish chronic ples, while disease surveillance continues to operate largely
infection [99]. HCV infection is currently treated with through public health channels, there is increasing interest
broad-spectrum combination antiviral therapy including in the use of alternative sources, including automated
94 N. Arinaminpathy et al.

tracking of Internet and social media trends [115, 116]. 11. Dietz K, Schenzle D. Mathematical models for infectious disease
Second, genetic sequencing and analysis complements statistics. In: Atkinson AC, Feinberg SE, editors. A celebration of
statistics. New York: Springer; 1985. p. 167–204.
existing epidemiological approaches. The advent of high- 12. Schenzle D. An age-structured model of pre- and post-vaccination
throughput whole-genome sequencing is expected to shed measles transmission. IMA J Math Appl Med Biol. 1984;1:169–91.
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 Part II
Viruses Causing Acute Syndromes
 Adenoviruses
6
Xiaoyan Lu, Amita Joshi, and Phyllis Flomenberg

1 Introduction among serotypes. Consequently, new types and variants of

established serotypes appear over time.
Adenoviruses are among the most widely distributed viruses Certain HAdV serotypes have caused epidemics of acute
in nature, infecting vertebrates as diverse as mammals, birds, respiratory disease (ARD) in military training camps. Live
fish, and reptiles [1]. Those adenoviruses that infect humans enteric HAdV vaccines have been used safely and effectively
(HAdVs) are ubiquitous among the general population and to prevent outbreaks of ARD in new military recruits since
have a worldwide distribution. Most HAdV infections occur the early 1970s. After the manufacture of the HAdV vac-
in early childhood. They are most frequently associated with cines was discontinued in 1996, outbreaks of ARD reemerged
febrile upper respiratory tract illnesses with pharyngitis, pha- in both military recruits and some civilians, prompting rede-
ryngoconjunctivitis, or coryza but can occasionally cause velopment of the vaccines.
pneumonia. Other manifestations of HAdV infections Adenoviruses are also under intensive investigation as
include gastroenteritis, keratoconjunctivitis, and rarely car- vectors to deliver foreign genes. Although the immunogenic-
diac, genitourinary, and neurologic diseases. Most HAdV ity of adenovirus vectors and the high prevalence of
diseases are self-limited and do not require therapy. However, adenovirus-specific memory immune responses have been
severe and sometimes fatal infections can occur in immuno- obstacles for long-term gene therapy, recombinant adenovi-
compromised hosts, neonates, and occasionally healthy chil- rus vectors have potential for use to immunize against other
dren and adults. Although there have been no clinical trials pathogens and as tumor vaccines.
performed to document efficacy, severe HAdV infections are
most frequently treated with cidofovir, an antiviral agent
licensed for the treatment of cytomegalovirus disease. 2 Historical Background
HAdVs can cause persistent infections, with the virus
being shed for months after acute infection. Additionally, In 1953, Rowe et al. [2] described an agent that caused spon-
HAdVs are heterogeneous and subject to recombination taneous degeneration of surgically removed human tonsils
and adenoids in tissue culture. In 1954, similar agents were
isolated from military personnel with febrile respiratory dis-
X. Lu, MS
Gastroenteritis and Respiratory Viruses Laboratory Branch, ease [3], and in 1956 the term “adenovirus” was first adopted
Division of Viral Diseases, [4]. Subsequent studies revealed that these newly discovered
Centers for Disease Control and Prevention, agents were a major cause of epidemics of acute respiratory
1600 Clifton Road NE Bldg 18, Rm 7/110, Atlanta,
disease (ARD) in military training camps [5].
GA 30329, USA
e-mail: [email protected] There is evidence that HAdV infections were present long
before the 1950s. Epidemics of ARD were described in mili-
A. Joshi, PhD
Division of Vaccines and Biologics Research, tary training camps as far back as the Civil War. The adeno-
Merck & Co., 770 Sumneytown Pike, viral syndrome epidemic keratoconjunctivitis (EKC) was
West Point, PA 19446, USA first described in German workers in the late 1800s [6].
e-mail: [email protected]
Outbreaks of EKC were commonly observed during World
P. Flomenberg, MD (*) War II in US shipyards, where the disease was referred to as
Division of Infectious Diseases, Department of Medicine,
“shipyard eye” [7]. Transmission likely took place in medi-
Thomas Jefferson University,
1015 Chestnut St., Suite 1020, Philadelphia, PA 19107, USA cal facilities where workers sought treatment for foreign
e-mail: [email protected] bodies or chemical irritation of the eye.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 99

DOI 10.1007/978-1-4899-7448-8_6, © Springer Science+Business Media New York 2014
100 X. Lu et al.

Epidemics of a febrile illness with conjunctivitis related to characteristics: phylogenetic distance, genome organization,
swimming activities were first described in the 1920s [8]. DNA homology (G + C%), number of virus-associated RNA
When diagnostic tools for HAdVs were developed in the genes, ability to recombine, cross-neutralization, oncogenicity
1950s, all swimming-pool-associated outbreaks of pharyn- in rodents, and differential ability to agglutinate human and
goconjunctival fever were traced to them [9]. HAdVs also animal erythrocytes [19]. Species B can be further divided into
became recognized as important causes of febrile respiratory two subspecies, B1 (3, 7, 16, 21, 50) and B2 (11, 14, 34, 35),
illnesses in infants and young children [10]. Continued out- based on homology of the viral genomes, tissue tropism, and
breaks of ARD in military training camps led to a large num- associated disease patterns [20, 21].
ber of epidemiologic studies of HAdV infections in the There are 51 recognized HAdV serotypes defined classi-
military [11, 12]. These investigations culminated in the cally by serum neutralization (SN) (Table 6.1), and several
development of HAdV vaccines that were introduced in 1971 new “types,” including one that belongs to the new species G
for use in military recruits to help prevent ARD [13, 14]. (HAdV-52), have been recently described by molecular anal-
yses [22]. For further discussion of the use of molecular and
immunotyping methods for classifying HAdVs, see Sects. 12
3 Virus Characteristics and 12.1, below.
Some HAdVs show considerable intraserotypic genetic vari-
3.1 Virion Structure and Antigenic ability, best documented by genome restriction fragment analy-
Composition sis [23, 24]. For example, more than 20 DNA variants or genome
types of HAdV-7 have been identified among strains isolated
Adenoviruses exhibit a distinct morphology and chemical worldwide, and regional shifts or replacement of predominant
composition. The virion consists of a 70–90 nm diameter genome types have been documented [25]. Designation of
nonenveloped icosahedral capsid that packages a single HAdV genome types is based on BamHI as the “type” defining
double-stranded linear DNA genome of approximately 35 kb restriction enzyme, with different genome types denoted with a
in length [15]. The capsid is composed of 252 capsomeres, character, e.g., “p” for the prototype strain and then “a” through
of which 240 are homotrimeric hexons and 12 are pentam- “k” for BamHI variants. Genome types that are further distin-
eric pentons located at the vertices of the icosahedron and guished by restriction pattern with additional enzymes are given
from which project a single, variable-length homotrimeric an Arabic numeral (e.g., Ad7p, p1, a, a1-6).
fiber (Fig. 6.1) [16]. The species F enteric HAdVs uniquely Many HAdV species and serotypes exhibit distinct tissue
possess two different lengths of fibers that occur alternately on tropisms and clinical manifestations, reflecting preferential
the vertexes. Four minor proteins (IIIa, VI, VIII, and IX) that infection of the respiratory, gastrointestinal, and urinary tracts
stabilize the structure are also elements in the capsid [17]. Six and eyes. Typically, HAdV species B (particularly serotypes
other proteins are located in the virus core, of which five (V, 3, 7, 14, and 21), C, and E cause respiratory tract infections,
VIII, Mu, IVa2, and the terminal protein) are associated with whereas the enteric species F serotypes 40 and 41 and species
the viral genome and the remaining component protease facil- A (particularly serotype 31) are more commonly associated
itates virion assembly. Type-specific antigenic determinants with infantile gastroenteritis. Species D serotypes 8, 19, and
that give rise to neutralizing antibodies are located primarily 37 are responsible for most outbreaks of EKC. Naturally
on the surface of the hexon capsomere, the fiber, and, to a occurring intermediate strains, which arise from intermolecu-
lesser extent, the penton protein. lar recombination [26] and possess the hemagglutinin (fiber)
Type-specific determinants on the hexon map to loops L1 of one serotype and serum-neutralizing determinants (hexon)
and L2 of the hexon monomer and are encoded by seven of another serotype, are well documented and may exhibit
hypervariable regions of the hexon gene, HVR1-6 (L1) and modified tropism and host pathology [20]. For example, spe-
HVR7 (L2) [18]. A genus-specific antigen is located on the cies B HAdV-11p, identified in the 1950s in association with
interior surface of the hexon. In addition, the fiber is a potent acute hemorrhagic cystitis, was later identified in outbreaks
hemagglutinin that also contains type-specific antigenic deter- of respiratory disease as a novel genome type, HAdV-11a
minants recognized by hemagglutination inhibition (HI). [27, 28]. Subsequent molecular analyses revealed that HAdV-
11a (also referred to as HAdV-11/H14 or HAdV-55) is a
recombinant virus that acquired the HAdV-14 fiber gene and
3.2 Adenovirus Classification possibly enhanced tropism for the respiratory tract [28–30].

Adenoviruses are classified within the family Adenoviridae

and further into five genera: Mastadenovirus, Aviadenovirus, 3.3 Adenovirus Replication
Atadenovirus, Siadenovirus, and Ichtadenovirus. HAdVs,
located within the genus Mastadenovirus, are divided into Adenoviruses exhibit a highly restricted host range.
seven species, A–G, based on several of the following Nonhuman primate species are poor hosts for HAdVs, as
6 Adenoviruses 101

Fig. 6.1 Schematic diagram of

the adenovirus virion and
associated structural proteins
(Reprinted with permission from
Russell [16])

Capsid proteins Minor proteins Core proteins

V
Hexon IIIa
VII

VI
Penton base Mu

VIII Terminal
Protein
Fibre
IX IVa2

Protease

shown by the inefficient replication of HAdVs in simian The adenovirus infectious cycle consists of an early and
cells unless coinfected with the helper papovavirus, SV-40 late phase separated by the onset of viral genome replication
[31]. Although most HAdVs do not induce clinically [15]. The early phase begins with virus entry into the host cell
apparent disease in common laboratory animals, hamsters, that proceeds by a two-step process: (1) binding of the fiber
cotton rats, rabbits, and mice have been used to model respi- knob domain to the cell receptor followed by (2) interaction
ratory and ocular infections with some HAdVs [32]. All between a specialized motif on the penton base (Arg-Gly-Asp
HAdVs can transform cultured rodent cells to a malignant (RGD) peptide sequence) and cellular integrin, as a second-
state, and species A, and to a lesser extent species C viruses, ary receptor, triggering virus internalization [33]. Two well-
can induce tumors in newborn hamsters. However, there is documented HAdV receptors are the membrane cofactor
no clear evidence that HAdVs can cause cancer in humans. protein (MCP or CD46), shown to bind most species B
For further discussion, see Sects. 12 and 12.4, below. HAdVs, and the coxsackievirus-adenovirus receptor (CAR),
102 X. Lu et al.

Table 6.1 Properties of human adenovirus serotypes

Oncogenicity Fiber DNA Most common

Hemagglutination in newborn length Number of SmaI disease
Speciesa Serotypes groupsb hamsters (nm) % homologyc % G + C fragments syndromesd
A 12, 18, 31 IV High 28–31 48–69/8–20 47–49 4–5 Gastroenteritis
B 3, 7, 11, 14, 16, 21, I Weak 9–11 89–94/9–20 50–52 8–10 URI, LRI,
34, 35, 50 cystitis
C 1, 2, 5, 6 III Negative 23–31 99–100/10–16 57–59 10–12 URI, LRI,
endemic viruses
D 8–10, 13, 15, 17, 19, II Negative 12–13 94–99/4–17 57–60 14–18 EKC,
20, 22–30, 32, 33, asymptomatic
36–39, 42–49, 51 rectal carriage
E 4 III Negative 17 4–23 58 19 URI, LRI
F 40, 41 III Negative ~29 62–69/15–22 52 10–12 Gastroenteritis
Adapted from Schnurr [278], Hierholzer [20], and De Jong et al. [21]
a
Species G and types identified solely by molecular methods are not included in the table. Please see Sects. 12 and 12.1
b
I complete agglutination of monkey erythrocytes, II complete agglutination of rat erythrocytes, III partial agglutination of rat erythrocytes, IV little
or no agglutination
c
Range of percent genome homology among serotypes of the same species (first listed) and between serotypes of different species (second listed)
d
URI upper respiratory illness, LRI lower respiratory illness, EKC epidemic keratoconjunctivitis

that binds HAdV serotypes from all other species [34]. centers, primary schools, groups of families, the broader
Integrins, CD80/CD86, sialic acid, and other cellular glyco- community, and other settings have allowed a detailed
proteins have also been implicated as receptors for some description of the epidemiology of HAdV infections. Global
HAdVs. Following virus entry and passage to the cell nucle- data assembled by the World Health Organization on type-
ase, selective transcription and translation of the early viral specific HAdV isolates in the early 1980s [35] and more
genes occur that facilitate viral genome replication. There are recent data from the United States on the prevalence and risk
five immediate early transcription units: early region 1A factors for severe HAdV disease from 2004 to 2006 are also
(E1A), E1B, and E2–E4. E1A encodes proteins that activate available [36].
transcription and induce the host cell to enter the S phase of
the cell cycle, and E1B proteins prolong cell survival and
block apoptosis. E2 proteins provide machinery for viral 4.2 Laboratory Tests
genome replication and ensuing transcription of late genes.
E3 proteins are nonessential for virus replication but help Because the disease syndromes caused by HAdVs are not
subvert the host’s defense mechanisms to prolong survival of easily distinguished clinically in the absence of epidemics,
the infected cell in vivo (see Sects. 7 and 8, below). E4 pro- laboratory testing is crucial for diagnosis. Laboratory
teins facilitate virus messenger RNA metabolism, genome diagnosis can be accomplished by virus isolation, poly-
replication, and shutoff of host protein synthesis. The late merase chain reaction (PCR) assay, antigen detection, or
phase consists of transcription of the late transcriptional units serology. Viral detection is greatly enhanced by proper
(L1–L5) that leads to production of the viral structural com- collection of specimens from affected sites in the early
ponents, assembly of the virus particles in the nucleus, and phase of illness. The type of specimen collected is dictated
eventual release from the host cell. Replication-deficient by the clinical presentation of the patient and the type of
mutants with deletions of the E1 transactivating region have test to be administered. Throat and nasopharyngeal speci-
been developed for use as vectors (see Sect. 11, below). mens are suitable for detection of HAdVs in the upper
respiratory tract. Fecal specimens or rectal swabs are the
specimens of choice for HAdV-associated gastroenteritis
4 Methodology Involved and may complement diagnosis of respiratory tract infec-
in Epidemiologic Analysis tions as virus shedding from the gastrointestinal tract may
persist for prolonged periods after disappearing from the
4.1 Sources of Data upper respiratory tract. Conjunctival swabs are suitable for
HAdVs causing ocular infections. Appropriately timed
HAdV disease is not reportable in the United States outside blood samples collected as soon as possible after the onset
of the military. Nevertheless, numerous studies conducted in of symptoms and 2–4 weeks later are needed to establish
military training camps, pediatric institutions, day-care diagnosis by serology.
6 Adenoviruses 103

4.2.1 Viral Culture epidemiology investigation [43, 44]. Quantitative real-time

Isolation of HAdVs in cell culture provides the most conclu- PCR assays for pan-HAdV detection have become popular
sive evidence of viral infection. Recovery of the virus is usu- [45, 46], particularly in the clinical diagnostic setting.
ally not very difficult in the acute phase of illness. With the Quantitation of viral load by real-time PCR has aided our
exception of the enteric HAdVs, most HAdV serotypes can be understanding of HAdV pathogenesis and has proven to be
easily isolated in continuous human cell lines of epithelial ori- an essential tool for early diagnosis and monitoring of antivi-
gin such as A549, HEp-2, and HeLa. Fibroblast cell lines like ral therapy in immunocompromised patients, especially in
MRC-5 and WI38 are generally less sensitive but can grow the posttransplant setting [47–49].
HAdVs after short adaptation. The enteric HAdVs can be iso-
lated in Graham 293 cells, a human embryonic kidney cell 4.2.3 Antigen Detection, Histology,
line that was stably transformed with HAdV-5 E1 transactivat- and Electron Microscopy
ing region [37]. Centrifugation of specimens directly onto cul- Antigen detection methods using monoclonal antibodies to
ture cells using the shell vial technique has greatly reduced the hexon group antigen permit rapid detection of HAdVs in
time for detection [38]. Adenoviruses assemble in the nucleus clinical specimens. Immunofluorescence (IF) assays are
of infected cells, and the nuclear morphologic changes can be widely used in clinical settings as they permit rapid, specific,
diagnostic on histologic examination. Typical cytopathic and relatively low-cost diagnosis of HAdV infections [50].
effect (CPE) appears as a rounding and clumping of cells in Commercial latex and enzyme immunoassays for detection
“grape-like” clusters within 1–2 weeks after inoculation. and identification of enteric HAdVs in stool specimens are
Adenovirus CPE does not immediately result in cell death, also available. However, antigen detection techniques tend to
however. Rounding and detachment are caused by the penton be less sensitive than culture and PCR, are not amenable to
base-induced cell detachment domain (RGD) located on the high throughput, and may be insufficiently informative for
penton capsomere that is produced in excessive amounts [39]. epidemiologic studies.
Histologic examination of biopsy or autopsy tissues can
4.2.2 PCR Assay help identify HAdV disease. HAdV-infected tissues often
In recent years, the application of molecular methods such as reveal cells with enlarged nuclei containing amphophilic or
PCR has significantly improved the sensitivity of HAdV basophilic inclusions with indistinct nuclear membranes
detection and shortened the time for reporting test results. known as “smudge” cells [51]. Confirmation of intranuclear
Numerous PCR methods have been developed for detection adenovirus antigen by immunohistochemical testing can
and identification of HAdVs. Conventional PCR assays tar- localize infection in affected organs, providing a more defin-
geting conserved regions in the hexon, fiber, or VA RNA itive diagnosis (Fig. 6.2) [52].
genes using broadly reactive primer sets are capable of Electron microscopy (EM) has been useful to visualize
detecting all recognized HAdV types [40–42]. Commercial HAdVs that are not easily grown in cell culture, such as the
multiplex PCR assays that can simultaneously detect multi- enteric HAdVs. Because of their unique morphology, HAdVs
ple respiratory pathogens including HAdV are now available can be rapidly identified by EM, but the method is insensitive
that greatly enhance the ability of laboratory diagnosis and and not widely available.

a b c

Fig. 6.2 Histologic section of the lung from a fatal case of adenovirus a degenerating, inclusion-bearing nucleus (smudge cell; ×1,000). (c)
pneumonia. (a) Low-power photomicrograph of lung tissue demon- Immunohistochemical stain showing multiple nuclei containing immu-
strating the presence of necrotizing exudates containing fibrin, inflam- noreactive adenovirus antigen (×400) (Reprinted with permission from
matory cells, and karyorrhectic nuclear debris (×200). (b) Same Barker et al. [52])
photomicrograph of lung tissue at higher magnification, demonstrating
104 X. Lu et al.

4.2.4 Serology 5 Descriptive Epidemiology

As noted above, detection of HAdVs in respiratory or gastro-
enteric specimens can be difficult to interpret, especially when HAdVs most commonly cause disease in infants and young
sensitive molecular methods are used. In such cases, serology children and are especially prevalent in day-care centers
can be helpful in distinguishing past from present infection. and households with young children. Longitudinal studies
Detection of HAdV-specific antibodies in single serum speci- performed in families and pediatric facilities documented
mens indicates prior infection with the virus. Seroconversion that species C HAdV infections occur early in life and are
or a ≥4-fold rise in antibody titer between acute- and convales- often endemic [64, 65]. Transmission among families was
cent-phase sera is considered evidence of recent infection or extensively investigated by the Virus Watch studies in the
possible reactivation of latent virus. Commonly used serologi- 1960s, during which biweekly collection of both fecal and
cal tests include enzyme immunoassay (EIA), serum neutral- respiratory specimens was performed [65, 66]. These stud-
ization SN, and hemagglutination inhibition HI. EIAs are easy ies identified a large number of asymptomatic infections
to perform, but do not distinguish between serotypes. Although with species C viruses and demonstrated a high frequency
more laborious to perform, SN and HI assays provide type- of fecal shedding of virus. HAdVs were found intermit-
specific information and have been useful for epidemiologic tently in fecal specimens for extended time periods, up to
and vaccine efficacy studies [53]. 1–2 years. Species F serotypes 40 and 41 cause gastroen-
teritis in infants and young children. In contrast, a large
4.2.5 Virus Typing proportion of species A and D adenoviral infections are
In the clinical setting, once a diagnosis of a HAdV infection asymptomatic [67].
has been made, further identification of the virus is often Species B (particularly serotypes 3, 7, 14, and 21) and
unnecessary. However, for research and epidemiologic pur- E (serotype 4) infections tend to occur later and cause
poses, species- and type-specific information may be useful symptomatic respiratory disease, including epidemics of
to strengthen association with disease and document emer- acute respiratory disease (ARD) in military recruits [12]
gence of new, potentially more virulent strains. For example, and pharyngoconjunctival fever in summer camps [68]. In
detection of species B or E viruses in specimens from persons a study of 1,653 clinical isolates collected from 22 hospital
with acute respiratory illness or species F viruses with gastro- laboratories in the United States between 2004 and 2006,
enteritis would be more meaningful than detection of species species B HAdV-3 was the most commonly identified
C viruses, due to the latter’s tendency to shed persistently (35 %), followed by HAdV-2 (24 %) and HAdV-1 (18 %)
from the upper respiratory and gastrointestinal tracts. Detailed [36]. These data are consistent with the fact that HAdV-3 is
molecular analyses documented genetic signatures unique to less prevalent than HAdV-1 and HAdV-2 but is more likely
the recently reemergent species B HAdV-14 [54] and revealed to cause a severe disease that requires hospitalization and/
patterns of virus transmission during outbreaks [55]. or testing.
Identification of a HAdV isolate is classically performed by
immunotyping (SN and HI), but these procedures are laborious
and time-consuming, and reference antisera are not widely avail- 5.1 Synopsis of Descriptive Epidemiology
able. In the early 1980s, molecular typing by genome restriction
fragment analysis as described above was used as an adjunct to 5.1.1 Incidence and Prevalence
immunotyping [24, 56], but this procedure also depends on cul- The incidence of infection is highest in infants and young
ture-grown virus for implementation. Molecular methods have children, during which time infections with the endemic
resolved many of these problems and are now used routinely species C HAdVs most commonly occur. In one family
for identification of HAdVs. PCR assays using species- or type- study, over 90 % of children under age 2 had SN antibodies
specific primers targeting the hexon, fiber, and VA RNA genes to HAdV-1 and HAdV-2 [66]. Young children also most
have been used for direct identification of HAdVs in clinical commonly shed virus in stool for prolonged periods after
specimens [41, 57–59]. PCR and amplicon sequencing of hexon the resolution of symptoms. Most transmission occurs
HVR1-6 [60] and HVR7 [61] have been shown to correlate with among families with young children, and the incidence is
SN. The addition of fiber gene sequencing permits identifica- higher in lower socioeconomic groups. Rates of HAdV
tion of some intermediate HAdV strains [62]. The availabil- infections are higher in day-care centers and pediatric
ity of reference hexon and fiber gene sequences in the public facilities.
domain makes preliminary type identification of HAdV strains
possible. Full genome sequencing combined with bioinformatic 5.1.2 Epidemic Behavior
analysis has allowed for more comprehensive characterization The species E HAdV-4 and species B serotypes 7, 14, and 21
of HAdVs, which has been useful for exploring pathoepidemi- occur mostly in epidemics; species B HAdV-3 is sometimes
ology and evolutionary relationships [63]. endemic and sometimes epidemic.
6 Adenoviruses 105

Table 6.2 Most common adenoviral disease syndromes associated among the most common causes of tonsillitis in young chil-
with specific patient populations
dren. Less frequently, HAdVs have been associated with
Patient population Clinical syndromes otitis media (in children under age one), a pertussis-like
Infants and young Pharyngitis, coryza, tonsillitis, otitis media syndrome, bronchiolitis, coryza without fever, or an
children Pneumonia exanthem.
Gastroenteritis, mesenteric adenitis The species B serotypes 3, 7, 14, and 21 have been associ-
Children Pharyngitis, coryza, conjunctivitis
ated with a more severe disease compared to the species C
Pharyngoconjunctival fever (PCF)
viruses [72, 73]. In particular, HAdV-7 has been observed to
Pneumonia
be one of the most pathogenic serotypes, causing a number
Gastroenteritis
of fatal lower respiratory tract infections [74–76]. Pneumonia
Adults Acute respiratory disease (ARD)
Epidemic keratoconjunctivitis (EKC)
is more severe in infants than older children and can be asso-
Immunocompromised Pneumonia ciated with extrapulmonary complications such as meningo-
hosts Gastroenteritis, hepatitis encephalitis, hepatitis, myocarditis, and nephritis [77–80].
Hemorrhagic cystitis, interstitial nephritis Other rare complications include disseminated intravascular
Meningoencephalitis coagulation and a toxic shock-like syndrome [73, 81].
Notably, there is a high incidence of pulmonary sequelae fol-
lowing HAdV pneumonia in children, especially bronchiec-
5.1.3 Geographic Distribution tasis [82, 83].
The species C HAdVs are endemic in most areas of the world
[35]. Certain other types such as the species D HAdV-8 are 5.2.2 Pharyngoconjunctival Fever
endemic in underdeveloped countries such as parts of the This HAdV syndrome is characterized by pharyngitis, con-
Middle and Far East and Africa but primarily occur in epi- junctivitis, and spiking fevers. Either one or both eyes can be
demics in developed countries. involved, and lymphadenopathy is often observed. Diarrhea
and coryza can sometimes occur, and tonsillar exudates can
5.1.4 Seasonality be observed. Pharyngoconjunctival fever is most commonly
HAdV infections occur throughout the year but are more caused by species B HAdV-3 and HAdV-7 and species E
commonly found in late winter, spring, and early summer in HAdV-4 [68, 84]. The disease can be seen sporadically and
temperate regions [65]. be transmitted among families. However, this syndrome is
best known as an epidemic disease in summer camps. Studies
5.1.5 Age suggest that infection is transmitted by direct contact with
Most children have been exposed to several types of endemic water from contaminated swimming pools and small lakes;
HAdVs by the time they enter school. Infections with the virus is likely introduced into the eye or upper respiratory
less common species E HAdV-4 and species B serotypes can tract [68]. Outbreaks of pharyngoconjunctival fever have
occur later in life. been specifically associated with inadequate chlorination of
swimming pools [85].

5.2 Epidemiologic and Clinical Aspects 5.2.3 Acute Respiratory Disease (ARD)
of Specific Syndromes ARD epidemics can occur under the special conditions of
fatigue and crowding present in military training camps
The clinical manifestations of HAdV disease vary according [13, 86]. Symptoms typically include fever, sore throat, and
to the HAdV serotype and the age and immune status of the cough, sometimes with coryza, headache, or chest pain.
host (Table 6.2) [32]. HAdVs are responsible for 5–10 % of Malaise is characteristic and lasts for approximately 10 days.
febrile illnesses in infants and young children [66]. Pneumonitis can also occur, resulting in rare fatalities
[87, 88]. ARD epidemics have been associated with species
5.2.1 Respiratory Illness E HAdV-4 and species B serotypes 3, 7, 11a, 14, and 21.
Infants and young children have the highest attack rates of Outbreaks in military training camps usually peak at about
endemic HAdV infections. These infections commonly 3–6 weeks after the onset of training, resulting in morbidity
present as mild upper respiratory tract illnesses and are rates as high as 6–17/100 per week.
most frequently caused by species C serotypes 1, 2, 5, and , Introduction of live, oral HAdV-4 and HAdV-7 vaccines
to a lesser extent, 6 [69, 70]. Pharyngitis is frequently asso- for US military recruits in the early 1970s significantly
ciated with conjunctivitis, laryngitis, or bronchitis. Fever is reduced the incidence of ARD [14]. After the manufacture of
common and may be associated with malaise, headache, HAdV vaccines stopped in 1996, however, there was a
myalgia, and occasionally abdominal pain [71]. HAdVs are prompt rebound in high rates of HAdV infections [89, 90].
106 X. Lu et al.

Rare new fatalities were reported in military recruits due to 5.2.5 Gastroenteritis
probable HAdV pneumonia with or without encephalitis Species F serotypes 40 and 41 are responsible for about
[91]. In a study of 584 clinical isolates from military recruits 5–10 % of acute diarrheal illnesses in infants and young chil-
collected between 2004 and 2006, 93 % were HAdV-4 [36]. dren. In one study of over 400 cases of acute infantile gastro-
HAdV transmission among new military recruits was studied enteritis, enteric adenoviruses were the sole recognizable
using active surveillance for illness and enrollment and end- cause of diarrhea in 7.2 % of cases [103]. Outbreaks have
of-study viral throat cultures and serology [86]. Febrile occurred in day-care centers and pediatric healthcare facili-
respiratory infections due to HAdV-4 were identified in 25 % ties. In a review of an outbreak in several day-care centers,
(67/271) of new recruits, and the percentage of recruits sero- 38 % of 247 young children tested had positive stool speci-
positive of HAdV-4 increased dramatically from 34 to 97 % mens for HAdV-40 or HAdV-41, although only half were
during the first 4 weeks of training. The authors proposed symptomatic [104]. These fastidious enteric adenoviruses
that introduction of recruits with asymptomatic viral shed- require special cell lines for growth but can be readily
ding into new training groups was likely a primary cause of detected by enteric adenovirus antigen assays. The species A
continual transmission of HAdV-4 in the camp. In 2006, spe- HAdV-31 is a less common cause of infantile diarrhea [105].
cies B adenoviruses became more prevalent, including sero- Most other serotypes have not been clearly associated with
types 3, 7, 14, and 21 [92]. diarrhea, but are commonly shed in stool for months after
Species B HAdV-14, which was first identified in the infection. However, non-enteric species C HAdVs have been
Netherlands in 1955 and caused sporadic outbreaks in associated with mesenteric adenitis, which may mimic
Europe, emerged in US military bases in 2005. Since the ini- appendicitis and occasionally cause intussusception in
tial reports, HAdV-14 has caused several outbreaks of ARD infants and young children [106, 107].
in both military recruits and civilians [88, 93, 94]. In a report
of 140 cases, 38 % were hospitalized, 17 % were admitted to 5.2.6 Uncommon Syndromes
intensive care units, and 5 % died [95]. An analysis of 99 HAdVs can cause acute hemorrhagic cystitis in children
isolates from military and civilian cases in the United States (species B serotypes 11 and 21) [108]. Rarely, HAdVs have
revealed that all isolates were identical and suggested that caused urethritis in adults (species D serotypes 19 and 37),
they arose from recombination between HAdV-11 and and sexual transmission has been postulated [109, 110].
HAdV-14 strains [54]. Meningitis and encephalitis have been reported occasion-
Due to the increasing incidence of ARD in military ally in association with HAdV infections [111]. Neurologic
recruits, live, oral HAdV-4 and HAdV-7 vaccines were rede- involvement usually occurs in association with severe pneu-
veloped by a new manufacturer and licensed in the United monia, especially with HAdV-7.
States in 2011 [96, 97]. HAdVs have been associated with acute myocarditis in
children [112]. In one study, 38 myocardial tissue samples
5.2.4 Epidemic Keratoconjunctivitis (EKC) from 34 children with acute myocarditis were tested by PCR
In contrast to the benign course of pharyngoconjunctivitis, for a panel of viruses [113]. HAdV was detected more com-
certain species D serotypes (8, 19, and 37) can cause a more monly (15 samples) than enterovirus (8 samples) and was
serious keratoconjunctivitis [98, 99]. This disease is charac- not detected in control samples.
terized by conjunctivitis, followed by the development of HAdVs have been occasionally associated with polyar-
corneal infiltrates that cause pain, edema of the eyelids, pho- thritis syndromes [114, 115]. Reye’s syndrome has also been
tophobia, and lacrimation. One or both eyes may be involved, reported in association with HAdV infections in infants [116,
and preauricular lymphadenopathy is common. Although 117].
this disease does not usually result in permanent corneal
damage, it can cause severe pain and blurry vision lasting up 5.2.7 Immunocompromised Hosts
to 4 weeks. HAdVs can cause severe, life-threatening infections in
A number of outbreaks of EKC have occurred in medical immunosuppressed persons, especially in the pediatric popu-
facilities, in particular ophthalmology offices. Infections can lation [118–122]. Disease can be caused by primary infec-
be spread from contaminated instruments, surfaces, or hands tion or reactivation of latent infection. In transplant recipients,
[100, 101]. Adenoviruses are relatively resistant to disinfec- HAdV infections can also rarely be transmitted from the
tants, so heat sterilization of ophthalmology instruments (in donated organ or bone marrow. Severe neonatal HAdV infec-
particular, tonometers) is preferred. Hand washing with soap tions occur primarily in premature infants, and vertical trans-
and water does not efficiently remove adenoviruses, so glov- mission may play a role [123].
ing is important. In one investigation of a HAdV-8 epidemic, Pediatric hematopoietic stem cell transplant (HSCT)
almost 50 % of patients diagnosed with EKC carried virus on recipients have the highest incidence of HAdV disease and
their hands, and the virus remained viable on inanimate sur- mortality [124, 125]. In one study of 204 pediatric HSCT
faces for up to 35 days [102]. recipients, 15.1 % had HAdV infections and 8.8 % developed
6 Adenoviruses 107

severe disease [126]. In contrast, in one study of 1,050 adult serotypes have been isolated from AIDS patients, including
HSCT recipients, 4.8 % had HAdV infections and only species B serotypes 11, 34, and 35 in urine and rare species
0.9 % had invasive disease [127]. D serotypes and intermediate strains in stool samples [140,
The clinical spectrum of HAdV infections in HSCT recip- 141]. Occasional fatal cases of adenoviral infection in AIDS
ients can range from asymptomatic shedding to fatal dis- patients have been reported, especially in the pediatric popu-
seminated disease. A wide range of clinical syndromes has lation [142, 143].
been reported, including pneumonia, colitis, hepatitis, hem-
orrhagic cystitis, tubulointerstitial nephritis, encephalitis,
and disseminated disease. Disease is most frequently associ- 6 Mechanisms and Routes
ated with the common species C serotypes 1, 2, and 5. of Transmission
Interestingly, there is also a preponderance of urinary tract
infections due to species B serotypes 11, 34, and 35, which Transmission of HAdVs primarily occurs by the fecal-oral
are infrequently isolated in the general population [127– and respiratory routes. Fecal-oral transmission is common in
129]. One hypothesis is that these types frequently cause households with young children [144]. Moreover, prolonged
latent infections of the genitourinary tract that can reactivate fecal shedding can sustain transmission in households and
following severe immunosuppression. day-care centers. HAdVs can be transmitted via inhalation of
In contrast to the HSCT population, HAdV disease typi- droplets dispersed in the air by coughing and via direct con-
cally involves the donor organ in solid organ transplant tact with infected secretions or contaminated surfaces. The
recipients. For instance, hepatitis is the most common mani- respiratory route is an important means of transmission in
festation of invasive adenoviral disease in pediatric liver epidemics of ARD in military training camps. In one study,
transplant recipients. In one large study of 484 liver trans- 50 % of air samples from recruit barracks were positive for
plant recipients, 14 (3 %) developed HAdV hepatitis [130]. HAdV DNA [86]. This study also detected viral DNA from
HAdV-5 was the most common isolate, and 43 % of patients 14 to 39 % of surfaces, suggesting that environmental con-
died. In another study, examination of pre- and posttrans- tamination of living quarters is a possible contributing factor.
plant sera from recipients and donors suggested that virus Direct contact with contaminated pool water plays a major
was transmitted from the donated organ [131]. role in causing outbreaks of pharyngoconjunctival fever in
In renal transplant recipients, HAdV can cause acute summer camps [68].
hemorrhagic cystitis, sometimes complicated by interstitial Nosocomial transmission has been well documented
nephritis [132]. The incidence has not been well defined but [145, 146] and can be challenging to prevent because HAdVs
is low, and this disease is exclusively associated with species are resistant to a number of common disinfectants and can
B serotypes 11, 34, and 34. The prognosis is generally good, survive on surfaces for weeks [100, 147]. In one investiga-
although infection occasionally results in fatal disseminated tion of a large community outbreak of EKC, HAdV-8 was
disease [133]. In one study of 339 adult renal transplant isolated from multiple ophthalmic solutions from one physi-
recipients, 17 (5 %) developed HAdV-related hemorrhagic cian’s office [148]. In another report, HAdV-8 conjunctivitis
cystitis [134]. Symptoms included dysuria, hematuria, fever, occurred in 7 premature infants who had undergone ophthal-
and bilateral testicular pain and lasted a mean duration of 2 mological examination in a neonatal intensive care unit;
weeks (range, 0.5–9.6 weeks). Reversible allograft dysfunc- infection was also transmitted to 9 staff and 12 family mem-
tion occurred in ten patients. One patient developed dissemi- bers [149]. There were 11 deaths from HAdV-7 infections
nated HAdV disease and died from bacterial sepsis. among 61 residents of a long-term care pediatric facility dur-
In a study of pediatric cardiac transplant recipients, ing an 8-week period; 23 staff members had febrile respira-
HAdVs have been implicated as a cause of graft loss and tory illnesses during this period, suggesting sustained
coronary vasculopathy [135]. HAdVs was detected by PCR transmission between patients and staff [150].
in myocardial biopsies from 24 of 149 patients (16 %) and Occasionally, neonatal infections have been caused by
was associated with reduced graft survival. vertical transmission of HAdV, especially with species B
Severe, frequently fatal neonatal infections have been HAdV-11 and HAdV-35 [136, 151]. Intrauterine infections
described [123, 136]. In addition to the usual routes of trans- have rarely been described, and ascending viral infection
mission, neonates can acquire HAdV infection from expo- from the cervix has been proposed as one mechanism [137].
sure to cervical secretions at birth [137]. Rare cases of
intrauterine infection have also been described [138].
Primary HAdV infections can also cause severe disease in 7 Pathogenesis
children with immunodeficiency syndromes such as severe
combined immunodeficiency disease (SCID) [139]. HAdVs are lytic DNA viral pathogens that usually cause
HAdVs are an uncommon cause of morbidity and self-limited localized disease but can disseminate in immu-
mortality in HIV-infected individuals. However, a number of nocompromised hosts and rarely in healthy children and
108 X. Lu et al.

adults, causing significant morbidity and mortality. HAdVs characterized as necrotizing bronchiolitis. Rosette formation,
cause pathology during the process of viral replication and mononuclear cell infiltrates, and focal necrosis of mucous
lysis of susceptible cells. Additionally, investigations of glands appear to be characteristic. Typical intranuclear virus
replication-defective adenoviruses in animal models and particles have been observed in alveolar lining and bronchio-
clinical studies have revealed that HAdVs can cause signifi- lar cells by electron microscopy [72]. In infants who recover
cant inflammation in the absence of viral replication. Thus, from adenovirus pneumonia, severe sequelae may follow,
the clinical manifestations of HAdV disease appear to result including bronchiectasis, radiolucent lung syndrome, persis-
from both the direct effects of the infection and the host tent lobar collapse, and bronchiolitis obliterans [161].
inflammatory responses. HAdVs can also cause pathology in the absence of repli-
cating virus. For example, replication-defective HAdVs can
trigger innate immune responses, primarily by recognition of
7.1 Tissue Tropism capsid proteins via pathogen recognition receptors (PRRs)
with activation of dendritic cells and macrophages [162].
Although certain HAdV serotypes are associated with dis- Capsid proteins can also activate the complement system via
tinct clinical manifestations, the basis for these differences is both classical and alternative pathways [163, 164]. This
not well understood. For example, the species D serotypes 8, results in a proinflammatory response with the release of
19, and 37 can cause a more severe eye disease than other multiple cytokines. The viral capsid proteins also trigger
types. The species E HAdV-4 and species B serotypes 3, 7, humoral and cell-mediated immune responses that further
14, and 21 have been associated with severe respiratory dis- exacerbate the developing proinflammatory response (see
ease. In contrast, the species B serotypes 11, 34, and 35 pri- Sect. 8, below). For instance, antiviral antibodies can increase
marily cause hemorrhagic cystitis. It is likely that differences Fc receptor-dependent viral internalization in macrophages
in the fiber and penton capsid proteins that mediate cell and amplify intracellular innate pathways [165]. The ability
receptor binding and cell entry contribute to serotype-specific of this virus to induce an inflammatory response in the host
differences in clinical manifestations. For instance, HAdV in the absence of replication has implications for the clinical
species display different cell receptor preferences that are use of replication-deficient adenovirus vectors. For further
mediated by the fiber. Most serotypes from species A, C, D, discussion, see Sect. 11, below.
E, and F bind to CAR (the coxsackie adenovirus receptor),
which is a member of the immunoglobulin superfamily
expressed on multiple cell types [152]. Most species B 7.3 Immune Evasion Mechanisms
viruses bind to a ubiquitously expressed membrane comple-
ment regulatory molecule CD46 [153], whereas the species In natural infections, HAdVs have evolved several mecha-
B HAdV-3 and HAdV-7 bind to the related molecules CD80 nisms to evade or downregulate the host immune response,
and CD86 [154]. Members of the group D viruses can also which can help counteract the above inflammatory responses.
utilize ubiquitous sialic acid receptors [155, 156]. Following The viral early region 3 (E3) encodes several small proteins
viral attachment, penton base protein binds to cellular ανβ3/ (range 6.7–19 kDa) that play a prominent role in evading
ανβ5 integrins [157], thus facilitating virus internalization host innate and acquired immune defenses [166]. The E3
via clathrin-coated vesicles into endosomes for further pro- promoter also has NFkB binding sites that can be induced by
cessing [158]. cytokines such as TNF-α, and as a result, E3 protein expres-
sion is directly upregulated during an inflammatory response
[167]. The E3-19K glycoprotein can block the transport of
7.2 Pathology major histocompatibility complex (MHC) class I antigens to
the cell surface, thereby inhibiting the recognition of virus-
In a natural infection of the human host, epithelial cells are infected cells by cytotoxic T lymphocytes (CTLs). E3-19K
the primary targets of adenoviral cytopathology [159]. The evokes two distinct mechanisms to inhibit MHC class I mol-
respiratory epithelial cells affected during adenoviral pneu- ecule presentation of viral peptides to CTLs. First, E3-19K
monitis can develop acidophilic intranuclear inclusions. directly binds to class I antigens in the endoplasmic reticu-
Infected cells with enlarged nuclei containing amphophilic or lum (ER) and inhibits their egress from the ER to the cell
basophilic inclusion bodies surrounded by thin rims of cyto- surface [168, 169]. Additionally, E3-19K can directly bind to
plasm referred to as “smudge cells” have also been described. TAP (transporter associated with antigen processing) and
There are no syncytia or multinucleated cells, as seen in her- block class I/TAP association [170]. As a result of this inter-
pes virus infection. In the rare case of fatal illness, the virus action, the transfer of viral peptides processed in the cytosol
has been recovered from most body organs [160]. In such to class I antigens is also inhibited.
cases, extensive pathology is found in the lungs, with micro- Other E3 proteins enable the virus to evade apoptosis by
scopic necrosis of tracheal and bronchial epithelium inhibiting signaling from death receptors. CTLs express Fas
6 Adenoviruses 109

ligand on their cell surface and induce apoptosis upon inter- mechanisms related to expression of E1 and E3 region pro-
action with the Fas ligand receptor CD95 on target cells. teins, as well as VA RNA (as described above). However,
Similar apoptotic pathways are triggered by TNF-α and its these immune evasion mechanisms are not activated by
ligand TRAIL (TNF-related apoptosis-inducing ligand). infection with replication-defective E1-deleted adenovirus
However, in the presence of the E3-10.4K and E3-14.5K vectors.
proteins, these pathways are inhibited. Two units of E3-10.4K
and one unit of E3 14.5K hetero-trimerize to form the RID
complex (receptor internalization and degradation), which 8.1 Innate Immune Responses
stimulates the degradation of receptors for Fas ligand and
TRAIL via endocytosis and lysosomal degradation [171– The innate response to adenovirus acts as a first line of
173]. Thus, RID inhibits apoptosis through the TNF receptor defense and serves to control infection locally and recruit
1 (TNF-Rl), Fas, and TRAIL-R1 pathways. The E3-14.7K effector leukocytes, such as granulocytes and monocytes/
protein is also a potent inhibitor of apoptosis mediated by macrophages, to the site of infection. The viral capsid pro-
TNF-α [174]. teins are primarily responsible for inducing innate immune
HAdVs also can counteract host antiviral immunity by responses and for activating and recruiting Kupffer cells,
interfering with the antiviral activity of type I interferons endothelial cells, neutrophils, splenic macrophages, and den-
(IFNs). About 1 % of the viral genome is transcribed into dritic cells [162, 179, 180]. Binding of capsid proteins to
noncoding virus-associated RNA (VA RNA). The synthesis complement, pattern recognition receptors (PRRs), erythro-
of VA RNA begins during the early phase of the infectious cytes, and other blood factors further enhances the ensuing
cycle and accelerates during the late phase. VA RNA inter- immune response [181, 182]. For example, recognition of
feres with IFN-dependent phosphorylation of cellular pro- HAdVs via Toll-like receptors (TLRs) triggers activation of
teins that inhibit viral peptide chain initiation [175, 176]. As NFκB and signal transduction via the mitogen-activated pro-
a result, the viral polypeptide chain synthesis proceeds unin- tein kinases (MAPKs), resulting in the transcription of host
hibited. E1A proteins also inhibit the function of type I IFNs chemokine and cytokine genes [183]. Overall, the inflamma-
by blocking signal transduction pathways via IFN-stimulated tory response generated from leukocyte recruitment provides
gene factor (ISGF3). Conserved regions of E1A bind directly additional local control of infection and facilitates antigen
to STAT 1, the transcription factor that mediates IFN- presentation by infiltrating cells such as macrophages and
stimulated transcription [177]. dendritic cells. A robust innate response is thus essential for
the development of adaptive immunity.
Capsid proteins induce the production of proinflamma-
7.4 Viral Persistence tory cytokines and chemokines such as TNF-α, IL-6, IL-1B,
IL-8, IL-12, RANTES, MIP-1, and MCP-1 [162, 184, 185].
Although most HAdV illnesses are acute and self-limited, While the primary role of these cytokines is to locally
infection may be prolonged, and asymptomatic infections enhance the antiviral activity (via recruitment of appropriate
are common. Viral shedding from the gastrointestinal tract effector cells) and enhance the immune cascade, overproduc-
may persist for weeks to months, thus increasing the risk for tion may result in acute toxicity. In one study, 38 children
horizontal transmission. By devoting about a third of the hospitalized with HAdV infections were found to have high
genome towards gene products that counteract host defense levels of IL-6, IL-8, and TNF-α without evidence of bacterial
mechanisms, HAdVs have evolved strategies (discussed infection. Very high levels of IL-8 were found in all 16 fatal
above) to help facilitate persistence after primary infection. cases of HAdV infection, comparable to the levels seen in
HAdVs can be isolated from at least 50 % of surgically septic shock in adults [186]. Similarly, TNF-α was more fre-
removed tonsils [178], occasionally from the kidney [131] quently detected in the fatal group. Thus, an association
and also from lymphocytes [58], suggesting that infection between high proinflammatory cytokine levels and the
may remain latent for a very long time, possibly for life. severity of a natural HAdV infection was noted in infected
Mechanisms for maintaining persistent and latent HAdV children.
infections are not well understood. For further discussion, Virus-induced innate responses are also clinically rele-
see Sect. 12.2 below. vant with regard to gene therapy using adenovirus vectors.
The inflammatory response to replication-deficient adeno-
virus vectors significantly reduces gene transfer efficiency
8 Host Immune Responses and vector persistence in both animal and clinical studies
[180, 187, 188]. Moreover, one of the initial gene therapy
Adenoviruses are highly immunogenic and elicit strong clinical trials to treat ornithine transcarbamylase deficiency
innate and adaptive immune responses. In natural infections, using a replication-deficient, E1-deleted HAdV-5 vector
the immune responses to HAdVs are moderated by multiple resulted in the unfortunate death of an 18-year-old patient.
110 X. Lu et al.

In this case, mortality coincided with the induction of a conserved epitope located in the COOH terminus of hexon
cytokine storm [189]. There was a massive release of proin- that is restricted by a HLA class II allele present in 75 % of
flammatory cytokines, including IL-6 and IL-10, causing a the population [204]. Lower frequencies of adenovirus-
systemic inflammatory response syndrome. The patient specific CD8+ T cells can be detected in most healthy adults.
developed disseminated intravascular coagulation and mul- Hexon is also recognized by CD8+ T cells, and multiple
tiple organ system failure, leading to death 98 h following cross-reactive hexon epitopes have been identified [205–
gene transfer. This case highlights the need to develop strat- 207]. In addition to the hexon, conserved regions of HAdV
egies to moderate HAdV vector-induced innate immune early proteins like the E2 DNA polymerase and DNA-
responses. In contrast, for the purposes of vaccine develop- binding protein (DBP) have been identified as CD8+ T cell
ment or cancer immune therapy, the innate immune response targets and are recognized by both healthy and acutely
to adenovirus capsid proteins can have a desirable adjuvant infected adults [208]. Thus, cross-reactive virus-specific
effect to enhance the host immune response or cause a memory CD4+ and CD8+ T cell responses can be detected in
bystander effect [179, 190]. For further discussion, see most individuals.
Sect. 11, below. Cross-reactive memory T cell responses to HAdVs pose a
challenge to the development of adenovirus vector applica-
tions (see Sect. 11, below). However, virus-specific memory
8.2 Adaptive Immune Responses T cells from HSCT donors have potential use for immuno-
therapy of severe HAdV infections in HSCT recipients.
The development of an adaptive immune response is critical Several reports have demonstrated an association between
in the resolution of HAdV infections. Virus-specific antibod- recovery of HAdV-specific T cells and resolution of acute
ies (mainly secretory IgA) are present in the upper respira- HAdV infections following HSCT [209, 210]. Specifically,
tory tract within 3 days of infection. Approximately 7 days HSCT recipients recovering from HAdV disease can mount
postinfection, antibodies can be detected in serum, and nasal CD4+ and CD8+ T cell responses to hexon, as well as CD8+
secretions contain both secretory IgA and IgG [191, 192]. T cell responses to the above early protein targets [208, 211].
Serotype-specific neutralizing antibodies (Nabs), predomi- Therefore, passive transfer of virus-specific donor T cells is
nantly directed against the hexon, fiber, and penton base, are being explored as an adjunctive therapy for severe HAdV
generated following the first exposure to each HAdV sero- infections in HSCT recipients [212, 213]. For further discus-
type [193, 194]. Anti-fiber and anti-penton Nabs prevent cel- sion, see Sect. 9 below.
lular entry of the virion, while anti-hexon antibodies prevent
virus uncoating and nuclear entry of viral DNA [195, 196].
Nabs inhibit HAdV transduction of cells by a variety of 9 Treatment
methods, including prevention of cell attachment and facili-
tation of virus aggregation and clearance [197]. In addition, The majority of HAdV infections are self-limited and do not
anti-hexon Nabs that allow virus entry but prevent virus gene require specific therapy. There are no agents licensed specifi-
expression have been identified [198]. Nabs generated cally for treatment of HAdV disease.
against hexon target hypervariable regions (HVRs), which
account for 80–95 % of virus-specific Nabs, appear to be
most critical for virus clearance. Studies have shown that the 9.1 Antiviral Agents
presence of Nabs prevents reinfection with the same sero-
type [66, 86], and Nabs have been demonstrated 10 years Of the available antiviral agents, only cidofovir has repro-
after documented infection [199]. However, the protective ducible in vitro activity against adenoviruses [214, 215].
effect of Nabs is serotype specific and does not allow for Cidofovir has been most commonly used to treat invasive
broad protection against multiple HAdV serotypes. infections in immunocompromised patients [126, 216, 217].
The severe HAdV infections observed in patients with a Treatment with cidofovir has been associated with clinical
suppressed cellular immune system reflect the importance of responses and declines in viral loads in some HSCT recipi-
cell-mediated immunity in limiting HAdV replication and ents with invasive HAdV disease [218, 219]. Early treatment
preventing dissemination of infection [118, 128, 200]. and immune reconstitution appear to be important factors
Adenovirus-specific memory CD4+ T cell responses can be associated with improved outcomes [220]. Although some
detected in the vast majority of healthy adults [201, 202]. pediatric HSCT programs perform active HAdV surveillance
These CD4+ T cell responses are Th1 type, based on the using blood PCR assays and treat preemptively [221, 222],
cytokine secretion pattern [203] and, unlike Nabs, are cross- the nephrotoxicity of cidofovir is often dose limiting. Less
reactive against different serotypes. Several CD4+ T cell epi- nephrotoxic lipid-ester derivatives of cidofovir are being
topes have been mapped from the hexon, including a highly investigated for the treatment of serious HAdV infections
6 Adenoviruses 111

[223, 224]. Disseminated adenoviral disease was success- pyl alcohol, 0.63 % quaternary ammonium compound alone,
fully treated with the cidofovir prodrug CMX001 in a pediat- 4 % chlorhexidine gluconate, and 10 % povidone-iodine,
ric stem cell transplant recipient [225]. were significantly less active. Additionally, one study found
that HAdVs are not completely eliminated from hands after
washing with soap and water [102]. These viruses are also
9.2 Other Therapies relatively resistant to UV light [231] but can be inactivated
by heat.
Pooled intravenous immune globulin has high titers of Nabs Infection control measures can reduce the risk of HAdV
against common species C HAdV and is commonly used as transmission in some settings. For instance, adequate chlori-
adjunctive therapy for severe HAdV infections in immuno- nation of swimming pools can help prevent pharyngocon-
compromised patients [226, 227]. A benefit from immune junctival fever outbreaks [68, 85]. The spread of EKC in
globulin was demonstrated in an immunosuppressed mouse ophthalmology offices can be prevented by heat sterilization
model where passive transfer of mouse adenovirus (MAV)- of instruments, the use of single-use ointments and solutions,
specific IgG caused a marked delay in mortality after MAV and the use of gloves and strict hand hygiene practices.
infection and survival correlated with levels of MAV-specific However, during a large community outbreak of EKC where
antibodies [228]. over 100 nosocomial cases occurred at a large ophthalmol-
Additionally, adoptive transfer of virus-specific donor T ogy center, transmission continued to occur despite the insti-
cells has been investigated in a small number of pediatric tution of strict hand washing, gloving, and bleach disinfection
HSCT recipients. In one study, virus-specific donor T cells of equipment [232]. Transmission was stopped only after
were isolated and infused into nine children with systemic patients were rigorously triaged and cohorted, highlighting
adenoviral infections [212]. Viral clearance was demon- the difficulties in preventing the nosocomial spread of HAdV
strated in 5 of 6 evaluable patients. In another study, patients infections.
were treated with donor lymphocytes stimulated in vitro with In healthcare facilities, the Centers for Disease Control
HAdV [213]. Reductions in viral loads were documented in and Prevention recommends to use contact precautions
all three patients with active infection, and one patient with (gloves and gown) for children with suspected or docu-
adenoviral pneumonia had a clinical response. Methods to mented adenoviral conjunctivitis or gastroenteritis (if incon-
optimize recovery and in vitro expansion of donor virus- tinent or in diapers). Both contact and droplet precautions
specific T cells for immunotherapy trials are being investi- (surgical mask) are recommended when caring for children
gated [211, 229]. with suspected or documented adenoviral respiratory tract
infections. Although there are no specific recommendations
regarding adults, isolation precautions should also be consid-
10 Prevention ered for adult patients with HAdV disease [146].
Immunization with live, oral, enteric-coated HAdV-4 and
Prevention of transmission of adenoviral infections is chal- HAdV-7 vaccines was used safely and effectively for years in
lenging. Transmission is common in households with young military training camps. After the sole manufacturer stopped
children and day-care centers. HAdVs can be intermittently production in 1996, outbreaks of ARD reappeared [90].
shed in stool for weeks to months and pose an ongoing Efforts to control the spread of HAdVs and prevent epidemics
source of infection. Nosocomial transmission can also occur, in new military recruits were largely unsuccessful. In response,
as discussed above. In an outpatient study, 126 (7 %) of new live oral HAdV-4 and HAdV-7 vaccines were produced
1,870 ophthalmology patients developed EKC due to [96] and were relicensed in 2011 for military use [97].
HAdV-8 [101]. Inadequate disinfection of instruments and
finger-to-eye transmission by healthcare workers were impli-
cated in this outbreak. 11 Adenovirus Vectors
Adenoviruses are nonenveloped viruses that can remain
viable on environmental surfaces for weeks and are rela- A large number of studies have investigated replication-
tively resistant to commonly used disinfectants. In one study, defective, E1-deleted adenovirus vectors for gene therapy.
only bleach (1:10 dilution) and 2.4 % glutaraldehyde were Species C HAdV-5 has been the primary platform for the
effective disinfectants against HAdV-8 under all test condi- construction of vectors. Adenovirus vectors can infect a vari-
tions [230]. The disinfectants 70 % ethanol and 65 % ethanol ety of cell types, including nondividing as well as dividing
plus 0.63 % quaternary ammonium compound also demon- cells, and can be prepared readily in large quantities in tissue
strated at least a 3-log10 reduction in titers, but their virucidal culture. These vectors can accommodate large inserts (up to
activity was significantly reduced in the presence of organic 8 kb) and express higher levels of recombinant proteins than
matter. Other common disinfectants, including 70 % isopro- most other viral vectors.
112 X. Lu et al.

A major obstacle to utilizing adenovirus-mediated gene study, sublingual administration of a HAdV-5 vector express-
therapy is the fact that adenovirus vectors are highly immu- ing HIV Gag in HAdV-5-immune mice elicited a broad CTL
nogenic, as discussed above. As a result, in vivo transgene response in both systemic and mucosal compartments [257].
expression is transient in both animal and clinical studies and
is associated with substantial inflammatory responses [233–
235]. Additionally, most individuals have serotype-specific 12 Unresolved Issues
Nabs to HAdV-5 and exhibit cross-reactive memory T cell
responses to HAdVs, which further limit transgene expres- 12.1 Typing Nomenclature
sion from HAdV vectors [205, 236]. Therefore, a number of
strategies to reduce immunogenicity in order to prolong Typing of HAdVs has historically been accomplished by
transgene expression have been investigated, including (1) establishing their immunologic distinctiveness by quanti-
making additional vector deletions to reduce adenovirus pro- tative neutralization with hyperimmune animal antisera.
tein expression [237], (2) preparing “gutless” or helper- Designation and numeric assignment of a new serotype
dependent vectors [238, 239], and (3) inserting or require demonstration of either no cross-reactions with other
upregulating proteins that inhibit immune responses such as serotypes or a homologous-to-heterologous titer ratio of ≥16
Fas ligand or the adenovirus E3 region [240, 241]. in both directions. For a HAdV with a borderline SN titer
Additionally, alterations in the fiber protein, which mediates ratio, distinctiveness is assumed if the hemagglutinin can
virus attachment to cells, have been engineered to enhance be distinguished by HI or if substantial biophysical, bio-
binding to cell types that are negative for CAR, the major chemical, or phylogenetic differences exist. As noted above,
HAdV cell receptor [242]. naturally occurring intermediate strains that possess hemag-
Since 1993, about 400 clinical protocols have been imple- glutinins of one or more serotypes and neutralizing antigens
mented using adenovirus vectors. Most clinical trials have of another have been designated with the SN component first
focused on the administration of E1-deleted HAdV vectors and the HI component following a slash (e.g., HAdV-11/
by direct injection into tumor masses. HAdV vectors express- H14) [20]. Other intermediate strains that are doubly neu-
ing the tumor suppressor p53 have shown modest activity in tralized by two prototype antisera have been described and
patients with head and neck cancer and non-small cell lung molecularly characterized [258, 259].
cancer [243]. HAdV vectors expressing the herpes simplex Because of the lack of immunotypic clarity with some
virus thymidine kinase have been used as a suicide gene ther- HAdV strains, the challenging requirements of virus cultiva-
apy in patients with glioblastoma or mesothelioma [244]. tion and antisera preparation, and the limited access to refer-
Additionally, HAdVs engineered with E1 mutations that ence prototype antisera, alternative typing schemes like
preferentially allow viral replication and cell lysis in tumor genome restriction analysis have been proposed as noted
cells, so-called “oncolytic” adenovirus vectors, have had above. With the increasing availability of cost-effective
modest activity in combination with chemotherapy in methodologies in DNA sequencing and computational anal-
patients with head and neck cancer [245, 246]. ysis, full genomic sequencing has become the agreed upon
Additionally, both live and E1-deleted replication- standard for future classification of adenoviruses [63].
defective adenoviruses are being investigated as vaccine vec- However, the criteria used for assigning new numbers based
tors for immunization against other infectious pathogens. solely on genomic sequences have been unclear [260], and in
A large phase II trial (the STEP study) of a replication- the last few years, a plethora of new HAdV numeric “types”
defective HAdV-5 vector expressing HIV-1 gag, pol, and nef have appeared in the literature (types 52–65) [259, 261, 262],
proteins failed to prevent HIV-1 infection or to reduce early many of which were previously described intermediate
HIV replication after infection [247, 248]. However, priming strains. To facilitate better integration into the existing sys-
with recombinant protein or DNA, followed by boosting tem, others have proposed retaining the preeminence of the
with an adenovirus vector, may improve vaccine efficacy by hexon gene as the primary identifier and the fiber gene as the
evading preexisting memory immune responses to adenovi- major determinant of tropism in designating a new virus type
ruses [249]. For instance, an influenza matrix protein 2 DNA [263]. However, the degree and character of sequence diver-
prime, followed by a recombinant adenovirus boost, con- gence necessary to consider a candidate virus as a new
ferred broad protection against influenza A infection (includ- numeric type has yet to be agreed upon.
ing H5N1 influenza) in mice [250]. Other strategies under
investigation to help evade preexisting immune responses
include (1) administration via the intranasal route [251, 252], 12.2 Mechanisms of Latency
(2) use of vaccines based on animal adenoviruses or rare
HAdV serotypes [253, 254], (3) insertion of immunostimu- HAdVs were originally isolated from tonsil and adenoid
latory proteins [255], and (4) incorporation of novel antigens explants. Using a sensitive real-time PCR assay, Garnett
into adenovirus capsid proteins [256]. For instance, in one et al. [264] detected a high prevalence of adenoviral DNA in
6 Adenoviruses 113

human tonsil tissues from children, but infectious virus was of 67, 22 %) [274]. However, a study in military personnel
isolated from only 13 of 94 donors (11 %). The average did not confirm a correlation between HAdV-36-specific
amount of viral DNA was 280 copies per cell, and all species antibodies and obesity but rather identified differences in
C serotypes were identified. Notably, adenoviral DNA repli- HAdV-36 seroprevalence related to race and sex [275].
cation could be induced in most samples by lymphocyte
stimulation in culture. In another study, analysis of lympho-
cyte populations from tonsil tissue indicated that adenoviral 12.4 Oncogenicity
DNA was associated with T cells, not B cells [58].
There is also evidence for HAdV reservoirs in lympho- All HAdVs can transform cultured rodent cells, a cell type in
cytes from other tissues and other cell types. HAdVs are fre- which infection is highly restricted. Species A HAdVs such
quently shed in feces, suggesting that the gastrointestinal as HAdV-12 and HAdV-18 and, to a lesser extent, species C
tract may be a source of persistent infection. In one study, viruses can induce tumors in newborn hamsters [276]. This
intestinal tissue from surgical specimens and the lympho- transformation process is mediated by the adenovirus E1
cytes isolated from these specimens were tested using a transactivating region [277]. However, there is no definite
nested adenoviral PCR assay [265]. Intestinal tissue from 21 evidence for oncogenicity in humans. In one study using a
of 58 specimens and intestinal lymphocytes from 21 of 24 highly sensitive quantitative PCR assay to test over 500 pedi-
evaluable specimens were positive for adenoviral DNA. atric tumor specimens, including leukemias, lymphomas,
Surprisingly, species E serotype 4 sequences were most and solid tumors, there was no evidence of HAdV DNA in
commonly detected – this virus has rarely been isolated from any tumor cell types except in brain tumors [266]. Although
stool samples in civilian epidemiologic studies or from clini- HAdV DNA was detected in 25 of 30 glioblastomas and 22
cal specimens in immunocompromised hosts. Species B and of 30 oligodendrogliomas, HAdV DNA was also detected in
C sequences were also detected. Rare reports implicating most control brain tissue samples. Predominantly, species B
HAdV transmission from donor organs (liver and kidney) and D HAdV DNAs were identified in both malignant and
suggest that these viruses can maintain reservoirs in other nonmalignant brain tissue specimens. These data suggest
tissue types [131, 133]. Additionally, adenoviral DNA was that the brain may represent another reservoir of latent HAdV
commonly detected by PCR assay in brain tissue specimens infection.
in one study [266] (see Sect. 12.4, below).
What are mechanism(s) of latency in lymphocytes?
Although HAdVs infect lymphocytes and monocytes very References
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 Alphaviruses: Equine Encephalitis
and Others 7
Scott C. Weaver and Ann M. Powers

1 Introduction transmission cycle takes from 1 day to more than a week

and constitutes the intrinsic incubation period. Biological
Arthropod-borne viruses (arboviruses) are an ecologically transmission should be distinguished from mechanical
defined set of viruses that share a common mode of transmis- transmission in which the virus contaminates the mouth-
sion involving arthropod vectors that transmit to vertebrate parts of the arthropod and can be transmitted immediately to
hosts. Most are biologically transmitted, requiring replica- a new vertebrate host without replication in the invertebrate.
tion in both arthropod and vertebrate host with transmission Also, nonviremic transmission has been described for a few
between vertebrate animals by the bite of mosquitoes, ticks, arboviruses whereby virus inoculated via the saliva is suffi-
sandflies, or midges [1]. The vast majority of arboviruses cient in amount to generate an immediate, nonreplicative
belong to one of five families: Togaviridae, Flaviviridae, viremia [3, 4].
Bunyaviridae, Reoviridae, and Rhabdoviridae. Information Arboviruses are usually maintained in a reservoir cycle
on their isolation, morphology, sensitivity to inactivation by that consists of both arthropod and vertebrate hosts. Both are
chemicals, arthropod vectors, vertebrate hosts, laboratory needed to maintain the virus in nature [5]. A subset of arbo-
propagation, serological reactions, geographic distribution, viruses, including members of the family Togaviridae [6, 7],
clinical manifestations, and epidemiology is found in the is transmitted vertically through the egg of the arthropod. In
International Catalogue of Arthropod-Borne Viruses, com- these cases of transovarial transmission, the arthropod alone
piled by the American Committee on Arthropod-Borne may be the reservoir of the virus and may maintain the virus
Viruses [2]. This exhaustive reference source has been used in the absence of a vertebrate animal (for a limited duration
freely in preparing this chapter. assuming the rate of vertical transmission is <100 %).
The biological transmission of arboviruses is character-
ized by their replication in the arthropod. The period from
ingestion of an infectious blood meal until the virus repli- 2 Sources of Mortality Data
cates, reaches the salivary glands, and can be transmitted is
called the extrinsic incubation period. After being fed upon Mortality data are collected systematically but passively by
by an infected arthropod that secretes saliva to block hemo- national governments for the encephalitic alphaviral infec-
static responses and often to limit pain, the vertebrate host tions and other diseases such as epidemic polyarthritis. Data
becomes infected and usually viremic. This part of the are published in the Morbidity and Mortality Weekly Report
of the US Centers for Disease Control and Prevention, in the
Weekly Epidemiological Report of the Pan American Health
S.C. Weaver, PhD (*) Organization, and the Weekly Epidemiological Report of the
Institute for Human Infections and Immunity,
World Health Organization. The mortality data are underre-
University of Texas Medical Branch,
6.200D Galveston National Laboratory, 301 University Blvd., ported, but may serve as a comparative database, since under-
Galveston, TX 77555-0610, USA reporting may be uniform throughout much of the world.
e-mail: [email protected] The information flow to the World Health Organization is
A.M. Powers, PhD sometimes facilitated by informal networks of scientists and
Arbovirus Diseases Branch, Division of Vector Borne Diseases, interested citizens. Nevertheless, the organization is con-
National Center for Emerging and Zoonotic Diseases, Centers for
strained from action until official reports are received. This
Disease Control and Prevention, Room NCEZID,
3156 Rampart Road, Fort Collins, CO 80521, USA constraint can mean a delay in control of a disease of regional
e-mail: [email protected] or world importance.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 123

DOI 10.1007/978-1-4899-7448-8_7, © Springer Science+Business Media New York 2014
124 S.C. Weaver and A.M. Powers

3 Sources of Morbidity Data virus from arthropods, wild or domestic animals, and people
is essential to determine the natural history of infection with
The same sources supply morbidity data as supply mortality these agents.
data. In the USA, of the arboviral diseases, only encephalitis is Positive findings on survey sera are greatly bolstered by
reportable. An observed trend of increased nonspecific enceph- virus isolations from the vector, nonhuman vertebrates, or
alitic cases during years of arbovirus epidemics may be due to humans. Virus isolation procedures for serum specimens
increased physician requests for more diagnostic tests, leading require inoculation of a small amount of serum into labo-
in turn to diagnosis in a higher percentage of cases in these ratory animals or cell cultures or both. Mice are observed
years. If so, this implies underdiagnosis in other years [8]. daily, or more than once a day, for evidence of illness. A
subpassage may be made to attempt to enhance the virulence
of the virus, and when one is assured that a virus has been
4 Serological Surveys isolated, a stock pool of virus is established and hyperim-
mune mouse serum or ascitic fluid is prepared if needed. In
Alphaviruses are distributed focally nearly throughout the fatal cases, 10–40 % suspensions of the tissue—brain, liver,
world [9]. The distribution of any given arbovirus is ecologi- lung, or spleen (purified by centrifugation)—may be inocu-
cally limited by the range of the vector and vertebrate hosts. lated as for serum.
Serological surveys are ideally suited for arboviruses to deter- Cell culture systems (vertebrate or insect cells) can also
mine the point prevalence in different geographic areas. Often be used for virus isolation. For certain alphaviruses and
distribution of the antibody will give clues to the ecological certain cell cultures, the cell culture systems, are as sensi-
conditions necessary for maintenance of the virus. Surveys of tive as laboratory animals (considering an equal volume of
different age groups will show if the virus is more prevalent total inoculum). Intracerebral inoculation of infant mice
as the population ages, typical of an endemically transmitted will serve to isolate a much wider total range of arboviruses
agent. Another prevalence pattern in which antibody is pres- than will any single cell culture system. Conversely, in the
ent only in persons born before a certain year may indicate an investigation of a specific virus, a cell culture system that
epidemic in that year. Alternatively, a relatively constant per- has been predetermined to be suited to the virus can be
centage of antibody in each age group may indicate a recent easier, cheaper, involve fewer regulatory hurdles, and
introduction of the virus causing a virgin soil epidemic. atleast as reliable than techniques that require laboratory
Broadly based serological surveys of large populations can animals.
provide extensive information about virus distribution in differ- In the study of material derived from patients, it is highly
ent geographic areas, rural versus urban populations, different desirable to have a pair of serum specimens to work with.
age and sex groups, and different occupational types. However, The first should be taken early in the course of illness and
arbovirus serosurveys have limitations. Cross-reactions occur can serve as material both for virus isolation and for the
among viruses of the same serogroup using certain tests. This determination of baseline serum antibody levels before
is especially true of the hemagglutination inhibition (HI) test the patient has developed antibodies to the infecting virus.
and enzyme-linked immunosorbent assay (ELISA). The neu- A second serum should be obtained at least 3 weeks after
tralization test is more specific and should be used where fea- onset of illness or as late as several months after onset.
sible. Surveys with HI or ELISA must be interpreted with A seroconversion (four-fold or greater rise in antibody titer)
caution unless one is certain that only one virus is extant in the demonstrable between the early and later specimens is a
region, or unless the results have been confirmed by neutraliza- strong evidence of a recent virus infection. Demonstration
tion test with a portion of the negative and positive sera. of IgM in ELISA permits a presumptive diagnosis with a
The serosurvey usually will not indicate when the infec- single serum as long as two etiologic agents that produce
tion responsible for the antibody occurred. If the antibody is similar disease are not circulating during the same time
suspected to be of recent origin, the IgM antibody-capture period [10, 11].
ELISA is useful for detection of infections originating within Details of the techniques of CF, HI, and virus neutral-
recent months. ization relating specifically to alphaviruses are available
in current references [11]. Fluorescent antibody (FA)
techniques; antigen detection ELISA, often coupled with
5 Laboratory Methods monoclonal antibody; and RT-PCR are widely used for
antibody, antigen, and RNA detection, respectively.
The laboratory is an all-important resource in the study of Additional specialized assays for the serotype determina-
the epidemiology of arbovirus diseases. Diagnosis can rarely tion and genetic characterization of specific alphavirus
be made with certainty by clinical examination. Isolation of groups are detailed below.
7 Alphaviruses : Equine Encephalitis and Others 125

6 Biological Characteristics of the Viruses eastern equine encephalitis (EEE) were reported during this
That Affect the Epidemiologic Pattern time, of which 12 cases and 6 deaths were in 1968; during
the same period, 1,030 cases of western equine encephalitis
A prerequisite for the survival of arboviruses in nature is a (WEE) were reported, of which 172 occurred in 1965, with
period of viremia in a vertebrate host at a level sufficient to four deaths, and 133 in 1975, with six deaths (http://www.
infect the arthropod vector. Even with alphaviruses that gener- cdc.gov/ncidod/dvbid/arbor/arbocase.htm). In contrast, from
ate high-titer viremias and highly susceptible vectors, a viremia 1988 to 2010, 159 EEE cases were reported, while only five
titer in excess of 10,000 infectious doses of virus per milliliter WEE cases occurred. Venezuelan equine encephalitis (VEE)
of blood is frequently required to sustain transmission. Such produced its first outbreak in Texas, USA, in 1971, with no
high levels of viremia are generally found in the natural host epidemic activity in the USA since that date.
and often are attained in the more commonly used labora-
tory animals such as mice, hamsters, guinea pigs, chicks, and
monkeys. For some alphaviruses, however, the natural hosts or 9 Geographic Distribution
natural vectors, or both, are either unavailable or not known,
so that the postulate of a level of viremia in the vertebrate ade- Alphavirus infections are nearly worldwide in distribution
quate to infect a susceptible vector remains undemonstrated. and may occur whenever the appropriate mosquito vectors
occur in sufficient numbers in proximity to humans and a
suitable amplifying host. Table 7.1 includes the geographic
7 Epidemiology distribution of alphaviruses.

The epidemiology of arbovirus infections in humans is influ-

enced by three major determinants: (1) the behavior of the 10 Temporal Distribution
arthropod vector, including the ecological setting in which
its larval habitats occur, its pattern and range of mobility, its In the USA, mosquito-borne alphaviruses produce human
biting habits and species preferences for feeding, its longev- infections primarily in late summer and fall. Tropical alpha-
ity, and the factors affecting the infection, replication, dis- virus infections tend to occur during rainy seasons when
semination, and secretion into the saliva of virus within the mosquito vectors are most abundant. However, chikungunya
arthropod host; (2) the frequency, nature, and duration of virus (CHIKV), which occurs both in enzootic, sylvatic
exposure of humans to the infected arthropod vectors, as primate-amplified and urban, human-amplified cycles, can
influenced by the presence, level, and specificity of humoral cause human disease throughout the year.
antibody and by use in the population of insecticides, insect
repellents, and protective clothing; and (3) the requirements
for the presence of a necessary and/or amplifying vertebrate 11 Age and Sex
host for the virus, such as horses, birds, or rodents, and of the
availability of humans as alternative hosts. Infections with alphaviruses can occur at any age. The
age distribution depends on the degree of exposure to the
particular transmitting mosquito relating to age, sex, and
8 Epidemic Behavior occupational, vocational, and recreational habits of the indi-
vidual or group of individuals. For most alphaviruses, there
Outbreaks of alphavirus infections in the USA involving is not a strong association between these groups and mark-
humans occur periodically and unpredictably, as evident edly higher rates of exposure. However, once humans have
from the activity from 1955 through 1987. Only 182 cases of been exposed and infected, the severity of the disease may

Table 7.1 Members of the genus Alphavirus

Antigenic complex Species Antigenic subtype Antigenic variety Clinical syndrome Distribution
Barmah Forest Barmah Forest virus Febrile illness, rash, Australia
(BFV) arthritis
Eastern equine Eastern equine I Febrile illness, encephalitis North, Central and
encephalitis (EEE) encephalitis (EEEV) South America
Madariaga virus II–IV
Middelburg Middelburg virus (MIDV) None recognized Africa
Ndumu Ndumu virus (NDUV) None recognized Africa
(continued)
126 S.C. Weaver and A.M. Powers

Table 7.1 (continued)

Antigenic complex Species Antigenic subtype Antigenic variety Clinical syndrome Distribution
Semliki Forest Semliki Forest virus Febrile illness Africa
(SFV)
Chikungunya virus Febrile illness, rash, Africa, Asia
(CHIKV) arthritis
O’nyong-nyong virus Febrile illness, rash, Africa
(ONNV) arthritis
Getah virus (GETV) None recognized Asia
Bebaru virus (BEBV) None recognized Malaysia
Ross River virus (RRV) Sagiyama Febrile illness, rash, Australia, Oceania
arthritis
Mayaro virus (MAYV) Febrile illness, rash, South America,
arthritis Trinidad
Una virus (UNAV) None recognized South America
Venezuelan equine Venezuelan equine I AB Febrile illness, encephalitis North, Central, South
encephalitis (VEE) encephalitis virus (VEEV) America
C Febrile illness, encephalitis South America
D Febrile illness, encephalitis South America,
Panama
E Febrile illness, encephalitis Central America,
Mexico
Mosso das Pedras virus F None recognized Brazil
(MDPV)
Everglades virus (EVEV) VEE-II Febrile illness, encephalitis Florida (USA)
Mucambo virus (MUCV) VEE-III A Febrile illness, myalgia South America,
Trinidad
C (strain Unknown Peru
71D1252)
D Febrile illness Peru
Tonate virus (TONV) VEE-IIIB Febrile illness, encephalitis Brazil, Colorado
(USA)
Pixuna virus (PIXV) VEE-IV Febrile illness, myalgia Brazil
Cabassou virus (CABV) VEE-V None recognized French Guiana
Rio Negro virus (RNV) VEE-VI Febrile illness, myalgia Argentina
Western equine Sindbis virus (SINV) Febrile illness, rash, Africa, Europe, Asia,
encephalitis arthritis Australia
(WEE) Babanki Febrile illness, rash, Africa
arthritis
Ockelbo Febrile illness, rash, Europe
arthritis
Kyzylagach None recognized Azerbaijan, China
Whataroa virus (WHAV) None recognized New Zealand
Aura virus (AURAV) None recognized South America
Western equine Several Febrile illness, encephalitis Western North, South
encephalitis virus America
(WEEV)
Highlands J virus (HJV) None recognized Eastern North
America
Fort Morgan virus (FMV) Buggy Creek None recognized Western North
America
Trocara Trocara virus (TROV) South America
Salmon pancreas Salmon pancreas disease Pancreatic disease Atlantic Ocean and
disease (SPD) virus (SPDV) (salmon), sleeping disease tributaries worldwide
(trout)
Southern elephant Southern elephant seal None recognized Australia
seal virus (SESV)
Eilat Eilata Incapable of infecting Middle East
vertebrates
a
Recommended for species designation [12] but not yet approved by the International Committee on the Taxonomy of Viruses
7 Alphaviruses : Equine Encephalitis and Others 127

also be influenced by age. For example, WEEV and VEEV (sometimes hemorrhagic), and involvement of the liver or
tend to produce the most severe clinical infections in young kidneys. In most arbovirus infections, only the first phase
persons. occurs, and the disease is “nonspecific.” In other instances,
the early phase may be missed, and only the severe mani-
festations occur. The early phase is often accompanied by
12 Mechanism and Route leukopenia and the second phase often by leukocytosis [16].
of Transmission Tissue injury may be the direct effect of viral multiplication
in susceptible cells, as is the case with lymphodepletion fol-
By definition, arboviruses must be transmitted by arthropod lowing VEEV infection.
vectors. There is often a high-level virus–vector specificity Humoral antibodies regularly appear early in the course
associated with biological transmission, usually manifested of alphavirus infection and constitute the major basis of
at the initial infection of the mosquito alimentary tract. This immunity. Such immunity may be lifelong. The role of cell-
biological transmission may be supplemented under some mediated immunity in alphavirus infections has been poorly
circumstances by mechanical transmission. studied but can protect mice deficient in antibody production
The duration of the necessary period of virus replication from lethal VEEV infection [17]. It is possible that it may be
within the arthropod host before it becomes infectious varies important in controlling virus persistence and in determining
from virus to virus and vector to vector and is also directly the immunopathological lesions suspected in certain mani-
temperature dependent. For some alphaviruses, the extrinsic festations of infection.
incubation period can be as little as 2 days under average
summer temperature conditions [13, 14]. However, mosqui-
toes generally must develop their eggs and oviposit before a 14 Patterns of Host Response
subsequent blood meal, resulting in transmission, occurs;
this gonotrophic cycle length may limit the minimum time 14.1 Clinical Features
until transmission if it is longer than the time for virus to
reach and replicate in the salivary glands after an infectious Inapparent and subclinical human responses predominate in
blood meal. Once infected, vectors often remain infected and most alphavirus infections, with the exception of CHIKV
able to transmit for life, which can be many weeks or even and VEEV. For example, infection with EEEV and WEEV
months. results principally in mild and inapparent infections; the rea-
Transmission of alphaviruses transovarially in mosqui- sons for these differences are unknown.
toes, often referred to as “vertical transmission,” has been
demonstrated for CHIKV [6] Sindbis [15] and Ross River
virus [7]. 14.2 Diagnosis

Cases of alphavirus infections in the USA are not likely to be

13 Pathogenesis and Immunity diagnosed unless there is a high degree of clinical suspicion.
Outbreaks of encephalitis in horses during the summer,
Most alphavirus infections are transmitted by the bite of a caused by EEEV, WEEV, or VEEV, serve to focus attention
mosquito vector, so that the skin represents the sole portal of on febrile illness in humans associated with symptoms or
entry. With early wide dissemination throughout the host, signs indicating involvement of the CNS. Recognition of the
multiplication follows in generally poorly determined target arbovirus infection acquired by the traveler outside the USA
cells and tissues. Viremia in the host then provides the seed- also depends on the alertness of the examining physician.
bed for infection of succeeding cohorts of biting arthropods. Rapid jet transport now permits exposed overseas travelers
The incubation period is usually 3–15 days. to reach home and fall ill even within the short incubation
The site of replication of most arboviruses remains period of such infections. This trend was exemplified by the
undetermined but is presumed to be in the vascular epithe- 35 CHIKV-infected patients diagnosed after their return
lium and the reticuloendothelial cells on the lymph nodes, from epidemic regions of the Indian Ocean and Asia during
liver, spleen, and elsewhere. Liberation of virus from these 2006 [18]. The physician must maintain a high degree of sus-
organs constitutes the “systemic phase of viremia,” resulting picion when seeing CNS infections or influenza-like ill-
after 3–7 days of symptoms such as fever, chills, and ach- nesses occurring in travelers recently returned from areas
ing. A number of arbovirus infections have two phases— endemic for alphaviruses. Testing for alphavirus infection is
this early phase and then a second phase with or without not widely available, so special testing must often be done
a few days of freedom from symptoms. The second phase through arrangements with regional, national, or interna-
may be attended by encephalitis, joint involvement, rash tional public health agencies and/or reference centers. It
128 S.C. Weaver and A.M. Powers

should be noted that arbovirus infections constitute only a two viruses are enzootic within the USA, Canada, and
small fraction of the encephalitis cases seen in the USA with Mexico, while VEE is enzootic and endemic only to Mexico
the majority of cases of undetermined etiology. but has spread into the USA on at least one occasion.
The laboratory diagnosis depends on the isolation of the
virus from the blood, a fourfold antibody rise in titer between
acute and convalescent sera, or the presence of specific IgM 15.2 Eastern Equine Encephalitis Virus
taken during the acute phase. Often, the suspicion of an
alphavirus infection in individual cases, especially in cases 15.2.1 Natural History/Transmission
of neurologic disease that occurs several days after the onset In North America, most human and equine EEEV infections
of illness, arises too late for virus isolation from the blood. occur near enzootic foci of transmission, which occur in
Under these circumstances, the presence of a high antibody hardwood swamps populated by the principal mosquito vec-
titer in a single serum may be significant if the infection is an tor, Culiseta melanura, and passerine birds that serve as
uncommon one in that region and particularly if antibody sur- amplification hosts (Fig. 7.1). These habitats occur along the
veys reveal a low antibody prevalence or if prior surveys have Atlantic and Gulf coasts but also at inland locations in the
demonstrated the absence of antibody in that community. The upper Midwest (Fig. 7.2).
appropriate procedure in suspected cases is to (1) notify the However, southeastern USA foci in locations where this
health department and seek background epidemiologic and mosquito is not abundant suggest the involvement as enzo-
clinical data and (2) send acute and convalescent serum sam- otic vectors of ornithophilic Culex spp. mosquitoes including
ples to the nearest public health laboratory (usually a state Culex erraticus [21]. Because Cs. melanura feeds primarily
laboratory), with a request for antibody tests for alphaviruses. on avian hosts, bridge vectors, probably including Aedes
Some state laboratories may not have all necessary testing vexans, Coquillettidia perturbans, Ae. canadensis, and Cx.
available, at which point a request for transshipment of sera salinarius, have been assumed to transmit from birds to
to the CDC for additional testing might be included. humans and horses [21, 22] (Fig. 7.3).
However, some studies suggest that Cs. melanura may bite
mammals at sufficient frequency to implicate them in human
14.3 Control and Prevention and other mammal infections [23, 24]. Passerine birds that
inhabit swamps are considered the principal enzootic amplifi-
Major control methods include (1) control of the arthropod cation hosts in North America. However, recent serological
vector, which may be by elimination of aquatic larval sites or and experimental infection data from the southeastern USA
their treatment with insecticides or by direct control of adult suggest that amphibians and reptiles could be involved in
female mosquitoes that are responsible for transmission, enzootic circulation [25, 26]. Although a wide variety of
through insecticide applications such as malathion; (2) domesticated mammals and birds suffer severe and often fatal
avoidance of exposure to mosquito vector bites by screening disease after EEEV infection, none is believed to be efficient
of houses, by using protective clothing, by staying indoors at amplification via mosquito transmission because viremia
during times of peak host seeking by vector mosquitoes, and levels are generally low [27–29]. Thus, unlike VEE outbreaks
by applying insect repellents when outside in high-risk areas. that can spread far from their origin due to efficient equine
Control of mosquito vectors through biological approaches amplification, cases of EEE rarely occur far from enzootic
ranging from larvicidal bacteria, predatory fish, and the foci, which tend to occur in relatively remote locations.
introduction of genes deleterious to the vector population Presumably because they receive greater exposure to
[19] or of Wolbachia bacteria that render adult female mos- mosquito feeding, equids suffer a higher incidence of EEE
quito vectors refractory to transmission [20] is receiving than humans in North America. EEE epizootics in horses and
much attention as an alternative control strategy. pheasants often precede human cases, and the former are
thus useful sentinels for detecting epizootic circulation [30].
Human and equine outbreaks are associated with excessive
15 Characteristics of Selected rainfall [31, 32] and usually peak in early fall in temperate
Arboviruses regions of North America.
EEEV strains closely related to North American strains iso-
15.1 Alphaviruses of Importance in the USA lates have also caused equine outbreaks in the Caribbean [33]
and northern Mexico [34]. Because there is no clear evidence
Of the alphaviruses found in North America, only eastern of enzootic circulation in these locations, these outbreaks may
(EEE), western (WEE), and Venezuelan equine encephalitis represent introductions followed by epizootics of limited dura-
(VEE) viruses are important human pathogens. The former tion. Similarly, it remains unknown if the detection of epizootic
7 Alphaviruses : Equine Encephalitis and Others 129

Culiseta melanura

Culiseta melanura

Aedes, Coquillettidia, etc.

Dead-end transmission

Fig. 7.1 Enzootic transmission cycle of eastern equine encephalitis virus in North America and epidemic or epizootic spillover to humans and
domestic animals, respectively

EEEV circulation as far north as northern Maine in 2009 [35] evidence of their exposure during epizootics and by living in
represents stable enzootic circulation or a temporary introduc- areas of enzootic transmission, only three cases of human EEE
tion. In Central and South America, strains recently assigned were reported in Latin America [37–39]. Genetic and anti-
to the species Madaringa virus (MADV have been isolated genic distinctions between EEEV strains that circulate in
repeatedly from mosquitoes, principally members of the sub- North versus South American strains (MADV), as well as dif-
genus Culex (Melanoconion), as well as from equids suffering ferences in sensitivity to and induction of interferons, suggest
from neurologic disease. The most important reservoir hosts in fundamental differences in human virulence [40, 41]. However,
South America remain unclear but may involve both small a 2010 EEE outbreak in Panama, involving both horses and
mammals and possibly birds [36]. Prior to 2010, despite clear humans, may reflect a change in human virulence there [10].
130 S.C. Weaver and A.M. Powers

VT
NH 10

MA 37
1 4
16
RI 6
2
CT
3
4 NJ 20
17
DE 3
13
7 28 MD 4
6
2 17 DC
70
WV

Fig. 7.2 Eastern equine encephalitis virus neuroinvasive disease cases reported by state, 1964–2010 (Adapted from http://www.cdc.gov/easterne-
quineencephalitis/tech/epi.html)

Fig. 7.3 Eastern equine 25

encephalitis virus neuroinvasive
disease cases reported as
encephalitis, meningoencephali-
tis, or meningitis, by year, 20
1964–2010 (Adapted from http://
www.cdc.gov/easternequineen-
cephalitis/tech/epi.html)
15

10

0
1964
1966
1968
1970
1972
1974
1976
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010

15.2.2 Impact on Human and Animal Health the 1950s involved more than 30 people [42], an average of
Clinical EEE in humans has remained sporadic with little only five to ten human cases has been documented annually
evidence of any overall change in incidence during the past in the USA since the 1970s (Fig. 7.3). Historic records sug-
several decades. Although some epidemics from the 1930s to gest that the principal enzootic vector of EEE virus (EEEV),
7 Alphaviruses : Equine Encephalitis and Others 131

Cs. melanura, may have become more abundant along the other mycobacterial or fungal diseases) etiologies. After
Atlantic seaboard following reforestation during the twenti- initial clinical suspicion, diagnosis is ideally established by
eth century, leading to increased virus circulation and peri- virus isolation from the serum, cerebrospinal fluid (CSF), or
odic epidemics. Although the average number of human brain tissue or the detection of viral RNA from these samples
cases remains low, the high EEE case-fatality rate, usually through reverse-transcription polymerase chain reactions
exceeding 50 %, leads to fear in the affected populations and [16]. However, human viremia is not typically of high titer,
considerable expenditures for mosquito vector control to so serological diagnosis is more common, including demon-
reduce the risk of infection [16]. Because EEE survivors stration of seroconversion (a fourfold or greater rise in anti-
often suffer permanent, debilitating neurologic sequelae, body titers between acute and convalescent serum) or the
health care and institutionalization costs were estimated at finding of high-titer IgM in serum. Because the background
$3 M per case in 1994 ($4.7 M in 2012 dollars) [43]. EEE prevalence of EEE in endemic areas is very low, a single
occurs predominantly in eastern and Gulf Coast states and in serum demonstrating high-titer antibodies is highly sugges-
northeastern inland states in the USA and Canada (Fig. 7.2). tive of etiologic infection. Evidence of an outbreak of dis-
ease involving birds and mosquitoes, and more particularly
15.2.3 Human Disease equids or other domesticated animals in the region, should
The incubation period in human beings is short, usually 4–10 alert clinicians.
days [16, 44]. Gender is not a major factor in risk for disease
[45], but patients <10 years of age are more likely to suffer 15.2.5 Prevention and Control
severe sequelae than older age groups. The ratio of inappar- Because there are no licensed human vaccines or treatments,
ent or mild infections to severe infections has been described prevention of exposure to infected mosquitoes is the only
as low in some studies based on very low human seropreva- way to prevent the disease. Passive surveillance relies on the
lence after epidemics [44]. However, following the 1959 finding that equine or avian disease usually precedes human
New Jersey epidemic, 3.1–3.6 % of persons surveyed exhib- infections. More active approaches include the use of chick-
ited complement fixing antibodies, suggesting many inap- ens to detect EEEV circulation through seroconversion and
parent infections [46], with inapparent (no recognized central monitoring mosquito populations for infection; [50] early
nervous system disease)—apparent ratios of 8–26 % in dif- season detection of the latter is predictive of late season epi-
ferent age groups, or 23 % overall [47]. Abrupt onset of zootics and human cases [51]. When high levels of EEEV
severe fever, nausea, myalgias, and intense headache are fol- circulation are detected, the control of adult female mosquito
lowed by encephalitis in 1–2 days. Infants and children often populations is often attempted using aerial insecticide appli-
present with convulsions. The encephalitis rapidly pro- cations. Avoidance of mosquito contact relies on wearing
gresses to a coma, particularly in small children. Between 30 long pants and long-sleeved shirts, applying repellents to
and 70 % of clinical cases are fatal. Patients with a short pro- exposed skin, and remaining indoors during the evening and
drome are more likely to develop encephalitis than those nighttime when mosquito vectors are most active. An inacti-
with a long prodromal illness accompanied by nonspecific vated virus vaccine has been administered to laboratory per-
signs and symptoms [48] a pattern observed in some animal sonnel at risk through the US Army Special Immunizations
models [49]. Pathological features include diffuse encephali- Program [52]. Equids and other domesticated animals can be
tis with evidence of scattered neuronal destruction. Intensive immunized with commercially available inactivated vac-
supportive care is essential, since there is no specific treat- cines [53]. However, these vaccines are poorly immuno-
ment for EEE. Immunity is probably lifelong, with no rein- genic, requiring annual boosters, and can carry the risk of
fections being described. disease from residual live, virulent virus [54]. Several
recently developed vaccine candidates have shown promise
15.2.4 Diagnosis in preclinical trials [55–57], but because of the small num-
EEEV infection is typically suspected based on the proxim- bers of natural human infections, they are unlikely to attract
ity of the patient to known swamp habitats that support enzo- a commercial market.
otic circulation in the eastern USA. Differential diagnoses
include a wide variety of noninfectious (e.g., tumor, stroke, 15.2.6 Viral Genetics and Evolution
Alzheimer’s disease, long-term alcohol abuse, and other Genetically, aside from the recombinant portion of WEEV
dementias, although these conditions are not usually accom- that was derived from an ancestral EEEV strain, EEEV is
panied by fever) as well as infectious (herpes simplex virus, most closely related to VEE complex alphaviruses [58, 59].
enterovirus, influenza virus, adenovirus, lymphocytic cho- Antigenically, it is also most closely related to the latter
riomeningitis virus, respiratory syncytial virus, rabies, or group. In North America and the Caribbean, EEEV is highly
Epstein–Barr virus infection, bacterial meningitis, myco- conserved genetically across time and space, with only less
plasma, Bartonella henselae, Rocky Mountain spotted fever, than 2 % nucleotide sequence divergence among all strains
leptospirosis, Lyme disease, HIV, syphilis, tuberculosis, and isolated since 1933 [60] (Fig. 7.4).
132 S.C. Weaver and A.M. Powers

GA01 GA01
TN08 1/98 1/86
GA97 TN08
5% nucleotide TX03 GA97
North America
FL93.969 TX03
Sequence divergence FL93.1637 FL93.969
FL91
MA06 1/62 FL93.1637
FL82 .98/52 FL91
MX97 MA06
TX91
TX95 FL82
MS83 .92/* TX91
GA91 1/98
.98/63 MX97
FL96 1/98 TX95
CT90 .92/*
MD85 .90/* MS83
WI80 GA91
MD90 FL96
FL93.939 1/74
MA77 CT90
.99/64
NJ60 1/76 MD85
VA33 WI80
Subtype I LA47
LA50 1/61 1/100 MD90
MA38 1/* FL93.939
GU68 MA77
BR56 .98/51
Subtype II NJ60
PE70 .86/71 VA33
1/100 PE18.0172
PE18.0140 1/100 1/76 LA47
PE3.0815 LA50
BR65
BR67 MA38
BR75 0.003
BR76
1/100 AR38
AR36
AR59
BR83
BR77
0.99/100 0.99/67 BG60
PE75
PE16.0050
PE0.0155
TR59
0.57/62 PA84
PA86
0.67/100 CO92
0.92/86 PA62
EC74
Subtype III VE80 South America
BR60
BR78
Subtype IV VE76
BR85

Fig. 7.4 Phylogenetic analyses of EEEV isolates using Bayesian meth- Scale bar shows a genetic distance of 5 % nucleotide sequence diver-
ods, with the complete structural polyprotein open reading frames. (a) gence. (b) Magnified version of NA EEEV phylogeny. Values for PP/
Left, tree of NA and SA EEEV. Bayesian posterior probability (PP) val- MP are shown only if either value is greater than or equal to 0.90 (PP)
ues and maximum parsimony (MP) bootstrap values are noted for all or 90 (MP) for the adjacent node. Asterisks indicate a polytomy in MP
major nodes of lineage divergence (PP/MP values). Within each SA bootstrap analysis. Scale bar shows a genetic distance of 0.3 % nucleo-
EEEV (MADV) lineage, values for PP/MP are shown only if either tide sequence divergence (Adapted from) [60]
value is less than or equal to 0.90 (PP) or 90 (MP) for the adjacent node.

This conservation and the evolution of EEEV as a single lineages have been identified in the same location at the same
ongoing lineage in North America presumably reflect the time [64], suggesting that EEEV population structure is not
efficient transport of strains via infected birds [61, 62], lead- completely spatially defined.
ing to periodic selective sweeps. However, phylogenetic
groupings of strains isolated from temperate foci over sev-
eral years also suggest that EEEV overwinters in the north- 15.3 Venezuelan Equine Encephalitis
eastern USA, where transmission does not occur during the
winter because Cs. melanura survives only in the larval stage 15.3.1 Natural History/Transmission
[62, 63]. In South America, EEEV strains (MADV) are much Venezuelan equine encephalitis (VEE) virus (VEEV) is
more diverse genetically and antigenically, with regionally one species in the VEE antigenic complex of alphaviruses,
defined viral clades that suggest the use of vertebrate ampli- which comprises several species, subtypes, and varieties that
fying hosts with limited dispersal potential [34, 60]. There is can be distinguished genetically and antigenically [9, 65].
no evidence of mixing of EEEV strains between the conti- Most of these viruses, including VEEV subtypes ID and IE,
nents. However, as with North American strains, two distinct circulate in enzootic transmission cycles involving rodent
7 Alphaviruses : Equine Encephalitis and Others 133

Fig. 7.5 Enzootic (above) and

epizootic/epidemic (below)
transmission cycles of most VEE
complex alphaviruses (enzootic)
and VEEV subtypes IAB and IC
Culex (Melanoconion) spp.
(epizootic/epidemic). Arrows
show transition from the
enzootic VEEV subtype ID
strains to the epizootic/epidemic
VEEV subtype IAB or IC strains
via adaptive mutations that
enhance equine viremia or from
enzootic subtype IE strains in
Pacific Coastal Mexico to an
epizootic subtype IE phenotype
that infects more efficiently
Aedes (Ochlerotatus) taeniorhyn-
chus mosquito vectors

Mutation
Selection

Aedes, Psorophora, etc.

reservoir/amplification hosts and mosquito vectors in the when surveillance activities were implemented (Fig. 7.6),
subgenus Culex (Melanoconion) (Fig. 7.5). they are probably widespread in South America.
Exceptions include the VEE complex alphaviruses Tonate Enzootic strains of VEEV, subtypes ID and IE, have been
and Bijou Bridge, which appear to utilize birds as vertebrate more intensively studied and are important causes of endemic
hosts in South and North America, respectively. Although disease among people who live in regions where they circu-
many members of the VEE complex have only been isolated late in forested or swamp habitats [66–70]. Studies of
in one or a few locations and at one or a few time points dengue-like illness suggest that they may cause a burden of
134 S.C. Weaver and A.M. Powers

Fig. 7.6 Known distributions of alphaviruses in the VEE antigenic complex

disease that is approximately 1/10 that of dengue in Latin hundreds to hundreds of thousands of people, with low rates
America and that fatal human infections occur regularly of fatality but with many long-term effects on survivors due to
[66]. Everglades virus, which is only found in Florida, occa- high rates of permanent sequelae, especially among infected
sionally causes human disease [71] but has never been asso- children. Endemic human VEE due to spillover directly from
ciated with equine outbreaks. enzootic cycles is rarely diagnosed because most infections
In addition to the enzootic strains that circulate continuously closely resemble dengue fever, which is endemic in most of
and in a widespread manner, VEEV variants in subtypes IAB Latin America [66, 67, 69]. However, if estimates that about
and IC, often called “epizootic” or “epidemic” strains, arise 3 % of dengue-like illness are caused by VEEV in Iquitos,
periodically from the enzootic strains to cause major outbreaks Peru [67], can be extended to other parts of Latin America,
of disease in people and equids [72, 73]. Phylogenetic and there could be tens of thousands of human cases annually,
reverse genetic studies indicate that these strains arise when with hundreds of fatalities assuming a case-fatality rate of
enzootic forms adapt via positive selection to increase their about 0.5 % [66].
viremia and virulence in equids, leading to highly efficient
amplification in rural locations [74], and/or to mosquito vec- 15.3.3 Human Disease
tors that undergo seasonal expansions [75]. However, most of Infection with VEEV or many VEE complex alphaviruses
these epizootic/epidemic strains are short lived, presumably typically leads to an undifferentiated acute febrile illness
due to their reliance on equids for amplification and the limited characterized by an abrupt onset of headache and fever, often
population turnover of these relatively long-lived hosts. accompanied by gastrointestinal signs and symptoms [16].
Unlike those of many other arboviruses such as EEEV, most
15.3.2 Impact on Human and Animal Health human VEEV infections are symptomatic and attack rates
VEE complex viruses have a severe impact on both human during outbreaks can exceed 50 % [65]. A small fraction of
and animal health. The former is affected both by spillover infections, primarily those in children, progress to central
of enzootic transmission cycles and by equine-amplified nervous system disease typically including convulsions, dis-
epidemics, while equids and other domesticated animals are orientation, and drowsiness and occasionally to coma and
affected primarily during epizootics [72]. Because equids death. Children who survive neurologic disease often experi-
remain important for agriculture and transportation in areas ence lifelong sequelae that affect motor function and cogni-
of Latin America where epidemics occur, epizootics also tion. In addition to its direct effects, VEEV is lymphotropic
have significant economic effects. Epidemics, which usu- and causes immune suppression, which leads to secondary
ally involve VEEV subtypes IAB and IC, typically affect infections at elevated rates for up to months after infection.
7 Alphaviruses : Equine Encephalitis and Others 135

There is no evidence that different strains of VEEV differ 15.3.6 Viral genetics and Evolution
greatly in human virulence, although virulence differs mark- Among the alphaviruses, VEEV and related VEE complex
edly in equids [76, 77]. viruses have received extensive study since the 1980s. Initial
studies focused on determining the relationships between
15.3.4 Diagnosis enzootic and epizootic/epidemic strains to determine the ori-
Diagnosis of VEEV infection largely follows the procedures gins of the latter. RNA fingerprinting [91] followed by par-
outlined above for EEEV [16]. However, in addition to the tial and complete viral genomic sequencing [78, 92, 93] first
basic diagnosis that generally relies on virus isolation, IgM supported the hypothesis that epizootic/epidemic strains
detection, or seroconversion (fourfold or greater ride in IgG evolve periodically from enzootic precursors. Sequencing
measures by ELISA or other assay), it is important to deter- studies also suggested that several outbreaks caused by
mine the serotype circulating because subtypes IAB and IC subtype IAB VEEV were initiated by incompletely inacti-
can spread rapidly due to equine amplification. Virus isolation vated equine vaccines made from early isolates [94]. Later,
followed by complete or partial genome sequencing with a reverse genetic studies demonstrated that a single mutation
focus on the E2 envelope glycoprotein is most informative in an enzootic subtype I strain was sufficient to transform the
[78], but more rapid, ELISA-based methods are also available ID serotype and equine amplification incompetent pheno-
to diagnose the serotype of infection [79]. Human viremia usu- type to an IC epizootic phenotype [74] (Fig. 7.5). Another
ally lasts 3–4 days and is relatively high in titer, so virus isola- mutation found in recent Mexican subtype IE strains appears
tion or viral RNA detection by RT-PCR is useful for definitive to have adapted the virus to more efficient transmission by
diagnosis. Once viral clearance from the blood has occurred, the mosquito vector, Ae. (Ochlerotatus) taeniorhynchus.
usually 5–6 days after infection, a blocking ELISA can still be Phylogenetic studies have also revealed spatially partitioned
used to determine the VEEV serotype of infection [80]. genetic lineages of VEEV within serotypes, presumably
reflecting the limited mobility of rodent and mosquito hosts.
15.3.5 Prevention and Control
Prevention of endemic VEE caused by spillover of enzootic
strains is challenging because the enzootic cycle involving 15.4 Western Equine Encephalitis
rodents and forest-dwelling mosquitoes would be difficult to
control due to its relatively remote location and widespread 15.4.1 Natural History/Transmission
occurrence in the neotropics [66]. Prevention of exposure to Western equine encephalitis virus (WEEV) is one species in
enzootic vectors, which generally are active during crepuscu- the WEE complex of alphaviruses [95]. Others include
lar time periods or during the nighttime, relies on wearing Highlands J Fort Morgan and Aura viruses in the New World
appropriate long-sleeved shirts, long pants, and other gar- and Sindbis virus in the Old World [96]. Western equine
ments to reduce skin exposure, applying repellents, and stay- encephalitis virus was first isolated from a horse in California
ing inside during the evening and night. Prevention of in 1930 [97], and the first human isolate was made nearby in
equine-amplified epidemics can be achieved by vaccinating 1938 [98]. Subsequent field studies determined that WEEV
equids with the live-attenuated TC-83 VEEV strain, which is mosquito-borne and that birds are the principal amplifica-
received its first widespread use during a 1971 Texas epi- tion hosts, especially house finches (Carpodacus mexicanus)
demic, where it may have limited spread of VEEV to other and house sparrows (Passer domesticus) [95, 99, 100]. In
parts of the USA [81]. However, TC-83 has several deficien- most North American locations, Cx. tarsalis, which is often
cies including reactogenicity in experimentally vaccinated abundant in irrigated farmland, is the most important enzo-
laboratory workers and poor immunogenicity in many people otic and epizootic vector mosquito, and human and equine
and its reliance on only point mutations for attenuation [82]. infections generally occur in agroecosystems from June to
Because it can be transmitted from vaccinated horses by mos- August (Fig. 7.7) [99]. A secondary enzootic cycle involving
quitoes, which occurred in 1971 [83], reversion to virulence leporids and other rodents and Ae. melanimon mosquitoes as
could occur and might allow equine vaccination to initiate an vectors in arid habitats has also been described (Fig. 7.7). In
epidemic. An inactivated version of TC-83, called C84 in South America, Ae. albifasciatus appears to be an important
human formulations, is administered to laboratorians and epizootic vector [101, 102].
horses in the USA. Several other vaccines have been devel- Genetic studies (see below) indicate that WEEV is
oped during the past decade, and one, live-attenuated strain readily transported between North and South America but
V3526, was tested in Phase I clinical trials but was associated that local overwintering and regionally defined evolution
with some reactogenicity and thus has been developed as an occur in temperate regions of North America. However,
inactivated formulation [84, 85]. Other DNA and live-attenu- experimental infections of birds have not generated chronic
ated vaccines appear promising in preclinical trials [86–90]. infections that could facilitate long-distance transport or
136 S.C. Weaver and A.M. Powers

Culex tarsalis

Aedes spp.

Aedes spp. Dead-end transmission

Fig. 7.7 Transmission cycles of western equine encephalitis virus in North America and epidemic or epizootic spillover to humans and domestic
animals, respectively

overwintering of the virus [103, 104]. Overwintering of 1938, 1941, 1944, and 1947 [95]. From 1937 to 1938, more
WEEV in infected mosquitoes undergoing reproductive than 300,000 equids were affected in the USA, and in 1941
diapause has been suggested by some laboratory studies but alone, 2,242 human cases were documented in the Dakotas,
not confirmed by virus isolation from field-collected, over- Nebraska, and Minnesota (Fig. 7.8).
wintering Cx. tarsalis [105]. Human attack rates during this epidemic were as high as
171/100,000 persons [95]. However, the last documented
15.4.2 Impact on Human and Animal Health human WEE case in North America occurred in 1999 in
During the mid-twentieth century, WEEV caused major Minnesota, and only one other case has occurred since 1988
epidemics and equine epizootics in the USA and Canada (in Colorado, 1989). Compared to the previous period from
involving up to thousands of people and tens of thousands of 1964 to 1988, when an average of 26 documented human
equids during a single summer transmission season [99, cases occurred each year, this suggests a dramatic reduction
100]. Major epizootics occurred in the USA in 1930, 1937, in severe human disease caused by WEEV. The most recent
7 Alphaviruses : Equine Encephalitis and Others 137

13
VT
27 78 NH
1 43
MA
40 2 1
16
RI
5
26
6 1 CT
53 173
36 7 NJ

3 DE
2 13
MD

94
DC

WV

Fig. 7.8 Western equine encephalitis virus neuroinvasive disease cases reported by state, 1964–2010 (Adapted from http://www.cdc.gov/ncidod/
dvbid/arbor/arbocase.htm)

estimates of human seroprevalence available from consis- usually one week from onset, to central nervous system dis-
tently enzootic regions of California were <3 %, indicating ease characterized by generalized weakness and tremulous-
infrequent exposure [106, 107]. In Latin America, serop- ness, especially of the hands, tongue and lips. Some cases
revalence studies have indicated that WEEV circulation is present with lethargy, drowsiness, nuchal rigidity, and pho-
widespread from Mexico [108] to Argentina [109], and tophobia, which can be followed by coma and death. Overall
equine epizootics have been described in Argentina since case-fatality rates usually range from 3 to 4 %. Children
the 1930s. Human cases, which are occasionally fatal [110], often exhibit muscular rigidity, involuntary movements, and
have also been detected. paralysis. Survivors of encephalitis often experience lifelong
neurologic sequelae, including fatigue, irritability, progres-
15.4.3 Human Disease sive mental and physical debilitation, headache, and trem-
After an incubation period of 5–10 days, human WEE disease ors, which can also follow infection in utero [95, 99, 100].
ranges from inapparent infection (by far the most common Because no specific treatment is available for any alphavi-
outcome, especially in adults where the apparent–inapparent rus infection, patient management focuses on supportive
ratio can be as low as 1:150,000) to a nonspecific flu-like care and treatment of acute complications, such as increased
illness to fatal encephalitis. The highest incidence and most intracranial pressure and seizures, as well as management of
severe infections generally occur in the youngest age groups, long-term sequelae [16].
especially in infants <1 year of age. Males are more likely
to be infected than females, possibly due to greater outdoor 15.4.4 Diagnosis
exposure in rural locations where the risk of infection is A diagnosis of WEEV infection is usually based on the sus-
highest [95, 99, 100]. Symptomatic infections are typically pected exposure to rural locations where WEEV transmis-
accompanied by a sudden onset of fever, headache, chills, sion occurs, often accompanied by knowledge of equine
nausea, and vomiting. A minority of adult cases progress, cases, which often precede those in humans. The diagnostic
138 S.C. Weaver and A.M. Powers

approach and methods outlined above for EEEV and VEEV

generally apply equally to WEE diagnosis. Like EEEV but Aedes furcifer/taylori,
A. africanus, etc.

Enzootic cycle
unlike VEEV, WEEV is rarely isolated from blood or CSF at
onset of symptoms because viremia titers are generally low
and the incubation period is long, but can be isolated from
brain samples taken at autopsy or from biopsies [95, 99, Non-human
100]. Antibodies are often detectable at first presentation due primates
to the relatively long incubation period preceding signs and
symptoms.

15.4.5 Prevention and Control

As for EEEV and VEEV described above, there are no
licensed vaccines or antiviral drugs to prevent or treat Humans

Epidemic cycle
WEE, so avoidance of contact with potentially infected
mosquito vectors, especially in rural, agricultural settings,
is critical.
Aedes aegypti,
A. albopictus
15.4.6 Viral Genetics and Evolution
Initial sequencing and phylogenetic studies of WEEV
revealed at least four major lineages [111]. Two of these Fig. 7.9 Enzootic and epidemic transmission cycles of chikungunya
were restricted to South America (Argentina and Brazil, virus
respectively), while the others were widely distributed in
both North and South America, suggesting that the latter are
readily transported between the continents, probably via sion cycle occurs when humans are bitten by infected Ae.
migratory birds. Enzootic WEEV strains from northern aegypti or Ae. albopictus. In this cycle, no other vertebrate
Argentina include an antigenic subtype [96] that is more reservoir is required (Fig. 7.9).
attenuated in certain laboratory animal models than others This is the only cycle that has been detected in Asia to
[112]. More detailed studies of WEEV in California suggest date, where a limited number of field studies have failed to
that introductions from outside the state are not common, identify an independent enzootic cycle. It is possible that
and little mixing of viral lineages occurs north versus south appropriate enzootic vectors are not present in Asia to estab-
of the Tehachapi and San Bernardino Mountains, indicating lish a zoonotic cycle such as that found in Africa.
regionally independent evolution [113]. However, within
southern California, WEEV appears to disperse more freely. 16.1.2 Impact on Human and Animal Health
Finally, all WEEV strains are descendents of a recombinant The first detection of CHIK fever occurred in 1952–1953
alphavirus that derived its nonstructural protein and capsid during a small epidemic in what is now Tanzania [116].
protein genes from an ancestral EEEV and the envelope gly- While there were periodic human infections in population
coprotein genes from an ancestral Sindbis virus strain [111, centers of East Africa, the first major urban outbreaks were
114]. All other members of the WEE complex of alphavi- recorded in Bangkok and India in the 1960s and 1970s [117–
ruses, including Highlands J and Fort Morgan viruses, but 119]. During the next 30 years, there were sporadic emer-
not Aura virus, are also descendents of this ancient recombi- gences of CHIKV resulting in febrile illness, but major
nation event [111]. epidemic activity returned only after the turn of the century.
At this time, reports of human cases began to increase in
frequency in areas with historical activity such as Indonesia,
16 Alphaviruses of Public Health while reports of the virus in new areas such as Central
Importance Outside of the USA African Republic were also occurring [120, 121]. A major
reemergence began in coastal Kenya in 2004 and moved to
16.1 Chikungunya Virus the islands of Comoros and Reunion off the coast of Tanzania
in 2005/2006 [122–124]. Over 1/2 million cases were esti-
16.1.1 Natural History/Transmission mated in these three areas in less than 2 years. The virus then
Chikungunya virus (CHIKV) is transmitted in two distinct spread from Africa to India (over 1 million cases) and
cycles. The enzootic maintenance cycle utilizes forest- throughout Southeast Asia (Fig. 7.10).
dwelling mosquitoes such as Ae. furcifer/taylori and Ae. afri- A viremic traveler also transported the virus from India
canus and nonhuman primates. This cycle has only been to Italy, where autochthonous transmission was detected
identified in Africa [115]. In contrast, the epidemic transmis- for the first time in a temperate region [125]. This dramatic
7 Alphaviruses : Equine Encephalitis and Others 139

Fig. 7.10 Approximate known geographic distribution of CHIKV, 2007–2012 (Map obtained from CDC: http://www.cdc.gov/chikungunya/map/
index.html)

geographic spread in less than 5 years with an estimated two conditions associated with infection including meningoen-
million cases led to serious concern that a global epidemic cephalitis, hepatitis, myocarditis, and uveitis. Additionally,
could ensue. Significantly, during this massive and lengthy mortality was associated with infection for the first time in
epidemic, the severe and chronic nature of CHIKV infec- 2006, and intrapartum transmission was shown to result in
tion was revealed, as was the tremendous economic drain on neonatal complications [127].
affected countries [126].
16.1.4 Diagnosis
16.1.3 Human Disease Because the symptoms associated with CHIKV infection are
Clinical disease begins between 3 and 7 days postinfection so nonspecific (fever, joint pain, rash) and similar to those
with the abrupt onset of a high fever (>101 ºF). The fever caused by other sympatric pathogens (including dengue and
typically lasts several days but usually subsides within 2 malaria), laboratory diagnosis is critical for confirmation of
weeks. Shortly after fever onset, additional signs and symp- infection. Fortuitously, viremia in humans is reasonably high
toms appear which almost always include a severe bilateral (up to ~107 pfu/ml of blood) and persists for up to 1 week,
arthritis of the small joints and may include a maculopapular allowing easy detection of the virus by either isolation or
rash. The rash, if present, lasts only a few days, while the RT-PCR methods [18]. For serum samples collected more
arthralgia may be highly incapacitating and can persist for than 1 week after the onset of illness, serological assays such
weeks or even months. Occasional reports of chronic joint as ELISA or plaque-reduction neutralization tests (PRNT) can
pain (>1 year) have been documented, but these are atypical be used to detect anti-CHIKV-specific antibodies. Typically,
and may be associated with comorbidities. both acute and convalescent samples must be tested, and a
Most CHIKV infections are symptomatic (75–97 %) and fourfold change in titer (for PRNT) or a change from negative
involve only the signs and symptoms noted above. However, to positive (in ELISA) must be detected for definitive diag-
recent outbreaks have revealed occasional, more serious nosis [128]. Commercial CHIKV diagnostic assays are now
140 S.C. Weaver and A.M. Powers

available in limited markets, but their quality and utility are not 16.2 O’nyong-nyong Virus
universally accepted [129, 130]. Because CHIKV has such a
broad geographic range, a complete travel history should also 16.2.1 Natural History/Transmission
be considered with samples submitted for laboratory testing. O’nyong-nyong virus (ONNV) is unique among the alphavi-
ruses in its transmission cycle; this virus is transmitted by
16.1.5 Prevention and Control anopheline mosquitoes (primarily Anopheles gambiae and
Like all mosquito-borne viruses, infection prevention can be An. funestus) during epidemics. Similarly, the few interepi-
accomplished using a limited number of approaches. These demic isolates have also come from anophelines, suggesting
include mosquito control, personal protective measures, or the virus is maintained enzootically by this genus as well
vaccination. In general, mosquito control is extremely chal- [144]. A single isolate of the virus was obtained from
lenging as efforts must be broadly distributed, sustained, and Mansonia uniformis, but this species was not abundant to
coordinated with surveillance efforts. While there is historic determine its role in epidemic transmission [145]. The verte-
evidence to show that CHIKV vector mosquitoes can be con- brate reservoir host for ONNV has not been determined, but
trolled and/or eliminated with intensive efforts, the mosqui- serosurveys suggest a role for domestic livestock and several
toes rapidly return when control measures are discontinued species of rodents.
[131]. Similarly, encouraging the use of personal protection
such as repellents or long-sleeved shirts and pants presents 16.2.2 Impact on Human and Animal Health
challenges because most individuals do not maintain rigor- The history of epidemic ONNV is one of the most intrigu-
ous efforts to prevent mosquito bites. This is particularly dif- ing of all alphaviruses. The virus was identified during the
ficult in the primarily tropical habitats where CHIKV first recorded outbreak that lasted 3 years beginning in 1959.
circulates because repellents may not be widely available The magnitude of the outbreak was staggering, with an esti-
and the wearing of long sleeves is uncommon because of the mated two million cases spanning the duration of the epi-
hot and humid climate. demic across Uganda and other parts of East Africa [146].
The ideal approach to control CHIKV would be by the With significant absenteeism due to the debilitating nature of
use of a vaccine. However, there are no commercially avail- infections, the outbreak had major impact on both the econo-
able options at this time. A live-attenuated vaccine devel- mies and public health resources of the numerous affected
oped by the US Army was evaluated as an investigational countries.
new drug [132], and several newer vaccine candidates have After this first epidemic subsided, there was virtually no
been generated by genetic engineering. These include virus- detection of ONNV for ~40 years with the rare exception
like particles, chimeric viruses, genetically modified attenu- of isolates from mosquitoes or febrile humans. A subtype
ated strains, DNA vaccines, and adenovirus-vectored options of ONNV, designated Igbo-Ora, was identified in West
and others [133–136, 137]. To date, none of these have Africa, expanding its known geographic range [147].
advanced into commercial development. However, no epidemic activity has been reported due to
this strain. The next major epidemic of ONN fever occurred
16.1.6 Viral Genetics and Evolution in Uganda in 1995–1996. Like the earlier outbreak, the
Three distinct genotypes of CHIKV have been identified virus moved rapidly through affected communities,
that, prior to the 2004 outbreak, were primarily aligned geo- impacting up to 70 % of the population [148]. No signifi-
graphically (Asian, East/Central/South African (ECSA), and cant epidemic activity has been reported since 1996,
West African genotypes). Strains within each genotype are although serological evidence of infection in vertebrates
up to ~22 % genetically divergent from the others, while has been documented.
strains within a genotype are much more similar genetically,
with a maximum of ~5 % divergence [138, 139]. 16.2.3 Human Disease
The large number of complete CHIKV genome sequences Like its close relative CHIKV, ONNV causes a febrile
publically available allowed close monitoring of the arthralgic syndrome. However, there are some distinctions
movement of the virus during the 2004–2009 outbreak. All from CHIKV infection such as the more prominent and fre-
strains from the 2004 to 2009 epidemics were the ECSA gen- quent appearance of cervical lymphadenopathy in ONN
otype, thus expanding the distribution of this genotype into cases. Additionally, the rash associated with ONNV infec-
Oceania, India, and Southeast Asia [139]. Studies of these tion is reported to be highly irritating but to cause little dis-
novel isolates also allowed identification of mutations asso- comfort in CHIKV cases [149]. No mortality has ever been
ciated with altered vector infectivity and provided insights reported to be associated with ONNV infection and long-
into the microevolution of the virus [140–143]. term sequelae appear to be rare.
7 Alphaviruses : Equine Encephalitis and Others 141

16.2.4 Diagnosis Among the very few strains of ONNV that are available
As noted for CHIKV, laboratory diagnosis is critical due to for evaluation, the degree of genetic diversity appears to be
the lack of specific symptomology associated with ONNV limited. Isolates obtained from the original outbreak have
infection, which precludes diagnosis based on clinical crite- fewer than 100 amino acid differences from isolates obtained
ria. Diagnosis is primarily accomplished by serological test- during the second Ugandan outbreak over 40 years later
ing, which includes testing for CHIKV infection since the [152]. This indicates that the virus may be maintained in a
two viruses have shared a close antigenic relationship and zoonotic cycle involving efficient virus dispersal, which
cross-reactions can be distinguished in only one direction, would retain a high degree of sequence similarity across a
i.e., CHIKV immune serum inhibits the formation of ONNV broad geographic range.
plaques to the same extent as CHIKV plaques, but the inverse
reaction is weaker [150]. Thus, when performing PRNT
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 Arenaviruses: Lassa Fever, Lujo
Hemorrhagic Fever, Lymphocytic 8
Choriomeningitis, and the South
American Hemorrhagic Fevers

Daniel G. Bausch and James N. Mills

1 Introduction shock [15, 16]. A few arenaviruses have been associated

with aseptic meningitis and other central nervous system
The family Arenaviridae (genus Arenavirus) derives its name (CNS) diseases, including congenital malformations when
from the Latin “arena” for “sand,” referring to the grainy transmitted in utero. Many arenavirus infections are asymp-
appearance of infected host cell ribosomes seen on electron tomatic or result in a nonspecific febrile illness difficult to
microscopy (Fig. 8.1) [1]. Arenaviruses are zoonotic, main- distinguish from many more common diseases.
tained in nature, with a few possible exceptions, by chronic Seven arenaviruses have been clearly established as
infection in rodents of the superfamily Muroidea [2]. The causative agents of HF via natural infection (i.e., exclud-
viruses are grouped serologically, phylogenetically, and geo- ing laboratory infections), Lassa and Lujo in Africa and
graphically into Old World/lymphocytic choriomeningitis Junín, Machupo, Guanarito, Sabiá, and Chapare in South
(i.e., Africa, Europe, Asia, and Oceania) and New World/ America (Table 8.1). Similar HF syndromes are caused by
Tacaribe (i.e., the Americas) complexes. Over 40 arenavi- members of the virus families Bunyaviridae, Filoviridae,
ruses have been identified, although less than half of these and Flaviviridae (see Chaps. 10, 15, and 17). As with other
are clearly recognized as human pathogens (Table 8.1 and HF viruses, arenaviruses are generally named after the geo-
Figs. 8.2 and 8.3). Arenaviruses continue to be discovered, graphic location of the first recognized case or place of
and at a quickening pace in recent decades [3–11]. Factors first virus isolation. For the New World arenaviruses, the
in disease emergence and the increased incidence of human
cases often relate to anthropogenic disturbance of natural hab-
itats, such as conversion of forest to cultivated fields, resulting
in loss of biodiversity and selection for opportunistic rodent
species that are frequently hosts for zoonotic pathogens [12].
Conversion to agriculture also prompts incursion of reservoir
rodents from the surrounding bush seeking food in croplands,
with resultant increased human exposure to rodents [13, 14].
Some reservoir species may also enter towns and homes, fur-
ther increasing risk to humans.
Arenaviruses are perhaps best known as agents of viral
hemorrhagic fever (HF), an acute systemic illness classi-
cally involving fever, a constellation of initially nonspecific
signs and symptoms, and a propensity for bleeding and

D.G. Bausch, MD, MPH&TM (*)

Department of Tropical Medicine, Tulane School
of Public Health and Tropical Medicine, SL-17,
1430 Tulane Avenue, New Orleans, LA 70112-2699, USA
Fig. 8.1 Electron micrograph of Lassa virus. The typical sandy appear-
e-mail: [email protected]
ance of arenaviruses from the internal ribosomes is evident, as well as
J.N. Mills, PhD the surface glycoprotein spikes. Magnification approximately ×55,000
Population Biology, Ecology and Evolution Program, (Micrograph courtesy of F.A. Murphy, University of Texas Medical
Emory University, Atlanta, GA, USA Branch, Galveston, Texas)

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 147

DOI 10.1007/978-1-4899-7448-8_8, © Springer Science+Business Media New York 2014
148 D.G. Bausch and J.N. Mills

Table 8.1 Arenaviruses known or suspected to cause human disease

Annual incidence Human disease-to- Human-to-human
Virus Associated human disease of human disease infection ratio transmissibility Case fatality
Dandenong Fever with encephalopathy 3 cases recognized Unknown All known cases 100 % of three
and multiorgan system failure infected through organ known cases
transplantation
Lassa Lassa fever Poor surveillance. 1:5–10 Moderate 25 %
Estimated
30,000–50,000
Lujo Lujo fever 5 cases recognized Unknown Moderate to high 80 % of five
known cases
Lymphocytic Nonspecific febrile illness/ Poor surveillance. Unknown but Congenital <1 %
choriomeningitis aseptic meningitis Estimated hundreds appears to be low transmission as well as
to thousands transmission through
organ transplantation
Chapare Chapare HF Unknown Unknown Unknown 1 fatality among
a “small cluster”
of cases. Few
details reported
Flexal Nonspecific febrile illness 2 laboratory Unknown Unknown 2 known
infections infections were
nonfatal
Guanarito Venezuelan HF <50 1:1.5 Low 30–40 %
Junín Argentine HF 50–100 1:1.5 Low 15–30 %
Machupo Bolivian HF <50 1:1.5 Low 15–30 %
Pirital Nonspecific febrile illness 1 laboratory infection Unknown Unknown Only known case
was nonfatal
Sabiá Brazilian HF 1 natural and 2 1:1.5 Low? 33 % of three
laboratory infections known cases
known
Tacaribe Febrile illness with mild CNS 1 laboratory infection Unknown Unknown Single nonfatal
symptoms case
Whitewater Arroyo Pathogenic potential unclear. Unknown Unknown Unknown Unknown
Three putative cases with fever
and some signs of hemorrhagic
fever

diseases are often named after the country where they were tendency to cause public panic and social disruption, some
first detected, with the virus named after the town or some HF-causing arenaviruses are considered “Select Agents”
other local geographic feature. For example, Junín virus was that could possibly be used as bioweapons. Attempts to
first isolated from a human in the town of Junín, in central weaponize various arenaviruses were reportedly made by
Argentina, and subsequently designated as the causative the Soviet Union during the Cold War era [18]. Many are-
virus of Argentine HF. Two Old World arenaviruses, lym- naviruses are classified as biosafety level four (BSL-4) or
phocytic choriomeningitis virus (LCMV) and an LCMV “maximum containment” agents [19].
variant named Dandenong virus, are typically associated
not with HF but rather with febrile illness and CNS disease
[17]. Whitewater Arroyo virus has been detected in three
sick persons in California but its role as a pathogen is con- 2 Biological Characteristics
troversial. Tacaribe, Pirital, and Flexal viruses have caused
nonspecific febrile illnesses after laboratory accidents but 2.1 Physical Properties
no natural infections have been recorded. Numerous arena-
viruses are thought to be nonpathogenic in humans, or at Arenavirus virions are pleomorphic, ranging from 60 to
least pathogenicity has yet to be recognized. Pichinde and 300 nM, and have a lipid envelope. On electron micros-
Pirital viruses are frequently used in animal models (guinea copy, host cell ribosomes show a grainy appearance inside
pig and hamster, respectively) of arenaviral HF. the cell, with surface glycoproteins seen as club-shaped
Because of their potentially high lethality, risk of sec- projections or spikes protruding from the envelope
ondary spread (although this is often overestimated), and membrane (Fig. 8.1) [1].
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 149

Mali

Kokodo Burkina Faso

Mus minutoides Guinea Benin
Cote
Sierra Leone Togo Nigeria
d’ Ivoire
Liberia Ghana Central African

Menekre
Gbagroube Hylomyscus species
Mus setulosus Lemniscomys
Lemniscomys rosalia
Tanzania
Ippy
Arvicanthis and Praomys species
Morogoro
Mastomys species
Mobala
Zambia
Mastomys natalensis
Mozambique
Luna
Mastomys natalensis
Meopeia
Praomys species
Lunk
Mus minutoides South Africa
Lujo
unknown
Merino Walk
Myotomys unisulcatus

Fig. 8.2 Geographic distribution of Old World arenaviruses. The virus of associated disease may vary significantly within each country. With
name and known or suspected rodent reservoir are listed. Although the the exception of Lassa and lymphocytic choriomeningitis viruses, most
virus has been isolated from the animal listed, the status of that animal Old World arenaviruses have been isolated on single or very few occa-
as the natural reservoir is not established in all cases. Countries where sions and the precise distribution of the virus beyond the place of first
Lassa fever, the major arenaviral disease in Africa, has been definitively identification is unknown. Not shown on the map: lymphocytic chorio-
shown are depicted in dark gray and the distribution of rodents of the meningitis virus (reservoir Mus musculus), which has a worldwide dis-
genus Mastomys is shown in light gray. Only Lassa, Lujo, lymphocytic tribution, and Dandenong virus (unknown reservoir), which is thought
choriomeningitis, and Dandenong viruses have been definitively asso- to be found in Eastern Europe
ciated with human disease. The distribution of the virus and incidence

2.2 Gene Organization, Replication, binding and cell entry. The L RNA encodes the viral poly-
and Transcription merase (L protein) and a small zinc-binding protein (Z),
which appears to play a regulatory role in virus replica-
Arenaviruses are bisegmented with a ~11 kb genome com- tion, particle formation, and budding, as well as having a
prised of two single-stranded RNA segments denoted small structural function as a matrix protein. The genes on the two
(S) and large (L) of 3.4 and 7.2 kb, respectively [20, 21]. RNA segments are separated by an intergenic region that
The S RNA encodes the viral nucleoprotein (NP) and a folds into a stable secondary structure.
precursor glycoprotein (GPC) that is posttranslationally Arenavirus genes are oriented in both negative and posi-
cleaved into GP1 and GP2 by the cellular subtilase SK1-1/ tive senses on the two RNA segments, a coding strategy
S1P. Proteolytic processing of GPC is necessary for are- called ambisense. Through this mechanism, GPC and NP
navirus infectivity. GP1 is a peripheral membrane protein gene expression are independently regulated. Viral RNA
while GP2 is transmembrane. Both are involved in receptor must be transcribed before GPC can be expressed, which
150 D.G. Bausch and J.N. Mills

Bear Canyon Portuguesa State

Peromycus californicus United States
Tamiami
Sigmodon hispidus
Whitewater Arroyo
Neotoma albigula
Guanario
Catarina Zygodontomys brevicauda
Tonto Creek Mexico Neotoma micropus
Neotoma albigula
Tacaribe
Pirital Artibeus bats (?)
Skinner Tank Sigmodon alstoni Barinas State Venezuela
Neotoma mexicana
Big Brushy Tank Trinidad and Tobago Beni Department
Neotoma albigula Ocozoautla de Espinosa Venezuela
Peromyscus mexicanus Chapare Province
Colombia Amapari
Real de Catorce Pinchindè Neacomys guianae
Neotoma leucodon Oryzomys albigularis

Allpahuayo Peru
Oecomys bicolor and Brasil Pinhal
paricola Calomys tener

Flexal Bolivia
Oryzomys species Bolivia
Cupixi
Oryzomys megacephalus Córdoba Santa Fe
Paraguay Province Province
Machupo
Calomys callosus

Aregentina Sabiá
Chapare Unknown
Unknown
Paraná
Oryzomys angouya

Latino
Calomys callosus Entre Ríos
Oliveros
Province
Necromys benefactus
Junín Buenos Aires
Calomys musculinus Province
Argentina

Fig. 8.3 Geographic distribution of New World arenaviruses. Left incidence of associated disease may vary significantly within each coun-
Panel: the virus name and known or suspected rodent reservoir are try. Many of the New World arenaviruses have been isolated on single or
listed, with viruses associated with natural infection and disease in very few occasions and the precise distribution of the virus beyond the
humans in bold. Although the virus has been isolated from the animal place of first identification is unknown. Right Panel: close-up of admin-
listed, the status of that animal as the natural reservoir is not established istrative regions in which Venezuelan, Bolivian, Chapare, and Argentine
in all cases. Countries with arenaviruses definitively associated with hemorrhagic fevers have been recognized. The distribution of the viruses
human disease are shaded gray. The distribution of the virus and and incidence of disease may vary significantly within the region

may be fundamental to the maintenance of persistent infec- 2.3 Phylogenetics

tion in the animal reservoir. Replication and transcription of
the genome occur in the cytoplasm and require the associa- Both Old and New World complexes are further divided into
tion of viral proteins with the viral RNA in the form of ribo- major lineages or clades (Fig. 8.4). Genetic diversity within
nucleoprotein (RNP) complexes. The NP and L proteins, virus species may vary with the host and geographic region.
together with virus RNA, are the minimal components of the Sequence diversity is generally higher among strains
RNP complex and are sufficient for genome replication and obtained from rodents than from humans; that pattern is
transcription. Purified RNPs are competent for RNA synthe- consistent with chronic infection and frequent intraspecific
sis in vitro and can initiate virus replication after transfection transmission among rodents [22, 23].
into cells. Naked RNA is not infectious. The Old World complex is grouped into two monophyletic
During genome replication, a full-length copy of the lineages that correlate with monophyletic genera within the
genome is synthesized yielding the corresponding antige- rodent family Muridae, subfamily Murinae (see Sect. 3). The
nomic S and L RNAs. Due to the ambisense coding strategy, most attention has been paid to characterizing Lassa viruses,
both genomic and antigenomic RNA serve as templates for which show considerable sequence heterogeneity across West
transcription of viral mRNA. The transcripts contain a cap Africa, with four recognized lineages—3 in Nigeria and 1 in
but are not polyadenylated. The viral RNA species that are the area comprising Sierra Leone, Liberia, Guinea, and Ivory
packaged into virions are defined as the genomic RNAs; Coast [24, 25]. There is also considerable genetic heterogene-
however, smaller amounts of antigenomic RNA and Z gene ity within lineages (up to 26 % on the nucleotide level of the L
mRNA are also packaged. In addition to viral RNA, ribo- and Z genes), especially in Nigeria [26]. Nigerian strains
somal RNA is present within virions. Virions mature by bud- appear to be ancestral to those found further west in Africa,
ding from the plasma membrane. with limited recombination in the evolution of Lassa virus.
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 151

WWAV (NC 010700.1)

TAMV (NC 010701.1)
OLW (NC 010248.1)
BCNV (NC 010256.1)
LATV (NC 010758.1)
Clade D

FLEV (NC 010757.1)

SABV (JN801474.1)
Clade C
PARV (NC 010756.1)

CHPV (NC 004293.1)

PIRV (NC 005894.1)

AMAV (NC 010247.1)

PICV (JN378747.1)
Clade B
Clade A
GTOV (NC 005077.1)

New world

CPXV (NC 010254.1)

LUJV (NC 012776.1)

TCRV (NC 006573.1)

Old world LCMV (NC 004294.1)
JUNV (NC 005081.1)

MACV (NC 005078.1) DANV (EU 136038.1)

LUKV (NC 018710.1)

MOPV (NC 006575.1) IPPV (NC 007905.1)

MORV (NV 013057.1)

LASV (NC 004296.1)
LUNV (NC016152.1)
MOBV (NC 007903.1)
0.1

Fig. 8.4 Phylogenetic relationships of arenaviruses inferred based on Ippy virus, JUNV Junín virus, LASV Lassa virus, LATV Latino virus,
full S segment nucleotide sequences. Phylogenies were reconstructed LCMV lymphocytic choriomeningitis virus, LUJV Lujo virus, LUKV
by neighbor-joining analysis applying the Jukes–Cantor model (1,000 Lunk virus, LUNV Luna virus, MACV Machupo virus, MOBV Mobala
replicates). The A, B, C, and D clades of the New World and Old World virus, MOPV Mopeia virus, MORV Morogoro virus, OLVV Oliveros
arenaviruses are delineated. Lujo virus (LUJV) falls between the two virus, PARV Paraná virus, PICV Pichinde virus, PIRV Pirital virus,
groups but appears to be closest to the Old World viruses, thus corre- SABV Sabiá virus, TCRV Tacaribe, TAMV Tamiami virus, WWAV
sponding to its site of isolation in Zambia. The scale bar indicates sub- Whitewater Arroyo virus. Viruses not shown due to incomplete
stitutions per site. GenBank accession numbers are shown after each sequence data: Allpahuayo, Big Brushy Tank, California Academy of
virus. Similar relationships are demonstrated based on L segment anal- Sciences, Catarina, Collierville, Gbagroube, Golden Gate, Kodoko,
ysis (not shown). Abbreviations: AMAV Ampari virus, BCNV Bear Lemniscomys, Menekre, Merino Walk, Ocozocoautla de Espinosa,
Canyon virus, CHPV Chapare virus, CPXV Cupixi virus, DANV Pinhal, Real de Catorce, Skinner Tank, and Tonto Creek
Dandenong virus, FLEV Flexal virus, GTOV Guanarito virus, IPPV

Field and laboratory data suggest variation in virulence theory are lacking. The virulence factors of the Lassa virus
among the four lineages and strains of Lassa virus. In labora- genome are not known, although for LCMV they are sus-
tory experiments, the virulence of Lassa virus strains in pected to map to the L segment [28].
guinea pigs roughly correlated with the severity of disease in The New World complex is classified into 4 distinct lin-
the humans from whom the viruses were isolated [27]. eages, A, B, C, and D, that generally correlate with mono-
Strains isolated from pregnant women and infants were phyletic genera of the rodent family Cricetidae (see Sect. 3).
benign in guinea pigs, suggesting that host factors such as All of the pathogenic New World viruses are in lineage B.
immunosuppression play a role in human disease. Strains of Viruses of lineage D appear to be the product of recombina-
Lassa virus from Nigeria may be more virulent than those tion between viruses of lineages A and other arenaviruses
from further west in Africa, but data to support or refute this and are thus also known as lineage A/Rec. Lineage A, B, and
152 D.G. Bausch and J.N. Mills

C viruses are restricted to South America, while lineage D is Chronically

exclusively North American. Infected rodent

There is also considerable genetic diversity among strains

of LCMV, Guanarito, and Mopeia viruses, but no clear rela-
Vertical
tionships between strain and pathogenicity in humans have and/or horizontal
been sought or recognized. Sequence diversity may also exist transmission between
for other arenaviruses but the matter has not been extensively rodents
Chronically
studied. Although pathogenic viruses generally cluster phylo-
Infected rodent
genetically, recently identified Lujo virus illustrates the limi-
tations in predicting clinical syndromes based on genetic
sequence alone; Lujo and Lassa viruses cause almost identi- Occasional transmission
cal HF syndromes, despite being genetically distinct (up to to humans
through mucous
38.1 % on the nucleotide level) [29]. Lujo virus is an outlier
membrane exposure,
between New World and Old World arenaviruses but appears skin breaches, ingestion
to be closest to the Old World viruses, corresponding to its and/or aerosol (routes
site of isolation in Zambia. unclear)
Rare sexual
transmission

Uncommon
2.4 Receptors human-to-human
transmission, most
Arenaviruses enter cells by attachment of the GP1 to one or likely through
more cellular receptors [30]; α-dystroglycan, a protein found mucous membrane
exposure
ubiquitously on primate and rodent cells, is a principal recep-
tor for Old World viruses and pathogenic viruses of the New
World clade C, while human transferrin receptor 1 protein is
a receptor for the New World clade B arenaviruses [31].
Orthologs of transferrin receptor 1 protein appear to be the Fig. 8.5 Transmission cycle of arenaviruses illustrating chronic infec-
major receptors in the respective rodent host of each New tion in rodents. Transmission between rodents may be vertical or hori-
zontal depending upon the specific arenavirus. Humans are incidental
World arenavirus and likely dictate species specificity [32].
hosts who play no role in virus maintenance in nature (Adapted with
permission from Enria et. al. [17])

3 Reservoirs and Patterns of Host

Response populations are usually not uniformly infected across their
entire geographic range; the distribution of the virus and dis-
Arenaviruses are generally maintained in nature by chronic ease is usually restricted to a small portion of the host range.
infection in rodents of the superfamily Muroidea (Fig. 8.5) [2]. The reasons for this are unclear but may relate to evolution-
Transmission may be vertical (dam to progeny) or horizontal, ary bottlenecks in dispersal of the virus, rodent reservoir, or
depending on the specific arenavirus. Infected animals may both. Landscape features are the most likely barriers to host
chronically shed virus in urine, feces, and saliva. Old World migration. Humans are dead-end hosts who play no role in
arenaviruses are maintained in rodents of the family Muridae, the natural maintenance of arenaviruses. There are generally
subfamily Murinae, and New World arenaviruses in the family few human cases relative to the frequency of infected rodents.
Cricetidae, subfamilies Sigmodontinae and Neotominae. There The incidence of human infection may vary with changes in
is a tight host–virus species pairing, thought to be the result of rodent abundance that relate to both climatic and seasonal
long-term rodent–virus coevolution [2]. Similar taxonomic weather changes and human-induced habitat alterations.
classifications and host–virus relationships exist for the rodent-
borne hantaviruses, suggesting a similar evolution. Occasional
findings of a given arenavirus in species other than its recog- 3.1 Old World Arenaviruses
nized rodent host are usually considered to result from spillover
infection (i.e., incidental transient infection of a non-reservoir 3.1.1 Lymphocytic Choriomeningitis Virus
host). These incidental and transient animal hosts are not The common “house mouse” (Mus musculus) is the reservoir
thought to play a role in long-term virus maintenance. of LCMV, with infection maintained through vertical transmis-
The endemic area of each arenaviral disease is restricted sion [33]. Infection causes glomerulonephritis in mice and
to the geographic distribution of its rodent reservoir. Rodent shortens their life-span by a few months, especially when
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 153

infected after birth. Renal deposition of virus–antibody com- Despite the occurrence of Mastomys species throughout
plexes is believed to be the underlying cause of the kidney dis- sub-Saharan Africa, for unknown reasons, Lassa virus and
ease [34]. Although house mice and LCMV are phylogenetically Lassa fever have been found only in West Africa (Fig. 8.2).
of Old World origin, the rodent and virus have been dissemi- Confusion over species identified in various regions as well
nated worldwide as a result of rodent passage, usually on ships, as potential differences in the competency of Mastomys sub-
over the last few centuries. Lymphocytic choriomeningitis species could account for this finding. Natal mastomys are
virus may also transiently (but for months) infect laboratory not typically found in large urban centers, so the risk of
mice and pet rodents, especially Syrian hamsters (Mesocricetus rodent transmission of Lassa virus to humans in these envi-
auratus) but also guinea pigs (Cavia porcellus), which can ronments is negligible.
transmit the virus to humans [17, 35–41]. Whether Dandenong Studies of transmission and maintenance of Lassa virus in
virus shares the same reservoir as LCMV is unknown. natal mastomys are few. Consequently, many conclusions
As its name implies, the house mouse is frequently found are based on extrapolations from experiments using LCMV
living in association with humans within homes and other man- in laboratory mice [58]. It is unknown how well this system
made structures in both rural and urban environments. The represents Lassa virus. Available data suggest that Lassa
prevalence of LCMV and LCMV antibody in house mouse virus is maintained in natal mastomys via vertical transmis-
populations is highly variable (0–60 %) and focal [42]; in inner- sion. In addition, the age of the animal, host genotype, and
city Baltimore, antibody-positive mice were clustered within route of inoculation affect the outcome of infection [59]; in
residential blocks [43]; the prevalence of infected mice on four the only published laboratory investigation of Lassa virus
farms in California only several miles apart was 0–27 % [44]. transmission in Mastomys species (reported to be natal mas-
tomys), Lassa virus persistence was achieved when animals
3.1.2 Lassa Virus were infected as neonates, while most animals inoculated as
Lassa virus is maintained in nature in the natal mastomys adults cleared the virus [60]. Infected neonates did not pro-
(Mastomys natalensis), also called the multimammate rat [45, duce Lassa virus antibody. Importantly, some laboratory
46]. Presumed spillover infection has been reported in other strains of rodents identified as natal mastomys were later
rodent species, including the reddish-white mastomys determined to be southern African mastomys (M. coucha),
(M. erythroleucus), the roof rat (Rattus rattus), and the south- although it is unclear which species was used for the afore-
ern African pygmy mouse (Mus minutoides), although the ani- mentioned study [61].
mal species could not be definitively confirmed in all cases In wild-caught Mastomys, Lassa virus antibody and anti-
[47–50]. (L. Moses, manuscript in preparation). Reddish-white gen are usually mutually exclusive [56]. Antibody-positive
mastomys were reported to also be a natural host of Lassa virus animals usually outnumber antigen-positive rodents, with a
but recent findings refute this. One explanation is the historical J-shaped curve of antibody prevalence (high at birth, decreas-
difficulty in identifying Mastomys to species; at least five mor- ing in early adolescence, and then gradually rising as ani-
phometrically identical species exist in sub-Saharan Africa mals age). This pattern is consistent with transmission of
that, until recently, could only be distinguished through karyo- maternal antibody to offspring, which is then lost as animals
typing, a technically cumbersome and perhaps error-prone wean, to be regained by animals exposed to Lassa virus as
technique. A reliable PCR-based assay to genotypically iden- they age. Although no pathology related to Lassa virus infec-
tify Mastomys to species has been recently developed and tion was observed in Mastomys infected in the laboratory,
become the standard [51]. Studies using this technique have Lassa virus-infected wild natal mastomys were smaller in
shown Lassa virus only in natal mastomys [50, 51]. size and weight and had higher frequencies of myocardial
Natal mastomys are almost always found in close asso- and perivascular inflammatory lesions compared to their
ciation with humans in rural villages and surrounding cul- uninfected counterparts [62].
tivated fields and, less commonly, in grasslands and at the
forest edge [52–54]. In highly endemic regions over 50 %
of rodents caught in houses are natal mastomys, with the 3.2 New World Arenaviruses
prevalence of Lassa virus infection ranging from 6 to 50 %
[48, 50, 51, 54–56]. Because the incidence of Lassa fever 3.2.1 Junín Virus
is similar in men and women, and because children are fre- Junín virus is maintained in the drylands vesper mouse
quently infected, peridomestic rodent exposure is thought to (Calomys musculinus), also called the corn mouse.
be most important [57]. Abundance of natal mastomys may Occasional findings of antibody in (or even virus isolation
be higher in houses than in surrounding agricultural fields or from) species sharing the same habitat, including Calomys
bush [48]. Open food storage and closed shutters during the laucha and Akodon azarae, are likely due to virus spillover.
day (a practice that favors prolonged rodent activity) may Although drylands vesper mice are common and widely
favor natal mastomys colonization of houses [48]. distributed in central and northwestern Argentina [63],
154 D.G. Bausch and J.N. Mills

Junín virus is restricted to a small fraction of the host range been associated with devastating outbreaks of Bolivian HF
(Fig. 8.3). The mice prefer more stable linear border habi- [74, 75]. This same commensal characteristic contributes to
tats such as roadsides, fence lines, and railroad rights of way relatively quick control of outbreaks by intensive trapping
but may move into more mature and post-harvest crop fields within and around homes in outbreak areas [73]. Both rodent
in summer and early autumn to feed on abundant corn and abundance and prevalence of infection are highly variable
soybeans [64, 65]. Unlike the natal mastomys, the drylands over space and time, with the prevalence of Machupo virus
vesper mouse lacks peridomestic affinities and very rarely infection in reservoir populations likely linked to population
enters human habitations or peri-urban areas. Population density. The prevalence of infection in rodents was 35 % in
densities of drylands vesper mice vary spatially and season- sites where human disease was present but much lower in
ally and appear to be dependent upon environmental condi- disease-free areas [76]. No long-term studies of host or host–
tions; the combination of a relatively mild winter followed virus dynamics have been conducted. Therefore, the specific
by a cool, moist summer was followed by extremely high factors influencing population dynamics and Machupo virus
mouse abundance and a subsequent 14-year peak in cases transmission within host populations are unclear but likely
of Argentine HF in 1990 [66]. The prevalence of Junín virus include environmental factors such anthropogenic distur-
infection in reservoir populations is also highly variable tem- bance of natural ecosystems, which favors opportunistic
porally (0–50 % monthly prevalence over 3 years of surveil- species [12]; flooding, which concentrates host and human
lance) and spatially (0–8 % average long-term prevalence populations together on high ground [77]; and rainfall, which
among sites within 30 km of each other) [67]. influences habitat quality and food availability.
Field studies have shown that male drylands vesper mice In the laboratory, infection with Machupo virus in big lau-
are more frequently infected with Junín virus than females, cha follows a similar pattern as Junín virus in drylands vesper
that the prevalence of infection increases with age, and that mice; females inoculated at birth were chronically infected
infection is positively associated with the presence of scars and sterile, indicating that vertical transmission would have
[67]. These characteristics suggest that transmission between a highly detrimental effect on the population. When infected
rodents is largely horizontal, between adult males, and may as adults, again there was a split response, with half of the
involve aggressive encounters and biting. In the laboratory, animals clearing the infection and half developing chronic
vertical infection resulted in highly deleterious effects on fit- infection and shedding virus in urine, feces, and saliva. It
ness [68]. Mice that were experimentally infected as adults has been proposed that this split response is genetically con-
showed a “split response”; half quickly developed antibod- trolled [78]. The mechanism of transmission among adult
ies and cleared the virus. The other half became chronically hosts in nature has not been studied, but presumably involves
infected and persistently shed virus in urine and saliva [69], many of the same modalities described above for Junín virus
a characteristic that would facilitate horizontal transmission and drylands vesper mice [78]. The split response caused by
via aggressive encounters with biting, allogrooming, vene- Machupo and Junín virus infections in their respective res-
real contact, contact with infected nest materials, and per- ervoirs has led to speculation that seasonal fluctuations in
haps inhalation of infectious aerosols. the incidence of Bolivian and Argentine HFs may be attrib-
uted to decreased populations of rodents caused by lowered
3.2.2 Machupo Virus fecundity in subsequent generations following chronic infec-
The host for Machupo virus has been described as the big lau- tion with these viruses.
cha (Calomys callosus), also called the large vesper mouse.
Recent studies have shown, however, that C. callosus sensu 3.2.3 Guanarito Virus
lato (s. l.) is a clade of related but independently evolved taxa Guanarito virus is strictly hosted by the short-tailed zygo-
[70]. The “Beni Department clade” is one of those taxa and dont mouse (Zygodontomys brevicauda), also referred to as
would be the host of Machupo virus [70, 71]. This new phylo- the cane mouse, on the plains of northwestern Venezuela.
genetic work helps to explain why Machupo virus and Bolivian A second Sigmodontine rodent species, Alston’s cotton rat
HF are restricted to a small area of Beni Department, north- (Sigmodon alstoni), shares the same habitat with short-tailed
eastern Bolivia, where C. callosus of the Beni Department zygodont mouse and hosts a second arenavirus (Pirital virus),
clade are found, while C. callosus s. l. occurs in a much wider yet spillover from one host to the other is rare [79, 80], illus-
area of Bolivia, Brazil, Paraguay, and Argentina [72]. trating the high specificity of rodent host–arenavirus rela-
The big laucha is an opportunistic species that frequents tionships. It is not known whether this lack of cross-infection
disturbed habitats where forest and grasslands meet. They is due to a lack of physical interactions between the two spe-
often seek higher ground during seasonal inundations, thus cies, despite their sharing the same habitat, or the poor abil-
increasing contact with humans [73]. Unlike its congener ity of each virus to infect the other host. Within the endemic
C. musculinus, C. callosus will readily enter human habi- area for Venezuelan HF, both rodent species are commonly
tations, sometimes in large numbers, and these events have captured in crop fields, borders, and roadside habitats but
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 155

rarely in the peridomestic environment. Although the short- nature on relatively few occasions, often as a serendipitous
tailed zygodont mouse is a savanna species ranging from result of field studies targeting other diseases. The animal
southeastern Costa Rica through most of Brazil north of the from which the virus was isolated is assumed to be the natu-
Amazon [81], the distribution of Guanarito virus (as deter- ral reservoir, but systematic confirmatory field or laboratory
mined by the occurrence of human cases) is restricted to a studies have generally not been conducted. A few arenavi-
9,000-km2 area in Portuguesa and Barinas States in north- ruses have been found only in animals other than rodents
western Venezuela [14]. Rodent community analysis [82] but the role of these animals as true natural hosts remains
suggests that the development of intensive mechanized agri- to be confirmed; Tacaribe virus has been isolated only once,
culture is associated with a relatively depauperate rodent from Artibeus species bats captured on the island of Trinidad
assemblage dominated by the two arenavirus host species, [84]. However, a definitive role of these bats as the natural
the short-tailed zygodont mouse and Alston’s cotton rat. reservoir of Tacaribe virus is far from certain and placed in
Laboratory studies [83] as well as detailed studies of tis- further doubt by failure to produce stable, nonlethal infection
sue and fluid samples from wild short-tailed zygodont mice after laboratory inoculation of these bats with Tacaribe virus
captured in the disease-endemic area [80] have provided a [85]. Three highly genetically divergent arenaviruses were
basic understanding of Guanarito virus infection dynamics recently discovered and putatively linked to inclusion body
in the host population. As with Junín and Machupo viruses, disease, a common infectious disease of captive snakes [86].
laboratory infection of newborn and juvenile rodent hosts
resulted in chronic infection and persistent viral shedding in
urine and saliva. There was no apparent detriment of infec- 4 Rodent-to-Human Transmission
tion to these animals after 3 weeks. Inoculation of adults
again resulted in a split response, with some animals quickly Transmission of arenaviruses to humans is believed to occur
clearing the virus and others developing a chronic infection, via exposure to rodent excreta, either from direct inoculation
shedding virus in urine, saliva, and respiratory secretions. to the mucous membranes or broken skin or from inhala-
However, infection in adult females was associated with tion of aerosols produced when rodents urinate [2, 45, 48,
significantly reduced reproductive success [83]. In field- 87]. The relative frequency of these modes of transmission
collected mice, infection was positively associated with host is unknown. Although the infectious dose for arenaviruses
age, suggesting horizontal transmission. However, virus was is thought to be low, transmission from rodents to humans
restricted to lung tissue (not spleen or kidney), suggesting appears to be inefficient, occurring infrequently even where
infection via the respiratory tract rather than wounding or infected rodents are common [56]. Transmission through
venereal routes as suggested for Junín and Machupo viruses. aerosolized rodent urine or virus-contaminated dust particles
Also unlike the Junín virus–drylands vesper mouse system, is often mentioned in the scientific literature, but there are
both sexes were equally infected, again arguing against few data to support or refute its occurrence [88]. Although
infection by fighting among males [80]. Studies involving household clusters of arenaviral HF cases occasionally
temporal patterns of host population dynamics and virus occur, single cases are much more common. This suggests
transmission and the influence of environmental factors on that aerosol transmission to humans is not common, since it
these processes are needed. would logically often simultaneously infect multiple people
in proximity to the aerosol source. Secondary aerosol gen-
3.2.4 Whitewater Arroyo Virus eration, such as what might be produced through sweeping
Numerous viruses antigenically and phylogenetically related an area contaminated by rodent urine, is inefficient and is
to Whitewater Arroyo virus are widely distributed through- thus a less likely mechanism of infection. However, infec-
out the southwestern United States in association with sev- tious and moderately stable aerosols of Lassa, LCMV, and
eral species of the genus Neotoma, including the southern Junín viruses have been artificially produced in the labora-
plains wood rat (N. micropus), Mexican wood rat (N. mexi- tory, so the possibility of primary aerosol transmission can-
cana), Stephen’s wood rat (N. stephensi), and bushy-tailed not be discarded [59, 89]. Regardless of their role in natural
wood rat (N. cinerea). infection, the artificial production of infectious aerosols has
obvious implications for the potential use of arenaviruses as
bioweapons [19, 90].
3.3 Other Arenaviruses Experimental data illustrate that arenavirus infection may
also occur by the oral route, perhaps through a gastric portal
The reservoirs of Sabiá, Chapare, Lujo, and Dandenong [91, 92]. Lassa virus has been contracted when rodents are
viruses are unknown but are presumed to be muroid rodents. trapped and prepared for consumption, a common practice
Most of the other arenaviruses from both the Old and New in some parts of West Africa, although it is rarely possible to
World complexes (Figs. 8.2 and 8.3) have been found in determine whether infection resulted from exposure during
156 D.G. Bausch and J.N. Mills

preparation or consumption [93]. Since arenaviruses are eas- where cases are more likely to be noted, laboratory confirmed,
ily inactivated by heating, eating cooked rodent meat should and reported) [42]. Older reviews of patients with CNS dis-
pose no danger [2]. Various arenaviruses have been found in ease showed LCMV infection in 8–11 % [42]. Infection with
rodent saliva, although there are no reports of human infec- LCMV was the confirmed diagnosis in about 10 % of patients
tion from bites [60]. with febrile neurological syndromes admitted to a tertiary care
hospital in Washington, DC, from 1941 to 1958, but the diag-
nosis was much less common recently [99, 100]. An investi-
5 Transmission and Disease in Humans gation of cases of encephalitis in California (91 cases from
1998 to 2000) and England (2,574 cases from 1989 to 1998)
With the exception of the LCMV reservoir, all arenavirus revealed LCMV infection in zero and less than 1 %, respec-
reservoirs occupy almost exclusively sylvatic rural habitats. tively [101, 102]. Antibody surveys showing much lower
Consequently, primary arenavirus infection in humans is prevalence in younger than in older patients support the notion
seen almost exclusively in rural and often remote settings. of declining incidence, although this could also be explained
Secondary human-to-human transmission may occur with by other factors that result in varied risk between age groups
some arenaviruses, most often to those caring for sick per- [97]. The incidence of LCMV infection peaks in the fall and
sons either at home or in healthcare centers. The combina- winter in the northern temperate zones, presumably reflecting
tion of the remote and geographically restricted endemic seasonal invasion of homes by house mice. The risk of infec-
areas (often in resource-poor countries), the apparent rarity tion is higher among rural dwellers and, in the United States,
of most arenaviral infections in humans, and the challenges in low-income groups and, as expected, varies with the degree
to both clinical and laboratory diagnosis (see Sect. 8) make of rodent infestation in a home [36, 97, 103].
surveillance for arenavirus infection difficult. Consequently, In 2003 and 2005, small clusters of LCMV infection and
reliable estimates of incidence are rare, except for Argentine highly fatal (seven of eight cases died) neurological disease
HF, for which intensive surveillance with supporting labora- with organ failure occurred in solid-organ transplant recipi-
tory diagnosis is applied in its circumscribed endemic area. ents in the United States [104]. A third transplant-related
cluster of three fatal cases occurred in Australia in 2009,
leading to the discovery of the LCMV variant Dandenong
5.1 Old World Arenavirus Diseases virus [105]. The donor died of cerebral hemorrhage 10 days
after returning to Australia from a 3-month visit to the former
5.1.1 Lymphocytic Choriomeningitis and Other Yugoslavia, where he had traveled in rural areas. None of the
Central Nervous System Disease (LCMV donors in these outbreaks had a recent history of acute febrile
and Dandenong Virus Infections) disease and no virus could be identified in them, although
Isolated during an investigation of encephalitis in St. Louis, the presence of IgG and IgM antibodies in the Australian
United States, in 1933, LCMV was the first recognized arena- donor confirmed recent infection. One donor in the United
virus as well as the first recognized cause of aseptic meningi- States had been exposed to a pet hamster from which LCMV
tis in humans [94]. Ironically, despite being the prototype was isolated and corresponded to the virus detected in the
arenavirus, LCMV is one of the only pathogenic arenaviruses transplant recipients. With the exception of congenital trans-
not associated with viral HF. Nevertheless, the virus has been mission and through transplanted organs, there is no human-
extensively employed in mouse models to study arenavirus to-human transmission of LCMV.
immunology. Given the great differences in associated clini-
cal syndromes and pathogenesis, the applicability of these 5.1.2 Lassa Fever (Lassa Virus Infection)
studies to other arenavirus infections is unknown [58]. Lassa fever is named after a village in northeastern Nigeria
Most human LCMV infections are thought to occur in where the first case was recognized in 1969 [106]. However,
homes, corresponding with the propensity of house mice to hospital records and anecdotal reports describe cases con-
inhabit human dwellings [42]. Although cases are sporadi- sistent with Lassa fever in Nigeria as early as 1952 [107].
cally reported worldwide, accurate estimates of incidence The disease is endemic exclusively in West Africa (Fig. 8.4).
are impeded by the usual presentation as a nonspecific After first recognition in Nigeria, Lassa fever was noted in
febrile illness for which [95] diagnostic testing is not com- Sierra Leone, Liberia, and Guinea in the wake of hospi-
monly requested or available. Most antibody surveys show a tal outbreaks and field studies in the early 1970s [57, 106,
prevalence in humans of 5–10 % [36, 95–98]. The incidence 108, 109]. Subsequent antibody prevalence studies and case
of disease appears to have declined in recent decades, per- reports indicate that Lassa virus is present in other West
haps as housing construction and sanitation have improved, Africa countries [110, 111]. However, the risk of exposure
decreasing human exposure to house mice (although there to Lassa virus varies significantly across a given country and
is perhaps a bias toward data from more developed settings often between regions or even villages within endemic areas.
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 157

The highest incidence of disease appears to be in areas of Consumption of rodents and poor-quality housing, which
eastern Sierra Leone, northern Liberia, southeastern Guinea, may reflect ease of rodent access to the home, are risk factors
and central and southern Nigeria (Fig. 8.4) [48, 57, 112, for Lassa fever [93, 121]. In Sierra Leone, the incidence is
113]. Reasons for the extreme heterogeneity in incidence consistently high in diamond mining areas, presumably due
are not clear, especially considering that natal mastomys are to increased contact with rodent excreta in the soil, although
often common in areas where little or no Lassa fever has poor hygiene in mining camps and consumption of rodents
been recognized. Varied intensity of surveillance may con- may contribute. Large nosocomial outbreaks of Lassa fever
tribute to the apparent heterogeneous distribution, but cannot have occasionally occurred in Africa following lapses in
completely explain it. proper infection control, including reuse of non-sterilized
Lassa fever is an extremely common infection among needles and use of contaminated multiuse antibiotic vials,
adults and children of both sexes in endemic areas of West but secondary transmission associated with imported cases
Africa. An annual incidence of 300,000–500,000 Lassa virus from returning travelers is extremely rare [122].
infections with up to 5,000 deaths is often quoted in the sci-
entific literature, but these figures are extrapolations from 5.1.3 Lujo Fever (Lujo Virus Infection)
surveillance data collected in the 1970s and 1980s in eastern The name “Lujo” reflects the two geographic areas involved
Sierra Leone where Lassa fever is clearly hyperendemic [48]. in the only outbreak recognized to date—Lusaka, Zambia,
Estimating the true incidence and mortality is extremely dif- and Johannesburg, South Africa. The first recognized case
ficult due to the nonspecific clinical presentation [57, 114], was infected near Lusaka and subsequently medically evacu-
logistical impediments presented by civil unrest [115], unsta- ated to Johannesburg, initiating a chain of four nosocomial
ble governments with underdeveloped surveillance systems infections (a medical evacuation attendant, two nurses, and a
[116], extensive human migration and perturbation of the cleaner) in South Africa in 2008 (Fig. 8.4). Four of the five
physical landscape, and lack of reagents and laboratories for (80 %) cases were fatal. The virus and disease have not been
diagnostic confirmation in West Africa [117]. seen since and no antibody or ecologic surveys have been
The incidence of Lassa fever is consistently highest during conducted to yield a greater understanding of the epidemiol-
the dry season [57, 118]. The reasons for this are not clear but ogy or epizoology of Lujo virus.
may relate to greater stability of Lassa virus at lower humid-
ity [87] and seasonal fluctuation in host abundance and the
prevalence of Lassa virus infection [54]. Natal mastomys may 5.2 New World Arenavirus Disease
enter human habitations more frequently during the dry sea-
son, when food in agricultural fields is scarce, but stored rice 5.2.1 Argentine Hemorrhagic Fever (Junín
and other staples in homes are abundant [54]. In a hospital Virus Infection)
in Guinea, the seasonal fluctuation in the incidence of Lassa In 1959, the first of the HF-causing arenaviruses, Junín
fever mirrored the general pattern of other febrile diseases (named after a village about 240 km west of Buenos Aires),
and hospital admissions, suggesting that nonbiological factors was described after a new disease, subsequently named
may be involved [57]. These may include cultural, economic, Argentine HF, emerged in the central Argentine pampas, an
and other logistical impediments, such as poor road condi- area of intensive agriculture. Annual outbreaks of up to 1,500
tions, the need to attend to crops, and lack of funds prior to cases continued in subsequent years. The endemic zone ini-
the seasonal harvest that often limit inhabitants’ ability to seek tially appeared confined to an area of approximately
medical care during the rainy season. 16,000 km2 but has steadily expanded to about 150,000 km2
Mild or asymptomatic Lassa virus infection appears to in parts of northern Buenos Aires, southern Santa Fe and
be frequent. In a hyperendemic area of Sierra Leone in the Entre Rios, and southeastern Córdoba Provinces as agricul-
1980s, less than 20 % of persons who recently developed ture expands (Fig. 8.5) [123]. The present endemic area is
antibody reported recent febrile illness [48]. However, since home to an estimated three million people. Transmission is
antibody reversion (loss of antibody after infection) was also typically high in the first few years of expansion into a new
frequent, some of the seroconversions and asymptomatic area and then declines.
infections may have represented re-exposure in persons with The endemic area is largely a mosaic of crop fields and
preexisting immunity from past Lassa virus exposure. The some cattle ranches crisscrossed by linear islands of rela-
possibility of cross-reacting antibodies from previous infec- tively stable border habitats that are the preferred habitat of
tion with other nonpathogenic arenaviruses in West Africa, drylands vesper mice. As expected, the annual incidence is
such as Kodoko, Gbagroube, or Menekre viruses, cannot positively correlated with local population densities of this
be excluded (Fig. 8.4). Studies of the rate of asymptomatic rodent. Young adult males are generally at highest risk due
transmission bear repeating with newer more sensitive and to their traditional involvement and exposure in agricultural
specific diagnostic modalities [119, 120]. activities [66]. Although cases occur throughout the year, the
158 D.G. Bausch and J.N. Mills

incidence is usually highest from March to June, correspond- incidence in male agricultural workers [14]. Recent surveys
ing to periods of peak agricultural activity. In the early years have demonstrated the presence of Guanarito virus geno-
the men affected worked in close contact with the land and types (some closely matching genotypes from case patients
harvested crops by hand, activities that presumably carried in the disease-endemic area) in host populations over a wide
a high risk of exposure to rodents and their excreta. Later, area of the Venezuelan plains (western llanos), including
mechanized agricultural practices became common, but areas in five states up to several hundred kilometers from the
cases continued to occur in field-workers, especially tractor recognized disease-endemic area [22].
drivers, probably through inhalation of infectious aerosols
generated during the mechanized harvesting process. 5.2.4 Brazilian Hemorrhagic Fever
An effective vaccine (see Sect. 10.6) was introduced in (Sabiá Virus Infection)
1991, decreasing the incidence of Argentine HF from 837 In 1990, a fatal case of HF was reported from outside of Sao
cases that year to an average of 122 cases yearly from 1992 Paulo, Brazil [127]. The victim was an agricultural engineer,
to 2009 [124]. The incidence has been steadily declining, although she worked primarily in the office. Although origi-
with less than 100 cases in most years since 2000, despite nally thought to be a case of yellow fever, a new arenavirus
increasing intensity of surveillance and quality of laboratory was subsequently isolated and named Sabiá after the commu-
diagnosis. As young male agricultural workers in the high- nity where the engineer was staying when she fell ill, although
risk provinces are increasingly protected by vaccination, the the name Brazilian HF has not been formally assigned. Only
proportion of cases fitting this demographic is decreasing. two cases of Sabiá infection, both nonfatal and due to labora-
tory accidents, have occurred since: one in Brazil and one in
5.2.2 Bolivian Hemorrhagic Fever the United States. The case in the United States resulted from
(Machupo Virus Infection) a centrifuge accident, illustrating the potential for aerosol
An outbreak of what was subsequently named Bolivian HF transmission of arenaviruses in the laboratory. Despite the
occurred in Bolivia’s El Beni Department in 1959. The etio- infected individual not reporting the incident for 12 days after
logic agent, Machupo virus, named after a river in the region the accident, no secondary transmission occurred.
of the outbreak, was described in 1965. Community outbreaks
of Bolivian HF continued during the 1960s but were eventu- 5.2.5 Chapare Hemorrhagic Fever
ally brought under control, in part by rodent trapping. There (Chapare Virus Infection)
were then no reported cases for decades, followed by sporadic The latest HF-causing New World arenavirus to be recog-
cases and small outbreaks reported again in the 1990s, which nized is Chapare virus, discovered in 2003 after a small out-
continue through the time of this writing in 2012. break in Chapare Province in Cochabamba Department,
Although Bolivian HF may be seen throughout the year, Bolivia. Few details have been reported and no cases have
like Argentine HF, the incidence is usually highest during the been reported since then, although surveillance is limited.
dry season (June–August) at the peak of agricultural activity.
Young men engaged in agricultural activities in rural areas 5.2.6 Whitewater Arroyo Virus Infection
are at highest risk. However, family and community clusters In 2000, in California, Whitewater Arroyo virus was detected
of Bolivian HF cases affecting both sexes and all age groups via RT-PCR in three fatal cases with symptoms consistent
have also occurred in towns and hospitals, either as a result with viral HF, with infectious virus isolated from one patient.
of epizootic conditions fostering high rodent population den- However, not all laboratory results could be confirmed, leav-
sities with invasion of towns and human dwellings [13] or ing doubt about the pathogenicity of this virus. No further
through person-to-person transmission [125]. cases have been recognized. Nevertheless, findings from
recent antibody surveys, including persons with significant
5.2.3 Venezuelan Hemorrhagic Fever increases in antibody titer associated with febrile illness,
(Guanarito Virus Infection) suggest a pathogenic role for Whitewater Arroyo or a similar
In 1989, an outbreak of viral HF occurred in Guanarito arenavirus in the southwestern United States [128].
District in the southern tip of Portuguesa State, Venezuela.
Although originally thought to be dengue HF, in 1991 the
disease was attributed to a new arenavirus named Guanarito, 5.3 Human-to-Human Transmission
the causative agent of Venezuelan HF [126]. Outbreaks of
Venezuelan HF have been reported every 4–5 years since its Most arenaviruses can be transmitted between humans
discovery, suggesting some cyclic climatic or social influ- through direct contact with infected blood or bodily fluids
ence. Similar to Argentine and Bolivian HFs, epidemics although, contrary to popular concept, secondary attack rates
appear to have a seasonal peak in November to January, are generally low, probably on the order of 5 % or less as
coinciding with maximum agricultural activity, with highest long as strict barrier nursing practices are observed. Tertiary
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 159

transmission is unusual. Large outbreaks are almost always Lassa virus back to their countries of origin, most frequently
fueled by nosocomial transmission, usually in resource-poor the United Kingdom and Europe, but occasionally as far away
regions where barrier nursing practices may not be main- as Japan [139]. Despite Lassa virus being one of the most
tained [109, 118, 122, 129]. communicable arenaviruses, in over 25 imported cases of
Viremia and infectivity of persons infected with arenavi- Lassa fever reported since 1969 with at least 1,500 cumula-
ruses generally parallels the clinical state, with highest infec- tive identified contacts, only a single putative and asymptom-
tivity late in the course of severe disease, especially when atic instance of secondary transmission has been noted [140].
bleeding is present. Data on the precise modes of human-to- Nevertheless, the risk is highlighted by the three secondary
human transmission are lacking, but infection presumably cases and one tertiary case following an index case of Lujo
results from oral or mucous membrane exposure, most often fever imported to South Africa; this transmission occurred in
in the context of providing care to a sick family member settings in which proper infection control practices may not
(community) or patient (nosocomial transmission). Although have been maintained.
aerosol spread of Lassa virus was speculated in the first rec-
ognized outbreaks in Nigeria, extensive field experience since
then has not suggested aerosol transmission of arenaviruses 6 Clinical Presentation
between humans in natural settings [118]. African funeral
rituals that entail the touching of the corpse prior to burial 6.1 Central Nervous System Disease
may also result in transmission of HF viruses, although this
has not been specifically recognized with arenaviruses [130]. Despite the name, a minority of patients infected with LCMV
Arenavirus clearance from immunologically protected sites manifest meningitis or CNS disease. Rather, most LCMV
such as the kidney and gonads may be delayed for weeks to infections are asymptomatic or result in a nonspecific febrile
months, potentially resulting in transmission during convales- illness [141].
cence [131–134]. Excretion may be intermittent [135]. Lassa The incubation period is about 1–2 weeks (typically
virus has been isolated from human urine up to 67 days after 8–13 days). After 7–14 days of nonspecific illness (fever,
onset of illness. Nevertheless, only a few incidences of trans- headache, malaise), and often with a brief period of efferves-
mission during convalescence have been reported, all consis- cence, CNS manifestations may ensue in a minority of
tent with sexual transmission of Lassa, Machupo, and Junín patients, running a spectrum from aseptic meningitis (the most
viruses months after recovery from acute disease [48, 136]. common) with headache, stiff neck, and photophobia to fulmi-
The risk of transmission during the incubation period or from nant encephalitis with cranial nerve palsies, abnormal reflexes,
asymptomatic persons is negligible. focal seizures, polyneuritis, flaccid paralysis, and papilledema.
New World arenaviruses appear to be less transmissible CNS symptoms can also occur without recognized febrile ill-
between humans than their Old World counterparts, although ness. Rarer manifestations and complications of LCMV infec-
human-to-human transmission of Machupo virus has been tion include hydrocephalus, transverse myelitis, Guillain–Barré
reported in both community and nosocomial settings [137, syndrome, hearing loss, arthritis, parotitis, orchitis, myocardi-
138]. Only one family cluster of Argentine HF is described, tis, mucosal bleeding, and pneumonia. Congenital infection
with the index case presenting with atypical skin lesions that has been associated with spontaneous abortion in early preg-
may have facilitated transmission. Person-to-person trans- nancy and, when occurring later in pregnancy, a variety of
mission or nosocomial infection has not been observed with neurological deficits, including psychomotor retardation,
Guanarito virus despite the fact that patients with Venezuelan microcephaly and macrocephaly, hydrocephalus, chorioretini-
HF are usually admitted to open wards with minimal isolation tis with visual loss, and seizures [17]. LCMV and Dandenong
precautions. It is unknown whether the perceived differences virus infection in immunosuppressed organ transplant recipi-
in transmissibility between the arenaviruses reflect true bio- ents has resulted in a highly fatal syndrome with graft dys-
logical properties or varied cultural and infection control function, CNS symptoms, and multiorgan system involvement,
practices in the endemic areas of each virus. in some cases resembling viral HF, within 3 weeks after trans-
Contrary to popular concept, the risk of imported arenavi- plantation, with case fatality approaching 90 % [104].
rus infections initiating outbreaks in industrialized countries,
where barrier nursing techniques are usually maintained, is
generally low. Because of the considerable travel between the 6.2 Arenaviral Hemorrhagic Fever
United Kingdom and their former West African colonies,
which also often happen to be the hyperendemic areas, Lassa Although there are differences in the pathogenesis and clini-
fever is easily the most exported arenavirus infection. Foreign cal manifestations produced by the various arenaviruses, they
military personnel, peacekeepers, aid workers, and tourist in are usually too subtle to allow for distinction on clinical
rural settings are occasionally infected, sometimes importing grounds, at least in the early stages of disease. Nevertheless,
160 D.G. Bausch and J.N. Mills

some notable distinctions can be made between the syn- the South American HFs and include disorientation, tremor,
dromes caused by Old World and New World viruses. The ataxia, seizures, and coma, particularly in the late stages,
disease caused by the various New World arenaviruses is usu- and usually portend a fatal outcome [142]. The cellular and
ally referred to simply as “South American HF.” Asymptomatic chemistry profile in CSF is often normal. Pregnant women
transmission and milder nonspecific febrile illness appear to with arenaviral HF often present with spontaneous abortion
be relatively frequent in Old World arenavirus infections, at and vaginal bleeding, with maternal and fetal mortality rates
least for LCMV and Lassa virus, while infection with the approaching 100 % in the third trimester [143]. Anasarca
pathogenic New World arenaviruses much more often results has been described in a single report of children with Lassa
in disease that can ultimately be recognized as HF. fever (termed the “swollen baby syndrome”) but may have
Arenaviral HF may be seen in both sexes and all age been related to aggressive rehydration [144]. Various clini-
groups, with a spectrum ranging from mild disease to shock, cal manifestations of arenaviral HF are illustrated in Fig. 8.6.
multiorgan system failure, and death. Most patients present Typical clinical laboratory findings in arenaviral HF include
with nonspecific signs and symptoms difficult to distinguish early leukopenia and lymphocytopenia, sometimes with atypi-
from a host of other febrile illnesses common in the tropics. cal lymphocytes, followed later by leukocytosis with a left
The incubation period is usually about 1 week (range shift; mild-to-moderate thrombocytopenia, hemoconcentra-
3–21 days). Illness typically begins with the gradual onset of tion; elevated aspartate aminotransferase (AST), alanine ami-
fever and constitutional symptoms, including general mal- notransferase (ALT), and amylase; electrolyte perturbations;
aise, anorexia, headache, chest or retrosternal pain, sore and proteinuria [134, 145]. Unlike classic viral hepatitides, in
throat, myalgia, arthralgia, lumbosacral pain, and dizziness arenaviral HF the AST is typically much higher than the ALT,
[57, 114]. The pharynx may be erythemic or even exudative suggesting that its source is not exclusively the liver. Since a
in Lassa fever, a finding which has at times led to misdiagno- broad range of tissues can release AST, it should be consid-
sis of streptococcal pharyngitis [114]. Gastrointestinal signs ered a marker of systemic organ damage [134]. Radiographic
and symptoms occur early in the course of disease and may and electrocardiographic findings are generally nonspecific
include nausea, vomiting, epigastric and abdominal pain and and correlate with the physical examination [146, 147].
tenderness, and diarrhea. Lassa fever has sometimes been
mistaken for acute appendicitis or other abdominal emergen-
cies. A morbilliform, maculopapular, or petechial skin rash is 7 Clinical Management
very frequent in South American HFs. Interestingly, although
rash almost always occurs in fair-skinned persons with Lassa Although data from clinical trials are sparse, most arenavi-
and Lujo fever, it rarely occurs in blacks. The reasons for this ruses show in vitro and in vivo sensitivity to the antiviral
observation are unknown, but prior infection with partial drug ribavirin, which should be considered in all severe are-
immunity and genetic differences have been postulated. navirus infections. Convalescent immune plasma has dem-
Conjunctival injection or hemorrhage is frequent but is not onstrated efficacy in Argentine HF and may be indicated,
accompanied by itching, discharge, or rhinitis. A dry cough, when available, in other New World arenavirus infections,
sometimes accompanied by a few scattered rales on ausculta- although it is associated with a late neurological syndrome in
tion may be noted, but prominent pulmonary symptoms are 10 % of cases. Detailed reviews of the clinical management
uncommon early in the course of the disease. Jaundice is not of viral HFs are available elsewhere [17, 145].
typical and should suggest another diagnosis.
In severe cases, patients progress to vascular instability,
which may be manifested by subconjunctival hemorrhage, 8 Diagnosis
facial flushing, edema, bleeding, hypotension, shock, and
proteinuria. Swelling in the face and neck and bleeding are 8.1 Clinical Diagnosis
particularly specific but not sensitive signs in Lassa and Lujo
fevers [114]. Despite the term “viral HF,” clinically discern- Difficulties in diagnosing arenavirus infection, both clini-
ible hemorrhage is not always seen, being less frequent in cally and in the laboratory, pose a major impediment to sur-
disease produced by Old World than New World arenavi- veillance and control. Even if more clearly recognized HF
ruses, and never in the first few days of illness [57, 114]. or neurological syndromes eventually develop, most patients
Hematemesis, melena, hematochezia, metrorrhagia, pete- present with a nonspecific febrile syndrome difficult to dis-
chiae, epistaxis, and bleeding from the gums and venupunc- tinguish from a host of other diseases that are usually more
ture sites may develop, but hemoptysis and hematuria are common in the area. The differential diagnosis of arenavi-
infrequent. Significant internal bleeding from the gastroin- ral HF includes a broad array of febrile illnesses, including
testinal tract may occur even in the absence of external hem- malaria, typhoid fever, leptospirosis, bacterial septicemia,
orrhage. Neurological complications are more common in rickettsial infections, dengue fever, and other viral HF
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 161

a b

Fig. 8.6 Clinical manifestations of arenaviral hemorrhagic fever. (a) Dinh), (d) gum bleeding in Argentine hemorrhagic fever, (e) petechial
Facial swelling and mild gum bleeding in Lassa fever (photo by Donald rash in Argentine hemorrhagic fever (Photos used with permission from
Grant), (b) conjunctival injection in Lassa fever (photo by Donald Blumberg et al. [210])
Grant), (c) maculopapular rash in Lujo hemorrhagic fever (photo by TH
162 D.G. Bausch and J.N. Mills

syndromes, depending upon the specific geographic region cerebrospinal fluid in LCMV infection [38, 95, 150, 151].
and patient history of exposures [145]. The classic presenta- Postmortem diagnosis of some arenavirus infections may be
tion of LCMV infection is aseptic meningitis occurring in established by pathology examination with immunohisto-
the fall, particularly if an initial prodromal period of fever, chemical staining of formalin-fixed tissue [152]. Although
perhaps with a remission, occurs before the CNS phase. extensive standardization and validation of these assays has
The differential diagnosis includes the arboviral meningiti- not been conducted, they generally appear to have high sen-
des and herpes encephalitis and, for congenital manifesta- sitivities and specificities when conducted by experienced
tions, the classic TORCH organisms (toxoplasma, rubella, technicians [119, 153]. Cross-reactions between antigeni-
cytomegalovirus, and herpes virus). The presence of throm- cally similar arenaviruses may pose a problem with the sero-
bocytopenia and leukopenia should enhance suspicion of logic assays, which sometimes can be resolved using
arenavirus infection. neutralization tests [59]. The diagnostic methodologies and
A diagnosis of arenavirus infection should be consid- their distinct advantages and disadvantages are reviewed
ered in patients with a clinically compatible syndrome who, elsewhere [154].
within 3 weeks prior to disease onset, (1) live in or traveled to
an endemic area (Figs. 8.2 and 8.3), (2) had potential direct
contact with blood or bodily fluids of a person with arena- 9 Pathogenesis and Immune
viral HF during their acute illness (this group most often is Response in Humans
comprised of healthcare workers or persons caring for fam-
ily members at home), (3) worked in a laboratory or animal 9.1 Central Nervous System Disease
facility where arenaviruses are handled, or (4) had sex with
someone recovering from arenaviral HF in the last 3 months. Overt cell damage by arenaviruses is minimal or modest, but
Recognized direct contact with rodents in endemic areas, they alter cell function and induce mediators of shock,
including laboratory mice and pet hamsters and guinea pigs directly or by immunopathologic mechanisms. Immune cell
if LCMV infection is suspected, should heighten suspicion activation is also the fundamental process in the pathogene-
but is rarely noted even among confirmed cases. Acts of bio- sis of LCMV, although without the same effects on endothe-
terrorism must be considered if arenaviral HF is strongly lial cell function and hemostasis as in HF (see Sect. 9.2) [17].
suspected in a patient without any of the aforementioned risk CNS involvement typically occurs a week or two after dis-
factors, especially if clusters of cases are seen [19]. Even ease onset, after virus has cleared from the blood, and is thus
persons who meet the above criteria most commonly have thought to be immune mediated, although virus can still be
a disease other than arenaviral HF, so alternative diagnoses recovered from the CSF at this time.
should always be aggressively sought.

9.2 Arenaviral Hemorrhagic Fever

8.2 Laboratory Diagnosis
As with all viral HFs, microvascular instability and impaired
The difficulty in clinical diagnosis makes prompt laboratory hemostasis are the hallmarks of arenaviral HF. Unchecked
testing imperative. Unfortunately, no commercial assays are viremia appears to be central to the pathogenesis of arenavi-
available, a situation further complicated by the BSL-4 des- ral HF [134]. Viremia is usually present at patient presenta-
ignation and Select Agent status of many arenaviruses that tion (presumably starting with disease onset, although
limit access and research potential even to many legitimate patients are rarely available for testing at this time), peaking
scientists. Various recombinant-protein- and viruslike- between days 4–9 and clearing within 2–3 weeks in survi-
particle-based assays are being developed which may even- vors. Cell-mediated immunity is thought to be the primary
tually relieve the diagnostic bottleneck, as well as improve arm of recovery in Old World arenavirus infection, while the
sensitivity and specificity, although interpretation and vali- humoral arm is important in disease caused by New World
dation of these assays are proving challenging [148, 149]. viruses [155–157]. Antibody titers are significantly lower in
Meanwhile, various “in-house” assays have been developed fatal cases relative to survivors [119, 134, 158] Neutralizing
and are performed in a few specialized laboratories. Common antibodies may be produced in Old World arenavirus infec-
diagnostic modalities include cell culture (restricted to tion, but usually months after recovery and often at a low
BSL-4 laboratories for most pathogenic arenaviruses), sero- titer. The continued increase in antibody titer to Lassa virus
logic testing by enzyme-linked immunosorbent assay or months after infection suggests a sustained B cell response
immunofluorescent antibody assay, and the reverse tran- that might be attributable to low-level virus persistence; in
scriptase polymerase chain reaction. Serum is the usual sub- vaccine experiments in monkeys, replication-competent
stance tested, but these assays may also be applied to virus was cleared within 14 days after Lassa virus challenge,
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 163

but detection of RNA up to 112 days suggested low-level For LCMV, Lassa, and Machupo viruses, whose hosts
viral persistence or the presence of defective interfering par- often colonize human dwellings, measures to improve “vil-
ticles [159]. The delayed clearance of Lassa virus from lage hygiene” are advocated to discourage rodent invasion,
immunologically protected sites could explain the finding. including assuring proper waste disposal, eliminating unpro-
In survivors, IgM antibodies begin to appear after about a tected storage of garbage and foodstuffs, reducing clutter and
week and progressively increase as virus clears, lasting at vegetation around houses that give rodents shelter, and seal-
least some months [119]. There are conflicting reports ing holes that allow entry into homes [55]. These measures
regarding the timing of IgM antibody appearance; this dis- may not always be possible with the rudimentary construc-
crepancy may reflect differences in the production of reagents tion of houses in some regions, especially with regard to
and their target epitopes, variations in assay sensitivity and Lassa fever in areas of West Africa that have been ravaged by
specificity, and experience of the persons conducting the war or civil unrest.
assay [119, 134, 160]. IgG antibody begins to appear Although complete elimination of rodents from the envi-
2–3 weeks after onset and recovery and lasts for years, ronment through trapping or poisoning is not feasible or
including being found in persons over 40 years after Lassa desirable, considering their importance to overall healthy
virus infection who left the endemic area for Lassa fever and ecosystems, short-term intensive rodent elimination in
had no opportunity for re-exposure [161]. defined areas may occasionally be useful to control out-
A long-standing mystery of arenaviral HF, especially Lassa breaks; an outbreak of Bolivian HF in 1963–1964 in the vil-
fever, is the extreme range of clinical severity. The reasons for lage of San Joaquin ended abruptly after 2 weeks of
this variation are unknown but may relate to heterogeneity in continuous trapping in homes during which 3,000 big laucha
the virulence of infecting Lassa virus strains, route and dose of were captured [73, 167]. However, a single trapping session
inoculation, genetic predisposition, underlying coinfections or in houses in Sierra Leone did not diminish the incidence of
premorbid conditions (e.g., malaria, malnutrition, or diabetes), human Lassa virus infection [55]. A sustained elimination
or misclassification of reinfection as new infection due to wan- program would be necessary for effective long-term control
ing of antibody. Most studies in nonhuman primates use chal- because rodents from surrounding fields and forests may
lenge doses designed to produce uniformly fatal disease and quickly recolonize villages and homes. Rodent elimination
thus are not particularly illustrative regarding the pathogenesis programs are unlikely to control transmission of Junín and
of the full spectrum of arenaviral HF. In a monkey model of Guanarito viruses to humans because their reservoir hosts
Lassa fever using the WE strain of LCMV, intravenous inocu- are widespread in agricultural fields, although targeting the
lation resulted in fatal viral HF while monkeys inoculated via linear habitats preferred by drylands vesper mice might be
the gastric route mostly had an attenuated infection with no plausible [2]. Crop replacement or periodic burning of tall
disease [92, 155]. Human genes encoding “like glycosyltrans- grassy areas that are in close proximity to agricultural fields
ferase” (LARGE), dystrophin (DMD), and IL-21 have appar- and human habitation has been recommended to control
ently undergone positive selection in populations in endemic these viruses, although the measures are untested [42].
areas for Lassa fever in Nigeria, suggesting a protective effect
[162, 163]. Readers are referred to recent reviews for more
details on arenavirus pathogenesis [21]. 10.2 Environmental Shedding
and Disinfection

10 Prevention and Control Little published literature exists on transmission dynamics or

viability of arenaviruses after shedding into the environment,
10.1 Rodent Control especially data applicable to settings other than research labo-
ratories. However, the lipid envelope of all HF viruses, includ-
In the absence of effective vaccines for most arenavirus ing arenaviruses, is easily disrupted, limiting their viability
infections, effective and sustainable control relies on avoid- outside a living host. Little concern is required regarding HF
ance of known reservoir host habitats or, when this is not viruses seeping into groundwater or posing any long-term
feasible, implementation of measures to prevent direct con- risk through casual exposures in the general environment,
tact with rodents and their excreta. Most successful control where harsh thermal and pH conditions would likely readily
programs entail principles of integrated pest management, inactivate them. The survival of HF viruses in animal excreta
integrating biological and chemical control measures with is likely affected by factors such as the animal’s diet and uri-
education or regulation to change human behaviors that put nary pH and the presence of protein. When shed naturally in
them at risk [164, 165]. Structural improvements to homes animal excreta or human body fluids, which would then usu-
may be important for arenaviruses whose rodent hosts ally dry, infectivity appears to last from a few hours to days,
invade houses [166]. varying with the specific virus and environmental conditions
164 D.G. Bausch and J.N. Mills

such as temperature and light [168–170]. However, other below freezing, which is unlikely in the endemic areas of
non-arenavirus HF viruses have been isolated from samples most arenaviral diseases. If corpses are to be exhumed, the
kept for weeks at ambient temperatures if stored hydrated in same protective measures used for burial of victims of viral
a biological buffer, such as blood or serum. This would likely HF, including placing the cadaver in a sealed body bag,
hold true for arenaviruses as well. The viruses are also stable should be used.
when frozen or freeze-dried [171].
Routine environmental surveillance (i.e., in soil, water, or
air) for the arenaviruses is generally not feasible or routinely 10.3 Occupational Exposure
conducted. However, concerns about bioterrorism have and Personal Protective Equipment
spurred interest in this area in recent years, especially around
specific events attracting large crowds, such as sporting 10.3.1 Healthcare Workers
events, that might be potential bioterrorist targets. Detection Since transmission of arenaviruses between humans requires
of an arenavirus outside its usual endemic area should cer- direct contact with blood and body fluids, infection can be
tainly raise suspicion about bioterrorism. The specificity of prevented by the isolation of the patient and use of routine
any test employed would have to be high to avoid the consid- barrier nursing. However, due to the high fatality rates asso-
erable alarm and expenditure of resources that a false- ciated some arenaviral HFs, so-called viral HF precautions
positive result would likely initiate. (surgical mask, double gloves, gown, protective apron, face
When contamination is suspected, such as in homes or shield, and shoe covers) are advised for added security [185].
hospitals treating persons with arenaviral HF, disinfection is Powered air-purifying respirators and other small-particle
warranted and effective. Arenaviruses can be inactivated by aerosol precautions should be used when performing pro-
exposure to temperatures above 60 °C for 1 h, acidic or basic cedures that may generate aerosols, such as endotracheal
pH, gamma irradiation, ultraviolet light (surface disinfection intubation. Items that were in direct contact with the patient
only), surfactant nanoemulsions, and a host of chemicals and should be decontaminated (see Sect. 10.2).
detergents, including chlorine compounds, alcohols, phe-
nols, quaternary ammonium compounds, formaldehyde, 10.3.2 Laboratory Workers
cupric and ferric ions, zinc finger-reactive compounds, pho- Fortunately, since most arenavirus infections are rare in
toinducible alkylating agents, and various proprietary humans and routine laboratory safety procedures are gen-
detergent-containing lysis buffers [172–184]. Toxicity and erally adequate to prevent transmission, infection in hospi-
corrosion may be concerns with some of these compounds tal laboratories from clinical samples is extremely rare. Of
depending upon the frequency of use and concentrations. more concern is the risk to laboratorians specifically working
Sodium hypochlorite (i.e., household bleach) is the most with cultured arenaviruses, especially the highly pathogenic
readily available effective inactivation method [185]. Bleach agents of HF. However, these agents are largely restricted
solutions should be prepared daily; starting with the usual to work in BSL-4 laboratories. No arenavirus infections
5 % chlorine concentration, a 1:100 (1 %) solution should be have been reported in laboratorians working in BSL-4 lab-
used for reusable items, such as medical equipment, patient oratories, although numerous accidents, sometimes fatal,
bedding, and reusable protective clothing before laundering. have occurred in lower-level containment laboratories.
A 1:10 (10 %) bleach solution should be used to disinfect Recognition of LCMV transmission associated with labora-
excreta, corpses, and items to be discarded. Workers clean- tory mice has led to guidelines and regulations in these set-
ing areas potentially contaminated by the excreta of small tings that have markedly decreased risk [187].
mammals should let the area aerate before entering, then
spray the area with the 10 % bleach solution and let it sit on 10.3.3 Field-Workers
the surface for at least 15 min before mopping or wet sweep- Field-workers such as agricultural workers, field biologists,
ing [186]. A site with appropriate security should be dedi- and pest control workers are potentially exposed to rodents
cated for waste disposal if routine autoclaving is not and their excreta in an occupational setting. Inadvertent
available. exposure to contaminated rodent excreta by agricultural
Specific guidelines exist regarding handling and burial of workers is difficult to prevent. Routine use of masks is prob-
corpses of victims of viral HF [185]. The question of the ably not feasible or cost-effective, but should be considered
safety of exhuming remains of persons who died of arenavi- when the risk of aerosols is high. Common dust or surgeon’s
ral HF overseas for transport back home occasionally arises. masks will not protect against small-particle aerosols; spe-
No data regarding the viability of arenaviruses under these cifically designed N-100 or P-100 face masks must be used
circumstances are available, but it is unlikely that the viruses for this purpose [188].
would survive long under the harsh pH conditions of a Field biologists and pest control workers who trap rodents
decomposing corpse unless the ambient temperature is merit special consideration; although the risk would seem to
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 165

be high, the limited evidence available suggests that arenavi- 10.4 Case Identification
rus infection in this group is rare, even in the absence of the and Patient Isolation
use of extensive personal protective equipment. Field biolo-
gists who handled rodents in North America showed a preva- Control of most arenaviral HF relies on classic public health
lence of exposure to HF viruses of less than 1 %, and not all principles of identification and isolation of cases and monitor-
infection could be clearly attributed to occupational expo- ing of their contacts. However, the early nonspecific presenta-
sure [189]. The risk may vary depending upon the prevalence tion of arenaviral HF poses a serious challenge to case
of the pathogen in the animals and the specific procedure identification. Fortunately, the low secondary attack rate affords
conducted. For example, simple elimination of pests via a measure of reassurance even when cases go unrecognized as
snap-trapping and disposal of intact dead animals would be long as proper barrier nursing is maintained. Furthermore,
less risky than live-trapping with subsequent euthanasia and since mild cases are not very infectious, missed or delayed
necropsy to procure samples for scientific research. diagnosis of these patients is unlikely to pose a problem from
In the absence of a clear consensus on the risk of infec- an infection control standpoint. All patients with a clinically
tion, a wide variety of biosafety practices are employed by compatible syndrome should be presumed infectious and kept
field-workers, ranging from no personal protective equip- under “viral HF isolation precautions” (see below) until a spe-
ment to use of gloves, gowns, safety glasses, and HEPA cific diagnosis is made [185]. If available, placement in a nega-
filter respirators or even positive-pressure respirators tive airflow room is prudent, but hermetically sealed isolation
[189]. While the inclination of those responsible for work- chambers are not required and may have severe adverse psy-
ers’ safety is to err on the side of caution, and thus require chological effects on both patient and staff. Access to the
the full complement of protective personal equipment, sig- patient should be limited to a small number of designated staff
nificant cost and practical implications must be consid- and family members with specific instructions and training on
ered. More research is needed to assess the true risk to the implementation of viral HF isolation precautions.
field-workers and thus the cost–benefit balance of the vari- Since the clinical status of patients with viral HF generally
ous protective measures if a widely agreed-upon standard correlates with the level of viremia and infectivity, patients
procedure is to be set. Because risk depends upon geo- who have recovered from their acute illness can safely be
graphic location, pathogens expected to be encountered, assumed to have cleared viremia and can be discharged with-
species handled, specific tasks, and worker training, any out concern of subsequent transmission. However, because
such standards will have to address multiple situations. In of potential delayed virus clearance in urine and semen,
the meantime, it seems prudent to follow established safely abstinence or condom use is recommended for 3 months
guidelines when handling known viral HF reservoir host after acute illness. Although transmission through toilet
species [190]. Full precautions, including positive-pres- facilities has not been noted, simple precautions to avoid
sure respirators, are warranted when sample centrifugation contact with excretions in this setting are prudent, including
or other potentially aerosol-generating procedures are separate toilet facilities and regular hand washing. Breast-
performed. feeding should be avoided during convalescence unless there
is no other way to support the baby.
10.3.4 Pet Store Workers and Pet Owners
Recognition of LCMV transmission associated with pet
animals has led to guidelines and regulations in these set- 10.5 Contact Tracing
tings [187]. Pregnant women and immunosuppressed per-
sons in particular must be protected from infection because Persons with unprotected direct contact with a patient during
of the potentially severe consequences in these groups the symptomatic phase of arenaviral HF should be monitored
[36]. Serologic testing of individual pet rodents is gener- daily for evidence of disease for three weeks (the longest
ally unreliable and not recommended. Educational materi- known incubation period) after their last contact. Contacts
als on safe handling of pet rodents should be made available should check their temperature daily and record the results
at pet stores. Pet stores with potentially infected rodents in in a log. Despite the lack of evidence for transmission dur-
stock should contact public health authorities for addi- ing the incubation period, exposed persons should avoid
tional information and guidance. Human infection with close contact or activities with household members that
arenaviruses other than LCMV from pet contact has not might result in exposure to bodily fluids, such as sex, kiss-
been reported. However, although not an arenavirus, mon- ing, and sharing of utensils. Hospitalization or other confine-
keypox virus, traced to the importation of infected ment of asymptomatic persons is not warranted, but persons
Gambian rats from Ghana, has infected humans in the who develop fever or other signs and symptoms consistent
United States and highlights the risk to those importing with arenaviral HF should immediately be isolated until the
and keeping exotic pets [191]. diagnosis can be excluded.
166 D.G. Bausch and J.N. Mills

10.6 Vaccines bodily fluids from a person with confirmed or highly suspected
arenaviral HF. Persons who develop manifestations of arena-
In Argentina, the use of a live attenuated vaccine for Argentine viral HF should be immediately converted to the intravenous
HF called Candid #1 began in 1991 and has resulted in a form. Prophylaxis should not be given if the only exposure
steady decrease in the incidence of cases, as described above. was during the incubation period. A review and specific guide-
The vaccine may also protect against Bolivian HF, although it lines have been published [209].
does not appear to cross-protect against other arenaviruses
[192]. However, Candid #1 is generally not available or
approved outside of Argentina, and even within Argentina 11 Unresolved Problems
supplies are insufficient to cover the population at risk. and Future Challenges
Furthermore, despite the efficacy and generally excellent
safety profile of Candid #1 for Argentine HF, fear of reversion The progress in our understanding of the arenaviruses in
to virulence with live-virus vaccines is a major disincentive to recent decades has primarily been driven by concern over
exploring its application to other arenavirus infections. the use of these agents as bioweapons [19, 117]. The focus
Other arenavirus vaccines are in the experimental stages. has been largely in the realm of the laboratory and basic sci-
A number of vaccine platforms have been explored in animal ences, with promising developments in treatments and vac-
models of arenaviral HF, primarily aimed at preventing Lassa cines. However, much remains to be elucidated regarding
fever. Approaches include inactivated or attenuated viruses arenavirus epizoology and epidemiology if improved pre-
[193, 194], recombinant vaccinia viruses [159], RNA repli- vention and control are to be achieved. The ultimate goal
con vectors derived from an attenuated strain of Venezuelan is to understand the complex epizoologic, epidemiologic,
equine encephalitis virus [195], recombinant Salmonella and ecologic interactions of arenaviruses and predict and
Typhimurium [196], arenavirus protein subunits [197], naked prevent their emergence and increased incidence of disease.
DNA [198], chimeric viruses using the yellow fever 17D vac- Significant barriers exist, including inadequate surveillance
cine strain [199, 200], reassortant Lassa/Mopeia viruses and lack of commercially available diagnostic reagents.
[201–203], viruslike particles [204], and recombinant vesicu- Population-based, longitudinal studies in both humans and
lar stomatitis virus [205, 206]. The arenavirus GP1 appears to rodents are necessary to understand transmission dynam-
be the most important immunogenic protein. ics, including reasons for patchy endemicity and seasonal-
The recombinant vesicular stomatitis virus platform is ity, and the genetic and evolutionary relationships between
perhaps the most promising candidate, providing 100 % pro- viruses and reservoirs. However, the often remote and some-
tection after a single dose in a monkey model of Lassa fever. times politically unstable endemic areas and the difficulty
Furthermore, this vaccine may be effective when given by the in securing long-term funding are significant challenges to
nasal or oral route, potentially facilitating its use in epidem- long-term studies. Laboratory studies on precise modes of
ics or as postexposure prophylaxis [207]. Although there are arenavirus transmission between rodents and humans are
concerns over the safety of an unproven live-vector vaccine in needed, especially the role of aerosol transmission. While
populations in Africa with a high frequency of immunosup- logical in terms of biosafety and biosecurity, the BSL-4 and
pressed persons due to HIV/AIDS or malnutrition, preliminary Select Agent status of many arenaviruses poses a significant
studies of the recombinant vesicular stomatitis virus vaccine impediment to research. Little attention has been paid to
platform in immunocompromised monkeys have not indicated community control, including the efficacy of measures such
problems, perhaps because the vector virus lacks the neuro- as regular rodent trapping or the use of rodenticides, perhaps
virulence genes and properties of the wild-type virus [208]. timed to coincide with the breeding season, rodent-proofing
The social, economic, and political barriers to development of of homes, and the introduction of predators, such as cats.
arenavirus vaccines are at least as formidable as the scientific Interdisciplinary programs bridging human, animal, and
ones, considering that most arenavirus diseases exist in geo- ecosystem health and conservation offer further opportuni-
graphically restricted areas with resource-poor populations. ties to understand ecosystem regulation of arenavirus trans-
mission and how human alterations of the environment and
biodiversity loss affect the risk of infection. Lastly, major
10.7 Postexposure Prophylaxis challenges include global economic distress that threatens
to blunt research funding, the lack of economic incentive to
Ribavirin has been used as postexposure prophylaxis for Lassa invest in research on agents that are primarily endemic in
fever, although there are no data on efficacy, dose, or duration resource-poor regions of the world, and the relatively weak
of administration of the drug for this purpose. Nevertheless, research and training infrastructure in developing countries
postexposure prophylaxis with oral ribavirin should be consid- that continue to experience loss of their scientists to more
ered for persons with direct unprotected contact with blood or lucrative jobs elsewhere.
8 Arenaviruses: Lassa Fever, Lymphocytic Choriomeningitis, and the South American Hemorrhagic Fevers 167

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 Bunyaviruses: Hantavirus and Others
9
Alexander N. Freiberg, Dennis A. Bente,
and James W. Le Duc

1 Introduction emerging and reemerging viruses, such as Rift Valley fever

virus, Crimean-Congo hemorrhagic fever virus, and the
The Bunyaviridae family is the largest family of RNA viruses hantaviruses, but also for recently emerged pathogens, such
with more than 350 named isolates [70]. Viruses in the family as the newly identified severe fever with thrombocytopenia
are divided into five genera (Orthobunyavirus, Phlebovirus, syndrome and Heartland, Shuni, and Schmallenberg viruses.
Nairovirus, Hantavirus, and Tospovirus) on the basis of sero- This chapter will discuss the most important human patho-
logical, molecular, antigenic, and structural characteristics. gens of the Orthobunyavirus, Phlebovirus, Nairovirus, and
The Bunyaviridae are a unique group of viruses whose mem- Hantavirus genera.
bers are able to infect invertebrates, vertebrates, and plants,
and they can be found worldwide. Multiple members are sig-
nificant pathogens with the ability to cause severe disease in 2 Overview of the Bunyaviridae Family
humans, such as encephalitis, hepatitis, or hemorrhagic fever.
Most bunyaviruses are spread through sylvatic transmission 2.1 Morphology and Morphogenesis
cycles between susceptible vertebrate hosts and hematopha-
gous arthropods, including ticks, mosquitoes, and phleboto- The morphological properties among the viruses in each of
mine flies. Unique are the members of the Hantavirus genus, the five bunyavirus genera vary; however, in general, virions
in that they are not infecting insect vectors but are maintained appear to have a spherical to pleomorphic morphology, with
in nature through persistent infection of rodents. Human and diameters ranging from 80 to 120 nm. Embedded in the lipid
animal pathogenic bunyaviruses can be found in four of the bilayered envelope are the two surface glycoproteins, Gn and
five genera, with tospoviruses being plant pathogens. The Gc, which can be up to 10 nm in length and form protrusions
large family of bunyaviruses is a pool not only for many or spikes. Recent structural studies have revealed that the
two surface glycoproteins of phleboviruses form the so-
called capsomeres (built by Gn/Gc heterodimers) and are
arranged on an icosahedral lattice with a triangulation num-
ber of 12 [86, 112, 176, 204, 223]. For hantaviruses each
A.N. Freiberg, PhD (*) glycoprotein spike complex is arranged in a square-shaped
Department of Pathology, assembly with fourfold symmetry [19, 111]. In contrast to
University of Texas Medical Branch, most other negative-strand RNA viruses, bunyaviruses do
301 University Blvd., Galveston, TX 77555-0609, USA
not contain a matrix protein, which facilitates the interaction
e-mail: [email protected]
between the surface glycoproteins and the ribonucleoprotein
D.A. Bente, DVM, PhD
(RNP) core. It has been demonstrated that the cytoplasmic
Galveston National Laboratory,
Department of Microbiology and Immunology, tails of at least one of the glycoproteins mimic the function
University of Texas Medical Branch, of a matrix protein [177, 194, 224]. The virion envelope is
Galveston, TX, 77555-0610, USA usually derived from cellular Golgi membranes but can occa-
e-mail: [email protected]
sionally also be derived from the cell surface membrane and
J.W. Le Duc, PhD contains 20–30 % lipids. The membrane encloses the viral
Department of Microbiology and Immunology,
tripartite single-stranded negative-sense RNA genome. The
University of Texas Medical Branch, 301 University Blvd.,
Galveston, TX 77555-0610, USA viral genomic RNA segments are only found encapsidated
e-mail: [email protected] by the nucleoprotein (N) in the form of RNP complexes and

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 173

DOI 10.1007/978-1-4899-7448-8_9, © Springer Science+Business Media New York 2014
174 A.N. Freiberg et al.

not as naked RNA molecules, indicating that encapsidation in stable panhandle structures and non-covalently closed cir-
and RNA synthesis occur concomitantly. The RNA- cular RNAs [105]. These terminal sequences are conserved
dependent RNA polymerase (L) is associated with the RNP among viruses within each genus but are different to mem-
complexes and they display a helical symmetry. At least one bers in the other genera. The 5′ NCRs of the genomic viral
of each of the three segments must be contained in a virion RNA segments are not modified. Viral mRNAs are not poly-
for infectivity. Virions contain 2–7 % carbohydrate by adenylated and are truncated relative to the 3′ NCR of their
weight, and asparagine-linked sugars on the glycoproteins genomic RNA counterpart. The 5′ ends of viral mRNAs have
Gn and Gc are largely of the high mannose type when viruses methylated caps and 10–18 non-templated nucleotides which
are grown in vertebrate cells. are derived from host cell mRNAs. The NCRs do contain sig-
nals not only for the N protein encapsidation but also for the
viral promoters, mRNA transcription termination, and prob-
2.2 Biochemical and Physical Properties ably the RNP-packaging signals [65, 81, 175, 258].
The four bunyavirus structural proteins (Table 9.2) are
Bunyavirus buoyant densities in sucrose and CsCl gradients are encoded in the complementary sense RNA of the three viral
1.16–1.18 g/cm3 and 1.20–1.21 g/cm3, respectively. The molec- segments. All bunyavirus L segments use conventional negative-
ular mass of the virions ranges from 300 to 400 × 106 and have sense coding strategies and encode for the RNA-dependent
a sedimentation coefficient of 350–500 S. Treatment with lipid RNA polymerase. The two envelope glycoproteins, Gn and Gc,
solvents or nonionic detergents results in loss of infectivity due are encoded in a single, continuous open reading frame (ORF)
to the removal of the envelope [173]. Bunyaviruses are also in the mRNA, and the primary gene products are then co-trans-
sensitive to heat and formaldehyde. lationally cleaved to give mature Gn and Gc proteins (referring
to the amino-terminal or carboxy-terminal coding of the pro-
tein). Orthobunya- and phleboviruses encode an additional non-
2.3 Genome Organization structural protein (NSm) in the virion-complementary-sense
and Viral Proteins RNA. Nairoviruses encode two proteins of unknown function, a
mucin-rich protein and glycoprotein GP38, which are the prod-
All members of the Bunyaviridae share a common tripartite ucts of posttranslational cleavage of glycoprotein precursor
single-stranded negative-sense RNA genome. The three RNA preGn. The N protein is encoded on the S segment.
molecules are designated as S (small), M (medium), and L Orthobunyaviruses and some hantaviruses encode a nonstruc-
(large) segments (Table 9.1). Each genome segment can be tural protein (NSs) in an overlapping ORF to that encoding the
divided into three regions, the 3′ noncoding region (3′ NCR), N protein in the 3′ half of the virion-sense S RNA. Phleboviruses
coding region, and 5′ NCR. The terminal nucleotides at the 3′ encode a NSs protein in an ambisense ORF in the 5′ half of the
and 5′ NCRs of each viral RNA segment are highly conserved virion-sense S RNA. The NSs proteins of orthobunya-, phlebo-,
and invertedly complementary to allow base pairing, resulting and hantaviruses have been shown to act as interferon antago-
nists (reviewed in [131]). The NSm protein of phleboviruses can
act as an apoptosis antagonist [196, 240, 262].
Table 9.1 Sizes of viral RNA segments in the bunyavirus genera (sizes
are given in kb)
RNA segment 2.4 Antigenic and Genetic Properties
Genus and virus S M L
Phlebovirus 1.7 3.5 6.4 One or both of the envelope glycoproteins display hemag-
Nairovirus 1.7 4.9 12.2
glutinating and neutralizing antigenic determinants.
Orthobunyavirus 1.0 4.5 6.9
Complement-fixing antigenic determinants are principally
Hantavirus 1.7 3.6 6.5
associated with the N protein.
Tospovirus 2.9 4.8 8.9

Table 9.2 Sizes of structural proteins in the bunyavirus genera (sizes are given in kDa)

RNA segment: viral Genus

protein Phlebovirus Nairovirus Orthobunyavirus Hantavirus Tospovirus
S: N 24–30 48–54 19–25 50–54 52
M: Gn 50–70 30–45 29–41 68–76 52–58
M: Gc 55–75 72–84 108–120 52–58 72–78
L: L 238–241 459 259–263 246–247 330–332
9 Bunyaviruses: Hantavirus and Others 175

Viruses with segmented genomes have the ability to complement fixation assays, and each serogroup contains
expand their genetic diversity through (a) slow genetic drift viruses that are antigenically distinct but related.
(i.e., accumulation of individual point mutations) or (b) sud- At least 30 members of the Orthobunyavirus genus have
den genetic shift (i.e., new arrangement of whole genome been associated with human disease, causing a range of
segments in progeny viruses produced during a coinfection symptoms such as encephalitis, hemorrhagic fever, or febrile
event). Genetic reassortment occurs in nature and in the lab- illness. In livestock, orthobunyavirus infections are mainly
oratory by exchange of genomic segments and genetic rela- associated with abortions or teratogenic effects.
tionships within the same serogroup and can affect The majority of the orthobunyaviruses are arthropod-
pathogenicity of RNA viruses. Genetic reassortment between borne viruses and have amplification cycles in a variety of
closely related bunyaviruses has been demonstrated in vertebrate hosts. Infection of the different species can result
experimentally coinfected tissue culture cells and arthropod in fundamentally different outcomes for the host cells.
hosts but also by analysis of naturally occurring virus iso- Mosquitoes (and derived cell lines) are persistently infected
lates [20, 26, 28, 43, 104, 140, 198, 201, 208, 212]. High- and shed virus.
frequency reassortment can occur when arthropod vectors
are infected simultaneously with two viruses. This can either 3.1.1 Oropouche Fever
happen in the same blood meal or through interrupted feed- Oropouche virus (OROV) is a member of the Simbu sero-
ing, when blood meals are taken within a relatively close group and the causative agent of Oropouche (ORO) fever, an
time window of each other [21]. urban febrile arboviral disease widespread in South America
[193]. OROV was first isolated in 1955 in Trinidad and
Tobago from the blood of a febrile forest worker from a pool
2.5 Biological Properties of Coquillettidia venezuelensis mosquitoes and named after
the Oropouche River [8, 134]. Recurrent epidemics have
Members of the Orthobunyavirus, Nairovirus, and involved tens of thousands of patients and are economically
Phlebovirus genera have the capability of replicating alter- significant, owing to loss of working hours. Over the past 50
natively in vertebrate and arthropod hosts. While they are not years, ORO fever has emerged as a major public health prob-
causing any cytopathic effects in their invertebrate hosts, lem in tropical areas of Central and South America [11].
cytolytic effects are generally observed in their vertebrate OROV is transmitted by culicoid midges and humans develop
hosts. Arthropod vectors can be mosquitoes, ticks, sandflies, a high viremia for an orthobunyavirus infection, such that
and phlebotomine flies, and transovarial and venereal trans- uninfected midges can acquire the virus after biting [101].
mission has been demonstrated. Hantaviruses are the excep-
tion, since they are the only rodent-borne bunyaviruses and Descriptive Epidemiology
are transmitted via the aerosol route of aerosolized rodent Members of the Simbu serogroup are distributed globally
excreta. In contrast to other bunyaviruses, hantaviruses rou- and transmitted by biting midges (Culicoides). Since the
tinely establish a persistent, non-cytolytic infection in sus- original isolation of OROV from a febrile patient in Trinidad
ceptible mammalian host cells, primarily in endothelial cells. and Tobago in 1955, the medical importance of the virus,
This observation is consistent with their nonpathogenic per- particularly in the Amazon basin regions of northern Brazil
sistence in their natural rodent host. and Peru, has become increasingly evident [193]. OROV has
been shown to be associated with more than 30 epidemics
reported in Brazil, Peru, Panama, and Trinidad and Tobago
3 The Viruses during 1960 and 2009, with more than 500,000 persons esti-
mated to be infected [193, 202]. Most of these outbreaks
3.1 Orthobunyavirus Genus occurred in relatively urban areas, leading to the suggestion
that OROV is maintained in two distinct cycles, an epidemic
The Orthobunyavirus genus is the largest of the five genera urban and a silent sylvatic cycle [61, 200]. High antibody
within the Bunyaviridae family and contains more than 170 prevalence in populations in endemic areas suggests that
named viruses, including medically important viruses, such human infection can be common in such areas [12, 202, 257].
as Oropouche, La Crosse, and Jamestown Canyon viruses. The principal urban vector in Brazil appears to be the tiny
Viruses within this genus are grouped into 18 serogroups biting midge, Culicoides paraensis [190, 200]. Isolates have
(Anopheles A, Anopheles B, Bakau, Bunyamwera, Bwamba, also been made from mosquitoes such as Culex pipiens quin-
Group C, Capim, California, Gamboa, Guama, Koongol, quefasciatus, but its vector competence is poor [61, 107].
Minatitlan, Nyando, Olifanstlei, Patois, Simbu, Tete, and The favorite larval breeding habitats of C. paraensis midges
Turlock). This classification is based on serological stud- are rotting banana tree stumps and cacao husks [192]. These
ies, using neutralization, hemagglutination inhibition, and decomposing plant materials remain moist even during dry
176 A.N. Freiberg et al.

periods and serve as an excellent growth medium for the investigated in cases of meningoencephalitis of unknown eti-
microorganisms on which the C. paraensis larvae feed. Adult ology in affected areas and that previous blood-brain barrier
midges feed predominantly in the early evening hours and damage might facilitate the entry of OROV into the central
are quite anthropophilic. It is assumed that humans entering nervous system. OROV is also suspected to be infectious by
the jungle become infected with OROV and develop viremia aerosol, based on reported laboratory infections [190].
sufficiently high to transmit the virus to uninfected midges
and appear capable of serving as the vertebrate amplifying Diagnosis
host during the urban cycle [192]. When they return to their The diagnosis of OROV is performed by virus isolation from
village or town, temporary urban transmission can occur, the blood of infected patients and by serological assays. The
resulting in an epidemic. Serological data also suggest that serological response is strongest in the acute phase against
forest animals (primates or sloths or even birds) may be the N protein, and recombinant expressed N protein has been
potential vertebrate hosts in the sylvatic cycle. Factors such used to develop an enzyme immunoassay for the diagnosis of
as deforestation, urbanization, and agricultural development ORO fever [206]. Further, RT-PCR-based assays are avail-
are probably contributing to the emergence of ORO fever as able for the detection of OROV [158, 259].
a human pathogen in South America. Phylogenetic studies
indicate that distinct OROV genotypes are present in differ- Treatment and Prevention
ent geographic areas: Genotype I viruses include the proto- There is no specific treatment for ORO fever and the disease is
type strain from Trinidad and Tobago and Brazilian strains normally self-limiting. However, treatment of symptoms with
from the states of Acre, Amazonas, Maranhão, Tocantins, anti-inflammatory drugs might be recommended. This consists
and Pará; Genotype II contains the Peruvian strains isolated of drinking plenty of fluids to prevent dehydration, as well as
between 1992 and 1998 and strains from the Brazilian states prescribing pain analgesics. A recent study indicated that ribavi-
of Amapá, Pará, and Rondônia; Genotype III includes virus rin does not have antiviral activity on OROV [144]. With the
strains isolated in Panama and the Brazilian states of Acre, ORO fever disease in Brazil and Peru, avoiding the buildup of
Minas Gerais, and Rondônia; and Genotype IV is formed rotting organic debris such as banana tree stalks or cacao husks
by the Brazilian strains isolated in Amazonas state [11, 171, in agricultural areas may help curtail population levels of C. par-
207, 250, 251]. aensis and reduce the risk of seasonal epidemics [192]. Avoiding
exposure (treated netting, DEET repellents) to these midges dur-
Clinical Features ing their early evening feeding hours is also recommended.
Several thousand cases of acute febrile illness can occur
during OROV epidemics observed throughout the Amazon 3.1.2 California Serogroup
basin regions of Brazil and Peru. Humans acquire the infec- The California serogroup has great relevance from a human
tion by bite from infected midges. In addition to large out- infection perspective, as they have been associated with
breaks, OROV can also cause sporadic infections in humans influenza-like illness in central Europe and are an important
[243]. The incubation period is approximately 4–8 days, cause of encephalitis in the United States. Other members
followed by an abrupt onset of fever, with arthralgia, gen- of the genus are of veterinary importance. The California
eralized myalgia, anorexia, nausea, vomiting, weakness, diz- serogroup includes 14 viruses and subtypes. All members
ziness, severe headache, chills, photophobia, and prostration. of the group are transmitted by mosquitoes. Although sero-
Occasionally, ORO fever patients also exhibit rash, menin- logical studies and analysis of genomic sequences have
gitis, or meningismus [161, 191]. Most of the symptoms demonstrated their relationship, each virus is only prevalent
resolve within 3–5 days, although a period of asthenia, myal- in a specific geographic area delimited by the presence of its
gia, and dizziness may persist for up to 9 months. Viremia mosquito vector and mammalian host. Hammon and Reeves
is detectable in the majority of patients 2–3 days post onset isolated California encephalitis virus from Aedes melani-
of illness. No fatalities have been reported with the disease, mon mosquitoes in 1943 in Kern County, California [100,
and lifelong immunity follows recovery. Approximately 199]; serological studies subsequently showed high inci-
half of the patients can exhibit recurrence of some disease dence of seropositivity. Nevertheless, California encepha-
symptoms 1–10 days after they become afebrile [252]. A litis virus has not been recently implicated with any human
recent study identified the presence of OROV in the cerebro- disease in California [199]. California serogroup viruses
spinal fluid of three samples from 110 meningoencephalitis sequentially replicate in striated muscle, cause viremia, and
patients, demonstrating the involvement of the central ner- invade and replicate in the central nervous system causing
vous system in OROV infections (Bastos et al. [17]). Two of encephalitis in their mammalian hosts. Each of these viruses
the three patients presented with other diseases affecting the has a limited geographic range, and members have been iso-
immune system and central nervous system (HIV and neuro- lated from several continents including North and South
cysticercosis). The findings suggest that OROV should be America, Europe, and Africa.
9 Bunyaviruses: Hantavirus and Others 177

La Crosse Virus mated that the incubation period ranges between 6 and 15
In 1960, La Crosse virus (LACV) was isolated from a fatal days. After mosquito bite, multiplication probably occurs in
case of a 4-year-old girl who suffered encephalitis in La vascular endothelial and reticuloendothelial cells; dissemi-
Crosse, Wisconsin [245]. LACV was shown to be closely nation occurs in the blood and lymph [203]. The symptoms
related but distinct from California encephalitis virus, and it of the central nervous system disease include stiff neck, leth-
was quickly recognized as an important virus causing human argy, nausea, headache, and vomiting in milder cases and
infections over a wide area of the Midwestern United States. seizures, coma, paralysis, and permanent brain damage in
Testing mosquitoes and more widespread serological diag- severe cases. Severe disease occurs most commonly in chil-
nosis of individuals with encephalitis identified a number of dren under the age of 16. Death from LACV encephalitis
other arboviruses that were related to California encephalitis occurs in less than 1 % of clinical cases. A small proportion
virus and LACV, and together these were designated as the of patients may develop persistent paresis or learning dis-
California serogroup. abilities and other cognitive deficits [203]. The cerebrospi-
nal fluid demonstrates elevated protein in 20 % of the cases.
Descriptive Epidemiology Unlike most viral encephalitis, many of the cells found in the
Approximately 80–100 cases of LACV encephalitis occur cerebrospinal fluid are polymorphonuclear neutrophils [97].
every year; however, the disease is gravely underreported
and underdiagnosed. The disease is primarily reported in the Diagnosis
upper Midwestern states (Minnesota, Wisconsin, Iowa, Diagnosis is based on clinical features and patient exposure
Illinois, Indiana, and Ohio) from late spring through early history. Laboratory testing is available in many state or local
fall; however, infrequently cases are also reported in winter public health laboratories and in some referral hospitals
in the Gulf states. Recently, more cases have been reported and is accomplished by testing serum or cerebrospinal fluid
from mid-Atlantic and southeastern states (West Virginia, to detect virus-specific IgM antibodies. Virus may also be
Virginia, Kentucky, North Carolina, and Tennessee) [99]. detected by nucleic acid amplification and in fatal cases by
LACV is transmitted to humans through the bite of an histopathology and virus-specific immunohistochemistry.
infected mosquito. LACV is maintained in nature by infec- Virus isolation may also be useful.
tions of two alternate hosts: mosquitoes and small mammals,
particularly chipmunks and squirrels. The main vector is a Treatment and Prevention
woodland mosquito, Aedes triseriatus (the eastern tree hole There are no vaccines against LACV infection or specific
mosquito), which preferably breeds in tree holes and in dis- antiviral treatment. Treatment is supportive. Prevention
carded tires and feeds during the day [133]. In the United is based on avoidance of vector mosquitoes through use
States, Aedes triseriatus is present in areas east of the of insect repellants, wearing protective clothing, keeping
Mississippi River, where numbers peak from June through screens in good repair, and effective vector control.
September [57]. Aedes albopictus, a mosquito introduced
into the United States from Asia, has been shown to be a 3.1.3 Ngari Virus
competent vector, at least in experimental studies. LACV An orthobunyavirus, originally designated Garissa virus, was
can be transmitted transovarially to the next generation or isolated from human hemorrhagic fever cases during a large
venereally from infected males to uninfected females [22]. disease outbreak in 1997 and 1998 in East Africa (Kenya
In endemic areas, a high proportion of chipmunks and squir- and Somalia) [28]. The disease was characterized by acute
rels have been found to be seropositive. Studies have shown onset of fever and headache, followed by hemorrhage (with
that these two species generate high enough viremia levels to gastrointestinal and/or mucosal bleeding). During the inves-
subsequently infect mosquitoes after feeding on the experi- tigations, a previously unidentified orthobunyavirus was iso-
mentally infected animals [267]. Incidence of California lated from two hemorrhagic fever cases. The genetic analysis
serogroup virus neuroinvasive disease has ranged from fewer of fragments of the virus S, M, and L genome RNA seg-
than 40 to over 160 cases per year since 1964. The highest ments revealed that the virus was subsequently recognized as
numbers of cases reported are from Ohio, West Virginia, being the same as the previously identified Ngari virus and
Wisconsin, and North Carolina, with an average annual inci- characterized as a naturally occurring reassortant between
dence above 1.0 per 100,000 population in some endemic Bunyamwera virus (S and L segment donor) and Batai virus
counties ([98]; www.cdc.gov/lac/tech/epi.html, accessed (M segment donor) [30, 93]. Interestingly, these two viruses
Sept 2012). do not cause a hemorrhagic febrile illness. During the out-
break in 1997–1998, an estimated 89,000 human infections
Clinical Features occurred with over 250 deaths. Of 231 febrile patients for
LACV causes acute encephalitis preceded by a nonspecific which clinical records existed, half met the case definition
febrile illness [47]. Based on experimental studies it is esti- of hemorrhagic fever [28]. Initially, it was thought that the
178 A.N. Freiberg et al.

hemorrhagic fever outbreak was associated with Rift Valley growth in floodwater Aedes mosquitoes [234], which can ini-
fever virus (RVFV) infections [263], a human pathogenic tiate an epizootic leading to viremic livestock that then act as
phlebovirus endemic in Africa. Of the hemorrhagic fever amplifying hosts and a source of the virus for feeding mos-
cases, 23 % had evidence of acute RVFV infection, whereas quitoes [234]. Once livestock is infected, the classical hall-
27 % had evidence of acute Ngari infection. mark of RVF epizootics is the large number of abortions
observed among pregnant ruminants. Usually 1–2 weeks
after these abortions, initial human infections can be detected,
3.2 Phlebovirus Genus especially in groups that are in close contact with livestock,
such as farmers, veterinarians, and abattoir workers. RVFV
The genus Phlebovirus comprises over 70 antigenically infection can be acquired either through the bite from an
distinct serotypes, which are divided into two groups, the infected mosquito or more importantly through direct con-
Phlebotomus fever viruses (sandfly group, transmitted by tact with infected livestock. People involved in the birth or
sandflies) and the Uukuniemi group (transmitted by ticks). abortions of livestock, butchering process of animals, or
Eight of these serotypes contain viruses known to cause abattoir workers are at high risk of infection during epizoot-
disease in humans (Alenquer, Candiru, Charges, Naples, ics [2, 39]. Aerosol exposure has been demonstrated to be
Punta Toro, Rift Valley fever, Sicilian, and Toscana viruses). another potential route of infection, especially in a labora-
Recently, two new human pathogenic tick-borne phlebovi- tory setting [14, 31, 84, 109, 159, 160, 229].
ruses have been identified, severe fever with thrombocytope-
nia syndrome virus and Heartland virus [152, 266]. Clinical Features
RVFV is one of the most severe human pathogen among the
3.2.1 Rift Valley Fever Virus phleboviruses and infections can result in a wide range of
Rift Valley fever virus (RVFV) is a member of the genus clinical features, varying from an asymptomatic (30–60 %),
Phlebovirus, endemic in sub-Saharan Africa and introduced to a weeklong undifferentiated febrile illness, to the severe
to Egypt and the Arabian Peninsula periodically. It is the development of hemorrhagic fever, encephalitis, retinitis,
causative agent of the mosquito-borne disease Rift Valley and potentially death (the overall case fatality rate is esti-
fever (RVF) in humans and ruminant animals. Infections mated to be between 0.5 and 2 %). The most frequent course
with RVFV generally result in self-limiting febrile disease of the disease is a self-limiting febrile illness; less than 2 %
but can also manifest in more severe illness, such as hemor- of human infections result in severe disease (hemorrhagic
rhagic fever, encephalitis, or retinitis. Currently, there is no fever, encephalitis, or retinitis). During the 1977–1978 out-
commercially available vaccine for human use. break in Egypt, approximately 200,000 human cases with
600 deaths were estimated to have occurred [153], and an
Descriptive Epidemiology estimated 27,500 RVFV infections occurred during the
The geographic distribution of RVFV covers much of Africa, 1997–1998 outbreak in the Garissa district of Kenya [263].
ranging from Egypt to South Africa and from Senegal to Infected patients can develop a mild form of RVF, which
Madagascar. The first confirmed outbreak of RVFV outside is characterized by a feverish syndrome with a sudden
Africa was reported in 2000 in Saudi Arabia and Yemen [3, onset of flu-like fever, muscle pain, joint pain, and head-
255]. RVFV has the potential to infect a remarkable number ache. Additional symptoms can include neck stiffness, pho-
of different vectors, including mosquitoes, ticks, and flies. tophobia, loss of appetite, and vomiting. These symptoms
However, mosquitoes, especially floodwater Aedes mosqui- normally last from 4 to 7 days. However, in a small percent-
toes, are thought to be the principal vector and play an age of patients, the development of one or more of the three
important role in RVFV epizootics, which frequently occur distinct syndromes can occur: ocular disease (0.5–2 %),
at times of unusual high precipitation [141]. In general, the meningoencephalitis (less than 1 %), or hemorrhagic fever
vectors can be classified into two groups: (1) reservoir vec- (less than 1 %). Ophthalmologic complications remain very
tors (Aedes species (spp.)) and (2) amplifying vectors (Culex important sequelae of human RVF disease and can result in
spp.) [82, 83, 91]. RVFV persists in the environment through long-lasting visual disturbance in affected patients [129]. It
vertical transmission in mosquitoes and horizontal transmis- has also been described that in its most severe form, RVF can
sion by mosquitoes among animals [174]. Dambos are lead to acute renal dysfunction [69].
thought to play a central role because they flood during A majority of RVF patients suffer from a self-limiting
heavy rainfall. During interepidemic periods RVFV was iso- and mild to often subclinical febrile illness, with an incuba-
lated from unfed mosquitoes, demonstrating that the virus tion time of typically 4–6 days [55, 80, 119, 147, 162, 232].
can be maintained between epidemics through transovarial Symptoms typically include severe chills, weakness, malaise,
transmission in mosquitoes [142]. Heavy rainfall and flood- throbbing headache, and dizziness [80, 120]. Further, the
ing increases the hatching of eggs and explosive population development of a sensation of fullness over the liver region
9 Bunyaviruses: Hantavirus and Others 179

has been described [80]. Following these symptoms, patients While in some cases a partial improvement in vision over
present with fever, decreased blood pressure, proximal large- time is established [210, 218, 226, 227], in many patients,
joint arthralgia (particularly shoulders, elbows, and knees), no complete recovery of vision occurred and chorioretinal
and anorexia followed by nausea and vomiting. A lowering scarring remained in macular and paramacular areas [4, 10,
of the body temperature can be observed between the 3rd and 59, 85, 210, 226, 227].
4th day after onset of symptoms, which then returns back While there is much documentation of the symptoms of
to a normal level. However, some patients can either main- severe RVF illness, much more work is still needed to better
tain their fever as long as 10 days, or within 1–3 days after describe specific manifestations of RVF in humans. This will
the recovery, a recurrence of high fever can be observed [80, be necessary to allow clinicians to recognize the disease in
162]. This biphasic febrile phase is normally combined with its early stages and to be able to limit and ultimately prevent
a severe headache. During the febrile period, viremia can be its spread.
detected, as well as neutralizing antibodies, which appear
around day 4 after onset of symptoms [80, 205]. Patients can Diagnosis
also develop a massive coronary thrombosis after the febrile RVFV belongs to a large group of RNA viruses endemic in
phase [162, 219]. Africa, which have the potential to cause viral hemorrhagic
Patients who develop one of the severe forms of RVF fever (VHF), including Lassa, Ebola, Marburg, Crimean-
(jaundice, hemorrhagic fever, or neurological disease) are at Congo hemorrhagic fever, and yellow fever viruses.
increased risk of fatality [130, 147]. Hemorrhagic manifesta- Infections by RVFV and other VHF viruses are clinically
tions are typically characterized by a sudden onset of illness, difficult to recognize, if no hemorrhagic or specific organ
including fever, throbbing headache, lethargy, malaise, vom- manifestations are present. Diagnosis of RVFV infection is
iting, nausea, vague mid-epigastric discomfort, and body often performed by combining the analysis of clinical signs
pain. Further, these patients may present with a low blood of disease and available diagnostic testing. RVF may be sus-
pressure, hematemesis, diarrhea, jaundice, macular rash over pected when there is an occurrence of abortions in domestic
the entire trunk, subconjunctival hemorrhage, and bleeding ruminants and death of young animals, associated with an
from the gums and/or gastrointestinal mucosal membrane outbreak of febrile illness in human with headache and myal-
[120, 237, 264]. Elevated aspartate aminotransferase (AST), gia. In the laboratory, the diagnosis of RVF is achieved by
lactate dehydrogenase (LDH), and alanine aminotransferase a variety of techniques, such as virus isolation from whole
(ALT) and reduction of platelet count and hemoglobin level blood, serum, or tissue samples [7]; viral antigen detec-
are typical [5, 237]. Death can occur within 3–17 days after tion [154, 168]; detection of specific antibodies [138]; and
onset of these symptoms, and fatal RVF cases show diffuse amplification of viral nucleic acids [64, 89, 115], which are
hepatic and gastrointestinal necrosis [1, 237]. preferable due to the rapid and reliable analysis of samples
RVF patients developing encephalitis may present with for RVFV. Multiplexed PCR, reverse transcription (RT)-PCR
a sudden onset of fever, rigor, retro-orbital and throbbing enzyme hybridization assays, quantitative (q)RT-PCR, and
headache, mild arthralgia of the knees and elbows, and real-time detection PCR are very useful techniques, since
bilateral retinal hemorrhage [6, 120, 145]. After onset of the detection can be performed simultaneously for many hem-
illness, patients eventually experience neck rigidity, confu- orrhagic fever viruses [25, 64, 89, 102, 115, 211]. Further,
sion, hypersalivation, fatigue, malaise, stupor, and coma, the real-time RT-loop-mediated isothermal amplification
and temporal or permanent vision loss has been described (RT-LAMP) assay demonstrated its practicability in a field
as well [6, 145, 249]. Indicative of a possible development laboratory, since it does not rely on thermocycler equip-
of meningitis or meningoencephalitis was the increased ment and only requires a one-step, single-tube reaction [132,
number of white blood cells (mainly lymphocytes) in the 188]. To prevent potentially false-negative results due to the
cerebrospinal fluid (CSF). Patients with a developing RVF- rather short viremic phase during which RVFV genomes can
induced encephalitis can also present with a decreased level be detected, diagnosis should be combined with the detec-
of consciousness [6]. Histopathological lesions in the brains tion of RVFV-specific antibodies. Viral antigen (such as the
of these patients were characterized by focal necrosis asso- RVFV nucleoprotein) can rapidly be detected in blood and
ciated with infiltration of lymphocytes and macrophages tissue samples by the use of immunohistochemistry staining
and perivascular cuffing [249]. of tissue samples or enzyme-linked immunosorbent assays
As described previously, in some cases, RVF patients can (ELISA) [117, 154, 168, 268]. Shortly after exposure to
develop a maculopathy or retinopathy (0.5–2 %), which can RVFV, serological tests, such as detection of type-specific
affect either one or both eyes and can occur either very early IgG and IgM antibodies, can also be performed [182, 183,
or months after infection [59, 210, 228]. Patients reported to 185, 186]. Additional serodiagnostic tests for the detec-
experience a blurry vision and noticed floating black spots tion of RVFV are hemagglutination inhibition, indirect
within their visual fields or even the loss of central vision. immunofluorescence, and virus neutralization test (VNT)
180 A.N. Freiberg et al.

[184, 185, 239]. However, VNT as a diagnostic tool has to epidemiology. However, infection with Toscana virus is the
be handled with care, because RVFV induces a long-lasting third most frequent cause of aseptic meningitis between May
neutralizing immunity and previously infected individuals and October in central Italy, where it was originally isolated
will also give a positive result. from sandflies in 1971. Toscana virus has since been detected
in many countries close to the Mediterranean area, such as
Treatment and Prevention France, Spain, Slovenia, Greece, Cyprus, and Turkey. In all
Currently, no licensed vaccines and specific treatments are Mediterranean countries Toscana virus should be included in
available for RVF in humans. Studies in monkeys and other the differential diagnosis of viral meningitis during the warm
animal models for RVF infection have demonstrated promise summer season.
for ribavirin as an antiviral drug for future use in humans
[187]. However, ribavirin might have unexpected side Descriptive Epidemiology and Clinical Features
effects. Further, studies have suggested that interferon, Toscana Virus
immune modulators, and convalescent-phase plasma may Toscana virus (TOSV) is an arthropod-borne bunyavi-
also help in the treatment of patients with RVF [187]. rus, which is transmitted to humans by Phlebotomus,
A formalin-inactivated vaccine (TSI-GSD-200) is the only Sergentomyia, and Lutzomyia genera, in particular by
RVFV vaccine presently available for use in humans [195] Phlebotomus perniciosus and P. perfiliewi. The virus was
but, currently, only for military personnel, veterinarians work- isolated in 1971 from P. perniciosus in Monte Argentario
ing in endemic areas, high-containment laboratory workers, (Tuscany) [235–255], but it was first recognized as a caus-
and others at high risk for contracting RVFV. Another strat- ative agent of neurological disease in humans only in 1983,
egy of controlling RVF outbreaks is vaccination of livestock when it was isolated for the first time from a young woman
to prevent epizootics. The currently used live Smithburn vac- with lymphocytic meningitis [167]. Transmission of the
cine is only partially attenuated and leads to high abortion virus occurs transovarially in the insect vectors, and it has
rates or teratology (10–25 %) of pregnant animals [50, 121, been suggested that the reservoir of TOSV is most likely
235] and exhibits pathogenicity in European cattle [27]. In the vector itself. However, a progressive decline of vector
addition, the risk of reversion to full virulence precludes its infected rates over many generations suggests that TOSV
use in countries where RVFV is not known to be endemic cannot be maintained exclusively by vertical transmission
[235]. A live attenuated vaccine (RVFV MP-12) is efficacious [48, 49, 242], but its animal reservoir has not been identified
in livestock. However, RVFV MP-12 may have similar safety yet. TOSV isolation from the brain of an insectivorous bat
concerns associated with the Smithburn strain, since RVFV (Pipistrellus kuhlii) has so far been the only evidence of the
MP-12 can be teratogenic in sheep [114]. RVFV MP-12 was possible involvement of this species in the ecology of the
recently tested in human clinical trials, to determine adverse virus [49, 254, 255].
effects in humans using a single injection dose, with prom- TOSV is the major cause of aseptic meningitis (95 %)
ising results [24]. A 95 % seroconversion rate was reported and meningoencephalitis (4.5 %) and influenza-like ill-
with a high titer in a plaque reduction neutralization test. In ness during the summer season, with a peak of incidence in
addition, a genetic analysis of RVFV MP-12 isolated from August [103, 214]. Most cases of the TOSV infections have
vaccinated individuals showed no reversions of vaccine virus been reported in either residents or travelers in central Italy
in attenuated regions compared to wild-type RVFV. Overall, or Spain. However, over the last years, an increased num-
many promising RVFV vaccines (including subunit and DNA ber of cases have also been recorded in other countries in
vaccines, as well as viruslike particles) and therapeutic con- the Mediterranean area, such as Portugal, France, Cyprus,
cepts are currently employed to generate an urgently needed Turkey, and Greece [67, 179, 189, 213, 215]. It has also been
safe and efficacious countermeasure against RVFV. described that TOSV can result in asymptomatic infection
Infection with RVFV can be prevented by decreasing the and infection without involvement of the central nervous
chance of contact with mosquitoes and other bloodsucking system, such as febrile erythema or influenza-like illness
insects through the use of mosquito repellents and bed nets. [103, 197]. Recent reports of TOSV infections included
An important protective measure for persons working with unusual clinical manifestations or severe sequelae, such as
animals in RVF-endemic areas is avoiding exposure to blood hydrocephalus, epididymo-orchitis, and ischemic complica-
or tissues of animals that may potentially be infected. tions [13, 103, 150, 197, 213].
Serological analysis conducted in Italy (particularly in
3.2.2 Sandfly Fever Viruses Tuscany) revealed a TOSV seroprevalence of 77.2 % in for-
Sandfly fever viruses are transmitted in the Mediterranean estry workers, compared to 22.7 % of seropositivity in the
area by the sandflies Phlebotomus papatasi, P. perniciosus urban population [247]. Moreover, this serological analysis
and P. perfiliewi. Human infections with Naples and Sicilian underlined the presence of asymptomatic infections of TOSV
sandfly fever viruses cause self-limiting disease of unknown in exposed people. Preexisting immunity also seems to play
9 Bunyaviruses: Hantavirus and Others 181

an important role in limiting TOSV-caused illness, and cases onset of symptoms and before the IgM seroconversion occurs).
with reinfection have not been detected so far [52, 53]. In most cases, the diagnosis is based on serological investiga-
A recent retrospective study on the antibody prevalence tion of acute and early convalescent sera. ELISA for the detec-
rates of TOSV among adults and children was performed in a tion of SFV antigen can be performed in reference laboratories.
population (n = 2,737) living in Tuscany during 1999 to 2006 Within the SFSV and SFNV antigenic complexes, serological
[241]. This study revealed that the seroprevalence rate was cross-reaction exists. VNT using early convalescent sera still
19.8 % in adults and 5.8 % in children, showing an age- remains the reference method to specifically identify the
dependent increase in TOSV-specific immunity. Furthermore, viruses or to assess the antibody response specificity.
the study indicated that asymptomatic TOSV infection is more
frequent in young people (91 %) than in adults (31.4 %). A Treatment and Prevention
correlation of the seroprevalence to the clinical profile showed There is no specific therapeutic available for infections
that a higher incidence of severe signs of neurological disor- with SFV and treatment is symptomatic. Individual pro-
ders was found in adults, but it is still unclear why this occurs. tective measures such as insect repellents and insecticide-
Interestingly, a seroprevalence study conducted in volunteer impregnated mosquito bed nets are recommended.
blood donors in France showed that 12 % of sera from healthy
donors and 18.9 % of sera from patients hospitalized for CNS 3.2.3 Severe Fever with Thrombocytopenia
infection were IgG positive for TOSV. These findings con- Syndrome Virus (SFTSV)
firmed that TOSV circulates in southeastern France and that a Recently, a new member of the Phlebovirus genus has been
significant proportion of healthy blood donors have a history of identified in China (Hubei and Henan provinces), after a
TOSV infection [56]. This indicates that a potential risk of heightened surveillance of acute febrile and life-threatening
transmitting the virus to naïve subjects is possible and can raise illness in China. The novel virus was isolated from patients
concerns about potential implications for blood donors. who presented with fever, thrombocytopenia, leukocytope-
nia, and multiorgan dysfunction and termed severe fever with
Sandfly Fever Sicilian and Naples Viruses thrombocytopenia syndrome virus (SFTSV) [266]. The clini-
The sandfly fever Sicilian virus (SFSV) and sandfly fever cal symptoms were initially considered to resemble those of
Naples virus (SFNV) serotypes have the widest geographic human anaplasmosis [270]; however, neither bacterial DNA
distribution which is related to the distribution of the vector (P. nor antibodies could be detected in blood samples.
papatasi), and these viruses have been isolated from sandflies
in Africa, Central Asia, and Europe [18, 51, 54, 60, 79, 116, Descriptive Epidemiology
128, 179, 209, 244]. Serological investigations performed on The majority of affected patients were farmers living in
the indigenous populations and tourists visiting temperate wooded and hilly areas, and they were working in the fields
regions demonstrated that sandfly activity normally peaks dur- before onset of clinical signs of disease. SFTSV RNA was
ing the summer months when the human population in these detected in ticks of the Ixodidae family (species Haemaphysalis
areas is exposed to sandfly-transmitted diseases [32, 40, 62, longicornis) that were collected from domestic animals where
66–68, 71, 72, 113, 231]. SFSV and SFNV were first described the patients lived, and these ticks may be a candidate vector for
in Italy in 1943–1944 during outbreaks of influenza-like ill- SFTSV. High seroprevalence of SFTSV was detected among
ness in US soldiers [244]. goats (up to 83 %) and cattle (up to 32 %) but also in pigs (up
SFSV and SFNV cause the so-called 3-day fever or pap- to 5 %), dogs (up to 6 %), and chicken (1 %) in SFTSV-
pataci fever. Infected patients present with influenza-like endemic regions [272]. SFTSV caused cytopathic effect in
symptoms, including fever, myalgia, malaise, and retro- DH82 cells after inoculation with white blood cells isolated
orbital pain [29, 256]. Usually, these patients recover fully from a 42-year-old patient were subsequently isolated. In cell
within 7 days. Even though infections with these viruses are culture, SFTSV can infect a variety of cells, such as Vero E6,
mild and self-limiting, patients are highly incapacitating dur- Vero, and L929 cells; however, cytopathic effect could only be
ing the course of the illness. detected in DH82 cells. Phylogenetic analysis of six isolated
Newly identified SFVs include the sandfly fever Turkey SFTSV strains indicated that all isolates cluster together and
virus [33, 127], which is an SFSV variant; Punique virus, a that they were nearly equidistant from the sandfly fever group
related virus to SFNV [273]; and Massilia virus, which is a and the Uukuniemi group, suggesting that SFTSV is the pro-
new member of the SFNV complex [41]. totype of a third group within the genus Phlebovirus.

Diagnosis Clinical Features

The direct diagnosis of SFVs can be performed by virus isola- The major clinical symptoms include fever (temperatures
tion or RT-PCR from the blood or CSF but is only possible of >38 °C), fatigue, conjunctival congestion, thrombocy-
during early stages of infection (i.e., within the first 2 days after topenia (platelet count of <100,000 per cubic millimeter),
182 A.N. Freiberg et al.

gastrointestinal symptoms, abdominal pain, diarrhea, pro- real-time RT-PCR assay [233]. Patient sera can also be
teinuria, hematuria, and leukopenia. Frequently, regional tested for the presence of antibodies to SFTSV by indirect
lymphadenopathy was observed. In most patients, multiorgan fluorescence assay (IFA), and sera reactive at a dilution of
failure develops rapidly, indicated by elevated levels of AST, ≥1:64 were considered to be positive [143]. Virus neutral-
ALT, CK, LDH, and CRP [58, 88]. The appearance of CNS ization assays (modified microneutralization assay) can be
symptoms (including apathy, lethargy, convulsions, muscu- performed to detect IgG-specific antibodies to SFTSV [16,
lar tremor, and coma) as well as hemorrhagic fever (bleeding 266]. High levels of neutralizing antibodies are generated
from the mouth mucosa, lungs, and gastrointestinal lumen) during the convalescent phase of the illness.
has been described [87, 88, 266, 269]. Dynamic profiling of
laboratory findings in SFTS patients showed that clinical pro- Treatment and Prevention
gression of the disease occurs in three stages [88]. The first There is no specific treatment available for infections with
stage or fever stage (days 1–7 after onset of illness) is char- SFTSV. Clinical management is directed toward a symptom-
acterized by marked thrombocytopenia and leukopenia and atic treatment. Individual protective measures such as insect
low platelet counts and peripheral white blood cell counts. repellents and avoidance of areas where ticks may be com-
Lymphocyte levels substantially decrease during the first 9 mon are recommended.
days, suggesting that these cells are mainly involved in leu-
kopenia. Coagulation tests indicated that prolonged aPTT 3.2.4 Heartland Virus
occurred on days 5–9. Cardiac and liver enzymes, such as Recently, a new pathogenic phlebovirus has been identified
ALT, AST, LDH, CK, and CK-MB, were elevated, indicative in northwestern Missouri (USA), named the Heartland virus.
for heart and liver impairment. The second stage (or multi- Two patients were independently hospitalized in 2009 with
organ dysfunction stage) of the disease is defined to occur fever, diarrhea, headache, anorexia, nausea, elevated hepatic
between days 7 and 13 after disease onset and characterized aminotransferase levels, thrombocytopenia, moderate neu-
by development of multiorgan dysfunction. In survivors viral tropenia, and leukopenia [152]. Both patients were bitten
load decreases, while it remains elevated (up to 108 viral by ticks approximately 5–7 days prior to onset of signs of
RNA copies/mL) in patients with fatal outcome. Further, in illness. After discharge from the hospital, both patients
fatal cases, platelet counts continued to decline, and serum reported fatigue and difficulties with short-term memory.
tissue enzymes further increased, reaching maximum levels Blood was negative for Ehrlichia and Rickettsia and elec-
before death. A recent study by Deng and colleagues ana- tron microscopy identified viruses isolated from patient
lyzed the cytokine and chemokine profile in 57 patients and leukocytes to be consistent with members of the bunyavi-
found that levels of TNF-α, IP-10, and IFN-γ were elevated rus family. Next-generation sequencing then revealed that
in patients with severe disease [58]. These findings suggest the viruses were new members of the genus Phlebovirus.
that a cytokine-mediated inflammatory response, mediated Isolates from both patients were highly identical, indicat-
through an imbalance in cytokine and chemokine production, ing that both patients were infected independently with the
might result in a severe disease progression. The third stage same virus strain. Further, phylogenetic analysis identified
or convalescent stage describes the recovery phase for surviv- that the Heartland virus is closely related to the recently
ing patients after day 13 and is characterized by the return identified tick-borne phlebovirus SFTSV in China [266].
of clinical parameters to normal physical levels. In summary, Interestingly, serum samples were strongly positive for IgG
the key risk factors contributing to a patient’s death have been antibodies 2 years after the onset of illness. Future studies
identified as increased viremia; elevated AST, ALT, LDH, will need to be performed to identify the vector and poten-
CK, and CK-MB levels past the fever stage; and the appear- tial hosts of Heartland virus, as well as epidemiologic and
ance of CNS symptoms and manifestation of hemorrhagic ecological studies.
tendency, DIC, and multiorgan failure. The average case
fatality rate is 12 % but can be as high as 30 % [266].
Initially, no epidemiologic evidence of human-to-human 3.3 Nairovirus Genus
transmission of SFTSV was detected [266]. However, recent
publications indicated that the virus can be transmitted from The Nairovirus genus was named after Nairobi sheep dis-
person-to-person through personal contact with the index ease, an acute hemorrhagic gastroenteritis in sheep, which
patient, while no exposure to suspected animals or vectors was first recognized in the early twentieth century in Nairobi,
was reported [16, 87, 143]. Kenya. The genus consists of at least 34 strains/serotypes
that have been divided into seven species/serogroups. All
Diagnosis viruses in the Nairovirus genus are exclusively transmitted
Direct diagnosis of SFTSV RNA in patient serum can be per- by ticks, although a few isolations have been made from
formed by real-time PCR from the blood or by quantitative Culicoides flies and mosquitoes.
9 Bunyaviruses: Hantavirus and Others 183

Annual Number of
Cases Reported to WHO
Virological or
= Serological Evidence
With Vector Presence

= 5-49 Cases Per Year

= 50+ Cases Per Year

Fig. 9.1 Geographic distribution of CCHF. Countries in red report atum and Hyalomma asiaticum is demonstrated by a gray, dashed line.
more than 50 human cases annually to the WHO, and those in orange The figure is based upon that created by the WHO (http://www.who.int/
report fewer than 50 cases. Countries in yellow have not reported csr/disease/crimean_congoHF/en/) and on tick distribution maps at
human cases, but CCHFV has been isolated, or its presence has been www.kolonin.org and http://www.efsa.europa.eu/en/efsajournal/
inferred from serological studies, and a transmission-competent tick pub/1723.htm
vector is also present. The northernmost limit of Hyalomma margin-

3.3.1 Crimean-Congo Hemorrhagic Fever Virus hemorrhagic fever virus (CCHFV) is extensively distributed
The most important human pathogen in this genus is in wild and domestic mammals, birds, and ticks throughout at
Crimean-Congo hemorrhagic fever, a zoonosis, which does least 30 countries in Western Asia, Southeast Europe, Middle
not cause disease in its animal host, but results in a severe East, and Africa regions [108, 23] (Fig. 9.1).
hemorrhagic syndrome in humans. The distribution pattern approximates that of the predominant
tick vector of the genus Hyalomma and is the largest among the
Descriptive Epidemiology tick-borne viruses, second of all medically important arboviruses
Crimean-Congo hemorrhagic fever (CCHF) is an acute, often only to that of the dengue viruses [90, 261]. The incidence of
fatal, tick-borne zoonosis and is among the most important CCHF cases has increased over the past decade, including
emerging viral hemorrhagic disease of man. Crimean-Congo reports of cases for the first time in some countries such as
184 A.N. Freiberg et al.

Fig. 9.2 Confirmed CCHFV cases in Turkey since 2002 1600

(Source: Turkish Ministry of Health) Deaths
1400
Cases
1200

1000

800

600

400

200

0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Turkey or India [123, 181]. This has been attributed to anthropo- gingival bleeding, and bleeding from the nose, vagina, uterus,
genic factors such as habitat fragmentation as well as possible or urinary tract may occur, as well as internal bleeding in
climate change. In affected countries, typically a few dozen con- abdominal muscles. Cerebral hemorrhage and massive liver
firmed cases are reported, but annual spikes three to four times necrosis indicate a poor prognosis. Hepatomegaly and sple-
higher occur sporadically with no apparent pattern or synchrony nomegaly may occur in up to 40 % of the patients. Deaths
between countries. Presumably, a combination of biological fac- generally occur on days 5–14 of illness and are attributed to
tors, both intrinsic and extrinsic, may trigger these spikes [75]. hemorrhages, hemorrhagic pneumonia, or cardiovascular dis-
Interestingly, the emerging epidemiologic characteristics of turbances. Lethal cases typically do not develop an antibody
CCHF in Turkey, where the disease was first diagnosed in 2002, response. The mortality rate has been reported to average
appear to differ from those reported previously. Since 2002, the around 30 %, but may range from about 5 % to more than 80 %
case numbers have increased exponentially and still continue to [261]. The variation in rates is believed to reflect the difference
remain high with a total of over 6,330 cases in the last 10 years in diagnostic capabilities or treatment of patients between out-
(Fig. 9.2). It is suspected that difference in ecology of the disease break areas as well as the genetic variation of CCHFV strains
might have contributed to this emergent trend [76]. resulting in different levels of virulence. The convalescence
period usually begins 10–20 days after the onset of illness and
Clinical Features recovery can take up to a year. Patients may experience gener-
CCHF viral apparent infection has been well documented with alized weakness, tachycardia, temporarily loss of all their hair,
an onset of a mild febrile illness that may progress to severe poor appetite, difficulty breathing, polyneuritis, loss of hearing
and often fatal hemorrhagic disease [148]. CCHF has a dis- and memory, and impaired vision [236]. In contrast, convalesc-
tinct course of infection in humans with four different phases: ing patients during recent outbreaks in Turkey and Iran did not
incubation, prehemorrhagic, hemorrhagic, and convalescence present with any of the latter symptoms [148]. The mortality
period [73]. The length of the incubation period for the illness rate is among the highest of all the viral hemorrhagic diseases,
varies and precise data is difficult to obtain. The incubation ranging from about 5 % to more than 80 %.
period appears to depend on the mode of acquisition and the
dose of the virus. Following infection via tick bite, the incuba- Diagnosis
tion period is usually short, ranging on average between 3 and Preliminary diagnosis is based on clinical features and rele-
7 days. The incubation period following contact with infected vant travel or exposure history. Laboratory diagnosis can be
blood or tissues is usually 5–6 days, with a documented maxi- made serologically by the detection of IgM-specific antibod-
mum of 13 days [73]. Clinical manifestations in the prehemor- ies and detection of viral nucleic acids or through virus isola-
rhagic period include fever, malaise, myalgia, dizziness, and, tion; however, appropriate biocontainment precautions should
in some patients, diarrhea, nausea, and vomiting lasting for an be taken. Virus-specific immunohistochemical testing of
average of about 3 days. The hemorrhagic phase usually begins autopsy tissues may aid in confirming the diagnosis [23].
on days 3–5 after the onset of illness and is usually short, last-
ing on average 2–3 days. The first evidence of disease is usu- Treatment and Prevention
ally a flushing of the face and the pharynx and a skin rash that At present, there is no internationally licensed vaccine, and
progresses to petechiae and ecchymoses, hemorrhage of the treatment is only supportive [74]. Ribavirin has been used
mucus membranes and conjunctiva, hematemesis, melena, epi- for treatment; however, efficacy has not been clearly estab-
staxis, hematuria, and hemoptysis [73]. Cerebral hemorrhage, lished [230]. Research has been hampered due to the need of
9 Bunyaviruses: Hantavirus and Others 185

a maximum biocontainment laboratory (BSL-4) to study the by acute fever, a tubular renal lesion, and in some instances
virus and lack of an animal model. a life-threatening capillary leak syndrome, often accompa-
nied by hemorrhagic manifestations. Hantaviruses found in
3.3.2 Other Nairovirus Infections the Americas cause hantavirus pulmonary syndrome, also
Dugbe Virus known as hantavirus cardiopulmonary syndrome, a severe
Dugbe virus (DUBV) was first isolated in 1964 in Nigeria acute disease associated with the onset of febrile illness that
from Amblyomma variegatum ticks [35]. Serologically, rapidly progresses to life-threatening respiratory failure and
DUBV is related to Ganjam virus, first isolated in 1954 in shock.
India from Haemaphysalis intermedia ticks and later also
from man. DUBV has since been recovered on many occa- 3.4.1 Descriptive Epidemiology
sions in Western Africa from ticks, domestic cattle, mosqui- Hantaviruses are maintained in nature through chronic
toes, and Culicoides. Despite the large number of isolations, infection of small mammals, primarily rodents, although a
serosurveys did not reveal widespread human infections, and number of novel hantaviruses have recently been found asso-
isolations from blood of humans were only made occasion- ciated with shrews and moles [122]. Transmission to humans
ally, mainly children, with benign febrile illness including a is by aerosol following exposure to infectious rodent excreta
laboratory infection. One patient had transient meningitis and occasionally by bite [94, 246]. Human infection is often
and virus was isolated from the cerebrospinal fluid [92, 157]. seasonal, occurring in temperate zones during the fall when
rodents may invade households, during warmer months
Nairobi Sheep Disease when summer cabins may be first occupied, and through
Nairobi sheep disease (NSD) is a noncontagious, tick-borne rural occupational exposure as seen among farmers, hunters,
infection of sheep and goats characterized by hemorrhagic gas- and field-deployed military. Hantaviruses are natural para-
troenteritis, abortion, and high mortality caused by NSD virus sites of small mammals, and this genus is apparently unique
(NSDV). Antibodies against NSDV have been detected in in the Bunyaviridae, which otherwise are uniformly bio-
human serum, but it is not known if these antibodies are the logically transmitted by arthropods. Routes of transmission
result of an NSDV infection or have been caused by a yet among small mammals are not known, although laboratory
unidentified agent. An apparently naturally acquired clinical experiments have clearly documented the susceptibility of
case was reported from Uganda in which a young man from laboratory rats to aerosol exposure [172], and a strong corre-
which the virus was isolated experienced transient clinical signs lation exists among free-living urban rats between increased
[125]. However, no serological conversion was demonstrated. evidence of wounding and rising antibody prevalence rates,
suggesting that transmission by bite may be important [94].
Erve Virus Depending on the virus and host, transmission may occur in
Erve virus (ERVEV) was isolated in 1982 from the tissues of rural, urban, or occupational (such as from laboratory rats)
a white-toothed shrew (Crocidura russula) collected in the settings. Person-to-person transmission is not known to exist
Erve river valley in northern France and is only distantly among Old World hantaviruses; however, the New World
related to the other nairoviruses [42]. The virus has been cause of HPS, Andes virus, has been associated with person-
described in the Czech Republic, Germany, France, and the to-person transmission [260]. Other New World hantaviruses
Netherlands. ERVEV has been suspected to cause severe do not appear to be transmitted in this manner. At least 20
headache and intracerebral hemorrhage in humans [42]. Due genetically and antigenically distinct hantaviruses are known
to the lack of commercial diagnostic test, only very little is to cause human disease (Table 9.3), and many other hanta-
known about the involvement in human disease. viruses with unknown pathogenic potential for humans have
been found associated with various species of small mam-
mals including shrews and moles.
3.4 Hantavirus Genus Systematic population-based surveys to establish anti-
body prevalence rates for hantavirus infection have not
Many distinct hantaviruses have been associated with human been widely attempted; however, sufficient examination of
illness since the isolation of prototype Hantaan virus in 1978 selected “at-risk” populations has been conducted to offer
and recognition of the new genus Hantavirus within the fam- a good indication of the global distribution of the hantavi-
ily Bunyaviridae [138, 217]. Hantaan virus is the causative ruses. Methods employed include immunofluorescent anti-
agent of Korean hemorrhagic fever (KHF) in Korea and body (IFA) assays, most often using prototype Hantaan
epidemic hemorrhagic fever (EHF) in China. Hantaviruses virus-infected cell cultures as antigen; enzyme immunoas-
likely occur around the world; however, to date they are best says (EIA); and radioimmunoassays (RIA). Populations
known in Asia, Europe, and the Americas. Human disease surveyed are generally rural, frequently with high potential
caused by hantaviruses in Asia and Europe is characterized for occupational exposure to rodents (farmers, woodcutters,
186 A.N. Freiberg et al.

Table 9.3 Old and New World hantaviruses causing hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS)
and their known distribution and hosts
Distribution and
subfamily of rodent host Virus Distribution Primary host species Human disease
Old World Hantaan virus China, Korea, Russia Apodemus agrarius HFRS
Murinae Dobrava (Belgrade) virus Balkans Apodemus flavicollis HFRS
Seoul virus Global Rattus sp. HFRS
Saaremaa virus Europe Apodemus agrarius HFRS
Amur virus Far East Russia Apodemus peninsulae HFRS
Arvicolinae Puumala virus Europe, Russia Myodes (Clethrionomys) glareolus HFRS (nephropathia
epidemica)
New World Sin Nombre virus North America Peromyscus maniculatus HPS
Sigmodontinae Monongahela virus North America Peromyscus leucopus HPS
New York virus North America Peromyscus leucopus HPS
Black Creek Canal virus North America Sigmodon hispidus HPS
Bayou virus North America Oryzomys palustris HPS
Choclo virus Panama Oligoryzomys fulvescens HPS
Andes virus Argentina, Chile Oligoryzomys longicaudatus HPS
Bermejo virus Argentina Oligoryzomys chocoensis HPS
Lechiguanas virus Argentina Oligoryzomys flavescens HPS
Maciel virus Argentina Bolomys obscures HPS
Oran virus Argentina Oligoryzomys longicaudatus HPS
Laguna Negra virus Argentina, Bolivia, Calomys laucha HPS
Paraguay
Araraquara virus Brazil Bolomys lasiurus HPS
Juquitiba virus Brazil Oligoryzomys nigripes HPS
Condensed from Jonsson et al. [118]

foresters, and others); patients with clinically compatible or NE is increasingly diagnosed as an important cause of acute
interchangeable disease (e.g., leptospirosis); or unselected disease [248]. Incidence of human infection with Puumala
blood donors. About 12,000 sera from the European parts virus increases following mast years, and recent studies in
of the Russian Federation were examined by RIA, with anti- Germany demonstrated multiple local outbreaks of distinct
bodies found in a geographically focal pattern, generally virus clades associated with simultaneous increases in densities
increasing in prevalence with age, and most common in oil and infection rates of voles in different regions [77]. Seoul
production and forestry workers or tractor and truck drivers virus, cause of a less severe form of HFRS, is thought to be
[163]. Men were more likely to have antibodies than women nearly global in its distribution based on serological surveys of
(1.3:1 to 2:1), and this ratio increased in the clinically ill to peridomestic rodents [135, 136], but extensive tests for this
3:1. In a subsequent study in Bashkortostan, antibody preva- specific virus in humans have not been widely attempted out-
lence among over 9,000 persons reached over 16 % in some side of China. In the United States, however, surveys were con-
adult age groups. Seropositive children have been infre- ducted among residents of Baltimore, Maryland, where inner-
quently identified in virtually all serological surveys. city rats are known to be heavily infected with a Seoul-like
The distribution pattern that has emerged from various sur- virus [44, 45, 46]. An antibody prevalence rate of 0.25 % was
veys suggests that Hantaan virus is most abundant in Asia, found among 6,060 persons with no known risk factors for
including China, where the endemic areas are thought to be hantavirus infection except residence in Baltimore [96]. This
expanding, and the Korean Peninsula. Puumala virus, cause of rate was significantly different from the rate found among
nephropathia epidemica, is most abundant across a broad band patients with proteinuria (1.46 %; OR, 3.23; p < 0.05) and the
from Norway, northern Sweden, Finland, and through the rate among dialysis patients with end-stage renal disease
Russian Federation to the Ural mountains. Antibody preva- (2.76 %; OR, 5.03; p < 0.05). Overall, 6.5 % of patients with
lence rates in these endemic areas approach or exceed 10 % end-stage renal disease due to hypertension were seropositive
[169, 170, 221]. Elsewhere in Western Europe, antibody prev- for a hantavirus, suggesting that hantavirus infection may be
alence rates are lower, but clinical cases and positive serologi- associated with hypertensive renal disease. Canadian blood
cal results have been found in most European countries; and in donors had 1.4 % prevalence of antibodies to hantaviruses
areas such as France, Belgium, and Germany, where active (3.5 % in the Maritime Provinces), but the exact strain of
research programs exist and clinicians recognize the disease, infecting virus was not determined [139].
9 Bunyaviruses: Hantavirus and Others 187

3.4.2 Clinical Features lated life risk of being hospitalized with NE, suggesting that
Hemorrhagic Fever with Renal Syndrome (HFRS) mild or asymptomatic infections are not uncommon [124].
Acute hemorrhagic and nephropathic clinical syndromes The case fatality rate for NE due to Puumala virus in Sweden
were described and variously named decades before their was estimated at about 0.2 % [220]. In the Balkan region of
causative agents were identified. As cited by Casals et al. Europe, the viruses that cause both severe HFRS and rela-
[34], records of severe, often fatal, hemorrhagic fever have tively mild NE coexist, although the total disease burden gen-
been discovered in Vladivostok, Eastern Siberia, as early as erally does not exceed several hundred cases annually. More
1912, and it seems likely that similar syndromes, albeit often than 100 clinical and subclinical infections associated with
confused with other causes of fever and hemorrhage, were occupational exposure to laboratory rats infected with Seoul
known in Asia and Europe several centuries prior to this time. virus have been recognized in Japan, and additional cases
Modern scientific descriptions are credited to Soviet and have occurred in Korea, the former Soviet Union, Belgium,
Japanese scientists working in Siberia and Manchuria in the and the United Kingdom. HFRS caused by Hantaan, Dobrava,
1930s [163]. English-speaking physicians first encountered or Amur virus is more severe, while Puumala and Seoul virus
this disease during the Korean conflict in the early 1950s and infections are generally milder.
named it Korean hemorrhagic fever (KHF). Modern Chinese Clinical HFRS in its classic severe form is highly distinct.
and Japanese authors prefer the term “epidemic hemorrhagic By far the best description of the disease in English is that writ-
fever” (EHF). A milder form of illness, with minor hemor- ten by a medical commission during the Korean conflict [222].
rhage and lower mortality, was recognized in Scandinavia, Distinct phases of evolution were described, and although not
also in the early 1930s, and termed “nephropathia epidemica” all patients exhibited each of them, the pattern accurately
(NE) [164]. Today, hemorrhagic fever with renal syndrome reflected significant physiological changes that characterized
(HFRS) is generally used to refer to the diseases caused by Hantaan virus infection (Table 9.4). The incubation period of
Old World hantaviruses. HFRS has been variously estimated at 10–35 days. The longer
Mortality in HFRS generally ranges from <5 to 15 % and intervals, in particular, are of significance both in terms of
is reduced by recourse to dialysis and proper management of pathogenesis and for their implications with respect to occur-
patients who experience renal shutdown [34]. Between 1950 rence of unusual illness among travelers who may be infected
and 2007, over 1.5 million cases of HFRS and over 46,000 on one continent and sick on another.
deaths (3 %) were reported in China with the highest case
fatality rates above 14 % in 1969; however, for the last decade Hantavirus Pulmonary Syndrome (HPS)
mortality rates for HFRS in China have maintained at approx- In May 1993, an outbreak of an apparently new disease char-
imately 1 % [271]. Case fatality rates of 10–15 % were acterized by unexplained adult respiratory distress syndrome
reported from the Russian Federation for the periods 1978– occurred among residents of rural southwestern United States.
1992, with peak annual incidence rates of over 11,000 cases This disease became known as hantavirus pulmonary syn-
(8.0 per 100,000 population) (World Health Organization drome (HPS) or sometimes hantavirus cardiopulmonary syn-
1993) and annual incidence rates of 50 cases per 100,000 in drome (HCPS). The original outbreak was centered in the Four
Bashkortostan [170]. Mortality in the former Yugoslavia dur- Corners region, where the states of New Mexico, Arizona,
ing an outbreak in 1989 was 6.6 % (15 deaths of 226 cases) Utah, and Colorado join, and primarily affected young, previ-
[95]. Dobrava virus is found in Greece, Bulgaria, Albania, the ously healthy adults. By October 1993, 42 cases had been con-
former Yugoslavia, and some foci elsewhere in Europe where firmed, including one retrospectively diagnosed from July
it occurs relatively infrequently but produces an especially 1991. Median age of patients was 32 years (range 12–69 years)
severe form of HFRS with mortality rates as high as 30 % in and 52 % were males. American Indians accounted for 55 %
some areas [9, 95, 135, 136]. Incidence rates have dropped in of the cases; of the remainder, 36 % were non-Hispanic whites,
recent years for prototype Hantaan virus in Korea to reports 7 % were Hispanic, and 2 % were black. The mortality rate
of a few hundred cases annually. Less severe NE-like disease was 62 %, and cases were identified from 12 states [36, 37].
occurs in several hundred patients each year in Scandinavia, Epidemiologic investigations quickly determined that small
and increasing numbers of cases are now recognized in rodents in the outbreak area were especially abundant in the
Western Europe. The highest incidence rates in Sweden spring and early summer of 1993, and virological examina-
exceeded 20 cases/100,000 population in endemic areas [169, tions of captured rodents found Peromyscus maniculatus to be
221], while the prevalence of serum IgG antibodies ranged the species most frequently infected. Route of transmission
from about 2 % in non-endemic areas to around 8 % in from rodents to humans was assumed to be by the aerosol
endemic regions [168] [169]. The incidence of clinical cases route, similar to other hantaviruses. The examination of PCR-
and antibody prevalence in an endemic area of Sweden were amplified products from human cases and from P. maniculatus
compared, and the antibody prevalence rate in the oldest age captured in and around patient’s homes found identical hanta-
groups was found to be 14–20 times higher than the accumu- viral sequences at a given site but surprisingly high levels of
188 A.N. Freiberg et al.

Table 9.4 Characteristic course of clinical disease for hemorrhagic fever with renal syndrome
Phase Duration Predominant signs and symptoms Laboratory findings
Febrile 3–7 days Fever, malaise, headache, myalgia, back pain, abdominal pain, WBC = normal or ↑
nausea, vomiting, facial flush, petechiae (face, neck, trunk), Platelets = ↓
conjunctival hemorrhage Hematocrit = ↑
Urine = proteinuria 1 + → 3+
Hypotensive 2 h–3 days Nausea, vomiting, tachycardia, hypotension, shock, visual WBC = ↑ with left shift
blurring, hemorrhagic signs, ± oliguria (late) Platelets = ↓↓
Bleeding time = ↑, PT may be ↑
Hematocrit = ↑↑
Urine = proteinuria 4
+ hematuria 1 +
Hyposthenuria
BUN and creatinine = increasing
Oliguric 3–7 days Oliguria ± anuria BP may ↑; nausea and vomiting may persist; WBC = normalizes
1/3 with severe hemorrhage (epistaxis, cutaneous, GI, GU, CNS) Platelets = normalize
Hematocrit = normalizes, then ↓
Urine = proteinuria 4 +
Hematuria 1 + → 4 +
BUN and creatinine = ↑↑
Na+↓, K+↑, Ca2+↓
Diuretic Days to weeks Polyuria = 4 + (3–6 l daily) BUN and creatinine =
Normalize electrolytes
Possibly abnormal (diuresis)
Urine = normalizes
Convalescent Weeks to months Strength and function regained slowly Anemia and hyposthenuria = may
persist for months
From McKee et al. [151] Reproduced with permission
Phases as seen in KHF. All phases may not be present in a given patient

genetic diversity among samples taken from different sites tic of HPS [146]. Incubation period is 9–33 days, with most
[166]. Investigators using the same PCR techniques on pre- onsets occurring between 14 and 17 days post exposure to
served tissues of P. maniculatus captured in 1983 in California small mammal reservoir hosts [265]. A detailed examination
likewise found infected rodents, indicating that this virus is not of HPS in Brazil was characteristic of HPS as seen elsewhere
“new” or recently mutated [222]. Peromyscus maniculatus is in the Americas and reported a typical clinical course associ-
one of the most common and widely distributed rodents in ated with a febrile prodrome with fever, myalgia, and worsen-
North America. Subsequently, many other hantaviruses have ing thrombocytopenia, headache, backache, and other
been identified from small mammals throughout the Americas, symptoms, followed by dyspnea, cough, and tachycardia along
from Canada to Argentina and Chile, many of which cause with low blood pressure, leading to shock in about a third of
HPS-like clinical disease, often resulting with fatalities. Each patients. More than half the patients developed renal failure and
recognized hantavirus appears to be predominantly associated about half had mild hemorrhagic disturbances. Reduced plate-
with a particular species of small mammalian host and occu- lets, metabolic acidosis, hemoconcentration, and leukocytosis
pies a distinct ecological habitat. were documented in more than half the patients seen. Diffuse
Hantavirus pulmonary syndrome is found in the New World bilateral rales were seen in the majority of cases. Patients were
and characterized by rapid onset of pulmonary edema, soon typically admitted 3–6 days post onset of symptoms, and fatali-
followed by respiratory failure and cardiogenic shock. Many ties occurred within a day or two after admission. Survivors
New World hantaviruses are capable of causing HPS, with the typically remained hospitalized for more than a week [118].
most severe disease typically associated with Sin Nombre
virus, isolated during the original North American outbreak in 3.4.3 Diagnosis
1993, and Andes virus, recognized predominantly in Chile and The serological diagnosis of hantaviral infection in humans
Argentina; however, many other New World hantaviruses is typically done by detection of IgM- and IgG-specific anti-
found elsewhere in the Americas are known to cause serious bodies, often using recombinant N protein as antigen, which
human infection similarly associated with severe morbidity is the most abundant viral protein and induces a strong
and high mortality rates. Case fatality rates for HPS are usually humoral response. Both traditional indirect IgG and IgM
about 40 %. New World hantaviruses infect lung microvascular enzyme-linked immunosorbent assays (ELISAs) and IgM
endothelium causing microvascular leakage that is characteris- capture ELISAs are used routinely. Virtually all patients are
9 Bunyaviruses: Hantavirus and Others 189

IgM positive by the onset of acute illness [137]. Rapid strip ratory function [155]. Beneficial effect of treatment with the
immunoblot assays have also been developed [106]. antiviral drug ribavirin has been reported for HFRS patients
Immunoblot and neutralization tests have been used for sero- in China when administered early in the course of illness
logical diagnosis, and focus reduction neutralization assays [110]; however, due to the rapid progression of HPS and
and plaque reduction neutralization assays have been used the fact that the diseases are rarely recognized until after
for virus-specific serological diagnosis and classification of the onset of cardiopulmonary involvement, ribavirin has
hantaviral isolates. Molecular diagnostics such as reverse not had a beneficial effect on the treatment of HPS patients
transcription PCR can rapidly detect hantaviral genomic [156]. A role for antiviral drugs in the prophylaxis of inti-
sequences in clinical specimens early in infection and even mate contacts of patients infected with Andes virus could
prior to onset of acute illness [78, 178]. Direct isolation of be beneficial, but has yet to be demonstrated. A number of
hantaviruses from acute human specimens or from tissues of hantavirus vaccines have been tested in humans, including
suspect small mammalian hosts is difficult and time- both rodent brain-derived and cell culture-derived formalin-
consuming. Hantaviruses are slow to adapt to growth in cell inactivated vaccines. Most have targeted Hantaan or Seoul
culture, and indeed only a few cell lines are known to support virus, either individually or in combination, although at least
hantaviral growth. Most infected cell cultures do not exhibit one candidate DNA vaccine has included Puumala virus. In
cytopathic effect, the exception being HEK293 cells [149]; addition, vaccinia-vectored vaccines expressing Gn, Gc, and
consequently, the presence of infectious virus must be estab- N viral proteins and plasmid DNA delivered by gene gun
lished by demonstration of specific viral antigen or nucleic have been tested (reviewed in [216]). Hantavax® is a com-
acids, typically by direct IFA assay or polymerase chain mercially available suckling mouse brain-derived formalin-
reaction (PCR). Primary isolation has been most successful inactivated vaccine marketed in Korea; however, its efficacy
when a combination of techniques was employed [126]. A has not been demonstrated [180]. Inactivated vaccines for
note of caution: Although asymptomatically infected, labo- Hantaan and Seoul viruses have also been produced in China
ratory animals may shed infectious virus in their excreta and with neutralizing antibody produced in over 90 % of those
saliva. Consequently, isolation attempts and experimentation receiving three doses [63].
that utilizes laboratory animals should only be carried out
under appropriate biocontainment conditions that minimize
the risk of aerosol transmission between animals and humans. 4 Unresolved Problems
BSL-2 practices, containment equipment, and facilities are
recommended for laboratory handling of sera from persons The bunyaviruses exist in nature through silent transmission
potentially infected with hantaviruses, and potentially cycles that typically involve an animal vector (arthropod or
infected serum or tissue samples should be handled in BSL-2 rodent) and only occasionally result in human infection, often
facilities following BSL-3 practices and procedures. Cell occurring in rural environments where human contact with
culture-virus propagation and purification should be carried potentially infectious vectors is more likely to occur. Those
out in a BSL-3 facility using BSL-3 practices, containment exposed are frequently residents of less developed regions of
equipment, and procedures. Experimentally infected rodent the world who may lack access to modern medical facilities
species known not to excrete the virus can be housed in and resources leading to a definitive diagnosis or effective
ABSL-2 facilities using ABSL-2 practices and procedures. treatment. Populations at greatest risk are often those least
Primary physical containment devices including BSCs able to afford preventive vaccines, diagnostic tests, or effec-
should be used whenever procedures with potential for gen- tive therapeutics should they be available for these low inci-
erating aerosols are conducted. All work involving inocula- dence diseases, and as a result few economic incentives exist
tion of virus-containing samples into rodent species for commercial investment in the development of the critical
permissive for chronic infection should be conducted at tools needed to address the diseases caused by bunyaviruses.
ABSL-4 [38]. Inexpensive, accurate diagnostic tests are needed to better
define the incidence of human infection. With the availabil-
3.4.4 Treatment and Prevention ity of better diagnostics, a greater appreciation of the full
The pathogenesis of both HFRS and HPS is complex and impact of bunyavirus infections on human and animal health
is associated with an intense immune activation that leads will become apparent and may lead to investments in vaccine
to changes in vascular permeability. Aggressive support- production or discovery of therapeutic interventions.
ive care and careful clinical management of fluid levels are Much remains to be learned regarding the natural history
essential to the successful treatment of critically ill patients. of the bunyaviruses, including how they exist in nature and
HPS cases may quickly progress to respiratory failure and interact with their vector; the molecular mechanisms devel-
shock that requires extracorporeal membrane oxygenation oped by the vector to limit virus spread; and why some vec-
(ECMO) and careful monitoring of cardiac output and respi- tors efficiently maintain and transmit these viruses, while
190 A.N. Freiberg et al.

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still needed to better understand how these viruses survive Saudi Arabia: a clinical study of severe illness in humans. Clin
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 Coronaviruses
10
Arnold S. Monto, Benjamin J. Cowling,
and J.S. Malik Peiris

1 Introduction encouraged more broadly based research on the agent and its
prevention and control. In the process, two new respiratory or
Coronaviruses of humans have been classified as a subfamily endemic coronaviruses have been identified as causing human
of the Coronaviridae family. The viruses are roughly spheri- infections, which appear to resemble in epidemiologic char-
cal, enveloped particles 120–160 nm in diameter. Their name acteristics those previously known. Very recently, MERS
derives from the characteristic “crown”-like projections on (Middle East respiratory syndrome) coronavirus, a novel
their surface, approximately 20 nm long. They are positive- human betacoronavirus lineage C virus, has been discovered
sense, single-stranded RNA viruses, are sensitive to heat and zoonotically infecting humans in Middle Eastern countries.
lipid solvents, and have a distinct replication strategy com- In light of the differences between the endemic respira-
mon to other viruses in the order Nidovirales [1–3]. tory and the SARS coronaviruses in terms of epidemiology
Coronaviruses have in the past been divided into three groups. and clinical expression, they will be covered separately in
In part, because of increasing work on coronavirus discovery much of this chapter.
in the wake of the outbreak of severe acute respiratory syn-
drome (SARS) in 2003, a number of new coronaviruses of
humans (hCoVs) have been identified. This has resulted in 2 Historical Background
the three groups being reclassified as genera of the
Coronavirinae subfamily. Before the SARS outbreak, while it Coronaviruses were first identified from domestic and labora-
was recognized that the coronavirus infected many different tory animals before they were identified in humans. Infectious
species, particularly domestic and laboratory animals, their bronchitis virus of chickens was actually isolated in embryo-
extreme diversity in nature was not fully appreciated. nated eggs in the 1940s. The late recognition of these viruses
Although severe diseases of differing characteristics were was, in large part, because of difficulty in recovering the
known to occur in animals, in humans the viruses were human viruses using standard cell culture techniques [4]. The
thought to cause only acute respiratory infection which were first human coronaviruses were identified by different tech-
generally mild. All this changed with the recognition that niques in the United Kingdom and the United States at approx-
SARS was caused by a coronavirus (SARS-CoV). This sig- imately the same time. The British Medical Research Council’s
naled that coronaviruses could produce lethal disease and Common Cold Research Unit had been studying fluids col-
lected from persons with natural respiratory infections by
standard cell culture isolation methods and by inoculating
A.S. Monto, MD (*)
them into human volunteers. Rhinoviruses or other cytopatho-
Department of Epidemiology, School of Public Health,
University of Michigan, 1415 Washington Heights, Ann Arbor, genic agents could be recovered from a portion of the fluids
MI 48109-2029, USA [5]. There was an additional, substantial portion from which
e-mail: [email protected] no agents could be isolated but that could still cause colds in
B.J. Cowling, PhD, FFPH the volunteers. Organ cultures of human embryonic trachea or
Division of Epidemiology and Biostatistics, nasal epithelium were then used in an effort to detect the recal-
School of Public Health, The University of Hong Kong,
citrant viruses present. A specimen, B814, that had been col-
21 Sassoon Road, Pokfulam, Hong Kong SAR
lected in 1960 from a boy with a common cold had not yielded
J.S.M. Peiris, DPhil
a virus on inoculation into cell culture. After the specimen had
Division of Public Health Laboratory Sciences,
School of Public Health, The University of Hong Kong, been passaged serially three times in human tracheal organ
21 Sassoon Road, Pokfulam, Hong Kong SAR culture, it could still cause colds on inoculation into volun-

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 199

DOI 10.1007/978-1-4899-7448-8_10, © Springer Science+Business Media New York 2014
200 A.S. Monto et al.

teers, which indicated that replication had taken place [6]. In cell culture as well; these distinct viruses have since been lost
Chicago during the winter of 1962, five agents were isolated in and may actually have been rediscovered recently.
primary human kidney cell cultures from specimens collected Work on the coronaviruses of humans proceeded slowly,
from medical students with common colds. The viruses were with debate about how frequently the viruses caused lower
ultimately adapted to WI-38 cultures and exhibited a type of rather than upper respiratory disease. The methodological
cytopathic effect (CPE) not previously seen. A prototype problems of working with them were a major impediment, as
strain, 229E, was selected for characterization and was found was their apparent involvement only in a relatively mild dis-
to be RNA containing, ether labile, and 89 nm in diameter but ease. All this changed when in 2002 SARS appeared first in
distinct serologically from any known ortho- or paramyxovi- China and then in other countries of the world. The near panic
ruses. Sera collected from the five medical students all exhib- resulted because of its transmission characteristics, case fatal-
ited a fourfold rise in neutralization antibody titer against ity, and the fact that the agent was initially unknown. That last
229E [7]. It became clear that these “novel” viruses were of aspect was solved quickly with the identification of the caus-
more than passing significance when organ culture methods ative agent as a new coronavirus. The knowledge that the virus
were added to standard cell culture techniques in a study of emerged from a zoonotic reservoir spurred investigation of its
acute respiratory infections of adults conducted at the National possible source. Small mammals (civet cats) in live game-ani-
Institutes of Health (NIH). Six viruses were found that grew in mal markets in Guangdong were identified carrying closely
organ but not cell culture and were ether labile; on electron related viruses. This led to the identification of these animals as
microscopy, the agents were shown to resemble avian infec- amplifier hosts and to the game-animal wet markets as an inter-
tious bronchitis virus (IBV) in structure, and these represented face where zoonotic infection of humans was being initiated
a distinct family of viruses [8]. The B814 and 229E strains [15]. The natural reservoir was later identified to be Chinese
were soon also demonstrated to have a similar structure on horseshoe bats [16, 17]. The SARS epidemic ended following
electron microscopy and to develop in infected cells by bud- the use of various population control measures and unexpect-
ding into cytoplasmic vesicles [9, 10]. As a result of the simi- edly has not recurred. However, increased attention to the coro-
larity of the human agents to IBV and also to mouse hepatitis naviruses continued globally. New coronaviruses were
virus (MHV), they were collectively considered to represent a identified in various animal hosts, and two additional coronavi-
group of vertebrate viruses distinct from the myxoviruses anti- ruses were identified in humans, the first since 229E and OC43.
genically and structurally [11]. The name coronavirus was NL63 was identified in Amsterdam in 2004 from a 7-month-
eventually adopted for the group to describe the fringe of pro- old child with febrile bronchiolitis [18]. The same virus was
jections seen around them on electron microscopy [1]. Except also isolated at almost the same time by the group in Rotterdam
for 229E, none of the human coronaviruses had been success- from an 8-month-old child with pneumonia [19]. The second
fully propagated in a system other than organ culture. McIntosh virus, HKU1, was detected in a specimen collected from a
et al. reported successful adaptation of two of the NIH isolates, 71-year-old man from China with pneumonia and then from
OC (organ culture) 38 and OC43, to the brains of suckling another adult with the same diagnosis [20]. With the develop-
mice [12]. These strains were shown to be essentially identical ment of real-time PCR techniques for all four human respira-
antigenically but quite distinct from MHV. Only OC38 and tory coronaviruses, it has now become possible to identify them
OC43 could be so adapted; the other four OC strains resisted in many situations. Such identification is now typically done
such attempts. The IBV was known to exhibit hemagglutina- not only for these viruses but also for a variety of other respira-
tion under certain conditions, but no such phenomenon had tory agents. This has meant that coronaviruses are now detected
been demonstrated for the human strains until OC38 and as much in clinical settings as in epidemiologic studies.
OC43 were adapted to mice. Kaye and Dowdle found that the
infected brain preparations would directly and specifically
agglutinate red cells obtained from chickens, rats, and mice 3 Methodology
[13]. This technique greatly expanded the ability to do sero-
epidemiologic studies, since it was simple and reproducible. 3.1 Sources of Mortality Data
Subsequent developments included adaption of OC38 and
OC43 to growth in cell monolayers; either mouse brain or Until the occurrence of SARS, coronaviruses of humans were
organ culture material could be used as sources of virus [9]. not thought to cause death, except, possibly, in those with
Not only was CPE available for reading of neutralization tests, underlying conditions. This was in contrast to the situation in
but also the OC38 and OC43 viruses were found to hemadsorb animals, where infections were sometimes fatal, depending
red cells of rats and mice, making available a more precise on the particular virus. Since the SARS episode, the major
means of evaluating endpoints in tests involving these organ change which has affected data on the respiratory coronavi-
culture-derived strains [14]. The other OC strains and B814 ruses has been more ready detection using the polymerase
that could not be adapted to mouse brain resisted adaptation to chain reaction (PCR) technique, so that infections are
10 Coronaviruses 201

now recognized in those who might previously have not been for determining the overall impact of illness [22–24]. While
studied. This has especially been the case in hospitalized indi- the older population-based studies were limited to 229E,
viduals, particularly those who will likely experience severe OC43, or both, they are useful since there are few recent
outcomes, such as the immunocompromised. It is also known investigations that give the same information. They were
from earlier studies that coronavirus frequently infects small conducted in different settings but in some cases contempo-
children and reinfects adults, including persons with chronic raneously which allows direct comparison. In fact, almost no
respiratory disease [21]. It would be logical to assume that studies include all four recognized coronaviruses and cover
deaths could occur in these most susceptible segments of the more than a single year. This makes it difficult to discuss
population, but they are probably not very frequent. year-to-year variation in frequency of activity as well as sea-
A problem during SARS was in defining the specificity of sonality, as was possible with the older data. Such studies are
infection, whether inapparent, mild, or fatal. This was related therefore of continuing interest, at least as background, in
to the lack of a readily available diagnostic test in many areas determining the long-term occurrence of the viruses. These
where outbreaks occurred. To a large extent, cases were clas- original investigations were typically broadly based, with
sified using a clinical case definition. In some cases that sur- coronavirus infection forming part of overall evaluations
vived, there was an attempt at serological confirmation. of the role of viruses in general in respiratory illnesses. As
indicated in the selected listing in Table 10.1, a variety of
different open and closed populations were used for these
3.2 Sources of Morbidity Data studies. The 229E strain was originally isolated from medi-
cal students in Chicago as part of a long-term study of respi-
Since the coronaviruses of humans, other than the SARS ratory illnesses in young adults [7, 25]. Employee groups
virus, usually produce illnesses indistinguishable from those were the source of specimens in the NIH [26, 27] and in the
caused by other respiratory viruses, it is not possible to define studies at Charlottesville, Virginia [30]. Infection was also
morbidity in the absence of laboratory identification. Before evaluated in children’s homes [28] and boarding schools [5],
PCR became available, it was difficult directly to identify among military recruits [34], and among children hospital-
the infecting virus; thus, besides anecdotal reports, most of ized for severe respiratory illnesses in various parts of the
the epidemiologic studies were based on identifying rises world [26]. Serological methods were used to detect occur-
in antibody titer. In contrast, since development of the PCR rence in persons with acute exacerbations of asthma [33] or
technique, direct identification has become relatively simple. chronic obstructive respiratory disease [21]. Patterns of coro-
However, this seeming advance has often been accompanied navirus infection were identified among the general popula-
by the use of the method to determine the incidence of infec- tion residing in the Tecumseh, Michigan, community as part
tion in population groups and to define characteristics of of a longitudinal study of respiratory illness [31, 32]. Also
associated disease or even seasonality. The major problem included in Table 10.1 are more recent studies using PCR to
is the short duration of many of the available studies and the identify infection. The previous studies, based on serology,
concentration on hospitalized individuals. As a result, while often did not characterize infections identified clinically; in
it is possible to say these viruses can sometimes cause hos- fact, challenge studies of volunteers were employed early, to
pitalization and to infer the particular clinical diagnoses they determine characteristics of illnesses, because of problems
produce, it is difficult to estimate what proportion of overall associated with direct isolation [35, 36]. The more recent
illnesses are severe. A small number of studies have been studies were able to characterize the illnesses clinically but
population based and have produced the only data available have other limitations as indicated above.

Table 10.1 Selected longitudinal studies on the epidemiology of coronavirus infection in humans
Location Population Virus studied Years
Chicago, IL [25] Medical students 229E 1961–1967
Washington, DC [26, 27] Hospitalized children 229E, OC43 1962–1967
Bethesda, MD [26, 27] Adult employees 229E, OC43 1962–1967
Atlanta, GA [28, 29] Institutionalized children 229E, OC43 1960–1967
Charlottesville, VA [30] Working adults 229E, OC43 1962–1970
Tecumseh, MI [31, 32] General community 229E, OC43 1965–1970
Denver, CO [33] Hospitalized asthmatic children 229E, OC43 1967–1969
N. and S. Carolina [34] Military 229E, OC43 1970–1972
Nashville, TN, and Rochester, NY Hospitalized children <5 years of age 4 viruses 2001–2003
Melbourne, Australia [23] Preschool children NL63 2003–2004
Edinburgh, Scotland [22] Medically attended illnesses—general population 4 viruses 2006–2009
202 A.S. Monto et al.

3.3 Serological Surveys monolayers of human embryonic lung fibroblast cells such
as WI-38 and MRC-5. A cytopathic effect of small, granular,
Relatively simple serological technique was available for the round cells appears at the periphery of the cell monolayer
first two coronaviruses identified (229E and OC43), and sur- [7]. Although these cells can be used for the cell-adapted
veys of antibody prevalence were carried out in various parts prototype 229E virus strain, primary isolation of new
of the world. Many surveys formed a part of studies directed 229E-like agents remains difficult. The human embryonic
mainly toward determination of incidence of infection. intestine (MA177) semicontinuous cell line has been used
Information on the prevalence of antibody was available for for the primary isolation of other 229E-like viruses [26].
populations in the United States [27, 30, 31], the United Human coronaviruses OC38 and OC43, not related to 229E,
Kingdom [36], Brazil [37], and other parts of the world. A were originally isolated in organ cultures of human trachea
special situation was determining the meaning of the pres- or lung [6, 8, 43, 44]. These two strains are similar and have
ence in man of antibody against coronaviruses of animals. been further adapted to replicate in suckling mouse brain and
The finding of mouse hepatitis antibodies in military recruits to primary monkey kidney and BS-C-1 cell cultures [9, 12,
and in children and adults from the general population was 14]. Another cell system, LI32, a heteroploid human lung
surprising when first described in 1964 [38]. It is now recog- line, has been reported to be suitable for primary isolation of
nized that this did not indicate past experience with MHV 229E, a related virus (LP), and the B814, the first-described
but rather with human coronavirus strains known to cross- organ culture agent [45, 46]. This last finding has not been
react with it. Similarly, antibodies in human sera against the confirmed by other workers [9]. Similarly, MRC-C cells
hemagglutinating encephalomyelitis virus of swine and the have been used for 229E-like viruses and human rhabdo-
coronavirus of calf diarrhea also appear to represent cross- myosarcoma cells for propagating 229E and OC43 [47, 48].
reactions with OC43 or related strains [29, 39]. In contrast, Human coronavirus NL63 was initially isolated in African
in a survey of antibodies to avian IBV, none could be found green monkey kidney cells (LLC-MK2) [18]. Human colon
in a military population. Low-level antibodies were detected carcinoma cells (CaCo-2) have recently been shown to be
only in a portion of subjects who had close contact with more susceptible to NL63 infection and show more promi-
poultry [40]. The virus is not known to cross-react with the nent cytopathic effect [49]. HKU1 was initially discovered in
human strains. More recently, following a gap in active work 2005 by “broad-range” (primers designed to detect all known
on the epidemiology of coronaviruses, ELISA methods have coronaviruses, rather than being specific for known corona-
been developed for at least some of the viruses [41, 42]. virus types) reverse transcriptase PCR (RT-PCR) [20], and
These have not been as much used as the older techniques, new isolates remain difficult to grow reproducibly in the
given the availability of PCR, and may not be as specific to laboratory. However, it has been successfully cultured in
the particular strain. human ciliated airway epithelial cells (HAE) [50].
SARS-CoV was initially isolated in 2003 on African
green monkey kidney epithelial cells (VeroE6) and in fetal
3.4 Laboratory Methods rhesus kidney cells [51, 52] during the SARS epidemic in
2003. VeroE6 cells, which are deficient in interferon induc-
3.4.1 Viral Identification tion, continue to be the cells of choice for culturing SARS-
Laboratory diagnosis can be achieved by identification of the CoV at present. Following isolation of this novel agent,
virus in clinical specimens, using virus isolation or antigen electron microscopy and molecular methods (random primer
or molecular detection techniques. Identification of infection PCR and virus detection arrays) followed by partial genetic
can also be accomplished by detecting a host antibody sequencing led to its identification as novel coronavirus.
response. Relevant specimens for virus detection are typi- Recently, MERS, a novel human coronavirus of the beta-
cally respiratory specimens, such as nasopharyngeal aspi- coronavirus lineage C, has been detected in patients from
rates, washes or swabs, nose or throat swabs, and, when Middle Eastern countries with severe pneumonia and renal
available, endotracheal aspirates or bronchoalveolar lavages. dysfunction. Vero and VeroE6 cells are suitable for primary
In the case of SARS, in which disseminated infection may isolation of this virus from clinical specimens [53].
occur, viral RNA could also readily be detected in the feces Continuous transformed cell lines do not mimic the phys-
and in the serum or plasma. iological state of cells in tissues in vivo, and this may be the
Although PCR-based methods are becoming the “gold reason why many coronaviruses are difficult to culture in
standard” in diagnosis, virus isolation is indispensable for vitro. The use of primary cells from the relevant species,
characterizing virus, studying pathogenesis, determining cells differentiated in vitro in air–liquid interface cultures
susceptibility to antivirals, and developing novel antivirals and ex vivo organ cultures (as was used in the early days of
and vaccines. The human coronavirus 229E was originally virology), may be needed for the isolation of more fastidious
isolated in cell culture and later adapted to roller culture viruses, since some animal viruses are more readily isolated
10 Coronaviruses 203

in culture although they are species-specific in their in vitro 3.4.4 Serological Tests
growth characteristics, especially on primary isolation [54– The demonstration of rising antibody titers between acute
57]. Embryonated egg culture has been used as a host system and convalescent sera to a specific viral antigen provides evi-
for avian coronaviruses [58]. However, none of the plethora dence of recent infection, while the detection of antibody in
of bat coronaviruses detected by RT-PCR have been readily seroepidemiologic surveys provides evidence of past infec-
cultured in vitro to date, even in primary bat epithelial cell tion. Methods that can be used for serodiagnosis have
lines [59]. included the complement fixation test, neutralization test,
indirect immunofluorescent (IF) assay, and enzyme-linked
3.4.2 Antigen Detection immunosorbent assay (ELISA). Hemagglutination-inhibition
Immunofluorescence tests on cells from the respiratory tract (HI) and gel-diffusion tests are less frequently used
(e.g., nasopharyngeal aspirates or swab) using commercially nowadays.
available reagents [60] or polyclonal [61] or monoclonal Neutralization test can also be used on coronaviruses that
antibodies to 229E and HKU1 [62, 63] have been reported, only grow in organ cultures [8]. Neutralizing antibodies
but these are not widely used. Such antigen detection tests could be detected as early as 5–10 days after symptom onset
can also be used for the diagnosis of SARS-CoV infection during SARS infection [76]. The seroprevalence for SARS-
[62]. Several ELISA systems have been developed to detect CoV in asymptomatic children and adults living in high- and
coronaviruses including coronavirus 229E [64] and the low-risk regions in Hong Kong in 2003 showed that subclini-
nucleocapsid (N) or spike (S) proteins of SARS-CoV in cal infection was rare [77, 78]. Pseudotyped virus expressing
respiratory samples [65, 66]. the SARS spike protein can also be used for detecting neu-
tralizing antibody to SARS-CoV without the need for han-
3.4.3 Molecular Diagnostic Methods dling the live SARS-CoV, which has to be carried out in
Since the majority of human coronaviruses cannot be readily BSL-3 containment [79].
cultured in vitro, reverse transcriptase PCR (RT-PCR) and Indirect immunofluorescence tests use virus-infected
real-time quantitative RT-PCR have become the methods of cells fixed to inactivate virus infectivity as the solid-phase
choice for detecting and quantifying coronaviruses in clini- antigen to bind antibody in serum specimens. Anti-SARS-
cal samples and for discovering novel viruses. RT-PCR CoV antibodies present in the serum would bind to the viral
methods were used for the detection of 229E and OC43 antigens expressed on the infected VeroE6 cells; these pri-
viruses in clinical specimens [67]. There are now a number mary antibodies could then be detected by adding secondary
of commercial assays that detect a range of respiratory antihuman antibodies labeled with FITC [80]. IgM subclass
pathogens (including coronaviruses) by the use of a combi- antibodies to SARS-CoV, though declining in titer, can be
nation of PCR amplification together with nucleic acid detectable for more than 6 months after onset of disease.
hybridization in Luminex bead assay formats. These meth- There is less of a decline in titers of IgG antibodies and neu-
ods provide the opportunity for the rapid detection of a panel tralizing antibodies to the virus. Such assays can also be
of over 15 respiratory viruses including a number of corona- adapted to detect low- and high-avidity IgG antibodies both
viruses in a clinical specimen. However, the sensitivity is for discriminating early from late antibody responses and for
generally less than that provided by individual RT-PCR distinguishing anamnestic cross-reactive antibody responses
methods [68, 69]. from primary specific responses. This may be useful in some
Apart from detection of known coronaviruses, RT-PCR is clinical situations [81].
useful in virus discovery and further characterization. This ELISA assays have been also developed for detecting
includes a wide range of coronaviruses in bats detected antibodies to coronaviruses [82, 83] and have been used to
solely by such broad-range RT-PCR methods because these investigate the epidemiology of coronavirus infections [84].
viruses cannot at present be cultured [70]. For example, the The elicited antibodies detected by ELISA predominantly
first identification of HKU-1 as a cause of human disease react with the viral surface proteins rather than the ribonu-
was initially based on detection of viral RNA in clinical cleoprotein [85], and infections of 229E- and OC43-like
specimens by broad-range RT-PCR with primers designed to viruses can be distinguished in ELISA assays [86]. ELISA
detect all viruses within the coronavirus family [20]. NL63 has been used to study the seroprevalence of HKU1, showing
was discovered using the VIDISCA (virus-discovery-cDNA- an increase from 0 % in age <10 years old to a plateau of
amplified restriction fragment length polymorphism) method 21.6 % in the age group of 31–40 years old [87]. Recombinant
[18]. Amplified sequences from RT-PCR permit viral protein-based ELISA assays for detecting antibody to SARS-
genome sequence analysis, which sheds light on virus struc- CoV have been developed [88, 89]. The duration of antibod-
ture, characteristics, biological properties, phylogeny, host ies by ELISA to the SARS-CoV spike protein was long-lived
and tissue tropism, epidemiology, cross-species transmission, and paralleled neutralizing antibody responses, while those
and drug design [53, 71–75]. to the nucleoprotein was less long-lived [90].
204 A.S. Monto et al.

A protein-based line immunoassay which individually 229E and OC43 coronaviruses [86]. However, persons with
detects antibody to HCoV 229E, NL63, OC43, HKU-1, and antibody to 229E and OC43 (most of the adult population)
SARS-CoV nucleocapsid protein has been evaluated [91]. did not have cross-reacting antibody to SARS-CoV even in
Paired sera from confirmed OC43 or 229E infections and 49 immunofluorescent tests, allowing these serological tests to
convalescent sera from SARS patients showed that there was be reliably used for diagnosis and seroepidemiology of
considerable cross-reactivity between the two betacoronavi- SARS. Patients who had OC43 infections without prior
ruses OC43 and HKU1 and between the two alphacoronavi- exposure to SARS-CoV had increases of antibodies specific
ruses 229E and NL63. However, 229E- or OC43-infected for the infecting OC43 virus but not to SARS-CoV. However,
patients did not develop cross-reactive antibody to SARS- antibody responses to SARS-CoV antibody were sometimes
CoV. It is important to keep in mind such cross-reactions associated with an increase in preexisting IgG antibody titers
when evaluating the results of serological assays. It is also for human coronaviruses OC43, 229E, and NL63 by immu-
relevant that immunofluorescent assays appear to manifest nofluorescent assays. This probably reflects anamnestic
the greatest cross-reactivity. Neutralization tests are likely to cross-reactive antibody responses to coronaviruses to which
be the most specific although this has not been systemati- the patient has had prior exposure (i.e., similar to the concept
cally studied with the recently discovered coronaviruses. of “original antigenic sin”) [96, 97]. The cross-reactivity is
Historically CF or HI tests were used in epidemiologic less when purifying nucleocapsid proteins are used in ELISA
studies of coronaviruses [7]. By this method, the CF test or Western blot assays.
detected antibody in low titer and for only a short time after
infection. However, if the antigen was highly concentrated,
antibody could be detected at a higher titer, and this antibody 4 Biological Characteristics
persisted in the population so that the CF method could be
employed in surveys of prevalence [92]. An indirect HI test for 4.1 Classification
229E virus using tanned sheep erythrocytes has been described
which appears to be highly sensitive with no cross-reactions All human coronaviruses (hCoVs) are enveloped, positive-
with OC43 virus [93]. CF tests can be satisfactorily performed strand RNA viruses and belong to the subfamily Coronavirinae
with OC43 virus using infected suckling mouse brain as anti- in the family Coronaviridae, order Nidovirales. The subfam-
gen [27]. The same mouse brain material can also be used in ily Coronavirinae is further divided into three genera, Alpha-,
the HI test for OC43 antibody. In this test, the hemagglutina- Beta-, and Gammacoronaviruses, corresponding to the previ-
tion titer has been higher for rat than for chicken erythrocytes ous informal classification groups I, II, and III, respectively;
but sufficient with the chicken cells so that HI testing could there is also a recently recognized Deltacoronavirus genus
generally be employed; this is of particular importance in view [98, 99] (Fig. 10.1). The genus Betacoronavirus consists of
of the wider availability of chicken erythrocytes and the spon- four separate lineages, designated from A to D, which cor-
taneous agglutination that often complicates working with rat respond to the former subgroup 2A to D. As viruses sharing
erythrocytes. Serum to be tested did not require treatment with more than 90 % amino acid (aa) sequence identity in the con-
receptor-destroying enzyme but rather standard heat inactiva- served replicase pp1ab domain are treated as the same spe-
tion at 56 °C. The agglutination took place equally at various cies, OC43 and human enteric coronavirus (HECV) are thus
temperatures including room temperature [94]. It has also now regarded as one species (Betacoronavirus 1).
been possible to demonstrate precipitin lines on gel-diffusion At present, only members of alpha- and betacorona-
tests with coronavirus antigens concentrated 10- to 50-fold. viruses are known to infect humans. They differ from
Two or three precipitin lines were observed by Bradburne [92] each other in nsp1 protein, which is distinct in size and
in tests with hyperimmune animal or human serum, but others sequence (gammacoronaviruses have no nsp1). In addi-
have identified only one such line [94]. tion, alphacoronaviruses commonly share an accessory
The neutralization, CF, HI, gel-diffusion, and immuno- gene designated as ORF3. The type species for alpha- and
fluorescent techniques have been used in the antigenic analy- betacoronaviruses are Alphacoronavirus 1 (equivalent to
ses of the older strains of 229E and OC43 [36, 95]. porcine transmissible gastroenteritis coronavirus, TGEV,
Cross-reactive antibody responses among hCoVs have been in older literature) and murine coronavirus (equivalent to
reported. When sera from individuals experimentally mouse hepatitis virus, MHV), respectively. The viruses
infected with 229E- and OC43-like coronaviruses, including that are human pathogens are the alphacoronaviruses
organ culture viruses, were tested, they are found to cross- 229E and NL63, the betacoronavirus lineage A viruses
react within each group but not across groups. Thus, it is betacoronavirus 1 (which comprises OC43 and human
possible that the ELISA test with 229E and OC43 antigens enteric coronavirus which are now regarded as variants
may be able to detect infection with most, if not all, human of the same species) and HKU1, and the betacoronavirus
10 Coronaviruses 205

95
Miniopterus bat coronavirus 1
Miniopterus bat coronavirus HKU8
59 Miniopterus bat coronavirus HKU7
60 Myotis bat coronavirus HKU6
76 Scotophilus bat coronavirus 512
Porcine epidemic diarrhea virus Alphacoronavirus
Human coronavirus 229E
100 76 Human coronavirus NL63
Rhinolophus bat coronavirus HKU2
Feline infectious peritonitis virus
100 Transmissible gastroenteritis virus
80 Human coronavirus HKU1
100 Murine hepatitis virus
A
51
Human coronavirus OC43
100 Bovine coronavirus
Rousettus bat coronavirus HKU9 D
Betacoronavirus
SARS-related bat coronavirus
100 B
Human SARS coronavirus
Tylonycteris bat coronavirus HKU4
100 Pipistrellus bat coronavirus HKU5 C
76 Human MERS coronavirus
70 Avian infectious bronchitis virus
Gammacoronavirus
Beluga whale coronavirus SW1

63 87 Munia coronavirus HKU13

Asian leopard cat coronavirus Deltacoronavirus
100 Bulbul coronavirus HKU11
78 Thrush coronavirus HKU12

0.05

Fig. 10.1 Phylogenetic relationships of Coronavirinae. A rooted Deltacoronavirus (orange). The distinct betacoronavirus lineages
phylogenetic tree generated from nucleotide alignments of RdRp A through D were denoted. Human coronaviruses are denoted in
gene by neighbor-joining method with equine torovirus Berne as an bold (Figure is based on Refs. [1–3] and includes novel human
out-group. Four genera of coronaviruses are indicated by different betacoronavirus coronavirus 2c)
colors, Alpha- (pink), Beta- (blue), Gamma- (green), and

lineage B virus SARS-related coronavirus [98]. Recently, 4.2 Genome and Structure
MERS (Middle East respiratory syndrome) coronavirus, a
novel human coronavirus in lineage C, has been isolated. The name coronavirus comes from its appearance under
Phylogenetically, it is closely related to bat viruses previ- the electron microscope with large 20 nm petal-shaped sur-
ously detected in China and in Europe [53]. face projections (“spikes”) on a 120–160 nm spherical or
206 A.S. Monto et al.

a b Nucleocapsid protein (N)

Membrane
glycoprotein (M)

Spike
protein (S)

Envelope
protein (E)

RNA

Nature Reviews Microbiology

Fig. 10.2 Morphology of coronaviruses. (a) Electron micrograph of comprising the spike (S), membrane (M), and envelope (E) protein
severe acute respiratory syndrome-related coronavirus (SARS-related cloaks the helical nucleocapsid, which consists of the nucleocapsid (N)
CoV) cultivated in Vero cells. Large, club-shaped protrusions (spike protein that is associated with the viral linear positive-stranded RNA.
protein) form a crown-like corona that gives the virus its name (Image The lipid envelope is derived from intracellular membranes of the host
courtesy of Dr L. Kolesnikova, Institute of Virology, Marburg, cells [100]
Germany). (b) Schematic representation of the virus. A lipid bilayer

pleomorphic body resembling a solar corona [100] (Fig. 10.2). endocytosis and uncoating of the virus genome, ORF1a/b of
Lineage A betacoronaviruses display an additional surface the genome is first translated to generate replicase proteins.
projection, the 5–7 nm homodimeric hemagglutinin-esterase These replicases use the positive viral genome as the tem-
(HE) glycoprotein. The interior ribonucleoprotein looks like plate to generate full-length negative-sense RNAs, which in
either a long strand with 1–2 nm diameter or a helix condensed turn serve as templates for generating additional full-length
into coiled structures with 10–20 nm diameter. The virions are genomes (i.e., replication). In viral protein synthesis, the 3′
sensitive to heat, lipid solvents, nonionic detergents, formalde- proximal genes of the viral genome are first transcribed to
hyde, oxidizing agents, and UV irradiation [98]. segmented subgenomic negative-sense RNAs by discontinu-
The genome of coronaviruses consists of a linear positive ous minus-strand RNA synthesis. The process is initiated at
single-stranded RNA between 26 and 32 kilobases and is the the 3′ end of the genome and proceeds until they encounter
largest RNA virus genome to cause infection in humans one of the transcriptional regulatory sequences (TRS) that
[101]. The infectious genome has multiple open reading reside upstream (5′) of most ORFs. Through base-pairing
frames (ORFs), six of which are conserved across the sub- interactions, the nascent transcript is transferred to the com-
family and are arranged (in 5′–3′order) as ORF1a and 1b plementary leader TRS, and transcription continues through
(which encode for two huge polyproteins, pp1a and pp1ab) the 5′ end of the genome. Therefore, all mRNAs of a corona-
and the ORFs for structural proteins, spike (S), membrane virus characteristically contain a common 5′ leader sequence
(M), envelope (E), and nucleocapsid (N). The two polyprot- fused to a downstream gene sequence. These subgenomic
eins pp1a and pp1ab from ORF1a/b are autoproteolytically RNAs then serve as templates for positive-sense mRNA pro-
cleaved into about 16 nonstructural proteins (nsp) which duction and subsequent translation into viral proteins [101].
subsequently form into the replicase. Between the structural Three structural proteins S, E, and M are found on the
proteins S, M, E, and N lies the ORFs coding for the acces- viral lipid-membrane envelope; these are acquired as the
sory proteins whose function is not essential for virus repli- virus buds into the endoplasmic reticulum, intermediate
cation in vitro and the functions of many of them in vivo are compartment, and/or Golgi complex of the host cell [98].
still unknown. The spike protein (S) is a large homotrimeric type I mem-
As a positive-sense RNA genome, genome of coronavi- brane glycoprotein (1,128–1,472 aa). The S protein carries
ruses serves as template for both replication and viral protein the receptor binding domain and is a class I fusion protein
synthesis. Following entry into the cell by receptor-mediated that triggers fusion between viral and host cell membranes
10 Coronaviruses 207

within the endocytic vesicle, thereby releasing the genome Table 10.2 Reported frequency of infection or respiratory illness with
into the cytoplasm. The envelope protein (E) is a pentameric 229E and OC43 in four locations
integral membrane protein (74–109 aa) that acts as ion chan- Mean incidence of infection with
nel for preferential transport of different ions into the virion. Study 229E OC43
Although present in low copy number in a virion, E is signifi- Chicago medical students 15/100/year —
[25]
cantly involved in virus budding, morphogenesis, and intra-
Tecumseh, MI [31, 32] 7.7/100/year 17.1/100/year
cellular trafficking [102]. The membrane protein (M) is an
Proportion of illnesses associated with
integral type III triple-spanning membrane protein (218–263
229E OC43
aa). Being the most abundant protein in the viral envelope, it
Charlottesville, VA, 1.7 % of illnesses 2.4 % of illnesses
is essential in virus assembly within the infected cell. It employees [10]
could also interfere with host immune responses by inhibit- Atlanta, GA, children [28, 4.3 % of illnesses 3.3 % of illnesses
ing type I interferon production [103, 104]. The nucleocap- 108]
sid protein (N) is a phosphoprotein that encapsidates the
RNA viral genome to form ribonucleoprotein complex and
regulates viral replication and translation (349–470 aa). It responsible for at least 14 % of all respiratory illnesses in a
has RNA chaperone activity and also functions as an inter- general population [109].
feron antagonist [105]. In addition to these, the group A
betacoronaviruses (includes OC43/HECV and HKU1) 5.1.1 Incidence and Prevalence of 229E Virus
express an extra accessory homodimeric type I envelope gly- The frequency of 229E illness and infection was determined
coprotein, hemagglutinin esterase (HE), on its surface. It is in several long-term investigations. The activity of 229E was
related to subunit 1 of the influenza C virus hemagglutinin- found to be high in three out of 6 years of a study among
esterase fusion protein (HEF) and mediates reversible virion Chicago medical students. The mean annual incidence of
attachment to O-acetylated sialic acids [106]. infection during the total period was 15 % where the crite-
rion for identification was a reproducible twofold serocon-
version determined by CF. There was marked year-to-year
5 Descriptive Epidemiology variation in infection frequency, ranging from a high of 35 %
of those tested in 1966–1967 to a low of 1 % in 1964–1965.
5.1 Incidence and Prevalence of However, nearly 97 % of the infections occurred during the
Respiratory Coronaviruses months from January to May, often at a time when isolation
of rhinoviruses was at a low and seroconversions for 229E
Coronaviruses are of major importance in common respira- were only rarely accompanied by a rise in titer against
tory infections of all age groups. Before the identification of another respiratory agent [25].
the newer endemic human coronaviruses, NL63 and HKU-1, The serological study of 229E activity in the community
it was recognized from the earlier studies that other unidenti- of Tecumseh, Michigan, initially covered 2 years, which
fied human coronaviruses existed. It is still unknown if addi- included one period of high prevalence. As with the study
tional unidentified viruses exist, so that it is still useful to in Chicago, routine blood specimens were collected so that
discuss burden in terms of both the individual virus and coro- infection rates could be determined; however, the study
naviruses in general. Incidence of infection with 229E and group was composed of individuals of all ages living in
OC43 exhibited a marked cyclical pattern, and reported rates their homes. Over the 2 years, infections were detected
can be expected to vary based on the number of seasons of in 7.7 % of individuals tested by CF, as shown in the curve
high viral activity included in a particular study, again indi- in Fig. 10.3. However, this appeared to be an underestimate
cating the need for long-term evaluation. Table 10.2 presents of the actual activity of the virus. Serum specimens had
a summary of results obtained in four studies which estab- been collected on a regular basis, 6 months apart; rises in
lished the patterns of infection and illness [25, 28, 30, 31]. titer by CF occurred most frequently in those pairs in which
Another approach toward developing a minimal estimate the second specimen was collected in April 1967, clearly
of the total role of coronaviruses in respiratory illnesses indicating the peak period of viral dissemination. Both CF
comes from a study involving exhaustive laboratory exami- and the more sensitive N test results were combined to give
nation, including organ culture, of specimens from 38 com- an overall infection rate for the population studied; this
mon colds. Coronaviruses were isolated from 18 % of the rate, 34 %, was remarkably similar to the 35 % observed in
specimens, but an additional 13 %, which were negative in Chicago at the same time. Because of the limited period of
the laboratory, produced colds when given to volunteers viral activity, it was possible to compare illness rates of
[108]. Based on these results, which came from a limited age those infected with persons not infected matched by age
group, it has been estimated that coronaviruses could be and sex; it was estimated that 45 % of the infections had
208 A.S. Monto et al.

Fig. 10.3 Serological incidence

by CF of infection with 229E
18
virus in Tecumseh, Michigan,

PERCENT WITH ANTIBODY RISE

1966–1967 16

14

12

10

0
NOV. DEC. JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEP. OCT.
1966 1967

produced clinical disease. Thus, the rate of 229E-associated hemagglutination test. The investigation involved collection
illnesses during the outbreak was 15 per 100 persons stud- of serum specimens related to illness and also routine collec-
ied. Activity in all age groups was apparent, including tion of sera from some non-ill individuals. Frequency of
those under 25 years of age [31]. infection showed marked variation from year to year. Overall,
In other investigations of 229E activity, attention has been 4 % of colds could be associated with 229E infection, with
directed mainly toward study of associated illnesses; in such greatest association in autumn, winter, and spring [28]. A
studies, sera have been collected before and after the illness more recent study took advantage of specimens sent from
rather than continually on a routine basis as done to deter- medical facilities in Edinburgh, Scotland, for laboratory
mine infection rates. Employees at State Farm Insurance identification of infection to study the incidence of a number
Company, in Charlottesville, Virginia, were studied during of respiratory viruses identified by PCR. Overall, 229E was
an 8-year period for rises in titer for both 229E and OC43. found in only 0.3 % of those sampled, lowest of any of the
By CF, 229E infection could be related to 3 % of the colds four coronaviruses studied. This may reflect that the source
that occurred in the winter–spring and to 0.4 % of colds that of the specimens was from illnesses seen in hospitals and
occurred in the summer–fall. There was some year-to-year primary care facilities, and coronaviruses are mainly involved
variation in activity, but differences in the number of speci- in milder illnesses [22].
mens tested from various years did not permit complete Surveys of prevalence of 229E antibody have been carried
identification of cyclical patterns [30]. Employees of the out to document past history of infection, often as parts of
NIH with respiratory illness were studied by both isolation longitudinal studies. A general finding is that antibody is
and serology for 229E infection over a 6-year period. Again, present in a significant portion of adults who, despite possess-
attention was specifically directed toward certain segments ing this antibody, can subsequently experience reinfection
of the 6 years, and no specimens were tested during other and illness. Reports of antibody prevalence in adults in the
segments. Of particular interest once more is the segment United States have varied from 19 to 41 %, depending on the
from December 1966 to April 1967. Isolation of rhinoviruses type of test used to determine antibody and the time of collec-
and myxoviruses was uncommon at this time, but respiratory tion of serum [27, 30, 110]. Children under 10 years of age
illness continued to occur. During this period, 24 % of those exhibited lower mean antibody titers than older children or
persons with colds studied had rises in titer for 229E. As part adults [27, 31]. Individual sera from normal healthy adults
of the same investigation, paired blood specimens collected collected serially in Britain from 1965 to 1970 were tested by
from infants and children admitted to the hospital with acute Bradburne and Somerset. It is of interest that the proportion
lower respiratory disease during the 1967 period of 229E of sera positive by CF increased from approximately 17 % in
activity were tested for rise in antibody against the virus, but specimens collected in October–December 1966 to 62 % in
none was found [26, 27]. Healthy children institutionalized those collected in July–September 1967. This suggests that
in Atlanta, Georgia, were studied from 1960 to 1968; anti- the spring 1967 outbreak that occurred in several parts of the
body response to 229E was determined by the indirect United States may have taken place in Britain as well.
10 Coronaviruses 209

5.1.2 Incidence and Prevalence of OC43 Virus more than 50 % had antibody present. Among adults, 69 %
Populations employed to study infection and illness caused could be demonstrated to have antibody; this indicates, in
by OC43 virus were generally been the same ones employed view of the high incidence of infection with the agents in all
to study the occurrence of 229E virus. Kaye et al. [107] used age groups, the frequency with which such infections must
the group of institutionalized children in Atlanta, Georgia, to represent reinfection. The high prevalence of antibody has
identify infection by means of their HI test. Infections with been confirmed in other studies [28, 30, 112]. In Britain,
the agent were detected in all years of the study, but with Bradburne and Somerset followed prevalence of antibody for
definite cyclical variation. Seasons most involved were the OC43 over time, as they also had done with 229E [36]. Each
winter and spring. Overall, 3 % of the illnesses recorded in year, the greatest prevalence of antibody was found in the
the 7-year period could be associated with OC43 infection, winter–spring period. The single highest point in antibody
with a high of 7 % in 1960–1961. Interestingly, testing of the prevalence was in January–March 1969, at the same time the
sera collected routinely from non-ill individuals indicated OC43 outbreak was occurring in some parts of the United
that an additional equal number of OC43 infections were States [30, 38].
occurring without the production of symptoms [107]. The
Charlottesville study of adult employees was of both OC43 5.1.3 Geographic Distribution
and 229E infections. Here, too, the emphasis was on illness, Occurrence of coronavirus infection has now been docu-
and in all years studied OC43 was associated with 5 % of mented, by isolation, PCR, or serology, throughout the
colds in the winter–spring and with no illnesses in the sum- world. In earlier studies, in the United States, in addition to
mer–fall. Again, there was cyclical variation from year to the studies listed in the first part of Table 10.1, a 229E-like
year in the number of rises in titer detected [30]. virus was isolated in California, and OC43 and 229E have
The original isolations of OC38 and OC43 were made in been demonstrated to be present in many regions of the
December and January 1965–1966 as part of the study car- country [21, 113]. Extensive studies have been carried out
ried out among NIH employees with colds. Testing of sera by the Common Cold Research Unit, which have demon-
collected from these employees indicated that during this strated the presence of the agents in Britain. The activity of
period, up to 29 % of the colds studied were accompanied by 229E virus has been documented in Brazil in an early study
rise in titer for OC43. In the children hospitalized with lower of children and adults with and without respiratory illness.
respiratory disease, up to 10 % of illnesses during this period Significant rises in antibody titer accompanied respiratory
were associated with such a titer rise. However, it was impos- infection in the nonhospitalized children. Prevalence of
sible to show that the relationship to disease was truly etio- antibody was determined by CF, and like the situation in
logic. This finding was in contrast to that seen with 229E, in some studies in the north temperate zone, children had little
which no rises in titer were detected in such cases [27, 111]. antibody, whereas 26 % of adults were antibody positive
In the Tecumseh study, occurrence of OC43 infection was [37]. Later investigations have confirmed the worldwide
determined in the community population over a 4-year distribution of these agents [112, 114]. In particular, the
period: CF and HI tests were used on all specimens, and N widespread use of PCR has now allowed easy documenta-
tests were used as an aid in evaluating these results in selected tion of the activity of all the coronaviruses. In fact, one of
specimens. During the total period, OC43-related infection the four viruses now recognized, HKU1, was first identified
was detected in 17.1 % of the 910 persons studied for 1 year. in the subtropical city of Hong Kong [20]. These findings
Most of the infections took place in the winter–spring months suggest that coronaviruses are worldwide in distribution
of 1965–1966, 1967–1968, and 1968–1969. The only win- and cause similar types of illness in different localities
ter–spring period without such activity was in 1966–1967, [115], as has been noted with many other respiratory viruses
when the 229E outbreak had taken place. The 1968–1969 [116, 117].
outbreak of OC43 infection was nearly as widespread as the The newly recognized human MERS coronavirus infec-
prior 229E outbreak, with 25.6 % of the population studied tions have only occurred in Middle Eastern countries (Jordan,
showing evidence of infection. Of special note was the fact Qatar, Saudi Arabia, the United Arab Emirates) with limited
that children under 5 years of age had the highest infection secondary transmission being reported in France, Italy,
rates [32, 112]. More recently, in the Edinburgh study involv- Tunisia, and the United Kingdom. The infection is probably
ing medical care, OC43 was the most commonly identified of zoonotic origin although other scenarios cannot be com-
coronavirus but only was identified by PCR in 0.85 % of pletely excluded at present. Persons with immunosuppres-
specimens. This again may be a reflection of the source of sive conditions and other underlying diseases appear to be
the specimens. Surveys of antibody prevalence have been particularly susceptible to infection. While there have been
conducted in several settings using OC43 antigens. McIntosh significant clusters of infection in some health-care facilities,
et al. [27] found that children began to acquire antibody to MERS coronavirus appears to have limited capacity for
this virus in the first year of life. By the third year of life, human-to-human spread at present [118–120].
210 A.S. Monto et al.

229E

Chicago Medical High Activity

Students Intermediate Act.

NH Employees

Charlottesville, Va.
Employees

Tecumesh, Mich.

OC-43

Atlanta, Ga.
Children

NIH Employees

Charlottesville, Va.
Employees

Tecumseh, Mich.

1960-′61 ′61-′62 ′62-′63 ′63-′64 ′64-′65 ′65-′66 ′66-′67 ′67-′68 ′68-′69 ′69-′70
YEARS

Fig. 10.4 Cyclic behavior of 229E and OC43 viruses observed in five longitudinal studies

5.1.4 Temporal Distribution virus types. In Fig. 10.4, data are summarized from five lon-
Because most illnesses caused by coronaviruses are similar gitudinal studies of coronavirus activity carried out in differ-
to those caused by other respiratory viruses, it is impossible ent parts of the United States. In all studies, some sporadic
to identify epidemic behavior of the viruses clinically. In activity did occur in nearly all years studied, but rises in anti-
early epidemiologic studies, there was, however, evidence of body titers were concentrated in certain years in which they
variation in the frequency of infection on both a seasonal and far exceeded the means for the entire studies. Those periods
a cyclical basis. In these investigations, isolation and rises in are indicated as solid black boxes in the figure. The times dur-
antibody titer for all types of coronaviruses were rare events ing which specimens were collected in each investigation are
outside the period from December through May. This is the indicated in the figure by the white boxes. Activity of 229E
portion of the year in which isolation rates for rhinoviruses was detected in all four studies at the same time, even though
often reach their lowest level. An exception to this rule was a two were in the Midwest and two in the eastern United States.
study in which frequent rises in titer were detected by ELISA It seems possible, on the basis of these data, to postulate a 2-
in summer as well [84]. More recent studies identifying the to 3-year cycle for this agent. The greatest number of infec-
viruses by PCR have largely confirmed the winter seasonal- tions in Chicago was seen in 1967, after the absence of the
ity of the viruses in the north temperate zone; one study agent for 3 years; that pattern suggests a role of herd immu-
observed that the timing of coronavirus identifications was nity in determining the time of reappearance of the agent.
similar to that of influenza in winter–early spring [22]. As With OC43, the situation is quite different. As with 229E,
expected, the seasonality appears to differ in places like in no investigation did 2 years with high rates of infection
Hong Kong, based on accumulating data [121]. or illness follow one another. A possible exception was in
In the earlier multiyear, population-based studies, a cycli- the Tecumseh study. However, the agent that caused the
cal pattern could also be seen in the occurrence of individual rises in titer in 1967–1968 did not appear as closely related
10 Coronaviruses 211

serologically to OC43 as the agent involved in the other two no data available on occupational or racial susceptibility to
outbreaks. This observation indicates a problem in identify- infection or on the role of socioeconomic status in influencing
ing cycling of OC43 using the serological test employed. rates. Occurrence of infection in closed or special populations,
More recent studies using PCR have mainly not been pop- such as military recruits or residents of children’s institutions,
ulation based or have focused on a limited number of the has been reported [5, 26, 34]. The role of the school-age child
four known respiratory coronaviruses, so that comparable in dissemination of coronavirus has not yet been clearly
observations across all of the viruses are not possible. defined, but it would be surprising if these infections differed in
However, it is possible to conclude that, in any winter sea- their transmission pattern so markedly from that documented
son, all four viruses may be identified in a single geographic with the other agents. Because of the high frequency of infec-
area [122, 123]. There are likely to be increases of one or tion in older children and adults, other sites of dissemination
more in specific years, but it is unlikely that any coronavirus may also be of significance. It has been possible to show that
disappears completely; this is somewhat similar to our grow- the family unit is of importance in transmission, since cluster-
ing realization, with better surveillance, of the long-term ing of 229E and OC43 infections in families was observed in
occurrence of influenza types and subtypes [22]. the Tecumseh and Seattle studies [31, 128].
Although nutritional and genetic factors have not been asso-
5.1.5 Age ciated with susceptibility to coronavirus infections, there are
There is little available evidence that the respiratory corona- clear indications that the viruses are associated with exacerba-
viruses behave differently than other respiratory viruses: tions of chronic obstructive respiratory disease. Such a finding
infections are most common in children and decrease with is hardly surprising in view of the high infection rates that have
increasing age. However, it is unclear whether the drop-off is been observed in unselected older adults [129]. It has not yet
modest or more extreme, as is the case with respiratory syn- been demonstrated whether this represents true increased sus-
cytial virus [124]. In the Tecumseh study, a total population ceptibility to infection or simply a more severe form of expres-
group was followed. During the 1968–1969 OC43 outbreak, sion of the infection when it occurs in an already compromised
infection rates were relatively uniform for all age groups, host. In addition to the situation in older individuals, there is
varying from a high of 29.2 per 100 person-years in the 0–4 evidence that both OC43 and 229E may trigger acute attacks of
age group to 22.2 in those over 40 years of age [32]. The wheezing in young asthmatics; in fact, in one study, coronavi-
reversal of the pattern of age-specific infection rates custom- ruses were the most common agent involved in episodes of
arily associated with the respiratory viruses becomes com- wheezy bronchitis [21, 33, 117, 130]. Recent studies using the
plete with 229E. Infection with this virus has been more PCR technique also associate the viruses with illnesses includ-
difficult to demonstrate in small children than in adults. In ing pneumonia in immunocompromised patients [63, 115,
Tecumseh, during the 1966–1967 outbreak, highest age- 131]. One study identified all the viruses over the course of a
specific infection rates by CF were found among those year but the newer viruses, NL63 and HKU1, most commonly.
15–29 years of age, following a steady increase in infection Again, this may be a reflection of these viruses being most
frequency from the 0- to 4-year-olds. However, when neu- common at that point in time; it should be noted that shortly
tralization tests were used to detect infection, the 15- to after the first identification of NL63 in one city in the
19-year-olds still had high infection rates, but the serial Netherlands, it was again identified in another, which may indi-
increase to that point among younger age groups was much cate increased circulation at the time [18, 132].
less steep [31]. This suggests that the apparent sparing of
small children with 229E may be an artifact resulting from
the relative insensitivity of the young to the serological pro- 5.2 Epidemiology of Severe Acute
cedures commonly employed. It would be surprising if two Respiratory Syndrome
different coronavirus serotypes behaved so differently [125].
In late 2002, the SARS coronavirus emerged in humans in
5.1.6 Other Factors southern China as a zoonotic pathogen [133]. Infection
There is little evidence for or against a sex differential in infec- spread in Guangdong province for approximately 3 months
tions with the coronaviruses. In Tecumseh, adult females expe- before an infected individual visited Hong Kong in mid-
rienced higher infection rates with OC43 than adult males, February 2003. That case infected a number of tourists,
which is in conformity with the usual patterns of all respiratory sparking a global outbreak, and also went on to initiate a
illnesses [126]. Similarly, female volunteers appeared to be large outbreak in Hong Kong [73]. The subsequent global
more susceptible to infection with 229E-like strains than males outbreak lasted around 4 months and had a substantial impact
in artificial challenge studies [127]. In the study by Candeias on global travel, trade, and economy [134]. Sustained epi-
et al. of antibody prevalence, the results were examined by sex, demics have not occurred since 2003, although there have
but no significant differences could be observed [43]. There are been a few sporadic events or minor outbreaks in Singapore,
212 A.S. Monto et al.

Taiwan, and mainland China in 2003–2004, with most of (RDS), (2) he/she was positive for SARS-CoV by laboratory
them linked to laboratory releases and only four cases from assays, or (3) his/her autopsy findings were consistent with
mainland China perhaps of animal origin [135]. the pathology of RDS without an identifiable cause [137].
SARS patients initially developed influenza-like prodro-
mal nonspecific symptoms including fever in the first week 5.2.1 SARS Epidemiology in Time, Place,
and usually presented cough, dyspnea, and diarrhea within 14 and Person
days. Severe illness developed rapidly progressing to respira- In total, 8,096 “probable” SARS cases were reported to the
tory distress and oxygen desaturation requiring intensive care World Health Organization by August 2003 [138]. The most
and potentially resulting in death [136]. The World Health affected areas were Hong Kong, with 1,755 “probable” cases
Organization (WHO) defined a suspected SARS case as a among a population of 6.8 million, and mainland China, with
person with high fever (>38 °C) and cough/breathing diffi- 5,327 “probable” cases among a population of 1.3 billion.
culty, who either had close contact with a suspect or probable Taiwan, Canada, and Singapore also experienced notable
case of SARS or resided in or traveled to an area with recent epidemics, with 346, 251, and 238 probable cases, respec-
local transmission of SARS in the 10 days prior to onset of tively, while altogether cases were reported in more than 25
symptoms. A suspected case became a probable case when different countries and administrative regions. Reported
(1) the patient’s chest X-ray (CXR) presented infiltrates con- cases of SARS globally and in Hong Kong by time of symp-
sistent with pneumonia or respiratory distress syndrome tom onset are shown in Fig. 10.5.

Worldwide
a
Updated report of cases
1000 from Nov−16 to Feb−28 in
Guangdong Province, China New cases
Deaths

800
No. of reported cases

600

400

First day of report

by WHO
200
Last day of report
by WHO

0
1 2 3 4 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2
Jan Feb Mar Apr May Jun Jul
Week of report

b Hong Kong
200
No. of cases

150
Symptom onset of first Symptom onset of last
100 (imported) case in Hong Kong case in Hong Kong
50
0
1 2 3 4 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2
Jan Feb Mar Apr May Jun Jul
Week of symptoms onset

Fig. 10.5 Probable cases of SARS by week of onset (Source: [138]). (a) Cases worldwide, (b) Cases in Hong Kong
10 Coronaviruses 213

A common feature of SARS outbreaks in different regions risk was estimated to be 9.6 % with 744 deaths among the
was the central role of hospitals and transmission among 8,096 probable cases [135]. However, this masks substantial
patients and health-care workers [139–143]. A peak in infec- variability between affected regions, from around 7 % in
tiousness was thought to occur around 10 days after illness Beijing to around 17 % in Hong Kong [150]. Reasons for vari-
onset [144], by which time cases would have been hospital- ation remain unclear but could partly be attributed to case defi-
ized, and certain medical procedures were particularly prone nitions, partly to case mix including age and underlying heath
to generating transmission [145]. Hospital transmission conditions [150] and partly to case management [162].
played a prominent role in the initial epidemic in Hong Kong, Few cases are thought to have been asymptomatic or sub-
with more than 250 cases attributed to an outbreak at the clinical. Serological studies were conducted in various
Prince of Wales Hospital in early March 2003 [146, 147]. The groups including health-care workers, close contacts of
Canadian outbreak began when a case returning from Hong cases, other patients, and the general community in affected
Kong was admitted to hospital, and 72 % of cases were sub- regions, and a review of these studies found that the average
sequently attributed to nosocomial transmission [143, 148]. seropositivity rate was just 0.1 % among more than 20,000
Old age and the presence of comorbidities including dia- individuals [163].
betes mellitus, hypertension, coronary artery disease, and
chronic obstructive pulmonary disease increased the risk of 5.2.3 Successful Control of the Global SARS
death or adverse outcomes, such as admission to an ICU Epidemic
requiring mechanical ventilation and development of ARDS Despite a basic reproductive number in the range 2–3, higher
[149, 150]. Sex (male), high lactate dehydrogenase concen- than influenza, the global epidemic of SARS was effectively
tration at presentation, and higher SARS-CoV viral load have controlled by appropriate nosocomial infection control
been found contributing to higher case fatality rate as well measures. A range of interventions contributed to contain-
[151]. Genetic factors may contribute to host susceptibility to ment [164], including the use of engineering controls such as
SARS infection [78, 152, 153]. To date, there have been no negative-pressure isolation rooms [143]; improved adherence
studies on the relationship of race, socioeconomic status, to the use of personal protective equipment such as gowns,
occupation, or nutrition to susceptibility to SARS infection. gloves, and masks [165]; as well as administrative measures
including patient triaging and isolation, visitor restrictions,
5.2.2 SARS Transmission Dynamics and establishment of dedicated SARS teams of staff [166].
Concerted efforts were made during the SARS epidemic to The importance of strict infection control was illustrated par-
determine the transmission dynamics and thereby support ticularly well by the experiences in Taiwan and Toronto,
control [154, 155]. Contact tracing exercises provided infor- where control of the initial outbreaks was followed by com-
mation to estimate the incubation period at around 5 days, placency and subsequent second waves [167, 168]. Despite
with around 95 % of infections leading to illness onset within the importance of infection control strategies, there are also
10–14 days [156–158]. Early in the epidemic, delays between examples of individuals with SARS who were hospitalized
illness onset and admission to hospital were typically 5–7 days, where infection control practices were lax and yet their infec-
and a measure of the success of public health control measures tion did not result in outbreaks [169]. Patient factors may also
was the reduction in onset-admission intervals to just 1–2 days have had a role in the risk of transmission [170].
by the end of the epidemic [150, 157]. On average, patients
remained in hospital for around 3–4 weeks [157].
The basic reproductive number, R0, an estimate of the aver- 6 Mechanism and Route
age number of secondary cases resulting from one infected of Transmission
case in a completely susceptible population, was estimated to
be in the range 2–3 [154, 155, 159]. The average time between The four endemic respiratory coronaviruses are presumably
successive cases was around 8.4 days [155]. Due to these fea- transmitted by the respiratory route. It has been possible to
tures taken together, in the early stages of outbreaks, the num- induce infection experimentally in volunteers by inoculating
ber of cases approximately doubled every week. virus into the nose [35, 44]. The virus is most stable at pH 6.0,
One issue of early controversy was the case fatality risk. and low temperature appears to protect it against varied rela-
Early in the epidemic, technical errors led to underestimation tive humidity [171, 172]. No other route of transmission for
[160]. For example, on March 25, the World Health coronaviruses seems involved in man, although animal coro-
Organization reported the case fatality risk to be around 4 %, naviruses are infectious by the fecal–oral route [57]. There is
based on 49 deaths among more than 1,000 cases at that time currently no direct evidence to aid in identifying the main
[161]. This estimate was erroneously low because cases had mechanisms of transmission. However, it is possible to com-
not yet recovered, and some cases would subsequently suc- pare the epidemiologic behavior of the coronaviruses with
cumb to the disease [160]. After the epidemic, the case fatality that of other respiratory agents, the transmission mechanisms
214 A.S. Monto et al.

of which have been more directly studied. Large-scale out- During the 1967 outbreak of 229E infection in Tecumseh,
breaks of coronavirus infections have taken place, as in Michigan, illness was significantly more common among
Tecumseh in 1967 [31]. This is much more analogous to the those with infection than among matched subjects without
situation seen with influenza than to that with the rhinoviruses infection [31]. Similarly, 229E infection among Chicago
[173]. Rhinoviruses are thought to be transmitted by large medical students was statistically associated with illness
droplet and may at times spread via fomites [174]. when those with rises in titer were used as their own controls
Unlike the situation with the SARS coronaviruses, there [25]. Furthermore, experimental inoculation of volunteers
is no evidence that any animal reservoir or vector is involved with strains of 229E and OC43 isolated in the laboratory has
in the maintenance of infection or transmission of the other resulted in clinical illness, fulfilling Koch’s postulates modi-
respiratory human coronaviruses. There has been a report of fied by Rivers [185] for attributing an etiologic role to a
antibody to avian IBV in the sera of poultry workers but not microbe as a cause of disease [35, 36, 47].
of controls, but no evidence of any further transmission [40]. SARS-CoV has also fulfilled the Koch–Rivers postulates
The SARS coronavirus was thought to spread through a for association with the disease of SARS. The virus was
number of different modes, most commonly via direct close detected in patients with SARS but not in those without dis-
contact. Health-care workers involved in direct patient care ease, and the virus was detected at the site of the pathology,
duties often had the highest attack rates [175, 176], while that is, lung [51]. The virus was isolated in pure culture from
contact precautions were effective in preventing transmis- the lung biopsy of a patient with SARS, and experimental
sion [165]. SARS coronavirus was capable of surviving on infection of cynomolgus macaques with this virus produced
dried, inert surfaces and was found on some hospital surfaces a comparable disease. A specific immune response to the
[177], and indirect contact was implied in the infections of virus was demonstrated, and the virus was successfully re-
some nonmedical staff [178]. Although there is substantial isolated from the site of pathology in the infected animal
evidence to support the role of transmission through droplet [186]. Fulfilling Koch–Rivers postulates for more recently
and direct contact and some evidence to support transmis- discovered human coronaviruses has been more challenging
sion by indirect contact, there is relatively little evidence for because of the lack of suitable animal models that recapitu-
airborne transmission [179]. In one large community out- late the disease in humans [187], and their etiologic associa-
break in Hong Kong, a computational fluid dynamics model tion with disease lies largely on epidemiologic grounds.
was used to demonstrate that airborne transmission was con- An important characteristic of the respiratory coronavi-
sistent with the observed pattern of infections [180], although ruses is their apparent high rate of reinfection, which in vol-
this hypothesis was not formally compared with other pos- unteers has now been documented to be possible within a
sible explanations. However, lacking other evidence of air- year of prior infection [188]. In the Tecumseh study, 81.5 %
borne transmission despite unprotected extended exposures of those infected with OC43 actually possessed prior N anti-
in health-care settings [169, 181, 182], the World Health body [189]. Possession of circulating OC43 HI antibody
Organization classified SARS as a disease with “opportunis- among the Atlanta children did not appear to play a role in
tic airborne” transmission to indicate that the disease natu- modifying severity of a subsequent illness [28]. With 229E
rally spreads by non-airborne routes but under special virus, Hamre and Beem [25] demonstrated that the frequency
environmental conditions may spread by the airborne route of rise in titer detected by N was inversely proportional to
[183, 184]. Diseases that are spread by opportunistic air- preinfection levels of N antibody, which would indicate that
borne transmission do not require special airborne infection this antibody exerted some protective effect. However, the
isolation measures, for example, negative-pressure isolation importance of this N antibody could not be confirmed when
rooms, but special precautions are recommended for high- infection was detected by CF. Thus, circulating N antibody
risk procedures. as measured at present may bear a relationship to modifica-
tion of infection, but this association is not a very strong one.
Since coronavirus infections involve mainly the surface of
7 Pathogenesis and Immunity the respiratory tract, it is likely that secretory IgA antibody
plays a more direct role in protection; this had in fact been
7.1 Etiology and Immunity demonstrated with a swine coronavirus [190] and subse-
quently with 229E in humans experimentally infected [191].
Data that demonstrate the etiologic role of coronaviruses in
respiratory infections are derived from laboratory and field
studies. Coronaviruses interfere with the action of cilia in 7.2 Virus Tropism and Pathogenesis
tracheal organ culture, which suggests that they could have
the same effect in vivo. Epidemiologic studies also have Interaction between coronavirus spike proteins and host cell
demonstrated association of 229E infection with disease. receptors determines specifically the host range, tissue
10 Coronaviruses 215

Table 10.3 Human coronaviruses and their major receptors

Human coronavirus Major receptor Receptor expression
229E Aminopeptidase N (APN) [4] Epithelial cells of kidney, intestine, and respiratory tract; granulocytes; fibroblasts;
endothelial cells; cerebral pericytes at blood–brain barrier; synaptic junctions;
macrophages; and dendritic cells [4, 193, 194]
Betacoronavirus 1 9-O-Acetylated sialic acid- Erythrocytes, neural gangliosides, gut mucins [193, 196]
(OC43/ HECV) containing receptors [195]
SARS-related CoV Angiotensin-converting enzyme Lung alveolar epithelial cells, enterocytes of small intestine, arterial and venous
2 (ACE2) [197] endothelial cells, arterial smooth muscle cells [198]
NL63 ACE2 [199] Same as above
HKU1 Unknown Not known
MERS coronavirus DPP4 (CD26) Found in many tissues including the respiratory epithelium [200]
From the wide range of receptor expression, one could appreciate that coronaviruses could cause illness in many parts of the body, including the
respiratory and gastroenteric systems [101, 201]

tropism, and pathogenesis [192]. Coronaviruses have a wide mutation (deletion) occurring in the spike gene led to a change
spectrum of susceptible host cell range, determined by the in virulence and was associated with a switch of virus tropism
expression of the relevant receptors (Table 10.3). Related from the gastrointestinal tract to the respiratory tract [207].
coronaviruses may use the same or similar receptors for
entry, and major receptors for hCoVs include aminopepti-
dase (APN) and ACE2. Most alphacoronaviruses bind to 7.3 Pathology and Pathogenesis
APN with the exception of NL63 and SARS-CoV which
binds to ACE2. Some of the betacoronaviruses (including There is limited data on the pathology of coronaviruses other
OC43) attach to 4- or 9-O-acetylated sialic acids via the than SARS-CoV because these infections are generally mild.
virus S and HE proteins. The HE protein also has enzymatic Electron micrographic changes from the nasal mucosa of a
activity to cleave sialic acid linkages and thus serves to child with a coronavirus infection showed minimal patho-
release virus from infected cells after replication is com- logical changes [208].
pleted. Some human coronaviruses have other binding recep- Although the clinically major pathology of SARS was that
tors that allow virus attachment to host cells, but these are seen in the respiratory tract, SARS-CoV caused a dissemi-
not sufficient as functional receptors to mediate viral entry nated infection with virus being found in the feces, urine, and
by themselves. These include calcium-dependent (C-type) plasma or serum [209]. Early disease was associated with
lectins such as L-SIGN (liver/lymph node-specific intercel- desquamation of the alveolar epithelium and disseminated
lular adhesion molecule-3-grabbing nonintegrin), which alveolar damage with hyaline membrane formation in the
may serve as a receptor for 229E and SARS [202, 203]. alveolar spaces. Viral antigen was demonstrated in alveolar
Recognition of a receptor from the same family in another and bronchial epithelial cells and alveolar macrophages
host species is possible and may allow cross-species trans- [210]. In intestinal biopsy specimens of patients with SARS,
mission. For example, 229E can use either human or feline virus infection of intestinal epithelium was demonstrated by
APN but not porcine APN [201], and human and palm civet electron microscopy with minimal cytopathic effect which is
ACE2 serve as receptors for epidemic strains of SARS-CoV, consistent with the watery diarrhea seen in these patients
but mouse and rat ACE2 do not [204, 205]. [211]. High serum levels of pro-inflammatory chemokines
The receptor for MERS coronavirus has been identified to (CXCL10, IL-8) and cytokines (IL-1 and IL-6) suggested a
be DPP4 (also known as CD26), a protein that is widely con- role for immunopathology although it is uncertain whether
served across mammalian species and found on the surface these inflammatory responses are causally relevant or an epi-
of several cell types, including the human upper airways. As phenomenon in the pathogenesis [212].
with APN and ACE2, DPP4 is an ectopeptidase that cleaves
amino acids from biologically active peptides [200].
The human coronavirus OC43 and bovine coronaviruses 8 Patterns of Host Response
share close genetic similarity suggesting that they arose from
a common ancestor less than 150 years ago [206]. 8.1 Disease Characteristics
Coronaviruses can undergo dramatic changes in tissue tropism
and virulence within the same host. For example, porcine The incubation period of coronavirus colds is relatively
enteric transmissible gastroenteritis virus caused a severe short. In studies involving volunteers, the mean period from
enteric disease in pigs. A spontaneously occurring genetic inoculation of virus to development of symptoms was from
216 A.S. Monto et al.

3.2 to 3.5 days, depending on the strain (range, 2–4 days) will decrease. As with other respiratory agents, a continuum
[35, 44]. Following exposure, the virus apparently multi- of severity of symptoms exists among those in whom infec-
plies superficially in the respiratory tract in a manner simi- tion results in disease, and this may also be related to past
lar to that in which multiplication occurs in vitro. Nasal experience with the viruses.
airway resistance and temperature of the nasal mucosa The mechanisms that lead to recovery from coronavirus
increase [213]. Virus excretion usually reaches a detectable infections have not been well defined. In volunteer studies, in
level at the time symptoms begin and lasts for 1–4 days. persons infected with 229E-like strains, it is clear that symp-
The duration of the illness is from 6 to 7 days on the aver- tomatic reinfection can occur after a period of about 1 year.
age but with some lasting up to 18 days. Serological It is not clear whether this is due to waning immunity or
response either to induced or to naturally acquired infection antigenic drift. Immunocompromised patients shed virus for
has been quite variable depending on the infecting strain a prolonged period and may sometimes be associated with a
and the serological test employed. For example, among fatal outcome.
those experimentally infected with OC38 or OC43 virus The novel MERS coronavirus initially presents with fever
who had a cold produced, only 46 % had rises in titer by HI and myalgia, sometimes with gastrointestinal symptoms,
and 23 % by CF. Fewer than half of those infected with rapidly progressing to severe viral pneumonia leading to
229E showed a CF rise. It is not clear how the existence of respiratory failure, with renal dysfunction observed in some
titer or preinfection antibody affects the magnitude of the patients. Virus was detectable in the respiratory tract as well
response detected by these tests. Rises in N antibody titer as the stool [119].
are easier to detect and have been found with sensitive tech-
niques in all volunteers experimentally infected [36, 92].
The use of the ELISA test has given added sensitivity in 8.2 Viral Antigens Associated
antibody detection; it is not as yet clear if decreased speci- with Immunity
ficity should be a concern.
The respiratory coronaviruses cause cold-like illness that Virus-neutralizing antibodies are those that react with the
on an individual basis is difficult to distinguish from illness virus S (and, where present, HE) protein although some anti-
caused by other respiratory viruses. They have also been bodies against the virus M protein can also neutralize the
reported to cause pneumonia and other severe respiratory virus in the presence of complement. Virus-neutralizing anti-
infections, such as croup and bronchiolitis [115, 123, 214]. bodies mainly bind to the N-terminal S1 part of the protein,
Most of the evidence of their involvement in severe disease which is also the part of the protein that manifests the great-
comes from reports from hospitals of the identification of the est amino acid sequence variation. The removal of glycans
viruses by PCR. It is thus impossible to say what proportion from the S protein greatly reduces the binding of neutralizing
of infections, which appear to be common, that result in hos- antibodies. Antibodies to the S protein have also been associ-
pitalization are in fact caused by these viruses. Another prob- ated with enhanced pathogenesis in feline infectious perito-
lem recently encountered is the frequent identification by nitis virus, but such immunopathology has not so far been
PCR of other viruses in those in whom a coronavirus is found convincingly demonstrated with human coronaviruses. The
[22]. This complicates determining the primary etiology. In nucleocapsid protein contributes to cell-mediated immune
induced infections in volunteers, the most prominent findings protection [209].
have been coryza and nasal discharge, with the discharge Neutralizing antibody responses to SARS-CoV appear in
being more profuse than that customarily seen with rhinovi- the second week of illness and peak at around 30 days of ill-
rus colds [35]. Sore throat has been somewhat less common ness and antibodies remain detectable for many years. The
and in children has been associated with pharyngeal injection major neutralizing epitope is in the region of the S protein
[215]. Experimental colds caused by B814 virus were about amino acid residues 441–700 [98, 99, 209].
as severe as those caused by 229E; however, natural OC43
infections caused illnesses with considerably more cough and
sore throat than did 229E infections [216]. The mean duration 9 Control and Prevention
of coronavirus colds, at 6.5 days, was shorter than that seen in
rhinovirus colds, at 9.5 days [35]. It is premature at present to think in terms of control of respi-
Clinical disease occurred in no more than 45 % of those ratory coronavirus infection by vaccination. Thus, prepara-
infected with 229E in Tecumseh during the 1967 outbreak tion of vaccines using conventional types is impossible. The
[31]. In Atlanta children, OC43 virus produced illness in frequency of reinfection observed is so high that control by
about 50 % of those infected [28]. It is likely that with vaccination may not be practical, but it is possible that future
increase in age and concomitant experience with these studies may allow further characterization of truly protective
agents, the ratio of clinically apparent to inapparent infection antibodies. Work on vaccines for the animal viruses is in
10 Coronaviruses 217

progress, and these studies may help in understanding issues still data suggesting that, in the temperate zones, the
of protection. Chemoprophylaxis and related measures may viruses are most active in late winter–spring. Ironically,
be a more practical approach; it has been shown that recom- with the PCR technique, it has become common to identify
binant α-interferon can prevent infections artificially pro- more than one virus from the same individual. This is not
duced in volunteers [217], and other approaches have been limited to the coronaviruses but includes many other respi-
under investigation [25, 217–219]. There remains environ- ratory viruses as well. There is a need to determine whether
mental control of infection; such efforts have rarely been these are true coinfections or whether there may be asymp-
useful for other respiratory agents, but they may be more tomatic or prolonged carriage involved. If they are real
efficacious if a practical barrier to transmission can be coinfections, there may be consequences of having more
devised [220]. than one agent present during an illness.
The situation is quite different in terms of the SARS-CoV, The SARS-CoV emerged from a zoonotic reservoir and
because of the severity of the disease produced. Here, the spread worldwide in a short period of time. We still do not
work on vaccines moved forward in the years immediately know how and why this happened, and therefore we must be
after 2003. Some of this activity of specific vaccine develop- concerned that such an event could occur again. We do not
ment continues but at a slower pace. Even when human cases have either vaccines or antivirals for the SARS-CoV; the
were occurring, it was unclear how such a vaccine, if avail- need is probably greater for antivirals, given the severity of
able, should be used, given the distribution of infection and the illness and the question of how a vaccine would be used
occurrence of clinical disease. With disappearance of human in the current situation. The recent identification of a novel
cases, it has become even more difficult to decide on the coronavirus gives increased urgency to this need; it is likely
appropriate vaccine target populations, except for those who that an anticoronavirus drug would be of use whatever the
might be exposed in a laboratory setting. particular type involved. Overall, the epidemiologic lesson to
During the SARS episode, as no virus-specific antiviral be learned from SARS is the need for good surveillance at
agent was available, a variety of treatments were used, the animal–human interface. The virus was probably trans-
including the broad-acting antiviral agents ribavirin and mitting locally from human to human for months before it
interferon as well as corticosteroids. Because of the severity escaped to the rest of the world. If this had been recognized,
of the disease, many were used in combination, and it was there could have been earlier efforts to contain spread, which
difficult to say retrospectively whether any individually or in was effectively accomplished later, but only after much dam-
combination had a positive effect [162]. Corticosteroid ther- age had been done.
apy was associated with both short-term (secondary infec- The emergence of a novel MERS coronavirus with poten-
tions, increased viral load) and long-term (osteoporosis, tial to cause severe human disease, though so far originating
avascular necrosis) adverse effects [221]. in the Middle East and manifesting limited human-to-human
transmission including transmission within health-care facil-
ities, is reminiscent of the emergence of SARS and is a con-
10 Unresolved Problems cern for global public health [120].
The numbers of laboratory-confirmed patients with MERS
The major problem in working with the respiratory coro- continues to increase with 837 laboratory confirmed cases
naviruses has been solved with the development of and 291 deaths being reported to WHO as of 23 July 2014.
RT-PCR. Previously, because of the difficulty in growing The median age of all cases is 52 years, but primary human
them in cell culture, epidemiologic and clinical studies had cases (those who have no exposure to other confirmed cases)
to rely on serology. While less limiting in epidemiologic are older (median age 58 years) compared to secondary cases
studies, where regular blood collections can be scheduled, (median age 45 years). The majority of confirmed cases have
it sharply constrained the ability to identify the role of underlying health conditions. All cases so far have a link to
hCoVs in causing severe respiratory infections. Now we the Middle East with primary human infections reported from
recognize the existence of four different coronaviruses, Jordan, Kuwait, Oman, Qatar, Saudi Arabia and the United
and there may be more that have not yet been identified. Arab Emirates. Cases reported from outside the Middle East
Paradoxically, the situation has now been reversed; there have either a history of travel to the Middle East or exposure
are many reports on the involvement of these agents in to a patient who acquired infection from that region. Clusters
hospitalized cases, but epidemiologic studies involving all of human cases and evidence of limited human-to-human
four hCoVs in different populations over time have been transmission have been reported. To date, more than half of
relatively scarce. It was previously thought that the viruses the secondary cases have been associated with health care set-
cycle in their appearance over a period of years, but recent tings, including health care workers. Health care workers
evidence is lacking, especially pertaining to the newly appear to have less severe disease in general, although deaths
identified viruses, NL63 and HKU1; however, there are have occasionally been reported [222].
218 A.S. Monto et al.

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 Enteroviruses and Parechoviruses:
Echoviruses, Coxsackieviruses, 11
and Others

M. Steven Oberste and Susan I. Gerber

1 Introduction spectrum of illnesses, and can be detected in the same clinical

specimens, so they are considered together here. The rhinovi-
The enteroviruses (genus Enterovirus, family Picornaviridae) ruses are described in detail in Chap. 29.
are among the most common viruses infecting humans. In The rate at which new enteroviruses and parechoviruses
addition to the human enteroviruses (polioviruses, coxsackievi- have been discovered has accelerated following the introduc-
ruses, echoviruses, and numbered enteroviruses), the genus tion of molecular typing methods [182, 184] and the ready
Enterovirus also contains viruses that infect nonhuman pri- availability of genome sequences for every known enterovi-
mates and livestock, as well as the human rhinoviruses, a recent rus type. In addition to the enteroviruses of humans, numer-
addition to the genus [117, 201] (Table 11.1). These viruses ous enteroviruses of other animals are known, including
share a number of clinical, epidemiologic, and ecological char- those of nonhuman primates and of livestock, primarily cat-
acteristics as well as physical and biochemical properties. tle, swine, and sheep. In some cases, human enteroviruses
Originally, the enteroviruses were classified into the subgroups and human parechoviruses have also been detected in nonhu-
of polioviruses (PV), coxsackie A viruses (CVA) and coxsackie man primates (Table 11.1) [89, 190].
B viruses (CVB), and echoviruses, based on the empirical Enteroviruses can cause a wide variety of illnesses in
observations of their association with certain clinical syn- humans, ranging from minor undifferentiated febrile illness to
dromes or disease, tissue tropism, nature of disease in suckling severe and permanent paralysis. For all members of the group,
mice, growth in certain specific cell cultures, and in some cases however, subclinical infection is far more common than clini-
antigenic similarities [46–48, 203] (Table 11.2). The viruses cally manifest disease. Although certain enteroviruses have
now known as “parechoviruses” share a number of physical been more frequently responsible for epidemics involving a
and biological properties with the enteroviruses and were origi- specific syndrome, the same serotypes may at other times and
nally classified in the genus Enterovirus. However, biological in other places be associated with infections having different
differences were apparent even 50 years ago, and, once molec- clinical manifestations or producing no symptoms. On the
ular diagnostics and genome sequences became available, it other hand, different viruses may produce the same syndrome.
was obvious that the parechoviruses were distinct from the For these reasons, clinical disease is not a satisfactory basis for
enteroviruses and they have been reclassified into a separate classification or, as a rule, for diagnosis.
genus, Parechovirus [45, 102]. Nevertheless, the enteroviruses Poliomyelitis is an acute infectious disease that in its seri-
and parechoviruses are very similar clinically, cause the same ous form affects the CNS. The destruction of motor neurons
in the spinal cord results in flaccid paralysis. Because polio-
viruses can cause the most severe disease of any for which
M.S. Oberste, PhD (*) enteroviruses are responsible, these agents have received the
Division of Viral Diseases, National Center for Immunization
most comprehensive study and have served as models in
and Respiratory Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Rd NE, Mailstop G-17, studies of other enteroviruses. The polioviruses are described
Atlanta, GA 30333, USA in detail in Chap. 13.
e-mail: [email protected] The disease manifestations caused by the coxsackieviruses,
S.I. Gerber, MD echoviruses, and numbered enteroviruses largely overlap. The
Epidemiology Branch, Division of Viral Diseases, National Center
for Immunization and Respiratory Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Rd NE, Mailstop A-34,, The findings and conclusions in this report are those of the authors and
Atlanta, GA 30333, USA do not necessarily represent the views of Centers for Disease Control
e-mail: [email protected] and Prevention.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 225

DOI 10.1007/978-1-4899-7448-8_11, © Springer Science+Business Media New York 2014
226 M.S. Oberste and S.I. Gerber

Table 11.1 Picornavirus genera, speciesa, and (sero)types

Genera and species # Types Comments
Genus Enterovirus 269
Human enterovirus Ab 22 Five have been found only in nonhuman primates
Human enterovirus Bb 60 Two have been found only in nonhuman primates
Human enterovirus Cb 21
Human enterovirus Db 4
Human rhinovirus Ab, c 77
Human rhinovirus Bb, c 25
Human rhinovirus Cb, c 49
Simian enterovirus A 1
Bovine enterovirus 2 Possible 3rd type detected in sheep
Porcine enterovirus B 3
Unclassified 5 Detected in nonhuman primates
Genus Parechovirusd 20
Human parechovirusb 16 Types 1 and 2 formerly classified in genus
Enterovirus
Ljungan virus 4
Genus Hepatovirusb, e 1
Genus Cardiovirus 12
Encephalomyocarditis virusb 1
Theilovirusb 11
Genus Kobuvirus 3
Aichi virusb 1
Bovine kobuvirus 1 Also found in sheep and pigs
Porcine kobuvirus 1
Unclassified 2 One virus detected in rodents and one in dogs
Genus Teschovirus 11
Genus Erbovirus 3
Genus Aphthovirus 11
Foot-and-mouth disease virus 7
Bovine rhinitis A virus 1
Bovine rhinitis B virus 2
Equine rhinitis A virus 1
Genus Sapelovirus 8
Simian sapelovirus 3
Porcine sapelovirus 1 Formerly porcine enterovirus A in genus
Enterovirus
Avian sapelovirus 1
Unclassified 3 One virus detected in rodents and two in sea lions
Genus Senecavirus 1
Genus Tremovirus 1
Genus Avihepatovirus 3
Proposed genus Aquamavirus 1
Proposed genus Cosavirusb 4
Proposed genus Megrivirus 1
Proposed genus Salivirusb 1
Unclassified picornaviruses 18 Viruses detected in bats, cats, rodents, sheep,
birds, fish, and reptiles
a
The classification scheme shown is from the Picornavirus Study Group of the International Committee on the Taxonomy of Viruses [117, 118].
The types that comprise the human enterovirus species are listed in Tables 11.3, 11.4, 11.5, and 11.6
b
At least one virus in the genus or species has been detected in humans
c
See Chap. 30
d
The human parechovirus types are listed in Table 11.7
e
See Chap. 18
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 227

Table 11.2 Classification of human enteroviruses

Traditional taxonomy Current taxonomy
Polioviruses [Human] enterovirus A (HEV-A)
PV1–3 CAV2–8, 10, 12, 14, 16; EV71, EV76 EV89, EV90, EV91, EV114, EV119 (EV92, SV19, SV43,
SV46, and BA13 are classified in HEV-A, but they have been detected only in nonhuman primates)
Coxsackie A viruses [Human] enterovirus B (HEV-B)
CVA1–22, 24 CAV9; CBV1–6; E1–7, 9, 11–21, 24–27, 29–33; EV69, EV73–75, EV77–88, EV100–101, EV106–
107 (EV110 and SA5 are classified in HEV-B, but they have been detected only in nonhuman
primates)
Coxsackie B viruses [Human] enterovirus C (HEV-C)
CVB1–6 PV1–3, CAV1, 11, 13, 17, 19–22, 24, EV95–96, EV99, EV102, EV104–105, EV109, EV113,
EV116–118
Echoviruses [Human] enterovirus D (HEV-D)
E1–7, 9, 11–21, 24–27, 29–33 EV68, 70, EV94, EV111 (EV120 is classified in HEV-D, but it has been detected only in nonhuman
primates)
Numbered enteroviruses Unassigned
EV68–71 EV122–123
In the new taxonomy, “Human” is in brackets because there is a pending taxonomy proposal to drop host species from picornavirus species names
[117]. The gaps in numbering result from changes in classification. Since the time of their discovery and initial classification, some serotypes have
been found to be identical to another enterovirus (i.e., coxsackievirus A15 is the same as coxsackievirus A11, coxsackievirus A18 is the same as
coxsackievirus A13, coxsackievirus A23 is the same as echovirus 9, echovirus 8 is the same as echovirus 1, and echovirus 34 is a variant of
CVA24). In addition, some serotypes have been reclassified as members of other picornavirus genera or other virus families. Echovirus 10 is reo-
virus 1 (genus Orthoreovirus, family Reoviridae), echovirus 28 is human rhinovirus 1A (genus Enterovirus, family Picornaviridae), enterovirus
72 is human hepatitis A virus (genus Hepatovirus, family Picornaviridae), and echoviruses 22 and 23 are now considered human parechoviruses
1 and 2, respectively (genus Parechovirus, family Picornaviridae). Additional types (SV6, EV103, EV108, EV112, EV115, and EV121) have been
detected only in nonhuman primates and are classified in the proposed species, Enterovirus J. Modified from Khetsuriani et al. (2006)

coxsackieviruses produce a variety of illnesses, including asep- antiquity, but it was first recognized as a clinical entity only in
tic meningitis, herpangina, epidemic myalgia (pleurodynia, the late eighteenth and early nineteenth centuries.
Bornholm disease), hand, foot, and mouth disease, myocardi- Poliomyelitis became the subject of intense study after large
tis, pericarditis, pneumonia, rashes, and common colds, with epidemics began to appear in Europe and North America. In
some differences between the group A and group B coxsacki- 1908, Landsteiner and Popper successfully transmitted the
eviruses. They may also have a role in some congenital mal- disease to nonhuman primates, marking identification of the
formations and in the development of type 1 diabetes. Aseptic first human “filterable agent” (virus) [123]. During the first
meningitis, febrile illnesses with or without rash, and common half of the twentieth century, many details of poliovirus biol-
colds are among the diseases caused by echoviruses. Among ogy and pathogenesis were elucidated, primarily by using
the newer enterovirus (EV) types, EV68 has caused lower nonhuman primates as a model system, though some strains
respiratory illness, EV70 is the agent of widespread epidemics could be adapted to growth in laboratory rodents, allowing for
of acute hemorrhagic conjunctivitis (as is a variant of CVA24), studies that were not feasible in larger animals. Despite the
and EV71 has caused aseptic meningitis, brainstem encephali- primary pathology occurring in the anterior horn of the spinal
tis, and hand, foot, and mouth disease in a number of countries. cord, the virus was shown to be excreted in stools of patients,
EV71 is described in detail in Chap. 12. Most of the numbered suggesting a role for fecal-oral transmission and enteric repli-
enteroviruses are also associated with the same illnesses as the cation. Certain nonhuman primates could be infected by the
coxsackieviruses and echoviruses. (Further details of clinical alimentary route, further aiding in the understanding of
manifestations of enterovirus infections are given in Sect. 8.) pathogenesis and transmission. Significant antigenic differ-
ences among poliovirus strains were documented, and neu-
tralization studies identified three antigenic types [24]. One of
2 Historical Background the key innovations—and, indeed, a key enabling technology
for all of animal virology—was the discovery that poliovi-
Enteroviruses have been studied in detail for over a century. ruses could be isolated and propagated in vitro, in cell cul-
As a result, a great deal of information is available in earlier tures derived from primate nonneural tissues [67].
reviews and textbooks [55, 65, 149, 150, 157, 166, 200–202, The first of the viruses in coxsackievirus group A was
225, 238, 251]. Consequently, only a few key highlights will isolated by inoculation of infant mice with fecal material
be addressed here (see also Chap. 14). from two paralyzed children during an epidemic of poliomy-
The history of the enteroviruses begins with the history of elitis in 1948 in Coxsackie, New York [54]. Additional cox-
poliovirus. Paralytic poliomyelitis appears in records of early sackie A viruses, as well as the first coxsackieviruses of
228 M.S. Oberste and S.I. Gerber

group B [144], were discovered shortly thereafter by similar The most recent enterovirus mortality data for the United
methods. Group B coxsackieviruses were associated with States is described in the Morbidity and Mortality Weekly Report
aseptic meningitis and with epidemic myalgia and pleuro- published by the Centers for Disease Control and Prevention
dynia [52]. Group A and B coxsackieviruses were distin- [113], with periodic updates [36]. The National Enterovirus
guished by their differing pathological effects in baby mice Surveillance System (NESS) is a voluntary passive reporting
as well as their antigenic properties (see Sect. 4). system that monitors trends in circulating enteroviruses since
Once human and monkey cell cultures were implemented 1961. The data in NESS are contributed by state and local public
in a number of virology laboratories, largely for poliovirus health laboratories and a few large clinical reference laboratories.
isolation from stool specimens of patients with acute flaccid Outcome was reported for 3,392 (15.9 %) of 24,654 cases during
paralysis [67], additional enteric viruses were discovered. 1983–1998. During this period, 131 (3.3 %) fatal outcomes were
While these new viruses readily produced cytopathology in reported. Of the 115 deaths with known age, 77 (67 %) occurred
cultured cells, they were not pathogenic for laboratory ani- among children <1 year. In 1998, the NESS reporting form was
mals, unlike the polioviruses and coxsackieviruses [146, simplified to encourage broader reporting by public health labo-
219]. These viruses could be isolated from healthy children ratories, and outcome data are no longer collected.
[84, 96, 146, 216] as well as from patients with aseptic men-
ingitis [146, 147], and multiple serotypes were identified
[147, 216]. Because they failed to produce illness in labora- 3.2 Sources of Morbidity Data
tory animals and were not yet clearly associated with human
disease, they were called “orphan” viruses or human enteric Sources of population morbidity data for enteroviruses are
viruses; later they became known as ECHO (enteric cyto- often not representative due to variable diagnostic capa-
pathogenic human orphan) viruses [47], a name subsequently bilities, lack of uniform reporting, and lack of necessary
simplified to “echoviruses.” “Orphan” viruses causing cyto- correlation between enteroviral excretion and association
pathology in culture were also identified in nonhuman pri- with disease. Most morbidity data are obtained from out-
mates and livestock. break investigations and case reports, with consequent
In addition to their characteristic mouse pathogenicity, extrapolations to the surrounding community.
certain of the coxsackieviruses were found to grow readily in Historically, the Virus Watch Program in the 1960s in the
tissue cultures; other strains, serologically identical with the United States provided prospective longitudinal data from a
mouse-pathogenic prototype, failed to produce disease in baby defined population [83, 241]. Families were observed for
mice. Conversely, certain strains of echoviruses were found acute illnesses for a period of years and periodically sampled
to be pathogenic for mice. As instances of such overlapping for viruses. Although an expensive study design, data col-
properties accumulated, blurring the initial distinction made lected about illness incidence with accompanying virologic
between coxsackieviruses and echoviruses, it was recom- sampling yielded important information about the associa-
mended that subsequently, as new enterovirus types were dis- tion of enterovirus identification with disease [49, 66, 83].
covered, they would simply be assigned sequential numbers, as Trends in enterovirus types and human parechovirus
enterovirus 68, enterovirus 69, and so on [222]. The currently (HPeV) types are reported through NESS in the United
accepted enterovirus types are listed in Tables 11.3, 11.4, 11.5, States [113]. Although only laboratory data is reported
and 11.6, and the parechovirus types are listed in Table 11.7. through NESS, descriptions of diseases associated with com-
mon serotypes may be apparent amidst outbreak reports
where detailed clinical information is collected. For exam-
3 Methods for Epidemiologic Analysis ple, in 2007 and 2008 CVB1 was the predominant serotype
reported, and in 2007 CVB1 was the cause of an outbreak of
3.1 Sources of Mortality Data severe neonatal infections in the United States [34]. In addi-
tion to NESS, the Infectious Agents Surveillance Report
In the United States, enterovirus infections are not generally from Japan’s National Institute of Infectious Diseases
notifiable in that they are not required to be reported to local, describes viral detections associated with specific diseases
state, and national public health authorities. In addition, such as herpangina and hand, foot, and mouth disease.
encephalitis and aseptic meningitis are not uniformly report-
able, although some jurisdictions within the United States
may collect information on these illnesses. Often, enterovi- 3.3 Serological and Clinical Surveys
ral infections may go unrecognized unless clinicians order
appropriate testing, and because there are no recommended Since there are many enterovirus and HPeV types, few
antivirals, virologic diagnosis does not lead to a specific anti- serological surveys exist that fully characterize seropreva-
viral therapy. lence in vulnerable populations. However, understanding
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 229

susceptibility to EV71 infection has received more attention specimens created a better understanding of circulating serotypes,
because of the potential for large outbreaks of hand, foot, which is important in preparing for large-scale outbreaks associ-
and mouth disease (HFMD) including a subset of children ated with the emergence of new enterovirus strains. Another sur-
with severe neurologic complications, particularly in Asia. In vey of clinical specimens obtained from hospitalized children in
Shanghai, after a large outbreak of HFMD in 2010, a serosur- Cyprus from 2003 to 2007 demonstrated changes in predominant
vey revealed that loss of maternal antibodies later in infancy serotypes over time; these changes were consistent with those
and lack of acquired anti-EV71 immunity were likely respon- in other European countries [254]. In the United States, NESS
sible for a large number of severe HFMD cases in the 1–2- has provided seasonal information on circulating serotypes
year age group in 2011 [285]. Likewise in Singapore, high (Fig. 11.1). Correlations between serotype detection and certain
EV71 neutralizing antibody titers were identified in a group specimen types may reflect clinical presentations observed in the
of children aged one to six years compared to older children, community [36, 113]. For example, echovirus 9 and echovirus
indicating that infections were acquired in early childhood [7]. 30, which are commonly associated with aseptic meningitis,
The predominantly circulating enteroviral serotypes may
change over time and may be associated with large outbreaks
of clinically recognizable syndromes. Enterovirus surveil- 25
lance systems vary greatly worldwide, but all contribute
information regarding circulating serotypes over geography 20
and time [201]. Molecular typing and phylogenetic surveys

% of reports
yield important information for detection of new viral strains 15

and outbreaks of clinical disease [169, 270].

10
Although enteroviruses are commonly identified among chil-
dren, especially in summer and autumn months, targeted epi- 5
demiologic surveillance may provide a snapshot of circulating
seasonal serotypes. Recently, specimens obtained clinically from 0
children were collected along with environmental samples in Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Month of specimen collection
the Republic of Georgia from 2002 to 2005 [114]. A wide range
of enteroviruses were identified, with notable genetic diversity, Fig. 11.1 Percentage of enterovirus reports by month, United States,
including some that have been rarely identified in the United by month of specimen collection, 1983–2005 (From Khetsuriani et al.
States. The information obtained from environmental and clinical [114], in the public domain)

Table 11.3 Enterovirus species A (EV-A)

Type Prototype strain Geographic origin Illness or source Accession number Investigator
CVA2 Fleetwood Delaware Poliomyelitis AY421760 Dalldorf
CVA3 Olson New York Meningitis AY421761 Dalldorf
CVA4 High point North Carolina Sewage of community AY421762 Melnick
with polio
CVA5 Swartz New York Poliomyelitis AY421763 Dalldorf
CVA6 Gdula New York Meningitis AY421764 Dalldorf
CVA7 Parker New York Meningitis AY421765 Dalldorf
CVA8 Donovan New York Poliomyelitis AY421766 Dalldorf
CVA10 Kowalik New York Meningitis AY421767 Dalldorf
CVA12 Texas-12 Texas Files in community with polio AY421768 Melnick
CVA14 G-14 South Africa None AY421769 Gear
CVA16 G-10 South Africa None U05876 Gear
EV71 BrCr California Meningitisa U22521 Schmidt
EV76 10226 France Gastroenteritis AY697458 Oberste
EV89 10359 Bangladesh Acute flaccid paralysis AY697459 Oberste
EV90 10399 Bangladesh Acute flaccid paralysis AY697460 Oberste
EV91 10406 Bangladesh Acute flaccid paralysis AY697461 Oberste
EV114 11610 Bangladesh Acute flaccid paralysis NA Oberste
EV119 C13 Cameroon NA Norder
NA information not available
a
An identical strain was isolated from the brain of a fatal encephalitis case in the same local outbreak of central nervous system disease
230 M.S. Oberste and S.I. Gerber

Table 11.4 Enterovirus species B (EV-B)

Illness yielding
Type Prototype strain Geographic origin prototype virus Accession number Investigator
CVA9 Bozek New York Meningitis D00627 Dalldorf
CVB1 Conn-5 Connecticut Meningitis M16560 Melnick
CVB2 Ohio-1 Ohio Summer grippe AF085363 Melnick
CVB3 Nancy Connecticut Minor febrile illness M16572 Melnick
CVB4 JVB New York Chest and abdominal X05690 Sickles
pain
CVB5 Faulkner Kentucky Mild paralytic disease AF114383 Steigman
with atrophy
CVB6 Schmidt Philippines None AF1 05342 Hammon
E1 Farouk Egypt None AF029859 Melnick
E2 Cornelis Connecticut Meningitis AY302545 Melnick
E3 Morrisey Connecticut Meningitis AY302553 Melnick
E4 Pesascek Connecticut Meningitis AY302557 Melnick
E5 Noyce Maine Meningitis AF083069 Melnick
E6 D'Amori Rhode Island Meningitis AY302558 Melnick
E7 Wallace Ohio None AY302559 Ramos-Alvarez
E9 Hill Ohio None X84981 Ramos-Alvarez
E11 Gregory Ohio None X80059 Ramos-Alvarez
E12 Travis Philippine Islands None X79047 Hammon
E13 Del Carmen Philippine Islands None AY302539 Hammon
E14 Tow Rhode Island Meningitis AY302540 Melnick
E15 CH 96-51 West Virginia None AY302541 Ormsbee
E16 Harrington Massachusetts Meningitis AY302542 Kibrick
E17 CHHE-29 Mexico City None AY302543 Ramos-Alvarez
E18 Metcalf Ohio Diarrhea AF317694 Ramos-Alvarez
E19 Burke Ohio Diarrhea AY302544 Ramos-Alvarez
E20 JV-1 Washington, DC Fever AY302546 Rosen
E21 Farina Massachusetts Meningitis AY302547 Enders
E24 DeCamp Ohio Diarrhea AY302548 Sabin
E25 JV-4 Washington, DC Diarrhea AY302549 Rosen
E26 Coronel Philippine Islands None AY302550 Hammon
E27 Bacon Philippine Islands None AY302551 Hammon
E29 JV-10 Washington, DC None AY302552 Rosen
E30 Bastianni New York Meningitis AF162711 Plager
E31 Caldwell Kansas Meningitis AY302554 Wenner
E32 PR-10 Puerto Rico Meningitis AY302555 Branche
E33 Toluca-3 Mexico None AY302556 Rosen
EV69 Toluca-1 Mexico None AY302560 Rosen
EV73 CA55-1988 California Unknown AF241359 Oberste
EV74 10213 California Unknown AY556057 Norder
EV75 10219 Oklahoma Unknown AY556070 Oberste
EV77 CF496-99 France Unknown AJ493062 Norder
EV78 W137-126/99 France Unknown AY208120 Norder
EV79 10384 California Unknown AY843297 Oberste
EV80 10387 California Unknown AY843298 Oberste
EV81 10389 California Unknown AY843299 Oberste
EV82 10390 California Unknown AY843300 Oberste
EV83 10392 California Unknown AY843301 Oberste
EV84 10603 Côte d’lvoire None DQ902712 Oberste
EV85 10353 Bangladesh Acute flaccid AY843303 Oberste
paralysis
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 231

Table 11.4 (continued)

Illness yielding
Type Prototype strain Geographic origin prototype virus Accession number Investigator
EV86 10354 Bangladesh Acute flaccid AY843304 Oberste
paralysis
EV87 10396 Bangladesh Acute flaccid AY843305 Oberste
paralysis
EV88 10398 Bangladesh Acute flaccid AY843306 Oberste
paralysis
EV93 38-03 Democratic Republic of EF127244 Junttila
the Congo
EV97 10355 Bangladesh Acute flaccid AY843307 Oberste
paralysis
EV98 T92-1499 AB426608 Yamashita
EV100 10500 Bangladesh Acute flaccid DQ902713 Oberste
paralysis
EV101 10361 Côte d'lvoire None AY843308 Oberste
EV106 10634 Bangladesh Acute flaccid NA Oberste
paralysis
EV107 TN94-0349 Thailand None AB266609 Yamashita
NA information not available
Echovirus types 1 and 8 share antigens, type 1 having the broader spectrum. Type 10 was soon excluded from this group: it turned out to be a larger
RNA virus and was reclassified as a prototypic reovirus. Type 28 was reclassified as rhinovirus type 1. Types 22 and 23 have been reclassified as
members of the genus parechovirus and are named parechovirus 1 and 2. Type 34, DN-19, is now considered a prime strain of CVA24, rather than
a distinct echovirus. Additional newer serotypes (EV73 and higher) are proposed new types defined on the basis of genetic sequence information

Table 11.5 Enterovirus species C (EV-C)

Type Prototype strain Geographic origin Illness in person with prototype Accession number Investigator
CVA1 Tompkins Coxsackie, NY Poliomyelitis AF499635 Dalldorf
CVA11 Belgium-1 Belgium Epidemic myalgia AF499636 Curnen
CVA13 Flores Mexico None AF499637 Sickles
CVA17 G-12 South Africa None AF499639 Gear
CVA19 NIH-8663 Japan Guillain-Barré syndrome AF499641 Huebner
CVA20 IH-35 New York Infectious hepatitis AF499642 Sickles
CVA21 Kuykendall; Coe California Poliomyelitis, mild respiratory AF546702 Lennette
diseases
CVA22 Chulman New York Vomiting and diarrhea AF499643 Sickles
CVA24 Joseph South Africa None D90457 Gear
PV1 Brunhilde Maryland Paralytic poliomyelitis AY560657 Howe
PV2 Lansing Michigan Fatal paralytic poliomyelitis AY082680 Armstrong
PV3 Leon California Fatal paralytic poliomyelitis K01392 Kessel
EV95 5-05 NA Norder
EV96 10358 Bangladesh Acute flaccid paralysis EF015886 Oberste
EV99 10461 Bangladesh Acute flaccid paralysis EF555644 Oberste
EV102 10424 Bangladesh Acute flaccid paralysis EF555645 Oberste
EV104 CL-1231094 Switzerland Acute respiratory illness EU840733 Tapparel
EV105 TW/NTU07 NA NA NA Chang
EV109 NICA08-4327 Nicaragua Acute respiratory illness GQ865517 Yozwiak
EV116 126/Russia/10 Russia NA JX514942 Lukashev
EV117 LIT22 Lithuania JX262382 Daleno
EV118 ISR10 Israel JX961708 Daleno
NA information not available
232 M.S. Oberste and S.I. Gerber

Table 11.6 Enterovirus species D (EV-D)

Type Prototype strain Geographic origin Illness in person with prototype Accession number Investigator
EV68 Fermon California Lower respiratory illness AY426531 Scheible
EV70 J670/71 Japan Acute hemorrhagic conjunctivitis D00820 Kono
EV94 E210 Egypt Detected in sewage DQ916376 Smura
EV111 KK2640 Cameroon Nonea JF416935 Harvala
a
The prototype strain was detected in a chimpanzee, but another strain of EV111 was detected in a human with acute flaccid paralysis in Democratic
Republic of the Congo

Table 11.7 Human parechoviruses

Type Prototype strain Geographic origin Illness in person with prototype Accession number Investigator
HPeV1 Harris Ohio L02971 Sabin
HPeV2 Williamson Ohio AJ005695 Sabin
HPeV3 A308/99 Japan AB084913 Ito
HPeV4 T75-4077 California AM235750 Al-Sunaidi
HPeV5 6760 Connecticut AF055846 Oberste
HPeV6 NII428-2000 Japan AB252577 Watanabe
HPeV7 PAK5045 Pakistan EU556224 Li
HPeV8 BR/217/2006 Brazil EU716175 Drexler
HPeV9 10902 Bangladesh NA Oberste
HPeV10 10903 Bangladesh NA Oberste
HPeV11 10905 Bangladesh NA Oberste
HPeV12 10904 Bangladesh NA Oberste
HPeV13 10901 Bangladesh NA Oberste
HPeV14 451564 Netherlands FJ373179 Benschop
HPeV15 11614 Bangladesh NA Oberste
HPeV16 11615 Bangladesh NA Oberste

were mostly detected from cerebrospinal fluid specimens. been developed to overcome this problem, including simple fil-
Epidemiologic assessments of enteroviral serotypes and clini- tration, adsorption to charged filters, and concentration by phase
cal disease worldwide are important for understanding outbreaks separation [228]. In some cases, concentration systems can be
of disease caused by emergent strains in new geographic areas. deployed in the field, to reduce the amount of sample that must
This type of surveillance is also important to identify modified be transported to the laboratory [155, 158, 159, 161, 261]. A
clinical presentations of common diseases such as HFMD caused second disadvantage is that sewage can often be expected to
by coxsackievirus A6 during 2010–2012 [22, 37, 76, 267, 277]. contain a mixture of enteric viruses, especially in areas with
Knowledge of circulating enteroviral serotypes in time and place high population density and poor sanitation. Separation of the
may allow for better recognition of large-scale outbreaks and con- virus mixtures may require passage on multiple cell lines, clon-
sequent approaches to control viral transmission. ing by limiting dilution, or plaque purification, steps which may
impact the feasibility of routine environmental surveillance.
Environmental sampling has been used to supplement
3.4 Detection of Viruses patient-based virus surveillance [20, 23, 43, 81, 110, 122, 135,
in the Environment 148], but it has also been applied to outbreak investigations, to
demonstrate the source of infection. For example, isolation of
Enteroviruses excreted in feces will end up in sewage in devel- coxsackievirus B5 from an open lake swimming area identified
oped countries and in surface waters and waterways in develop- the lake as a source of an epidemic at a summer camp [91].
ing countries that lack a closed sewage collection system. As a
result, virus isolation from sewage, whether in a closed or open
system, provides an opportunity for a relatively unbiased sam- 3.5 Laboratory Methods
pling of enteroviruses circulating in the population. Typical
sewage treatment systems may not fully remove or inactivate Detailed descriptions of the principles and procedures for
viruses, including enteroviruses [72]. The primary disadvantage diagnosis of enterovirus infections have been published
of sewage testing is the extra effort required to remove solids [166, 187, 202, 224].
and other potential inhibitors of cell culture isolation or molecu- Understanding the epidemiology of enterovirus infections
lar detection methods. However, a number of methods have and disease depends largely on the ability of the laboratory
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 233

to identify the specific virus involved and to integrate that The enteroviruses can generally be propagated in either cell
information in the context of other studies of the same sero- culture or suckling mice [156, 201, 202], but some of the
type or the same disease. Traditionally, enterovirus identifi- newer enterovirus and parechovirus types have been identified
cation was based on virus isolation in culture and typing by only by direct detection in stool specimens. Most types can be
serotype-specific antisera, either individually or in specific grown in at least one human or primate continuous cell culture
intersecting pools, a system which depends on shared, stan- [119, 125, 180, 196, 205, 206, 211, 229, 234]; however, no
dardized reagents [47, 154]. While this system was sufficient single cell line can be used to isolate or propagate all cultivable
for most purposes, and was the standard for more than four enteroviruses. Despite many years of work, a few types (e.g.,
decades, it became increasingly complicated as more entero- CVA19) have been propagated only in suckling mice. The
virus serotypes were discovered. It was recognized as early typical host range of human enterovirus in cell cultures or ani-
as 1962 that “the rate of making new [enterovirus] discover- mals is not clearly associated with a given virus species.
ies has slowed, probably only because the labors involved in Infection of target cells depends on virus binding to specific
establishing ‘new’ serotypes are now so very great.” [269] receptors on the cell surface. Collectively, the enteroviruses
Since the late 1990s, molecular typing methods have become use at least eight different receptors, including two different
the standard for identifying enterovirus serotype (often now integrins (αvβ3 and α2β1), decay-accelerating factor (DAF;
referred to as simply “type”), and the methods are equally CD55), the coxsackievirus-adenovirus receptor (CAR), intra-
adept at identifying new enteroviruses, more than 50 of cellular adhesion molecule 1 (ICAM-1), scavenger receptor B2
which have been discovered since 2000. (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and
the poliovirus receptor (CD155) [174, 223, 236, 279]. Some
3.5.1 Virus Isolation and Identification enteroviruses may use more than one receptor (e.g., EV71 uses
The specimens that most frequently yield enterovirus or both SCARB2 and PSGL-1; some group B coxsackieviruses
parechovirus in culture are stools, rectal swabs, throat swabs, can use both CAR and DAF), and other, unidentified recep-
and naso- or oropharyngeal swabs [156, 202]. In addition, tors may also exist. The parechoviruses use two different integ-
cerebrospinal fluid (CSF) may yield virus in cases of neu- rins, αvβ3 and αvβ1, as well as at least one additional, unknown
rologic illness, such as aseptic meningitis. In children, even receptor. The procedure for virus isolation involves inoculation
after development of symptoms has led to hospitalization, of appropriate specimens onto susceptible cultured cells. In
a variety of non-polio enteroviruses may be detected in the clinical laboratories, it is common to simultaneously inocu-
blood, either free in the serum or in mononuclear leukocytes late several types of human and primate cells to increase the
[212]. Virus may also be isolated from vesicle fluids (e.g., spectrum of viruses that can be detected [156, 240]. The cox-
from HFMD cases), urine, conjunctival swabs (EV70 and sackieviruses, including those that do not grow in cell culture,
CVA24 variant), and nasal secretions. In general, virus may can be isolated and propagated by intracerebral inoculation of
be recovered from throat swabs up to about 2 weeks after suckling mice [234].
onset of symptoms and from stool for up to 8 weeks [6]. It is usually not clinically necessary to identify the specific
In fatal cases of suspected enteroviral etiology, virus may serotype for every enterovirus isolate; simply knowing an
be detected in the organ system affected as well as in colon enterovirus has been detected is usually sufficient. However,
contents. Of course, other viruses may also be isolated from knowing the type can be important in outbreak investiga-
feces or respiratory secretions, including rotaviruses, reovi- tions, to determine whether a case is part of the outbreak
ruses, adenoviruses, rhinoviruses, and the viruses of measles, or part of the sporadic enterovirus background. Knowing
mumps, rubella, and herpes simplex. Many of these agents the type can also be of interest in cases that are particularly
produce distinctive cytopathic effects (CPE), which at once severe or that have an unusual presentation.
differentiate them from enteroviruses. Many clinical laboratories no longer perform cell culture
Traditionally, experienced laboratory personnel could isolation for suspected enterovirus cases in routine diagnos-
make a presumptive diagnosis of enteroviral infection on tic testing. When isolates are obtained, they may be con-
the basis of the nature of the associated illness (if any), the firmed as a specific enterovirus type by neutralization with
time of year when the specimen was obtained, the culture type-specific antisera. The most widely used reagent antisera
(or mouse) system in which the virus isolate grew, and the were prepared in horses and can be obtained from the World
characteristic CPE observed in the cultures or the character- Health Organization (WHO) [85, 86, 151–153]; however,
istic pathology induced in the mice. There was some value supplies are now quite limited. Type-specific monoclonal
in reporting a presumptive identification without waiting antibodies may also be used for typing, usually in indirect
for specific typing as early recognition of probable entero- immunofluorescence assays to identify viruses isolated in
viral infection could provide information for the manage- culture [130, 218]. Commercially available monoclonal
ment of a patient or of a community outbreak and serve to antibodies can be used to detect relatively common sero-
contraindicate administration of unnecessary or undesirable types, including PV1 to 3, CVA9, CVA24, CVB1 to 6, E4,
antibiotic therapy. E6, E9, E11, E30, EV70, and EV71. Additional monoclonal
234 M.S. Oberste and S.I. Gerber

antibodies have recently been developed for CVA2, CVA4 to nucleic acid sequence of a virus represents its ultimate char-
5, and CVA10 [130]. Indirect immunofluorescence is faster acterization. All important information about a virus could
and easier to perform than neutralization, and the reagents potentially be obtained directly by PCR in conjunction with
can be produced in large quantity as needed, but the method nucleic acid sequencing if all the molecular correlates of
still suffers from the same limitations as other antigenic typ- viral phenotypic determinants were understood. The genetic
ing methods, namely, the requirement for a virus isolate in correlates for many enterovirus properties remain uncertain,
culture prior to typing and the need for a large number of but it is possible to use sequence information to assign a
reagents to identify all serotypes. Despite these limitations, sequence to a particular type [30, 31, 175, 179, 182, 183,
the method has been adopted as the standard typing method 187]. The most common molecular typing system is based
in a large number of clinical and reference laboratories. on RT-PCR and nucleotide sequencing of a portion of the
genomic region encoding VP1 [175, 182, 184]. The type is
3.5.2 Molecular Detection and Identification inferred by comparison of the partial VP1 sequence with a
Because of distinct advantages in speed, molecular detec- database containing VP1 sequences for the prototype and
tion techniques have already supplanted traditional meth- variant strains of all human enterovirus types [187]. Using
ods of detection and characterization as the gold standard. this approach, strains of homologous types can be easily dis-
Specifically, molecular procedures are now the methods of criminated from heterologous types, and new types can be
choice for enterovirus detection in CSF and are widely used identified. This method can greatly reduce the time required
for diagnosis of patients with a clinical presentation of men- to type an enterovirus and can be used to type samples that
ingitis. Several of these are licensed for clinical use in the are difficult or impossible to type using standard immuno-
United States or Europe. logic reagents or that fail to grow in culture [175, 184]. The
By far the most common use of reverse transcription technique is also useful to rapidly determine whether viruses
(RT) PCR for enterovirus diagnosis is the direct detection of isolated during an outbreak are epidemiologically related.
virus in clinical specimens [189, 220, 231]. The individual
details of the procedures may vary, but all methods that can 3.5.3 Tests for Antibody
generically detect enterovirus are similar in their key fea- Testing for the presence of type-specific antibody against
tures. The most important property of these tests is that the enteroviruses is appropriate only when (i) a known enterovi-
primers target conserved sequences in the 5′ non-translated rus isolate from the patient is available and confirmation of
region (NTR) of the virus genome. Many different primers the infecting serotype is necessary or (ii) a seroepidemio-
targeting this region have been published, recognizing differ- logic survey is being conducted to determine the community
ent sequence motifs and slightly different sequences within or study-group history of experience with a particular sero-
those sites [187, 226]. Many of these assays, however, have type or group (e.g., polioviruses). For routine diagnosis in a
not been completely evaluated on a large number of clinical single patient or a locality, virus detection by RT-PCR is far
isolates to confirm reactivity with all enterovirus types and/ simpler and faster and is the recommended approach, with
or with multiple strains within a given type. Therefore, they virus isolation and/or molecular typing as options depending
have not been validated sufficiently for diagnostic use [187]. on the individual circumstances. For any purpose except a
The major advantage of these pan-enterovirus RT-PCR serological survey, paired serum specimens are required; the
assays is that rapid detection is possible, even with very small first sample must be taken as early as possible in the course
amounts of clinical specimens such as CSF. Such assays also of the illness or infection, the second 3–4 weeks later.
facilitate detection of enteroviruses that do not readily grow The neutralization test [156, 160] is accurate and type
in cell culture. As with all RT-PCR, the sensitivity of amplifi- specific; complement fixation and agglutination (or agglu-
cation of RNA from biological specimens is highly variable, tination inhibition) tests are no longer commonly used and
depending on the nature of the specimen. Many assays can are not recommended. Acute and convalescent sera are usu-
be shown to give a positive result even from only a few cop- ally tested simultaneously, using various dilutions of serum
ies of viral RNA in a “clean” specimen such as CSF, but it is against a constant amount (usually 100 CCID50) of the spe-
not uncommon for the sensitivity to be many orders of mag- cific virus [266]. A fourfold or greater rise in type-specific
nitude lower in other specimen types (e.g., stool). The intro- neutralizing antibody titer is considered diagnostic of recent
duction of “real-time” PCR methods has greatly improved infection. Antibody, however, may already be present at
sensitivity, as the fluorescence detection systems generally the time the original specimen is obtained because of the
increase the sensitivity significantly. extended incubation period and prodromal period of many
A common goal in virus identification is knowledge enteroviral illnesses, which complicates interpretation of
of the sequence of the viral genome. Encoded within this results. If neutralizing antibody titers are found to be equally
sequence are determinants for all the biological properties high in both acute and convalescent specimens, the infec-
that are attributable to a given virus. In theory, therefore, the tion might have taken place either recently or many years
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 235

before, since neutralizing antibody to any of the entero- enteroviruses, human parechoviruses were traditionally
viruses persists for years if not for life. In addition to the detected and identified by virus isolation and antigenic typ-
homologous antibody, antibodies against other enterovirus ing [127]. By these methods, HPeV1 (formerly echovirus
types may appear transiently and at low levels. 22) consistently accounted for 2–4 % of “enteroviruses”
For diagnosis and study of acute hemorrhagic conjuncti- reported to the CDC from 1975 to 2005 [113]. RT-PCR began
vitis caused by enterovirus type 70, isolation of the virus has to supplant virus culture as the method of choice for entero-
been difficult, and most of the recent outbreaks have been virus detection in clinical diagnostic laboratories in the mid-
identified solely by serological means, though RT-PCR can 1990s. Since that time, the number of HPeV1 reports has
also be used to detect and identify virus directly in conjunc- declined to under 1 %, probably because HPeV-containing
tival swab samples. specimens are usually reported as enterovirus PCR-negative
Many serological studies rely on the detection of IgM and not further characterized.
antibody as evidence for recent enterovirus infection, and More recently, a number of investigators have developed
this is sometimes used as an alternative to the neutralization real-time RT-PCR assays to detect HPeVs [11, 16, 17, 51,
test, especially in epidemiologic studies. Several groups 60, 108, 176, 178]. These methods target conserved sites in
have developed an enzyme-linked immunosorbent assay the parechovirus 5′ NTR that are analogous to those targeted
(ELISA) for enterovirus-specific IgM [14, 61, 78, 94, 248]. by enterovirus-specific RT-PCR assays. Like the enterovirus
These tests have been found positive for nearly 90 % of assays, the HPeV RT-PCR assays vary in level of validation;
culture-confirmed group B coxsackievirus infections and can in particular, several have not been shown to detect the more
be performed rapidly. The ELISA has been successfully recently identified types. Despite this possible limitation, a
applied for epidemiologic investigations of outbreaks [79] as number of recent studies have applied molecular methods to
well as for specific diagnostic use [57, 264]. Depending on the detection of human parechoviruses in patients with enter-
the configuration and sensitivity of the test, from 10 % to itis, respiratory illness, and neonatal sepsis-like syndrome
nearly 70 % of serum samples show a heterotypic response [3, 11, 19, 105, 257, 265]. As these methods are increasingly
caused by other enterovirus infections. This heterotypic integrated into the diagnostic routine of clinical and refer-
response has been exploited to measure broadly reactive ence laboratories, a better estimate will emerge of the burden
antibody, and the assay has been used to detect enterovirus of disease attributable to this group of picornaviruses.
infection generically [26, 245]. It is clear that the human
immune response to enterovirus infection includes antibod-
ies that react with both serotype-specific epitopes and shared 4 Biological Characteristics
epitopes [74]. Despite this problem, there is a reasonably
high concordance of results between assays of different con- 4.1 General Properties
figurations [94]. The IgM assays that are used in epidemio-
logic studies have very good sensitivity and appear to be very Enteroviruses share the basic properties of picornaviruses,
specific for enterovirus infection; however, these assays including a ~7,500 nt genome of single-stranded, positive-
detect heterotypic antibodies resulting from other EV infec- sense RNA, small size (diameter 22–30 nm), lack of an enve-
tions and, therefore, cannot be considered strictly serotype lope (i.e., a “naked” nucleocapsid), and insensitivity to ether
specific. A positive result with either the neutralization test and other lipid solvents, indicating lack of essential lipids
or IgM ELISA indicates a recent viral infection; however, (Fig. 11.2) [117, 201]. The genome encodes a single long
the infecting serotype found with the IgM assay may not be open reading frame, flanked by non-translated regions at
the same one determined by the neutralization test, and the 5′ and 3′ ends. The virus replicates in the cytoplasm of
because the duration of IgM is relatively long and variable infected cells.
among individuals, the presence of enteroviral IgM is not a The enterovirus infectious particle is an icosahedral virion
definitive test for current infection. Therefore, IgM data must consisting of 60 copies of each of four capsid proteins (VP1–
be interpreted with some caution. 4) and a molecule of single-stranded genomic RNA. The
molecular biology of enterovirus replication is typical for
3.5.4 Parechovirus Diagnostics that of other plus-strand RNA viruses: uptake into the host
Despite their original classification, sequencing and PCR cell through attachment to a specific cellular receptor, release
studies demonstrated that echoviruses 22 and 23 are distinct of genomic RNA, protein synthesis, genome replication, and
from the enteroviruses, resulting in their reclassification as encapsidation. The molecular mechanisms underlying each
members of a new picornavirus genus, Parechovirus [45, of these steps are under intensive investigation. Because
102, 117, 242]. Since then, 14 additional human parecho- genomic RNA is of positive polarity, infectious virus can be
virus types have been identified (Oberste MS, unpublished recovered by transfection of naked RNA into appropriate cell
data, 2008) [3, 11, 17, 18, 62, 105, 126, 265]. Like the cultures. Full-length cDNA clones have been generated for
236 M.S. Oberste and S.I. Gerber

infectivity rapidly when heated to 50 °C, but 1 M magnesium

chloride can make the virions resistant to treatment at 50 °C
for 1 h [259]. Enteroviruses are stable at freezing tempera-
tures for decades, at 4 °C for weeks, and at room temperature
for days. Virus in stool is stable at room temperature for at
least 4 weeks and is also resistant to multiple freeze-thaw
cycles. Desiccation quickly renders enteroviruses noninfec-
tious. They are also inactivated by treatment with ultraviolet
light, and vital dyes, such as neutral red, acridine orange,
proflavine, can be incorporated into the structure of the
viruses, making them susceptible to visible light [232, 260].

4.3 Antigenic Properties

Fig. 11.2 Transmission electron micrograph of coxsackievirus B4 par-
ticles (From the Public Health Image Library, Centers for Disease The enterovirus serotypes were originally defined by neu-
Control and Prevention, 1981, in the public domain) tralization assays, either in cell culture or in susceptible ani-
mal models [46–48, 203]. Antigenicity by neutralization in
many enteroviruses, facilitating a reverse genetics approach vitro correlates well with human immunity. Initially, entero-
to understanding the function of individual viral proteins and viruses were identified by neutralization with a standard set
RNA elements. of antisera that recognized the “known” serotypes. When a
Although much is known about the enteroviruses, still virus was not neutralized by the standard panel, an antiserum
unresolved is the biochemical basis of virus stability associ- was raised against the new virus and used in neutralization
ated with its portal of entry through the enteric tract, the deter- tests with standard reference strains. Strains which exhibited
minants of virus spread in the host, and knowledge of how the no cross-neutralization were considered putative new types.
virus penetrates its target tissue, thereby causing disease [167]. However, most of the newer types have been defined strictly
The key to the early events of infection is determined by on the basis of capsid sequences [185]; hence, their antigenic
unique cell surface receptors. The receptor plays a key role in relationships to one another and to the older types is
the binding, penetration, and uncoating of the virus [223]. unknown. The extensive use of the neutralization assay in the
The human enteroviruses use at least eight different 1950s and 1960s also revealed a number of minor cross-
receptors to bind and gain entry into the host cell (CD155, reactions and other antigenic relationships, both within and
DAF [CD55], CAR, ICAM-1, SCARB2, PSGL-1, and two between the established types.
different integrins) [174, 223, 236, 279]; however, the recep- The neutralizing epitopes are located primarily at the
tors for many enteroviruses remain unknown. The receptors edges of the “canyon” on the capsid surface, an area of
for the polioviruses and rhinoviruses are members of a large virus-receptor interaction. These epitopes tend to be “con-
group of normal cellular proteins known as the immunoglob- formational,” i.e., they are not contiguous amino acids but,
ulin gene superfamily [87, 177, 217, 223]. The receptors for rather, are composed of residues on adjacent loops of one or
echovirus 1 receptor and CVA9 are integrins [21, 252], pro- more of the capsid proteins, VP1–3 (the VP4 protein is
teins known to play a role in the interactions between cells internal to the capsid and does not contribute to antigenic-
and the extracellular matrix. ity). In the polioviruses, where the neutralization sites have
been studied most extensively, sites on VP1 appear to be a
major component of the key epitopes, with additional con-
4.2 Reactions to Chemical and Physical tributions by VP2 and VP3. Enterovirus capsids also con-
Treatment tain a number of non-neutralizing epitopes, some of which
are shared widely among the different types [74]. These can
Enteroviruses are resistant to most laboratory disinfectants be readily detected by ELISA.
and to lipid solvents (e.g., ether), but they are rapidly inacti- Despite the high error rate inherent in the enterovirus
vated by treatment with 0.3 % formaldehyde, 0.1 N HCl, or RNA-dependent RNA polymerases [1], the antigenic iden-
free residual chlorine at a level of 0.3–0.5 ppm. However, the tity of enteroviruses is relatively stable (e.g., unlike influenza
presence of organic matter (e.g., in stool or sewage) is pro- virus). Indeed, polio vaccines, developed using strains iso-
tective, so caution should be exercised when attempting to lated over 50 years ago, are still effective against strains cir-
extrapolate from laboratory sensitivity data, often generated culating today, even with considerable amino acid variation
using purified virus, to real-world situations. The viruses lose in the capsid (up to about 12 % difference in VP1).
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 237

4.4 Host Range to yield the mature viral proteins. A number of viral proteins
interact with cellular proteins and structures to effectively
Almost by definition, humans are the natural host for the inhibit cellular transcription and translation, shifting the cel-
human enteroviruses. There is no evidence for zoonotic lular machinery to produce virus [53]. Once sufficient viral
transmission, though at least some human enteroviruses can proteins are produced, viral synthesis shifts from an empha-
naturally infect certain nonhuman primate species [190]. In sis on translation to RNA replication, catalyzed by the virally
areas where humans and nonhuman primates are in frequent encoded RNA-dependent RNA polymerase and using intra-
and close contact, such as in South Asia, infected primates cellular membranes derived from the rough endoplasmic
presumably may participate in transmission, forming a sin- reticulum and Golgi as a scaffold for the replication com-
gle human and primate reservoir. Swine vesicular disease plexes [15, 170]. RNA synthesis is primed by the genome-
virus, an important livestock pathogen primarily because it is linked viral protein (“VPg”) which remains covalently linked
part of the differential diagnosis for foot-and-mouth disease to the genomic RNA [243]. Unlike cellular organisms and
virus, is genetically and antigenically related to coxsackievi- most DNA viruses, viral RNA-dependent RNA polymerases
rus B5, presumably through infection of swine with the do not have proofreading and error-editing functions capable
human virus at some time in the recent past [115, 287]. of correcting viral polymerase errors, and a large number of
Many enteroviruses can infect or be adapted to infect faulty nucleotide incorporations accumulate during RNA
common laboratory animal species, including rodents and replication [272]. Mature virions are assembled in the cyto-
monkeys, though blind passages may be needed in some plasm through a mechanism that is not yet fully understood.
cases. Indeed, the polioviruses and coxsackieviruses were The time required from initiation of infection to comple-
first discovered by inoculation of laboratory animals. In tion of virus assembly ranges from 5 to 10 h, depending on
newborn mice, the group A coxsackieviruses induce flac- pH, temperature, host cell, and number of particles to which
cid paralysis and extensive degeneration of skeletal muscle, the cell is exposed. Yields may be up to 100,000 particles per
with no involvement of the tongue, heart, and CNS; the ani- cell, but only 0.1–1 % of particles may be infectious.
mals usually succumb to infection within a week. By con- Complete genome sequences have been determined for at
trast, infection with group B coxsackieviruses proceeds more least one strain of every enterovirus serotype, except for the
slowly and is characterized by spastic paralysis and tremors absolute newest—even those can be expected soon, given the
associated with encephalomyelitis, focal myositis, necrosis ease with which viral sequences can be generated [116]. Full-
of brown fat pads, myocarditis, hepatitis, and acinar cell length cDNA clones are available for many serotypes, often
pancreatitis. With few exceptions, the echoviruses do not downstream of a bacterial promoter than can be used to synthe-
generally cause disease in mice. Most enteroviruses can be size infectious RNA. Such clones have been invaluable in eluci-
isolated and propagated in a wide range of cell cultures, usu- dating the function of individual viral proteins, as well as RNA
ally of human or monkey origin, including cells derived from structures that regulate viral translation and replication [192].
kidney, lung, and other tissues. However, some enteroviruses
have been grown in only one or a few cell lines (e.g., some of
the coxsackie A viruses have been propagated only in human 5 Descriptive Epidemiology
rhabdomyosarcoma cells) [234]. The range of cells that can
be infected is probably related to receptor usage, though dif- 5.1 Key Features of Epidemiology
ferentially expressed intracellular factors necessary for virus of Enteroviruses
replication could also play a role.
Over 100 enterovirus types have been identified, and many
cause overlapping spectra of illness presentations, from sub-
4.5 Replication of Enteroviruses clinical or asymptomatic infections to central nervous sys-
tem manifestations and sepsis. In particular, neonates and
Most of the details of enterovirus replication have been very young children are susceptible to more severe illness
elucidated through the use of poliovirus as a model [214]. due to antigenic inexperience, and most children are exposed
When a virus particle binds to its cellular receptor, a con- to enteroviruses early in life. However, some enterovirus
formational change is induced in the virion, resulting in infections may preferentially infect older age groups, and
extrusion of the genomic RNA into the cell cytoplasm prognoses may vary. Parechoviruses are known to cause
[27, 95]. The viral RNA is translated through a cap-indepen- more severe disease in neonates and young children.
dent mechanism facilitated by direct interaction of the viral Enteroviruses are common and are primarily transmitted
internal ribosome entry site (“IRES”) in the 5′ NTR with via the fecal-oral route. They may also be spread via respira-
cellular ribosomes [10]. Picornaviruses encode a single poly- tory droplets and from mother to infant prenatally and during
protein, which is subsequently processed by viral proteinases the peripartum period. Enteroviruses may survive on surfaces
238 M.S. Oberste and S.I. Gerber

long enough that fomites may play a role in transmission. In in subsequent years. There are two general patterns of circu-
addition, vesicle fluid may be another important route of trans- lation that describe enteroviruses in a population, endemic
mission for children who have HFMD or other exanthems. and epidemic activity. Endemic circulation within a popula-
Outbreaks of enterovirus infections may be identified in tion is best described as sporadic activity each year over a
communities and group settings. Families may spread long period of time such that children acquire infection early
enteroviruses among each other, and outbreaks often occur in life. Most individuals have acquired antibodies within the
in day-care and school settings. Hygiene practices play an first few years of life. Because of constant sporadic activity, a
important role in transmission so viruses are often spread large population of nonimmune children will not occur; thus,
from child to child. Fecal shedding of enteroviruses may per- it is unlikely a wide epidemic will follow. For example, in the
sist for several weeks, thus allowing children to potentially United States between 1970 and 2005, coxsackieviruses A9,
remain infectious for long periods. B2, B4, and enterovirus 71 were identified consistently at
In temperate climates, enteroviral infections are more low levels of circulation with few distinct peaks during this
often identified during summer and fall months, usually time period [113]. The epidemic pattern is best described
June–October (Fig. 11.1). However, in tropical climates, as large waves of circulation with longer periods of time
where sanitation may be poor and population density high, between waves such that a nonimmune and therefore sus-
children may become infected with multiple enterovirus ceptible population accumulates, potentiating an outbreak
types and strains simultaneously and year-round. Because of when the virus is later introduced. Epidemic patterns of cir-
the opportunity for increased transmission of enteroviruses culation in the United States from 1970 to 2005 were best
in this setting, most neonates are born with maternal antibod- exemplified by echoviruses 9, 13, and 30 and CVB5. Each
ies to a broader range of enterovirus types and are thus pro- of these viruses peaked and was among the most prevalent
tected from illness. However, these children will acquire enteroviruses reported in a given year [113]. Interestingly,
multiple enteroviral infections when maternal antibodies enteroviruses associated with endemic and epidemic patterns
wane, usually in infancy and preschool ages. of circulation in one geographic area or country may exhibit
a different pattern of circulation in another part of the world.
Thus, enterovirus circulation patterns and surveillance pri-
5.2 General Epidemiology of Enteroviruses orities are unique to different areas in the world, and trends
in enterovirus activity and identification of outbreaks should
5.2.1 Incidence and Prevalence be an important priority such that information can be shared
Seasonality of enteroviruses may be an indicator of disease with other countries where a specific type may not yet be
incidence for common disease presentations such as HFMD. In circulating.
summer and fall months, the percentage of specimens testing Outbreaks of infections caused by enteroviruses and
positive for enterovirus by NESS is elevated as compared to parechoviruses have occurred frequently in a wide variety of
other seasons [36, 113]. Occurrences of clinical syndromes geographic regions at different times. Enterovirus 71, first
associated with specific enteroviral serotypes may serve as a isolated in California in 1969 from stools from an infant with
marker of incidence of a particular enterovirus type in the com- encephalitis, is known to cause large outbreaks of HFMD,
munity. Because surveillance is passive and dependent on case most frequently affecting young children. Over the next 3
finding, the true incidence and prevalence of enteroviruses is years, 19 patients were identified with EV71 [5]. Notably,
unknown, and estimates of circulating enteroviruses are often EV71 activity has been identified to some degree in many
obtained from outbreak investigations. For example, an epi- countries of the world, but the largest outbreaks have occurred
demic of encephalitis in Uttar Pradesh, India, in 2008 was more recently in the Asia-Pacific region. Large outbreaks of
found to be associated with echovirus 19 and coxsackievirus EV71 were identified in this region in the late 1990s, with the
B5 [121]. In 2007 in the United States, coxsackievirus B1 was first report from Malaysia in 1997, followed by several out-
the predominant type at the same time an outbreak of serious breaks in other countries, including Singapore, Taiwan, and
neonatal infections was reported [36, 270]. Outbreak investiga- China [38, 141, 263, 288]. Although most affected children
tions of clinical syndromes and associated enterovirus types will have mild illnesses consistent with HFMD, a subset of
will often correlate with predominant strains circulating in the children will have more serious clinical presentations with
community. Understanding trends of circulating serotypes may neurologic manifestations, including aseptic meningitis,
help predict increases in incidence and prevalence. acute flaccid paralysis, and brainstem encephalitis. The World
Health Organization has drafted guidelines for clinical man-
5.2.2 Epidemic and Endemic Behavior agement and public health response to HFMD [275]. In Hong
Outbreaks of enterovirus infection/illness occur throughout Kong, enterovirus 71 infection has been a notifiable disease
the world. One set of types may be predominant for a time in since March 2009 [136]. These large outbreaks with associ-
a given geographic area, only to be succeeded by other types ated severe neurologic disease appear to be restricted to the
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 239

Asia-Pacific region, while in other parts of the world EV71 is was associated with severe central nervous system complica-
generally associated with uncomplicated HFMD with few tions [263]. During an investigation of enterovirus-associated
serious cases. The reasons for this geographic disparity are acute respiratory infections in older children and adults in
unclear but could include genetic differences or cultural dif- China from 2006 to 2010, the two most frequently detected
ferences in treatment. Enterovirus 71 is discussed in more types were identified in two distinct age groups. The median
detail in Chap. 13. age of patients with CVA21 was 22 (15–67) years, while the
Enteroviruses that cause acute hemorrhagic conjunctivitis median age for EV68 detection was 34 (18–67) years [278].
(AHC) may cause large epidemics in specific regions of the Male preponderance has been documented among sur-
world. AHC, primarily caused by EV70 and CVA24 vari- veillance data and descriptions of outbreaks of enteroviral
ant, is a rapidly progressive and highly contagious disease. illnesses. In the United States from 1970 to 2005, males pre-
AHC was first recognized in 1969 in Ghana [40] and, at dominated in the <20-year age group, but no gender prepon-
the time, was sometimes termed “Apollo eye disease” as its derance was seen in the ≥20-year age group (male/female
emergence coincided with the first moon landing. During a ratios, 1.4 and 0.9, respectively) [113]. Surveillance data for
4-month period, 13,664 cases were seen at one clinic, and all echovirus 6 and echovirus 9 infections in Taiwan from 2000
had subconjunctival hemorrhages associated with conjunc- to 2008 revealed a male preponderance for both types; 57
tival inflammation. A subsequent pandemic occurred from and 69 % of echovirus 6 and echovirus 9 cases, respectively,
1969 to 1971, when hundreds of millions of people were were male [124]. Among pediatric HFMD cases in Shanghai
estimated to have developed AHC. Since then, epidemics from 2009 to 2010, 61 % were male [286].
have been periodically identified, mostly in tropical regions
of the world. Outbreaks of AHC caused by CVA24v have 5.2.5 Occurrence in Families and Closed
occurred in Singapore, Brazil, Uganda, Southern Sudan, Ecological Units
China, and Cuba [35, 70, 246, 280, 284]. Transmission of enteroviruses is a frequent event in families
and contained group settings. The New York Virus Watch
5.2.3 Geographic Distribution and Climate Program in the 1960s demonstrated that spread of coxsacki-
Enteroviruses are most often identified during the summer eviruses within families was relatively high, 76 %, as com-
to fall months in temperate climates. In the United States, pared to spread of echoviruses at 43 % [118]. The reason
77.9 % of enterovirus cases were reported from June to for this discordance is unknown. Outbreaks of enteroviruses
October [113]. Likewise, NESS reported the majority of may occur in day-care settings, preschools, and summer
enterovirus detections during July–October in 2008 [36] camps; in particular, clusters of illnesses will be more likely
(Fig. 11.1). However, seasonality is less pronounced in tropi- identified in group settings of younger children. Outbreaks
cal climates, with the peak incidence generally occurring have been described at summer camps [91, 140], where chil-
during the rainy season. Prevalence tends to be highest in dren were involved with multiple group activities and lived
the tropics and lowest in cold regions. Children who live in in cabins or crowded dwellings. Enterovirus outbreaks char-
tropical climates and who may be exposed to poor hygienic acterized by HFMD or aseptic meningitis have occurred in
circumstances may transmit enteroviruses year-round. schools for younger children [2, 276]. Huang et al. reported
an outbreak of EV71 infection among infants in a newborn
5.2.4 Age and Sex nursery [99]. Seven of 19 infants were documented with clin-
Enteroviruses may cause disease in any age group, although ical illnesses that included fever, poor activity, and drowsi-
young children are the primary reservoir for these infections. ness. Another nosocomial outbreak involved transmission of
In the United States during 1970–2005, among reports where echovirus 30 from an index patient to five babies within a
age was known, 44.2 % of cases were aged <1 year [113]. neonatal nursery [9]. In addition, a nosocomial outbreak of
Similarly, from 2006 to 2008 from reports where age was HPeV1 was described in a neonatal postintensive care unit in
known, 47 % of laboratory detections of enteroviruses and Croatia [132]. Seven patients, six of whom were born prema-
parechoviruses were from children aged ≤1 year [36]. In turely, were documented to have had HPeV1. Transmission
a recent study performed in Spain, 76.8 % of all meningi- of enteroviruses among older children and adolescents in a
tis cases where a pathogen was isolated were positive for defined group has also been documented, including an out-
enteroviruses and 65 % of the meningitis cases identified break of CVB2 infection identified among members of a
were in children ≤15 years [58]. high school football team [4].
Children may be most susceptible to enteroviral infec-
tions, but epidemiologic and clinical features of disease can 5.2.6 Socioeconomic Setting
vary across age groups. EV71 causes more severe neurologic Low socioeconomic status has been associated with increased
manifestations in young children, particularly in Asia-Pacific transmission of enteroviruses, most probably due to poor
countries. For example, in 2008 in Taiwan, a younger age hygiene and high population density. Low socioeconomic
240 M.S. Oberste and S.I. Gerber

Fig. 11.3 Routes of enteric virus Excreta from Man

transmission (From Melnick et al.
[156], used with permission)

Land Runoff Sewage Solid Waste

Landfills

Oceans and Estuaries Rivers and

Groundwater Irrigation
Lakes

Shellfish Recreation Water Supply Crops Aerosols

MAN

settings have played a role in increased acquisition of entero- Transmission from mothers to babies may occur pre-
viruses in both temperate and tropical climates. Neonatal natally, during the peripartum period, and possibly
acquisition of enteroviruses was found to be associated with through breastfeeding. Mothers who have symptomatic
lower socioeconomic status in an urban area in the United or subclinical infections may transmit virus to neonates
States [107]. In tropical climates, where enteroviruses are which can result in aseptic meningitis or sepsis-like dis-
transmitted continuously throughout the year, poor sanita- ease. A study performed in Taiwan among pregnant
tion has been shown to be a factor in frequency of virus women who had herpangina demonstrated an increased
infection among infants [195]. risk of infection in low birth weight and small for gesta-
tional age babies, as well as preterm delivery [41].
Enteroviruses have been detected in breast milk, though
6 Mechanisms and Routes the role of breastfeeding in transmission from mothers to
of Transmission babies is unclear [139].
Shedding of enteric viruses, such as enteroviruses, in
Enteroviruses are primarily spread by the fecal-oral and the stool raises the possibility of contamination of recre-
respiratory routes (Fig. 11.3). Lack of hygiene will facilitate ational waters. Enteroviruses have been identified in
spread of virus person to person especially in areas of poor clams or oysters, and in recreational hot spring waters, but
sanitation. Because individuals may shed virus in the stool the role of shellfish and water in direct transmission to
for several weeks, fecal contamination of fingers and hands humans remains unclear [42, 98, 134]. Outbreaks of viral
may lead to efficient spread particularly in young children. meningitis have been associated with swimming in ponds
Droplets or aerosols may play a role in transmission and will and pools [33, 109]. In 2001 in Germany, an outbreak of
also contribute toward contamination of the hands of young echoviruses 13 and 30 was found to be associated with
children. Under optimal conditions, which include tempera- swimming in a pond [90]. An investigation of an echovi-
ture and pH, enteroviruses may survive for weeks in the envi- rus 30 outbreak among children in Italy in 1997 showed
ronment, so fomites may play a role in viral spread as well. the risk of meningitis-like illness to be higher among chil-
Select enterovirus types may be spread from person to dren attending a common school, among children who
person by other specific routes. EV70 and CVA24 variant are swam at any public pool, and those who swam at a spe-
causes of acute hemorrhagic conjunctivitis. Transmission of cific pool [68]. In Mexico in 2004, an outbreak of echovi-
these viruses from person to person is most likely through rus 30 and coxsackievirus A1 infections among travelers
eye secretions, as virus has been isolated from conjunctival was associated with swimming in sewage-contaminated
swabs in outbreak settings [280]. In addition, enteroviruses seawater [12]. Enteroviruses have the potential to contam-
that cause rashes may be transmitted from person to person inate recreational waters and may pose a health risk to
via vesicular fluid which contains infectious virus. swimmers.
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 241

Fig. 11.4 Serum and secretory antibody response to oral Serum IgG
administration of live attenuated polio vaccine and to 512
intramuscular inoculation of killed polio vaccine (From Ogram
et al. [192], used with permission)
Killed parenteral

Reciprocal poliovirus antibody titer

128
Live oral

32
Serum IgM Nasal IgA

Serum IgA
8

Duodenal IgA
2
Nasal and
duodenal IgA

1
Vaccination
16 32 48 64 80 96
Days

7 Pathogenesis and Immunity exposures to other enterovirus types and the presence
of shared, cross-reactive epitopes [61, 208]. Enterovirus
7.1 Pathogenesis immunity in humans has been most easily studied using
polio vaccine as a model system (Fig. 11.4) [191].
The enteroviruses and parechoviruses are transmitted pri- Antibody plays the major role in protection from disease.
marily by the fecal-oral route, with entry through the alimen- A type-specific poliovirus serum neutralization titer of
tary tract, though respiratory transmission may also occur. 1:8 protects from paralytic disease; however, even very
The incubation period is generally 7–14 days, but it can high titers do not protect from reinfection by the homolo-
range from as few as two to as long as 35 days. Initial repli- gous virus. Maternal antibody passively transferred to the
cation occurs in the pharynx and gut, probably in lymphoid infant gradually wanes during the first 6 months of life,
tissues such as Peyer’s patches. In some cases, a brief vire- with a half-life of approximately 28 days. Type-specific
mia may be followed by replication at secondary sites in spe- immunity is also transferred from mother to infant via
cific target organs such as the spinal cord and brain, meninges, milk [145]. Upon exposure to virus, neutralizing antibody
myocardium, muscle, or skin, with some variability by strain appears within a few days, often before the onset of symp-
or type. Virus is excreted in stool for 4–8 weeks postinfec- toms; antibody may persist for life [209]. The tonsils and
tion and in respiratory secretions for 1–2 weeks [6]. Mixed adenoids appear to play a role in development of immu-
infections and detection of multiple enteroviruses in a single nity, as surgical removal decreases secretory antibody
stool sample are not uncommon, especially in areas with levels in the nasopharynx and resistance to poliovirus
poor sanitation and high population density [188, 190, 204]. infection is also decreased.
EV70 and certain strains of CVA24 are unique in that they Persons with primary immunodeficiency disorders,
can cause explosive outbreaks of acute hemorrhagic con- especially those with profound antibody deficits, are at
junctivitis, sometimes of pandemic proportions. These increased risk for enterovirus disease, as they lack the
viruses are rarely detected in stool or respiratory specimens, ability to clear the infection [93, 142, 173, 181, 213, 235,
but they can be readily detected in conjunctival swabs [199]. 271, 290]. Such persons may become chronically infected
and excrete virus over an extended period [93, 142]. There
has been some suggestion that treatment with intravenous
7.2 Immunity immune globulin is helpful in some cases, but, overall,
results have been mixed. Patients treated for B-cell lym-
Enterovirus infection elicits a strong humoral immune phoma with monoclonal antibody drugs such as rituximab
response. In young children, the response tends to be type appear to be at increased risk for persistent enteroviral
specific, whereas older children and adults tend to mount infection and disease [8, 198], probably through depres-
a more heterotypic response, probably due to previous sion of B-cell function [255].
242 M.S. Oberste and S.I. Gerber

8 Patterns of Host Response EV71 is known to cause brainstem encephalitis and acute
flaccid paralysis, particularly in young children in Asia-Pacific
8.1 Clinical Syndromes countries. Brainstem encephalitis is often associated with car-
diorespiratory symptoms which may include neurogenic pul-
Enteroviruses may cause a wide spectrum of clinical syn- monary edema. Severe cases of EV71 disease may also include
dromes, with severity ranging from asymptomatic presenta- acute flaccid paralysis as the presenting feature and anterior
tions to acute flaccid paralysis, encephalitis, and myocarditis. horn cell destruction may be identified as well [193].
Specific enterovirus types may be associated with a particular
clinical syndrome or multiple clinical presentations. In addi- 8.1.3 Pleurodynia (Epidemic Myalgia,
tion, a particular clinical syndrome may be caused by multiple Bornholm Disease)
enterovirus types. The ability of individual enterovirus and Pleurodynia is described as fever with accompanying chest
parechovirus types to cause overlapping clinical syndromes and upper abdominal pain. It is characterized by abrupt onset
emphasizes the notion that typing in the laboratory is neces- of fever and lower chest pain, sometimes preceded by mal-
sary for diagnosing a potential cause of an enterovirus illness. aise, headache, and anorexia [13, 97, 268]. The chest pain
may be located on either side or substernally, is intensified by
8.1.1 Asymptomatic Infections movement, and may last from 2 days to 2 weeks. Abdominal
The vast majority of enterovirus infections are asymptomatic pain resulting from involvement of the diaphragm occurs in
or result in only very minor symptoms such as lethargy and approximately half the cases; in children, this often takes the
general malaise, transient low-grade fever, or minor upper place of chest pain and may be the chief complaint. The syn-
respiratory tract illness [77, 171, 201, 225]. Despite the lack of drome was first described after an outbreak on the island of
discernible illness, persons with asymptomatic infections may Bornholm in Denmark. Epidemic pleurodynia or Bornholm
excrete virus for long periods and participate in transmission. syndrome is usually caused by group B coxsackieviruses.
Less commonly, this syndrome may be caused by echovi-
8.1.2 Meningitis, Encephalitis, and Acute ruses and group A coxsackieviruses. A recent outbreak of
Flaccid Paralysis epidemic pleurodynia caused by coxsackievirus B3 included
Neurologic manifestations caused by non-polio enteroviruses clinical descriptions of pain with deep inspirations [100].
and parechoviruses include aseptic meningitis, encephalitis, These findings may indicate inflammation of the pleura as
and acute flaccid paralysis. Aseptic meningitis is characterized part of this syndrome. The illness is self-limited, and recovery
by fever, myalgia, nuchal rigidity, nausea, and vomiting [227]. is complete with no long-term sequelae.
In small children <2 years of age, fever and irritability are the
predominant symptoms [221]. In a prospective study of viral 8.1.4 Herpangina
infections of the central nervous system in Spain from March Herpangina is caused by a wide variety of different group A
1, 2008 to February 28, 2009, the most common cause of men- coxsackievirus types [82]. The illness is characterized by an
ingitis was an enterovirus [58]. Aseptic meningitis may be abrupt onset of fever and sore throat. There may be anorexia,
caused by many enterovirus types including echoviruses 4, 6, dysphagia, vomiting, and abdominal pain. The pharynx is
9, 11, 14, 16, 25, 30, 31, and 33 and coxsackieviruses B1–B6, usually hyperemic, and a few (not more than 10–12) charac-
A7, and A9. EV71 has frequently been associated with aseptic teristic tiny discrete vesicles with a red areola occur on the
meningitis [193]. Ultimately, virtually all enteroviruses are anterior pillars of the fauces, the posterior pharynx, the pal-
capable of causing aseptic meningitis at some frequency. ate, the uvula, the tonsils, or the tongue. The illness is self-
Meningoencephalitis, aseptic meningitis with inflamma- limited and occurs most frequently in small children.
tion involving the brain parenchyma, and encephalitis with-
out aseptic meningitis may also be caused by enteroviruses. 8.1.5 Hand, Foot, and Mouth Disease
Although an etiology is not associated with most cases of Hand, foot, and mouth disease (HFMD) is a common illness
encephalitis, enteroviruses are consistently identified. From in young children. HFMD is characterized by almost univer-
1998 to 2005, the California Encephalitis Project reported sal occurrence of enanthem, usually on the buccal mucosa,
nearly 1,600 encephalitis cases, of which 4.6 % were either generally followed by exanthem on the palms of the hands
confirmed as enterovirus etiology by detection in CNS sam- and soles of the feet. The oral lesions are ulcerative, while
ples or of possible enterovirus etiology, by detection in other lesions on the hands and feet are usually vesicular. CVA16
specimen types [71]. Enterovirus encephalitis cases tended and EV71 are the most common causes, though other group
to be younger and had less severe symptoms than those with A coxsackieviruses are also frequently associated with
other identified etiologies. Similarly, the New York State HFMD, especially CVA6 and CVA10. The disease is usually
Department of Health identified 21 cases of encephalitis due mild and self-limiting, but more severe illness has accompa-
to enteroviruses from June 2004 to September 2007 [63]. nied large HFMD outbreaks due to EV71, primarily in Asia.
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 243

In 2010–2012, there were large outbreaks of hand, foot, and For both forms of enteroviral conjunctivitis, virus can be
mouth disease due to coxsackievirus A6 in Asia, Europe, and readily detected in conjunctival swab and throat swab speci-
North America [37, 69, 76, 80, 133, 162, 194]. These out- mens by PCR. While CVA24 is excreted in feces, EV70 is
breaks were somewhat unusual in that illness was often more only rarely detected in stool [163].
severe, with a more pronounced, often pustular-appearing
rash that involved the face and trunk as well as hands and 8.1.8 Cardiac Diseases
feet; in addition to young children, adults were also affected, Acute Cardiac Disease
often in the context of day-care centers or other exposures to Group B coxsackieviruses are known to be associated with
very young children [37]. HFMD may sometimes be accom- myocarditis. CVB3 is the most common cause of human
panied by onychomadesis (shedding of nails) sometime after viral myocarditis, although group A coxsackieviruses and
resolution of initial symptoms [56, 194, 267]; however, the echoviruses have been identified in association with cardiac
nails usually regrow without complication. disease [274]. Most cases of group B coxsackievirus myo-
carditis occur in young adults, though severe coxsackievirus
8.1.6 Respiratory Illness B myocarditis can also occur among neonates. Although a
Many different enteroviruses, from each of the traditional number of serological studies have attempted to correlate
taxonomic groups and each of the current viral species, have myocarditis with enterovirus seropositivity or antibody titer,
been associated with mild upper respiratory tract illness [55, there has not been a clear association between viral antibody
165]. CVA21 has caused outbreaks of pharyngitis in military and causality of myocarditis. Endomyocardial biopsy is the
recruits and has induced respiratory tract disease in normal diagnostic test of choice to determine etiology of viral myo-
adult volunteers. For the most part, severe disease is rare, but carditis [137].
EV68 was initially isolated from children with bronchiolitis
and pneumonia [233]. More recently, EV68 has been associ- Chronic Cardiovascular Disease
ated with sporadic cases and outbreaks of bronchitis, bron- The pathogenesis of chronic heart disease due to enterovirus-
chiolitis, and pneumonia [103, 104, 106, 143, 186, 215]. associated dilated cardiomyopathy has been thought to begin
with acute myocarditis and progress over time. Although
8.1.7 Eye Disease enteroviruses have not been isolated directly from these
Infection with a number of different enteroviruses has been chronic cardiac disease patients, the presence of enteroviral
occasionally accompanied by conjunctivitis. However, in RNA has been detected [138].
1970, an explosive epidemic of acute conjunctivitis occurred
in Singapore, with some 60,000 cases reported. The caus- 8.1.9 Neonatal Disease
ative agent was identified as an antigenic variant of CVA24 Acquisition of enteroviruses is common during the first
[128, 163]. Additional large outbreaks occurred elsewhere in month of life [164]. In one study, 12.8 % of neonates were
Asia in subsequent years [39, 129, 282, 283], and CVA24 found to have enterovirus infections, although most of these
variant conjunctivitis has been reported globally since then infections were asymptomatic [107]. Of over 26,000 entero-
[28, 29, 32, 64, 70, 111, 253]. Conjunctivitis associated with virus detections reported to NESS during 1983–2003, 11.4 %
CVA24 variant is generally mild, with complete recovery in of those with known age were from neonates (age ≤ 1 month)
1–2 weeks. The only potentially serious sequelae are due to [112]. The 10 enterovirus types most frequently reported
secondary bacterial infections. were echoviruses 11, 9, 6, and 30 as well as coxsackieviruses
Beginning in 1969 and extending to 1971, a somewhat B1–B5 and CVA9. The risk of a fatal outcome was higher for
different form of conjunctivitis emerged in Africa, India, neonates as compared to reports where age was noted to be
Southeast Asia, and Japan, reaching pandemic proportions, ≥1 month. Infection with CVB1–4 and echoviruses 11 or 25
with tens of millions of cases. This pandemic of acute hem- was significantly associated with increased risk for fatal out-
orrhagic conjunctivitis (AHC) was due to a new enterovirus, come, with odds ratios of 1.4–2.3. Similarly, neonates com-
EV70 [120, 163, 281]. About 10 years later, AHC reap- prised 9.5 % of patients from whom enterovirus was isolated
peared, causing outbreaks in several of the countries initially during a 2-year study in the Netherlands [256].
affected and spreading to the Caribbean and tropical Latin Severe illnesses caused by enteroviruses in this age group
America, with a few cases reported in the continental United include sepsis, meningoencephalitis, hepatitis, and myocar-
States [207], as well as several Pacific islands. AHC is char- ditis [247]. A study of neonatal enterovirus infections in a
acterized by severe eye pain, photophobia, and blurred Taiwan children’s hospital revealed 43 cases of nonspecific
vision, accompanied by subconjunctival hemorrhage which febrile illness, 61 cases of aseptic meningitis, and 42 cases of
can vary in extent from discrete petechiae to large blotches. hepatic necrosis with coagulopathy [131]. Of these 42 cases,
The incubation period is about 24 h, onset is sudden, and 10 were fatal; eight of the 10 fatalities were associated with
recovery is usually complete within less than 10 days. group B coxsackievirus infections (four CVB1, three CVB3,
244 M.S. Oberste and S.I. Gerber

and 1 CVB2), with the remaining two cases due to echovirus significantly more likely than controls to have IgM to one or
4 and an untypable enterovirus. In the United States, from more of the enteroviruses tested, with the strongest associa-
2007 to 2008, CDC received reports of six neonatal deaths tion in the age group 13–18 years. A second study used
associated with CVB1 infection, all of which were due to RT-PCR to assess the presence of enterovirus RNA in three
multisystem organ involvement [270]. During the outbreak, groups of children: 47 with newly diagnosed type 1 diabetes,
a cluster of eight neonates with severe myocarditis due to 50 positive for at least one common islet autoantibody, and 50
closely related strains of CVB1 was also described [258]. An negative for islet autoantibodies [168]. Enteroviral RNA was
additional two infants, one with a diagnosis of CVB1, were detected in 36 % of those with newly diagnosed diabetes,
identified. Of these ten neonates, one patient died and one 20 % of the autoantibody-positive group, and only 4 % of the
patient required cardiac transplantation. In the Netherlands, autoantibody-negative group, again suggesting an association
Freund et al. reported a case series of seven patients with between enterovirus infection and islet autoimmunity or dia-
severe neonatal myocarditis, of which two died and the betes. Similarly, another RT-PCR study detected enteroviral
remaining five infants had cardiac sequelae [73]. RNA in serum of 26 % of children with newly diagnosed type
Human parechoviruses have been associated with severe 1 diabetes versus 3 % of matched controls [230]. The latter
disease in young infants [19, 25, 88, 257, 273]. In Spain, eight study is significant in that it was conducted in Cuba, a country
(3 %) of 264 CSF samples collected from children <2 months with high enterovirus prevalence and low rate of type 1 diabe-
from January 2006 to September 2009 were positive for tes, a situation in which one might expect it to be difficult to
human parechoviruses [210]. Human parechovirus was iden- demonstrate the association.
tified in 7.4 % of CSF samples tested in 2006–2008 in a
regional children’s hospital in the Midwestern United States 8.1.12 Summer Minor Illnesses with or Without
[237]. Associated clinical syndromes were mostly associated Exanthems
with sepsis and meningitis. Notably, CSF pleocytosis was Enteroviruses are often isolated from patients with undiffer-
rarely documented. Likewise, another case series identified entiated acute febrile illnesses of short duration occurring
ten children, aged 6–59 days, with parechovirus type 3 asso- during the summer or fall. In young children, the illness may
ciated with workups for sepsis and meningitis [262]. HPeV3 be accompanied by a rubelliform, maculopapular rash on the
was identified in 51 (13 %) of 388 CSF samples from children face, neck, and chest. The incidence of rash tends to be
aged <6 months from a children’s hospital in Kansas City in higher in young children and decreases with age.
2009 [239]. CSF pleocytosis is often absent [239, 257]. Conjunctivitis may also be present. Echovirus 16 has been
HPeV3 has been specifically associated with more severe dis- associated with outbreaks of “Boston exanthem disease,”
ease in neonates, compared with HPeV1 [19]. characterized by fever and maculopapular rash [171, 172].

8.1.10 Gastrointestinal Diseases

Group B coxsackieviruses have been associated with inflam- 9 Control and Prevention
mation of the pancreas. Coxsackievirus-associated pancre-
atitis has been described in case reports [50, 197]. Mouse There are currently no licensed antiviral therapies available
models of pancreatitis induced by group B coxsackieviruses to treat enterovirus or parechovirus infections or illness, but
have demonstrated pancreatic damage that may be similar to it is an area of active research and a number of compounds
human disease [101]. are in preclinical development [59, 249, 250]. Similarly,
there are no vaccines available to prevent disease due to non-
8.1.11 Diabetes polio enteroviruses or parechoviruses. However, there is
While the connection is not yet conclusive, a number of stud- interest in developing a vaccine for EV71, particularly in
ies have demonstrated an association between enterovirus Asia, where large outbreaks have occurred over the last 15
infection and development of prediabetic autoimmunity or years, with a considerable burden of severe disease and hun-
onset of type 1 diabetes in individuals at genetic risk for dia- dreds of fatalities. One EV71 vaccine candidate has recently
betes [44, 92, 168, 230]. Several ongoing longitudinal stud- completed a phase II immunogenicity trial, with favorable
ies have also been designed to address the association results [289]. There has been apparent success with the use
between a variety of potential environmental triggers with of immunoglobulin for prophylaxis and control of an out-
diabetes onset, including enterovirus or other infections, break of enterovirus infection in a nursery [75].
foods, and other factors [reviewed in reference 244]. Prevention and control generally depend on stressing
Helfand et al. tested sera from 128 children with new onset good personal hygiene, including hand washing and thor-
insulin-dependent diabetes mellitus and 120 matched con- ough cleaning of potentially contaminated items such as
trols [92]. The sera were tested for enterovirus-specific IgM toys, etc., especially in settings such as day cares where very
using 14 different serotypes as antigen. Case children were young children may be together. In a hospital setting, enteric
11 Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others 245

precautions are important, as nosocomial infections can be Acknowledgments We are indebted to the late Dr. Joseph Melnick,
particularly serious in newborns and immunocompromised enterovirus pioneer and author of the previous edition of this chapter. His
outstanding text has helped guide the development of the current edition.
patients. Cohorting of patients and staff can also be useful to
control transmission during an outbreak. In some cases, fur-
ther virus spread has been limited by closing newborn nurser-
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 Enteroviruses: Enterovirus 71
12
Mong How Ooi and Tom Solomon

1 Introduction and Historical Background 2 Descriptive Epidemiology

The global polio eradication campaign has eliminated this 2.1 Initial Epidemiological Observations
devastating enteroviral disease from Europe, the Americas,
and most of Africa and Asia. However, in the past 15 years, a Although EV71 was first isolated from the stool of a 9-month-
related virus, human enterovirus 71 (EV71), has emerged old infant with encephalitis in California in 1969, a retrospec-
across Asia, where it threatens to become the “new polio” tive analysis of samples from the Netherlands showed that
[120]. EV71 first appeared in California in the 1960s and sub- EV71 was circulating as early as 1963 [128, 147]. Before long
sequently caused sporadic cases or small outbreaks of hand- small outbreaks of neurological infection including encephali-
foot-and-mouth disease (HFMD) [120], neurological disease, tis and aseptic meningitis attributed to the newly identified
or both. In 1997, the virus caused an unexpectedly large and neurotrophic virus were reported in New York, Melbourne,
severe outbreak with many fatalities in Sarawak, Malaysia. and Sweden in the early 1970s [9, 43, 74]. The dermotropic
Since then, countries of the Asia-Pacific region have been hit properties of EV71 were first recognized during epidemics of
by regular epidemics of EV71, including an epidemic in HFMD in Japan in 1973 [54, 67]. In the 1970s, two large and
Taiwan in 1998, in which millions of people were thought to severe EV71 epidemics occurred in Europe. The first was in
have been infected, and an outbreak of HFMD in China in Bulgaria in 1975, initially attributed to polioviruses because
2009, during which nearly 500,000 cases were reported [13, its epidemiological, clinical, and pathological characteristics
16, 33, 55, 56, 76, 98, 112, 146, 171, 177]. Although the virus mimicked those of poliomyelitis [40, 137]. In fact, at the
circulates worldwide, the largest outbreaks of disease have so height of the epidemic, nationwide administration of live-
far been largely confined to the Asia-Pacific region, for rea- attenuated poliovirus vaccine was instituted. Isolation of EV71
sons that are incompletely understood [12, 13, 16, 17, 33, 56, later confirmed this virus as the etiological agent in 347 (77 %)
70, 76, 98, 136, 146, 176, 178]. The neurological manifesta- of 451 children who presented with nonspecific febrile illness
tions range from aseptic meningitis to acute flaccid paralysis or neurological disease, of whom 44 died. The second major
and brainstem encephalitis. They are often accompanied by European epidemic occurred in Hungary in 1978, with 1,550
systemic complications such as severe pulmonary edema and cases (826 aseptic meningitis and 724 encephalitis) and 47
shock, which are thought to be neurogenic in origin [22, 63]. deaths. Unlike the Bulgarian epidemic, the Hungarian one
included a small number of patients with HFMD [103].

M.H. Ooi, MD (*)

Institute of Health and Community Medicine, Universiti Malaysia 2.2 EV71 Activity in the Asia-Pacific Region
Sarawak Kota Samarahan, Sarawak 94300, Malaysia
e-mail: [email protected]
After the Australian and Japanese EV71 epidemics of the
T. Solomon, BA, BM, BCh
1970s, further small epidemics and sporadic clusters occurred
Brain Infections Group, Institute of Infection
and Global Health, University of Liverpool, Liverpool, UK in Hong Kong (1985) and Australia (1986) [51, 126]. Then in
1997, a large outbreak in Sarawak, Malaysia, heralded the
Department of Neurological Science,
Walton Centre NHS Foundation Trust, Liverpool, UK beginning of a series of large outbreaks across the Asia-Pacific
region. These continuing epidemics have established EV71 as
NIHR Health Protection Research
Unit in Emerging and Zoonotic Infection, Liverpool, UK a major public health threat across the region (Table 12.1) [11,
e-mail: [email protected] 12, 27, 39, 64, 66, 71, 79, 82, 97, 136, 146, 177, 178].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 253

DOI 10.1007/978-1-4899-7448-8_12, © Springer Science+Business Media New York 2014
 254

Table 12.1 EV71 genogroups circulating in Asia-Pacific region between 1973 and 2012 [11, 12, 27, 39, 64, 66, 71, 79, 82, 88, 97, 105, 121, 125, 136, 143, 146, 166, 177, 178]
Countries 1973 1980 1986 1988 1990 1993 1994 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Singapore – – – – – – – – B3, B3, B3 B4 B4 C1, B4 – – B5 – B5, – – – –
B4 C1 B4 C2
Peninsular – – – – – – – – 3, C1 B4, B4, – – – – B5, – – – – – – –
Malaysia B4, C1 C1 C1
C1a,
C2
Sarawak, – – – – – – – – B3 C1 None B4, None C1 B5, None B5 B5 – B5 B5 – – B5
Malaysia C1 C1
Perth, – – – – – – – – – – B3, C1 None None – – – – – – – – – –
Australia C2
Australia – – – – – – – – – C2 B3, B4, B4, C1 C1 C4 – – – – – – – –
C2 C1 C1
Japan B1 – – – B2, B2 C3 B3, C2 C2 B4 C2 B4, C4, C4 – – – – – – – –
C1 B4, C2 B5
C2
Taiwan – B1 B1 – – – – – – C2, B4 B4 B4 B4, B4, C4 C4, C5 B5, B5, B5 C4 – –
B4a C4a B5a C5a C5 C2a,
C4a,
C5a
Korea – – – – – – – – – – – C3 None None C4 – – – – – C4 – – –
Brunei – – – – – – – – – – – – – – – – – B5 – – – – – –
Vietnam – – – – – – – – – – – – – – – – C1, – – – C4 – C4 –
C4,
C5
Thailand – – – – – – – – – – – – – C1 C1 C1 – B5, B5, B5a, B5a, – B5 –
C1, C1, C1, C1a,
C2, C2, C2a, C2a,
C4 C4, C4 C4
C5
China – – – – – – – C2 – C4 – C4 C4 C4 C4 C4 – – C4 C4 C4 C4 C4 –
Cambodia – – – – – – – – – – – – – – – – – – – – – – – C4
Hong Kong – – – C1 – – C1 – C2 C4 B3, C1, B4, C1, C4 C4 C4 C4 C2, B5, – – – –
C1, C4 C4 C2 C4 C2,
C4 C4
Subgenogroups underlined are those which caused large outbreaks
a
Subgenogrous that were isolated in a smaller number
None: No HEV71 was detected despite active surveillance
– No data
M.H. Ooi and T. Solomon
12 Enteroviruses: Enterovirus 71 255

Table 12.2 Clusters and outbreaks of EV71 infection occurring outside the Asia-Pacific region, 1988–2010
Country Year Ages N Clinical features and comments Reference
Brazil 1988–1990 Children 90 IgM to EV71 in 20 blood samples of patients with [42]
flaccid paralysis
Finland 1994–2010 Children <11 years 928 Neutralizing antibodies in 1.6 % of serial serum [58]
samples
1996–2008 Children <6 years 359 PCR positive in 0.3 % of serial stool samples
Central African 1997–2006 Children 93 EV71 in stool samples of patients with flaccid paralysis [7]
Republic
Canada 1998 Children 20 EV71 Aseptic meningitis; respiratory symptoms [100]
Brazil 1998–2001 Children ≤15 years 389 Neutralizing antibodies in 222 serum samples [14]
Kenya 1999–2000 Children – – [15]
France 2000–2009 Mostly children 81 EV71 Neurological disease, HFMD, fever rash, [141]
gastrointestinal symptoms, respiratory symptoms,
and gingivostomatitis; 2 fatal: cardiorespiratory
failure and encephalitis
Austria 2001–2004 Children 181 with aseptic 16 with EV71 in stool [113]
meningitis
United Kingdom 2001–2006 – 32 EV71 Most with neurological disease or HFMD [8]
Norway 2002–2003 Children 19 EV71 Asymptomatic [161]
Denver, USA 2003, 2005 Children: 4 weeks 15 EV71 1 fatal [116]
to 9 years
Denmark 2005–2008 Mostly infants 29 EV71 Aseptic meningitis, gastroenteritis, HFMD [130]
Netherlands 2007 – 58 EV71 Fever, gastrointestinal, and neurological symptoms [147]
Greece 2009–2010 Children 6 EV71 Fever, rash, or HFMD [138]

During the Sarawak outbreak, between May and July The clinico-epidemiological features of the EV71 epidem-
1997, a total of 2,618 HFMD cases and 34 deaths were ics in Asia in recent years differ considerably from earlier epi-
recorded. EV71 also caused four deaths in peninsu- demics in that in addition to HFMD, aseptic meningitis, and
lar Malaysia and a number of cases of severe neurologi- flaccid paralysis, brainstem encephalitis associated with car-
cal disease in Japan [13, 76, 92]. Another large epidemic diopulmonary dysfunction also occurs, which has been the
occurred in Taiwan in 1998 when 405 children were hospi- principal cause of death in most fatal cases in Asia [13, 63,
talized for serious neurological complications and 78 died. 120, 146, 178]. The affected children have typically presented
Epidemiological studies estimated that almost 1.5 million with a short febrile illness accompanied by subtle neurological
people were infected with the virus [56]. The largest epi- signs, following which they develop signs of tachycardia, poor
demic to date occurred in China in 2008; approximately perfusion, and tachypnea, which rapidly develops into acute
490,000 children including 126 deaths were reported intractable cardiac impairment and fulminant, often fatal, pul-
nationwide. At the epicenter in Anhui Province, more than monary edema or hemorrhage [111]. Diagnostic imaging and
6,000 HFMD cases and 22 deaths were reported [178]. autopsy examination indicate that this is associated with
Surveillance improved following designation of HFMD encephalitis in the brainstem, especially the medulla, and neu-
as a notifiable disease in China in May 2008, and regular rogenic pulmonary edema is thought to be the primary patho-
massive epidemics with alarming number of deaths were genic process [22, 63, 91, 92]. Such rapidly fatal HFMD was
recorded. There were 1.1 million cases (353 deaths), 1.8 not observed in epidemics caused by EV71 in the 1970s and
million cases (905 deaths), 1.6 million cases (509 deaths), 1980s, where aseptic meningitis was the predominant clinical
and 2.2 million cases (567 deaths) reported in 2009, 2010, manifestation [9, 74].
2011, and 2012, respectively (http://www.chinacdc.cn/tjsj/
fdcrbbg/index_1.html, accessed 15/7/2013). In addition
to these very large outbreaks, many countries, including 2.3 EV71 Circulation Outside
Sarawak, Taiwan, Singapore, Vietnam, and Japan, have the Asia-Pacific Region
experienced cyclical epidemics at 2- to 3-year intervals
[59, 101, 119]. The most recent EV71 epidemic occurred in Outside the Asia-Pacific region, EV71 has continued to circulate
Cambodia in 2012. The outbreak, first labeled as a mysteri- at low levels of activity in America, Europe, and Africa, produc-
ous illness by local media, was associated with more than ing sporadic cases or small outbreaks. The outbreaks involve
50 sudden deaths within 24 h of hospitalization in children mainly young children who develop aseptic meningitis, hand-
who were 3 years of age and below [129]. foot-and-mouth disease, and other complications (Table 12.2).
256 M.H. Ooi and T. Solomon

Clearly, EV71 has spread across the world. Why the huge A genetically distinct EV71 strain (R13223, GenBank acces-
epidemics have been confined to the Asia-Pacific region, and sion no. AY179600 to AY179602), with no genetic relation-
whether they might be seen elsewhere in the future is not ship to other strains recently isolated in the Asia-Pacific
fully understood, though recent molecular epidemiological region, was isolated from a child with acute flaccid paralysis
work on of the evolution of the different genotypes of virus in India in 2001; it has been assigned as the only member of
has offered some key insights into this important question. genogroup D [44]. In short, two major EV71 genogroups (B
and C) have co-circulated and coevolved into subgenogroups
in the Asia-Pacific region over the past 15 years. In Malaysia
3 Molecular Epidemiology and Singapore, the genogroup B viruses have dominated,
whereas in East Asia, particularly in mainland China and
3.1 Virus Genogroup, Evolution, Vietnam, genogroup C viruses have dominated.
and Geographical Distribution Interestingly, EV71 isolates of genogroup A were reported
from 5 of 22 children presenting with HFMD in Anhui province
Before 1999, data about the molecular epidemiology of EV71 of Central China during the 2008 outbreak that was the first ever
were sparse. However, systematic laboratory surveillance estab- reported detection of genogroup A viruses since the original
lished in several Asian countries following the first epidemics in prototype was isolated in America [174]. The nucleotide
the late 1990s has provided invaluable information on the geo- sequence of the complete VP1 gene of the isolates clusters very
graphical distribution, spread, and evolution of the virus. closely with that of the prototype genogroup A virus, with mini-
The first complete phylogenetic analysis of EV71 based mal divergence. This highly unexpected occurrence could indi-
on the structural VP1 gene identified three independent lin- cate that the genogroup A virus has been circulating undetected
eages of EV71 and designated them as genogroups A, B, and in central China with very little evolutionary change for four
C [11]. A sequence diversity of at least 15 % in the VP1 gene decades or it could raise doubts about the source of the virus
was used to distinguish genogroups. Genogroup A consists templates that were sequenced. Surveillance from the same out-
of a single member, the prototype BrCr strain, which was break by the Chinese Center for Disease Control does not
first identified in California in 1970 and until very recently appear to have identified any genogroup A viruses [178]. Hence,
had never been reported outside the USA (see below). The it is important to await confirmation by other laboratories before
genogroup B viruses, subdivided into subgenogroups B1 and concluding that genogroup A viruses have reemerged. Another
B2 with divergence of 12 % at the nucleotide level, were the genetically distinct EV71 strain (NMA-03-008, GenBank acces-
predominant circulating strains in the 1970s and 1980s. The sion no. JN255590) belonged to a previously unknown novel
genogroup C viruses, also similarly subdivided into C1 and genogroup has been reported in Central African Republic; this
C2, were only identified later in the mid-1980s (Fig. 12.1). A indicates the existence of an additional African genogroup,
recent phylogenetic analysis suggests that EV71 may have which may have restricted geographical distribution so far [7].
emerged from the genetically closely related coxsackie virus Clearly, good surveillance programs are required in many dif-
(CV) A16 as recently as 1940 [144]. ferent geographical regions in order to gather accurate and rel-
A number of new subgenogroups within genogroups B evant information about EV71 transmission and evolution.
and C have emerged in the Asia-Pacific region in recent
years. Subgenogroup B3 and B4 viruses are thought to have
co-circulated in the region since 1997 [12, 97, 136]. 3.2 Transmission and Epidemic Potential
Subgenogroup B5 was first isolated in Sarawak and Japan in of Genogroups
2003 and was responsible for epidemics in Sarawak, Taiwan,
and Brunei in 2006 [1, 66, 101, 119]. Except for the major Following the initial outbreaks in Sarawak and Taiwan in the
community outbreak in Sydney in 1986, subgenogroup C1 late 1990s, surveillance systems for EV71 were established in
viruses have mostly been isolated from sporadic cases since a number of countries in the Asia-Pacific region; while pri-
the mid-1980s, suggesting a low level of endemic circulation marily aimed at monitoring transmission and spread, they
across the globe [11, 127]. Subgenogroup C2 viruses were have also provided invaluable insights into how the virus
responsible for the large EV71 epidemic in Taiwan in 1998 evolves in the community during outbreaks. For example, a
and the Perth outbreak in 1999 [12, 82, 96, 98]. Subgenogroup virological surveillance system in Sarawak, Malaysia, has
C3 was isolated in Japan in 1994 and Korea in 2000 [12, 68, shown that increased EV71 circulation occurred every 3 years
70]. Since 2000, subgenogroup C4 has been the predominant (1997, 2000, 2003, 2006, and 2008/09), closely paralleling
circulating genogroup in mainland China, through the most increased reports of HFMD in the community [119]. The phe-
recent epidemic in 2008, and has occurred in Japan, Vietnam, nomenon of regular cyclical epidemics has also been observed
and Taiwan [82, 136, 146, 178]. Subgenogroup C5 has been in Fukushima Prefecture, Japan [59]. Such cyclical activity is
reported from Southern Vietnam and Taiwan [66, 146]. presumed to relate to the availability of new birth cohorts of
12 Enteroviruses: Enterovirus 71 257

Fig. 12.1 Phylogenetic

analysis of EV71 VP1
gene sequences. A
neighbor-joining tree
constructed using the
Kimura-2 parameter as a
model for nucleotide
substitution. The
robustness of the tree was
determined by bootstrap-
ping using 1,000
pseudoreplicates.
Sequences are labeled
according to the
following convention:
“GenBank accession
number” – “Country of
origin” – “Year of
isolation.” The scale bar
represents nucleotide
changes per site per year
258 M.H. Ooi and T. Solomon

susceptible children who have not been exposed to the virus

during the earlier epidemics [23, 89]. Trying to make predic-
tions about the epidemic potential of specific subgenogroups
has proved difficult, beyond the observation that some sub-
genogroups, such as C1, appear to be less virulent and cause
endemic disease rather than large epidemics, whereas other
subgenogroups, particularly those that appear to have emerged
in Asia in recent years, are associated with large epidemics.
More than one subgenogroup may co-circulate, and a shift
from one dominant genogroup to another was described during
outbreaks in Sarawak and Vietnam [12, 119, 146]. In Japan and
Taiwan, subgenogroup B and C viruses have caused epidemics
at different times (Table 12.1) [66, 82, 149]. On the contrary in
the Netherlands, a permanent shift appears to have occurred,
from genogroup B viruses prior to 1986 to genogroup C viruses
since 1987; cross-neutralization among the genogroup B viruses,
but not with genogroup C viruses, is postulated as one explana-
tion for this, although other experimental cross-neutralization
data did not support it [77, 102, 147, 148]. While the older sub-
genogroups of virus have been circulating and causing low levels
of disease for many years, some of the more recently evolved
subgenogroups such as genogroup B5, which possess distinctive
antigenicity from other viruses may have the potential to cause
massive epidemics [64, 148]. Although these have been confined
to the Asia-Pacific region so far, the fact that humans are the
natural reservoir for the virus and that international air travel is
increasing means that every region is at risk of EV71 epidemic.

4 Biological Characteristics

EV71 is a small, non-enveloped virus with a positive-stranded

RNA genome about 7.4 kb in size [169]. The genome has a
single open reading frame encoding a polyprotein that is
cleaved into 11 proteins: the four capsid proteins (P1 – VP1,
VP2, VP3, VP4) and seven nonstructural proteins (P2 – 2A,
2B, 2C; P3 – 3A, 3B, 3C, 3D) [99]. The VP1 open reading
frame has considerable genetic variability, which confers anti-
genic variability and enables investigators to differentiate
EV71 strains [53, 100]. VP1 is also important for attachment to
cellular receptors and for viral virulence [169]. Other details
regarding the biological characteristics of EV71 were provided
earlier in the molecular epidemiology section; additional gen- Fig. 12.2 Mucocutaneous lesions in hand-foot-and-mouth disease.
eral information regarding the characteristics of enteroviruses This Malaysian child has ulcers that are seen inside the upper lip (top)
can be found in Chap. 11. and has vesicular and macular lesions on the wrists (middle) and soles
(bottom) (Photos from T. Solomon) (Reproduced from Ooi et al. [111])

5 Clinical Features 5.1 Mucocutaneous Manifestations

EV71 infection may present with a wide spectrum of clinical HFMD is a common childhood exanthema characterized by
features, although central nervous system (CNS) infection a short, usually mild, febrile illness with papulovesicular
and HFMD are the two most commonly recognized disease rashes over the palms and soles and multiple mouth ulcers
manifestations [114]. (Fig. 12.2).
12 Enteroviruses: Enterovirus 71 259

Table 12.3 Neurological Purely neurological manifestations

syndromes associated with
Encephalitis, especially brainstem Common
EV71 infection
Acute flaccid paralysis (anterior myelitis) Common
Encephalomyelitis Common
Aseptic meningitis Very common
Cerebellar ataxia Uncommon
Transverse myelitis Rare
Neurological plus systemic manifestations
Brainstem encephalitis with cardiorespiratory failure Common
Manifestations indicative of immune-mediated (para-infectious) mechanisms
Guillain-Barré syndrome Uncommon
Acute disseminated encephalomyelitis Rare
Opsoclonus-myoclonus syndrome Rare
Benign intracranial hypertension Rare
Modified from McMinn [96]

Herpangina, a closely related childhood enanthema, is char- cardiorespiratory dysfunction. This feature is also seen in
acterized by a febrile illness associated with multiple mouth poliomyelitis and has been attributed to neurogenic pulmo-
ulcers at the anterior pharyngeal folds, uvula, tonsils, and soft nary edema [6], although the mechanism of disease remains
palate. Although the classical HFMD picture is typically seen controversial.
in older children with EV71, widespread and atypical rashes During a 7-year prospective clinical study of HFMD in
may occur in children aged 2 years and below. Besides EV71, Sarawak that covered several epidemics, 10–30 % of hospital-
another picornavirus, CVA16, is also a principal causative ized children with HFMD due to EV71 had CNS complica-
agent of HFMD in both sporadic and epidemic forms. The tions [108, 112]. Brainstem encephalitis was the most common
virus is not normally associated with neurological complica- presentation, accounting for about 60 % of neurological syn-
tion [114], but the rash it causes is indistinguishable from that dromes, followed by aseptic meningitis (36 %) and brainstem
caused by EV71. There may be other clues that HFMD is due encephalitis with cardiorespiratory dysfunction (4 %). Most
to EV71 rather than CVA16; for example, studies from Sarawak children with CNS disease will also have features of HFMD;
and Taiwan show that children with EV71 are more likely to however, a small proportion may present with neurological
have a longer duration of fever (≥3 days), a higher peak tem- disease only. Myoclonic jerks are seen more often with EV71
perature (≥38.5 °C), lethargy, and myoclonus [108, 152]. A than with other enterovirus infections. This sign may be an
household contact study in Taiwan during the 2001–2002 epi- early indicator of neurological involvement, particularly in the
demic showed that, in addition to HFMD and herpangina, there brainstem [90]. However, it is not pathognomonic for entero-
is a broad range of mild clinical manifestations, including virus infection; myoclonus has also been reported in other
upper respiratory tract infection, gastroenteritis, and nonspe- virus infections of the CNS including Japanese B encephalitis,
cific rashes [24]. Other respiratory manifestations in young subacute sclerosing panencephalitis, and Nipah virus, herpes
children include acute exacerbation of bronchial asthma, bron- simplex virus, varicella-zoster virus, and HIV infection.
chiolitis, and pneumonia [100]. More than 20 % of adult con- Myoclonic jerks are also often seen in otherwise healthy
tacts during one Taiwanese outbreak had symptoms of an upper young infants, particularly when they are asleep and may
respiratory tract infection, while asymptomatic infection occur spontaneously or be provoked by a loud noise.
occurred in more than 50 %, indicating that adults may be Seizures, if they occur at all in EV71 infection, tend to be
important source of infection for younger children [24]. seen in younger children and are short-lived with rapid
recovery of consciousness, suggesting that they are febrile
convulsions, rather than due to CNS infection itself. Unlike
5.2 Neurological, Respiratory, and Systemic those seen in other viral encephalitides, recurrent and pro-
Manifestations longed seizures are very rare with EV71 infection, a distinc-
tion probably reflecting predominantly brainstem rather than
Similar to other enteroviruses, EV71 can cause aseptic men- cortical involvement.
ingitis, acute flaccid paralysis, encephalitis, and other rarer Brainstem encephalitis with associated pulmonary
manifestations (Table 12.3) [96]. edema has been the hallmark of EV71 CNS infection in
Encephalitis typically affects the brainstem, and unlike Asia since the late 1990s. This distinctive clinical syn-
most other enteroviruses, it is often accompanied by marked drome has a stereotypic clinical course characterized by a
260 M.H. Ooi and T. Solomon

a b

Fig. 12.3 MRI changes in EV71 encephalomyelitis (Modified from dependent. (a) Sagittal section showing high signal intensity in the pos-
Shen et al. [131]). T2-weighted images of a 10-month-old female who terior portion of the pons and medulla (black arrows) and anterior cer-
presented 3 months earlier with somnolence tachycardia, tachypnea, vical cord (white arrows). (b) Axial section showing the high signal
and coma and who recovered consciousness but remained ventilator intensity in the two anterior horns of the cervical cords (black arrows)

prodromal illness of HFMD followed by a sudden deterio- HFMD with autonomic system dysregulation, and HFMD
ration that typically occurs after 3–5 days of fever. Children with cardiopulmonary failure [160].
then develop acute rapidly progressing cardiorespiratory Findings on magnetic resonance imaging (MRI) of children
failure presenting as shock and pulmonary edema or hem- with brainstem encephalitis correlated well with those of
orrhages. Without critical care support, most of these chil- autopsy examination; both procedures demonstrate frequent
dren die within 24 h of hospital admission and in some even involvement of the medulla oblongata, reticular formation,
before arriving at the hospital. A system of clinical staging pons, and midbrain in several studies (Fig. 12.3) [29, 131, 162].
(stage 1 through stage 4) has been used to help monitor the Acute flaccid paralysis is the primary presenting feature
progress of the affected children during the clinical course of a number of neurological syndromes caused by EV71
of EV71 infection and to guide management, from uncom- including poliomyelitis-like paralysis, Guillain-Barré syn-
plicated febrile illness to CNS involvement to cardiorespi- drome, and transverse myelitis. Poliomyelitis-like paralysis
ratory failure and development of neurological sequelae is probably the most common of these, though it may be less
[20, 85, 86]. Such staging systems have not been adopted severe than that caused by polioviruses, with a higher recov-
widely, possibly because they are not always easy to ery rate [96]. Other respiratory manifestations in young chil-
remember and they imply sequential progression through dren include acute exacerbation of bronchial asthma,
stages that do not always occur. In 2010, a WHO Informal bronchiolitis, and pneumonia [100]. More than 20 % of adult
Consultation on Hand Foot and Mouth Disease proposed contacts during one Taiwanese outbreak had symptoms of an
the use of a simple clinical description of disease manifes- upper respiratory tract infection, while asymptomatic infec-
tation to assess the disease severity. It included uncompli- tion occurred in more than 50 %, indicating that adults may
cated HFMD/herpangina, HFMD with CNS involvement, be important source of infection for younger children [24].
12 Enteroviruses: Enterovirus 71 261

6 Clinical Management 6.2 Virological Diagnosis

During outbreaks of EV71, tens of thousands of children Laboratory diagnosis of EV71 is established primarily
develop symptoms, and while most of them have mild through virus isolation or molecular detection of the virus
self-limiting illness, in a small proportion of apparently nucleic acid in appropriate clinical specimens.
well children, the condition can rapidly deteriorate to
severe and fatal neurological and systemic complications 6.2.1 Choice of Sample
over days or even hours. Whereas in the past children A wide range of samples may be available, depending on the
with mild HFMD tended to be managed at home, with disease manifestations; these include throat and rectal swabs,
increasing parental awareness about the risk of fatal com- serum, urine, and, when taken, cerebrospinal fluid (CSF), as
plications, many are now brought directly to hospital, well as fluid from vesicles and swabs from ulcers, if they are
and health services can easily become overwhelmed. The present. The sensitivity, specificity, and usefulness vary
challenges faced by primary care clinicians are recog- according to the sample [109]. In particular, virus detection
nizing which patients are likely to deteriorate, knowing in sterile sites such as vesicular fluid, CSF, serum, urine,
which investigations yield the best diagnostic informa- serum, or autopsy material more reliably indicates a caus-
tion, and deciding which treatments might be appro- ative organism than does detection from non-sterile sites
priate, without the benefit of guidance from controlled such as throat or rectum, which may indicate coincidental
clinical trials. carriage. However, many of the sterile sites only occasion-
ally yield virus. For example, virus is isolated from only
0–5 % of the CSF of patients with neurological disease [25,
6.1 Laboratory Tests 40, 51, 67, 74, 103, 108, 112], because, as for poliomyelitis,
the viral load in the CSF is very low [48]. The yield for serum
In mild disease, the blood count is usually normal, but in is similarly low [112, 159]. Vesicular fluid, when present, is
severe disease, the white blood cell count is often high more useful. Although throat and rectal swabs are more
with a neutrophilia [25]. Blood urea and electrolytes are likely to have an enterovirus detected, one study from
typically unaffected, but there may be hyperglycemia in Malaysia found that this was not always the same enterovirus
severe disease [25]. Creatine kinase is sometimes elevated as that isolated from a sterile site: using the isolate from ves-
in patients with cardiac involvement [46] and elevated car- icle swabs as a reference, 10 % of positive throat swabs gave
diac troponin I has been reported as a predictor of immi- a different isolate, and for rectal swabs the figure was 20 %
nent cardiopulmonary failure in children with brainstem [109]. Presumably, the isolate from the non-sterile site repre-
encephalitis [65]. Chest X-ray characteristically shows a sented coincidental carriage, whereas that from the vesicles
normal heart size, even in the presence of marked pulmo- was actually pathogenic.
nary congestion, indicating that neither acute viral myo- Prolonged viral shedding from the gastrointestinal tract
carditis nor congenital heart disease is causing the illness. (throat, rectum, or stool) may occur after complete resolution
There are often nonspecific ECG changes [46], and con- of EV71 infection, as it does for other enteroviruses; a study
tinuous monitoring may show abnormal beat-to-beat vari- in Taiwan showed that EV71 may be detected in the throat up
ability, which may predict imminent cardiovascular to 2 weeks after recovery from HFMD or herpangina; in the
collapse [83]. Echocardiography shows generalized left stool, it can be detected up to 11 weeks later [41]. During an
ventricular hypokinesia, occasionally accompanied by outbreak, so many samples could potentially be positive that
mitral regurgitation, in children who are hemodynamically laboratories can soon become overwhelmed. In one study, the
unstable with tachycardia, hypotension, or pulmonary most efficient approach was to examine throat swabs for all
edema [46]. Pericardial effusion is rare. patients plus swabs either from at least two vesicles when
Lumbar puncture is essential in children who are unwell present or from the rectum when vesicles are absent [109].
with suspected CNS involvement. In some patients, the
clinical features, such as meningismus or myoclonic jerks, 6.2.2 Virus Isolation, Serotyping, and Nucleic
may clearly point to CNS involvement. However in other Acid Detection
children particularly those younger than 2 years of age, The gold standard for diagnosis of enterovirus infection is
there may just be high fever, vomiting or lethargy, but a virus isolation. Several human and nonhuman primate cell
lumbar puncture reveals CNS disease. There is typically a lines may be used: rhabdomyosarcoma (RD), which is most
mild CSF lymphocytic pleocytosis of 10–100 cells per efficient; human lung fibroblast cells (MRC5); and African
mm3, but not always [116]. The CSF-to-plasma glucose green monkey kidney cells (Vero) [114]. In RD cells, a char-
ratio is usually normal but it can be low. acteristic cytopathic effect is typically observed 7–10 days
262 M.H. Ooi and T. Solomon

after inoculation. However, to improve the yield, blind pas- 6.4 Diagnostic Imaging
sage may be necessary before cytopathic effects become
apparent. Once a cytopathic effect is observed, the virus is Computer tomography scans are almost always normal in
identified by neutralization tests using intersecting pools of EV71 encephalitis, where the pathology is mostly in the
type-specific antisera, by EV71-specific antisera, or by an brainstem. Conventional MRI may be normal in the early
indirect immunofluorescence assay using EV71-specific phase of EV71 encephalitis. Conversely, it may show charac-
monoclonal antibodies [114]. More recently, a molecular teristic high signal intensities on T2-weighted and fluid-
“serotyping” approach has been devised. It involves ampli- attenuation inversion recovery (FLAIR) images in the dorsal
fying part of the VP1 gene of the cultured virus, using pons and medulla, most of the midbrain, and the dentate
polymerase chain reaction (PCR) and pan-enterovirus EV71- nuclei of the cerebellum. Similar high-signal lesions may
specific primers, and then sequencing the product [104]. To also be found in the anterior horn cells of cervical spinal cord
this end, several sets of primers directed at different regions (Fig. 12.3) [63, 131, 175]. Gadolinium-enhanced MRI exam-
of the VP1 gene of human enterovirus have been developed ination improves the results [175]. Diffuse-weighted imag-
[10, 104, 115]. ing (DWI), a sensitive tool for detecting of early changes in
EV71-specific primers are now also being used to per- brain cellular function, seems to be better at detecting EV71
form PCR directly on clinical samples. The advantage of encephalitis than conventional MRI [81]. However, the value
this approach over virus culture is that it can provide rapid of MRI screening has yet to be demonstrated. In children
diagnosis in the midst of explosive EV71 outbreaks where with acute flaccid paralysis, MRI typically shows unilateral
urgent public health intervention is needed. Several sets of high-signal lesions in the anterior horn cells of spinal cord on
real-time RT-PCR protocols directed to detect EV71 and T2-wieghted images and contrast-enhancing ventral root on
CVA16 in primary clinical samples have been published T1-weighted images [28, 63, 132].
recently; however, their disadvantage is that the technique
detects only the suspected virus for which primers are
available, but they will miss any agents that are unexpected 6.5 Predictors of Severe Disease
or for which no primers have been generated [34, 115, 142,
167]. DNA microarray technology is a powerful, though Several clinical features and laboratory abnormalities have
expensive, new tool designed to detect multiple pathogen been associated with neurological and fatal EV71 disease,
targets by hybridization of pathogen-specific probes. Two but few have been prospectively validated as prognostic indi-
groups have recently reported using such an approach to cators [25, 60, 65]. Younger age is associated with increased
distinguish EV71 and CVA16 infection in primary clinical risk of severe disease [21]. A prospective clinical study of
specimens [34, 134]. nearly 1,500 children presenting to one hospital in three
EV71 outbreaks in Sarawak over 7 years suggested that peak
temperature of 38.5 °C or higher, duration of fever for 3 or
6.3 Serology more days, and a history of lethargy were useful clinical pre-
dictors for neurological involvement. The presence of at
Serological diagnosis of an acute virus infection is classi- least two of those three factors was strongly associated with
cally established by demonstrating a fourfold rise in specific the subsequent development of neurological disease [108].
neutralizing antibody between the acute and convalescent The study corroborated the findings from early retrospective
samples [114]. However, in the case of EV71, very high studies. However, it did not confirm other findings from ear-
levels of neutralizing antibodies are often detectable within lier studies, such as the association between the absence of
the first few days of illness, and thus a fourfold rise cannot mouth ulcers and development of complicated or fatal dis-
be demonstrated [40, 103]. Furthermore, although homolo- ease [38, 112]. Hyperglycemia and leukocytosis have also
gous antibody is produced when young children encounter been associated with fatal EV71 disease in a retrospective
their first enterovirus infection, heterologous cross-reacting evaluation [25], and although these findings were confirmed
IgG and IgM antibodies are produced by older children and in the prospective study, because they were late changes
adults following repeated infection with different enterovi- occurring about the same time as the fulminant disease, they
rus serotypes; this reduces their diagnostic usefulness [87]. were not helpful clinically in identifying children at high risk
Several rapid IgM ELISA tests for EV71 have recently been of complications and death [108].
developed to try to overcome some of these limitations Not all children with CNS involvement in EV71 infection
[153]; however, cross-reactivity remains an issue [168], and will progress to cardiorespiratory collapse. A recent study in
the duration of detectable EV71-specific IgM following an Southern Vietnam revealed that, in general, about 6 % of
infection is also uncertain. children with CNS involvement would develop autonomic
12 Enteroviruses: Enterovirus 71 263

system dysregulation. Children with frequent myoclonus 7 Pathogenesis

observed by doctors and healthcare workers, with or without
other focal neurological signs, were at higher risk of disease 7.1 Viral Determinants of Virulence
progression compared with those children with no history of
myoclonus (22.2 % vs 4.4 %). A small study involving 46 The factors that determine whether EV71 infection will be
patients showed that children developed abnormal heart rate asymptomatic or result in HFMD or severe neurological dis-
variability, an index of autonomic nervous system disease, ease remain unknown. For poliovirus, the 5’UTR and VP1
about 7 h before the clinical onset of cardiorespiratory insta- genes are known to contain virulence determinants [72].
bility [83]. The authors proposed that screening children with Several studies have therefore examined the nucleotide
CNS involvement for abnormalities in heart rate variability sequence of these genes, or the whole genome, comparing
may provide early warning of imminent cardiorespiratory EV71 isolates from fatal and nonfatal cases, but for the most
failure and allow timely institution of appropriate interven- part the isolates have been nearly or entirely identical, and sig-
tions. Cardiac troponin I is a cardiac-specific biomarker for nificant changes have not been found [135, 139]. The inci-
myocardial damage, used for early diagnosis of acute coro- dence of CNS disease and other severe complications of EV71
nary syndrome in adults. Elevated cardiac troponin I has been infection seems to have varied among the recent outbreaks in
observed in children with brainstem encephalitis and cardio- Asia, leading to the postulation that differences in the viru-
pulmonary failure [65]; in some cases, it was elevated prior to lence of the various genotypes may have a role. However,
the development of cardiopulmonary failure, suggesting that comparisons between outbreaks have been hampered by the
serial measurement of troponin may be helpful in identifying retrospective nature of many of the studies as well as differ-
children at risk of left ventricular failure. However, neither ences in inclusion criteria, definitions of disease severity, and
the evaluation of heart rate variability nor the measurement of viral diagnostic approaches and capabilities. Perhaps the
troponin I level has become routine clinical practice in the strongest data supporting the hypothesis that strain virulence
management of EV71 infection, probably because the former determinants play an important role in the pathogenesis of
requires relatively sophisticated equipment not widely avail- severe neurological disease come from studies in Perth and
able and the latter is relatively expensive in developing coun- Sarawak. During the Perth epidemic in 1999, two subgeno-
tries. Overall simple clinical parameters such as length of groups, B3 and C2, co-circulated, thus providing a unique
illness, height of fever, and lethargy are probably more useful opportunity to examine the role of virulence determinants in a
indicators of potentially severe disease. single epidemic setting [96, 97]. In this outbreak, subgeno-
group C2 viruses linked to the Taiwan epidemic of 1998 were
almost exclusively isolated from children with severe neuro-
6.6 Outcome logical disease, and only a single isolate came from a case of
uncomplicated HFMD. Conversely, subgenogroup B3 viruses,
Follow-up for as long as 7 years after infection shows that similar to those from the Sarawak 1997 epidemic, were iso-
children who present with aseptic meningitis generally have lated mainly from children with uncomplicated HFMD, asep-
a favorable outcome, although a recent report documented tic meningitis, or post-infectious neurological disease, none of
the incidence of attention-deficit/hyperactivity disorder- whom died [98]. A detailed prospective clinical study of EV71
related symptoms, as reported by parents and teachers, is disease in Sarawak, which included 277 children with EV71-
higher (20 %) in children who had recovered from CNS associated HFMD, provided further clinical and epidemio-
infection compared with the matched controls (3 %) [49]. logical evidence for different biological behavior of EV71
Approximately one-fifth of those with more severe neuro- subgenogroups, with regard to risk of CNS disease and trans-
logical disease, including encephalitis, poliomyelitis-like missibility within families [112]. Two discrete EV71 epidem-
paralysis, and encephalomyelitis, have sequelae, particularly ics, caused predominantly by subgenogroup B3 and B4,
focal limb weakness and atrophy [21, 110, 145]. Cerebellar respectively, but with small numbers of cases caused by sub-
dysfunction is observed in about 10 % of patients who had genogroup C1, occurred [112]. Children infected with sub-
moderately severe brainstem encephalitis, including cranial genogroup B4 viruses were less likely to present with CNS
neuropathies, myoclonus, tremor, and ataxia. However, only infection than those infected with C1 or B5 viruses, and they
a quarter of those with more severe brainstem encephalitis were also less likely to be part of a family cluster. On the other
associated with fulminant cardiorespiratory failure make a hand, children infected with B5 were more likely to be part of
full neurological recovery. Common sequelae include focal a family cluster, and there was a trend toward a greater inci-
limb weakness and atrophy, swallowing difficulties requiring dence of CNS disease in these patients. These results suggest
nasogastric feeding, central hypoventilation, facial nerve that subgenogroups do indeed vary in their propensity to cause
palsies, seizures, and psychomotor retardation. neurological disease.
264 M.H. Ooi and T. Solomon

7.2 Dual Infection leukocytosis, and neutrophil counts when compared with
patients with CC + CT genotypes [93]. Another Chinese
During the first of the Asian EV71 epidemics in Sarawak in study showed increased frequency of Th17 cells, as well as
1997, which was due to subgenogroup B3, an adenovirus elevated serum IL-17 and IL-23, in peripheral blood of chil-
type 21 was also implicated in the fatal cases as well as in dren infected with EV71 when compared to the healthy con-
some cases with acute flaccid paralysis [13, 110]. The adeno- trols [32]. Interferon gamma, a Th1 antiviral cytokine, and
virus was isolated from sterile sites such as CSF, brain, and IL-10, a potent anti-inflammatory cytokine that suppresses
heart in fatal cases and indeed was more frequently detected innate host defense, including interferon gamma production,
than EV71 itself; this led to the suggestion that the fatalities have been implicated in the pathogenesis of severe EV71
were due to dual infection, rather than EV71 alone [13]. infection. Susceptibility to EV71 encephalitis has been
However, subsequent detailed studies, including longitudinal reported in children with the IFN-gamma + 874 T/A geno-
studies from Sarawak, have not found evidence of adenovi- type, previously associated with reduced IFN-gamma pro-
rus 21 infection in other HFMD or neurological cases, duction, and in children with the IL-10-1082G/A genotype,
though dual infection of EV71 with other viruses, including previously associated with reduced IL-10 production [172].
dengue and Japanese B encephalitis, has been found [112]. All of these associations require replication.
Furthermore, adenovirus 21 has never been isolated in
Sarawak since 1997.
7.4 Viral Entry and Spread

7.3 Host Susceptibility EV71 is transmitted predominantly via the feco-oral route,
with respiratory spread also implicated [114]. As for other
A range of host factors may affect pathogenesis, particularly enteroviruses, initial viral replication is presumed to occur in
partial cross-protective immunity from prior outbreaks. Lack the lymphoid tissues of the oropharyngeal cavity (tonsils) and
of cross protection may partially explain why young age is a small bowel (Peyer’s patches), with further multiplication in
risk factor for severe disease [23, 24, 89]. The potential for the regional lymph nodes (deep cervical nodes, mesenteric
host genetic variants to explain differential susceptibility, nodes), giving rise to a mild viremia. The majority of infections
clinical severity, and outcome of EV71 has also been studied. are controlled at this point and remain asymptomatic. However,
One study in Taiwan reported that HLA-A33 is associated in vivo studies show that if enteroviruses disseminate further,
with increased susceptibility to EV71, although the role of they reach target organs, particularly the reticuloendothelial
the major histocompatibility complex in the virus infection is system (liver, spleen, bone marrow, and lymph nodes), heart,
still unknown [19]. HLA-A33 is more frequently found in lung, pancreas, skin, mucous membranes, and central nervous
the populations of Asian ancestry than in those of European system, coinciding with the onset of clinical features.
ancestry, and a causal association of this allele could help The mechanism by which enteroviruses enter the CNS is
explain the higher frequency of EV71 epidemics in Asia. In not completely understood. A number of epidemiological
the same study HLA-A2 was linked to the risk of cardiopul- and experimental animal studies on polioviruses indicate that
monary failure often observed in fatal cases [19]. HLA-G, an the virus can invade the CNS system by permeation through
important immunotolerant molecule, is involved in the sup- a disrupted blood-brain barrier or by retrograde axonal
pression of T lymphocytes, NK cells, and antigen-presenting spread along cranial or peripheral nerves. For EV71, this lat-
cells and in the induction of regulatory T cells and tolerant ter route has been implicated both in mouse models and by
dendritic cells. Increased levels of cell surface bound HLA-G examining the distribution of virus and inflammation in fatal
and plasma sHLA-G were found in EV71-infected children human cases [31, 107, 163].
and in children with pulmonary edema [179]. Cytotoxic T
lymphocyte antigen-4 (CTLA-4) is an important regulator of
T cell cytotoxicity and is involved in the regulation of 7.5 Pathological Findings
immune response. Studies of polymorphisms in the gene
encoding CTLA-4 in children with meningoencephalitis The topographical distribution of CNS inflammation has
have yielded conflicting results [19, 173]. Th17 cells are been stereotypic and is observed predominantly in the neuro-
effector cells in human immune response, and its related nal areas of spinal cord, the entire medulla oblongata except
cytokines, IL-17 and IL-23, are important mediators in pro- the pyramidal areas, the tegmentum and floor of fourth ven-
inflammatory response. Chinese children with the IL-17 F tricles in the pons but not the anterior pons, and the whole
7488C allele, which has been associated with a blunted pro- midbrain sparing the cerebral peduncles. In addition, the
inflammatory response, were more likely to have milder hypothalamus, subthalamic and dentate nuclei, and to a
EV71 infection, and patients who were homozygous for the lesser degree motor cortex of the cerebrum and meninges are
T allele had significantly higher level of C-reactive protein, involved (Fig. 12.4) [62, 63, 91, 92, 131, 133, 163].
12 Enteroviruses: Enterovirus 71 265

a b

c d

e f g

Fig. 12.4 Pathological findings in enterovirus 71 encephalitis macrophages/microglial (e), CD8-positive lymphocyte adjacent to a
(Modified from [163]). Parenchymal inflammation (arrows) and peri- neuron (f). Viral RNA in the anterior horn cells of the spinal cord
vascular cuffing in the medulla (a); more severely inflamed areas (b), (g). (a–d: hematoxylin and eosin stains; e, f: immunohistochemistry/
with edema (*) and neuronophagia (c, arrows). More subtle inflamma- peroxidase/DAB; g: ISH/nitroblue tetrazolium/5-bromo-4-chloro-3-
tion in the motor cortex with mild perivascular cuffing (arrow) and indolyl phosphate stains. Original magnification: (a) 4×; (b, d) 10×;
parenchymal inflammatory cells (circle) (d). Numerous CD68-positive (c, f) 40×; (g) 20×
266 M.H. Ooi and T. Solomon

Inflammatory changes were absent in the cerebellar cor- increased cardiac-specific troponin I level suggests some
tex, thalamus, basal ganglia, mammillary body, hippocam- degree of cardiac injury [22, 46, 65, 91, 92]. A detailed echo-
pus, temporal lobe, peripheral nerve, and autonomic ganglia. cardiographic study of 11 children with EV71 brainstem
The histopathological changes, characterized by perivascular encephalitis shows that their cardiac function was impaired,
cuffing of macrophages, lymphocytes, neutrophils and with significantly reduced left ventricular ejection fraction
plasma cells, variable edema and necrosis, focal neurono- [46]. Two children whose cardiac output was supported with
phagia, and microglia nodules, are similar to those in enceph- a left ventricular assist device survived, whereas all the others
alitis caused by other viruses [50]. Neither virus inclusion died [45].
nor vasculitis has been observed, and viral antigens and RNA Although there is no myocardial inflammation, histologi-
can only be seen in a small number of neuronal processes cal examination of heart ventricular tissue from six fatal
and phagocytic cells [163, 164]. cases and one survivor, obtained though a biopsy, revealed
significant coagulative myocytolysis, myofibrillar degenera-
tion, and cardiomyocyte apoptosis which are characteristic
7.6 Pathogenesis of Severe Pulmonary of catecholamine cardiotoxicity [46, 47]. Thus it is argued by
Edema and Heart Failure some that the massive release of catecholamine caused by
brainstem encephalitis may have a direct effect on cardiac
While it is clear that fulminant pulmonary edema is closely function, as well as causing pulmonary edema through raised
associated with, and preceded by CNS involvement, there is pulmonary pressures.
no consensus on its cause, especially the relative contribu- The other potential contributor to pulmonary edema is
tions of neurogenic pulmonary edema, cardiac dysfunction, increased vascular permeability, which is postulated to have
increased vascular permeability, and a cytokine storm occurred following the systemic inflammatory response.
(Fig. 12.5). Early studies that examined a narrow range of cytokines and
Neurogenic pulmonary edema is classically seen follow- chemokines have shown that interleukin (IL)-6, IL-1B, IL-10,
ing a head injury, where the associated raised intracranial IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ
pressure is thought to be important. Although the patho- are all significantly higher in EV71 patients with pulmonary
genesis is not completely understood, studies from animal edema than in those with uncomplicated encephalitis, and
models suggest that the hypothalamus, and vasomotor cen- several of these, including IL-1, IL-6, IL-13, and IFN-γ, are
ters of the medulla, and nuclei in the cervical spinal cord known mediators of increased vascular permeability [84–86,
are important; lesions to various nuclei in these regions can 151]. In addition, increased plasma levels of several chemo-
increase activity along the sympathetic trunk, resulting in kines including interferon gamma-induced protein (IP-10),
profound systemic and pulmonary hypertension and conse- monocyte chemoattractant protein (MCP)-1, monokine
quent pulmonary edema [57]. Pulmonary edema was also induced by interferon gamma (MIG), and IL-8 have been
seen in poliomyelitis, and because it was associated with found in children with pulmonary edema when compared to
damage to brainstem nuclei, it was thought to be neurogenic those with uncomplicated brainstem encephalitis [156]. A
in origin [6]. Thus when severe pulmonary edema was study of 30 cytokine and chemokine levels in EV71-infected
first seen in EV71 encephalitis, and brainstem inflamma- Malaysian children confirmed that cardiorespiratory impair-
tory changes were observed too, the development of edema ment was associated with a widespread elevation of both pro-
was attributed to neurogenic origin. Autopsy examination inflammatory and anti-inflammatory mediators in the serum
and magnetic resonance imaging studies of children with and CSF. In addition, the study also showed that serum
EV71 brainstem encephalitis showed that there was exten- IL-1Ra and G-CSF levels were significantly elevated in
sive inflammation of gray matter of the spinal cord and the patients who died, with a serum G-CSF/IL-5 ratio of >100
whole medulla oblongata, as described above [62, 63, 91, being the most accurate prognostic marker [52]. Children
92, 131, 163]. The observations of hyperglycemia and leu- with edema also had depleted lymphocyte population particu-
kocytosis were also postulated to be due to increased sym- larly in CD4, CD8, and natural killer cells [151, 173].
pathetic discharges [25]. Thrombocytosis, neutrophilia, and hyperglycemia are all
However, detailed hemodynamic observations of children thought to be indicative of a systemic inflammatory response
with EV71 and pulmonary edema have not always shown the [84, 151]. Fewer studies have looked at cytokines in the CSF.
profound systemic and pulmonary hypertension that would Elevated CSF IL-1b was found in patients with encephalitis
be expected [46, 86, 89, 165] This may be because the complicated by edema when compared to those with enceph-
changes in vascular pressures in neurogenic pulmonary alitis alone [84]. It has previously been proposed that the CNS
edema are only transient, as was shown for one child with may be the source of the inflammatory cytokines detected in
EV71 [165]. Others have argued that cardiac impairment is a the serum of patients with EV71-associated cardiac dysfunc-
major contributor to the pulmonary edema. Although there is tion [156]. A study examining the relative abundance of
no histological or virological evidence of a viral myocarditis, inflammatory mediators in the serum and CSF samples from
12 Enteroviruses: Enterovirus 71 267

Severe EV71 infection

? viral entry via blood brain barrier

? viral entry via spinal neural route

Systemic Viral sepsis Virus entry into CNS

Marked systemic Marked CNS

Brainstem encephalitis
inflammatory inflammatory
response response

Extensive damage to
medulla oblongata

Imbalance between sympathetic and

“Cytokine storm” parasympathetic discharge activities

Excessive sympathetic hyperactivity

“Sympathetic storm”

Massive catecholaminerelease ↑↑SVR; ↑↑SBP; ↑↑HR

Increased pulmonary Catecholamine cardiotoxicity

vascular permeability

Cardiomyocyte apoptosis

Cardiac damage

Acute LV dysfunction

Acute pulmonary edema(hemorrhage)

Fig. 12.5 The postulated pathogenesis of enterovirus 71-associated hypothetical but unproven steps or evidence from animal models only.
acute pulmonary edema Major postulated pathogenic pathways are EV71 human enterovirus 71, BBB blood-brain barrier, CNS central ner-
shown with thick lines; lesser contributory pathways are shown with vous system, ൹൹ markedly high, SVR systemic vascular resistance,
thinner lines. The solid boxes indicate strong supporting evidence from SBP systemic blood pressure, HR heart rate, LV left ventricular
human clinical or pathological studies, while the dotted boxes indicate (Reproduced from Solomon et al. [140])
268 M.H. Ooi and T. Solomon

88 Malaysian children showed two distinctive immune and nonspecific anti-inflammatory properties might be thera-
response patterns occurring independently in these two com- peutic [112, 152]. Retrospective comparisons of patients
partments [52]. Because the development of pulmonary who received IVIG with those who did not suggested possi-
edema in patients with EV71 encephalitis appears to be ble benefit from IVIG if given early [20, 108]; for example,
strongly associated with a dysregulated systemic and CNS in Sarawak over 3 seasons, 204 (95 %) of 215 children who
inflammatory response, this observation has at least partly survived despite severe CNS complications had timely IVIG
formed the basis for treatment with intravenous immunoglob- treatment, typically once severe disease occurred, whereas
ulin, which appears to be effective [20, 85, 86, 108, 112, 154]. only one (11 %) of nine fatal cases received this treatment
In summary, although still ill-defined, brainstem inflam- (OR 148.36, 95 % CI 16.34–6609.04, p < 0.0001) [108].
mation appears to be an important neurogenic mechanism Proinflammatory cytokines measured before and after IVIG
for pulmonary edema in EV71 encephalitis. However, simi- treatment were significantly lower in EV71 patients with
lar pathological changes are also observed in other encepha- autonomic dysfunction than patients with less severe disease
litides without pulmonary edema as a prominent feature. [84–86, 151, 154]. IVIG has since become routine treatment
Cardiac compromise and the effects of the systemic inflam- for severe EV71 disease, and it has been recommended in the
matory response on the vascular endothelium may also make national treatment guidelines in Taiwan and Vietnam [20, 75,
an important contribution. In vivo mouse and nonhuman pri- 85, 86, 108, 112, 150]. While remaining uncertainty over the
mate models have replicated some of the features of severe efficacy of this treatment warrants randomized placebo-
EV71 disease, such as neuroinvasiveness with marked controlled trials, they would be logistically and ethically
inflammatory changes; however, no model has yet repro- challenging to conduct in the face of the wide acceptance of
duced the severe systemic features such as pulmonary edema IVIG as the current standard of care and the beliefs in their
[5, 31, 36, 107, 158, 163]. value strongly held by some. Carefully designed phase II tri-
als would need surrogate end points for disease progression
(e.g., failure of resolution of tachycardia).
8 Control and Prevention
8.1.3 Milrinone
8.1 Treatment Milrinone is a cyclic nucleotide phosphodiesterase (PDE)
inhibitor currently used in the treatment of congestive heart
8.1.1 Antiviral Agents failure. Inhibition of PDE subtype III by milrinone results in
There are no established antiviral treatments for EV71. an increase in intracellular cyclic adenosine monophosphate
Pleconaril is an antiviral drug that inhibits entry into cells for (cAMP), which in turn leads to increased cardiac output and
a number of enteroviruses by blocking viral attachment and decreased peripheral vascular resistance. In a small nonran-
uncoating. It has been used in clinical trials of aseptic men- domized retrospective comparison involving 24 children
ingitis [122–124]; however, it is not active against EV71 with EV71-induced pulmonary edema, those treated with
[35]. Several other newer capsid-function inhibitors have milrinone had reduced tachycardia and lower mortality [150,
been investigated, and some have demonstrated promising 155]. Intriguingly, the peripheral white blood cell count,
anti-EV71 activities in preclinical studies [35]. Two human platelet count, and plasma IL-13 were also lower [155], sug-
transmembrane proteins, P-selectin glycoprotein ligand-1 gesting an immunomodulatory as well as a cardiovascular
(PSGL-1) and scavenger receptor class B member 2 effect of the drug. More recently a prospective, open-label,
(SCARB2), have recently been identified to be functional randomized controlled trial conducted by the same authors,
receptors of EV71 [106, 170]. Analysis of the much-awaited involving 41 Vietnamese children 5 years of age and below,
crystal structure of EV71 revealed important structural dif- showed that milrinone significantly reduced the 1-week mor-
ferences from other enteroviruses. The mature virion of tality from EV71-induced cardiovascular collapse without
EV71 has a shallower canyon (believed to be the receptor adverse effect. This encouraging finding has tentatively
binding site) on the viral surface and a relatively exposed raised hope that earlier milrinone treatment might be useful
“pocket factor” (which stabilizes the virus) compared to in halting disease progression of cases with severe brainstem
other enteroviruses [118, 157]. These discoveries are major encephalitis [37].
steps forward and will guide rational design of antivirals
against EV71. 8.1.4 Fluid Balance and Ionotrope Support
Hypovolemia and dehydration are the commonest causes of
8.1.2 Intravenous Immunoglobulin shock in children and are treated with rapid fluid resuscita-
Intravenous immunoglobulin (IVIG) was used during the tion with good results. However, similar approaches used in
initial epidemics of EV71 in Sarawak and Taiwan in the late the early EV71 epidemics in Asia precipitated massive pul-
1990s on the basis that its anti-EV71 neutralizing antibodies monary edema. Judicious use of intravenous fluid and early
12 Enteroviruses: Enterovirus 71 269

institution of inotrope support are critical in children with Transmission of enteroviruses including EV71 is most
severe EV71 infection. Where fluid management could be efficient in overcrowded settings, and most countries in the
guided by central venous pressure monitoring, in Taiwan, region including Malaysia, Singapore, Taiwan, Hong Kong,
management algorithms based on this approach appear to and China have adopted “social distancing” measures during
have improved outcome [20]. epidemics. These measures include closures of childcare
facilities and schools and cancelation of public functions
8.1.5 Novel Treatment Approaches involving children [2, 3]. Although there has been little sys-
Although better recognition of early signs of CNS involve- tematic research to examine the effectiveness of public health
ment and the disease progression has helped improve the interventions, a few studies of school closure or other
clinical outcome, many children continue to succumb to approaches from Singapore and Hong Kong appeared to
severe EV71 infection because of late presentation or disease show some benefit [2, 95]. It nevertheless remains uncertain
progression despite intervention. A left ventricular assist whether social distancing measures are effective or what the
device with extracorporeal membrane oxygenation was asso- optimal timing for instituting them may be—at the onset of
ciated with a higher survival rate in a small number of recent an HFMD outbreak or at the time of laboratory confirmation
cases than in historical controls, but with significant neuro- of an EV71 etiology. Although this widely adopted empirical
logical sequelae [69]. Hemofiltration has been employed to measure has considerable socioeconomic impact, the cost of
treat children with cardiovascular collapse in Vietnam [117]. the disease and its control measures have not yet been stud-
ied. If experience with other directly transmissible viruses
applies to EV71, then such measures as school closure could
8.2 Prospects for Prevention decrease the peak incidence but prolong the outbreak, with
no reduction in the overall number of cases (Fig. 12.6) [140].
8.2.1 Surveillance Further epidemiological work to elucidate the transmis-
The only measures available currently for disease control at sion dynamics of the virus will guide the formulation of
the population level are public health approaches. Countries evidence-based interventions to control the spread of EV71.
in Asia (Singapore, Taiwan, Japan, and Vietnam) have imple- Critical pieces of information such as precise estimates of
mented heightened surveillance for EV71 [2, 3, 33, 101, 119]. the incubation period, ratio of asymptomatic to symptom-
HFMD has now become a notifiable disease in many places, atic cases, and time and duration of infectiousness are
including Malaysia, Singapore, Thailand, Taiwan, Vietnam, needed. The reproduction number (R0) for EV71 has been
and China [3, 4]. However, HFMD may be caused by a num- estimated to be higher than that of CVA16 (median 5.48,
ber of enteroviruses including CVA8, 10, and 16, and concur- IQR 4.20–6.51 compared with 2.50 [1.96–3.67], respec-
rent virological surveillance is necessary. Virological tively; p = 0.002) [94].
surveillance also provides invaluable molecular epidemio-
logical data about the circulating EV71 genotype and may 8.2.2 Vaccine Development
thus help to track the spread of the virus across the region. The success story of the control of poliomyelitis indicates
Because humans are the only known natural hosts of that vaccines would be the best approach for future disease
human enteroviruses, outbreak control measures are targeted control, and the target population should be younger chil-
primarily at interrupting person-to-person virus transmission dren, especially those less than 3 years of age, because
via contact with throat and nose secretions, saliva, stool, and they are at highest risk of severe disease. In fact, an inacti-
vesicular fluid but also at minimizing contact with contami- vated EV71 whole virus vaccine was developed in Russia
nated surfaces, toys, or fomites. Because of the lack of a lipid in 1976 after the Bulgaria epidemic. But it received no fur-
envelope, EV71 has considerable stability in the environ- ther evaluation because no further outbreaks of EV71
ment. It can remain viable at room temperature for several occurred [78]. More recent candidates for an EV71 vac-
days and has been recovered from surface and ground water cine include inactivated whole virus, live-attenuated,
and hot spas [30, 61]. Hence, health education focuses on recombinant viral protein, virus-like particle, and DNA
personal hygiene and good sanitation including frequent vaccines. These are at different stages of the development
hand washing, proper disposal of soiled diapers, and disin- in China, Taiwan, and Singapore [78, 176]. Among these,
fection of soiled surfaces with sodium hypochlorite [73]. inactivated whole virus vaccine candidates, the develop-
Like other enteroviruses, EV71 is resistant to alcohol. ment of which modeled that of inactivated polio vaccine,
Consequently, use of the widely available alcohol-based are at the final stages of the clinical evaluation. A phase 3
(70 % ethanol or isopropanol) disinfectants alone for hand randomized double-blinded, placebo-controlled trial of
hygiene is ineffective in preventing EV71 transmission [26]. inactivated alum-adjuvant EV71 vaccine (Beijing Vigoo
A recent study showed that EV71 can be destroyed by viru- Biological, EV71 strain FY7VP5/AH/CHN/2008, geno-
cidal disinfectants such as Virkon [18]. group C4) involving 10,245 Chinese children between the
270 M.H. Ooi and T. Solomon

300

250
2003

200
2006
Cases reported

150

100

50

0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
50
51
52
53
epid week

Fig. 12.6 The effect of public health interventions on hand-foot-and- response was limited, and the 2006 outbreak, when more rigorous
mouth disease outbreaks, comparing sentinel surveillance data from social distancing measures were encouraged. (Reproduced from
the 2003 outbreak in Sarawak, Malaysia, when the public health Solomon et al. [140])

ages of 6 and 35 months has shown encouraging vaccine 9 Concluding Remarks and Unsolved
efficacy, immunogenicity, and safety [180]. Another alum- Problems
adjuvant inactivated whole virus candidate (EV71 strain
H07, genogroup C4) produced by Sinovac Biotech Co., The emergence of EV71 in the Asia-Pacific region over the
LTD) was reported to be well tolerated and highly immu- last 15 years has had a major public health impact. Molecular
nogenic in a phase 1 trial in infant populations in China epidemiological studies suggest that some subgenogroups
[80]. While an eagerly awaited vaccine will likely be avail- appear to have massive potential for explosive epidemics,
able for routine use in the near future, it remains uncertain while others circulate in a more indolent pattern. However,
whether the vaccine developed from a specific genogroup the biological determinants of these differences are poorly
would provide adequate cross protection against all geno- understood. The epidemiological differences observed
groups. This concern is critical because genogroup B and between EV71 in the Asia-Pacific region and the strains
C virus so far appear to have different geographical distri- found in Europe and USA also represent an unsolved puzzle.
butions in Asia, and the data to date on cross protection There are no reliable and easy-to-use clinical tools to predict
between genogroups are conflicting. Another important who will develop neurological complications and which
issue pertaining to manufacturing processes of inactivated patients with CNS involvement are at risk of disease progres-
whole virus vaccine is that no international reference stan- sion. The virological and host determinants of the wide-
dards for potency assays and quantification of EV71 vac- ranging clinical phenotypes in those infected remain unclear.
cine antigens exist [78]. Establishment of such standards is There are relatively good animal models of neurological dis-
urgent in Asia, where the vaccine will likely be used soon- ease caused by EV71, but there is an urgent need for an ani-
est and most extensively. mal model of cardiorespiratory dysfunction to advance
12 Enteroviruses: Enterovirus 71 271

understanding of its pathogenesis. Despite lack of solid evi- 12. Cardosa M, Perera D, et al. Molecular epidemiology of human
dence for its efficacy, the wide use of IVIG for severe EV71 enterovirus 71 strains and recent outbreaks in the Asia-Pacific
region: comparative analysis of the VP1 and VP4 genes. Emerg
infection in many Asian countries will make efficacy trials Infect Dis. 2003;9(4):461–8.
difficult. There is still no specific antiviral drug available for 13. Cardosa MJ, Krishnan S, et al. Isolation of subgenus B adenovirus
EV71 infection although the determination of its crystal during a fatal outbreak of enterovirus 71-associated hand, foot, and
structure and identification of several EV71 receptors should mouth disease in Sibu, Sarawak. Lancet. 1999;354(9183):987–91.
14. Castro CM, Cruz AC, et al. Molecular and seroepidemiologic
accelerate drug discovery. An inactivated whole virus vac- studies of Enterovirus 71 infection in the State of Para, Brazil. Rev
cine is nearing clinical availability, but important steps must Inst Med Trop Sao Paulo. 2005;47(2):65–71.
be taken to ensure that it ultimately reaches the populations 15. Chakraborty R, Iturriza-Gomara M, et al. An epidemic of entero-
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and pathomorphological characteristics of epidemic poliomyelitis- Biol. 2012;19(4):424–9.
like disease caused by enterovirus 71. J Hyg Epidemiol Microbiol 158. Wang YF, Chou CT, et al. A mouse-adapted enterovirus 71 strain
Immunol. 1979;23(3):284–95. causes neurological disease in mice after oral infection. J Virol.
138. Siafakas N, Attilakos A, et al. Molecular detection and identifica- 2004;78(15):7916–24.
tion of enteroviruses in children admitted to a university hospital 159. Welch J, Maclaran K, et al. Frequency, viral loads, and serotype
in Greece. Mol Cell Probes. 2011;25(5–6):249–54. identification of enterovirus infections in Scottish blood donors.
139. Singh S, Poh C, et al. Complete sequence analyses of enterovirus Transfusion. 2003;43(8):1060–6.
71 strains from fatal and non-fatal cases of the hand, foot and 160. WHO. A guide to clinical management and public health
mouth disease outbreak in Singapore (2000). Microbiol Immunol. response for Hand, Foot and Mouth Disease (HFMD, 2011).
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140. Solomon T, Lewthwaite P, et al. Virology, epidemiology, patho- calmanagementofHFMD.pdf. Accessed 6 Aug 2013.
genesis, and control of enterovirus 71. Lancet Infect Dis. 2010; 161. Witso E, Palacios G, et al. Asymptomatic circulation of HEV71 in
10(11):778–90. Norway. Virus Res. 2007;123(1):19–29.
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162. Wong KT, Lum LC, et al. Enterovirus 71 infection and neurologic 174. Yu H, Chen W, et al. Genetic analysis of the VP1 region of entero-
complications. N Engl J Med. 2000;342(5):356–8. virus 71 reveals the emergence of genotype A in central China in
163. Wong KT, Munisamy B, et al. The distribution of inflammation 2008. Virus Genes. 2010;41(41):1–4.
and virus in human enterovirus 71 encephalomyelitis suggests 175. Zeng H, Wen F, Gan Y, Huang W. MRI and associated clinical
possible viral spread by neural pathways. J Neuropathol Exp characteristics of EV71-induced brainstem encephalitis in chil-
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Japanese encephalitis can be distinguished by topographic 176. Zhang D, Lu J, et al. Enterovirus 71 vaccine: close but still far.
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170. Yamayoshi S, Yamashita Y, et al. Scavenger receptor B2 is a
Suggested Reading
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798–801. Nishimura Y, Shimizu H. Cellular receptors for human enterovirus spe-
171. Yang F, Ren L, et al. Enterovirus 71 outbreak in the People’s cies A. Front Microbiol. 2012;3:105. doi:10.3389/fmicb.2012.00105.
Republic of China in 2008. J Clin Microbiol. 2009;47(7):2351–2. Wong KT, Ng KY, et al. Enterovirus 71 encephalomyelitis and Japanese
172. Yang J, Zhao N, et al. Association of interleukin 10 and interferon encephalitis can be distinguished by topographic distribution of
gamma gene polymorphisms with enterovirus 71 encephalitis in inflammation and specific intraneuronal detection of viral antigen
patients with hand, foot and mouth disease. Scand J Infect Dis. and RNA. Neuropathol Appl Neurobiol. 2012;38(5):443–53.
2012;44(6):465–9. Wu K, Ng M, et al. Developments towards antiviral therapies against
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tions in children with complicated enterovirus 71 infections in Zhang D, Lu J, et al. Enterovirus 71 vaccine: close but still far. Int
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 Enteroviruses: Polio
13
Olen M. Kew

1 Introduction Initiative (GPEI), established by a landmark 1988 resolution

of the World Health Assembly (the governing body of the
Six decades ago, every child faced the threats of lifelong paral- WHO) [15], has grown to become the largest public health
ysis or death from poliomyelitis. Poliomyelitis, an infectious program in history [16], engaging key segments of both the
disease dating back to antiquity (Fig. 13.1), suddenly appeared public and private sectors [17]. The launch of the GPEI was
in epidemic form in the late nineteenth century in northern made in light of dramatic progress by PAHO toward achiev-
Europe and the United States and emerged as one of the great ing its regional eradication goal, attained in 1991 [18]. Like
epidemic diseases of the twentieth century [1–3]. The threat of the PAHO initiative, the GPEI achieved early rapid prog-
poliomyelitis quickly receded in developed countries follow- ress, reducing poliomyelitis incidence worldwide by >99 %,
ing the introduction of the inactivated poliovirus vaccine (IPV) from an estimated 350,000 cases in 125 countries in 1988 to
in 1955 [4, 5] and the oral poliovirus vaccine (OPV) in 1961 a low of 493 cases reported in 10 countries in 2001, raising
[6, 7] and had all but disappeared in high-income countries by the long-held hope that a polio-free world would soon be
the early 1970s [8, 9]. In sharp contrast, poliomyelitis remained realized [19]. Optimism was fueled by the eradication of
largely uncontrolled in the developing countries of Latin wild poliovirus type 2 and reinforced by the cessation of
America, Asia, and Africa and continued to threaten the wild poliovirus transmission in many highly challenging
majority of the world’s children with crippling disease [8, 9]. settings. However, progress stalled between the years 2000
Today, poliomyelitis is on the verge of eradication, and its and 2010 as the global incidence poliomyelitis stabilized at
etiologic agents, the three poliovirus serotypes, are on the brink ~500–2,000 cases per year (Fig. 13.2a) [10, 19, 20]. With
of extinction from the natural environment (Figs. 13.2 and 13.3) intensified efforts, the GPEI steadily reduced the number of
[12]. Circulation of indigenous wild type 2 poliovirus ceased in endemic reservoirs, such that by the end of 2012 the global
1999 [13], and wild type 3 poliovirus may be nearing eradica- poliomyelitis case count again fell to a new all-time low of
tion [12]. Wild type 1 poliovirus circulation is localized to a 223, and only three countries (Nigeria, Pakistan, and
small and decreasing number of districts in parts of three coun- Afghanistan) had never stopped wild poliovirus transmis-
tries (for updates see http://www.polioeradication.org/) [12]. sion (Figs. 13.2 and 13.3) [12, 21]. Despite setbacks, the
This brightening picture is the direct result of the initia- GPEI achieved many landmark successes: coordinating the
tives launched in 1985 by the Pan American Health vaccination of 2.5 billion children, many of them among the
Organization (PAHO; the Regional WHO Office for the most vulnerable living in the most under-resourced commu-
Americas) to eradicate poliomyelitis in the Americas by nities in the world, and saving more than ten million people
1990 [14], and subsequently by the World Health (mostly children <2 years of age) from lifelong paralysis
Organization (WHO) to eradicate poliomyelitis worldwide and sparing the lives of more than 250,000 others [22]. The
by the year 2000 [15]. The Global Polio Eradication WHO GPEI is now developing a detailed endgame strategic
plan to secure forever the many gains achieved by polio
eradication (http://www.polioeradication.org/portals/0/doc-
ument/resources/strategywork/endgamestrat-
O.M. Kew, PhD plan_20130414_eng.pdf) [22].
Polio and Picornavirus Laboratory Branch,
Division of Viral Diseases, National Center for Immunization and
Respiratory Diseases, Centers for Disease Control and Prevention, The findings and conclusions in this chapter are those of the author and
Atlanta, GA 30329-4018, USA do not necessarily represent the views of the Centers for Disease
e-mail: [email protected] Control and Prevention.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 277

DOI 10.1007/978-1-4899-7448-8_13, © Springer Science+Business Media New York 2014
278 O.M. Kew

a b

Fig. 13.1 Thirty-three centuries of poliomyelitis. (a) Stele from the dia.org/wiki/File:Polio_Egyptian_Stele.jpg). (b) Poliomyelitis in
Eighteenth Dynasty of Egypt (c.1550 to c.1292 BCE) portraying a Delhi, India, 2002. The last case of poliomyelitis associated with wild
young man with an atrophied right leg and flaccid foot drop character- poliovirus in India had onset in January 2011. The photograph was
istic of the long-term sequelae of paralytic poliomyelitis. The stele is in downloaded from the WHO Media Centre (http://www.who.int/media-
the Ny Carlsberg Glyptotek, Copenhagen, Denmark; the photograph centre/multimedia/2002/ind_polio211460.jpg)
was downloaded from Wikimedia Commons (http://commons.wikime-

Polioviruses are members of the Enterovirus genus of the the fecal–oral or respiratory routes [23, 26]. The abrupt
family Picornaviridae (pico, L., small; rna, RNA genome) appearance of large poliomyelitis outbreaks generated
(Chap. 11) [23]. The Enterovirus genus, comprising >100 intense interest in the disease and prompted intensive studies
serotypes, is divided into 12 species (enterovirus species A of poliovirus epidemiology, pathology, immunology, and
to J and rhinovirus species A to C); poliovirus, along with virology, leading to the development and worldwide deploy-
>20 other serotypes are members of human enterovirus spe- ment of effective poliovirus vaccines and many ground-
cies-C (for updates see: http://www.ictvonline.org/) [24, 25]. breaking contributions to public health, medical science, and
Enteroviruses inhabit the intestinal tracts and/or the naso- basic research [3, 19, 23, 27].
pharyngeal tissues of humans and other mammals.
Polioviruses, for which humans are the only natural reservoir
host, occasionally invade the central nervous system (CNS) 2 Historical Background
and cause destruction of motor neurons in the spinal cord,
resulting in acute flaccid paralysis (AFP). However, poliovi- The rich history of research on poliomyelitis and poliovi-
rus invasion of the CNS occurs in less than 1 % of infections ruses has been chronicled in numerous excellent books,
and represents a dead end for transmission, which occurs by chapters, and reviews and in an extensive scientific literature
13 Enteroviruses: Polio 279

450
2500
a
400 2000

1500
350

Cases
1000

300
500
Cases (Thousands)

250 0

2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
AMR

200 Year

Last WPV2
150
Worldwide
Estimated cases
Reported cases
100
WPR
EUR
50

0
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Year

b c
2000 2000
Last WPV3: world wide
Last WPV: India
1500 1500
Cases

bOPV
Cases

1000 1000
bOPV
500 500

0 0
2005

2006

2007

2008

2009

2010

2011

2012

2005

2006

2007

2008

2009

2010

2011

Year Year 2012

2500 2500
d e PAK-Im
2000 2000 PAK-En
NIE-Im
1500 1500
Cases

NIE-En
Cases

IND-Im
1000 1000
IND-En
500 500

0 0
2005

2006

2007

2008

2009

2010

2011

2012

2005

2006

2007

2008

2009

2010

2011

2012

Year Year

Fig. 13.2 (a) Incidence of paralytic poliomyelitis cases associated VAPP per year worldwide. (b) Wild poliovirus type 1 (WPV1) poliomy-
with wild poliovirus (WPV) infections worldwide, 1985–2012 (source elitis cases, 2007–2012. Introduction of bivalent OPV (bOPV; types
http://www.polioeradication.org/). Estimated cases are shown as gray 1 + 3) in late 2009 is indicated by the arrow. (c) Wild poliovirus type 3
bars; reported, clinically confirmed, and virologically confirmed cases (WPV3) poliomyelitis cases, 2007–2012. (d) Poliomyelitis cases from
are shown as black bars. Starting in 2001, all WPV case counts were endemic (solid bars) and imported (hatched bars) WPV, 2007–2012.
based on virologic confirmation by the GPLN. Arrows below three- (e) Poliomyelitis cases associated with endemic (En) and imported (Im)
letter codes for WHO regions (AMR Americas, EUR Europe, WPR WPV, 2007–2012 (PAK Pakistan [and Afghanistan], NIE Nigeria, IND
Western Pacific) indicate year of last detection of indigenous WPV. Red India). (e) Poliomyelitis cases associated with endemic and imported
dashed lines in inset indicate estimated number (250–500) of cases of WPV3, 2007–2011 (Modified from reference Kew [10])
280 O.M. Kew

1988

2001

2012

Fig. 13.3 Progress toward global polio eradication, 1988 to 2012. Red countries with indigenous wild polioviruses (WPVs), yellow countries with
one or more case associated with imported WPV, green polio-free countries (Modified from reference Kew and Pallansch [11])
13 Enteroviruses: Polio 281

dating back over a century [1–3, 7, 23, 28–35]. The disease monthly poliomyelitis case counts to the Surgeon General’s
probably emerged at the dawn of civilization, when popula- Office [46]. The Drinker respirator, commonly known as the
tion centers grew in size and density sufficient to support “iron lung,” was introduced in 1929 as a device to save the
continuous endemic circulation. The earliest evidence for lives of patients with respiratory paralysis (many of whom
endemic poliomyelitis comes from Egypt, recorded on a would subsequently recover unassisted respiratory function)
small funerary stele from the Eighteenth Dynasty (c.1550 to [47]. In 1931, reinfection experiments in monkeys by Burnet
c.1292 BCE), depicting a crippled young man, standing with and Macnamara provided the first evidence of more than one
the aid of a staff, with an atrophied right leg and flaccid foot poliovirus serotype [48]. Seven years later, President Franklin
drop characteristic of the long-term sequelae of paralytic D. Roosevelt, who had been paralyzed in both legs by polio-
poliomyelitis (Fig. 13.1) [1]. Three millennia later, in 1789, myelitis in 1921, cofounded with Basil O’Connor the National
Underwood in England wrote the first clear clinical descrip- Foundation for Infantile Paralysis and the March of Dimes
tion of poliomyelitis as a “debility of the lower extremities” campaigns, providing a critical source of support for poliovi-
[36]. In 1840, the German orthopedist, von Heine, described rus vaccine research [1, 3]. Adaptation of the type 2 Lansing
“Spinale Kinderlähmung” (infantile spinal paralysis) and strain to cotton rats and mice by Armstrong in 1939 opened
postulated that the disease could be contagious [37]. In the way for much broader and more quantitative virologic and
Sweden, Medin conducted the first investigations of the epi- serologic studies on a scale previously unattainable with titra-
demiology of poliomyelitis during the outbreak in Stockholm tions in monkeys [49]. By the early 1940s, Trask and Paul [50,
in 1887 [38]. Sporadic small outbreaks of paralytic disease 51] and Sabin and Ward [52] recognized that poliovirus repli-
had been described in the United States since 1841 [33, 39], cated in the tissues of the intestinal tract as well as the CNS,
but the first large outbreak (132 cases) occurred in Rutland, confirming the earlier observations of Kling. In 1949, Enders,
Vermont, in 1894 [40]. In northern Europe, major epidemics Weller, and Robbins cultivated the type 2 Lansing strain of
(>500 reported cases) erupted in Norway and Sweden poliovirus in nonneural cells from human embryonic tissue—
(1905), Austria (1908–1909), Germany (1909), and England including skin, muscle, and intestine—yielding large quanti-
and Wales (1911) [34, 39]. During this period, Wickman in ties of virus, thereby accelerating the pace of poliovirus
Sweden firmly established that poliomyelitis was transmitted research and opening the way for expanded vaccine develop-
by person-to-person contact, that the disease spread along ment and large-scale vaccine production [53]. That same year,
the major lines of transportation, and that it gave differing Bodian and colleagues established that there were only three
clinical presentations. Wickman hypothesized that all infec- poliovirus serotypes [54, 55]. In 1953, following the peak year
tions, both severe and mild, contributed to spread, and that for poliomyelitis cases reported in the United States (57,628)
the incubation time was 3–4 days [41]. In Vienna in 1908, [56], Hammon et al. demonstrated that administration of
Landsteiner and Popper demonstrated that monkeys became immune gamma globulin was protective against paralytic dis-
paralyzed after intraperitoneal injection of a filtered homog- ease [57], and the following year Horstmann et al. showed that
enate from the spinal cord of a 9-year-old boy (who died viremia preceded paralysis in humans [58]. With these strong
within 3 days of onset of paralysis), and that they developed experimental underpinnings, the stage was set in 1954 for
neural lesions similar to those observed in paralyzed humans Francis to conduct a field trial enrolling 1,800,000 children in
[42]. Landsteiner and Popper were unable to passage the the United States [59], demonstrating the safety and efficacy
virus, but several groups, including Flexner and Lewis in of the IPV developed by Salk and colleagues [4]. The new IPV
1909, achieved serial passage of poliovirus by nasal inocula- was promptly licensed and distributed following announce-
tion of monkeys [43]. With continued passage in monkeys, ment of the field trial findings in April 1955 [1, 3, 35]. In 1959,
they selected a strictly neurotropic type 2 variant, MV, lead- large field trials of the OPV of Albert Sabin were conducted in
ing them to postulate that poliovirus grew only in neural tis- the Soviet Union, Poland, and Czechoslovakia [60], leading to
sues. However, in 1912, Kling, Wernstedt, and Pettersson the licensure and distribution of monovalent OPV types 1 and
isolated poliovirus not only from neural tissues but also from 2 in 1961 and type 3 in 1962 [6].
the oropharynx and small intestine, as well as from intestinal
contents and throat swabs [44], but the views of Flexner and
colleagues prevailed, and the critical findings from human 3 Methodology Involved
pathology were overlooked for nearly three decades [1, 2]. in Epidemiologic Analysis
After 1906, large epidemics appeared in the northeastern
and north central states of the United States, culminating in the 3.1 Sources of Data
epidemic of 1916 centered around New York City, far larger at
23,000 reported cases than any previous outbreak [34, 45]. Poliomyelitis has been a notifiable disease in the United States
Nationwide poliomyelitis surveillance had begun in the United since 1910 [46], when case reports from state and territorial
States in 1910, when all states were requested to forward boards of health and health departments were summarized
282 O.M. Kew

monthly (1910–1926) and then weekly (1927–1951) in CFRs (27 % in the 1916 New York epidemic) [45], and more
Public Health Reports [61]. Starting in 1952 [62], reports recent outbreaks from importation of wild poliovirus into
were regularly published in the CDC (Communicable previously polio-free countries have also been characterized
Disease Center, later Centers for Disease Control and by high CFRs in older age groups: Albania, 1996 (18 % for
Prevention) Morbidity and Mortality Weekly Report ages 19–24 years) [78]; Cape Verde Islands, 2000 (57 % for
(MMWR) [63]. Special annual poliomyelitis surveillance ages >15 years) [79]; Namibia, 2006 (31 %; most paralytic
summaries were also published by CDC through 1974 [64]. cases were among adults) [80]; the Republic of Congo,
In addition to the United States, many European countries 2010–2011 (43 %; most paralytic cases were among adults)
and Canada established systems early in the last century for [81]; and Xinjiang, China, 2011 (10 %) [82]. During the out-
reporting cases of poliomyelitis, allowing epidemiologists to break year of 2006 in India, highly sensitive surveillance
monitor rising disease incidence up to mid-century [65, 66] documented a CFR of 7.1 % among children <2 years [77].
and the sharp decline after the introduction of poliovirus vac-
cines [8, 9]. In contrast, data on poliomyelitis incidence in
developing countries was very incomplete, with only a small 3.2 Serologic Surveys
fraction of cases reported and with many populous countries
not reporting any cases at all [8, 9]. In 1969, the World Health Serologic studies of infectious diseases were initially applied
Assembly adopted a resolution that placed poliomyelitis to the diagnosis of individual cases [83]. The first applica-
“under international surveillance” [67]. However, systematic tions of serology to epidemiology were the studies by Aycock
and sensitive surveillance for poliomyelitis in developing and Kramer in 1930, who used the newly developed neutral-
countries only followed the launch of polio eradication ization test to show that antibodies to poliovirus appeared at
efforts in the Americas [14, 18] in 1985 and the GPEI in younger ages in urban compared with rural populations [84].
1988 [15] and the establishment of field surveillance for Despite the methodological limitations (neutralization tests
cases of acute flaccid paralysis (AFP) [68] closely integrated were performed in monkeys and predated recognition of
with virologic testing of clinical specimens [69]. The quality more than one poliovirus serotype), these early studies her-
of the integrated surveillance data improved gradually, usu- alded a powerful new tool to address fundamental questions
ally in step with improvements in OPV coverage, and the about the epidemiology of poliomyelitis. In the pre-vaccine
findings were published regularly in reports by PAHO [70], era, serologic surveys played an indispensable role in defin-
WHO [71], and CDC [72]. Current weekly and monthly ing the prevalence of poliovirus infection, the intensity of
reports are posted on the WHO website (http://www.polio- transmission of each poliovirus serotype, the age profiles of
eradication.org/Dataandmonitoring/Poliothisweek/.aspx), exposure in different settings, the years and the associated
and lists of wild polioviruses by country and year are posted serotypes of past outbreaks in isolated populations, the dura-
on http://www.polioeradication.org/Dataandmonitoring/ tion of type-specific immunity, the identification of suscep-
Poliothisweek/Wildpolioviruslist.aspx. tible populations, and key aspects of the pathogenesis of
Over the past century, the large majority of case counts paralytic disease—including estimates of age-specific case/
were based on clinical diagnoses, and it was soon recognized infection ratios [23, 28, 85, 86]. In the post-vaccine era, sero-
that the most accurate counts were obtained in outbreak set- epidemiology has been used to detect immunity gaps in
tings [73]. Cases not associated with large outbreaks were underserved populations [87, 88], to estimate the extent of
more likely to be underreported. In the PAHO and WHO wild poliovirus circulation in populations [89], to determine
eradication initiatives, AFP cases were systematically the efficacy of different OPV formulations in inducing neu-
reported and investigated, and as national and regional labo- tralizing antibodies [90], and to provide evidence for eradi-
ratory networks developed, all specimens from AFP cases cation of indigenous wild polioviruses [91]. In the GPEI (and
were tested for the presence of poliovirus [69, 74, 75]. in the earlier PAHO initiative), serology was found not to be
Countries and regions shifted from a clinical case definition useful in the diagnosis of individual cases, because the
to a virologic case definition once field surveillance for cases response to detection of an AFP case was prompt administra-
of AFP was tightly integrated with laboratory investigations tion of trivalent OPV (tOPV) to the patient and to the com-
for poliovirus. By 2001 global poliomyelitis case counts munity (“mop-ups”; Sect. 10.4), such that many initially
were based on virologic findings. seronegative children had seroconverted to all three poliovi-
Data on case/fatality (CFR) ratios are not routinely avail- rus serotypes by the time of the second blood sample [18].
able as ratios vary with the age distribution of population However, seroprevalence studies continue to be important in
susceptibility and by setting [9, 23]. CFRs increase with age measuring the immunogenicities of different OPV formula-
and are generally on the order of 2–5 % in children <5 years tions [92], in detecting otherwise inapparent spread of OPV-
of age and 10–30 % in adults [23, 76, 77]. The epidemics derived viruses in unimmunized populations [93, 94], and in
early in the twentieth century were associated with high providing objective data on population immunity [95, 96],
13 Enteroviruses: Polio 283

including in polio-free countries where OPV coverage rates from at least 80 % of patients with AFP were tested for the
have fallen and the rising risks of outbreaks might not other- presence of poliovirus (Sect. 3.6) [69]. The successful PAHO
wise be recognized [22]. In recent outbreaks, primarily asso- strategy was adopted by the GPEI and implemented in all
ciated with poliovirus type 1 (Sect. 10.6), determination of polio-endemic countries [68, 74, 102]. The benchmark AFP
the prevalence of neutralizing antibodies to poliovirus type 2 rate was raised to at least 2 cases per 100,000 population
is a surrogate for vaccine-induced population immunity <15 years in endemic areas, and the global rate has been >4
when the initial immunization response is deployment of since 2007 (http://apps.who.int/immunization_monitoring/en/
type 1 monovalent OPV (mOPV1). diseases/poliomyelitis/afpextract.cfm). In the last stages of
polio eradication in India, AFP surveillance sensitivity reached
the extraordinarily high levels of >25 AFP cases per 100,000
3.3 Lameness Surveys population <15 years in the remaining polio-endemic states of
Uttar Pradesh and Bihar (http://www.npspindia.org/bulletin.
Severe underreporting of poliomyelitis cases in many devel- pdf) [91]. Only a small fraction of the AFP case-patients had
oping countries led to the misperception that the disease was wild poliovirus infections, and the integrated AFP and poliovi-
not a source of serious morbidity in the tropics [8, 66]. rus surveillance system was approximating a community stool
Lameness surveys conducted in the 1970s and 1980s in sampling survey of a population of ~300 million. It was criti-
Africa, Asia, and the Middle East confirmed the high preva- cal to integrate AFP surveillance with laboratory-based polio-
lence of paralytic disease in developing countries and virus surveillance because AFP has multiple etiologies
prompted many countries to begin polio vaccination pro- (including Guillain–Barré syndrome, transverse myelitis,
grams [97, 98]. For example, in India in 1981–1982, esti- infections by other neurotropic viruses, and traumatic neuritis;
mates of the incidence of poliomyelitis from lameness Sect. 8.1) [7], and the large majority of wild poliovirus infec-
surveys were as high as 200,000 cases per year, more than tions are inapparent.
tenfold higher than officially reported case counts, with 83 %
of cases occurring before 3 years of age [99]. Methods to
improve the comparability of lameness surveys in different 3.5 Environmental Surveillance
settings, including the use of standardized case definitions,
has been reviewed [97]. Sewage sampling was used as early as the 1940s to monitor
the seasonal variation of poliovirus circulation in urban com-
munities [51, 103]. Because most poliovirus infections are
3.4 Acute Flaccid Paralysis (AFP) inapparent (Sect. 3.4 and 8.1), sewage sampling can greatly
Surveillance increase the overall sensitivity of poliovirus surveillance. For
example, during the pre-vaccine era in the United States,
AFP is the most serious clinical manifestation of wild poliovi- poliovirus could be detected in sewage shortly before and
rus infection (Sect. 8.1) [100]. Poliomyelitis outbreaks are after the seasonal appearance of paralytic cases, and the com-
readily recognized, but low-level circulation in interepidemic bined clinical data and environmental poliovirus isolation
periods may be missed in the absence of a sensitive surveil- rates permitted estimation of the ratio between inapparent
lance system. This is especially true for endemic circulation of infections and paralytic cases [103]. Sewage sampling is
poliovirus types 2 and 3, which have much lower paralytic widely implemented in Europe (conducted by 20 countries,
case/infection ratios than type 1 (estimated case/infection including Israel [104–108]) and Japan [109] as a component
ratios [assuming an overall case/infection ratio of 1/150]: type of enterovirus (and poliovirus) surveillance. During the 1984
1, ~1/190; type 2, ~1/1,900; type 3, ~1/1,150) [34, 101]. poliovirus outbreak in Finland, environmental surveillance
Recent importation of wild poliovirus or emergence of circu- demonstrated widespread circulation of the wild type 3 out-
lating vaccine-derived polioviruses (cVDPVs; Sect. 10.8) may break virus and provided a basis for the estimate of the occur-
also be missed by the AFP surveillance system unless high rence of at least 100,000 inapparent infections despite the
sensitivity is maintained. Starting in 1985, PAHO built an AFP appearance of only nine paralytic cases [104, 110]. Wild
surveillance system to support the regional Polio Eradication poliovirus type 3 was found to be present in the environment
Initiative [18]. Performance indicators for reporting of AFP 3 weeks before the appearance of the first paralytic case dur-
cases were established assuming a background rate of nonpo- ing the 1992–1993 outbreak in the Netherlands, and circula-
lio AFP of at least 1 case per 100,000 population <15 years. In tion of the outbreak virus was found to be localized to
addition, surveillance sites were required to report weekly, communities that refused immunization [106]. Environmental
including “zero reporting” when no AFP cases were identified surveillance coupled with sequencing of wild poliovirus iso-
during the previous week [75]. AFP surveillance was closely lates was introduced on a limited basis in the PAHO program
integrated with virologic surveillance whereby stool samples [111, 112]. Sampling of wastewater in a high-risk community
284 O.M. Kew

in Cartagena, Colombia, revealed close sequence relation- Environmental surveillance has also helped inform the
ships between sewage isolates, stool survey isolates, and endgame strategy for the GPEI (Sect. 11.2). A key question
paralytic case isolates obtained from the community over the is the persistence of vaccine-related viruses in the population
same period, but also detected circulation of lineages not and environment following cessation of OPV use. In Cuba,
found by AFP surveillance [111]. Sewage sampling in Israel where OPV is delivered only in mass campaigns in the form
and adjoining Palestinian territories detected outbreaks of of two rounds of National Immunization Days (NIDs),
wild poliovirus infections in Gaza, Ashdod, and the West vaccine-related viruses were detected in stool surveys for up
Bank in 1990 (type 3), 1991 (type 1), 1994–1995 (type 1), to 8 weeks and in the environment for up to 15 weeks after
and 1996 (type 1) [107]. The outbreaks were described as the second NID round [121]. New Zealand shifted from OPV
“silent” because no poliovirus-associated paralytic cases had to IPV in February 2002, and vaccine-related viruses were
been detected by the AFP surveillance systems. All of the regularly detected in sewage samples until May 2002.
wild poliovirus sewage isolates were found to be related to Sporadic vaccine-related isolates detected subsequently
viruses circulating in Egypt. The continued detection in showed very limited sequence divergence from the parental
Gaza of wild polioviruses of Egyptian origin at times when OPV strains, indicating that they were recent imports from
none were reported by the AFP surveillance system in the OPV-using countries rather than persistence of vaccine-
source reservoir communities prompted the implementation related viruses in the community [122].
of environmental surveillance in Egypt in September 2000, An important difference between environmental sampling
which by 2004 sampled 33 sites in 18 governorates [113– and AFP surveillance is that environmental sampling is most
115]. This approach, combined with strengthened AFP sur- sensitive in locations with developed sewage systems or
veillance, improved the overall sensitivity of the poliovirus open sewage canals in large slums and therefore is usually
surveillance system, and wild polioviruses disappeared from established in more urbanized settings. Sewage sampling
the environment soon after their disappearance in specimens sites in urban centers are selected to include communities of
collected from patients with AFP [113, 115]. Implementation migrant populations from rural areas. Environmental surveil-
of sewage sampling in the open canals in the large slum com- lance is necessarily localized, targeted, and intermittent, in
munities of Mumbai, India [116], coupled with sequencing contrast to a well-functioning AFP surveillance system that
(section “Nucleotide sequencing of poliovirus isolates”), monitors the entire population on a continuous basis.
confirmed the disappearance of the local wild polioviruses Consequently, poliovirus isolates obtained by the two sur-
and the repeated importation of wild polioviruses from veillance approaches may yield different kinds of public
known reservoirs in the northern Indian states of Uttar health information. For example, a wild poliovirus isolate
Pradesh and Bihar [91]. Because no suitable sampling sites from an AFP case is directly linked to a specific patient from
were available in the highest-risk rural reservoir communi- a specific locale and usually signals many other inapparent
ties, additional sites were established in Delhi and Patna, infections in the community. In contrast, the high sensitivity
Bihar, which receive migrants from the endemic rural areas of environmental sampling can result in multiple poliovirus
[91]. As in Egypt, the findings from the environmental and isolations from a single infected person but does not yield
AFP surveillance systems were in agreement: the last envi- further information about the specific source of infection.
ronmental wild poliovirus isolate (a type 1) was found in However, some information about the extent of poliovirus
Mumbai sewage in November 2010, and the last wild polio- circulation can be obtained from the extent of genetic diver-
virus isolate (a type 1) from an AFP case-patient was in West sity of polioviruses obtained at a sampling site [111, 115].
Bengal in January 2011 (http://www.npspindia.org/bulletin.
pdf) [91]. The GPEI and the Global Polio Laboratory
Network (Sect. 3.6.1) have extended environmental surveil- 3.6 Laboratory Methods
lance to six cities in Pakistan and three cities in Nigeria and
are planning further expansions in countries at high risk of 3.6.1 The Global Polio Laboratory Network
reestablishment of poliovirus circulation either by importa- (GPLN)
tion or by the emergence of cVDPVs (see below and The GPLN was established by the WHO to support the GPEI
Sect. 10.8) [22]. [123]. Currently the GPLN consists of 145 laboratories, ini-
VDPVs closely resembling those excreted by individuals tially organized in three tiers: (1) National and Subnational
with primary immunodeficiencies (Sect. 10.8.2) have been Laboratories (n = 122), (2) Regional Reference Laboratories
detected in sewage in Israel [117], Estonia [118], Slovakia (n = 16), and (3) Global Specialized Reference Laboratories
[119], and Finland [120]. Despite efforts in each country to (n = 7) (Fig. 13.4). As the GPLN developed, activities once
identify the source of the excreted virus, no poliomyelitis assigned to Regional Reference Laboratories are frequently
cases or chronically infected individuals have so far been performed by many National and Subnational Laboratories,
identified. and Regional Reference Laboratories currently perform
13 Enteroviruses: Polio 285

Global Specialized Reference Laboratory

Regional Reference Laboratory
n=145
National/Sub-national Laboratory

Fig. 13.4 Distribution of laboratories of the Global Polio Laboratory Network (GPLN). Triangles National and Subnational Laboratories, circles
Regional Reference Laboratories, stars Global Specialized Reference Laboratories

many functions (such as genomic sequencing) originally tion of (or in response to) increasingly focused surveillance
assigned to Global Specialized Reference Laboratories. questions posed by the GPEI [22]. All GPLN laboratories
These trends have strengthened the GPLN, permitted con- participate in a formal accreditation process which includes
tinuous technical innovation, and moved many of the diag- review of performance in standardized proficiency tests as
nostic activities closer to the endemic areas of highest well as routine diagnostic work as confirmed by GPLN refer-
priority. The GPLN was patterned after the PAHO Regional ence laboratories [128]. Well-characterized cells, reference
Laboratory Network established in parallel with develop- OPV virus stocks, serologic reagents, and molecular reagents
ment of AFP surveillance [69]. Close integration of AFP and are distributed by GPLN reference laboratories to ensure a
poliovirus surveillance was facilitated by the use of stan- high degree of standardization, and internal quality control
dardized case (“EPId”) numbers accessible to surveillance procedures are regularly implemented by all GPLN laborato-
officers, virologists, and program managers [100, 124]. ries to ensure high routine performance [128]. GPLN labora-
Methods for poliovirus isolation, identification, and serol- tories participate in annual regional and global meetings to
ogy have been described in detail previously [23, 125, 126]. review performance, discuss effective implementation of new
Laboratory manuals were developed by the GPLN to stan- methods, develop approaches to improve coordination, and
dardize methods for detecting and characterizing polioviruses plan research and other future activities (http://www.polio-
in clinical specimens and environmental samples [124], and a eradication.org/Dataandmonitoring/Surveillance/
GPLN Quarterly Update was published by the WHO to keep GlobalPolioLaboratoryNetwork.aspx) [128]. The GPLN is
GPLN virologists and others abreast of new developments interdependent, applying common approaches to problem
and innovations [127]. Because the overriding emphasis of solving, parallel testing as needed, training, and other kinds
the GPLN is to monitor poliovirus circulation, less attention of technical support. The GPLN is guided by expert WHO
has been given to the routine typing and characterization of virologists who serve as global and regional laboratory
nonpolio enterovirus isolates, and readers are referred to coordinators. The GPLN, with its close integration with
Chap. 11 for details on those methods. The WHO Polio program, has served as the model for newer regional and
Laboratory Manual [124] is regularly updated as new meth- global networks supporting laboratory-based surveillance for
ods are developed and tested for suitability for use by GPLN other viral and bacterial vaccine-preventable diseases [129–
laboratories. New methods, designed to increase sensitivity, 131]. Many of the methods and reagents used by the GPLN
specificity, and work efficiency, are developed in concert with have also been shared with state laboratories in the United
the rising technical capabilities of the GPLN and in anticipa- States [132].
286 O.M. Kew

3.6.2 Virus Isolation and Identification Virus Isolation in Cell Culture

Categories of Poliovirus Isolates The most critical and basic procedure is virus isolation in cell
The primary purpose of infectious disease surveillance is to culture. Polioviruses can be grown in a wide range of human
identify agents that present potential public health risks. cells (RD, HeLa, HEp-2, WI-38, MRC-5, HEK293) and sim-
Poliovirus isolates of each serotype are grouped into three ian cells (from rhesus macaques and African green monkeys;
categories, correlating with the risk of transmission and primary monkey kidney cells, Vero, LLC-MK2, BGM)
spread, and based on the extent of divergence of the VP1 (http://www.atcc.org/) [23], but two cell lines are routinely
nucleotide region compared to the corresponding OPV used in combination by the GPLN for virus isolation [124]:
strain: (1) wild polioviruses (no genetic evidence of deriva- (1) RD cells (a continuous line from human rhabdomyosar-
tion from any vaccine strain and demonstrated capability of coma [136]) which are highly sensitive to poliovirus infection
continuous person-to-person transmission); (2) vaccine- and yield virus at high titers [23] and (2) L20B cells (a deriva-
derived polioviruses (VDPVs) (vaccine-related polioviruses tive of the mouse L cell line engineered to express the human
that are >1 % divergent [types 1 and 3] or >0.6 % divergent poliovirus receptor, CD155) which are highly selective for
[type 2] from the corresponding OPV strain and potentially growth of poliovirus [124, 137]. Viruses that grow in L20B
capable of causing paralytic disease and establishing person- are usually polioviruses (although some Coxsackie A viruses
to-person transmission) (Sect. 10.8); and (3) “OPV-like” can grow in L20B cells [124, 138]) and are further character-
polioviruses (vaccine-related polioviruses that are ≤1 % ized by molecular identification methods.
divergent [types 1 and 3] or ≤0.6 % divergent [type 2] from
the corresponding OPV strain) that are ubiquitous wherever Molecular Characterization and Intratypic
OPV is used [133]. VDPVs are further categorized as (1) Differentiation of Isolates
circulating VDPVs (cVDPVs), when there is evidence of The original methods for identification of polioviruses and
person-to-person transmission in the community; (2) other enteroviruses were based on antigenic properties. Virus
immunodeficiency-associated VDPVs (iVDPVs), which are isolates were typed by testing for growth in the presence of
isolated from persons with primary immunodeficiencies who pools of antisera containing different combinations of high-
have prolonged VDPV infections; and (3) ambiguous titer neutralizing antibodies [23]. Typing of individual polio-
VDPVs (aVDPVs), which are either clinical isolates from virus (or enterovirus) isolates was then confirmed by use of
persons with no known immunodeficiency or sewage iso- type-specific antisera. Heterotypic poliovirus mixtures were
lates whose ultimate source is unknown [133]. resolved by growth in the presence of different pairs of type-
specific neutralizing antisera.
Clinical Specimens Intratypic differentiation (ITD; distinguishing wild polio-
The specimens of choice for AFP and poliovirus surveillance viruses from vaccine-related isolates) was originally based
are stool samples collected as soon after onset of paralysis as on antigenic or phenotypic properties [139]. Before the
possible. The GPEI has defined “adequate” stool specimens development of molecular methods, the most reliable of
as “two stool specimens of sufficient quantity (~8 g) for lab- these were the antigenic methods, and isolates were described
oratory analysis, collected at least 24 h apart, within 14 days as “vaccine-like,” “non-vaccine-like,” or “intermediate.”
after the onset of paralysis, and arriving in the laboratory in Most assignments based on antigenic methods were con-
good condition and with proper documentation” (http:// firmed by the more precise molecular methods. It is remark-
www.polioeradication.org/Dataandmonitoring/Surveillance. able that the antigenic methods worked so well in view of the
aspx). Two samples are collected because poliovirus shed- fact that the Sabin OPV strains undergo frequent antigenic
ding is often intermittent [134]. The GPEI has established evolution toward “non-vaccine-like” antigenicity during rep-
clear performance guidelines and training procedures for lication in the human intestine [139–141], and that the wild
proper specimen transport to the laboratory via a “reverse polioviruses themselves are antigenically diverse [139, 142].
cold chain” similar to the forward cold chain used for deploy- Although the Sabin type 1 OPV strain (Sabin 1) has multiple
ment of OPV [100, 124]. Such importance has been assigned non-consensus antigenic changes in its neutralizing anti-
to specimen collection from AFP cases that runners with genic sites [139, 143, 144], Sabin 2 and Sabin 3 are usually
specimens in insulated backpacks were allowed safe passage less antigenically divergent from the corresponding wild
through combat lines in war zones where use of motorized polioviruses [139]. ITD based on antigenic properties was
transport was very hazardous. improved by use of highly specific cross-absorbed mono-
Poliovirus may be isolated at lower frequencies from rec- typic sera which contained antibodies that reacted specifi-
tal swabs [135] and throat swabs (if taken within the first few cally with “vaccine-like” or “non-vaccine-like” antigens
days of infection) and only rarely from cerebrospinal fluid [143]. ITD using cross-absorbed antisera was adapted to an
(CSF); however, none of these specimens are recommended ELISA format [145] and widely used by the GPLN, espe-
by the GPEI and the GPLN [100, 124]. cially in recent years for the characterization of VDPVs
13 Enteroviruses: Polio 287

Table 13.1 Poliovirus identification by real-time RT-PCR

RT-PCR Poliovirus/enterovirus isolate categorya
primer–probe
set NPEV WPV1b WPV3b S1 S2 S3 WPV1/WPV3 VDPV1 VDPV2
panEV + + + + + + + + +
panPV – + + + + + + + +
Sero1 – + – + – – + + –
Sero2 – – – – + – – – +
Sero3 – – + – – + + – –
Sab1 – – – + – – – + –
Sab2 – – – – + – – – –
Sab3 – – – – – + – – –
VDPV1 – – – – – – – + –
VDPV2 – – – – – – – – +
VDPV3 – – – – – – – – –
Based on data from references [151, 153, 157–159]. Reagents for identification of VDPV3 isolates have also been prepared. All suspected wild
poliovirus and VDPV isolates are routinely further characterized by VP1 sequencing. Special sequencing primer sets have been developed to
resolve both heterotypic and homotypic mixtures. The combinations shown, along with others (especially different combinations of Sabin vaccine-
related isolates), have been observed in clinical isolates
a
Abbreviations: NPEV nonpolio enterovirus, WPV1 wild poliovirus type 1, WPV3 wild poliovirus type 3, S1 Sabin type 1 vaccine-related, S2 Sabin
type 2 vaccine-related, S3 Sabin type 3 vaccine-related, VDPV1 vaccine-derived poliovirus type 1, VDPV2 vaccine-derived poliovirus type 2
b
As previously described for other wild poliovirus genotypes [152], wild genotype-specific real-time RT-PCR primers and probe sets have been
prepared for the Nigeria wild type 1, Nigeria wild type 3, Pakistan–Afghanistan wild type 1, and Pakistan–Afghanistan wild type 3 genotypes
(D. Kilpatrick, manuscript in preparation)

[133]. Other ITD methods based on antigenic properties Currently the GPLN uses real-time RT-PCR for ITD
used panels of neutralizing monoclonal antibodies [140, 142, [124]. A series of primer pairs and specific fluorescent probes
145, 146]. However, none of the antigenic ITD methods have been developed that identify isolates hierarchically: (1)
could overcome the basic biological limitations arising from as enteroviruses (panEV), (2) as polioviruses (panPV), (3)
the antigenic evolution of the OPV strains [140, 141, 145], by poliovirus serotype (Sero1, Sero2, Sero3), and (4) whether
and antigenic methods have been replaced by methods based vaccine-related (Sab1, Sab2, Sab3) (Table 13.1) [153, 157,
on the nucleotide sequence properties of poliovirus isolates. 158]. The sets of real-time RT-PCR reagents are deployed as
The earliest molecular method for routine ITD was oligo- kits for routine use by the GPLN [160] and can be supple-
nucleotide fingerprinting [147, 148]. This approach had the mented with additional real-time RT-PCR reagents that fur-
high reliability required for poliovirus surveillance in sup- ther identify wild polioviruses by genotype (Sects. 3.6.3 and
port of eradication, but it was also laborious, expensive, and 10.7) and can facilitate screening for genetically divergent
difficult to scale up and required the use of radioisotopes. VDPVs (VDPV1, VDPV2, VDPV3) [133, 161]. The rapid
Therefore, oligonucleotide fingerprinting was not appropri- evolution and high genetic diversity within and across polio-
ate for developing country laboratories. Consequently, oligo- virus serotypes presented special challenges to development
nucleotide fingerprinting was replaced by nucleic acid probe of the panPV and serotype-specific primer and probe sets
hybridization [149, 150], and the transfer of this ITD method (Table 13.1), as it was necessary to use degenerate and
to the PAHO Polio Laboratory Network commenced in the inosine-containing oligonucleotides to base pair with the
late 1980s. The reverse transcriptase-polymerase chain reac- appropriate specificities at positions of codon degeneracy
tion (RT-PCR) offered specificities and sensitivities unattain- [157, 158]. Although the nondegenerate Sabin vaccine
able with probe hybridization, and it became the method of strain-specific RT-PCR reagents can be used in a multiplex
choice within the GPLN [151, 152], although full deploy- format, the complexity of the degenerate reagents limits the
ment awaited adaptation to a real-time format, which greatly number of reactions that can be combined in multiplex.
reduced the risks of contamination by PCR products [153].
RT-PCR coupled to restriction fragment length polymor- Nucleotide Sequencing of Poliovirus Isolates
phism analysis was also widely used, because this routine ITD screens for wild polioviruses and VDPVs and screens
test provided insights into the origins of wild poliovirus iso- out OPV-like polioviruses that are unlikely to be of current
lates [154]. An elegant approach based on microarrays was epidemiologic importance. Since 2001, all wild poliovirus
also developed [155, 156], but it was less readily transferra- and VDPV isolates are sequenced by GPLN laboratories fol-
ble to developing country laboratories at the front lines of lowing standardized procedures and using standardized
global poliovirus surveillance. sequencing primer sets. The ~900-nucleotide interval
288 O.M. Kew

(representing ~12 % of the total genome) encoding the major immunity and where demographic and environmental condi-
capsid protein, VP1, is routinely sequenced. VP1 sequences tions favor poliovirus circulation. During the peak months of
are used for routine comparisons because they encode sev- poliovirus circulation, virus spreads from the reservoir com-
eral serotype-specific antigenic sites [162] and evolve pri- munities to adjacent non-reservoir indicator communities
marily by successive fixation of nucleotide substitutions (where the density of nonimmune susceptible children can
rather than by recombination [163, 164]. Wider genomic support some poliovirus circulation during the peak trans-
intervals, up to the complete genome, may be sequenced to mission season). This has led to a refinement in the concept
obtain higher epidemiologic resolution or to address specific of virus importation, which in previous usage referred to
virologic questions [165–168]. Serotype- and genotype-spe- virus transmission across national boundaries. Although
cific sequencing primers have been developed to specifically many importations over long distances have been docu-
amplify components of heterotypic and homotypic poliovirus mented [10, 180], reservoir communities and their associ-
mixtures, bypassing selective cultivation in the presence of ated indicator communities frequently overlap international
neutralizing antibody or incubation at supraoptimal tempera- borders [10, 184], underscoring the importance of regional
tures [169]. Sequence relationships among poliovirus iso- synchronization of NIDs and Subnational Immunization
lates are summarized in phylogenetic trees and genotypic Days (SNIDs). Equally important are the patterns of impor-
maps that are distributed monthly by GPLN laboratories to tation from reservoir communities to indicator communities
Ministries of Health, WHO country and regional offices, within a country [168, 184, 185]. High vaccine coverage in
WHO-Geneva, and other GPLN laboratories. the reservoir communities, especially in mass campaigns
conducted during the low transmission season, prevents the
3.6.3 Molecular Epidemiology of Polioviruses subsequent spread to indicator communities.
The application of genomic sequencing of poliovirus isolates Sequence analysis led to the recognition of highly diver-
has added a new dimension and resolving power to the gent iVDPVs [165, 186] and cVDPVs [166, 187, 188]
understanding of the epidemiology of poliomyelitis [170]. (Sect. 10.8), and it has been used to resolve at high-resolution
Because poliovirus genomes evolve rapidly (typically just chains of cVDPV transmission [132, 188–190] and separate
over 1 % nucleotide substitutions per site per year at all sites, iVDPV lineages in individual immunodeficient patients with
equivalent to one to two nucleotide substitutions per week) prolonged infections [165, 173, 186, 191, 192].
[164, 165, 168, 171–174], links between poliomyelitis cases Molecular epidemiologic methods have also opened a
can be determined with precision, and the sources and timing new avenue for detecting gaps in polio surveillance. In areas
of importations from the remaining poliovirus reservoirs can with good surveillance, poliovirus isolates representing fre-
be established [11, 163, 164, 168, 175–178]. Sequence anal- quent sampling of a single chain of transmission are typi-
yses offer an additional tool to monitor the progress of the cally closely related (usually >99.5 % VP1 sequence identity
GPEI and has shown that poliovirus genotypes (viruses among the closest relatives). These closely related viruses
within a genotype differ by <15 % in their nucleotide are routinely visualized as sequences connected by short
sequences) and genetic clusters within genotypes (viruses branches on phylogenetic trees [193]. Long-branch connec-
within a cluster differ by <5 % in their nucleotide sequences) tions between isolate sequences indicate missing informa-
disappear sequentially through intensive immunization tion. If the virus is imported, the missing information may be
efforts (Sect. 10.7) [11]. Experience in the Americas has recovered from the sequence relationships to viruses from
found that in settings of sensitive surveillance, a genotype the source reservoir [11]. However, in many other circum-
that is not detected for more than a year has probably become stances, no closely related viruses can be found, and the
extinct [11, 170]. Molecular epidemiology has established recent virologic history of the isolate lineage is indetermi-
the existence of numerous poliovirus genotypes endemic to nate. For example, gaps in AFP surveillance in southern
different regions of the world (Sect. 10.5.3) [11, 170], dem- Egypt were inferred from the sequence data, because indig-
onstrated that poliovirus type 2 is usually the first serotype to enous type 3 isolates in 1999 appeared as “orphan lineages”
be eliminated [13], that poliovirus type 3 appears to circulate at the tips of long branches on phylogenetic trees, and the
more locally than type 1 [10], and that poliovirus type 1 closest relatives were isolated nearly 3 years earlier [194],
appears to be most commonly associated with importations observations that highlighted the importance of environmen-
from neighboring countries and with intercontinental or tal surveillance to improve sensitivity. Orphan lineages have
global spread of the virus (Sect. 10.6) [10, 11, 163, 175, 176, been repeatedly found in areas with insensitive surveillance.
179–181]. In some settings, different genotypes of poliovirus The GPLN regularly monitors for the appearance of poliovi-
type 1 have been found to have co-circulated in a geographi- rus orphan lineages as a means to assess surveillance
cally limited area [163, 179, 182, 183]. sensitivity.
Molecular epidemiologic methods are routinely used to A serious challenge to the integrity of poliovirus surveil-
help identify reservoir communities with low population lance data is the occurrence of poliovirus contamination of
13 Enteroviruses: Polio 289

cultures. High workloads in many GPLN laboratories poten- encoding the capsid proteins are unique to polioviruses, as
tially increase the risk of contamination. Fortunately, sequence the flanking sequences are frequently exchanged by recom-
analysis can distinguish contaminants from true clinical iso- bination with the closely related species-C enteroviruses
lates. Contaminants are easily recognized when they are stan- during circulation (Sect. 4.6.2) [24, 166, 188, 205, 206]. The
dard wild reference strains, such as Mahoney, MEF-1, or poliovirion consists of 60 copies each of 4 capsid proteins
Saukett (OPV-like contaminants are usually of little current (VP1–4) that form a highly structured capsid shell [203].
programmatic importance), but are more difficult to recognize The three major proteins (VP1, VP2, VP3) share a similar
when they are the wild polioviruses indigenous to a country or basic architecture and were probably derived from a com-
community. However, when wild polioviruses isolated at dif- mon ancestral protein [203]. The smallest protein, VP4,
ferent times and locations have identical VP1 sequences, con- internalized in the native virion, is formed by the cleavage of
tamination is suspected, because such sequence identities are the precursor VP0 (VP4 + VP2) during final maturation of
inconsistent with the rapid rate of evolution of the poliovirus the virion. The external surface of the poliovirion is deco-
genomes. Contamination can be definitively confirmed (or rated by peptide loops extending from VP1, VP2, and VP3,
ruled out) by complete genomic sequencing. At the advanced which form the neutralizing antigenic sites (Fig. 13.5) [162,
stages of polio eradication, laboratory contamination could 207]. Polioviruses attach to and enter cells via the specific
have severe programmatic consequences if unrecognized, poliovirus receptor (PVR) on the cytoplasmic membrane; the
prompting the diversion of resources into unnecessary immu- PVR was later identified as CD155, a glycoprotein of the
nization campaigns mobilizing large populations and costing immunoglobulin superfamily [208–210]. The key distin-
many millions of dollars. guishing properties of poliovirus capsids are their antigenic
surfaces and their abilities to specifically bind to CD155, as
3.6.4 Tests for Antibody the sequences and structures of the internal capsid domains
Because precise and detailed epidemiologic information is are largely conserved among species-C enteroviruses.
routinely obtained from characterization of poliovirus iso-
lates, virologic methods are the mainstay for global poliovi-
rus surveillance [195]. However, antibody tests, especially 4.2 Physical Properties
those measuring population immunity or vaccine efficacy,
have assumed greater prominence in recent years [92, 95, Poliovirus capsids contain no essential lipids, and infectivity
196–200]. The “gold standard” is the neutralization test, as is insensitive to inactivation by detergents and lipid solvents
the presence of neutralizing antibody is regarded as the key such as ether, chloroform, or alcohol [23]. The viruses are
indicator of protective immunity to poliovirus [7]. Current stable at pH 3–5 for 1–3 h and can therefore pass through the
automation methods permit tests for neutralizing antibody to stomach without inactivation. Exposure to 0.3 % formalde-
be performed at scales previously unattainable. hyde, pH <1, pH >9, or free residual chlorine at 0.3–0.5 ppm
causes rapid inactivation. Infectivity is stable indefinitely at
–20 °C or lower, and stable for weeks at 4 °C, but is rapidly
4 Biological Characteristics inactivated at temperatures above 50 °C [211]. Molar con-
of Poliovirus centrations of MgCl2 significantly increase the thermal sta-
bility of poliovirions [212], both at elevated and ambient
4.1 General Properties temperatures, and MgCl2 is added to many OPV preparations
to preserve potency [7, 213]. High intensity ultraviolet light
Polioviruses, as members of species-C of the Enterovirus or desiccation inactivate infectivity by causing an irrevers-
genus, share most properties with other members of that spe- ible conformational transition from D-antigenicity to
cies and genus (Chap. 11) [201, 202]. Polioviruses are small C-antigenicity (Sect. 4.3) [211, 214]. The three-dimensional
(~30 nm in diameter [203]), non-enveloped viruses with cap- crystal structures of representatives of all three serotypes
sids of icosahedral symmetry enclosing a single-stranded, have been determined [203, 215, 216]. Poliovirions have a
positive-sense RNA genome. The genome is ~7,500 nucleo- buoyant density of 1.34 g/ml and a sedimentation coefficient
tides long, has a small (22-amino acid) basic protein, VPg, of 160S, properties that can be exploited to obtain highly
covalently linked to the 5′-end, and is polyadenylated at the purified virus preparations [217].
3′-end (Fig. 13.5). The single open reading frame (ORF) is
flanked by a long (~740 nucleotides) 5′-untranslated region
(5′-UTR) and a short (~70 nucleotides) 3′-UTR. Complete 4.3 Antigenic Properties
genomic sequences have been determined for numerous rep-
resentatives of each of the three serotypes, including those of There are three poliovirus serotypes [54, 55]. Three (or four)
the three Sabin OPV strains [204]. Only the sequences neutralizing antigenic sites have been identified by patterns
290 O.M. Kew

5´-UTR P1/CAPSID P2/NONCAPSID P3/NONCAPSID 3´-UTR

VP4

3B
VP2 VP3 VP1 2Apro 2B 2C 3A 3Cpro 3Dpol
IRES
AAAn

0 1 2 3 4 5 6 7
kb

Fig. 13.5 Schematic of the poliovirus genome. The single open read- (catalyzes cleavage between VP1 and 2Apro; the cleavage site is indi-
ing frame (ORF) is indicated by a rectangle, flanked by the 5′- and cated by a dashed arrow) and 3Cpro (catalyzes all other cleavages except
3′-untranslated regions (UTRs); the small protein VPg (encoded by the the VP4/VP2 maturation cleavage; the cleavage sites are indicated by
3B sequence interval) is covalently attached to the 5′-UTR and is rep- the solid arrows). Mature cleavage products are bounded by dashed
resented by a circle at the 5′ end. The internal ribosome entry site lines. Protein 3Dpol is an RNA-dependent RNA polymerase (RdRP).
(IRES; nucleotide positions 130–600) in the 5′-UTR is shown as a Colored bars symbolize virion surface loops forming neutralizing anti-
shaded rectangle. A single polyprotein is translated from the ORF, genic sites 1 (red), 2 (green), and 3 (blue) (Redrawn from reference
which is cotranslationally processed by virus-encoded proteases 2Apro Kew et al. [94])

of reactivity with neutralizing murine monoclonal antibod- Within each serotype there are two basic antigenic con-
ies (Fig. 13.5) [162], and the assignments have been con- formations: D-antigen (“dense”; sometimes also called N or
firmed by high-resolution x-ray crystallography [216, 218, “native” antigen) and C-antigen (“coreless”; corresponding
219]. Neutralizing antigenic site 1 is continuous and formed to H or “heated” antigen) [211, 229]. The D-antigen corre-
by a loop in VP1; sites 2 and 3 are discontinuous and formed sponds to that of the intact native virion, and IPV potency is
from loops contributed by different capsid proteins. The measured in D-antigen units [5]. The C-antigen contains no
major type-specific differences in the capsid polypeptides RNA and is not cross-reactive with the D-antigen [211].
primarily reside on the most surface-accessible peptide Transitions between the D and C conformations are rapid in
loops, which represent less than 4 % of the total capsid pro- empty capsids, but D-antigen is stabilized by RNA packag-
tein [204]. Although the neutralizing antigenic sites vary ing [214].
within each serotype [139, 142, 164, 165, 174, 220–223], Poliovirus antigenic properties have been reviewed by
the range of variability is constrained, possibly because of Minor [162].
steric requirements for interaction with CD155 [224, 225],
such that all polioviruses within a serotype can be neutral-
ized by type-specific antisera, and poliovirus vaccines (both 4.4 Host Range In Vivo and In Vitro
IPV and OPV) can induce protective immunity to all known
antigenic variants. Poliovirus antigenic evolution differs Humans are the only reservoir host for poliovirus [230].
importantly from that of influenza virus in that there is no Chimpanzees, gorillas, and orangutans have been infected
cumulative antigenic divergence from ancestral viruses dur- while in captivity [231, 232], and chimpanzees can be experi-
ing person-to-person transmission, and genetically unre- mentally infected by the oral route [233]. Poliomyelitis cases
lated viruses may have similar antigenic properties and appeared in a natural chimpanzee colony following an out-
shared epitopes. break in an upstream African village [234]. Old World mon-
Limited cross-neutralization has been observed for all keys are susceptible to experimental poliovirus infection upon
three poliovirus serotypes [226], and a shared epitope intraspinal or intracerebral injection, and macaques (cynomol-
between types 1 and 2 has been identified by mapping escape gus, rhesus, and bonnet) can be infected by the oral route, but
mutants to cross-reactive neutralizing monoclonal antibody high virus titers are required for infection [235–238]. New
[227]. Recently, chimeric chimpanzee–human monoclonal World monkeys are not susceptible to poliovirus infection by
antibodies have been produced showing patterns of strong any route of administration because of substitutions in the
cross-neutralization [228]. Epitope mapping with these pri- variable domain of their CD155 orthologs [239, 240]. Paralytic
mate monoclonal antibodies have identified shared determi- attack rates in humans differ by poliovirus serotype in the
nants not previously recognized by studies using murine order type 1 > type3 > type 2 (Sect. 3.4) [8, 9, 34, 241].
monoclonal antibodies [228], suggesting that the poliovirus Susceptibility to oral infection is in the order of humans > chim-
antigenic surface may be more complex than previously panzees > macaques, whereas neural susceptibility is in the
thought. order macaques > chimpanzees > humans [242–244].
13 Enteroviruses: Polio 291

Poliovirus variants of all three serotypes have been 4.5 Poliovirus Replication Cycle
selected for growth in mice [245–247] and a type 2 variant
has also been selected for growth in chick embryos [248]. An overview of the poliovirus replication cycle is shown in
Polioviruses normally cannot directly infect cultured mouse Fig. 13.6. Virus attaches to cells through specific interactions
or chick cells, but can replicate efficiently when viral RNA is between the amino-terminal variable domain 1 of CD155 and
introduced by transfection, an observation that led to the a “canyon” that surrounds the fivefold axis of the virion [225,
concept of a specific viral receptor [249]. The CD155 PVR is 253–257]. After endocytosis, viral RNA is uncoated and
a transmembrane glycoprotein with three extracellular released into the cytoplasm [257], VPg is cleaved from 5′-end
immunoglobulin-like domains, encoded by a gene mapped of the RNA, and the RNA is translated. Translation is under
to human chromosome 19 [250]. The normal function of the control of the internal ribosome entry site (IRES;
CD155 is as a receptor for establishment of intercellular Fig. 13.5), an element (nucleotides ~130–600) within the
junctions between epithelial cells, a function that is “mis- 5′-UTR that has a highly conserved stem-loop structure [258,
used” by poliovirus to gain entry into human cells [251]. 259]. The translation product is a single polypeptide, the

parental virion 9

2 viral RNA
progeny virions
VPg
3

pUp

VPg

polyprotein

+ –
4 + – 8
5
+ –
non-strucural proteins RF
structural proteins + –
RI 6

replication complex

Fig. 13.6 Overview of the poliovirus replication cycle: 1 attachment of strands of viral RNA by RdRP in replication intermediates (RI), 7
polio virion to poliovirus receptor (PVR; CD155) on cytoplasmic mem- cleavage of VPg from some + RNA strands for programming as mRNA,
brane, 2 endocytosis and uncoating of RNA, release into cytoplasm, 8 encapsidation of other + RNA strands into virions, and 9 release from
and cleavage of VPg from 5′-end of RNA, 3 translation of viral proteins cytoplasm of infected cell. In cell culture, the entire infectious cycle is
from viral RNA serving as mRNA, 4 proteolytic processing viral pro- complete within ~6 h with release of up to 10,000 infectious virions per
teins, 5 replication of negative (–) strands of viral RNA by poliovirus cell (Reproduced from reference De Jesus [252] with permission from
RNA-dependent RNA polymerase (RdRP), 6 replication of positive (+) BioMed Central)
292 O.M. Kew

polyprotein, which is cleaved by virus-encoded proteinases, evolutionary relationships among poliovirus genotypes are
2Apro and (primarily) 3Cpro, into mature viral proteins [260]. obscured by saturation of variable nucleotide sites [164].
Host protein synthesis is rapidly inhibited by the cleavage by Poliovirus populations in cell culture and in humans [173]
2Apro of the translation initiation factor eIF4G, required for are a spectrum of mutational variants termed “quasispecies”
initiation of translation of capped host messenger RNA but [273, 274]. On average, each genome in a virus population
not for the internal initiation of translation from the poliovirus contains one nucleotide substitution difference from the con-
IRES [259, 261]. One cleavage product is 3Dpol, an RNA- sensus “master sequence” of the quasispecies population.
dependent RNA polymerase (RdRP), that catalyzes the syn- Two important consequences are that the virus populations
thesis of negative-polarity (–) RNA strands from the genomic in the live, attenuated OPV contain preexisting variants of
and mRNA-polarity (+) strands forming a duplex called the higher potential neurovirulence [275], and that antigenic
replicative form (RF) [262, 263]. Multiple copies of positive variants can be rapidly selected in cell culture [218, 276] and
RNA strands are produced from negative-strand templates in in humans [141, 220–223].
replicative intermediates (RI) arrayed in intracellular mem-
brane complexes [262, 263]. VPg is cleaved from some newly 4.6.2 Recombination
synthesized positive RNA strands for programming as mRNA Recombination occurs continuously during poliovirus infec-
and further translation [260]. Other positive strands are tion of cultured cells [271, 277, 278] and individuals [173,
encapsidated during the maturation step in which the VP0 279]. Wild polioviruses undergo frequent recombination
precursor to VP4 and VP2 is cleaved followed by release of with the closely related human species-C enteroviruses dur-
infectious virions from the infected cell. The entire replica- ing circulation [24, 167, 205, 206]. Indeed, the 5′-UTR and
tion cycle takes place within the cytoplasm, and poliovirus P2/P3 noncapsid sequences (Fig. 13.5) of wild polioviruses
can replicate in anucleate cells. Infected cells show cytopathic are drawn from a potentially large and constantly exchang-
effects within 6 h and can release up to 10,000 infectious ing genetic pool that includes the locally circulating human
virus particles upon cell lysis and death. This rapid rate of species-C enteroviruses [167, 206, 280]. Crossovers usually
cellular destruction accounts for the rapid progression of map outside the capsid region when the exchange partners
paralysis when poliovirus infects motor neurons [264]. are different poliovirus or enterovirus serotypes, but cross-
Poliovirus (and picornavirus) replication has recently overs may occur within the capsid region when the partners
been reviewed in depth [27, 202, 259]. are of the same poliovirus serotype [173, 271, 277]. The bio-
logical role of recombination in poliovirus is unclear.
Recombination may facilitate maintenance of replicative fit-
4.6 Poliovirus Genetics ness by countering the accumulation of deleterious muta-
tions [281]. However, natural selection apparently maintains
4.6.1 Rapid Evolution of Poliovirus Genomes wild poliovirus near its fitness optimum, and multiple recom-
Poliovirus is one of the most rapidly evolving viruses known binational variants can co-circulate locally [168]. Children
[147, 164, 171, 172, 265]. Most of the nucleotide substitu- fed tOPV regularly excrete vaccine/vaccine recombinants
tions generate synonymous codons [164], and the basic bio- [279] and most circulating VDPVs (Sect. 10.8) are vaccine/
logical properties of wild polioviruses remain unchanged, non-vaccine recombinants [133, 166–168].
although the Sabin OPV strains can undergo important The genetics of poliovirus and other RNA viruses has
phenotypic changes (Sects. 9.1.4 and 10.8). Estimates of the been comprehensively reviewed [268, 273, 274, 277, 282,
rates of total nucleotide substitution into poliovirus capsid 283].
regions average ~10−2 substitutions per site per year [164–
166, 171–173, 188, 191]. The rates appear to be similar across
the three poliovirus serotypes and for both circulating polio- 5 Descriptive Epidemiology
viruses and polioviruses associated with chronic infections,
and constitute a robust poliovirus molecular clock. Underlying The epidemiology of poliomyelitis remained obscure until
the rapid pace of poliovirus genomic evolution are the high inapparent infections and mild cases were recognized [40,
rates of base misincorporation (in the range of 10−5 to 10−3 per 41]. Unlike smallpox, where every infection of a susceptible
base per replication) by the poliovirus RdRP [266–271]. person is associated with overt and characteristic clinical
These high mutation rates are attributable to the absence of signs [284], the first poliomyelitis outbreaks erupted with no
3′ → 5′ exonuclease proofreading mechanisms for the viral evident source [1, 34]. Five phases in the natural history of
RNA polymerases [267], although other mechanisms may poliomyelitis can be recognized: (1) the endemic phase, (2)
also be involved [272]. This exceptionally rapid rate of the epidemic phase, (3) the vaccine era, (4) the eradication
genomic evolution has facilitated high-resolution molecular era, and (5) the post-eradication era [22, 23]. Most developed
epidemiologic studies (Sects. 3.6.3 and 10.7), even as deeper countries eradicated their indigenous wild polioviruses four
13 Enteroviruses: Polio 293

to five decades ago [8, 9, 33, 34]. Similar progress has now poliovirus circulation (“force of infection”) was high, espe-
been achieved by all but three developing countries cially in areas with high population densities [180]. For
(Fig. 13.3), parts of which remain in the endemic and epi- example, up to the 1990s in Mumbai, India, all three sero-
demic phases because the vaccine era has not yet been fully types of wild poliovirus could be found in the community,
implemented [12]. and children in high-risk urban slums were occasionally
found to be concurrently infected with all three wild poliovi-
rus serotypes [285, 286]. In recent years, some children in
5.1 Endemic, Epidemic, Vaccine Era, low-coverage endemic communities were coinfected with
and Eradication Phases wild poliovirus types 1 and 3.
As with other enteroviruses, poliovirus circulation had a
During the endemic phase, virtually all children were distinct seasonality in temperate zones. Paralytic cases
exposed to wild polioviruses at an early age. Large out- peaked during summer and early autumn and could disap-
breaks were rare because large cohorts of nonimmune sus- pear altogether in winter. Sewage sampling could detect the
ceptible children rarely accumulated. Frequent exposure to presence of virus before and after the appearance of cases,
wild polioviruses maintained population immunity and had but generally not throughout the year in smaller communities
the potentially important additional beneficial effect of in cooler climates [103]. In both temperate zones and tropi-
boosting the immunity of women of childbearing age. cal areas, different serotypes predominated in different years.
Outbreaks were more likely to occur in smaller, more iso- Poliovirus circulation would stop completely in small, rural
lated populations than in large populations that could sup- populations, which would then be subject to outbreaks once
port continuous poliovirus circulation. The endemic phase poliovirus was reintroduced into the community [28].
was inevitably followed by an epidemic phase [1, 8, 9, 23, Very isolated communities had no poliovirus infections
28, 29, 33, 34, 66]. In the United States and Europe, out- for years. For example, age-stratified seroprevalence data
breaks of increasing size and severity occurred for six have shown that Eskimo communities in Canada and the
decades until the mid-1950s and were halted only by the United States experienced sharp outbreaks covering a broad
introduction of IPV [8, 9, 23, 29, 34]. Unfortunately, the age distribution preceded and followed by many years with
vaccine era arrived unequally in the world, starting first with no serologic evidence of poliovirus circulation [85, 287]. A
the most developed countries of Europe, North America, similar pattern of infrequent outbreaks also occurred in iso-
Australia, and New Zealand and progressing to Japan, the lated tropical communities. Following introduction of polio-
Soviet Union and Eastern Europe, and the countries of tem- virus from the mainland, a large outbreak in the Andaman
perate South America [8, 9]. As the vaccine phase pro- and Nicobar Islands in the Bay of Bengal in 1947–1948
gressed in more developed countries, periodic epidemics affected a broad age distribution, with an overall paralytic
appeared in less developed countries [8, 9]. Incomplete vac- attack rate of 10 % and a CFR of 14 % [288].
cine coverage in some developing countries had the per- In the vaccine era, poliovirus type 1 had the widest geo-
verse effect of reducing, but not eliminating, poliovirus graphic distribution, and poliovirus type 2 the most restricted
circulation, potentially increasing the risk of explosive epi- [180]. It is difficult to separate out the effects of immuniza-
demics following the buildup of nonimmune susceptible tion, even at low rates of coverage, from the intrinsic biologi-
persons in the population. The eradication phase has been cal properties of wild polioviruses. For example, type 1 is
permanent in most countries, but continued wild poliovirus most frequently associated with large outbreaks and appears
circulation in a few areas carries ongoing global risks, and to be able to spread over wider geographic areas than type 3
some countries have allowed immunity gaps to widen after [180] and (especially) type 2. Wild poliovirus type 2 was the
eradication of indigenous wild polioviruses and suffered first to be eradicated globally (Fig. 13.3) [13] but was also
outbreaks from imported wild polioviruses [10, 12] the first to disappear regionally. In the United States, for
(Sect. 10.6) or from the emergence and spread of cVDPVs example, wild poliovirus type 2 disappeared long before type
(Sect. 10.8) [133]. The global post-eradication phase 3 and finally type 1. By the mid-1980s, no wild type 2 polio-
(Sect. 11.2) is far more complex than originally envisioned viruses could be found in several large countries, including
and is a key element of the current WHO strategic plan [22]. Brazil and China, where the other two serotypes were still
endemic [170]. Only wild poliovirus type 1 was found in the
Caribbean during the 1970s and 1980s, whereas both types 1
5.2 Geographic Distribution and 3 could be found in the larger island populations of the
Philippines and Indonesia until the mid-1990s [289, 290].
Before the vaccine era, all three poliovirus serotypes had vir- With the introduction of OPV, OPV-like viruses of all
tually a worldwide distribution. Virus circulated continu- three serotypes became ubiquitous in areas of high coverage.
ously in populous tropical areas, and the intensity of wild Unlike wild polioviruses, vaccine-related strains usually do
294 O.M. Kew

not persist. For example, in Cuba, where OPV was adminis- In Brazil, polio was rapidly controlled in the south, but the
tered only twice a year in campaigns, vaccine-related viruses reservoirs persisted in the northeast, with its more tropical
disappeared from the environment within 4 months of the climate as well as poor sanitation [70]. Polio had already
second campaign round [291]. been eradicated from the temperate Southern Cone
(Argentina, Chile, Paraguay, Uruguay) before the launch of
the PAHO Polio Eradication Initiative [18]. In China, polio
5.3 Seasonality persisted in the provinces of the southeast but not in the
coastal northeast [172, 297, 298]. One caveat is that the level
In temperate zones, circulation of polioviruses, like that of of immunization (and OPV efficacy) is usually higher in
all enteroviruses, is seasonal [23]. The summer-fall seasonal- temperate zones than in tropical zones.
ity of poliomyelitis outbreaks was clearly described in the
early reports of epidemics in Europe and the United States
[41, 45]. Seasonality was most pronounced in temperate 5.4 Age and Sex
zones and gradually decreased toward the equator, where
intense poliovirus circulation could occur throughout the In endemic areas, children are chiefly responsible for main-
year [9, 34, 56]. In the tropics, the residual poliomyelitis sea- taining poliovirus circulation. The primary ages of first
sonality was variable and circulation tended to increase dur- infection (as indicated by appearance of cases) was 2 years
ing the rainy season. The typical summer-autumn wild or younger in the pre-vaccine era [9, 23, 28, 29, 66]. Older
poliovirus seasonality peak was offset by 6 months between individuals may have added to poliovirus transmission, but
the northern and southern temperate zones [9, 292]. their contributions were likely to have been relatively small
Poliomyelitis outbreaks have occurred on rare occasions dur- because most would have had prior exposure to poliovirus
ing the winter months [110]. and the effect of reexposure would have been to boost muco-
Poliovirus seasonality is a reflection of the fluctuation in sal immunity and thus limit poliovirus excretion. In both
the number of transmission chains during the year [168, developing and developed countries, the hygiene of children
193]. However, the underlying mechanisms for poliovirus <2 years of age favors enteric virus dissemination, but in the
seasonality are unknown. Seasonal patterns of human asso- pre-vaccine era the likelihood of exposure to wild poliovirus
ciation do not appear to be major factors because the peaks was much higher in settings with poor sanitation [29].
of the poliovirus and rotavirus (another non-enveloped The age distribution of poliomyelitis cases has shifted
enteric virus) seasons are offset by 6 months in the United dramatically to older age groups over the past century [9, 23,
States [34]. One hypothesis for poliovirus seasonality is 28, 29, 66]. During the 1916 epidemic in New York, 80 % of
based on the observation that poliovirus is more stable when cases were among children <5 years of age. By the mid-
relative humidity is above 40 % [293]. In temperate zones, 1950s, peak cases were in children 5–9 years of age and two-
indoor relative humidity is highest in the summer, and in the thirds of deaths were in patients >15 years of age [23]. In
tropics humidity is high during the rainy season and through- developing countries, poliomyelitis remained a disease of
out the year in coastal areas, a pattern closely correlated with younger children, with >75 % of cases <2 years of age and
the observed patterns of poliovirus seasonality. However, >95 % of cases <5 years of age [299, 300].
seasonal patterns of human migration can also facilitate In the endemic phase, which represented all countries
poliovirus dissemination. For example, the spread of wild before the late nineteenth century, many children would be
poliovirus from the tropics to more temperate zones was cor- infected by the then-prevalent wild polioviruses while still
related with the seasonal migration of underimmunized farm protected by maternal antibody, and thus could become
worker families moving with the harvest season. Reaching immune without risk of paralytic disease. As sanitation
underimmunized children in migrant populations remains a improved, the first exposures to poliovirus were delayed to
key eradication strategy in developing countries [22]. later in life, when the risk of severe disease is increased, and
Seasonality has been an important facilitating factor for after protective maternal antibodies had waned. Delayed
the eradication of wild polioviruses in developing countries. infection also resulted in expansion of the pool of nonim-
Mass immunization campaigns in the cooler months, when mune susceptible individuals, increasing the potential for
the transmission chains of poliovirus (and potentially com- explosive outbreaks once wild poliovirus was reintroduced
peting enteroviruses) are at their seasonal low [193], have into the population. Paul had first proposed this process to
been a mainstay of eradication efforts [294–296]. In the explain the sudden appearance of epidemic poliomyelitis,
northern Andean region, polio circulation ceased in cities in first in the most developed parts of Europe and North
the temperate highlands years before it ceased in cities of the America and then elsewhere [1]. Paul’s hypothesis has been
tropical coastlands [164]. In temperate Bolivia, OPV cover- repeatedly confirmed [23, 34], and it predicted an irrevers-
age rates of only 50 % were sufficient to eradicate polio [70]. ible shift from the endemic to the epidemic phase. This had
Sequence data showed that wild poliovirus was imported been the pattern in all countries until poliomyelitis was
into the Bolivian highlands from tropical coastal Peru [170]. finally brought under control through immunization.
13 Enteroviruses: Polio 295

It has been known for decades that males are more sus- 5.6.2 Poliomyelitis in the Vaccine Era
ceptible than females to paralytic poliomyelitis and to more The high-income countries of North America, Western
severe forms of the disease [9, 23, 66, 301]. The reasons for Europe, and the southwest Pacific were quick to adopt wide-
this are unknown, although it has been suggested that greater spread immunization with IPV. The impact was rapid and
physical exertion by boys might be a factor [23]. A majority dramatic. In the United States, for example, the incidence of
of the paralytic cases occurred in males in recent outbreaks paralytic poliomyelitis fell from 13,850 in 1955 to 829 in
in Namibia (89 %) [80], Republic of Congo (68 %) [302], 1961 (Fig. 13.7) [308]. With the availability of OPV in 1961,
and Tajikistan (66 %) [178]. more countries adopted immunization against poliomyelitis,
and the downward trend continued. In the United States,
Canada, Australia, and New Zealand, combined cases fell
5.5 Occurrence in Families and Contact >700-fold from 44,378 in 1951–1955 to 62 in 1968 [8], and
Groups in Europe cases fell >50-fold from 28,359 in 1951–1955 to
529 in 1968 [8]. Dramatic progress was also made in the
Polioviruses, like other enteroviruses, are highly communi- Soviet Union as some republics reported no cases by 1968
cable [23]. In the pre-vaccine era, virus was spread effi- [8]. Poliomyelitis cases in Japan, which did not use IPV and
ciently by young children to other family members, many of introduced OPV in 1961 [309], fell >120-fold from 2414 in
whom had prior exposure and were not susceptible to dis- 1951–1955 to 20 in 1968 [8]. Apart from Singapore, the pic-
ease. In the vaccine era, most cases of contact vaccine-asso- ture for the rest of Asia was not encouraging. Several coun-
ciated paralytic poliomyelitis (VAPP; Sect. 9.1.4) were tries in Latin America (Costa Rica, Uruguay, Chile, and
within the family unit [303, 304]. More recently, concerns Argentina) reduced cases by 4- to 50-fold [8], and Cuba
have focused on extended family units in developing coun- eradicated indigenous polioviruses in 1962 [8, 310], as did
tries, especially among groups that refuse immunization and Jamaica in 1968 [8]. However, many other countries in Latin
who could represent an interconnected, socially defined res- America and the Caribbean made little progress [8]. Only
ervoir of poliovirus transmission within an otherwise ade- Israel in the Middle East made evident progress (>35-fold
quately immunized population. Attention is also being given reduction by 1968), and poliomyelitis in Africa remained
to nomads and other mobile populations who could facilitate virtually uncontrolled [8].
dissemination of poliovirus originating from fixed reservoir The downward trends continued in the United States and
communities [22]. other higher-income countries through the 1970s (Fig. 13.7)
[9]. Circulation of wild polioviruses indigenous to the United
States apparently ceased after the 1970 outbreak along the
5.6 Epidemiologic Patterns of Poliomyelitis Texas-Mexico border [163, 311]. Subsequent sporadic polio-
myelitis cases (and a small outbreak in 1972) were usually
5.6.1 Epidemiologic Patterns in Developed associated with wild type 1 polioviruses imported from
Countries in Temperate Zones Mexico [163]. An outbreak associated with type 1 virus orig-
The shift from endemic to epidemic phase was first observed in inating in Turkey spread to underimmunized religious com-
countries with the highest standards of community sanitation munities in the Netherlands and Canada, and to the Amish
and personal hygiene [23, 28, 29, 33, 34]. As outlined above, community in the United States in 1978–1979 [147], was
the epidemic phase appears to have been the consequence of followed in August 1979 by one last sporadic case associated
delaying poliovirus exposure to later age groups who are prone with wild type 1 poliovirus imported from Mexico [163]. A
to more severe paralytic disease and to the accumulation of similar picture emerged in Europe. Outbreaks associated
nonimmune susceptible populations poised for large outbreaks. with imported wild polioviruses occurred in the Netherlands
As sanitation conditions continued to improve, the poliomyeli- in 1971 (type 1) [312, 313], 1978 (type 1) [163, 312, 313]
tis outbreaks steadily shifted in size and severity and peaked in and 1992 (type 3) [314, 315]; in Sweden in 1977 (type 2)
increasingly older age groups. In some settings the shift was [105]; in Spain in 1983 (type 3) [180]; in Finland in 1984
gradual; in others it was abrupt [23]. (type 3) [110, 316]; and repeatedly in the Balkans and the
The mechanism proposed by Paul [1] for the shift from southern Republics of the Soviet Union (types 1 and 3) [180,
endemicity to epidemicity received further support by the 317]. Also, a large outbreak in Taiwan in 1982 (1,031 cases)
observation that families with the highest socioeconomic from wild type 1 poliovirus (probably imported from
advantages were at highest risk for severe poliomyelitis, Indonesia) highlighted the risk to poliomyelitis-free coun-
whereas less advantaged families living in communities with tries in Asia of reinfection by importation from neighboring
poor sanitation were at reduced risk [23]. Indeed, the last countries [318].
outbreaks in the United States in the epidemic pre-vaccine In many developed countries using OPV, the only cases of
era included parents in more advantaged families whose poliomyelitis each year were from VAPP (Sect. 9.1.4) [24, 319–
children carried wild poliovirus into the home [305]. 323]. However, the risks of importation of wild polioviruses
296 O.M. Kew

Total Paralytic Poliomyelitis IPV Total No. of Paralytic

10000 Poliomyelitis Cases, 1953-2003
VAPP
OPV
110 1000

No. of Cases
100
100
90
No. of Paralytic Poliomyelitis Cases

80 10

70 1
1955 1961 1967 1973 1979 1985 1991 1997 2003
60
Year
50

40

30

20

10

0
1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003
Year

Fig. 13.7 Reported cases of poliomyelitis, United States, 1953–2003. derived poliovirus (VDPV) infections occurred in Minnesota in 2005
Arrows indicate years of introduction of inactivated poliovirus vaccine (type 1) [132] and a separate immunodeficiency-associated VDPV
(IPV; 1955) and oral poliovirus vaccine (OPV; 1961–62). The last wild (iVDPV) case occurred in Minnesota in 2008 (type 2) [223]. Note that
poliovirus (type 1) case occurred in August 1979 [163, 306]. Bars indi- abscissa of inset is a logarithmic scale and abscissa of main graph is a
cate cases of vaccine-associated paralytic poliomyelitis (VAPP). New linear scale (Source: Centers for Disease Control and Prevention
VAPP cases stopped after the shift to IPV in 2000 [307], but vaccine- (CDC). Reproduced from reference Alexander [307])

remained, and the sharp divergence between the poliomyelitis- 6 Mechanisms and Routes
free and poliomyelitis-endemic worlds was unsustainable. of Transmission

5.6.3 Poliomyelitis in Developing Countries As with other enteroviruses, poliovirus is spread by person-
By 1980, many developing countries had made little prog- to-person contact via two routes of transmission, fecal–oral
ress in controlling poliomyelitis in the nearly two decades and respiratory [23, 29]. The relative importance of these
since the widespread availability of OPV [9, 324]. With their two routes varies by setting. Fecal–oral is the more efficient
growing populations, the problem of epidemic poliomyelitis route because fecal shedding continues for up to 6 weeks,
in developing countries, well recognized by the mid-1950s and the quantities of virus shed in stool may be as high as
[292], was increasing [98, 324]. In addition, lameness sur- 300 million infectious particles per day [328]. Under experi-
veys revealed that the incidence of poliomyelitis in Africa mental conditions, vaccine virus was shown to spread to con-
and other developing countries was far higher than originally tacts even when administered in gelatin capsules, thereby
believed [97, 98, 319, 325]. Poliomyelitis cases in much of bypassing throat infection and respiratory transmission
Africa and Asia had been grossly underreported [324], with [329]. In areas of poor sanitation and hygiene, fecal–oral
the actual global incidence being tenfold greater than the transmission (via contaminated fingers, food or water, uten-
officially reported counts. For example, lameness surveys in sils, or toys) is most likely the dominant route, as young chil-
1981–1982 suggested that India had half of the estimated dren are continually exposed to unsanitary conditions and
world total of 400,000 poliomyelitis cases per year [326, live in close proximity to contaminated soil and open sewers.
327]. Throughout the 1980s, the city of Mumbai alone Respiratory transmission probably played a more important
reported ~1,000 cases per year [285, 286], more than 100 role in developed countries with high standards of hygiene
times the rate (from VAPP) for the United States over the and sanitation, and where in the pre-vaccine era exposure to
same time period (Fig. 13.7) [303, 304]. Moreover, large out- poliovirus in higher socioeconomic communities was typi-
breaks, some recurring, occurred in many developing coun- cally delayed by several years. The effectiveness of IPV,
tries as they steadily shifted to the epidemic phase [180]. which blocks oropharyngeal but not intestinal infection
Clearly, remedial action was urgently needed. [330], to stop wild poliovirus transmission is evidence of the
13 Enteroviruses: Polio 297

importance of respiratory transmission in settings where 7 Pathogenesis and Immunity

fecal–oral transmission is less prominent.
There is no evidence for an extrahuman reservoir for 7.1 Pathogenesis
poliovirus, apart from virus stored in laboratory freezers
[331]. Sequence analyses show close genetic relatedness Studies on the pathogenesis of poliomyelitis in humans date
among the polioviruses obtained in the same locales from to the nineteenth century [1]. The availability of a primate
clinical cases, stool surveys, and sewage sampling [111, model for pathogenesis in 1909 accelerated the pace of
113]. Virus imported over short or long distances can consis- pathogenesis research up to the mid-1950s [1, 242, 243].
tently be linked by molecular epidemiologic methods to Interest in the pathogenesis of poliomyelitis waned with the
infections recently occurring in the source communities [10, availability of poliovirus vaccines and the increased focus on
11, 163, 193, 332]. molecular virology, even though pathology studies contin-
On very rare occasions, poliomyelitis infections and ued to address key unanswered questions [337, 338]. Two
outbreaks have started by mechanisms other than direct major models of pathogenesis, one by Bodian [242]
person-to-person transmission. In the spring of 1955, 204 (Fig. 13.8) and the other by Sabin [243], were proposed in
vaccine-associated cases occurred in the United States 1955 and 1956. The principal difference between the two
following injection of children with IPV preparations models is whether the major primary sites of poliovirus rep-
which contained residual infectious wild poliovirus (the lication are in lymphatic tissues (Bodian) or mucosal tissues
Cutter incident) [333, 334]. The majority of case isolates (Sabin) [244]. According to Bodian’s model, orally ingested
were derived from the neurovirulent type 1 Mahoney poliovirus first replicates locally in lymphatic tissues at the
strain used in IPV production [1, 333, 334]. A small num- sites of initial virus implantation (tonsils, intestinal microfold
ber of cases in Uttar Pradesh, India, in 2000 and again in epithelial [M] cells of the Peyer’s patches in the ileum).
2002–2003 were found to be associated with the type 2 Within 1–2 days virus spreads via lymphatic pathways from
reference strain, MEF-1, found in contaminated lots of the small intestine to the mesenteric lymph nodes and from
OPV [335, 336]. the tonsils and adenoids to deep cervical lymph nodes.

SITES OF POLIOMYELITIS VIRAL MULTIPLICATION AND PATHWAYS OF VIRAL SPREAD

SHOWN SEQUENTIALLY IN CHIMPANZEES AFTER VIRUS FEEDING
(OR PARENTERAL INJECTION)

INGESTED VIRUS
1 - ALIMENTARY PATHWAY
OF VIRUS SPREAD

TONSILS PEYER’S PATCHES

VIRUS IN VIRUS IN
THROAT SECRETIONS FECES

2 - LYMPHATIC PATHWAYS DEEP CERVICAL LYMPH NODES MESENTERIC LYMPH NODES PARENTERAL
INJECTION

3 - BLOOD VASCULAR INVASION OF BLOOD STREAM AND SPREAD TO SUSCEPTIBLE

PATHWAY “TARGET ORGANS”

CENTRAL NERVOUS SYSTEM LYMPHATIC STRUCTURES BROWN FAT

4 - NEURAL PATHWAYS (INCLUDING TONSILS AND PEYER’S PATCHES)

NERVE FIBER SPREAD WITHIN CNS AND CENTRIFUGALLY TO SENSORY GANGLIA

Fig. 13.8 David Bodian’s scheme of the pathogenesis of poliovirus (Copyright 1955, American Association for the Advancement of
infection based on studies in monkeys, chimpanzees, and humans. Science (http://www.sciencemag.org).) CNS central nervous system
Reproduced with permission from the original article by Bodian [241].
298 O.M. Kew

By days 1–3, virus appears in the feces and throat. In the next variety of tissues, including tissues that do not support polio-
viremic phase, virus invades the bloodstream and infects virus replication [323]. Therefore, CD155 expression is nec-
other susceptible “target organs,” including further spread to essary but not sufficient for poliovirus replication in vivo,
systemic lymph nodes, brown fat, and occasionally motor and a stage in the replication cycle after initiation of transla-
neurons of the CNS. The first 1–2 days of infection are tion determines tissue tropism [323]. One potential mecha-
asymptomatic. During viremia, all tissues are exposed to nism for the observed patterns of tissue tropism is that a
virus, and about 10 % of infected persons experience “minor barrier of innate immunity in extraneural tissues blocks
illness” at days 3–4, with malaise and fever accompanying poliovirus replication [232]. In support of this view, it was
systemic viral infection. Clinical signs subside after day 4. found that in PVR-transgenic mice deficient in the interferon--
Viremia ends with the appearance of antibody by day 6, and α/β receptor, poliovirus replication occurred not only in the
virus bound to antibody can be detected for a few days lon- CNS but also in extraneural tissues such as the liver, pan-
ger [320]. Incubation periods (time from exposure to disease creas, and small intestine [343, 344].
onset) vary in different individuals, but they are usually from The mechanism of axonal retrograde transport has been
7 to 17 days but range from 2 to 35 days [23]. Virus repli- reexamined in transgenic mice [345]. At the molecular level,
cates in the oropharynx for 1–2 weeks and is shed in the stool the cytoplasmic domain of CD155 was found to specifically
for 3–6 weeks [23, 321]. bind to Tctex-1, a light chain of the dynein motor complex (a
Progression to “major illness,” including nonparalytic driver for retrograde transport), and that the rate of Tctex-1
poliomyelitis (aseptic meningitis) and paralytic poliomyelitis, transport is similar to the rate of poliovirus ascent along
occurs within 8–30 days of exposure [28]. The biphasic nature nerve fibers [232, 346].
of the disease, with minor illness followed by major illness, has New developments in the pathogenesis of poliomyelitis
been termed the “Dromedary” form [322] to imply two humps have been the subject of several recent reviews [26, 232, 244,
(even though the dromedary is a one-humped camel) [1]. 323, 338].
Invasion of the CNS may occur by either penetration of the
blood–brain barrier or by retrograde axonal transport. Paralytic
illness follows directly from the lytic infection of motor neu- 7.2 Immunity
rons, constituting the gray matter of the spinal cord. In fatal
cases, virus replication in motor neurons rises sharply the day Neutralizing antibody protects against paralytic disease [57].
preceding paralysis, peaks to high titers at day 3, and disap- Type-specific immunity from natural infection is lifelong
pears by day 7 [29]. In spinal poliomyelitis, motor neurons in [85], and immunity also appears to be permanent for indi-
the anterior horn of the spinal cord are rapidly destroyed, with viduals who have produced neutralizing antibodies after
the severity of paralysis correlated with the extent of neuronal receipt of OPV or IPV. Infants are protected against disease
destruction [29]. In bulbar poliomyelitis, motor neurons in the during the first few months of life by maternal antibody,
medulla in the brainstem are primarily affected. Lesions may which has a half-life of approximately 28 days and falls to
also occur in the reticular formation in the brainstem and in the undetectable levels after 6 months [347]. In the endemic
thalamus and hypothalamus just above the brainstem. Lesions period, children <6 months of age rarely contracted paralytic
in the brain occur in the roof nuclei of the cerebellum and in the disease, but could induce protective immunity when infected
precentral gyrus of the motor cortex [23, 29]. Other regions of with wild poliovirus [28]. It has long been known that even
the cortex are usually resistant to infection [264]. Some motor low levels of circulating antibodies are protective [57],
neurons may lose function because of edema, but the damage because virus titers during viremia are low [58].
is temporary and motor function is restored once the inflamma- Neutralizing IgM and IgG antibodies appear within a few
tion subsides [23]. Local secretory IgA immunity can block days of exposure to virus. IgM titers decline after 10 days,
poliovirus replication in tonsils and the intestinal tract, and neu- but IgG titers continue to rise for at least 2 months [348].
tralizing IgG and IgM antibodies can prevent virus spread to Mucosal immunity in the gastrointestinal tract and orophar-
motor neurons of the CNS. ynx, in the form of secretory IgA, rises more slowly and per-
Poliovirus pathogenesis has enjoyed a renaissance [26, sists for at least several months, but the duration of mucosal
232, 244, 323, 338] after cloning and identification of the immunity to poliovirus has not been intensively studied [7,
PVR, CD155 [208], and the development of transgenic mice 349]. Children who have had tonsillectomy are more suscep-
expressing CD155 [340–342]. The transgenic mice are sus- tible to poliomyelitis than children with intact oropharyngeal
ceptible to poliovirus infection upon intraspinal, intracere- lymphoid tissue, a finding that points to the importance of
bral, intravenous, intranasal, intraperitoneal, or intramuscular oropharyngeal mucosal immunity in protection from disease
inoculation and exhibit clinical signs and neural lesions typi- [350–352]. OPV induces both serum antibody and intestinal
cal of human poliomyelitis [232]. However, the transgenic immunity, whereas intestinal immunity from IPV is low
mice are usually resistant to infection by the oral route [232, (Sect. 9.1.5) [7, 348].
340, 343]. It has been found in both humans and transgenic Additional evidence for the critical importance of neutral-
mice that CD155 RNA and protein are expressed in a wide izing antibody to immunity is that individuals with primary
13 Enteroviruses: Polio 299

(B-cell) immunodeficiencies are at a 3,000-fold higher risk 8.1.3 Major Illness: Nonparalytic Poliomyelitis
than immunocompetent individuals of contracting VAPP (Aseptic Meningitis)
when exposed to OPV (Sect. 9.1.4) [353], and some may Aseptic meningitis occurs in 1–2 % of infections. It may be
develop prolonged poliovirus infections [354]. Patients with preceded by minor illness and is characterized by signs of
primary immunodeficiencies are treated with intravenous inflammation of the meninges, including severe headache, and
immunoglobulin (IVIG), and preparations used in the United stiffness and pain in the back and neck. Aseptic meningitis
States must meet minimum neutralization titers to poliovirus lasts 2–10 days and is usually followed by complete recovery.
[7]. However, IVIG neutralizing titers become undetectable In rare instances, the disease progresses to paralytic poliomy-
after 3–5 weeks [23], and several immunodeficient individuals elitis. Other viral infections, including those of many nonpolio
with chronic poliovirus infections have contracted poliomyeli- enteroviruses (Chap. 11), may cause aseptic meningitis [23].
tis despite IVIG treatment, presumably after titers of neutral-
izing antibodies fell below protective levels (see Sect. 10.8.2) 8.1.4 Major Illness: Paralytic Poliomyelitis
[186, 223]. Newly developed chimpanzee–human neutralizing Paralytic poliomyelitis occurs in <1 % of wild poliovirus infec-
monoclonal antibodies may hold promise for the availability tions (Sect. 3.4) and is characterized by acute flaccid paralysis
of high-titer preparations for immunotherapy of immunodefi- (AFP) usually accompanied by fever. It may be preceded by
cient patients with prolonged poliovirus infections [228]. minor illness with apparent recovery followed a few days later
by rapid onset of paralysis. Minor illness preceding paralysis
may be absent in adolescents and adults, but pain in the affected
8 Patterns of Host Response extremities during onset of paralysis may be severe. Paralysis
and Diagnosis progresses rapidly and is descending (i.e., moving proximally
to distally), with loss of deep reflexes but no sensory loss.
8.1 Clinical Features Paralysis is usually asymmetric and affects the legs more fre-
quently than the arms, especially in young children. Residual
The patterns of host response were described in Sect. 7.1 in paralysis persists beyond 60 days. The affected muscles and
the context of the pathogenesis of poliomyelitis. Paul recog- severity of paralysis depends on the location and concentration
nized four categories of response to poliovirus infection in of neuronal lesions [29]. Paralytic poliomyelitis is further
susceptible individuals: (1) inapparent (asymptomatic) infec- divided into three categories based on the CNS lesions and the
tions, (2) abortive poliomyelitis, (3) nonparalytic poliomyeli- corresponding affected muscle groups.
tis (aseptic meningitis), and (4) paralytic poliomyelitis [28].
Abortive poliomyelitis is described as minor illness, and non- Spinal Poliomyelitis
paralytic and paralytic poliomyelitis constitute major illness. Spinal poliomyelitis (~79 % of paralytic poliomyelitis cases)
The proportion of each category of host response depends on occurs when lesions are localized to the spinal cord and
the patient age, the poliovirus serotype, and other physiologic cause weakness in the legs, arms, back, or abdominal mus-
factors such as tonsillectomy, pregnancy, recent injections, cles. Spinal poliomyelitis may also paralyze the diaphragm
trauma, or recent strenuous physical exertion [29]. and intercostal muscles, causing respiratory failure.

8.1.1 Inapparent (Asymptomatic) Infections Bulbar Poliomyelitis

Most (up to 95 %) wild poliovirus infections of nonimmune Bulbar poliomyelitis (~2 % of paralytic poliomyelitis cases)
susceptible individuals are without fever or other symptoms. occurs when lesions form in the cranial nerve or medulla and
Infected individuals produce protective neutralizing antibod- results in paralysis of the pharynx, vocal cords, and facial
ies and have permanent resistance to paralysis upon reinfec- nerves. Bulbar poliomyelitis may rapidly progress to respira-
tion by the same poliovirus serotype. Infections that occur tory failure when the respiratory centers of the medulla are
under the cover of maternal antibody are asymptomatic. attacked.
Individuals with inapparent infections shed virus in their
stool and can participate in the spread of infection. Bulbospinal Poliomyelitis
Bulbospinal poliomyelitis (~19 % of paralytic poliomyelitis
8.1.2 Minor Illness: Abortive Poliomyelitis cases) is a combination of the above two forms and is most
Abortive poliomyelitis is the most frequent disease manifes- frequently seen in adults [29].
tation, occurring in about 8 % of poliovirus infections [23,
28]. Symptoms are indistinguishable from those of many 8.1.5 Post-Polio Syndrome
other infections and include fever, malaise, drowsiness, Post-polio syndrome is a recrudescence of weakness, pain,
headache, vomiting, and sore throat. Abortive poliomyelitis fatigue, and atrophy in the muscles originally affected by acute
occurs during the viremic phase a few days after infection. paralytic poliomyelitis [355]. Symptoms progress over a
Complete recovery occurs within a few days, although virus period of years and first appear on average 30–35 years after
shedding may continue for up to 6 weeks postexposure. recovery from the initial acute attack [356]. It is estimated that
300 O.M. Kew

25–50 % of patients with acute paralytic poliomyelitis will 8.2.2 Virologic Testing
develop some signs of post-polio syndrome, the risk being Virologic testing has high sensitivity provided that adequate
highest among survivors with more severe paralysis from the specimens are collected soon after onset of paralysis and
original attack. In the United States, over 440,000 survivors of received by the laboratory in good condition (section
paralytic poliomyelitis were recognized in 1994–1995, but no “Clinical specimens”). Because wild polioviruses can be
updated counts have been obtained. Post-polio syndrome is a accurately identified by molecular methods, and contami-
neurologic condition whose underlying mechanisms are nants ruled out by sequence analysis, specificities approach
unknown. Current evidence indicates that the syndrome results 100 %. Classification of cases of VAPP requires the inclu-
from the denervation of the peripheral neuromuscular junc- sion of clinical, epidemiologic, and laboratory findings [303,
tions that expanded during recovery to compensate for neuro- 304, 307, 359]. In countries using OPV, most isolations of
nal damage from the original infection [355]. It is thought that OPV-like virus from patients with AFP are independent
the neurologic dysfunction progresses subclinically for many events and do not signal the occurrence of VAPP. Laboratory
years until a critical threshold is reached beyond which the detection of genetically divergent vaccine-derived poliovi-
remaining motor neurons cannot maintain the extended num- ruses (VDPVs; Sect. 10.8) indicate either prolonged replica-
ber of neuromuscular junctions [355]. Post-polio syndrome tion in an immunodeficient individual or community
does not appear to result from persistence of the original polio- circulation of VDPVs [133]. The distinguishing genetic
virus infection [357]. Polio eradication is the most effective properties of the more divergent VDPVs, coupled with infor-
way to prevent post-polio syndrome. mation about the rates of poliovirus vaccine coverage in the
source community, are strong predictors of the VDPV cate-
gory [133].
8.2 Diagnosis
8.2.3 Residual Neurologic Deficit
Most clinicians trained since the late 1960s in the United The clinical case definition for paralytic poliomyelitis
States and other developed countries have never seen a case includes residual paralysis at 60 days after onset of paralysis
of poliomyelitis. Similarly, clinicians in most developing [100, 133]. In less severe cases of paralytic poliomyelitis,
countries no longer see poliomyelitis, apart from rare residual paralysis may be difficult to discern because of
sporadic cases of VAPP (Sect. 9.1.4). Nonetheless, the risk functional compensation by intact muscles [7].
of importation of wild poliovirus (or cVDPVs; Sect. 10.8.1)
and the potential for outbreaks remain as long as poliovirus
circulation continues anywhere in the world [10, 12, 80–82, 9 Control and Prevention
132, 175–178, 181]. Diagnosis of paralytic poliomyelitis is of Poliomyelitis
primarily based on three criteria: (1) clinical signs and
course, (2) virologic testing, and (3) residual neurologic defi- 9.1 Poliovirus Vaccines
cit at 60 days after onset of paralysis [7].
Attempts to inactivate poliovirus infectivity through forma-
8.2.1 Clinical Signs and Course lin treatment date to 1911, but when the results from leading
The most obvious sign of poliomyelitis is the appearance of investigators were consistently disappointing, interest in
AFP. However, AFP has multiple etiologies, including immunization against poliomyelitis waned [1]. Interest was
Guillain–Barré syndrome, transverse myelitis, enterovirus briefly rekindled during the field trials of 1935, conducted by
infections (especially EV71; Chap. 11), other viral infec- two groups who took different approaches to experimental
tions, traumatic neuritis, Bell’s palsy, hypokalemia, and poliovirus vaccines, one applying formalin inactivation [360]
toxin exposure [7]. Studies in the Americas comparing clin- and the other using live virus that was claimed to be “attenu-
ical findings with the isolation of wild poliovirus from stool ated” through treatment with detergent [361]. However, the
specimens reported a sensitivity of 75 % and specificity of appearance of poliomyelitis cases—including fatalities—
73 % for poliomyelitis when the diagnostic criteria for AFP linked to the field trials [362] cast a long shadow on further
cases were age <6 years and fever at onset of paralysis [68]. attempts to develop a poliovirus vaccine. The experimental
Inclusion of progression to complete paralysis in <4 days poliovirus vaccines of 1935 were developed before the
increased sensitivity [68], whereas inclusion of specific pat- advent of cell culture, before the identification of three dis-
terns of paralysis (descending, asymmetric, absence of tinct poliovirus serotypes, before the outlines of poliovirus
paralysis in all limbs) increased specificity but reduced sen- pathogenesis were firmly established, and, in the case of one
sitivity [7]. In India, which achieved AFP rates >25 per investigator, a failure to distinguish between chemical inacti-
100,000 children <15 years of age, residual paralysis at 60 vation and attenuation of neurovirulence through genetic
days was most strongly correlated with wild poliovirus selection [361]. Prospects for a poliovirus vaccine again
infection [358]. brightened in 1948 with the successful immunization of
13 Enteroviruses: Polio 301

monkeys with formalin-inactivated poliovirus [363]. could be produced by serial passage of virus in chick embryo
Moreover, the rising incidence of paralytic polio in devel- tissues [367]. Theiler applied this basic approach to develop
oped countries in the postwar years greatly increased the an experimental attenuated variant of the poliovirus type 2
urgency of developing and deploying effective poliovirus Lansing strain in 1946 [368]. In 1952, Enders, Weller, and
vaccines. Robbins developed the attenuated poliovirus type 1
Brunenders strain by serial passage in cultured cells [364].
9.1.1 Inactivated Poliovirus Vaccine (IPV) Two years later, Li and Schaeffer developed a highly attenu-
The IPV of Salk and colleagues was the first poliovirus vac- ated derivative of the neurovirulent type 1 Mahoney strain,
cine to be licensed [4, 5]. Its development followed several LS-c, by passage at 35 ºC in cultured rhesus or cynomolgus
key advances in virology, pathology, and immunology [5]: monkey kidney cells and in monkey skin [369]. The LS-c
(1) the cultivation of poliovirus in nonneural cells [53], (2) strain was further treated by three consecutive single-plaque
the identification of three poliovirus serotypes [54, 55], (3) passages by Albert Sabin to produce the LS-c, 2ab strain,
the finding that viremia precedes paralysis [58], and (4) the generally known as Sabin type 1 (Sabin 1) [370]. Sabin also
demonstration that administration of immune globulin pro- developed attenuated strains for serotypes 2 and 3. The Sabin
tected against paralytic poliomyelitis [57]. IPV is prepared 2 OPV strain (P712, Ch, 2ab) was derived from virus (P712)
by formalin inactivation of three wild, virulent reference that was isolated from a healthy child and shown to have low
strains: Mahoney (type 1), MEF-1 (type 2), and Saukett (type neurovirulence [370]. In contrast, the attenuated Sabin 3
3). A less virulent type 1 strain, Brunenders [364], is used in strain (Leon 12 a1b) was produced by rapid passage in mon-
IPV production in Sweden and Denmark. Although anti- key kidney cell culture of a highly neurovirulent strain iso-
genic sites 1 (Fig. 13.5) of types 2 and 3 are modified by lated from the spinal cord of a child who had died of
formalin inactivation [365], immunization with IPV can bulbospinal poliomyelitis [370]. In addition to Sabin, two
induce high titers of neutralizing antibodies protective other groups, led by Koprowski [329] and Cox [371], devel-
against all poliovirus strains. After exposure of some IPV oped attenuated poliovirus vaccine strains for each serotype.
recipients with live wild poliovirus contained in production All three sets were carefully evaluated for low neuropatho-
lots of incompletely inactivated vaccine (the Cutter incident) genicity for monkeys, immunogenicity, genetic stability on
[333, 334], conditions for IPV manufacture were modified, human passage, safety (inability to cause paralysis in man),
resulting in a reduction in the immunogenicity of IPV prepa- and restricted capacity to spread [372, 373]. The Sabin
rations [23]. However, improvements in cell culture technol- strains, which had the lowest neuropathogenicity and an
ogy in the 1970s led to development of an enhanced-potency excellent safety record from large-scale field trials [6, 60],
IPV (eIPV), similar in immunogenicity to the original prod- were approved for worldwide distribution. In the United
uct [5, 35, 366], which has replaced the second- States, OPV was licensed sequentially (Sabin 1, August,
generation IPV. 1961; Sabin 2, October, 1961; Sabin 3, March, 1962).
IPV was licensed for use in the United States, Canada, Licensure of Sabin 3 was delayed because of concerns about
and Western Europe in 1955 and was the only poliovirus vac- its undesirable genetic instability and relatively low immu-
cine available until licensure of OPV in 1961–1962. IPV use nogenicity [1]. OPV was initially administered serially in
in the United States declined after the introduction of OPV, monovalent form, frequently in mass immunization cam-
but it has been used continuously by some countries in paigns (SOS; “Sabin on Sunday”), but successful tOPV field
Western Europe (Finland, Iceland, Sweden, and the trials in Canada led to licensure of a trivalent formulation in
Netherlands) and some provinces of Canada [5, 35]. In 1997, 1963 [7]. OPV and its application to poliomyelitis eradica-
in response to the eradication of wild polioviruses in the tion has been recently reviewed [7].
Americas and the continuing occurrence of cases of VAPP OPV was developed in the 1950s using the well-
(Sect. 9.1.4), the United States shifted from an all-OPV established empirical approach of rapid passage of virus at
immunization schedule to a sequential IPV/OPV schedule in suboptimal temperatures in cells and tissues of nonhuman
1997, which was replaced in 2000 by an all-IPV schedule origin. It would be another two decades after licensure of
[5, 307]. OPV that the molecular basis of OPV attenuation became
amenable to systematic investigation.
9.1.2 Oral Poliovirus Vaccine (OPV)
Early attempts to produce live-virus vaccines date to the 9.1.3 Genetic Determinants of Attenuation
work of Jenner, who “vaccinated” with cowpox virus to pro- of the Sabin OPV Strains
tect against smallpox in the 1790s, and Pasteur, who devel- Identification of the genetic determinants of attenuation of
oped an “attenuated” rabies vaccine in the 1880s [35]. the Sabin OPV strains has been comprehensively reviewed
Experimental live poliovirus vaccines were tested in mon- [207, 209, 268, 374]. The first reports of the sequences of
keys as early as 1910 [29]. In the 1930s, Theiler demon- complete poliovirus genomes in the early 1980s [375, 376]
strated that an effective attenuated yellow fever vaccine and the development of infectious poliovirus cDNA clones
302 O.M. Kew

5´-UTR P1/CAPSID P2/NONCAPSID P3/NONCAPSID 3´-UTR

VP4
VP2 VP3 VP1 2A 2B 2C 3A 3C 3D AAAn

A1106T
SABIN 1 A4065S L3225M L1134F Y3D073H

A480G G935U U2438A C2879U U6203C

G2795A

SABIN 2 T1143I

G481A C2909U

SABIN 3 S3091F I1006T

C472U C2034U U2493C

0 1 2 3 4 5 6 7
kb

Fig. 13.9 Location of principal attenuating nucleotide (lower bars) protein (4, VP4; 2, VP2; 3, VP3; 1, VP1; 3D, 3D-polymerase) and
and amino acid (upper bars) substitutions in each of the three Sabin numbered consecutively from residue 1 of each protein. For example, a
OPV strains. Abbreviations of nucleotide residues: A adenine, C cyto- guanine (Mahoney) → uracil (Sabin 1) substitution at RNA position
sine, G guanine, U uracil. Abbreviations of amino acid residues: A ala- 935 (G935U) encodes an alanine (Mahoney) → serine (Sabin 1)
nine, C cysteine, F phenylalanine, H histidine, I isoleucine, L leucine, replacement at residue 65 of VP4 (A4065S). The Y3D073H substitu-
M methionine, S serine, T threonine, Y tyrosine. Substitutions are tion in Sabin 1 and S3091F substitution in Sabin 3 are important deter-
shown as nonattenuated parent–position–Sabin strain; nucleotide posi- minants of temperature sensitivity (Figure summarizes findings from
tions are numbered consecutively from residue 1 of the RNA genome; references [379–384] (Redrawn from reference Kew et al. [94])
amino acid positions are indicated by the abbreviated name of the viral

[377] opened the way for systematic investigation of the VP1), and a substitution contributing to the temperature-
critical mutations responsible for the attenuated and sensitive phenotype (but not to the attenuated phenotype)
temperature-sensitive phenotypes of the Sabin OPV strains. mapped to the 3Dpol region encoding the RdRP (Fig. 13.9) [7,
A common feature of the Sabin strains is the presence of 378, 379, 385].
nucleotide substitutions in the IRES, which in serotypes 1
and 3 have been clearly shown to be critical attenuating Sabin 2
mutations. Additional mutations encoding amino acid sub- Only two nucleotide substitutions (G481A in the IRES and
stitutions in the capsid region contribute to and stabilize the C2909U encoding a threonine → isoleucine substitution at
attenuated phenotype. position 143 of VP1) appear to be main determinants of the
attenuated phenotype of Sabin 2 (Fig. 13.9) [381, 382].
Sabin 1 Because P712 has inherently low neurovirulence [370],
The 57 nucleotide substitutions distinguishing the Sabin 1 identification of critical attenuating sites in Sabin 2 involved
strain from its neurovirulent parent, Mahoney, are scattered determination of the effects of introduction of sequences
throughout the genome [144]. Six map to the 5′-UTR, 49 derived from a minimally divergent neurovirulent revertant
map to the coding region (21 of which encode amino acid of Sabin 2 (obtained from a case of VAPP) into infectious
substitutions), and 2 map to the 3′-UTR. Infectious cDNA cDNA constructs derived from Sabin 2 [381, 382]. The neu-
constructs containing different combinations of blocks of rovirulence of Sabin 2 is remarkably stable in routine tests of
Sabin 1 and Mahoney sequences were tested for neuroviru- OPV lots in monkeys, because the G481A substitution (not
lence in monkeys or transgenic mice expressing the CD155 found in the IRES sequences of wild type 2 polioviruses)
receptor, for temperature sensitivity, and for other pheno- does not increase neurovirulence in monkeys, as it does in
typic properties distinguishing the two strains [378, 379]. transgenic mice [386].
The single most important determinant of the attenuated
phenotype of Sabin 1 was the A → G substitution at position Sabin 3
480 (abbreviated A480G) in the IRES [380]. Four other sub- Detailed analysis of the attenuated phenotype of Sabin 3 has
stitutions contributing to the attenuated phenotype mapped been possible because the neurovirulent parental strain, Leon,
to the capsid region (one in VP4, one in VP3, and two in differs from Sabin 3 by only ten nucleotide substitutions
13 Enteroviruses: Polio 303

[387]. In addition, numerous neurovirulent revertants of the 9.1.4 Vaccine-Associated Paralytic

Sabin 3 strain have been isolated from patients with VAPP Poliomyelitis (VAPP)
[383] and from healthy OPV recipients [388]. Only three After over 40 years of use and many billions of doses distrib-
substitutions (C472U in the IRES, C2034U encoding a ser- uted worldwide, OPV has been associated with few adverse
ine → phenylalanine substitution at position 91 of VP3, and events. The most commonly recognized adverse event is
U2493C encoding an isoleucine → threonine substitution at VAPP, which is clinically indistinguishable from poliomyeli-
position 6 of VP1) appear to be the main determinants of the tis caused by wild polioviruses. The first cases of VAPP were
attenuated phenotype (Fig. 13.9) [207, 383, 388]. recognized within a year of licensure of OPV, and most of
the early cases were associated with the Sabin 3 strain [396].
In all three Sabin strains, the attenuated phenotype is The Sabin 3 association was unambiguous because OPV had
determined by multiple substitutions. The substitutions in been delivered in monovalent form [7, 301, 396]. VAPP rates
the IRES, which alter stem-loop structures [207, 389–391] are very low and similar worldwide [7, 359, 397–400]. In the
and reduce the efficiency of initiation of translation of the United States, the risk of VAPP in first-dose OPV recipients
poliovirus RNA template [390, 392], contribute most to the is about 1 case per 1.4 million children immunized [7, 301,
attenuated phenotype of the Sabin 1 and Sabin 3 strains. 303, 304, 307, 401]. In immunologically normal recipients,
Mutations restoring the original stem-loop structure in the the risk of VAPP decreases sharply (>tenfold) for subsequent
IRES (Sabin 1, G480A [back mutation] or U525C [suppres- doses. VAPP cases are sporadic and occur in both OPV
sor]; Sabin 2, A481G; Sabin 3, U472C) are frequently recipients and their unimmunized household and non-
found in vaccine-related isolates from healthy OPV recipi- household contacts. A small proportion (~7 %) of VAPP
ents [389, 393] and patients with VAPP [207] as well as cases in the United States are classified as “community-
from the environment [109]. In Sabin 3, the critical C472U acquired,” indicating no known exposure to OPV. VAPP in
substitution reduces the efficiency of binding of the polypy- OPV recipients and household contacts is most frequently
rimidine tract-binding protein (PTB), required for initiation associated with Sabin 3 (71 % of cases), followed by Sabin 2
of translation, to the IRES [394]. The translational deficit (26 % of cases) [304]. VAPP in non-household contacts and
for Sabin 3 is moderate in intestinal cells, where PTB levels in community-acquired cases is most frequently associated
are high, but severe in neurons, where PTB levels are low. with Sabin 2 (50 % of cases), followed by Sabin 3 (33 % of
The precise mechanisms by which the capsid mutations cases) [304]. Sabin 1 is rarely associated with VAPP in
contribute to the attenuated phenotype are less clear. immunocompetent individuals when administered in tOPV
Impairment of the efficiency of binding to the CD155 recep- [303, 304, 398].
tor [379] and reductions in the stability of the capsid [215] Poliovirus isolates from immunocompetent VAPP case-
may play a role. patients show only limited genetic divergence from the
Sabin and other developers of OPV strains struck a bal- parental OPV strains, although the key substitutions confer-
ance between low neuropathogenicity, good immunogenic- ring the attenuated phenotype have frequently reverted [393,
ity, and acceptable levels of genetic stability [373]. The high 402]. Many isolates from VAPP cases, AFP cases with inci-
genetic stability of the Sabin type 1 strain is probably attrib- dental isolation of vaccine-related virus (much more fre-
utable to the greater number of substitutions contributing to quent than VAPP cases), and healthy OPV recipients are
the attenuated phenotype. This property is especially impor- vaccine/vaccine recombinants [161, 279, 403, 404]. The bio-
tant for the Sabin 1 vaccine strain, because wild type 1 polio- logical and genetic properties of viruses isolated from
viruses typically have high paralytic attack rates and can healthy OPV recipients/contacts are often indistinguishable
spread over wide geographic areas in explosive outbreaks from viruses isolated from patients with VAPP [393, 405].
[10, 170, 175–177, 180, 181]. Sabin 2 may revert more rap- Persons with primary B-cell immunodeficiencies
idly, but its immunogenicity is very high [7, 373] and the (Sect. 10.8.3) [406] should not be given OPV because they
paralytic attack rates of wild type 2 polioviruses are low are at a much higher (~3,000-fold) risk for VAPP [353, 354].
(Sect. 3.4) [34, 101]. Sabin 3 is associated with the highest However, some children have received OPV before their
rates of VAPP (Sect. 9.1.4), which is probably a consequence immunodeficiency was recognized. Immunodeficiency-
of low genetic stability of the critical attenuating substitution associated VAPP (iVAPP) differs markedly from VAPP in
[275], relatively low immunogenicity [395], and an interme- immunocompetent individuals, as it is rarely associated with
diate paralytic attack rate for type 3 polioviruses [34, 101]. Sabin 3 (14 % of cases), and is more frequently associated
Nonetheless, all three Sabin strains normally have very low with Sabin 2 (72 % of cases) and Sabin 1 (31 % of cases)
pathogenic potentials, and incidence of VAPP in countries (some patients were infected with more than one serotype)
with high rates of OPV coverage [304] are several orders of [354]. On the other hand, persons with T-cell immunodefi-
magnitude lower than the incidence of paralytic poliomyeli- ciencies, including those infected with HIV, do not appear to
tis in areas with circulating wild polioviruses. be at elevated risk for VAPP [304, 353, 354, 407].
304 O.M. Kew

Each case of VAPP is an independent event. It is esti- lytic polio fell from 13,850 in 1955 to 829 in 1961 [308].
mated that 250–500 cases of VAPP occur worldwide, most in Although transition to OPV in the United States was not com-
countries free of circulating wild poliovirus (Fig. 13.2a) [7]. plete until the mid-1960s, the last peak year (>100 cases) for
VAPP is a direct clinical consequence of the genetic instabil- polio was 1966 (Fig. 13.7), and all domestic reservoirs for polio-
ity of the Sabin OPV strains, and the most effective means to virus circulation apparently had been eliminated after 1970 [163,
prevent VAPP is to stop OPV use after cessation of wild 413]. Similar highly favorable results were achieved in other
poliovirus circulation [22, 307]. developed countries [8, 9, 309, 414, 415]. IPV was not widely
used in developing countries (and some developed countries,
9.1.5 Sequential Use of IPV and OPV such as Japan [309]), partly because of cost and also because of
The relative merits of IPV and OPV have been compared for the challenges of administering an injectable vaccine to large
many years [23, 408]. Key advantages of IPV are (1) it effi- populations. Thus, the possibility of widespread immunization
ciently induces serum immunity protective against paralytic against polio had to await the availability of OPV, where its key
disease, (2) it is unaffected by interference by other enterovi- advantages were decisive. The synchronous induction of intesti-
ruses or among vaccine components, (3) it can be used in com- nal immunity through mass OPV campaigns efficiently blocks
bination with other injectable vaccines, and (4) it presents no person-to-person transmission of wild poliovirus, thereby pro-
risk for reversion to virulence. The main disadvantages to IPV tecting both individual vaccine recipients and the wider commu-
are (1) it provides much reduced levels of intestinal immunity; nity. Despite its advantages, OPV coverage in most developing
(2) until recently, most preparations had been produced from countries of Asia, Africa, and the Americas remained low, and
virulent wild poliovirus strains; and (3) its costs of production by 1970 a widening divide separated high-income countries
and delivery are higher than OPV [24, 409]. Key advantages where wild poliovirus circulation had stopped from most low-
of OPV are (1) it is easily administered, (2) it confers both income countries where wild poliovirus circulation continued
serum immunity and intestinal immunity (the latter important unabated [8, 9]. By 1985, poliomyelitis had all but disappeared
to limiting poliovirus transmission), (3) it can be easily used in in developed countries, while poliomyelitis crippled a child on
mass campaigns in developing country settings, (4) it is suit- average every 90 s in developing countries (Figs. 13.2 and 13.3).
able for use in outbreak control, and (5) it has low costs of
production and delivery. Disadvantages to OPV include (1) it
presents a continuous, low-level risk of VAPP, (2) it may have 10 The Global Polio Eradication Initiative
low per-dose efficacy rates of seroconversion in some high- (GPEI)
risk settings, (3) it is subject to interference by other enterovi-
ruses and among OPV strains (especially by the type 2 strain), 10.1 Prelude to the GPEI
(4) it has the potential to establish chronic infections in per-
sons with primary immunodeficiencies, and (5) it has the A compelling case for global poliomyelitis eradication has
potential to spread person-to-person and cause poliomyelitis been advanced for decades [294] and receives continued sup-
outbreaks when used at low rates of coverage [23, 94, 133, port from the perspectives of social equity [17, 416], eco-
408, 410]. A sequential IPV/OPV immunization schedule can nomic benefit [417], and technical feasibility [12, 418].
combine the major advantages of each vaccine and mitigate However, the launch of a vertical program to eradicate polio-
some disadvantages. A sequential schedule can virtually elim- myelitis was controversial in the early 1980s [30] and
inate VAPP in OPV recipients and reduce it in OPV contacts, remained so nearly two decades later [419]. Several key fac-
because shedding of OPV-related viruses is reduced. This was tors described below set the stage for the 1988 WHA resolu-
observed in the United States when it shifted to a sequential tion establishing the GPEI [30].
IPV/OPV schedule between 1997 and 2000, as no cases of
VAPP were observed in persons using the sequential IPV/ 10.1.1 Poliomyelitis Eradication in Cuba, 1962
OPV schedule (Fig. 13.7) [307]. Mucosal immunity induced Sabin advocated mass OPV campaigns as the most effective
by an IPV/OPV schedule is much better than with IPV alone means of polio control [294]. Cuba adopted his approach in
[411]. However, global implementation of any schedule using 1962 and within a year had stopped all wild poliovirus circu-
IPV will require much higher rates of routine immunization lation [295, 310], becoming the first country to eradicate
coverage than currently exist (Sect. 11.2.1) [22, 412]. poliomyelitis. The experience in Cuba conclusively demon-
strated that mass campaigns were effective in a tropical
developing country setting.
9.2 Impact of Immunization
10.1.2 Smallpox Eradication, 1977
As described in Sect. 5.6.2, the high-income countries of North Smallpox was the first infectious disease to be eradicated.
America, Western Europe, and the southwest Pacific were quick Global smallpox eradication was launched in 1966 following
to adopt widespread immunization with IPV, with dramatic a WHA resolution to eradicate the disease by 1976. The last
impact. In the United States, for example, the incidence of para- natural case of smallpox was reported in Somalia in 1977,
13 Enteroviruses: Polio 305

and global eradication was certified in 1980 [284]. In addition workers, communications, regular engagement of national
to the complete disappearance of a universally feared viral leaders, infrastructure development, support for surveillance
disease, the successful smallpox program accrued many other and the GPLN, and support for research (http://www.endpo-
public health benefits. The program forged strong interna- lio.org/) [17, 30, 424].
tional cooperation in public health, trained a generation of
international public health professionals who would lead the
way to polio eradication, and set an irreversible precedent that 10.2 The 1988 WHA Declaration to Eradicate
infectious diseases could be controlled on a global scale [30]. Polio Worldwide
In light of the smallpox eradication initiative, the Expanded
Program on Immunization (EPI) was established by the WHO In view of the rapid progress attained in the Americas, the
in 1974 to make immunization against six diseases (diphthe- WHA resolved in 1988 to eradicate polio worldwide by the
ria, pertussis, tetanus, poliomyelitis, measles, and tuberculo- year 2000 [15], launching the WHO GPEI. At the time of the
sis) available to every child in the world by 1990 [420]. The World Health Assembly resolution, wild polioviruses were
core EPI strategy was high routine immunization coverage, circulating unabated in much of the developing world
which was to prove insufficient to control poliomyelitis in (Figs. 13.2 and 13.3). The Western Pacific Region set a goal
some developing country settings. of eradication of indigenous wild polioviruses by 1995, a
goal reached in 1997 and certified in 2000 [425]. China, after
10.1.3 National Immunization Days (NIDs) years of outbreaks [426], adopted the strategy of NIDs and
in Brazil, 1980 Subnational Immunization Days (SNIDs) which, when cou-
Brazil began routine immunization with OPV in the early pled with strengthened routine immunization, stopped wild
1960s [296]. The impact of immunization was initially unclear poliovirus transmission by early 1994 [297, 427]. Other
because poliomyelitis was not a notifiable disease until 1968. developing countries soon followed.
Although mass campaigns in 1971–1973 reduced poliomyeli-
tis cases, they were replaced by routine immunization in 1974.
Cases sharply increased until 1980, when Brazil reestablished 10.3 Biological Principles of Poliovirus
mass campaigns in the form of biannual NIDs, targeting all Eradication
children <5 years of age (regardless of prior immunizations)
for immunization with OPV in a single day. Poliovirus circula- The key biological requirements for poliovirus eradication are
tion dropped sharply in Brazil after implementation of NIDs the following: (1) the absence of a persistent carrier state, (2)
and set Brazil on the path to polio eradication [18, 296]. virus is spread by person-to-person transmission, (3) immuni-
zation interrupts virus transmission, (4) the absence of any non-
10.1.4 The PAHO Regional Polio Eradication human reservoir hosts for the virus, and (5) finite virus survival
Initiative, 1985 time in the environment [230]. An additional important nonbi-
The dramatic success of mass campaigns in Cuba, Brazil, ological requirement for any disease eradication effort is politi-
and Mexico led PAHO to resolve in 1985 to eradicate polio cal will, arising from the perceived benefits of eradication, and
from the Americas by 1990 [14]. PAHO modeled their expressed internationally through resolutions passed by the
regional initiative on Brazil’s successful strategy of NIDs WHA. Essential to the success of the GPEI has been the strong
and used poliomyelitis eradication as the vanguard of efforts alliance among the four “spearheading” partners, WHO (http://
to revitalize childhood immunization in the Americas. The www.polioeradication.org/), UNICEF (http://www.unicef.org/
PAHO approach of coordinated NIDs to supplement health/index.html), Rotary International (http://www.endpolio.
improved rates of routine OPV coverage eradicated indige- org/), and the United States Centers for Disease Control and
nous wild polioviruses within 6 years [18]. PAHO set the Prevention (CDC) (http://www.cdc.gov/polio/), as well as
standard for global efforts and built an infrastructure for the strong support from national governments and new partners,
subsequent elimination of indigenous measles and rubella including the Gates Foundation (http://www.gatesfoundation.
viruses from the Americas [421–423]. org/What-We-Do/Global-Development/Polio) and the UN
Foundation (http://www.unfoundation.org/news-and-media/
10.1.5 Rotary PolioPlus, 1985 press-releases/2013/fight-against-polio.html).
Rotary International launched their global PolioPlus cam-
paign in 1985, with the goal of helping eradicate poliomyeli-
tis worldwide by 2005, the centennial of Rotary’s founding. 10.4 Basic Strategy for Global Polio
In their initial drives for support of PolioPlus, Rotarians Eradication
more than doubled their target goal of $120 million for OPV
and have raised more than $1.2 billion since 1985. Rotarians The four pillars of the GPEI strategy are (1) high routine
have been active in all phases of polio eradication, including immunization coverage of infants with OPV, (2) supplemen-
active participation in mass campaigns, training of health tary OPV immunization activities in the form of NIDs and
306 O.M. Kew

SNIDs, (3) targeted door-to-door “mop-up” OPV immuniza- and SNIDs. For example, Colombia and Peru were reservoirs
tion in areas of focal transmission, and (4) sensitive surveil- for all three wild poliovirus serotypes in the early 1980s.
lance for poliovirus [102]. Very high rates of routine OPV Active cross-border migration of high-risk populations among
immunization are required to block poliovirus circulation in the northern Andean countries facilitated wide dissemination
areas where the risk factors converge, conditions under of wild poliovirus [164, 170] and required close synchroniza-
which routine OPV coverage rates exceeding 90 % may be tion of NIDs among the affected countries [18]. A guerilla
insufficient to block poliovirus circulation [7, 180, 296, 428]. movement in the interior of Peru targeting health clinics and
Such rates are currently unattainable through routine immu- health personnel required active engagement by public health
nization in the least developed countries. Supplementary leaders to ensure access to unimmunized children. The final
immunization is the mainstay of polio eradication in devel- chains of wild poliovirus transmission in the Americas were
oping countries and has been instrumental in raising popula- broken in the interior of Peru in 1991 [18]. In Sri Lanka, major
tion immunity rates above the thresholds required to block military campaigns in a devastating civil war were suspended
poliovirus transmission [429]. Supplementary immunization during the NIDs, and wild poliovirus circulation stopped after
strategies are driven by poliovirus surveillance, which is 1993. In the 1990s, Vietnam and Cambodia were still recover-
used to guide the intensified SNIDs and mop-up campaigns ing from decades of conflict. Detailed aerial mapping of the
to the reservoir communities where the chains of poliovirus migratory at-risk populations in the water courses linking
transmission continue to survive and propagate. Cambodia and Vietnam (with active cross-border poliovirus
transmission) set the stage for house-to-house and boat-to-
10.4.1 AFP and Poliovirus Surveillance boat immunization campaigns that stopped wild poliovirus
Surveillance for circulating polioviruses has two arms: (1) AFP transmission in 1997 [290, 435]. The Democratic Republic of
case investigations and (2) virologic studies of polioviruses the Congo and Angola have experienced shattering civil con-
obtained from clinical specimens or the environment. Even flicts over the past two decades. Nonetheless, both countries
though most wild poliovirus infections are inapparent, over eradicated their indigenous wild polioviruses in 2000 and
time, all effectively performing AFP surveillance systems can 2002, respectively [11]. Both countries appear to have eradi-
detect endemic poliovirus circulation. In suspected high-risk cated polio twice, the second time was a widely disseminated
areas lacking effective AFP surveillance, supplementary sur- wild type 1 poliovirus imported from India around 2002 [177].
veillance activities, such as sampling community contacts of Similarly, Somalia eradicated the indigenous wild poliovi-
AFP cases, stool surveys of healthy children, or environmental ruses in 2002 [436], and again eradicated wild poliovirus type
sampling, have been implemented to increase sensitivity for 1 imported from Nigeria in 2005 [177, 437].
detecting wild polioviruses [104, 106, 107, 111, 113, 116, 315].
10.5.2 Polio Eradication in Very High-Risk
Settings
10.5 Increasing Momentum Toward Successful control of poliomyelitis in settings of intrinsically
Eradication, 1988–2001 high biological risk demonstrated the feasibility of global
polio eradication. However, conditions varied widely, and cre-
The global incidence of poliomyelitis was on a virtually ative local solutions repeatedly had to be found and imple-
unbroken downward trend between 1987 and 2001 (Figs. 13.2 mented [434]. The critical test case for the PAHO polio
and 13.3). Three WHO regions reported their last indigenous eradication initiative was Brazil, the most populous country in
cases before 2000: the Americas (last indigenous case: Peru, Latin America. Control of wild poliovirus transmission was
1991) [430], the Western Pacific Region (last indigenous case: more difficult in the tropical northeast (with its lower levels of
Cambodia, 1997) [425], and the European Region (last indig- hygiene and sanitation), and migration from the endemic
enous case: Turkey, 1998) (Fig. 13.3) [431]. Wild poliovirus northeast to the rapidly growing megacities of Rio de Janeiro
type 2 was last detected in October 1999 in Uttar Pradesh, and São Paulo presented the continuous risk of reinfection
India [13]. By 2001, the tide of global polio eradication, with wild polioviruses. Nonetheless, steady pressure from
although delayed in some places, seemed unstoppable [432]. NIDs, SNIDs, and mop-up campaigns stopped all wild polio-
virus transmission by early 1989 [75]. Success in the Americas
10.5.1 Polio Eradication in Conflict Countries did not necessarily presage success elsewhere because the
The sense of momentum was reinforced by control of polio- Americas were often seen as having several key advantages:
myelitis in countries with armed conflict such as Colombia, (1) greater resources and infrastructure than many developing
Peru, the Philippines, Sri Lanka, Cambodia, Angola, the countries elsewhere, (2) a tradition of strong public health
Democratic Republic of the Congo, and Somalia [433, 434]. leadership in many countries, (3) geographic isolation protec-
In many countries, conflict was suspended during “Days of tive against re-importation of wild polioviruses, and (4) a cul-
Tranquility” allowing children to be immunized during NIDs tural unity conducive to a common approach.
13 Enteroviruses: Polio 307

China, comprising 22 % of the world population, pre- (Figs. 13.10 and 13.11). Diversity within the surviving geno-
sented challenges of scale not encountered in the Americas types continues to decline as genetic clusters steadily disap-
[426]. Climate varied widely from the temperate north to the pear (Sect. 10.7), and wild poliovirus type 3 may be very
tropical south, and levels of infrastructure and sanitation also near eradication in both Asia and Africa.
varied widely in this rapidly developing country. Population
size and density across wide areas favored efficient wild
poliovirus transmission, and massive economic migration 10.6 Resurgence of Wild Polioviruses,
from the countryside into the increasingly prosperous cities 2002–2011
presented additional challenges of reaching unimmunized
children. However, China had already established good cov- After years of steady decline, poliomyelitis cases increased
erage through routine immunization with tOPV, and a series sharply from 483 cases in 2001 to 1918 cases in 2002
NIDs and SNIDs in 1992–1993 broke the remaining chains (Fig. 13.2) [436]. This increase was the result of a large epi-
of indigenous wild poliovirus transmission by early 1994 demic in India (1,600 cases), primarily associated with wild
[297, 427]. poliovirus type 1, centered in Uttar Pradesh (78 % of total
Other successes in high-risk settings in Asia and Africa cases) [409], and a rise in reported cases in Nigeria [440].
followed. Indonesia and the Philippines have humid tropical Global cases fell to 784 in 2003, as the epidemic in India was
climates and very high population densities in their most controlled by large-scale NIDs and SNIDs [441], but wild
populous islands. A combination of strengthened routine poliovirus type 1 was beginning to spread from northern
immunization and NIDs at high rates of coverage stopped Nigeria to neighboring countries [442]. The upsurge in
wild poliovirus types 1 and 3 transmission in both countries Nigeria followed suspension of NIDs and SNIDs in several
by 1995 [289, 290]. Bangladesh has one of the highest popu- northern states in 2003 and 2004 in response to false rumors
lation densities in the world, and wild poliovirus was deeply about OPV safety [443]. By 2006, wild poliovirus type 1 had
entrenched in multiple endemic reservoirs with poor infra- spread from northern Nigeria to 18 countries, across a wide
structure. High-quality NIDs built upon a solid national pro- importation belt in Africa from Guinea in the west to Somalia
gram of routine immunization stopped wild poliovirus in the Horn of Africa, and into Asia from Saudi Arabia and
transmission in 2000 [425]. Neighboring India has more Yemen in the west to Indonesia at the southeastern rim,
children <5 years of age than any other country. Despite the sparking large outbreaks (>100 cases) in Sudan, Somalia,
biological challenges of high population densities, tropical Yemen, and Indonesia [10, 176]. Wild poliovirus type 1
conditions, and poor infrastructure in many areas, the south- introduced into Angola from Uttar Pradesh caused cases that
ern states stopped indigenous wild poliovirus transmission were first reported in 2005 [176], and Nepal experienced
through a combination of high rates of routine immunization repeated importations from northern India [176]. The major-
coverage and mass campaigns. Progress was much slower in ity of cases in 2005 were associated with imported virus, but
the large and populous states of Uttar Pradesh and Bihar most of the outbreaks outside of the core endemic reservoirs
(with a combined monthly birth cohort >500,000), but all of Nigeria, India, Pakistan, and Afghanistan were largely
wild poliovirus transmission stopped in India in 2011 [12, controlled by the end of 2005 (Fig. 13.2d, e) [176], except
91]. Eradication was especially challenging in these two for Kenya and Uganda where virus originating from Nigeria
Indian states because the per-dose efficacy of OPV was low, persisted until 2011 [10]. By 2006, most wild type 1 poliovi-
as many children with poliomyelitis had received multiple rus cases occurred in the core reservoir countries, but virus of
OPV doses. Mass campaigns (NIDs, SNIDs, and large-scale Nigerian origin continued to circulate in Niger, Ethiopia,
mop-ups) overcame weaknesses in routine immunization Yemen, and Kenya [444], and virus of Indian origin spread to
[438]. The most populous parts of Afghanistan (northern Bangladesh and from Angola to Namibia [80] and the
provinces) [184], Pakistan (Punjab province) [184], and Democratic Republic of Congo [444]. Instrumental in rolling
Nigeria (southern states) [185] stopped wild poliovirus cir- back the wave of imported wild poliovirus type 1 was the
culation several years ago, only to be subject to reinfection widespread use in NIDs and SNIDs of monovalent type 1
primarily from domestic endemic reservoirs. OPV (mOPV1), which has a higher type-specific per-dose
efficacy than tOPV because interference from the robust type
10.5.3 Genotypic Indicators of Progress 2 component of OPV is eliminated [196, 410]. However,
Progress in polio eradication has been accompanied by a exclusive use of mOPV1 in successive campaigns led to the
steady reduction in the genetic diversity of circulating wild development of growing immunity gaps to poliovirus types 2
polioviruses. Of the 20 wild poliovirus type 1 genotypes, 5 and 3.
wild poliovirus type 2 genotypes, and 17 wild poliovirus In India, as wild poliovirus type 1 cases declined 87 %
type 3 genotypes found in 1988 [439], only 4 (or fewer) wild from 646 in 2006 to 87 in 2007, wild poliovirus type 3 cases
poliovirus genotypes remain (two type 1 and two type 3) surged from 28 in 2006 to 787 in 2007 [445]. Widespread
308 O.M. Kew

TYPE 1 2008 and 2009 [10, 177]. During 2007–2011, wild poliovi-
Sudan/09 China/94 Pakistan/12
Nigeria/01
Nigeria/12
rus type 3 from Nigeria spread east to Chad, Cameroon, and
Angola/09
India/11
the Central African Republic and north and west to Niger,
Zambia/02
Mali, Côte d’Ivoire, and Guinea [10, 177].
Brazil/88
A second wave of wild poliovirus type 1 from Nigeria
Philippines/93
began in 2008 and spread to 14 countries, reinfecting many
Guatemala/87 of the same countries that experienced the 2003–2006 out-
Indonesia/95
breaks [177]. Wild poliovirus type 1 imported into Chad
China/93
from northeastern Nigeria in 2010 caused a large outbreak
South Africa/89 that continued into 2012, resulting in reestablished transmis-
Sabin 1
sion in Chad and further export of wild poliovirus type 1 into
Peru/91 Myanmar/96 the Central African Republic [10]. However, effective NIDs
Iraq/00 Vietnam/92 and SNIDs in the countries of West Africa again rolled back
Ethiopia/01 Tunisia/85
Somalia/00 the imported Nigerian virus [10, 16].
Egypt/04
While wild poliovirus type 1 was disappearing from
TYPE 2 Vietnam/89 India, virus originating from Uttar Pradesh was associated in
India/99 2010 with a large outbreak (458 cases) in Tajikistan [178],
with further spread to Turkmenistan, Kazakhstan, the
Russian Federation, and probably Uzbekistan [181]. During
2010–2011, wild poliovirus type 1, originally imported sev-
eral years earlier into Angola from Uttar Pradesh, sparked a
Côte d’Ivoirie/94 large outbreak (~441 cases) in the Republic of the Congo
with a very high CFR (Sect. 3.1) [81]. These outbreaks were
controlled by aggressive mass immunization campaigns, and
all wild polioviruses of Indian origin appear to have been
eradicated worldwide.
Peru/89 Wild poliovirus type 1 circulation continued at much
Sabin 2
reduced levels in Pakistan and Afghanistan [184]. However,
TYPE 3
in 2011 wild poliovirus type 1 from southern Pakistan was
DR Congo/09 Mexico/90
Pakistan/12 imported into Xinjiang in western China, causing an out-
India/10
Nigeria/12 Sabin 3 break of 21 cases [10, 82]. Sensitive surveillance in China
detected the outbreak in its early stages, and a series of inten-
Turkey/90
sive mass immunization campaigns halted poliovirus circu-
Sudan/04 Vietnam/93 lation. Although China and the Western Pacific Region are
again free of wild poliovirus circulation, the risk remains of
Somalia/02
Philippines/93 re-importation from neighboring Pakistan even as poliomy-
Egypt/00
Colombia/88 elitis is in decline in that country.
Syria/91
Indonesia/95
Angola/00
Brazil/88
10.7 Wild Poliovirus in Retreat, 2011–2012
Turkmenistan/90 China/93
0.1
A major milestone in global polio eradication was the eradi-
Fig. 13.10 Radial neighbor-joining trees of VP1 sequence relation- cation of indigenous wild poliovirus in India, once the
ships of representatives of wild poliovirus genotypes detected since the
launch of polio eradication activities in the Americas in 1985. Genotypes world’s most intense wild poliovirus reservoir [326, 327] and
believed to be extinct are represented by stop signs at the branch tips. the source of repeated dissemination of wild poliovirus into
Sequences representing the extinct genotypes usually are from the last countries in four continents [11, 170, 176, 177, 179, 315,
known isolate of that genotype (Source: WHO Global Polio Laboratory 415, 446]. The last wild poliovirus type 3 in India was iso-
Network. Modified from reference Chumakov and Kew [439])
lated in October 2010 and the last wild poliovirus type 1 was
isolated in January 2011 (Figs. 13.10 and 13.11) [12, 91]. In
circulation of wild poliovirus type 3 continued in Uttar neighboring Pakistan and Afghanistan, which constitute a
Pradesh and Bihar through 2009 [438]. Wild poliovirus type common epidemiologic block, the diversity of wild poliovi-
3 imported from Uttar Pradesh into Angola caused 24 cases rus types 1 and 3 has been in steady decline, despite fluctua-
in 2008 and spread to the Democratic Republic of Congo in tions in case counts (Fig. 13.12) [12].
13 Enteroviruses: Polio 309

TYPE 1

TYPE 2

TYPE 3

Fig. 13.11 Progressive eradication of wild poliovirus genotypes, 1986 cate, respectively, countries with reestablished transmission and spo-
to 2012. Stop signs indicate the year and location where the last isolate radic importation of West Africa genotype viruses (Source: WHO
was obtained for each extinct genotype. Surviving type 1 and type 3 Global Polio Laboratory Network. Modified from reference Kew and
poliovirus genotypes are color-coded; hatch and shading patterns indi- Pallansch [11])
310 O.M. Kew

Fig. 13.12 Spot maps of genetic WPV1 WPV3

clusters based upon VP1 sequence
relationships among isolates of
wild poliovirus type 1 (WPV1 left 2010
column) and wild poliovirus type
3 (WPV3 right column), endemic
to Pakistan and Afghanistan,
2010–2012. Isolates within a
genetic cluster share ≥95 % VP1
nucleotide sequence identity, and
the cluster count provides an
indication of the genetic diversity
of circulating wild polioviruses.
Genetic clusters are color-coded
to facilitate visualization of
endemic reservoirs, transmission Cases = 142 Cases = 32
pathways (including cross-border Clusters = 15 Clusters = 4
transmission), and decline in
genetic diversity. In 2011,
poliovirus type 1 from southern
Pakistan was imported into
Xinjiang, China, and caused an 2011
outbreak of 21 cases [82]. (Map
prepared by Elizabeth Henderson
[CDC] based on sequence data
produced by the Regional Polio
Reference Laboratory at the
National Institute of Health,
Islamabad, Pakistan, and by
CDC). As of April 2014, the last
wild poliovirus type 3 isolate in
Asia was from a child in the
Federally Administered Tribal
Area of Pakistan who had onset of Cases = 276 Cases = 2
AFP in April 2012 Clusters = 8 Clusters = 1

2012

Cases = 92 Cases = 3
Clusters = 4 Clusters = 1

One measure of genetic diversity is the number of genetic disappearance of genotypes is a measure of global progress
“clusters,” defined as isolates sharing ≥95 % VP1 nucleotide toward eradication (Figs. 13.10 and 13.11), the stepwise disap-
sequence identity (isolates within a genotype share ≥85 % VP1 pearance of clusters is a measure of regional and local progress
nucleotide sequence identity [163]). Distinct clusters originate (Figs. 13.12, 13.13, and 13.14). Poliovirus genetic clusters are
from different local endemic reservoirs, and in areas of sensi- color-coded and mapped to facilitate visualization of patterns
tive surveillance, the disappearance of clusters within a geno- of circulation and to monitor progress toward eradication.
type is an indication that source reservoirs within geographic The number of wild poliovirus type 1 clusters in Pakistan
area are being cleared of wild polioviruses. Just as the stepwise and Afghanistan fell from 15 in 2010 to 4 in 2012 (Fig. 13.12).
13 Enteroviruses: Polio 311

Fig. 13.13 Spot maps showing 2010 Cases = 46 Clusters = 10

spread and retreat of genetic clusters
of wild poliovirus type 1 from
Nigerian reservoirs to neighboring
countries in West and Central
20° N
Africa, 2010–2012 (Map prepared
by Elizabeth Henderson (CDC)
based on sequence data produced by
the Regional Polio Reference
Laboratory at the National Institute
for Communicable Diseases,
Johannesburg, South Africa, and
CDC). As of April 2014, the last
wild poliovirus type 3 isolate in 10° N
Asia was from a child in the
Federally Administered Tribal Area
of Pakistan who had onset of AFP
in April 2012

2011 Cases = 178 Clusters = 8

20° N

10° N

2012 Cases = 100 Clusters = 6

20° N

10° N

Wild poliovirus type 3 clusters decreased from 4 in 2010 to within Afghanistan appear to have been cleared of wild
1 in 2012 (Fig. 13.12), and the last case associated with wild polioviruses.
poliovirus type 3 in Pakistan (and in Asia; Fig. 13.2c) was Although progress in Nigeria has been less dramatic than
reported in April 2012 [12]. By the end of 2012, reservoirs in Pakistan and Afghanistan, wild poliovirus type 1 clusters
312 O.M. Kew

Fig. 13.14 Spot maps showing 2010 Cases = 31 Clusters = 9

spread and retreat of genetic
clusters of wild poliovirus type 3
from Nigerian reservoirs to
neighboring countries in West and 20° N
Central Africa, 2010–2012 (Map
prepared by Elizabeth Henderson
based on sequence data produced
by the Regional Polio Reference
Laboratory at the National Institute
for Communicable Diseases,
Johannesburg, South Africa, and
CDC). As of April 2014, the last
wild poliovirus type 3 isolate in the 10° N
world was from a child in
northeastern Nigeria who had
onset of AFP in November 2012

2011 Cases = 66 Clusters = 6

20° N

10° N

2012 Cases = 20 Clusters = 2

20° N

10° N
13 Enteroviruses: Polio 313

Table 13.2 AFP and wild poliovirus cases by WHO region and globally, 2001–2012
AFP cases (virologically confirmed wild poliovirus cases) reporteda
WHO regionb
Year AFR AMR EMR EUR SEAR WPR Global
2001 8,542 (69) 2,207 (0) 3,865 (143) 1,764 (3)c 10,612 (268) 6,529 (0) 33,519 (483)
2002 8,587 (208) 2,168 (0) 4,625 (110) 1,717 (0) 12,900 (1,600) 6,835 (0) 36,832 (1,918)
2003 8,181 (446) 2,229 (0) 5,290 (113) 1,529 (0) 11,289 (225) 6,397 (0) 34,915 (784)
2004 9,719 (934) 2,309 (0) 6,176 (187) 1,516 (0) 16,270 (134) 6,521 (0) 42,511 (1,255)
2005 11,683 (879) 2,213 (0) 8,849 (727) 1,479 (0) 31,530 (373) 6,680 (0) 62,434 (1,979)
2006 12,472 (1,189) 2,151 (0) 8,739 (107) 1,481 (0) 36,665 (701) 7,011 (0) 68,519 (1,997)
2007 12,080 (367) 2,111 (0) 9,394 (58) 1,449 (0) 46,124 (894) 6,237 (0) 77,395 (1,315)
2008 14,256 (912) 2,063 (0) 10,799 (174) 1,360 (0) 50,509 (565) 6,417 (0) 85,404 (1,651)
2009 15,127 (691) 1,873 (0) 10,611 (176) 1,363 (0) 54,962 (741) 6,291 (0) 90,227 (1,604)
2010 16,500 (657) 2,006 (0) 11,338 (169) 2,087 (478)d 60,456 (48) 6,401 (0) 98,788 (1,352)
2011 16,636 (350) 1,704 (0) 11,742 (278) 1,544 (0) 65,331 (1) 7,303 (21)e 104,260 (650)
2012 18,110 (128) 2,437 (0) 11,100 (95) 1,529 (0) 66,176 (0) 7,585 (0) 106,937 (223)
Source: WHO (http://apps.who.int/immunization_monitoring/en/diseases/poliomyelitis/afpextract.cfm). Starting in 2001, all wild poliovirus case
counts were based on virologic confirmation by the GPLN
a
cVDPV outbreaks not included
b
WHO regions: AFR Africa, AMR Americas, EMR Eastern Mediterranean, EUR Europe, SEAR South-East Asia, WPR Western Pacific
c
Importations of wild poliovirus type 1 into Bulgaria (from Pakistan) and Georgia (from India) [11]
d
Outbreaks in Tajikistan, Turkmenistan, Kazakhstan, and Russian Federation following importation of wild poliovirus type 1 from India [178]
e
Outbreak in Xinjiang, China, following importation of wild poliovirus type 1 from Pakistan [82]

fell from 10 in 2010 to 6 in 2012 (Fig. 13.13) and wild polio- type 3 circulation in areas where coverage rates in mass cam-
virus type 3 clusters fell from 9 in 2010 to 2 in 2012 paigns were high (Figs. 13.2c, 13.12, and 13.14) [12, 16, 82,
(Fig. 13.14). In addition, the surge of wild polioviruses from 451]. However, a remaining challenge is to prevent widening
Nigeria was again rolled back between 2010 and 2012 immunity gaps to poliovirus type 2.
(Figs. 13.13 and 13.14). In 2012, only Chad (5 cases in 2012 In 2012, more than two billion doses of OPV were admin-
compared with 132 cases in 2011) and Niger (1 case in 2012 istered to 448 million people, primarily to children <5 years
compared with 5 cases in 2011) had residual circulation of the of age in NIDs and SNIDs in 46 countries [12]. Only 223
wild poliovirus type 1 imported from Nigeria [12]. Although cases of poliomyelitis associated with wild poliovirus infec-
wild poliovirus transmission in Africa is localized to northern tion were reported in 2012, an historic low, from a total of
Nigeria, the risk for resurgence remains until immunization 106,937 reported cases of AFP, the most ever investigated
activities in key northern states are intensified [12]. (Table 13.2) [12]. The number of wild poliovirus cases in
Important factors in improving OPV coverage have been 2012 fell below the estimated number of worldwide VAPP
intensified community engagement, targeting mobile and cases (250–500) (Fig. 13.2a). For the first time, the risk of
migrant populations for immunization, and high-resolution poliomyelitis from the use of OPV exceeded the annual case
mapping of underserved communities [12, 22]. An addi- burden from wild poliovirus infection.
tional critical factor for eradication of wild polioviruses has
been the widespread use of monovalent OPV type 1 (mOPV1)
and mOPV3 in NIDs, SNIDs, and mop-ups. However, in 10.8 Emergence of Vaccine-Derived
countries where both wild poliovirus types 1 and 3 were co- Polioviruses (VDPVs), 2000–2012
circulating, balancing the use of mOPV1, mOPV3, and
tOPV in NIDs and SNIDs was difficult. Priority was usually In principle, all clinical and environmental vaccine-related
given to the use of mOPV1 because of the higher risk of wild poliovirus isolates are VDPVs. However, as described in sec-
poliovirus type 1 spread compared with wild poliovirus type tion “Categories of poliovirus isolates”, for the purposes of
3 [10, 176, 177, 180, 181] and the greater risk of large out- poliovirus surveillance, vaccine-related isolates have been
breaks [181]. Routine OPV immunization exclusively uses classified into two broad categories: (1) OPV-like isolates,
tOPV, but coverage rates in the core reservoirs remain low which have limited divergence from their parental OPV
[12], such that immunity gaps to types 2 and 3 could develop strains and are ubiquitous wherever OPV is used, and (2)
with near exclusive use of mOPV1 [447, 448]. The introduc- VDPV isolates, whose higher level of VP1 sequence diver-
tion of bivalent OPV (bOPV; types 1 and 3) [92, 449, 450] in gence from their parental OPV strains (>1 % [types 1 and 3]
late 2009 and early 2010 sharply reduced wild poliovirus or >0.6 % [type 2]) indicates prolonged replication (or
314 O.M. Kew

transmission) of the vaccine virus. VDPVs are further cate- VDPVs are heterogeneous: some represent the initial iso-
gorized as (1) circulating VDPVs (cVDPVs), (2) lates from cVDPV outbreaks, whereas others, such as highly
immunodeficiency-associated VDPVs (iVDPVs), and 3) divergent aVDPVs detected in sewage, are probably iVD-
ambiguous VDPVs (aVDPVs) [133]. PVs from inapparent chronic infections.
Although the vast majority of vaccine-related isolates are
OPV-like, VDPVs are of particular interest because of their 10.8.1 Outbreaks of Circulating VDPVs (cVDPVs)
implications for current and future strategies for global polio Among the two well-defined categories of VDPVs, cVDPVs
eradication [22, 94, 133, 428, 450]. VDPVs can cause para- are of the greatest current public health concern [22, 450,
lytic polio in humans and have the potential for sustained 455]. Since 2000, cVDPV outbreaks have occurred in 18
circulation. The clinical signs and severity of paralysis countries, with the large majority (86.6 %) of reported cases
associated with VDPV and wild poliovirus infections are associated with type 2 (Fig. 13.15; Table 13.3). Type 1 cVD-
indistinguishable. VDPVs resemble wild polioviruses phe- PVs were associated with outbreaks in Hispaniola in 2000–
notypically [133] and differ from the majority of vaccine- 2001 and Indonesia in 2005 [166, 183]. In contrast, type 3
related poliovirus isolates by having genetic properties cVDPV outbreaks are rare, accounting for only 1.7 % of
consistent with prolonged replication or transmission. known cVDPV cases, an unexpected finding because the
Because poliovirus genomes evolve at a rate of approxi- type 3 OPV strain is a major contributor to VAPP in OPV
mately 1 % per year (Sect. 3.6.3), vaccine-related viruses recipients [94]. Because the case/infection ratio for poliovi-
that differ from the corresponding OPV strain by >1 % of rus type 2 infections is low (Sect. 3.4), the number of type 2
VP1 nucleotide positions are estimated to have replicated for cVDPV infections worldwide since 2000 is estimated at
at least 1 year in one or more persons after administration of nearly one million [168, 461].
an OPV dose. This is substantially longer than the normal During 2005–2012, a polio outbreak of 411 cases associated
period of 4–6 weeks of vaccine virus replication in an OPV with cVDPV2 was reported from 11 northern and 3 north cen-
recipient [321]. The demarcation for type 2 VDPVs was low- tral states of Nigeria (Fig. 13.16; Table 13.3) [447, 448, 455].
ered to 0.6 % to increase sensitivity for early detection of The outbreak peaked at 153 cases in 2009, but 27 cases were
type 2 cVDPV outbreaks [133, 168, 452]. detected in 2010, 35 cases in 2011, and 7 cases in 2012. Genetic
It seems likely that many OPV-like isolates have recovered analysis resolved the outbreak into >20 independent type 2
the capacity for higher neurovirulence and possibly increased VDPV emergences that occurred during 2004–2012, at least 7
transmissibility. The small number of substitutions control- of which established circulating lineages [168, 448]. The out-
ling neurovirulence (Sect. 9.1.3; Fig. 13.9) was found to have break occurred in states where routine immunization coverage
reverted among many OPV-like isolates, especially among with tOPV is low and NIDs and SNIDs using tOPV were infre-
isolates of types 2 and 3 [382, 388, 393]. Because the critical quent [447, 448]. Spread of the type 2 cVDPV from northern
attenuating mutations in the 5′-UTRs of the Sabin strains also Nigeria has been very limited, with only six cases in Niger from
affect fitness for virus replication in the human intestine multiple importations and one case in Chad from an importation
[393], it appears possible that revertants at these sites would in 2010 (Fig. 13.16; Table 13.3). The limited spread may indi-
have a higher fitness for person-to-person spread. However, cate that surrounding areas have higher levels of population
spread is normally limited by high OPV coverage, and the immunity to poliovirus type 2 (because of the high immunoge-
VDPVs represent viruses whose potentials for prolonged rep- nicity of Sabin 2 and its higher tendency to infect OPV contacts)
lication or transmission have been clearly actualized, as dem- and that poliovirus type 2 may have an intrinsically lower capac-
onstrated by their genetic prioperties. ity to spread than types 1 and 3 (Figs. 13.13 and 13.14).
The three categories of VDPVs differ in their public Most of the other recent outbreaks have also been associ-
health importance. Circulating VDPVs pose the same public ated with type 2 cVDPVs (Table 13.3), several of which
health threat as wild polioviruses because they have recov- (e.g., Afghanistan, the Democratic Republic of Congo, India,
ered the biological properties of wild polioviruses, have the Pakistan, and Yemen) were associated with multiple inde-
potential to circulate for years in settings where poliovirus pendent emergences [133]. Other type 2 cVDPVs have
vaccination coverage to prevent that particular type is low, emerged successively in the same geographic area [459]. In
and require the same control measures. Immunodeficiency- several settings, this reflects the continued weakness in rou-
associated VDPVs may be excreted by persons with certain tine immunization with tOPV and the extensive use of
primary immunodeficiencies for many (>10) years with no mOPV1 and bOPV in mass campaigns. However, it also
apparent paralytic signs [223, 453]. Persons infected with reflects the greater tendency of the Sabin 2 OPV strain to
iVDPVs without paralysis are at risk of developing paralytic revert and spread to contacts [94]. Three separate type 3
poliomyelitis [186, 223, 454] and may infect others with cVDPV emergences were detected in Ethiopia in 2009–
poliovirus, presenting the potential risk of outbreaks in areas 2010, and a single type 1 cVDPV emergence was detected in
with low poliovirus vaccine coverage [132]. Ambiguous Mozambique in 2011 (Table 13.3).
13 Enteroviruses: Polio 315

22 14 3
2
21 1 1
12
383 9 3 17 3
4 7 5 2

71 21
46
2 9

cVDPV1 cVDPV2 cVDPV3

200
180
cVDPV1: 11.7%
160

140 cVDPV2: 86.6%

cVDPV Cases

120 cVDPV3: 1.7%

100 cVDPV1

80 cVDPV2
60 cVDPV3
40
20
0
2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

Year

Fig. 13.15 Circulating vaccine-derived poliovirus (cVDPV) out- Hispaniola (Haiti and the Dominican Republic) and the limited spread
breaks, 2000–2012. Map: Location of cVDPV outbreaks, color-coded of the cVDPV2 from Nigeria to Niger and Chad, all other outbreaks are
by serotype (red cVDPV type 1 [cVDPV1], yellow cVDPV2, blue independent events. Some countries had successive (e.g., Madagascar)
cVDPV3). The independent emergences of cVDPV2 and cVDPV3 in or concurrent (e.g., Nigeria and D. R. Congo) cVDPV2 outbreaks. Bar
Ethiopia and Yemen are indicated by yellow and blue diagonal patterns. chart: cases associated with cVDPV outbreaks, 2000–2012, color-
Apart from the 2000–2001 cVDPV1 outbreak on the island of coded by serotype (Modified from reference Kew [10])

In 2013, cVDPV2 from the outbreak in Chad continued rus serotype, (3) emphasis on use mOPV and bOPV in NIDs
with 4 new cases and spread to Cameroon (4 cases), Niger (1 and SNIDs [133, 447], and (4) insensitive AFP surveillance.
case), and Nigeria (4 cases). The cVDPV2 outbreak in Many of these factors exist in areas of insecurity and where
Pakistan continued with 47 new cases and spread to rates of routine tOPV coverage remain low, such as in parts
Afghanistan (4 cases in 2012−2013). of northern Nigeria, Somalia, and Yemen. In this context,
Key risk factors for cVDPV emergence and spread are (1) type 2 VDPVs present the greatest threat for emergence
development of immunity gaps arising from low OPV cover- [133], and routine immunization should be strengthened
age, (2) prior elimination of the corresponding wild poliovi- and, for the immediate future, whenever possible regular
316 O.M. Kew

Table 13.3 Circulating vaccine-derived poliovirus (cVDPV) outbreaks, 2000−2012

Estimated
% VP1 independent
Country Sero-type Years Reported cases divergence emergences References
Haiti/Dominican Republic 1 2000−2001 21 1.9−2.6 1 [166]
Madagascar 2 2001−2002 4 2.5−3.0 1 [187]
Philippines 1 2001 3 3.1−3.5 1 [189]
China 1 2004 2 1.0−1.2 1 [456]
Laos 2 2004−2005 1 (2 contacts) 1.1−1.2 1 [94]
Cambodia 3 2005−2006 2 1.9−2.4 1 [457]
D R Congo 2 2005−2009 7 1.0 >10 [458]
Indonesia 1 2005 46 1.1−3.0 1 [183]
Madagascar 2 2005 5 1.1−2.7 1 [459]
Nigeriaa 2 2005−2012 385 0.7−7.2 >20 [168, 447, 448]
China 1 2006 1 (6 contacts) 1.4−2.2 1 [190]
Myanmar 1 2006−2007 5 1.5−2.2 1 [457]
D R Congo 2 2008−2012 64 0.7−3.5 >10 [133, 455]
Ethiopia 2 2008−2009 4 1.3−3.1 2 [458]
Somaliab 2 2008− 18 0.7−4.0 4 [133, 455]
Ethiopia 3 2009−2010 7 1.1−1.2 1 [455]
India 2 2009−2010 17 1.3−1.6 5 [455]
Afghanistan 2 2010− 22 0.9−5.5 2 [455]
Madagascar 2 2011 0 (2 contacts) 3.3−3.7 1 [133]
Mozambique 1 2011 2 3.0−4.3 1 [133, 455]
China 2 2011−2012 3 0.7−1.8 1 [133]
Yemen 2 2011 9 0.6−1.6 4 [133]
Yemen 3 2011− 3 2.0−3.0 1 [460]
Chad 2 2012− 12 0.7−2.1 2 [460]
Pakistanc 2 2012− 14 0.7−2.9 2 [460]
a
cVDPV2 from Nigeria was exported to Niger (2006, 2009–2012; 7 total cases) and Chad (2010 and 2012; 2 cases)
b
cVDPV2 from Somalia was exported to Kenya (2012; 3 cases)
c
cVDPV2 from Pakistan was exported to Afghanistan (2012; 3 cases)

immunization campaigns using tOPV should be conducted oligonucleotide fingerprinting [186, 462] and genomic
to close any immunity gaps. sequencing [165, 186, 454], to poliovirus diagnostics. The
All of the cVDPVs shown in Table 13.3 except those from extent of sequence divergence is correlated with the duration
China have vaccine/non-vaccine recombinant genomes, of the prolonged infection [165, 173, 186, 223, 454]. Not all
which are very rare among OPV-like and iVDPV isolates isolates from immunodeficient patients are classified as iVD-
[94]. Recombination with other species-C enteroviruses fre- PVs. Some isolates are from specimens taken early in the
quently occurs during wild virus circulation and may be prolonged infection and no subsequent specimens were
interpreted as an indication of person-to-person transmis- taken. In other situations, either the prolonged infections had
sion. Whether recombination facilitates cVDPV emergence resolved spontaneously or the patient died from complica-
is unclear, because type 1 cVDPVs from China had nonre- tions of the immunodeficiency (including fatal poliomyeli-
combinant genomes [456] and recombination continues after tis) [354]. Prolonged iVDPV infections are independent
emergence, similar to what is observed with wild poliovi- events, and the isolates obtained from such infections trace
ruses [94]. separate pathways of divergence from the original OPV
strains.
10.8.2 Immunodeficiency-Associated VDPVs Since the introduction of OPV in 1961, >70 persons with
(iVDPVs) primary immunodeficiencies have been found worldwide to
It has long been recognized that patients with primary B-cell be excreting iVDPVs; the majority of these immunodeficien-
immunodeficiencies (defects in antibody production) could cies were detected only after onset of AFP (Table 13.4). The
become chronically infected when exposed to OPV [353]. extent of sequence divergence is correlated with the duration
Unambiguous demonstration that vaccine-related poliovirus of the prolonged infection [165, 173, 186, 223, 454]. Four of
isolates from immunodeficient patients had unusual sequence the iVDPV isolates are highly divergent (>10 % VP1
properties awaited the application of molecular tools, such as sequence divergence from the parental OPV strain),
13 Enteroviruses: Polio 317

2005 2006

2007 2008

2009 2010

2011 2012

Fig. 13.16 Emergence and spread of type 2 cVDPV in Nigeria, 2005– cVDPV emergences that occurred in Chad in 2012 are represented by
2012. At least 20 independent emergences of type 2 cVDPVs (green purple dots (Map prepared by Elizabeth Henderson based on sequence
dots) occurred in Nigeria between 2005 and 2012. Independent type 2 data produced by CDC)
318 O.M. Kew

Table 13.4 Documented prolonged iVDPV excretors, 1962–2012

Maximum %VP1
divergence from
Country Year detected Immune deficiencya Paralysis Serotype Sabin References
UK 1962 HGG No 1 2.5 [463–465]
UK 1962 HGG No 3 2.3 [165, 466]
Japan 1977 XLA Yes 2 – [462]
USA 1980 AGG Yes 2 1.3 [354]
USA 1981 CVID Yes 1 10.0 [186]
USA 1986 XLA Yes 2 2.0 [467]
USA 1986 CVID No 1 5.4 [467]
1992 2 11.8
UK 1987 CVID No 2 4.1 [464, 468]
USA 1989 AGG Yes 1 1.1 [354]
Germany 1990 CVID Yes 1 8.3 [454]
USA 1990 SCID Yes 2 1.9 [354]
USA 1991 CVID Yes 2 1.4 [354]
Iran 1995 HGG Yes 2 2.2 [469]
UK 1995 CVID No 2 12.9 [453, 464]
USA 1995 SCID Yes 2 2.1 [354]
Argentina 1998 XLA Yes 1 2.8 [470]
Germany 2000 CVID Yes 1 3.5 [467]
UK 2000 CVID No 2 6.3 [464]
Taiwan 2001 CVID Yes 1 3.5 [173]
Kazakhstan 2002 HGG Yes 2 2.3 [467]
Kuwait 2002 MHC II def No 2 2.0 [467]
UK 2002 ICF syndrome No 2 2.5 [464]
Peru 2003 AGG Yes 2 1.2 [467, 471]
Thailand 2003 HGG Yes 2 2.2 [472]
China 2005 XLA Yes 2; 3 4.2; 3.9 [457, 467]
Iran 2005 MHC II def Yes 1; 2 1.1; 1.4 [469, 473]
Moroccob 2005 SCID Yes 2 4 [467]
Syria 2005 HGG Yes 2 1.3 [457, 467]
USA 2005 SCID Yes 1 >2.3 [132]
Iran 2006 SCID Yes 2 1.7 [469]
Iran 2006 XLA Yes 3 2.1 [192, 469]
Kuwait 2006 SCID Yes 3 1.2 [457]
Syria 2006 HCI Yes 2 2.2 [457]
Tunisiac 2006 SCID No 2 2.0 [457, 467]
Belarus 2007 HGG Yes 2 1.9 [195]
Egypt 2007 SCID Yes 3 1.1 [457]
Iran 2007 SCID Yes 1; 2 1.7; 1.7 [469]
Iran 2007 XLA Yes 3 2.0 [469]
Russia 2007 HGG Yes 1 1.0 [195]
Iran 2008 XLA Yes 2 1.2 [195]
Argentina 2009 HGG Yes 1 3.8 [458]
Colombia 2009 AGG Yes 2 1.5 [455]
India 2009 CVID Yes 1 6.2 [455]
USA 2009 CVID Yes 2 12.3 [223]
Algeria 2010 HLA-DR Yes 2 1.8 [455]
China 2011 PID Yes 3 2.0 [455]
China 2011 PID Yes 2 1.9 [455]
India 2010 PID Yes 2 1.6 [455]
Iraq 2010 PID Yes 2 1.2 [455]
13 Enteroviruses: Polio 319

Table 13.4 (continued)

Maximum %VP1
divergence from
Country Year detected Immune deficiencya Paralysis Serotype Sabin References
Sri Lanka 2010 SCID No 2 1.3 [455, 474]
Egypt 2011 PID No 2 1.4 [133]
Egypt 2011 AGG Yes 1 2.1 [133]
Egypt 2011 PID Yes 3 4.2 [133]
Iran 2011 SCID Yes 2 2.0 [133]
Iran 2011 XLA Yes 2 2.7 [133]
Iran 2011 PID Yes 1; 2 2.7; 3.3 [133]
South Africa 2011 AGG Yes 3 1.9 [475]
Sri Lanka 2011 CVID Yes 3 1.9 [133, 474]
Turkey 2011 PID No 2 1.8 [455]
West Bank 2011 SCID No 2 1.2 [133]
China 2012 CVID Yes 2; 3 1.4; 1.6 [133]
Egypt 2012 PID No 2 1.0 [460]
India 2012 HGG Yes 2 2.8 [133]
Iran 2012 PID Yes 2 1.4 [133]
Iran 2012 PID Yes 2 1.1 [460]
Iraq 2012 PID Yes 2 1.0 [460]
a
Ab deficiency antibody deficiency, AGG agammaglobulinemia, CVID common variable immunodeficiency (CVID is shown in bold font because
it is most frequently associated with chronic iVDPV excretion and highly divergent iVDPV isolates), HCI humoral and cellular immunodeficiency,
HGG hypogammaglobulinemia, HLA-DR HLA-DR-associated immunodeficiency, ICF immunodeficiency-centromeric instability-facial abnor-
malities, MHC II def major histocompatability complex class II molecule deficiency, SCID severe combined immunodeficiency, XLA X-linked
agammaglobulinemia
b
Patient treated in Spain
c
Patient treated in France

suggesting that the chronic poliovirus infections had per- the prevalence of long-term iVDPV excretors might be
sisted for ~10 years (Table 13.4). Patients with the most higher than suggested by existing surveillance of persons
divergent isolates and the most prolonged (chronic) infec- with primary immunodeficiencies, and several aVDPVs have
tions have common variable immunodeficiency (CVID), a properties closely resembling iVDPVs (Sect. 10.8.3). The
group of late-onset immunodeficiencies that have multiple development of treatment for prolonged iVDPV infections
etiologies [476]. CVID is the most prevalent (~1 in 50,000) might facilitate detection of and access to those with pro-
[476] of the primary immunodeficiencies, but only a small longed infections [133, 477].
proportion of CVID patients exposed to OPV become chron- Type 2 iVDPVs are the most prevalent (63 %), followed
ically infected with iVDPVs. by type 1 (22 %) and type 3 (15 %). Some patients have het-
Unlike cVDPV outbreaks, prolonged iVDPV infections erotypic iVDPV infections, with the isolates having similar
cannot be prevented by high OPV coverage. Persons with degrees of divergence from the parental OPV strains, consis-
prolonged iVDPV infections can transmit poliovirus to oth- tent with a common OPV dose initiating the infections
ers, raising the risk for VDPV circulation in settings of low (Table 13.4). In addition to the occasional heterotypic infec-
population immunity to the corresponding poliovirus sero- tions, iVDPV isolates generally have distinguishing genetic
type. So far, all reports of persistent iVDPV infections have properties, including heterogeneity at sites of nucleotide
been from countries with high to intermediate levels of variability within serotypes (indicative of mixed virus popu-
development, where the rates of vaccine coverage are high lations), extensive antigenic variability, and either nonre-
and where the survival times of immunodeficient patients combinant or vaccine/vaccine recombinant genomes [94,
may be extended by treatment with intravenous immune 133].
globulin. The survival rates for persons with primary immu-
nodeficiencies are probably very low in developing countries 10.8.3 Ambiguous VDPVs (aVDPVs)
at highest risk for poliovirus spread. The population of pro- Unlike the well-defined cVDPV and iVDPV categories,
longed iVDPV excretors is declining in developed countries aVDPVs are a heterogeneous collection of isolates (Tables
because some patients have died (Table 13.4), some have 13.5 and 13.6). Some aVDPVs have diverged by just over
cleared their infections, and no new iVDPV infections have 1 % from the parental OPV strains, have no detected prog-
been found in countries that have shifted to IPV. However, eny, and may reflect the extremes of the usual distribution of
320 O.M. Kew

Table 13.5 Other vaccine-derived poliovirus (VDPV) with genetic evidence of community circulation, 1965–2012
Reported cases Estimated independent
Country Serotype Years (contacts) % VP1 divergence emergences Reference
Byelorussia 2 1965–1966 0 (9) 1.6–2.1 3 [156]
Romania 1 2002 1 (7) 1.1–1.3 1 [479]
Laos 2 2004 1 (1) 1.1 1 [94]
United States 1 2005 0 (5) 2.3–2.6 1 [132]
China 1 2006 1 (6) 1.4–2.2 1 [190]
Madagascar 2 2011 0 (2) 3.3–3.7 1 [133]

Table 13.6 Highly divergent aVDPVs from the environment, 1998–2012

Country Years isolated Serotype % VP1 divergence Reference
Israel 2009–2012 1 8.0–13.8 [117, 133, 455, 460]
1998–2012 2a 6.6–16.2
2006–2011 2b 10.7–11.2
Finland 2008–2012 1 12.4–14.0 [133, 455, 460, 480]
2008–2012 2 13.0–15.5
2008–2010 3 13.7–14.6
Slovakia 2003–2004 2 13.4–15.0 [119]
Estonia 2008–2012 2 13.5–16.2 [455, 460]
2002–2008 3 12.6–14.9
a
Group 1 aVDPV2 isolates from Israel are genetically distinct from Group 2 isolates, and are probably derived from two different chronic excretors
b
Group 2

vaccine-related variants in countries using OPV [133]. modeled on the International Smallpox Certification
Detection of other aVDPVs have foreshadowed subsequent Commission, which certified in December 1979 that small-
cVDPV outbreaks [133, 478], others indicate limited person- pox had been eradicated [482]. However, polio certification
to-person spread of OPV virus in small communities with differs in important ways from the smallpox model. PAHO
gaps in OPV coverage [94, 132, 156, 190, 479], whereas oth- established its Regional Certification Commission (RCC) in
ers appear to indicate more prolonged circulation (Table 13.5) 1990 and certified regional eradication of indigenous wild
[133]. Most of these latter aVDPVs have vaccine/non- polioviruses in 1994 [112, 430], setting the standard for cer-
vaccine recombinant genomes typical of cVDPVs. tification by individual regions and implicitly considering
Many of the aVDPVs are isolated from the environment, genetic evidence to distinguish indigenous wild polioviruses
and some have genetic properties (mixed VDPV populations, from imported virus. Because of the high proportion of inap-
extensive antigenic changes, and nonrecombinant or vaccine/ parent wild poliovirus infections, regional certification
vaccine recombinant genomes) typical of iVDPVs. Highly requires a period of at least 3 years of sensitive surveillance
divergent aVDPVs have been detected in Israel [117], Estonia since the last reported case associated with indigenous wild
[455], Slovakia [119], and Finland [120], countries with high poliovirus. Small outbreaks from imported virus occurred on
rates of polio vaccination coverage that are unlikely to have the eve of certification in the Western Pacific (in China in
extensive VDPV transmission (Table 13.6). The sequence 1999 [483, 484]) and in Europe (in Bulgaria [485] and
divergence of some of these isolates is consistent with more Georgia [11, 431] in 2001), but they were quickly controlled
than 15 years of replication from the original initiating OPV and followed by intensive surveillance. The certification of
dose. It is likely that the sources of these viruses are immuno- eradication of indigenous wild polioviruses by three WHO
deficient persons with asymptomatic VDPV infections, regions—the Americas in 1994 [112, 430], the Western
although the infected persons remain to be identified. Pacific in 2000 [486], and Europe in 2002 [431]—has stood
the test of time. No indigenous wild poliovirus circulation
has been detected in these regions post-certification. Cases
10.9 Certification of Polio Eradication and outbreaks associated with imported wild polioviruses
and cVDPVs (in addition to cases of VAPP) have occurred
The WHO GPEI has established a formal process for the cer- subsequently (Sects. 10.6 and 10.8.1), but circulation has
tification of polio eradication to ensure that future been promptly stopped by effective outbreak control mea-
immunization policy decisions are based on the best avail- sures [12, 82, 166, 178, 189, 190, 456]. The South-East Asia
able evidence [481]. The certification process for polio is Region was certified by the RCC as polio-free in March 2014
13 Enteroviruses: Polio 321

(http://www.searo.who.int/entity/campaigns/polio-certifica- identify those storing materials containing (poliovirus

tion/en/). Key to certification was the observation that >3 stocks) or potentially containing (clinical specimens or
years have passed since the last wild poliovirus case in India environmental samples taken at locations during times of
where surveillance has been maintained at high levels of sen- known poliovirus circulation) wild and vaccine-derived
sitivity [12]. Similarly, documentation in all countries polioviruses, (2) encourage destruction of all unneeded
except Pakistan and Afghanistan has been accepted by the materials, (3) develop inventories of laboratories retaining
Eastern Mediterranean RCC. Several countries in the African these materials and report to the RCC, (4) encourage imple-
Region have also prepared detailed documentation of polio- mentation of biosafety level 2 (BSL-2) procedures in all
free status [22]. enterovirus laboratories, and (5) plan for Phase 2. The
Global Eradication Phase (Phase 2) will begin 1 year after
10.9.1 The Certification Process detection of the last wild poliovirus or cVDPV at which
Review of documentation to assess polio-free status is orga- time countries will (1) notify biomedical laboratories that
nized on three tiers [481]. National Certification Committees wild poliovirus and cVDPV transmission has been stopped;
(NCCs) meet to critically assess national data and prepare (2) require laboratories on national inventories to select
detailed reports to Regional Certification Commissions among the three options to (a) render materials noninfec-
(RCCs), who in turn report to the Global Certification tious, (b) transfer all materials to high containment facili-
Commission (GCC). The NCCs, RCCs, and GCC meet on a ties, or (c) implement the appropriate laboratory procedures
regular basis, and in the regions already certified as free of (BSL-2/polio or BSL-3/polio); and (3) document that all
poliovirus circulation, the RCCs working with NCCs are pri- containment requirements have been met for global certifi-
marily responsible to oversee and support activities to main- cation [487]. The Post-Global Certification Phase (Phase 3)
tain the polio-free status. The NCC reports contain current was developed in response to the WHO goal of stopping all
information on the following: (1) national demographic data, OPV use post-certification and proposes to minimize
including high-risk populations, (2) poliovirus vaccine cov- facility-associated poliovirus risk by destruction of both
erage and any changes in vaccine policy, (3) surveillance wild and vaccine-related polioviruses in all facilities,
data and indicators of surveillance sensitivity, (4) descrip- except for an absolute minimum needed for vaccine pro-
tions of any poliomyelitis cases, (5) information on the per- duction, quality control, reference, and research [22].
formance of laboratories serving each country, and (6) Phase 1 containment was completed in the Americas sev-
progress toward poliovirus containment. NCC, RCC, and eral years after certification of polio-free status. In the United
GCC members are charged to serve as independent experts. States, among the 105,356 laboratories surveyed, 180 labo-
Following certification of all WHO regions, the GCC will ratories in 122 institutions were found to be storing materials
review all RCC reports and other relevant information to containing or potentially containing wild poliovirus; 87 held
determine when the world can be declared free of all poliovi- wild poliovirus stocks, 56 stored materials potentially con-
rus circulation [481]. Special provisions are planned to rec- taining wild polioviruses, and 37 stored both [489]. Europe
ognize the global eradication of wild poliovirus type 2 completed Phase 1 containment in 2006 [96]. Among the
(Sect. 11.2) [22]. An important prerequisite for certification 55,748 biomedical facilities surveyed, 265 laboratories in
is poliovirus containment. 164 institutions were found to be storing materials contain-
ing or potentially containing wild poliovirus; 116 held wild
10.9.2 Laboratory Containment of Polioviruses poliovirus stocks and 149 stored materials potentially con-
Containment is integral to poliovirus eradication [487, taining wild polioviruses. In 20 European countries, 1 or
488]. Unlike smallpox, where very few facilities stored more laboratories reported destroying all previously stored
materials containing (virus stocks) or potentially contain- wild poliovirus materials during the survey. The Western
ing (clinical specimens) smallpox virus (variola), many Pacific Region completed Phase 1 containment in 2009
facilities worldwide store materials containing poliovi- [490]. Of the 77,260 biomedical facilities surveyed, 45 labo-
ruses. The purpose of containment is to minimize, as much ratories (27 of which are members of the GPLN) retained
as possible, the risk of reintroduction into the community materials containing or potentially containing wild poliovi-
of polioviruses stored in laboratories and vaccine produc- ruses. Surveys have been completed in 155 of the 194 WHO
tion facilities. In 1998, the GCC approved the WHO Global member states, with the remaining surveys to be completed
Action Plan for Laboratory Containment of Wild in countries in sub-Saharan Africa (these countries are
Polioviruses [487] and subsequently established that satis- thought unlikely to have many facilities with wild poliovirus
factory completion of containment activities be a require- materials). Worldwide ~550 facilities, including 6 IPV man-
ment for regional certification. Containment will be ufacturers, have been found to be storing wild poliovirus-
implemented in three phases. During the Laboratory infectious materials or potentially infectious materials [22].
Inventory and Survey Phase (Phase 1) countries will (1) VDPVs are stored in GPLN laboratories and a small number
conduct national surveys of all biomedical laboratories to of collaborating laboratories.
322 O.M. Kew

11 Unresolved Problems the environment (Fig. 13.12) [184]. Several wild poliovirus
type 1 reservoirs have been cleared and genetic diversity
The immediate and crucial unresolved problem is to finish continues to decline. In recent years, >70 % of cases have
the task of eradicating wild polioviruses from the last remain- been in the Pashtun minority, many of whom migrate widely
ing reservoir communities. The second unresolved problem within Pakistan and into Afghanistan. The NEAP focuses
is implementation of the “polio endgame” to ensure that the on (1) the implementation of an aggressive polio campaign
promise of polio eradication is forever secured. The third schedule in the remaining reservoirs, (2) immunizing chil-
challenge is to build upon the legacy of polio eradication. dren of migrant and mobile Pashtun communities, (3) closer
integration of immunization campaigns with community
engagement, (4) closer civil–military cooperation in inse-
11.1 Eradication of Wild Polioviruses in Last cure areas, and (5) increased (>1,350) human resources for
Global Reservoirs deployment in high-risk areas [22, 184]. Campaigns in some
highest-risk areas (with ~165,000 children) have been sus-
In May 2012, the WHA declared that polio eradication was a pended because of repeated fatal attacks on young women
“programmatic emergency for global public health” and vaccinators from the community [184]. However, the attacks
called for intensification of eradication activities in the have been met by the young vaccinators with courage and
remaining reservoir areas [22]. A Global Emergency Action resolve, and campaigns have continued elsewhere, with
Plan and National Emergency Action Plans (NEAPs) were large numbers of children receiving OPV in the key reser-
developed and implemented in the remaining three reservoir voir areas.
countries [22]. All three countries face challenges of conflict Polio cases in Pakistan increased from 58 in 2012 to
and insecurity, as well as underperformance of immunization 93 in 2013; all cases in 2013 were associated with wild
activities in high-risk areas. Afghanistan and Pakistan made poliovirus type 1. Virus spread from Pakistan to Syria (33
clear improvements in 2012, but the pace of eradication has cases in 2013) (http://www.polioeradication.org/), and to
been slower in northern Nigeria. Egypt and Israel where wild poliovirus type 1 was detected
in sewage [491].
11.1.1 Afghanistan
Polio has been controlled in most of Afghanistan except for 11.1.3 Nigeria
the southern region, where intense armed conflict contin- All three poliovirus serotypes circulated in northern
ues. The last case associated with wild poliovirus type 3 Nigeria in 2012: wild type 1, type 2 cVDPV, and wild type
had onset in April 2010. Wild poliovirus type 1 had spread 3. In 2011–2012, 25 % of poliomyelitis case-patients had
from the southern region provinces of Kandahar and never received a dose of OPV [185]. Progress has been
Helmand in 2012 (Fig. 13.12), but the only 2013 cases were gradual in the northern states, and although polio cam-
associated with wild poliovirus type 1 imported across the paign quality has improved, serious gaps remain in rural
northeastern border from reservoirs in the insecure Tribal and hard-to-reach communities. In addition to insecurity
Areas of Pakistan. Only 2–5 % of children in southern in the northeast, 18 % of missed children are because their
Afghanistan were inaccessible in 2012 compared with parents refuse them to be vaccinated because of anti-OPV
6–21 % in 2010 and 2011 [184]. In 2012, 65 % of case- rumors propagated by some clerics [22]. The Polio
patients were underimmunized and 35 % had received no Emergency Action Plan calls for the following: (1)
OPV dose. The Afghanistan NEAP calls for reaching improved detailed planning of mass campaigns; (2) better
missed children through (1) improved detailed planning of selection, training, monitoring, and support of vaccination
mass campaigns; (2) better selection, training, monitoring, teams; (3) enhanced community engagement; (4) increased
and support of vaccination teams; (3) enhanced community (~2,500) human resources for deployment in high-risk
engagement; (4) robust response to poliomyelitis out- areas; (5) real-time data monitoring; (6) focused interven-
breaks; (5) expanded field staff in high-risk areas; and (6) tions in hard-to-reach rural and nomadic communities; (7)
use of permanent polio teams of trusted local people in GIS mapping of settlements and GPS tracking of vaccina-
insecure areas who visit homes and immunize children on a tion teams; and (8) strengthened surveillance [22, 185].
regular basis [22, 184]. Full implementation of this plan should break all remain-
ing chains of poliovirus transmission, as had been achieved
11.1.2 Pakistan a decade earlier in the more densely populated and tropical
Pakistan made dramatic progress in 2012 with implementa- southern states. As in Pakistan, fatal attacks on young
tion of the NEAP. Wild poliovirus type 3 was highly local- women vaccinators have been an impediment to complete
ized to the tribal areas in 2012 and was last detected in April implementation of the plan across the northern Nigerian
2012 from an AFP case, with no subsequent detections in states.
13 Enteroviruses: Polio 323

Polio cases in northern Nigeria decreased from 122 in completed, along with Phase 1 containment of type 2 vac-
2012 to 53 in 2013; all cases in 2013 were associated with cine-related isolates. When all prerequisites are met, coordi-
wild poliovirus type 1. Wild poliovirus type 1 spread from nated global cessation of tOPV use in routine immunization
Nigeria to Somalia (194 cases in 2013), and from Somalia to and mass campaigns will occur over a short time period,
Kenya (14 cases) and Ethiopia (9 cases). Wild poliovirus replaced with bOPV, the remaining tOPV stocks will be
type 1 of Nigerian origin spread from Chad to Cameroon in withdrawn worldwide, and the process carefully docu-
2013 (http://www.polioeradication.org/). mented [22]. After the switch, the WHO will stockpile
“stand-alone” IPV (IPV is usually formulated as a compo-
nent of multivalent injectable vaccines) for emergency
11.2 Implementation of the Polio deployment to areas peripheral to outbreak communities.
Eradication Endgame The tOPV–bOPV switch will precede the global bOPV ces-
sation and withdrawal projected for 2019.
Compared with the straightforward smallpox eradication
endgame, the polio eradication endgame is much more com- 11.2.2 Transition to IPV
plex and includes the following elements, some operating on Many previously OPV-using countries, including the
parallel tracks: (1) cessation of wild poliovirus transmission, United States, have transitioned to exclusive use of
(2) outbreak response (especially cVDPVs), (3) strength- IPV. Several countries have maintained a sequential IPV/
ened routine immunization, (4) IPV introduction and shift OPV schedule (Fig. 13.17), similar to what the United
from tOPV to bOPV, (5) sequential IPV/bOPV in routine States employed in 1997–1999 (Sect. 9.1.5). The WHO has
immunization, (6) completion of poliovirus containment obtained extensive experience in the introduction of new
(ultimately including vaccine-related isolates), (7) global vaccines into low- and middle-income countries. However,
certification, (8) cessation of bOPV use, and (9) mainstream- worldwide use of IPV will be greatly facilitated by reduc-
ing of polio activities into national and global disease alert ing the cost of IPV, which is substantially higher than
and response systems. Several of these activities are dis- OPV. Steps to reduce IPV costs could include (1) the use of
cussed above. A key new element is the worldwide with- intradermal fractional (1/5th) IPV doses [492], (2) develop-
drawal of use of the type 2 component of OPV. ment of new adjuvanted intramuscular IPV products, and
(3) replacement of conventional IPV (using neurovirulent
11.2.1 Withdrawal tOPV wild strains as seeds) with IPV based on the Sabin OPV
Since 1999, all poliomyelitis cases associated with poliovi- strains [493–495], more suitable for production in develop-
rus type 2 (apart from a few cases in India associated OPV ing countries [22]. New-generation biotechnology tools are
contaminated with MEF-1 [335, 336]) have been associated being explored to develop genetically stable IPV seeds that
with the continued use of tOPV. The rising incidence of type would substantially enhance biological containment in IPV
2 cVDPV outbreaks (Sect. 10.8.1; Fig. 13.15), representing production facilities [496–499]. However, the many options
nearly 90 % of all reported cVDPV cases, prompted the for immunization products and schedules present increas-
WHO GPEI to plan for the logistically challenging step of ingly complex policy decisions for national public health
worldwide withdrawal of tOPV and replacement with bOPV authorities [500].
[22]. The tOPV–bOPV switch, targeted for 2016, would be
predicated on the complete cessation of type 2 cVDPV
transmission and will require continued sensitive AFP and 11.3 The Legacy of Polio Eradication
poliovirus surveillance. Under the new strategic plan, by the
end of 2014, polio-funded field staff will devote >50 % of The GPEI is the largest public health initiative in history,
their time assisting countries to strengthen routine immuni- engaging communities, governments, health professionals,
zation. Large stockpiles of mOPV2 (500 million doses; in and private donors throughout the world. During the past
addition to 300 million doses each of mOPV1 and mOPV3) quarter century, the GPEI has trained millions of volunteers,
will be maintained, and a robust surveillance and response social mobilizers, and health workers to reach and immunize
capacity established. In addition, steps will be taken to children in the most marginalized and vulnerable communi-
secure affordable IPV and introduce at least one dose of IPV ties in the world [22]. In all but a few places, poliomyelitis,
into the routine immunization programs of all countries by increasingly a disease of the poorest of the poor, has gone the
the end of 2015. Before tOPV withdrawal, the GCC must way of smallpox. Moreover, other health interventions have
“validate” (certification will be for all poliovirus serotypes) directly benefited from polio immunization campaigns. For
the eradication of type 2 wild polioviruses and the elimina- example, vitamin A supplements delivered during OPV cam-
tion of type 2 cVDPV transmission. Also, Phase 2 contain- paigns are estimated to have prevented more than one mil-
ment of type 2 wild polioviruses and cVDPVs must be lion childhood deaths [22], and the administration of
324 O.M. Kew

OPV Only
Sequential
IPV Only

Fig. 13.17 Countries using exclusively oral poliovirus vaccine (yellow OPV), exclusively inactivated poliovirus vaccine (green IPV), or a combi-
nation of IPV and OPV (hatched pattern) (Modified from reference Sutter [7])

anti-helminthics and the distribution of bed nets have spared References

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 Filoviruses: Marburg and Ebola
14
Thomas G. Ksiazek

1 Historical Overview name to the family to which the virus belongs, the Filoviridae,
because of the filamentous appearance of the virus.
Because of the inordinate media attention given to the filovi- Marburg virus appeared again in 1975 when a young
ruses, particularly Ebola virus, a brief historical overview is Australian man, touring with his female companion in
in order. Southern Africa, became ill in South Africa after visiting
Rhodesia (now Zimbabwe). He was hospitalized in
Johannesburg 4 days after onset of illness and died there
1.1 Marburg Virus with rash and significant bleeding after 4 days of hospital-
ization. His companion, who remained at the side of the ini-
In 1967 the first known appearance of the filoviruses occurred tial case constantly throughout his early illness and
in Germany (Marburg and Frankfurt) and Yugoslavia hospitalization, became ill 2 days after the death of the
(Belgrade) [1, 2]. The disease was associated with the import index case as did a nurse caring for him, 8 days after the
of green monkeys (Cercopithecus aethiops, now Chlorocebus death of the initial case. The companion and the nurse sur-
aethiops) from Uganda as a source of monkey kidney cells vived their infections [4]. The exact origin of the infection
for the manufacture of polio virus vaccine. There were 29 of the index patient was not clearly elucidated in the subse-
cases who had direct contact with the monkeys or tissues quent investigations [5].
from the monkeys and an additional 6 cases among individu-
als who had contact with the initial patients. Seven fatalities
occurred, all among the individuals with exposure to the pri- 2 The Dawn of Ebola Virus
mates or their tissues. The disease was initially called green
monkey disease and the virus named after the town, Marburg, In 1976 two outbreaks of a disease with similar signs and
in which the initial cases were noted. symptoms occurred more or less simultaneously in north
Over the ensuing months the virus was isolated by serial central Zaire (now the Democratic Republic of the Congo or
passage in guinea pigs and also produced disease in two spe- DRC) and in southern Sudan [6–8]. The outbreaks were
cies of monkey (Chlorocebus aethiops and Macaca mulatta) larger than the initial Marburg incidents and were clearly
[3]. Interestingly, early reports suggested that there were no comprised of chains of person-to-person transmission fol-
cytopathic effects in a variety of cell cultures, while there lowing the introduction of the infection into medical care
was suggestion of replication in the cultures by inoculation facilities. The reuse of needles and syringes without proper
of guinea pigs with supernatant fluids of the cultures. Later sterilization was eventually thought to play a role in much of
reports do indicate the presence of cytopathic effect in a the transmission from patient to patient, at least within medi-
number of cell lines. cal care facilities, in these outbreaks.
The virus had a distinct, long pleomorphic appearance in Material from patients was sent to a number of interna-
electron micrographs; this trait would eventually give the tional laboratories, and it was soon discovered to be related
to Marburg virus by its similar appearance in electron micro-
graphs. In spite of the similarity of the appearance between
T.G. Ksiazek, DVM, PhD Marburg virus and this new virus, there was no serological
Department of Pathology, Sealy Center for Vaccine Development,
cross-reaction when reference sera from Marburg virus were
University of Texas Medical Branch,
301 University Blvd., Galveston, TX 77555-0610, USA reacted with this newer one or laboratory animals were
e-mail: [email protected] cross-challenged [9].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 337

DOI 10.1007/978-1-4899-7448-8_14, © Springer Science+Business Media New York 2014
338 T.G. Ksiazek

Field investigations of the Zaire and Sudan outbreaks did caused was centered. Although next-generation sequencing
not yield evidence of a direct link between the outbreaks at sped up the characterization of this new virus, its novelty was
the two locations. The virus was given the name Ebola virus indicated when targeted PCRs for the epidemic-prone viruses
after a river that is near the site of the original outbreak in Ebola Zaire and Ebola Sudan failed to react, but an antigen
Zaire. There was a single case in Zaire in 1977 [10] and detection assay reacted with initial patient bloods submitted
another small outbreak in southern Sudan in 1979 [11, 12]. to the Special Pathogens Branch at CDC in Atlanta [61]. As
The viruses causing the outbreaks in Zaire and the outbreaks is common in Ebola outbreaks, much of the transmission
in Sudan are in fact quite distinct; however, they were not occurred within medical facilities, where some of the initial
determined to be separate viruses until the early 1980s when patients sought care, and many of those subsequently infected
a radioimmunoassay suggested that antibodies to the two included health-care workers [62]. In addition, the mortality
viruses could distinguish between them [13] and then molec- associated with infection with this virus appears to be less
ular biology had reached some ability to distinguish the two than the Zaire and Sudan Ebola viruses [48].
viruses from each other [14]. Following the 1979 outbreak in Another filovirus has been associated with deaths in
southern Sudan, there were no reported African outbreaks insectivorous bats in Spain, but the virus has not been iso-
until 1995 when the outbreak in Kikwit, Zaire, occurred. lated [63]. Its genetic signature appears to be between the
In 1989 Ebola virus came to the USA in, similar to the cir- existing Ebola viruses and Marburg virus, but its taxonomic
cumstances for Marburg virus in 1967 in Europe, in monkeys status remains unsettled, and the host range and pathogenic-
(cynomolgus macaques, Macaca fascicularis) in quarantine in ity of the virus remain unknown and will probably remain so
Reston, Virginia, that were being imported for biomedical until a virus is either isolated or reconstructed from the
research into the USA from the Philippines. The monkeys were sequence derived from the tissues of the dead bats.
found to be dying of Ebola virus, although a second virus,
Simian hemorrhagic fever virus, was also present [15]. By
1989, the ability of molecular biology to sequence and deter- 3 The Virus
mine the relationships of viruses more easily indicated that this
was a novel Ebola virus, given the name Ebola Reston, related The filoviruses are negative-sense single-stranded RNA
but distinct from the two strains which had been the etiology of viruses. They are members of the family, Filoviridae, so
the Zaire and Sudan outbreaks in the 1970s, Ebola Zaire and named after the long filamentous appearance of the viri-
Ebola Sudan. The outbreak was traced back to a facility in the ons in electron micrographs. They belong to the order
Philippines where the imported monkeys had originated [16, Mononegavirales. Marburg virus comprises one genus of
17]. There were to be further instances of monkeys from this the family (Marburgvirus), while the Ebola viruses comprise
same facility being infected with Ebola Reston, including 1992 the other genus (Ebolavirus). The genomes are the largest
into Italy [18, 19] and 1996 into the USA once more [20]. of the viruses in this order having a genome of just over 19
In 1994, a fourth Ebola virus, Ebola Ivory Coast, emerged kilobases. The order of the seven genes is similar to other
when a female scientist studying mortality among chimpan- members of the Mononegavirales, the rhabdoviruses and the
zees in the Tai Forest National Park in Ivory Coast became ill paramyxoviruses. The nucleoprotein is located at the 3’ end
following a postmortem exam that she had performed upon a of the genome and is followed by VP35, VP40, glycoprotein,
chimp that had died. Her illness was a dengue-like syndrome, VP30, VP24, and the L (polymerase) open reading frames
and the virus was isolated from her blood [21]. Serological [64]. Several of these proteins have the ability to interfere
testing at the Institute Pasteur in Paris and sequencing soon with the immune response of the host and are believed to play
demonstrated that this was another novel Ebola virus. a role in the high pathogenicity of the filoviruses [65–67].
Starting in the mid-1990s the number of Ebola virus out-
breaks was more frequent and precludes individual discus-
sion. Some of these outbreaks will be discussed in the context 4 The Disease
of their contribution to our knowledge of the diseases and
viruses, but a complete listing of the occurrence of known Infection with Marburg and Ebola viruses leads to similar
outbreaks can be found in Table 14.1 (Marburg) and diseases which are marked by inappropriate innate immune
Table 14.2 (Ebola viruses). The relative size of each of these responses which both downregulate the useful antiviral
outbreaks and its location can also be seen in maps provided effects expected in the early postinfection period and induce
in Fig. 14.1 (Marburg) and Fig. 14.2 (Ebola). vigorous responses of certain cytokines and chemokines,
Briefly, a fifth Ebola virus was discovered in late 2007 inducing a sepsis-like syndrome with high mortality. The
when an outbreak in western Uganda turned out to be caused mortality ranges between a high of approximately 90 %
by another novel and distinct virus, now known as Ebola seen with Ebola Zaire virus infection, followed by approxi-
Bundibugyo, after the Ugandan town in which the outbreak it mately 55 % seen with Ebola Sudan, and approximately
14 Filoviruses: Marburg and Ebola 339

Table 14.1 Marburg outbreaks through 2012

Reported
number of Reported number
Apparent or human (%) of deaths among
Year(s) Country suspected origin cases cases Situation
1967 Germany and Uganda 31 7 (23 %) Simultaneous outbreaks occurred in laboratory
Yugoslavia workers handling African green monkeys imported
from Uganda [22]. In addition to the 31 reported
cases, an additional primary case was retrospectively
serologically diagnosed [23]
1975 Johannesburg, Zimbabwe 3 1 (33 %) A man with a recent travel history to Zimbabwe was
South Africa admitted to a hospital in South Africa. Infection
spread from the man to his traveling companion and a
nurse at the hospital. The man died, but both women
were given vigorous supportive treatment and
eventually recovered [24]
1980 Kenya Kenya 2 1 (50 %) Recent travel history included a visit to Kitum Cave
in Kenya’s Mount Elgon National Park. Despite
specialized care in Nairobi, the male patient died. A
doctor who attempted resuscitation developed
symptoms 9 days later but recovered [25]
1987 Kenya Kenya 1 1 (100 %) A 15-year-old Danish boy was hospitalized with a
3-day history of headache, malaise, fever, and
vomiting. Nine days prior to symptom onset, he had
visited Kitum Cave in Mount Elgon National Park.
Despite aggressive supportive therapy, the patient died
on the 11th day of illness. No further cases were
detected [26]
1998–2000 Democratic Durba, DRC 154 128 (83 %) Most cases occurred in young male workers at a gold
Republic of mine in Durba, in the northeastern part of the country,
Congo (DRC) which proved to be the epicenter of the outbreak.
Cases were subsequently detected in the neighboring
village of Watsa. This is actually a series of small
epidemics originating with miners who were working
in a galleried gold mine [27]
2004–2005 Angola Uige Province, 252 [28] 227 Outbreak believed to have begun in Uige Province in
Angola October 2004. Most cases detected in other provinces
have been linked directly to the outbreak in Uige [29]
2007 Uganda Lead and gold mine 2 2 (50 %) Small outbreak, with 2 cases of young males working
in Kamwenge in a mine. To date, there were no reported cases
District, Uganda among health workers [30]
2008 USA ex Cave in 1 0 (0) A 44-year-old woman who resides in Colorado
Uganda Maramagambo returned on January 1, 2008, from a Safari in Uganda.
forest in Uganda, at She became ill on January 4 and consulted her
the southern edge of physician on January 6 and 8 and was hospitalized on
Queen Elizabeth January 8 with a diagnosis of acute hepatitis. She was
National Park discharged on January 19 with no serious sequelae.
Testing of a sample drawn 10 days post-onset was
initially negative by serology, virus isolation, and
real-time RT-PCR. After the Dutch case, a
convalescent serum was submitted which was found
to be Marburg IgG positive by ELISA. A nested
RT-PCR of the original sample was found to be
positive for Marburg RNA [31]
2008 Netherlands Cave in 1 1 (100 %) A 40-year-old Dutch woman with a recent history of
ex Uganda Maramagambo travel to Uganda was admitted to a hospital in the
forest in Uganda, at Netherlands. Three days prior to hospitalization, the
the southern edge of first symptoms (fever, chills) occurred, followed by
Queen Elizabeth rapid clinical deterioration. The woman died on the
National Park 10th day of the illness [32, 33]
(continued)
340 T.G. Ksiazek

Table 14.1 (continued)

Reported
number of Reported number
Apparent or human (%) of deaths among
Year(s) Country suspected origin cases cases Situation
2012 Uganda Kabale, Ibanda, 23 8 (35 %) As of November 29, 2012, the Ugandan Ministry of
Kampala, and Health reported 15 confirmed and 8 probable cases of
Mbarara Districts Marburg virus infection, including 15 deaths, in the
Kabale, Ibanda, Mbarara, and Kampala Districts of
Uganda. Testing of samples by CDC’s Viral Special
Pathogens Branch is ongoing at the Uganda Virus
Research Institute in Entebbe. Working with the
Ministry’s National Task Force, a CDC team is
assisting in the diagnostic and epidemiologic aspects
of the outbreak. Note that Kabale District, on the
border with neighboring Rwanda, is distinct from
Kibaale District, the site of the recently ended Ebola
outbreak; both districts are in Uganda’s Western
Region [34]
Adapted from CDC. http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/marburg/marburgtable.htm

Table 14.2 Outbreaks caused by Ebola viruses through 2012

Reported no. of Reported no. (%) of
Year(s) Country Ebola subtype human cases deaths among cases Situation
1976 Zaire Ebola Zaire 318 280 (88 %) Occurred in Yambuku and surrounding area. Disease was
[Democratic spread by close personal contact and by use of
Republic of contaminated needles and syringes in hospitals/clinics.
Congo(DRC)] This outbreak was the first recognition of the disease [8]
1976 Sudan Ebola Sudan 284 151 (53 %) Occurred in Nzara, Maridi, and the surrounding area.
Disease was spread mainly through close personal contact
within hospitals. Many medical care personnel were
infected [7]
1976 England Ebola Sudan 1 0 (0 %) Laboratory infection by accidental stick of contaminated
needle [35]
1977 Zaire Ebola Zaire 1 1 (100 %) Noted retrospectively in the village of Tandala [10]
1979 Sudan Ebola Sudan 34 22 (65 %) Occurred in Nzara, Maridi. Recurrent outbreak at the
same site as the 1976 Sudan epidemic [36]
1989 USA Ebola Reston 0 0 (0 %) Ebola Reston virus was introduced into quarantine
facilities in Virginia, Texas, and Pennsylvania by monkeys
imported from the Philippines [15]
1990 USA Ebola Reston 0 0 (0 %) Ebola Reston virus was introduced once again into
quarantine facilities in Virginia and Texas by monkeys
imported from the Philippines. Four humans developed
antibodies but did not get sick [37]
1989– Philippines Ebola Reston 4 (asymptomatic) 0 (0 %) High mortality among cynomolgus macaques in a primate
1990 facility responsible for exporting animals in the USA [17].
Three workers in the animal facility developed antibodies
but did not get sick [16]
1992 Italy Ebola Reston 3 (asymptomatic) 0 (0 %) Ebola Reston virus was introduced into quarantine
facilities in Sienna by monkeys imported from the same
export facility in the Philippines that was involved in the
episodes in the USA. No humans were infected [18]
1994 Gabon Ebola Zaire 52 31 (60 %) Occurred in Mékouka and other gold-mining camps deep
in the rain forest. Initially thought to be yellow fever;
identified as Ebola hemorrhagic fever in 1995 [38]
1994 Ivory Coast Ebola Ivory 1 0 (0 %) Scientist became ill after conducting an autopsy on a wild
Coast chimpanzee in the Tai Forest. The patient was treated in
Switzerland [21]
14 Filoviruses: Marburg and Ebola 341

Table 14.2 (continued)

Reported no. of Reported no. (%) of
Year(s) Country Ebola subtype human cases deaths among cases Situation
1995 Democratic Ebola Zaire 315 250 (81 %) Occurred in Kikwit and surrounding area. Traced to index
Republic of case patient who worked in the forest adjoining the city.
the Congo Epidemic spread through families and hospitals [38, 39]
(formerly
Zaire)
1996 Gabon Ebola Zaire 37 21 (57 %) Occurred in Mayibout area. A chimpanzee found dead in
(Jan– the forest was eaten by people hunting for food. Nineteen
April) people who were involved in the butchery of the animal
became ill; other cases occurred in family members [38]
1996– Gabon Ebola Zaire 60 45 (74 %) Occurred in Booué area with transport of patients to
1997 Libreville. Index case patient was a hunter who lived in a
(July– forest camp. Disease was spread through close contact
Jan) with infected persons. A dead chimpanzee found in the
forest at the time was determined to be infected [38]
1996 South Africa Ebola Zaire 2 1 (50 %) A medical professional traveled from Gabon to
Johannesburg, South Africa, after having treated Ebola
virus-infected patients and thus having been exposed to
the virus. He was hospitalized, and a nurse who took care
of him became infected and died [40]
1996 USA Ebola Reston 0 0 (0 %) Ebola Reston virus was introduced into a quarantine
facility in Texas by monkeys imported from the
Philippines. No human infections were identified [20]
1996 Philippines Ebola Reston 0 0 (0 %) Ebola Reston virus was identified in a monkey export
facility in the Philippines. No human infections were
identified; one animal handler has Ebola antibody [41]
2000– Uganda Ebola Sudan 425 224 (53 %) Occurred in Gulu, Masindi, and Mbarara Districts of
2001 Uganda. The three most important risks associated with
Ebola virus infection were attending funerals of Ebola
hemorrhagic fever case patients, having contact with case
patients in one’s family, and providing medical care to
Ebola case patients without using adequate personal
protective measures [42]
2001– Gabon Ebola Zaire 65 53 (82 %) Outbreak occurred over the border of Gabon and the
2002 (Oct Democratic Republic of the Congo [43]
1–March
2)
2001– Democratic Ebola Zaire 57 43 (75 %) Outbreak occurred over the border of Gabon and the
2002 (Oct Republic of Democratic Republic of the Congo. This was the first time
1–March Congo that Ebola hemorrhagic fever was reported in the
2) Democratic Republic of the Congo [43]
2002– Democratic Ebola Zaire 143 129 (89 %) Outbreak occurred in the districts of Mbomo and Kéllé in
2003 Republic of Cuvette Ouest Département [44]
(Dec Congo
2–April
03)
2003 Democratic Ebola Zaire 35 29 (83 %) Outbreak occurred in Mbomo and Mbandza villages
(Nov– Republic of located in Mbomo District, Cuvette Ouest Département
Dec) Congo [45]
2004 Sudan Ebola Sudan 17 7 (41 %) Outbreak occurred in Yambio county of southern Sudan.
This outbreak was concurrent with an outbreak of measles
in the same area, and several suspected EHF cases were
later reclassified as measles cases [46]
2007 Democratic Ebola Zaire 264 187 (71 %) Outbreak occurred in Kasai Occidental Province. The
Republic of outbreak was declared over November 20. Last confirmed
Congo case on October 4 and last death October 10 [47]
Dec Uganda Ebola 131 42 (32 %) Outbreak occurred in the Bundibugyo District in western
2007–Jan Bundibugyo Uganda. First reported occurrence of a new strain [48, 49]
2008
(continued)
342 T.G. Ksiazek

Table 14.2 (continued)

Reported no. of Reported no. (%) of
Year(s) Country Ebola subtype human cases deaths among cases Situation
Nov 2008 Philippines Ebola Reston 6 (asymptomatic) 0 (0 %) First known occurrence of Ebola Reston in pigs. Strain
closely similar to earlier strains. Six workers from the pig
farm and slaughterhouse developed antibodies but did not
become sick [50, 51]
Dec2008– Democratic Ebola Zaire 32 15(47 %) Outbreak occurred in the Mweka and Luebo health zones
Feb 2009 Republic of of the Province of Kasai Occidental [52]
the Congo
May Uganda Ebola Sudan 1 1(100 %) Single case in Luwero District, Uganda [53]
2011
July 2012 Uganda Ebola Sudan 24 7(29 %) On July 28, 2012, the Uganda Ministry of Health reported
an outbreak of Ebola hemorrhagic fever in the Kibaale
District of Uganda. A total of 24 human cases (probable
and confirmed only), 17 of which were fatal, have been
reported since the beginning of July. Laboratory tests of
blood samples, conducted by the Uganda Virus Research
Institute (UVRI) and the US Centers for Disease Control
and Prevention (CDC), confirmed Ebola virus in 11
patients, four of whom have died [54]
2012 Democratic Ebola 77 36 (47 %) The DRC Ministry of Health has declared an end to the
Republic of Bundibugyo most recent Ebola outbreak in DRC’s Province Orientale.
the Congo The November 26 press release reports a final total of 77
cases, including 36 laboratory-confirmed cases, 17
probable, and 24 suspect cases, with a total of 36 deaths.
CDC assisted the Ministry of Health in the epidemiologic
and diagnostic aspects of the investigation. Laboratory
support was provided both through CDC’s field laboratory
in Isiro and through the CDC/UVRI lab in Uganda. The
Public Health Agency of Canada (PHAC) also provides
diagnostic support through its field lab in Isiro. The
outbreak in DRC has no epidemiologic link to the
near-contemporaneous Ebola outbreak in the Kibaale
district of Uganda [55–57]
2012 Uganda Ebola Sudan 7 4 (57 %) As of December 2, 2012, the Ugandan Ministry of Health
reported 7 cumulative cases (probable and confirmed) of
Ebola virus infection, including 4 deaths, in the Luweero
District of central Uganda. CDC is assisting the Ministry
of Health in the epidemiologic and diagnostic aspects of
the outbreak. Testing of samples by CDC’s Viral Special
Pathogens Branch is taking place at the Uganda Virus
Research Institute in Entebbe [58]
2014 Guinea and Ebola-Zaire (See situation) (See situation) Guinea [59]
Liberia As of 18:00 on 26 April 2014, the Ministry of Health
(MOH) of Guinea has reported a cumulative total of 224
clinical cases of Ebola Virus Disease (EVD), including 143
deaths. To date, 202 patients have been tested for ebolavirus
infection and 121 cases have been laboratory confirmed,
including 74 deaths. In addition, 41 cases (34 deaths) meet
the probable case definition for EVD and 62 cases (35
deaths) are classified as suspected cases. A revised number
of 25 health care workers (HCW) have been affected (19
confirmed), with 16 deaths (12 confirmed); the number of
HCW was previously reported as 26.
Liberia [60]
From 13 March, the date of onset of the first laboratory
confirmed case in Liberia, to 24 April, the Ministry of
Health and Social Welfare (MOHSW) of Liberia has
reported a total of 35 clinically compatible cases of EVD; 6
confirmed cases, 2 probable cases and 27 suspected cases.
The date of onset of the most recent confirmed case was 6
April. The MOHSW has started to reclassify suspected cases
against their laboratory test results. Most of the suspected
cases are expected to be discarded at the end of this process
Adapted from CDC. http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola/ebolatable.htm
14 Filoviruses: Marburg and Ebola 343

Fig. 14.1 Map of African Marburg virus outbreaks and year in which same location, Kitum Cave on Mt. Elgon. The 1998 outbreak in DRC
they occurred. The circles are proportional to the number of cases in the was actually a series of small outbreaks associated with a mine near the
outbreak (see Table 14.1 for the number of cases). Note that the 1980 village of Durba which continued through 2000
date obscures the date of the 1987 case, both of which originated at the

35 % seen with Ebola Bundibugyo, the three viruses that Ebola Zaire in 1995, less than 50 % of the lab-confirmed
seem to have epidemic potential. Ebola Ivory Coast has had cases had bleeding signs. The viruses are considered to be
too few infections, one or perhaps two, to allow a useful pantropic, infecting many tissues and also infecting many
estimate of the mortality, while Ebola Reston has so far not cell lines from a great many mammalian species [68]. Among
caused mortality in the 10s of persons that have been those with bleeding signs, petechiae, ecchymoses, and gas-
infected. Marburg virus outbreaks have tended to be smaller, trointestinal bleeding along with failure of venipuncture sites
often with only one or two individuals infected, but there to clot are the most common forms of bleeding. Nevertheless
also may be strain differences in the mortality. Among the the disease is severe and vascular permeability, loss of fluids
three outbreaks with significant numbers infected, the initial from the GI tract from diarrhea and vomiting, and diminished
outbreak in Germany produced an overall mortality of fluid intake all combine with the effects of the inappropriate
approximately 23 % (7 of 30 or 31) [22, 23], while the small immune response to induce shock and eventual multiorgan
clusters constituting the 1998–2001 composite cases in the failure.
DRC outbreak had an overall mortality of 82 % (123/149) Patients who do survive often have prolonged recover-
[27], and the 2005 outbreak in Uige, Angola, had a mortal- ies with hair loss and desquamation of areas initially
ity of 90 % (227/252) [29]. affected by rash. Weakness, myalgias, and arthralgias were
In human cases, there is an incubation period of 5–7 days also common among survivors of the Kikwit outbreak in
followed by onset of fever, weakness and often muscle pains, which convalescent survivors were compared to controls.
and abdominal discomfort. Rash is also a common feature, In human cases, lesions are common throughout the tissues
but in dark-skinned patients, it is not always apparent. with endothelial cells and macrophages in many organs
Bleeding is not the common manifestation that many believe having demonstrable virus antigens as well as histological
is the hallmark of the disease. In the Kikwit outbreak of changes and virus antigens in the parenchyma [69–71].
344 T.G. Ksiazek

Fig. 14.2 Map of African Ebola virus outbreaks and the year in which show sufficient detail of the area in which the majority of outbreaks
each occurred. The circles are proportional to the number of cases in the have occurred, the 1994 single case of Ebola Ivory Coast is not shown,
outbreak (see Table 14.2 for the number of cases). The color of circles and the 1996 South African imported case of Ebola Zaire is not shown.
indicates which Ebola virus caused the outbreak: Ebola Zaire (red), The 1976, 1979, and 2004 Ebola Sudan outbreaks overlap each other
Ebola Sudan (burnt orange), and Ebola Bundibugyo (blue). In order to and the dates are obscured in the figure

5 Laboratory Diagnosis 1989, a number of newer technologies were applied to the

diagnosis of Ebola virus infections. An antigen detection
As for all virus diseases, detection of the virus (or its anti- assay, utilized extensively in the Reston outbreak and its
gens or nucleic acids) or detection of the resulting antibody investigation, allowed for a rapid and specific identification
response in patients is the primary means of confirming a of Ebola virus in the blood of acutely ill individuals even in
specific diagnosis. Virus antigens are also found in the skin, remote areas [72]. Continued difficulties with the nonspe-
a feature which has been exploited to allow for diagnosis cific detection of antibodies by the indirect fluorescent anti-
using skin biopsies fixed in formalin, which may aid in body test in primates during the Reston investigation led to
surveillance for the filoviruses. Until the early 1990s, virus the adaption of enzyme-linked immunosorbent assays to the
isolation and identification by electron microscopy or refer- detection of both IgM antibody in acute Ebola infections
ence immune reagents remained the definitive means of and IgG antibody in individuals surviving infection [73] and
definitive diagnosis. This was difficult as high-containment the application of these assays to human patients in the
laboratories, of which there were very few in the world, Kikwit outbreak [74]. These assays also had the advantage
were required to carry out this work safely. Detection of that they could be adapted for use in the field during out-
antibodies was also somewhat fraught with issues as the breaks or outbreak investigations by utilizing appropriate
indirect fluorescent antibody test was prone to yield false personal protective equipment and inactivating the clinical
positives in the sera of individuals who had no previous specimens using heat and detergent. This allowed the diag-
exposure to filoviruses and the antibody response requires nosis of patients quickly in either onsite or at a local labora-
some time to become apparent, and in many instances tory, depending on the logistical support and demands of the
patients died without yet having detectable antibodies. particular outbreak. At about the same time that the antigen
Following the outbreak of a new Ebola virus in Reston in detection and serological assays were being developed, the
14 Filoviruses: Marburg and Ebola 345

use of reverse transcriptase-polymerase chain reactions for ample testament to the association of infection to exposure to
the detection and diagnosis of RNA viruses advanced where seriously ill patients or the cadavers of individuals who had
it too began to play a role in Ebola diagnosis [75] which died of Ebola infection [78, 79]. Nevertheless, some care and
eventually led to it being deployed to the field setting along education to avoid transmission via blood-borne routes by
with the ELISA tests [29, 76]. Another means of diagnosis, traditional medicine practices or reuse of needles in local
particularly useful in remote areas, was described and shown pharmacies remain a concern.
to be of utility during the Kikwit outbreak and employs the Even though the filoviruses can be readily transmitted by
use of formalin-fixed skin snips as a means of avoiding inva- the experimental creation of small particle aerosols, the role
sive postmortem exams and the safety and convenience of of aerosol transmission in outbreaks is, at most, minimal as
using formalin for transport of the biopsy to a suitable labo- attested by the lack of transmission in the community, other
ratory for diagnostic testing [72]. than by direct contact, and by studies of family members
sharing small enclosed spaces with infected patients [78].

6 Epidemiology and Ecology

6.2 Ecology
6.1 Epidemiology
The reservoirs of the filoviruses are just beginning to be
The epidemiology of the filoviruses is largely comprised of understood, and only recently has the primary host for
the person-to-person transmission that occurs once the virus Marburg virus, rousette fruit bats (Rousettus aegyptiacus),
has been introduced into the human population. The distribu- been established with a degree of certainty [80–82]. The res-
tion of the virus and, for the most part, the disease is dictated ervoir host(s) for the Ebola viruses is less certain, but indica-
by the underlying ecology of these zoonotic viruses that tions are that, like Marburg virus, bats are the leading
reside in reservoir or maintenance hosts. candidates [83, 84]. Anecdotal accounts of outbreaks going
The high mortality associated with filovirus infection has back to the Sudan outbreaks of the 1970s, in which the initial
created an aura of fear associated with the outbreaks. At a cases in both 1976 and 1979 worked in a cotton factory in
local level, particularly in the developing world where the which there were resident bats [85], have suggested to many
virus appears to be resident, this is understandable. The his- in the field that bats were leading candidates, but hard scien-
tory of outbreaks is marked by caregivers as early victims of tific data was lacking until more recent circumstances pro-
the disease. However, intense investigations during more vided stronger circumstantial and scientific support for this
recent outbreaks have provided valuable information on how contention.
transmission occurs and the level of protection that is neces- The big break for Marburg virus came when an outbreak
sary to protect caregivers and aid in stopping the chain of in Durba in the Democratic Republic of Congo (formerly
transmission that continues to fuel the epidemics. This was Zaire) which began in 1999 provided a combination of scien-
afforded by an earlier response than had occurred in the ini- tific and circumstantial evidence that led to further concen-
tial Ebola outbreaks of the 1970s and was particularly taken tration on bats as the reservoir. The outbreak in this instance
advantage of during the Kikwit outbreak in 1995. was not a typical filovirus outbreak with a single introduc-
The principle lessons are that transmission of the filovi- tion of the virus and a serial passage of that virus through a
ruses is by direct contact with infectious material and that chain of human cases; rather, in this instance, it was a series
moderate infection control practices, rigorously applied and of small outbreaks. This could be discerned because the
controlled, can almost immediately stop transmission [77]. viruses that caused each of the mini-outbreaks could be
In most of the endemic areas, this is a matter of providing genetically differentiated and the epidemiology of each
resources and training to health-care facilities where they small chain leads back to a single source of the virus. This
may be available on only a limited basis and are not routinely source was a former commercial mine now being exploited
used in the everyday practice of health care. In the developed by free-lance miners who were at the beginning of the trans-
world, standards of care and the employment of standard mission chain for each of the small mini-outbreaks [27].
precautions for patient contact have addressed the majority Investigation of the mine found that bats were the principal
of the risks associated with caring for patients with these fauna and genetic evidence of Marburg virus could be found
infections as has been demonstrated in a number of instances in animals collected during the investigation [86]. These
of imported cases where no, or, at worst, very limited, trans- findings were further advanced by investigations of cases
mission to care givers has occurred [31, 40]. occurring or originating in Uganda that were associated with
Risks to individuals in the community who do not have large concentrations of bats first at the Kamwenge mines [28,
direct contact with infected patients are practically nonexis- 30], where miners were infected, and then at a cave in Queen
tent. Assessment of risks during the outbreak in Kikwit gave Elizabeth Park, where two tourists became infected [31, 32].
346 T.G. Ksiazek

Subsequent investigations of the bat populations have focal that the cost of immunization would not be economi-
allowed the repeated isolation of the virus from the rousette cally viable; at best they might be used in the midst of an
bats that are the principal fauna of these two locations and outbreak to provide protection to at-risk individuals, particu-
the demonstration of the persistence of the virus in the bat larly if the protection is rapid, as the postexposure treatment
populations [80, 81]. use of certain of these vaccines is effective as pointed out
above. One added note about the use of vaccines, there is at
best limited cross-protection among the Ebola virus vaccines,
7 Prevention and no cross-protection between Marburg virus and Ebola
viruses; multivalent vaccines will have to be developed or the
Prevention of filovirus outbreaks is possible. In the first vaccines targeted at outbreaks with known filovirus etiology.
instance, now that the reservoirs of the viruses are becoming
apparent, education of local populations should enable them
to avoid the source of the virus. However, this may be cultur- 8 Treatment
ally difficult in hunter-gatherer populations that have strong
traditional values that may be difficult to change, particularly Aside from vaccines used postexposure, there have been
in economically developing areas where hunting remains an some advances in therapeutics for filoviruses.
important source of nutrition.

8.1 Monoclonal Antibodies

7.1 Infection Control
Monoclonal antibodies have recently been shown to protect
Outbreaks of disease are more often the product of exposure nonhuman primates against Ebola Zaire infection [92–95].
of individuals to the primary case who is infected by interac- Efforts are underway to improve the utility of some of these
tion with the reservoir. The lesson of the last 20 years is that preparations by making them more compatible for use as
infection control in health-care facilities, often the source of human therapeutics. As was the case for the vaccines, the
amplification of outbreaks, can stop the transmission of the monoclonal preparations are directed against specific filovi-
viruses from patients in these settings. Unfortunately, this ruses and would be most effective when used in situations
does require that these facilities have the basic supplies that where the specific virus causing disease was known.
are part of the daily routine of medical care in the developed Monoclonal antibody cocktails would be needed to broaden
world, what have come to be known as standard precautions. protection against multiple viruses.
Without the ability to use these simple measures, including
routine hand washing and single use of needles and syringes,
outbreaks are likely to continue to be amplified by health- 8.2 iRNAs
care facilities in the endemic areas.
Specific iRNAs have recently been shown to be effective in the
treatment of Ebola virus-infected animals [96, 97]. However,
7.2 Vaccines like vaccines, these are targeted and exhibit specificity of
action which means that a cocktail of iRNAs would be required
Vaccines are being developed and have reached a stage where for situations in which the virus was not yet identified or if a
nonhuman primates can be protected from infection by Ebola new filovirus were to appear. Closely related to the use of
and Marburg viruses [87–91]. Some of these vaccines have iRNAs has been the use of antisense phosphorodiamidate
even demonstrated their potential use as therapeutics for morpholino oligomers (PMOs) which are also targeted at spe-
treatment of individuals following exposure to a filovirus if cific RNA sequences but increase the stability, affinity, and
the vaccine is administered relatively soon after infection access into cells of the antisense nucleotides. In a recent study,
[92]. While almost all funding for the development of filovi- they have been used and demonstrated to successfully treat up
rus vaccines is driven by biodefense concerns, the vaccines to 75 % of rhesus macaques from Ebola Zaire infection when
are of practical use in providing protection to individuals who used either pre- or post-virus challenge [98].
have occupational exposure because they work with the
agents in the laboratory and to individuals who respond to
outbreaks that have similar risks of exposure from caring for 8.3 Small Molecule Inhibitors
patients in the field. There, in reality, is probably little use for
filovirus vaccines as a means of protecting populations in Other small molecule inhibitors of the viruses have been
endemic areas because the outbreaks are so sporadic and described from in vitro screening exercises or testing of
14 Filoviruses: Marburg and Ebola 347

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 Flaviviruses: Dengue
15
Stephen J. Thomas, Timothy P. Endy,
and Alan L. Rothman

1 Introduction (3) increasing DENV distribution (travel), and (4) the conver-
gence of the these three: urbanization, poverty, and decaying
Dengue is the world’s most important human arboviral dis- infrastructure [10–12].
ease with indigenous and endemic transmission in more than DENV infection produces a spectrum of clinical presen-
100 tropical and subtropical countries. There are numerous tations from asymptomatic infection to a nonspecific febrile
other locales that experience non-sustained epidemic trans- illness termed dengue fever (DF) to severe dengue known
mission with cases in returning travelers or military per- as dengue hemorrhagic fever (DHF) and dengue shock syn-
sonnel [1, 2]. More than half the population of the world drome (DSS) [13]. The original World Health Organization
is at risk of being infected with a dengue virus (DENV). (WHO) clinical classification scheme has recently been
Despite its importance dengue is under-recognized and under review and an alternative classification has been pro-
underreported with current literature estimating 400 mil- posed to account for instances where biochemical and clin-
lion infections each year with 100 million being clinically ical markers of severe disease are present in cases of
apparent [3]. The human, community, country, and regional otherwise uncomplicated dengue fever. Reconciling the
cost of dengue in terms of mortality, morbidity, and health value of the new scheme for both research and patient care
care resource utilization is significant and growing in scope purposes has been controversial [14–18]. Why some
[4–9]. There are numerous factors that are believed to con- human-vector-virus interactions result in infection and oth-
tribute to the increase in dengue burden, which include (1) ers do not and why some infections result in no disease and
rising number of susceptible hosts (population growth), (2) others severe multisystem organ failure and death are
expanding Aedes mosquito vector populations (ineffective incompletely understood. The pathophysiology of dengue
vector control, increasing breeding sites, changing ecology), is multifactorial with host (age, gender, clinical comorbidi-
ties, immunologic background), vector (salivary proteins),
and virus (type and genotype virulence, escape mutants)
factors and the interplay between them all playing a role
S.J. Thomas, MD (*)
[19–23]. There is no licensed anti-dengue antiviral or vac-
Viral Diseases Branch,
Walter Reed Army Institute of Research, cine to treat or prevent dengue disease. Vector control,
503 Robert Grant Ave, Silver Spring, MD 20910-7500, USA except in rare circumstances, has been insufficient to sig-
e-mail: [email protected] nificantly curtail dengue expansion. Fortunately, there are
T.P. Endy, MD MPH numerous efforts underway to explore methods to strategi-
Infectious Disease Division, Department of Medicine, cally and effectively reduce vector populations or alter their
State University of New York, Upstate Medical University,
ability to become infected and/or transmit dengue [24, 25].
725 Irving Avenue, CPOB Suite 304, Syracuse, NY 13210, USA
e-mail: [email protected] Anti-dengue therapeutic development efforts have focused
primarily on reducing viral burden (as measured in the
A.L. Rothman, MD
Laboratory of Viral Immunity and Pathogenesis, peripheral circulation) or reducing the magnitude of the
Institute for Immunology and Informatics, host pro-inflammatory response. New drug development
College of the Environment and Life Sciences, and seeking new indications for existing drugs are both
University of Rhode Island, FCCE Room 334D,
being actively explored [26–28]. The dengue vaccine
80 Washington Street (Shepard Bldg.),
Providence, RI 02903-1803, USA pipeline is abundant with candidates at various stages of
e-mail: [email protected] preclinical and clinical development [29–32].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 351

DOI 10.1007/978-1-4899-7448-8_15, © Springer Science+Business Media New York 2014
352 S.J. Thomas et al.

2 Historical Background lated since the 1600s, associated with dengue virus introduc-
tion into port cities by international commerce. Vector
Dengue’s nonspecific clinical features make the interpreta- eradication efforts initiated in the 1940s were initially suc-
tion of historical records for evidence of past epidemics dif- cessful, but the program ultimately ended and reinfestation
ficult. David Bylon is sometimes credited with the first and the return of dengue occurred in the region by the early
clinical description of dengue [33]. In 1779, he observed an 1980s [54]. Dengue has been increasingly reported from the
epidemic of febrile disease in Batavia, a disease he termed Middle East and Africa reaffirming the global spread of den-
“knokkel-koorts,” knuckle or joint fever [34]. At the same gue [55, 56].
time (1779), August Hirsch described a similar epidemic in
Cairo [35]. Dr. Benjamin Rush provided one of the first in-
depth clinical descriptions of dengue after observing numer- 3 The Dengue Viruses
ous patients during a 1780 epidemic in Philadelphia,
Pennsylvania [36]. Rush wrote, “…its more general name 3.1 Evolution of the Dengue Viruses
among all classes of people was, the Break-bone fever.” The
term “dengue” is derived from the Swahili phrase “Ka dinga The evolution of the flaviviruses has been covered in another
pepo,” where Ka means “a kind of”; dinga, “sudden cramp- chapter (Chap. 16). This section covers specifically the evolu-
like seizure”; and pepo, “plague.” The phrase is believed to tion of the dengue viruses (DENV) and why understanding its
have crossed from Africa to the Caribbean in 1827 [37, 38]. evolution is important in how these viruses have evolved into
Cubans identified this phrase with the Spanish word “den- a global health problem and challenging dengue vaccine
gue,” which represented the painful and stiff gait of people development. The dengue viruses evolved in fundamentally
afflicted with the disease. Spanish archives indicate the use different ways from their flavivirus brothers though retaining
of “quebranta huesos” (breakbone) by a physician in Puerto the properties responsible for the characteristics of flaviviral
Rico describing a febrile illness in 1771 and the use of “den- infection in humans such as fever, myalgias, headache, hepa-
gue” by the Queen of Spain in 1801 to describe an acute titis, encephalitis, and in particular hemorrhagic fever. The
febrile illness with bone and joint pains, hemorrhage, and continent of origin of the dengue viruses is not known, though
jaundice [39]. circumstantial evidence suggests an African origin based on
Early research into the etiology of dengue implicated bac- the many flaviviruses that circulate there now, its principal
teriological, protozoan, and spirochetal causes [40, 41]. mosquito vector Aedes aegypti is thought to have originated
Ashburn and Craig provided evidence for the viral etiology in Africa, and DENV circulates in an African sylvatic cycle
of dengue making it the second human viral pathogen identi- with nonhuman primates [57]. An argument can be made that
fied after the yellow fever virus [40, 42, 43]. Siler and col- the DENVs originated in Asia or perhaps evolved into four
leagues built upon the work of Graham in exploring the separate serotypes in Asia based on the hyperendemicity of
dengue vector and transmission dynamics [41, 44]. Large DENV in this geographic region and the phylogenetic rela-
outbreaks during World War II and human infection experi- tionship of the Asian sylvatic strains to current circulating
ments resulted in the isolation of dengue virus types 1 and 2, DENV strains [58, 59]. It is estimated that DENV evolved
identified the presence and possible protective role of anti- into four antigenically distinct serotypes approximately
dengue virus neutralizing antibodies, and described the pro- 1,000 years ago, and each of these four serotypes emerged
tective and disease attenuating capacity of homotypic and independently into a non-sylvatic endemic cycle of transmis-
heterotypic immunity, respectively [43, 45, 46]. In 1956, a sion between humans and Aedes aegypti approximately 125–
dengue epidemic occurred in Manila resulting in the identifi- 320 years ago [59, 60]. The Asian relationship of the DENV
cation and naming of dengue virus type 3 and 4 viruses [47]. is historically important as the first cases of the more severe
Dengue has a consistent presence in Asia and a sporadic form of DENV infection, dengue hemorrhagic fever (DHF),
pattern in the Americas. In 1897, North Queensland reported made its appearance in the 1950s first in the Philippines and
a dengue outbreak clinical manifested as epistaxis, then in Thailand [61]. The current Asian strains of each sero-
hematemesis, and gingival bleeding [48]. Severe dengue was type are considered more severe than the American strains
implicated in major disease outbreaks in Greece in 1928 and [62]. The evidence suggests that Asia plays a pivotal role his-
in Formosa in 1931 [49–51]. After World War II, intensified torically and currently in creating viruses that produce severe
and sequential transmission of multiple dengue virus types illness and due to its population growth and urbanization,
began in Southeast Asia, leading to hemorrhagic fever out- contributing to the increase in genetic diversity which has
breaks. In the 1950s, the dengue viruses would be associated increased by a factor between 14 and 20 in the last 30 years
with Philippine hemorrhagic fever and Thai hemorrhagic [63]. In prospective cohort studies in Northern Thailand and
fever, both later identified as dengue hemorrhagic fever hospitalized children in Bangkok, rates of genetic diversity
(DHF) [52, 53]. Outbreaks in the Americas have been postu- have been high among all serotypes in any given year,
15 Flaviviruses : Dengue 353

Fig. 15.1 The evolutionary II

history of the DENV as
determined by phylogenetic I
analysis [68]
126 DENV–1
(96–209) III

III

100 DENV–3
(90–116)
II

IV
American

Asian/
American

Asian I
321 DENV–2
(280–378) Asian II

Cosmopolitan
1115
(NA) Sylvatic

II

DENV–4

I
195
(152–265)
III
– 0.01 substitutions / site Sylvatic

restricted flight of the vector has accounted for spatial genetic geographically distributed throughout the world, and DHF
conservation, and major genetic clade replacements have has become a global health problem.
occurred over time [64–67]. The change of DENV from a
sylvatic cycle to a primarily human-vector cycle, especially in 3.1.1 DENV Phylogeny
Asia, changed the viruses in a fundamental way. Each sero- The phylogeny of DENV demonstrates the genetic diversity
type adapted to infecting and replicating in a human popula- of these viruses and its evolution as displayed in Fig. 15.1
tion with high levels of flavivirus antibody from previous [68]. The four serotypes diverge from each other by 30 %
DENV infections or in Asia from DENV and Japanese across their polyproteins, conferring their serotypic distinc-
encephalitis virus (JEV) infections. As a result, DENV have tion. Variation among genes among the viruses range from
become adept at escaping heterologous neutralizing antibody highly variable for the C and E proteins to relatively con-
and using it as a means to attain high viral load levels and served for NS3 and NS4B. Within a serotype there is a high
more severe disease, i.e., antibody enhancement. Today the degree of genetic variation with the formation of genotypes.
DENVs continue to evolve at a rapid rate, Asian strains are There are three genotypes (I, II, and III) for DENV-1, four
354 S.J. Thomas et al.

genotypes (I, II, III, and IV) for DENV-3, six genotypes tein that has dimers arranged in a herringbone pattern con-
(American, Asian/American, Asian I, Asian II, sisting of three domains termed the structurally central
Cosmopolitan, and Sylvatic) for DENV-2, and four geno- N-terminal domain I, domain II which contains the hydro-
types (I, II, III, and sylvatic) for DENV-4. Understanding phobic “fusion” peptide that allows virus-cell fusion, and the
the genotypes is important in comprehending the epidemiol- C-terminal domain III [71, 72]. Domain III is important in
ogy of the DENVs because genotypes arise in specific geo- virus binding and as a neutralizing site for human antibody
graphic regions and can be tracked as they spread to other and important in protection following infection [73, 74].
areas of the world, as occurred with the Asian strains of Figure 15.3 demonstrates the crystal structure of the E pro-
DENV-2 introduced into the Americas. The other important tein and its conformation under varying conditions [70]. In
reason for understanding the genotypes is that the occur- panel (a) is the structure of the immature virion at a neutral
rence of severe dengue illness is associated with specific pH where it exists as a heterodimer with 60 trimeric spikes
genotypes, the Asian strain of DENV-2, for example. Finally that extend from the surface and the structure that exists as it
there may be a valid concern for understanding the geno- develops in the endoplasmic reticulum (ER) of the host cell.
types for vaccine development as certain circulating geno- Panel (b) displays the immature virion at a low pH as it
types may not be covered by potential dengue vaccines that encounters the trans-Golgi network (TGN). In this form the
are being developed currently. heterodimers form dimers giving a smooth surface to its
structure. In panel (c) the prM protein is shown cleaved into
the M protein by the host endoprotease, furin. In (d) the
3.2 Flavivirus Genome and Structure mature virion is seen secreted in the extracellular space. This
figure is an elegant schematic of the processing of the E pro-
The DENV genome is typical of all the flaviviruses and con- tein and the effects of pH on its conformational structure as
sists of a single-stranded, positive sense RNA of 11 kilo- it develops into a mature virion.
bases in length [69]. The DENV genome has an open The E protein’s structure was crystallized and its confor-
reading frame (ORF) of over 10,000 bases that encodes mational structure revealed by cryoelectron microscopy by
polyproteins of 3,386–3,434 amino acids with the order of Kuhn and colleagues and demonstrated in Fig. 15.4 [75].
proteins 5′ –C-prM(M)-E-NS I -NS2A-NS2B-NS3-NS4A- Domain I is red, domain II in yellow, and the neutralizing
NS4B-NS5-3′ [69]. The structural proteins consist of the domain III in red. The fusion peptide is shown in green.
proteins C (capsid) and M (membrane or its precursor prM)
and the E (envelope) protein. The nonstructural proteins
consist of the NS1, NS2A, NS2B, NS3, NS4A, NS4B, and 3.4 The Nonstructural Proteins
NS5 proteins. The reader is referred to an excellent review
of the DENV genomic structure and the protein structures The nonstructural proteins of dengue virus play an impor-
from each gene and their function by Perera and Kuhn [70]. tant role in viral replication in the host cell and in immune
Figure 15.2 displays the organizational structure of the evasion allowing viral replication. As shown in Fig. 15.2,
DENV genome and the functions of each protein and its there are five nonstructural dengue virus proteins, NS1 to
structure from this publication. NS5. NS1 is involved in RNA replication of the virus and
thought to act with NS4A as an RNA replicase; it is secreted
in the extracellular space by infected cells and can be mea-
3.3 The E Protein sured in the serum of patients infected with DENV. NS2A
binds to NS3 and NS5 and is involved in the recruitment of
The E protein of DENV is the receptor that mediates virus- RNA templates to the membrane-bound replicase. NS2B is
host cell binding and entry into cells by membrane fusion, a membrane-associated protein and a cofactor for the serine
and it is the target of serotype-specific neutralizing antibody protease function of NS3. NS3 is a cytoplasmic protein
during the human immune response following infection. The involved in the polyprotein processing of the virus and RNA
E protein is therefore the major target for vaccine developers replication. NS4A and NS4B are hydrophobic proteins that
in developing a protective tetravalent vaccine. As the DENVs are membrane associated and function in RNA replication.
evolved to infect the host in the presence of preexisting anti- NS5 is the largest of the nonstructural proteins and serves as
body, the E protein developed properties with which to evade the viral polymerase and methyltransferase. Host immune
the host response by utilizing non-neutralizing or sub- evasion is performed by the inhibition of interferon α and β
neutralizing concentrations of antibodies to mediate the phe- production in human dendritic cells in which the nonstruc-
nomenon of antibody-dependent enhancement (ADE) of tural proteins play a major role [76, 77]. The nonstructural
infection. This enhancement phenomenon is thought to result proteins (NS2A, NS4A, NS4B, and NS5) in particular
in severe hemorrhagic disease. The E protein is a glycopro- inhibit the mammalian interferon signaling pathway [78].
15 Flaviviruses : Dengue 355

a
5’UTR 3’ UTR
CAP Structural Nonstructural

ER lumen E
NS1

pr
NS4B
NS2B NS4A

M
C NS2A COOH–
NS3
+
Cytoplasm NH3
NS5

Signal Stop-transfer Transmembrane Membrane NS2BNS3 Signalase Furin Unknown

sequence sequence domain assocated (cytoplasm) (ER) (ER lumen) (ER lumen)
c pr M

C prM E NS1 2A 2B NS3 4A 4B NS5

Capsid Pr peptide
Methyltransferase Polymerase

Protease-helicase
Envelope (E)

NMR structure

X-ray structure

Fig. 15.2 Organizational structure of the DENV genome and its structural and nonstructural proteins [70]

NS5, for example, binds to STAT2 and reduces its expres- 3.5 Virus-Host Interactions
sion essential for host cell interferon signaling [78]. The
multiple ways DENV evades the host immune response is This section discusses the complex interplay of viral evolu-
illustrated by findings that suggest DENV may use preexist- tion and E protein assembly and its structural components
ing enhancing antibodies not only to promote its entry into that influence the host immune response and the role of the
Fc receptor-bearing cells but also to form DENV-antibody nonstructural proteins in virion replication and assembly and
complexes inside human monocytic cells [79]. These com- are equally important in evading the host innate immune
plexes downregulate type 1 interferon production and response. The readers are referred to several excellent reviews
upregulate cytokine IL-10, which in turn downregulates sec- referenced in this section and in addition an excellent sum-
ondary antiviral responses, such as nitric oxide radical pro- mary of these complexities illustrated in Fig. 15.5 by Rothman
duction [79]. [80]. This figure demonstrates the human antibody response
356 S.J. Thomas et al.

a Immature virus b Immature virus c Immature virus d Mature virus

non-infectious non-infectious non-infectious infectious

pr pr
prM-E + 180 pr
prM-E
Furin M-E
M-E

pH 7.0 pH 6.0 pH 6.0 pH 7.0

ER TGN TGN Extracellular
Current Opinion in Microbiology

Fig. 15.3 Structures of the E protein and its conformations under vary- (TGN). (c) Structure of the E protein as an immature non-infectious
ing conditions. (a) Structure of the E protein as an immature non-infec- virus in the TGN after furin digestion. (d) E protein structure in a
tious virus in the endoplasmic reticulum (ER). (b) Structure of the E mature infectious virus (Current Opinion in Microbiology) [70])
protein as an immature non-infectious virus in the trans golgi network

in Fig. 15.3. The virion is internalized through endocytosis,

and in the resultant lysosomes, acidification drops the pH,
leading to a conformational change of the E protein as shown,
and the virions are internalized through endocytosis. The
acidification of the endocytic vesicle leads to the rearrange-
ment of the surface E protein as shown in Fig. 15.2, fusion of
the viral and vesicle membranes, uncoating of the virus, and
release of viral RNA into the cytoplasm. Viral genomic RNA
is then translated to produce viral proteins in the endoplasmic
reticulum, and the viral proteins and newly synthesized viral
RNA assemble into immature virions within the lumen. The
cleavage of the viral precursor membrane occurs by the host
cell enzyme furin, forming mature virions that are then
secreted from the cell. In this figure also is shown how NS1
forms and secreted from the plasma membrane of the cell. As
demonstrated, host antibody forms against both mature and
immature virions that can neutralize virus or result in ADE.
Anti-NS1 antibodies can cause complement-dependent lysis
of virus-infected cells. In (b), the structure of the dengue virus
E glycoprotein ectodomain and its three domains are shown
with antibody specific to each. In (c), the mechanisms of neu-
Fig. 15.4 Structure of the DENV E protein demonstrating domains I, II, tralization and enhancement by dengue virus-specific anti-
and III and the fusion peptide [75]. Domains I, II, and III are in red, yel- bodies are shown. Neutralization can occur when high levels
low, and blue, respectively. The fusion peptide is shown in green. The
C-terminal residue 395 is shown as a white asterisk for monomers within of epitope-specific antibody block the binding of virions to
the defined icosahedral asymmetric unit. Scale bar represents 100 Å the cellular receptor or can block fusion at the post-binding
stage. At lower concentrations, antibodies can enhance the
to dengue proteins as the virion enters the host cell and repli- uptake of virions into cells by interacting with immunoglobu-
cates. In (a), the mature virion binds to the cell surface recep- lin (Fc) receptors and enhancing binding and cellular uptake
tor via the E protein in its conformational structure as shown of virus.
15 Flaviviruses : Dengue 357

Virion Unknown host

receptor for virus
Envelope
protein
Release of Antibody
Endocytosis Neutralization,
mature virions specific for
envelope antibody-dependent
protein enhancement
Formation of Mature
mature virions virion
Endosome
Pre-M
Acidification protein
of endosome Assembly Secretion of
of virions immature virions Antibody
specific Antibody-dependent
for pre-M enhancement
Viral RNA
protein
ER Immature
virion
Translation of Viral proteins
viral genoric RNA
NS1-specific Complement-
antibody dependent lysis
Some NS1 expressed NS1
on plasma membrane
or secreted
Nucleus

Cytoplasm

b c
Antibodies Antibodies can enhance
block binding uptake of virions
DIII DII-fusion of virions to
loop cell receptor

DII
DI
Fc
receptor

Domain Function Serotype specificity Antibody function

I Hinge region Cross-reactive > specific Weak neutralization

Antibody-dependent
enhancement
Antibodies
II Fusion peptide Cross-reactive > specific Weak neutralization block fusion
dimerization Antibody-dependent Endosome
enhancement

III Receptor Specific> cross-reactive Potent neutralization

bindng Antibody-dependent
enhancement

ER

Fig. 15.5 Human antibody responses to dengue virus protein targets protein and their domains. (c) Mechanism of antibody mediated neu-
and antibody functions. (a) DENV binds to host cell and internalized tralization and enhancement by DENV specific antibodies [80]
with viral protein production and virus assembly. (b) Structure of the E

criteria [81]. The reliance on syndromic surveillance is in

4 Methodology Involved large part due to the lack of affordable DENV diagnostics
in Epidemiologic Analysis of Dengue available and the lack of infrastructure in which to perform
them. There are several problems with using syndromic sur-
4.1 Sources of Data veillance, and these include an inability to detect other causes
of fever in susceptible populations from other diseases with
A variety of sources of data are used to estimate the occur- similar presentation such as leptospirosis and an underesti-
rence of dengue in countries where it is endemic. These mation of the true burden of dengue infection as the majority
include hospital admissions, clinic visits, and prospective of cases are subclinical or of less severity, not requiring hos-
studies. The majority of countries rely on syndromic surveil- pitalization or presentation to a health clinic. Incidence of
lance diagnosing patients by clinical symptoms consistent dengue burden can be measured more reliably in prospective
with dengue fever or dengue hemorrhagic fever using WHO cohort studies, but these studies are expensive and require
358 S.J. Thomas et al.

technical expertise often lacking in endemic countries [82]. risk for infection. Several early prospective serologic surveys
To compensate for this underestimation of dengue burden by conducted in Thailand estimated risk factors for DHF and
syndromic surveillance, much has been written about using the incidence of dengue burden in defined populations. They
expansion factors (also called multiplication factors in some established the important contribution of serologic surveys
manuscripts) to estimate the true burden of infection. to the understanding of the pathogenesis of DENV infection
Expansion factors use a combination of incidence data from [89, 90]. In one of the first prospective studies on DENV
prospective studies to correct for the underestimation of dis- infection, Burke and colleagues prospectively studied 1, 757
ease burden by syndromic surveillance. For example, inci- children in a Bangkok school in June of 1980 [91]. At the
dence data from cohort studies performed in children in start of the study, 50 % of children had DENV antibodies to
Thailand and Cambodia were used as expansion factors to at least 1 DENV serotype by the age of 7 years. Sequential
calculate the national burden of infection [83]. From these serologic surveys of this population demonstrated that 87 %
cohort studies the annual incidence of laboratory-confirmed who became infected during the study period were asymp-
dengue was 23/1,000–25/1,000 in Thailand and 41/1,000 in tomatic; of those remaining who were symptomatic, 53 %
Cambodia. Age-specific rates of confirmed dengue were were clinically recognized as cases of DHF requiring hospi-
compared to the same national data and an expansion factor talization. Incidence rates ranged from 5.5 % in children
based on the degree of underestimation calculated and used with preexisting DENV antibody to 6.3 % in those who were
to readjust the national rate. In Thailand, the median read- serologically naïve. None of the children with primary den-
justed provincial number of dengue cases was 229,886 gue infection required hospitalization as compared to 12 %
(range 210,612–331,236) annually during 2003–2007, and of secondary infections. The authors concluded that preexis-
in Cambodia, the median was 111,178 (range 80,452– tent dengue immunity was a significant risk factor for the
357,135). The degree of underestimation of the burden of development of DHF.
dengue nationally was 8.7-fold in Thailand and 9.1-fold in
Cambodia. Similar approaches have been used to estimate
the true burden of infection in Southeast Asia and South 4.3 Dengue Diagnostics
America [84–87]. While using expansion factors to calculate
national burden is well meaning, it is still inherently inaccu- 4.3.1 Introduction
rate due to the variable nature of DENV transmission spa- A variety of laboratory methods and diagnostic tests are
tially and temporally. Prospective studies have demonstrated available for diagnosing past or current DENV infection.
a high degree of spatial and temporal variation on the inci- They can be divided into two broad categories, those that
dence and burden of dengue among neighborhoods and dis- detect the virus (viral isolation, RNA detection) and those
tricts and from year to year [88]. To capture the true burden that detect the host response via antibody production spe-
of dengue infection in endemic countries requires an invest- cific to DENV. Understanding the viral-host interaction and
ment in training, infrastructure development, and accurate pathogenesis during a DENV infection is essential in
yet inexpensive dengue diagnostics combined with ongoing understanding the predictive value of each assay by clinical
prospective studies to reflect national incidence. illness day.

4.3.2 Virus Isolation and RNA Detection

4.2 Serologic Surveys Since the first isolation of a virus, tobacco mosaic virus in
1935 by Stanley, viral isolation has been the mainstay by
Serologic surveys to estimate serotype-specific DENV inci- which to detect viral infection [92]. DENV (DENV-1) was
dence have played an important role in understanding DENV first isolated during an outbreak in the Nagasaki-Sasebo area
transmission and burden of infection. The methods of sero- of Japan brought in from returning seamen in 1942 by
logic surveys and their importance have been discussed more Kumura and Hotta and subsequently by Sabin and Schlesinger
extensively in Chaps. 3, 4, and 16, to which the reader is the following year [43, 45]. DENV isolation was performed
referred for more information. Serologic surveys involve by taking serum from ill patients within 48 h of their first
sampling a defined population and measuring the amount of fever and inoculating mice intracerebrally. Serial mouse
specific antibody to the targeted DENV protein which will brain passage isolated the virus, which was then identified
indicate past or current infection. This is performed by a through neutralization by convalescent sera of patients clini-
number of assays, but all currently employ a variation of the cally ill with DENV infection. Though cell lines had been
enzyme-linked immunosorbent assay (ELISA) or plaque used to propagate viruses such as vaccinia virus and yellow
reduction neutralization titer tests (PRNT). The results give a fever virus since the early 1900s, the development of tissue
point prevalence of past or current DENV infection in differ- culture lines for viral isolation by Weller and colleagues, first
ent geographic areas or subpopulations or those at particular used for poliovirus culture, transformed the ability to isolate
15 Flaviviruses : Dengue 359

and study viruses in the laboratory [93, 94]. Today a variety DENV infection where antigenic sin forms the basis for
of cell lines are used to isolate and study DENV and include severe dengue illness [102].
but are not limited to the mammalian monkey kidney cell It is not within the scope of this section to discuss every
lines, Vero cells and LLC-MK2 cells, as well as the mosquito available assay for detecting DENV infection. Most are
cell lines from Aedes albopictus (C636) and Aedes pseudos- experimental and performed in research laboratories and
cutellaris (AP61) [95, 96]. All have similar sensitivity and only a few assays have been licensed and are commercially
specificity for isolation, and their usefulness is limited to the available. The traditional DENV serologic diagnostics will
viremic period of patients discussed below. be discussed in detail as most assays are variations of these
Amplification of the DENV ribonucleic acid (RNA) and are essential in understanding the complexities of serol-
genome by polymerase chain reaction (PCR) provides a ogy during and after infection.
highly sensitive and specific assay in which to isolate the
viral genetic material [97–99]. Specific primers and probes Plaque Reduction Neutralization Test (PRNT)
and nested PCR permits DENV serotyping with a reaction The plaque reduction neutralization test utilizes the ability of
time of 4 h and represents a rapid means of detecting DENV a DENV to infect a single cell in a cell culture under agar,
both for diagnosis and for epidemiologic characterization of forming areas of cell death and clearing (plaques), and the
serotype-specific transmission. Today DENV PCR has ability of neutralizing IgG antibody to form complexes with
become the mainstay technique to diagnose and serotype the virus, preventing cell entry and thus plaque formation.
dengue-infected patients. Real-time quantitative PCR allows The PRNT was first adapted to DENV by Russell and col-
an estimation of the amount of virus (i.e., viral load) during leagues in 1967 [103]. The endpoint for the PRNT was cal-
clinical illness, thereby providing information on the patho- culated as the titer of neutralizing antibody that inhibits 50 %
genesis of severe dengue illness [100]. of the plaque formation known as the PRNT50. Originally
used with LLC-MK2 cells, a variety of PRNT assays exist
4.3.3 Serologic Tests now utilizing the same principles but with differing cell lines
Serologic testing for past or current DENV infection relies such as Vero cells and assay formats including the use of
on the host response to produce specific antibody to the tar- microwell plates and a micro-focus assay [104]. The PRNT
geted DENV proteins that indicates host recognition of is an important assay for determining previous and current
infection. Current serologic testing involves detection of DENV infection. Two critical issues limit the interpretability
either IgM or IgG antibody, the former indicating current of the results. The first is antibody cross-reactivity, making
active infection and the latter either current or past infection. serotype-specific interpretation difficult, and the second is
Two important concepts are involved in understanding sero- intra and inter-assay variability. The latter issue has been dis-
logic testing for DENV infection. The first is that IgG anti- cussed extensively by Thomas and colleagues, demonstrat-
body from current or past infection displays a high degree of ing with one sera set a high degree of variability of the PRNT
cross-reactivity to other DENV serotypes or other depending on the cell lines used, the type of prototype
flaviviruses. IgG antibody to a specific infecting DENV sero- DENV, and the use of complement in the assay [105]. This is
type is termed “homotypic” and antibody to another non- an important issue as the PRNT has been used by DENV
infecting serotype “heterotypic” or cross-reactive. This is vaccine developers in determining protective immunity from
particularly important as DENV occurs as four distinct sero- vaccines.
types. Infection from one serotype confers long-term immu-
nity to that serotype but not to the others. Thus, the human Hemagglutination Inhibition (HAI)
host can be infected from multiple DENV serotypes during a The hemagglutination inhibition assay (HAI) is a technique
lifetime. The second concept is that the host response to the widely used to measure antibody to a variety of viruses
first DENV infection differs from the response to subsequent including DENV and first described by Hirst in 1942 to mea-
DENV infections. The first infection is termed primary den- sure influenza virus antibody [106]. As described in Chap. 3,
gue and the second or additional DENV infection termed the assay utilizes the ability of viruses to adhere to red blood
secondary dengue. The second or subsequent DENV infec- cells (RBC) and form clusters thereby agglutinating them
tion is characterized by a blunted IgM response, a rapid and from solution. The HAI assay was adapted to DENV using
heightened IgG response, and cross-reactive T-cell responses goose RBCs and originally a powerful assay to measure
inherently involved in the pathogenesis of DHF discussed in DENV antibody though not much in use today except in spe-
more detail below. This heightened host response during a cialized DENV research laboratory The HAI is distinct from
secondary infection is termed “antigenic sin” and was first the PRNT as it measures any DENV antibody that adheres to
described by Francis and defined as the ability of the host the serotype-specific DENV, not just neutralizing antibody.
immune system to utilize immunologic memory based on a The advantages of the DENV HAI are that it is a high-
previous similar infection [101]. This has been applied to throughput assay that can accommodate several hundred
360 S.J. Thomas et al.

samples in a short period of time, the reagents are relatively Lateral Flow Immunochromatographic Assay
low cost, and intra- and inter-assay variabilities are well con- This assay use porous paper to allow the wicking and conju-
trolled. The disadvantage of the HAI test is similar to that of gation of the test sample; it is in essence an ELISA assay on
the PRNT in that cross-reactive antibody makes it difficult to paper. A number of DENV lateral flow assays have been
determine serotype-specific infection. Unlike the PRNT, the developed, and some (e.g., the NS1 rapid lateral flow assay)
HAI can be used to determine acute primary or secondary are commercially available in endemic countries. Studies
DENV infection by WHO criteria: a fourfold rise in HAI have demonstrated that this assay is less sensitive (72–73 %)
antibody titer between acute and convalescent sera is diag- than its ELISA format counterpart [108, 109]. The lateral
nostic for an acute DENV infection; the sera pair that is ≥7 flow assay has been developed to detect dengue IgM and
days and the convalescent titer that is ≤ to 1:1,280 for an IgG, potentially broadening the time window for clinical
acute primary DENV infection; the convalescent titer that is diagnosis of infection to several weeks while IgM is
≥ than 1:2,560 for an acute secondary infection; and no four- Detectable [110]. The lateral flow tests, also known as rapid
fold rise in HAI titer between the acute and convalescent titer diagnostic tests (RDTs), have a number of distinct advan-
and the convalescent that titer is > 1:2,560 for a recent sec- tages. The first is that they are potentially point-of-care diag-
ondary flavivirus infection [81]. nostic devices that can be done rapidly at the bedside or in
villages where dengue infection is suspected. The second
Enzyme-Linked Immunosorbent Assay (ELISA) and third are that a cold chain is not needed to store material
The enzyme-linked immunosorbent assay (ELISA) is an eas- and interpreting the result requires nothing beyond the abil-
ily reproducible high-throughput assay in which to measure ity to determine the color lines that indicate a positive or
both IgM and IgG DENV antibody. In general, the indirect negative test. The disadvantage is that in general they are less
ELISA is the most widely performed assay for measuring sensitive and specific than an ELISA format and PRNT-, or
DENV antibody. Like the HAI test, all DENV IgG antibod- PCR-based assays. Table 15.1 summarizes the advantages
ies are quantified and not just neutralizing antibody. Innis and limitations of available DENV diagnostic tests [111].
and colleagues were the first to develop an ELISA specifi-
cally to measure DENV IgG and also IgM, the latter known 4.3.4 Dengue Diagnostics
as an IgM antibody capture ELISA or MAC-ELISA [107]. During Clinical Illness
This assay was unique as it capitalized on the observation Figure 15.6a, b summarizes the importance of knowing the
that in secondary DENV infection the IgM to IgG ratio was day of clinical illness, whether the suspected dengue infection
lower than in primary DENV infection. In sera of children is primary or secondary and the appropriateness of the diag-
infected with DENV in Thailand, this assay demonstrated a nostic assay for the time of collection. In general, assays that
sensitivity of 78 % in admission sera and 97 % in paired sera. detect the virus, whether by isolation or RNA detection,
Dengue infections were classified as either primary or a sec- require viremia in the patient and are limited to the first week
ondary DENV infection by determining the ratio of units of of infection. Assays that rely on host antibody detection,
dengue IgM to IgG antibody where an IgM to IgG ratio of whether IgG or IgM, differ in primary and secondary detec-
≥1.78 was indicative of primary dengue and <1.78 second- tion with IgM higher and longer lasting in the first infection
ary infection. The dengue IgM/IgG ELISA assay is commer- as compared to secondary infection. Figure 15.6 also illus-
cially available and offers the advantage of being trates the importance of obtaining an acute and a convalescent
reproducible, technically within the scope of clinical labora- sample in both primary and secondary infections to determine
tories; high throughput; and relatively inexpensive to per- a fourfold rise in IgG antibody as a criterion for diagnosis.
form. A characteristic feature of DENV infection is that the
virus in infected cells secretes free NS1 protein into the
extracellular space. NS1 can be measured in the serum of 5 Descriptive Epidemiology
patients and correlates directly with viral replication and
viral load. The NS1 ELISA assay is commercially available 5.1 Incidence and Geographic Distribution
and uses an antibody capture of NS1 protein and a secondary
enzyme-linked detection antibody to produce a colorimetric DENV is considered the most common arboviral infection in
reaction that measures the amount of NS1 in the sample. The tropical and subtropical regions of the world. As mentioned
NS1 ELISA assay was 82 % sensitive in diagnosing patients in a previous section, the frequency of dengue infection is
with acute dengue [108]. The advantages of the ELISA assay underestimated due to the emphasis on reporting of hospital-
for NS1 are its high-throughput capacity and ease of perfor- ized cases by syndromic surveillance by most countries
mance in most clinical laboratories. The disadvantage is that endemic for dengue. A recent analysis with mathematical
NS1 correlates to DENV viremia, and thus, detection is lim- modeling of the geographic distribution and global incidence
ited to the first 7–10 days of clinical illness. of dengue in 2010 demonstrated that DENV is endemic
15 Flaviviruses : Dengue 361

Table 15.1 Summary of DENV diagnostic assaya

Diagnostic tests Advantages Limitations
Viral isolation and Confirms infection Requires acute sample during the viremic period (first 7
identification days of illness)
Is serotype specific Needs specialized expertise and laboratory
Allows additional scientific investigation into the Requires several weeks to isolate virus and is not high
virus throughput
Does not differentiate between primary and secondary
infection
Can be expensive
RNA detection (PCR format) Confirms infection Has potential false-positives due to contamination
Sensitive and specific Requires acute sample during the viremic period (first 7
days of illness)
Identifies serotype and genotype Needs specialized expertise and laboratory
Has rapid results in 24–48 h Does not differentiate between primary and secondary
infection
Antigen detection
ELISA format for NS1 for Confirms infection and sensitive and specific Is not as sensitive as virus isolation or RNA detection
example Is easy to perform and high throughput Requires basic clinical laboratory and training
Is less expensive than virus isolation or RNA Requires acute sample during the viremic period (first 7
detection days of illness)
Does not differentiate between primary and secondary
infection
Lateral flow assay for NS1 for Confirms infection Is not as sensitive as virus isolation, RNA detection, or
example ELISA format
Is easy to perform and high throughput Requires acute sample during the viremic period (first 7
days of illness)
Is less expensive than virus isolation or RNA Does not differentiate between primary and secondary
detection infection
Point of care and does not require specialized lab
equipment or cold chain
Serologic tests
Plaque reduction Confirms infection Is expensive and time-consuming to perform
neutralization test (PRNT) Is serotype specific in primary infection Requires specialized training and laboratory
Is considered the “gold standard” Does not differentiate between primary and secondary
infection
Is considered a correlate for protection Has high degree of antibody cross-reactivity
Hemagglutination inhibition Confirms infection Requires specialized training and laboratory
(HAI) Confirms primary and secondary infection Reagents such as goose red blood cells may be difficult to
obtain
Is relatively inexpensive Confirmation may require two or more serum samples
Is high throughput
IgM/IgG ELISA Is easy to perform IgM levels can be low in secondary infections
Confirms infection and sensitive and specific Requires specialized training and laboratory
Is high throughput
Confirms primary and secondary dengue
Lateral flow assay for IgM/ Confirms infection Is not as sensitive as ELISA
IgG Is easy to perform and high throughput Requires acute sample
Is less expensive than ELISA Does not differentiate between primary and secondary
Point of care and does not require specialized lab infection
equipment or cold chain
a
Adapted from Peeling et al. [111]

throughout the tropics with spatial variations and influenced 284–528) of which 96 million (67–136) are symptomatic [3].
by climate such as rainfall and temperature as well as urban- This estimate is greater than three times the dengue burden
ization [3]. The authors estimated that the number of DENV as determined by the WHO and further emphasizes impor-
infections per year is 390 million (95 % confidence interval tance of this infection for global health [18].
362 S.J. Thomas et al.

Fig. 15.6 (a) Types of dengue

diagnostics appropriate by day
a
Hemagglutination Inhibition
of infection in primary dengue. lgM and lgG ELISA
(b) Types of dengue diagnostics Virus isolation Plaque Reduction Neutralization test
appropriate by day of infection Mosquito inoculation Rapid tests
in secondary dengue Cell culture (C6/36, AP61)

Molecular techniques Anti-dengue lgM

Blot hybridization
Polymerase chain reaction (PCR)
Taqman Anti-dengue lgG
NASBA
Manifestations:

Headache
Fever
Myalgias
Viremia

0 2 4 6 8 10 12 14 16
Days after infection

b
Hemagglutination Inhibition
lgM and lgG ELISA
Virus isolation Plaque Reduction Neutralization test
Mosquito inoculation Rapid tests
Cell culture (C6/36, AP61)

Molecular techniques Anti-dengue lgG

Blot hybridization
Polymerase chain reaction (PCR)
Taqman Anti-dengue lgM
NASBA
Manifestations:
Shock Pathology:
Fever Hemorrhage Dengue Ag staining
Liver injury PCR
Viremia

0 2 4 6 8 10 12 14 16
Days after infection

5.2 Epidemic Behavior and Contagiousness in thrombocytopenia, leukopenia, coagulopathy and plasma
leakage [112]. Research on severe DENV resulted in the
5.2.1 Studies of DENV in Thailand observation that both secondary DENV infection and primary
During the 1950s, collaboration between the United States infection of infants with declining maternal antibody were
and Southeast Asian countries through the Southeast Asian risk factors for DHF, and the theory of ADE as an important
Treaty Organization (SEATO) created a number of host- force in the pathogenesis of DHF was born [113]. Prospective
country laboratories to respond to and study endemic dis- studies of DENV in children living in Bangkok established
eases. In Thailand the SEATO General Medical Research the role of enhancing antibody in the peripheral blood mono-
Project originally was located at Mahidol University in nuclear cells of children in producing severe dengue illness
Bangkok and later became a joint Thai and US military proj- and DHF [114]. Additional prospective studies demonstrated
ect named the Armed Forces Research Institute of Medical the importance of viral load and T-cell responses in disease
Sciences (AFRIMS) located on the Thai military medical severity and the circulation of all DENV serotypes and their
complex. Bangkok and Thailand became the epicenter for spatial and temporal diversity [88, 115–117].
severe dengue illness and allowed Thai and US investiga-
tors to study all aspects of DENV, an effort that has contin-
ued for more than half of a century and produced seminal 5.3 Spatial and Temporal Distribution
advances in DENV epidemiology, vector transmission,
and pathogenesis. Early studies of hospitalized children in DENV transmission, being vector-borne, is dependent on
Bangkok characterized the clinical severity of DHF resulting both the propagation of the Aedes aegypti vector mosquito
15 Flaviviruses : Dengue 363

and the availability of viremic individuals from whom the transmission in Bangkok using the location of dengue
mosquito can become infected. Thus, DENV transmission is patients’ homes and the infecting serotype found evidence of
seasonal and has both spatial and temporal distributions. The localized transmission at distances of under 1 km [121]. In
experience in Thailand in many ways illustrates how DENV summary, there is a diverse spatial and temporal distribution
can emerge in a population and through the combination of of DENV transmission illustrating the complexities of trans-
increasing urbanization, a high birth rate providing a con- mission between the host and the mosquito vector. Aedes
tinuous pool of susceptible individuals, and climate changes, aegypti, as an urban mosquito, has a limited range of approx-
evolved from a seasonal disease with periodic epidemics to a imately 200–500 m from its breeding site as determined by
hyperendemic one involving transmission of all four sero- capture-release studies [122]. Infected viremic individuals
types. A classic paper by Nisalak and colleagues illustrates move from one A. aegypti area to another and introduce the
this concept nicely [118]. Dengue virus serotype was deter- virus to feeding vectors that then become infected. After an
mined by viral isolation and PCR in hospitalized children extrinsic incubation period of approximately 14 days, mos-
with suspected dengue at the Queen Sirikit National Institute quitoes can then feed and infect the susceptible human host,
of Child Health in Bangkok, Thailand, from 1973 to 1999. typically within a 500 M radius, thereby propagating a
During that period, acute dengue was diagnosed in 15,569 restricted spatial outbreak. A useful metaphor is to imagine a
children with 4,846 viral isolations. All four DENV serotypes large-scale DENV outbreak as throwing a series of small
circulated with DENV-3 the most frequent serotype in pri- pebbles in a pond. Each pebble represents the location of one
mary dengue and DENV-2 in secondary infection and DHF. infected individual that infects an A. aegypti mosquito. The
However, the change in predominant serotype from year to waves from the one pebble represent individuals who are
year was striking, with DENV-1 the major cause of the out- infected from that mosquito producing a spatially confined
break from 1990 to 1992, DENV-2 from 1973 to 1986 and outbreak. As more and more pebbles are thrown in the pond,
1988–1989, DENV-3 in 1987 and 1995–1999, and DENV-4 the numerous resulting waves create more and more spatially
from 1993 to 1994. In Thailand there has been a progressive confined outbreaks producing a large outbreak across a
increase in the number of dengue cases from several thou- country or region. This wave concept was illustrated by a
sand per year in the 1960s to 200–300,000 cases per year in mathematical model of how DENV travels across Thailand
the last decade. In the Americas the more recent pattern during the epidemic season in each of several years [123].
resembles the experience in Thailand during the 1970s and The authors demonstrated the existence of a spatial-temporal
1980s. DENV is introduced in a country producing localized traveling wave in the incidence of DHF with the wave ema-
epidemics and through the movement of infected individuals nating from Bangkok moving radially at a speed of 148 km
and transportation of the mosquito vector, the disease spreads per month.
and becomes endemic in a region. Continued urbanization
and an increase in birth rate provide high vector burden and
a pool of susceptible individuals for all four serotypes, and 5.4 Age and Sex Distribution
the country becomes hyperendemic for DENV. Like in
Thailand, one serotype emerges as the predominant serotype In countries with endemic DENV where the force of infec-
causing an outbreak in any given year. As the population tion is high, dengue illness becomes primarily a pediatric
becomes infected and develops herd immunity to that sero- disease. Maternal DENV antibody is transplacentally
type, another serotype becomes predominant and thus the transferred to the infant conferring a degree of protection
cycle is repeated every year. Long-term prospective cohort that lasts for approximately 12 months [124]. The first
studies of children in Northern Thailand from 1998 to the infection with DENV typically occurs before the age of
present illustrate the fine-scale spatial and temporal distribu- five as a primary infection and goes largely unrecognized
tion of DENV transmission in a fixed geographic area. Using as just another febrile illness that infants experience. The
a school-absence surveillance system, absent students were second infection often occurs between the age of 5 and the
evaluated for fever or a history of fever. If present, acute and teenage years and as a secondary infection produces severe
convalescent blood samples were tested and DENV serotype DENV illness. The age for severe infection ranges between
determined by isolation and PCR [88, 119, 120]. DENV 5 and 15 years and both sexes are equally affected [115].
infection incidence and serotype circulation varied geo- In a recent study of children in Bangkok with severe infec-
graphically and from year to year with one school having a tion, ages ranged from 18 months to 15 years with a mean
DENV-3 outbreak and several miles away another school age of 8.6 years. The mean age of children diagnosed with
having a DENV-2 outbreak. The following year the same DF was 8.5 years and 8.7 years for children with DHF
schools had outbreaks with different DENV serotypes. These [125]. Similar age and sex distributions are seen in the
studies demonstrate the complexity and remarkable diversity regions of the Americas where DENV infections are
in DENV serotype transmission. A recent study of DENV endemic [126].
364 S.J. Thomas et al.

5.4.1 Phenomenon of the Increasing Median symptomatic illness, 53 % were classified as having DHF.
Age in SE Asia Overall incidence was 6.3 % and a symptomatic-to-
First in Thailand and then in other Southeast Asian countries, asymptomatic ratio of 1:8. The risk for DHF in secondary
it was observed that the median age of severe dengue illness infection was ≥6.5. Table 15.2 summarizes the incidence of
was increasing over time. To determine the reasons for this infection and the symptomatic-to-asymptomatic ratio in the
observation, an analysis was performed from 72 provinces of various cohorts. An important result of these prospective
Thailand to examine the force of infection and demographic studies has been a better understanding of the full burden of
and climactic variables [127]. The force of infection was subclinical infection, particularly in those patients who con-
found to have declined by 2 % each year, with the strongest tribute to the pool of potential hosts for mosquito infections
predictor for this change being the median age of the popula- but go unreported in most surveillance programs. Another
tion. The increase in median age was explained by a reduced value of the prospective studies is to determine the full eco-
birth rate and a shift in the population age structure to include nomic burden of dengue disease. In the Thai studies, inci-
older individuals. Lower birth and death rates are thought to dence and economic impact were used to calculate the
decrease the number of susceptible individuals in the popula- disability-adjusted life years (DALYs) lost to DENV infec-
tion as a result of increased longevity of immune individuals. tions [117]. The mean DALYs per million population was
If true, other countries with shifts in the age of the population 465 per year which accounted for 15 % of DALYs lost to all
to older individuals may also see an increase in the age of febrile illnesses. Nonhospitalized patients were an underap-
severe dengue illness. preciated economic burden that represented a substantial
proportion of the overall disease burden; 44–73 % of the
total DALYs were lost to dengue each year. During high inci-
5.5 Occurrence in Different Settings dence years, the number of DALYs lost to dengue was
greater than that calculated for important tropical diseases
5.5.1 Prospective Studies in Families such as meningitis and hepatitis B and was three times
and Schools greater than reported by WHO [128].
A number of prospective studies conducted in families and
schools have played an important role in increasing our 5.5.2 Military
understanding of DENV transmission and the pathogenesis The US military considers developing a DENV vaccine a
of subclinical and severe infection. The readers are referred high priority and has sustained an effort to develop an effec-
to a review that discusses these studies in more detail [82]. tive vaccine for over 50 years [129]. The reason for this effort
Table 15.2 summarizes these studies and their salient study is the impact that DENV infection has had on US military
findings. Burke and colleagues performed a 2-year (1980– operations since the Spanish-American War [2, 130]. Before
1981) school-based study involving 1,757 children, ages WWII soldiers stationed in Panama and the Philippines
4–16 years, in Bangkok, Thailand [91]. DENV-antibody test- experienced major outbreaks of DENV infection. During
ing demonstrated a high antibody prevalence with 50 % of WWII several hundred soldiers stationed in Queensland,
the children having evidence of preexisting dengue antibody, Australia, were hospitalized with dengue, and outbreaks
indicating infection prior to the age of 7 years. The majority occurred in soldiers throughout the South Pacific. Severe
(87 %) of the students became infected during the study outbreaks occurred in US soldiers landing in Saipan in July
period but remained asymptomatic. Of those who developed 1944; the initial incidence of 300 cases per 1,000 rose to

Table 15.2 Prospective studies in families and schoolsa

Incidence (average) Symptomatic:
Population Age range Study Dengue Symptomatic Hospitalized asymptomatic
Study site size (years) period infection (%) dengue (%) dengue (%) ratio
Bangkok, Thailand [91] 1,757 4–16 1980–1981 11.8 0.7 0.4 1:8
Rayong, Thailand [90] 1,056 4–14 1980–1981 9.4 0.1
Yangon, Myanmar [167] 12,489 1–9 1984–1988 5.1 0.3
Yogyakarta, Indonesia [341] 1,837 4–9 1995–1996 29.2 0.6 0.4
Kamphaeng Phet I, Thailand [115] 2,119 7–11 1998–2002 7.3 3.9 1.0 1:0.9
Managua, Nicaragua [342] 1,186 4–16 2001–2002 9.0 0.85 1:13–1:6
Kamphaeng Phet II, Thailand [119] 2,095 4–16 2004–2006 6.1 2.0 0.5 1:3.0
West Java, Indonesia [343] 2,536 18–66 2000–2002 7.4 1.8 0.1 1:3
Adapted from Endy et al. [82]
a
Number in cohort tested for dengue antibody (incidence denominator)
15 Flaviviruses : Dengue 365

3,500 per 1,000 [2]. After WWII dengue was a significant previous stroke that contributed to the severity of dengue
cause of illness and noncombat injuries in soldiers stationed illness. In a recent study of 309 adults with DHF, causes of
in Vietnam and most recently during operations in both Haiti fatality were massive gastrointestinal (GI) bleeding, dengue
and Somalia. Soldiers deployed to Haiti in particular suf- shock syndrome (DSS), subarachnoid hemorrhage, and bac-
fered from acute dengue illness, which was responsible for teremia from Klebsiella pneumoniae [136]. The influence of
30 % of all febrile illness [131]. DENV infections continue comorbidities and the pathogenesis of severe dengue illness
to be documented in deploying military personnel. in adults should be a high priority for future research.

5.5.3 Risk to Travelers

Dengue poses a significant risk to travelers vacationing or 6 Pathogenesis and Immunity
working in dengue-endemic countries and is now considered
the most common cause of febrile illness in returning travel- Most human infections with DENV originate from a trans-
ers [132]. Information from the US Centers for Disease mission cycle between infected humans and mosquitoes of
Control and Prevention’s laboratory-based Passive Dengue the genus Aedes, of which A. aegypti constitutes the most
Surveillance System (PDSS) described trends in travel- important vector for epidemic dengue worldwide [137].
associated dengue from January 1, 1996 to December 31, Sylvatic transmission of DENV between nonhuman primates
2005. Of 1,196 suspected dengue infections in returning and other Aedes species has been documented in forest areas
travelers, 334 (28 %) were confirmed laboratory positive. of Africa and Asia [138, 139]. Some evidence suggests that
Regions where travelers were at particularly high risk for this cycle served as the original source of the viruses now
infection were the Caribbean, Mexico, Central America, and circulating in the human population, but the latter viruses
Asia [132]. Dengue in returning travelers was examined by have since evolved as separate lineages with little evidence
the GeoSentinel Surveillance Network [133]. Dengue cases of ongoing interaction between these transmission cycles.
demonstrated regional and seasonal peaks—June to Mosquitoes acquire DENV through feeding on the blood of
September for Southeast Asia, October for South Central viremic hosts. Viral infection of the mosquito midgut epithe-
Asia, March for South America, and August to October for lium leads to dissemination and later infection of salivary
the Caribbean. In Southeast Asia, for example, the annual gland tissue [140]. At that point, the mosquito is capable of
incidence of dengue in travelers increased from 50 cases per transmitting the virus to a vertebrate host during subsequent
1,000 in non-epidemic years to 159 cases per 1,000 during blood feedings and remains infectious for the remainder of
epidemic years [133]. its lifetime. The time required for the mosquito to become
infectious, or extrinsic incubation period, is modulated by
5.5.4 Adult Symptomatic Dengue Infection ambient temperature, with higher temperatures resulting in a
As noted, DENV infection in endemic countries due to the shorter extrinsic incubation period and therefore a greater
high force of infection is primarily a pediatric disease. potential for transmission [141].
However, DENV infection occurs at all ages. In countries DENV injected into the dermis during mosquito feeding
that experience sporadic DENV outbreaks because of recent most likely initially infects cells locally. Many different cell
introductions, such as islands, all ages are infected and severe types can be infected with DENV in vitro, including human
illness will occur in adults. Current knowledge of the patho- fibroblasts, but Langerhans cells or tissue macrophages are
genesis of adult DHF is limited to a few studies. A prospec- considered more likely initial targets of infection [142].
tive study performed in Thai patients in Phetchabun Additional components of the mosquito saliva may enhance
Provincial Hospital demonstrated significant clinical differ- infectivity, based on experiments in animal models, through
ences in adults as compared to children [134]. More com- mechanisms that are poorly defined [143]. The intrinsic (in
mon in adults than in children were manifestations such as humans) incubation period typically ranges from 4 to 9 days
petechiae, melena, headache, retro-orbital pain, joint pain, but can extend up to 14 days [141]. In experimental models,
myalgia, nausea, and emesis. A retrospective study of DENV is detected in local and regional lymph nodes and
Taiwanese adults and children during a DENV-2 outbreak in likely undergoes some replication there [144, 145]. Following
2002 demonstrated that adult patients presented more fre- the local/regional replication of DENV, systemic dissemina-
quently with arthralgias, myalgias, headache, and abdominal tion occurs. Viremia is detectable in nearly all symptomatic
pain as compared to children [135]. Adults had more severe infections, often reaching very high titers within the first few
thrombocytopenia, elevated alanine aminotransferase (ALT) days of symptoms [146, 147]. Dendritic cells, monocytes,
levels, and greater incidence of upper gastrointestinal bleed- and macrophages are considered the principal cellular tar-
ing and DHF. Clearly different in adults than in children are gets; there is also substantial evidence of infection of B lym-
the higher rates of comorbidities such as diabetes mellitus, phocytes and hepatocytes [148–150]. Virus is detected
hypertension, chronic obstructive pulmonary disease, and principally in the liver and tissues of the reticuloendothelial
366 S.J. Thomas et al.

system including the bone marrow [151]. There is limited increased, particularly in severe cases [164, 165]. Bleeding
evidence for infection of other cells types in vivo, including does not correlate well with the severity of thrombocytope-
mast cells, vascular endothelial cells, neurons, myocytes, nia, however. Other contributing factors may include coagu-
and platelets; however, the frequency and significance of lopathy due to clotting factor consumption and inhibitory
these phenomena remain controversial. antibodies recognizing cross-reactive determinants on clot-
Innate immune responses, especially the production of ting or hemostatic proteins [166]. The pathophysiologic
type I interferons (IFN), are induced in both infected cells basis of dengue-associated plasma leakage has been and
and in natural IFN-producing cells such as plasmacytoid remains a source of controversy. The competing hypotheses
dendritic cells [152, 153], and these responses likely account of a viral mechanism versus an immunologic mechanism for
for many of the systemic symptoms and signs of infection. this syndrome have largely been decided in favor of a prin-
Plasma levels of IFN follow kinetics similar to plasma viral cipally immunologic mechanism on the basis of strong epi-
titers and are often at or near peak levels at the onset of fever demiologic data showing an association with prior DENV
and systemic symptoms [154]. Although DENV has evolved exposure [91, 167]. Nevertheless, viral factors are obviously
several immune evasion mechanisms, inhibiting both IFN also involved, as some DENV genotypes appear to be inca-
production and response [155], studies in laboratory mice pable of triggering plasma leakage [168]. Elevated produc-
suggest that these innate responses are critical to the control tion of factors promoting vascular permeability—TNF-α
of DENV infection [156]. and vascular endothelial growth factor (VEGF) among
Activation of adaptive immune responses can be detected others—is clearly correlated with the extent of plasma leak-
within the first few days, with an increase in the circulating age [158]. The sources of these factors and reasons for their
levels of activated T lymphocytes, plasmablasts, NK cells, increased production remain unclear, however. High level
DENV-specific T lymphocytes and antibodies, soluble mark- viremia appears to be necessary to initiate this cascade of
ers of immune activation, and a wide array of both pro- events, but is not sufficient to cause plasma leakage.
inflammatory and anti-inflammatory cytokines [157–160]. Furthermore, plasma leakage occurs several days after the
This immune activation coincides with a rapid clearance of peak of viremia and instead closer in time to the period of
virus from both plasma and peripheral blood monocytes and rapid clearance of viremia; these observations support the
lymphocytes, as well as the most serious symptoms and immune-mediated model of pathogenesis [147]. Both
signs of dengue, including maximal thrombocytopenia, DENV-infected cells, such as monocytes, macrophages,
bleeding, elevation of hepatic transaminases, and plasma dendritic cells, and mast cells, and responding effector cells,
leakage. such as NK and T cells, produce factors that are capable of
Leukopenia, affecting both neutrophils and lympho- contributing to vascular permeability under experimental
cytes, is present throughout much of the symptomatic conditions [152, 169–171]. Given this variety of potential
phase, and its severity does not correlate with other mea- mechanisms, it is possible that no one single pathway
sures of disease severity [161]. Other common laboratory explains plasma leakage in all cases.
abnormalities include thombocytopenia and elevations in Patients who have recovered from acute dengue illness
serum aspartate aminotransferase (AST) and alanine ami- demonstrate antibodies and T lymphocytes that recognize
notransferase (ALT); these abnormalities reach their both serotype-specific and serotype-cross-reactive determi-
extremes late in illness, coincident with plasma leakage, nants. The principal targets of DENV-specific antibodies are
and correlate with the overall severity of illness. In fatal the E, pre-M, and NS1 proteins [172–174]. Antibodies to the
cases, the principal findings are serous effusions in pleural E protein mediate neutralization in vitro and in vivo to vari-
and peritoneal cavities, widely distributed microhemor- ous degrees determined by both the antibody affinity and the
rhages, and hepatocellular injury that can range from mild epitope targeted; antibodies directed at domain III of the pro-
to severe [162]. The paucity of histologic findings in the tein tend to display greater serotype specificity and neutral-
vasculature has suggested that the enhanced vascular per- ization activity [172]. Antibodies with lower affinity and
meability represents physiologic disturbance to a greater those with high affinity when present at concentrations below
extent than structural damage to blood vessel walls. Edema the threshold for neutralization form immune complexes with
of the lung parenchyma is uncommon, even in patients with virions and facilitate uptake into and productive infection of
severe plasma leakage, and may reflect overly aggressive immunoglobulin receptor-bearing cells in vitro and in vivo,
fluid administration rather than a consequence of DENV i.e., ADE [175]. In addition to increasing the mass of infected
infection per se. Inflammation of the brain, heart, and other cells, infection via ADE may also modify infection by (a)
tissues has been described in isolated cases or small case increasing the number of progeny virions produced by each
series but is atypical. infected cell and (b) inducing IL-10 and other cytokines via
DENV infection of bone marrow cells may in part explain signaling from the immunoglobulin receptor(s) [176].
the cytopenias [163], but platelet consumption is also Antibodies to pre-M bind only to immature or partially
15 Flaviviruses : Dengue 367

mature virions. Although a pre-M-specific monoclonal anti- infections [190]. However, a greater proportion of DENV-
body was reported to protect mice from a lethal DENV chal- specific T lymphocytes target more conserved regions of the
lenge [177], other studies have found these antibodies to have DENV proteome and show serotype cross-reactivity in their
poor neutralizing activity but to be capable of enhancing the responses.
infectivity of immature virions [178]. Antibodies to NS1, in The immune responses induced by DENV infection
contrast, are incapable of binding to virions; these antibodies described above can have either protective or pathological
bind both to soluble NS1, which is released from infected effects during the second or subsequent DENV infection.
cells and is found in the circulation, and to NS1 on the surface Protective immunity is mainly serotype specific. Protective
of infected cells [179, 180]. Binding of anti-NS1 antibodies immunity can be ascribed to high-avidity E protein-specific
to cells can trigger complement-mediated and/or cell-medi- neutralizing antibodies based on experimental challenge
ated cytotoxicity [181, 182]. Under some conditions, soluble studies in animal models [193]. However, in vitro assays of
NS1 can attach to the surface of uninfected cells, thereby neutralizing antibody are a poor measure of protective immu-
causing uninfected cells to be targeted by the same processes nity, especially in individuals who have had several prior
[183]. Some antibodies to NS1 also bind in a cross-reactive DENV infections, in whom antibody responses are highly
fashion to determinants on components of the coagulation serotype cross-reactive [116]. Antibodies to the pre-M and
pathway or platelet/endothelial cell surface proteins [166]. NS1 proteins have also shown protective efficacy in animal
Epitopes recognized by CD4 and CD8 T lymphocytes models, but the applicability of these findings to human
have been identified in all of the DENV proteins [184–186]. infection is unclear. Studies of protection mediated by
Several studies have found a concentration of epitopes in the DENV-specific T lymphocytes are more limited. However,
NS3 protein, however, raising the possibility that this protein one prospective cohort study conducted in Thailand found an
has features associated with higher levels of MHC class I and association between higher baseline DENV-specific T-cell
II presentation or has sequence constraints that limit its abil- IFN-γ responses and subsequent subclinical DENV infection
ity to mutate toward reduced immunogenicity. Upon contact [194], and a survey of healthy adults from Sri Lanka found
with DENV-infected cells, DENV-specific T cells secrete higher DENV-specific T-cell IFN-γ responses in subjects
predominantly type 1 cytokines (IFN-γ, TNF-α, and IL-2) with HLA alleles that were associated with a lower risk of
and lyse antigen-presenting cells [187, 188]. An intriguing DHF [186].
feature of the serotype-cross-reactive T-cell response is that Except for a short (several months) period of resistance to
the sequence differences between serotypes (usually 1–3 challenge with other serotypes, individuals who have had
residues of the 9 amino acids in the epitope) result in both one (primary) DENV infection are not only at risk for infec-
quantitative and qualitative changes in the effector functions tion with any of the other three serotypes, but they are actu-
expressed by T cells, sometime referred to as heterologous ally at increased risk for development of dengue-related
immunity [170, 171, 189, 190]. In particular, the production plasma leakage compared to individuals who have never had
of the antiviral cytokine IFN-γ is reportedly diminished, prior exposure to DENV [20, 82, 91]. This finding is the
while the production of pro-inflammatory cytokines such as principal basis for proposing the existence of pathologic
TNF-α and MIP-1β is unchanged. immune responses to DENV. Both DENV-specific antibod-
The immune response differs substantially between the ies and T cells have pathologic potential, through mecha-
first (primary) DENV infection and subsequent (secondary, nisms discussed above. The observation by Halstead of
tertiary, and quarternary) DENV infections. The differences enhanced DENV infection due to ADE (see above) provided
are attributed to two factors. First, at the time of the second- a model for antibody-mediated immunopathology [195] and
ary and subsequent infections, memory T and B lymphocytes has also been invoked to explain dengue-associated plasma
are already present, having been induced by the earlier pri- leakage among infants in endemic countries, who acquire
mary infection. Second, because reinfection with the same DENV-specific antibodies transplacentally from their
serotype is not known to occur, secondary and subsequent DENV-immune mothers [114]. At odds with this model are
infections involve antigenic differences from the earlier the observations that (a) plasma leakage occurs several days
infection. Anti-DENV IgG antibodies rise earlier and reach after the peak viremia, (b) anti-DENV antibodies are neither
dramatically higher peak titers, while IgM antibody responses necessary nor sufficient for the occurrence of plasma leak-
are blunted, and the antibodies produced are predominantly age, and (c) preinfection serum ADE activity has not been
serotype cross-reactive. Most of the serotype-cross-reactive correlated with disease severity [196, 197]. Immune activa-
antibodies display lower affinity for epitopes on the newly tion and cytokine production thus appear to be a critical link
infecting DENV than for the previously encountered DENV, in the pathogenetic cascade. One study has suggested an
however [174, 191, 192]. The frequencies of circulating association with T-cell production of TNF-α [198], but the in
DENV-specific T lymphocytes and their expression of acti- vivo evidence for a specific T-cell mechanism remains
vation markers are similarly high in primary and subsequent extremely limited.
368 S.J. Thomas et al.

7 Patterns of Host Response 7.2 Dengue Hemorrhagic Fever

7.1 Common Clinical Features Plasma leakage is the most worrisome manifestation of
DENV infection, although other less common syndromes
Many DENV infections resolve without a recognized illness, can be life threatening. For historical reasons, this syndrome
particularly in children [115]. Among patients with is referred to as dengue hemorrhagic fever (DHF) and has
laboratory-confirmed dengue illness, constitutional symp- been defined based on the presence of four cardinal features
toms are most often reported, including fever, headache, eye [3, 5, 18]: fever lasting 2–7 days; increased vascular perme-
pain, body pain, and joint pain (60–90 % of patients) [199]. ability as evidenced by hemoconcentration (20 % or greater
Rash, typically macular or maculopapular, is reported in rise in hematocrit above baseline value), pleural effusion, or
approximately half of the subjects. Gastrointestinal symp- ascites [15]; marked thrombocytopenia (platelet count
toms—nausea, vomiting, or diarrhea—occur in 30–50 % of 100,000 cells/mm3 or lower); and a hemorrhagic tendency as
patients, and respiratory symptoms, including cough, sore demonstrated by a positive tourniquet test or spontaneous
throat, and nasal congestion, are somewhat less common bleeding. The term dengue shock syndrome (DSS) is used
(~1/3 of patients). The frequency of symptoms is influenced when shock is present along with these four criteria [18].
by the patient’s age, sex, and previous history of DENV The increase in vascular permeability leading to plasma
exposure (primary vs. secondary DENV infections) [199]. leakage in DHF develops rapidly, over a period of 24–48 h,
All symptoms are less frequent in children. Joint pain, body and characteristically occurs between 3 and 7 days after the
pain, and rashes are more commonly reported in females. onset of illness [204]. This time point coincides with the dis-
Rashes have been more frequently reported in primary appearance of fever, the nadir of platelet count, and the peak
DENV infections, whereas constitutional and gastrointesti- of serum aminotransferase elevations [205]. Abdominal pain
nal symptoms have been more common in patients experi- is reported shortly before the onset of plasma leakage in
encing a secondary DENV infection. ~60 % of patients with DHF [206–208]. Intense abdominal
Fever accompanied by prominent headache, retro-orbital pain, persistent vomiting, change from fever to hypothermia,
pain, and muscle and joint pains forms the syndrome of clas- and marked restlessness or lethargy, especially occurring
sical dengue fever (DF), which had historically evoked more during this window of time, have been proposed as “alarm
descriptive terminology such as “breakbone fever” [39]. signs” for clinicians of possible impending shock [209].
However, these classic symptoms have been reported in a The hemorrhagic manifestations are variable in severity
minority of cases in studies of travelers or military personnel among patients with DHF. Spontaneous petechiae or ecchy-
[131, 200–202]. The duration of symptoms in DF typically moses have been reported in approximately one half of cases
ranges 5–7 days [202], with some patients showing a bipha- with DHF [206, 208]. The tourniquet test, performed by
sic (“saddleback”) fever curve. Periods of marked fatigue inflating a blood pressure cuff on the arm to a level halfway
lasting days to weeks after the acute illness are occasionally between systolic and diastolic pressure for 5 min, elicits
reported, especially in adults. petechiae in the remaining cases. Less common sites of
Hemorrhagic manifestations occur often in patients with spontaneous bleeding include the nose, the gastrointestinal
DF, with spontaneous bleeding in as many as two thirds of tract (hematemesis more often than melena), and genitouri-
cases [158]. The skin (petechiae) and nose are the most com- nary tract (more often in women).
mon bleeding sites; gastrointestinal or genitourinary bleed- Leukopenia and thrombocytopenia are common labora-
ing is less common [202]. In rare cases, bleeding is severe tory findings in DHF, the latter being a component of the
enough to be life threatening, but this clinical presentation WHO case definition. Serum transaminase levels are signifi-
needs to be differentiated from dengue hemorrhagic fever cantly different between DF and DHF, and they have been
(DHF), described below. proposed as useful markers of illness severity prior to clini-
The physical examination in patients with DF is generally cally evident plasma leakage [203, 210].
nonspecific, with fever and rash being the most common find- There continues to be debate as to whether DF and DHF
ings. Typical laboratory findings include leukopenia, throm- are different diseases, implying distinct pathogenesis mecha-
bocytopenia, and elevated hepatic transaminases [131, 161, nisms, or whether DHF represents an extreme along a con-
203]. Thrombocytopenia can be profound, with platelet counts tinuous spectrum of disease severity encompassing both
<100,000 cells/mm3 observed in one quarter to one half of patient populations. Some investigators have highlighted
patients. Serum aspartate transaminase (AST) levels are char- cases that have been difficult to classify, usually where
acteristically more elevated than alanine aminotransferase plasma leakage is apparent, but the other three criteria of
(ALT) levels; elevations are usually modest (2–5 × upper limit DHF were not all met [211]. Although such cases occur, they
of normal), but marked elevations (5–15 times the upper limit clearly represent a minority of patients, as most patients with
of normal) are occasionally noted [202, 203]. plasma leakage display the full picture of DHF [158].
15 Flaviviruses : Dengue 369

7.3 Atypical Dengue Syndromes dengue illness, based on the model of immune-mediated
pathogenesis described above. In controlled clinical trials,
In rare cases, liver failure or neurologic dysfunction is the corticosteroids have not proven effective either in patients
predominant clinical feature in a patient with DENV infec- with established shock [221] or when given earlier in illness
tion [212–215]. Liver failure may have been a consequence [222]. Other adjunctive therapies, for example, pentoxifyl-
of prolonged shock in some cases, rather than a direct viral line or intravenous immunoglobulins, have only been tested
effect. A wide range of neurological manifestations have in uncontrolled case series or small clinical trials.
been described in patients with laboratory-confirmed DENV
infection, including encephalopathy, seizures, pure motor
weakness, mononeuropathies, polyneuropathies, Guillain- 8.2 Vector Control
Barré syndrome, and transverse myelitis [212, 213, 216,
217]; some patients have none of the characteristic features Efforts to control endemic and epidemic dengue transmis-
of DF or DHF. A causal association has been supported by sion and reduce disease burden have been limited largely to
the occasional detection of DENV in cerebrospinal fluid. vector control and personal protective measures to reduce
Myocarditis, cholecystitis, and retinal vasculitis [218], have human-vector interaction [223]. The World Health
been reported in isolated patients with serologic evidence of Organization (WHO) promotes a concept of Integrated
acute dengue, but a causal association with DENV is not Vector Management (IVM) with the aim to improve efficacy,
definitively established since subclinical DENV infections cost-effectiveness, ecological soundness, and sustainability
are common in endemic areas. of disease-vector control. Program pillars include environ-
mental management (i.e., reduce potential breeding contain-
ers, modify human behavior), chemical control (i.e.,
7.4 Differential Diagnosis larvicides/adulticides and residual and space spraying), and
biologic control (i.e., fish, predatory copepods) [224]. There
The clinical features of DF are largely nonspecific. The dif- are historic examples of successful vector control campaigns.
ferential diagnosis for this syndrome includes influenza, In the late 1940s, Dr. Fred Soper led an A. aegypti eradica-
enteroviral infection, measles, and rubella. In residents of or tion effort in the Americas; between 1948 and 1962, the spe-
travelers to endemic areas, malaria, leptospirosis, and cies was eliminated from 21 countries. Unfortunately,
typhoid fever are also relevant for consideration [219]. Over success was short lived due to reduced funding, lack of pro-
90 % of patients in dengue-endemic areas who meet all of gram leadership, lack of political will, and a deficit of highly
the criteria in the case definition for DHF have serologic evi- trained people to design and execute the programs [223]. In
dence of DENV infection [158]. Bacterial sepsis, leptospiro- 1973, Singapore completed the implementation of a vector
sis, malaria, and other viral hemorrhagic fevers are worthy of control program to reduce the premise index (i.e., the per-
consideration, however. centage of houses infested with A. aegypti larvae and/or
pupae), bringing the index to ~2 % with an associated
15-year period of low dengue disease incidence. However, in
8 Control and Prevention the 1990s, despite maintaining the premise index ~2 %, den-
gue surged [225]. Singapore’s experience highlighted the
8.1 Treatment complexity of managing vector control programs and the
need for a more evidenced-based and data-driven approach
There is no licensed therapeutic agent or vaccine to prevent to designing and implementing control programs. In addi-
dengue disease. The recommended treatment for patients tion, a more strategic application of tools capable of defining
with suspected acute DENV infection includes supportive and modeling dengue disease and vector population dynam-
care, acetaminophen for relief of fever and aches, and moni- ics in space and time was needed.
toring to detect plasma leakage and other severe manifesta- Interesting advances have been made in vector-based
tions at the earliest stage possible. Aspirin and nonsteroidal interventions to include biocontrol and genetic modification
anti-inflammatory drugs are best avoided to minimize the of vector populations [226]. One example is the proposed
risk for bleeding and Reye syndrome. There are no antiviral use of Wolbachia, an endosymbiotic bacterium estimated to
drugs available at the present time with demonstrated effi- be present in up to 66 % of insect species [227]. The bacte-
cacy in dengue. Chloroquine, which shows some antiviral rium infects the gonads, where it ensures transmission to the
activity in vitro, was ineffective in a high-quality clinical next host generation (from mother to egg) and manipulates
trial [220]. reproductive capabilities [226]. The manipulations directly
There has been substantial interest in the potential value or indirectly benefit the infected females assisting with its
of immunomodulation as a strategy to reduce the severity of spread through host populations [228]. Other changes
370 S.J. Thomas et al.

associated with Wolbachia infections include a shortened experienced early success, but tetravalent formulation and
lifespan, altered locomotor activity, and poor blood feeding balancing immunogenicity (i.e., neutralizing antibody
in mosquitoes [229–231]. Wolbachia use to control insect responses to each dengue virus type) with safety proved dif-
populations has been explored since 1967 [232]. Three ficult [251–254].
Wolbachia strains have been successfully introduced into the The Walter Reed Army Institute of Research (WRAIR)
A. aegypti populations, setting the stage for release studies also developed LAV candidates by serial PDK passage of
[230, 233, 234]. Mosquito population reduction and suppres- each dengue virus type with final passages in fetal rhesus
sion, decreased pathogen transmission, and/or decreased lung cells (FRhL). Early development efforts identified den-
pathogen replication in mosquito species are desirable out- gue virus strains and promising formulations combining
comes [226]. Recent, small-scale releases demonstrated the variations of attenuation and dengue virus antigen concen-
ability for genetically modified males to persist in nature and trations [255–263]. A single formulation was tested in Thai
reduce wild-type populations [235, 236]. Regulatory issues, schoolchildren and toddlers. Vaccination was well tolerated
proof-of-concept trial design, and sustainability are a few of and, although variable neutralizing antibody responses were
the issues requiring extensive thought prior to large-scale use observed across cohorts, was sufficiently immunogenic to
[237]. pursue continued development of the candidate [264, 265].
Vector control through any means will require community Newly derived vaccine lots were tested in adults in the United
buy-in and, in some cases, active participation. Political will States and in Thailand and Puerto Rico across a broad age
and sustained financial resources will be required for any range (12 months to 50 years). There were no overt safety
effort to be effective in reducing disease burden. Providing signals in over 300 vaccine recipients, and rates of serocon-
scientists and operational personnel state-of-the-art educa- version were moderate to high [264, 266]. Although the can-
tion and access to advanced epidemiologic tools (global didate demonstrated promise, the WRAIR and its corporate
positioning systems, modeling software, etc.) will increase partner indefinitely suspended development in pursuit of a
the likelihood of program success. superior target product profile (i.e., shorter dosing schedule).
The US National Institutes of Health (NIH) constructed
viable cDNA clones of DENV-4 and induced a 30-nucleotide
8.3 Vaccines deletion in the 3′ untranslated region (i.e., directed mutagen-
esis). The result was lower viremia levels than the parental
The dengue vaccine pipeline in 2013 is robust; the following strain and retained immunogenicity. Numerous monovalent
is a review of the approaches currently being tested in human vaccine candidates were tested in phase 1 clinical trials to
trials. Initial development efforts began in 1929 with unsuc- identify optimal candidates for tetravalent formulation [267–
cessful attempts to produce an inactivated virus vaccine 275]. Tetravalent candidates (admixtures, TetraVax-DV) were
using phenol, formalin, or bile [46]. During World War II, prepared and are being tested in phase 1/2 clinical trials [276].
live attenuated virus (LAV) vaccines were explored by seri- The insertion of dengue preM and E genes into the cDNA
ally passaging DENV-1 and DENV-2 strains in suckling backbone of YF 17D as a vaccine development platform (i.e.,
mouse brain [45, 238, 239]. Mouse brain-derived candidates chimera) was pioneered at the St. Louis University Health
were eventually abandoned for cell culture-based vaccines. Sciences Center, further developed by Acambis, Inc., and
Currently, six different vaccine approaches have been tested licensed for manufacture to Sanofi Pasteur [277–281]. Phase
in human clinical trials, and a single candidate is in phase 3 1 clinical trials of monovalent vaccine preparations in
clinical trials. YF-naïve and YF-primed volunteers demonstrated accept-
Flaviviral live attenuated virus (LAV) vaccines (yellow able safety and immunogenicity profiles (i.e., neutralizing
fever (YF), Japanese encephalitis) have proved to be safe and antibody responses) [282, 283]. Expanded trials with tetrava-
durably efficacious [240–243]. LAV vaccines replicate in the lent preparations in volunteers of various ages, genetic back-
recipient, presenting all viral antigens in the vaccine construct, grounds, and flavivirus priming status (i.e., preexisting
and eliciting both antibody and T-cell responses, resembling immunity to YF, dengue, Japanese encephalitis) demon-
those seen after natural infection. A tetravalent vaccine strated a consistently excellent safety profile and robust,
approach (i.e., DENV-1 + DENV-2 + DENV-3 + DENV-4) is balanced neutralizing antibody profiles [284–288]. Sanofi’s
designed to induce primary-type immune responses to all ChimeriVax (a construct containing yellow fever and dengue
four dengue viruses simultaneously. The first major effort components) was the first dengue vaccine candidate tested in
at live attenuated dengue vaccine development was made at a clinical endpoint efficacy study. The phase 2b trial was an
Mahidol University in Bangkok; investigators used the clas- observer-blinded, randomized, controlled, single-center trial
sical method of serial passage of virus in dog (PDK) and in healthy Thai schoolchildren (N = 4,002) aged 4–11 years
nonhuman primate (PGMK) cell lines [244–250]. The effort who were randomly assigned (2:1) to receive three injections
15 Flaviviruses : Dengue 371

of dengue vaccine or control (rabies vaccine or placebo) at 0, PIV formulation adjuvanted with GSK’s proprietary adju-
6, and 12 months. All subjects were followed for dengue ill- vant systems. Phase 1 trials in the United States and Puerto
ness. The primary objective was to assess protective efficacy Rico are underway.
against virologically confirmed, symptomatic dengue, irre- DNA vaccines consist of one or more plasmids containing
spective of severity or serotype, occurring 1 month or longer DENV genes reproduced to high copy number in bacteria
after the third injection. Although the vaccine was safe and such as E. coli. The plasmid contains a eukaryotic promoter
neutralizing antibody responses were moderate to high, and termination sequence to drive transcription and presenta-
overall efficacy was 30.2 % (95 % CI −13.4 to 56.6) and dif- tion to the immune system. DNA vaccine advantages include
fered by serotype [289]. The results of this trial were disap- ease of production, stability and ability to be transported at
pointing and highlighted the numerous, still unanswered, room temperature, ability to accept new genes, ability to
questions which exist regarding immuno-protective profiles immunize against multiple pathogens with a single construct,
(see below). Phase 3 trials in Latin America and Asia are and reduced reactogenicity [309]. A DENV-1 monovalent
ongoing. DNA vaccine trial enrolled 22 flavivirus-naïve US volun-
The US Centers for Disease Control and Prevention teers. None of the low-dosage recipients and only five of 11
(CDC) developed a tetravalent chimeric dengue vaccine by high-dosage recipients developed neutralizing antibodies.
introducing DENV-1, DENV-3, and DENV-4 prM and E The safety profile was acceptable and supported exploration
genes into cDNA derived from an attenuated LAV DENV-2 of an adjuvanted DNA dengue vaccine [310].
component [290–294]. Dengue-dengue chimeras were for- In addition to the dengue vaccine candidates listed above
mulated as a DENV-1/DENV-2, DENV-2, DENV-3/DENV- in clinical development, there are also numerous in preclini-
2, and DENV-4/DENV-2 tetravalent vaccine candidates cal development (i.e., mice and nonhuman primate studies).
(DENVax) and licensed to Inviragen, Inc. [295, 296]. Phase Without an animal model of disease, a human challenge
1 clinical trials in dengue-endemic and dengue-non-endemic model, or a correlate of protection, it will be a slow and ardu-
areas are underway [297]. Another approach to producing ous process to advance candidates through efficacy trials.
viral vaccines is the use of inactivated whole virus or viral Despite this, the field is hopeful a dengue vaccine can be
subunits. Such vaccines have potential advantages to include licensed in an endemic country within 5 years.
the inability to revert to a more pathogenic phenotype, they
are unlikely to produce immune interference when combined
into a tetravalent formulation, and, theoretically, there could 9 Unresolved Problems
be fewer safety issues. Inactivated flaviviral vaccines have
been licensed and are in wide use to prevent Japanese Unraveling the virologic and immunologic complexities of
encephalitis and tick-borne encephalitis [298–300]. the dengue viruses, virus-vector-host interactions, and den-
These vaccines have certain potential disadvantages. gue disease has been challenging. The existence and co-
Killed or subunit vaccines raise antibodies to only a portion circulation of four antigenically distinct viruses, the absence
of the structural proteins and normal virion-based structural of an animal model of disease, the absence of a dengue
conformation. High concentrations of antigens and multiple human infection model, and gaps in our comprehensive
doses may also be required to induce a potent and protective understanding of what constitutes immunopathologic ver-
immune response. Another concern is the potential for an sus immuno-protective profiles have contributed to difficul-
adverse response resembling the occurrence of atypical mea- ties in prognosticating, in a clinically relevant way, dengue
sles and respiratory syncytial virus (RSV) infection follow- disease severity and the development of anti-dengue virus
ing vaccination and subsequent wild-type exposure [301, therapeutics and protective vaccine candidates. The discon-
302]. Merck & Co. is developing a dengue vaccine candidate nect between vaccine immunogenicity, as measured using a
produced in a Drosophila S2 cell expression system [303– standard plaque reduction neutralizing antibody test, and
305]. A single DENV-1 monovalent trial has been completed clinical outcome following wild-type virus exposure further
and a phase 1 tetravalent trial is under way [297]. complicates the field. The following are a few of the knowl-
The WRAIR developed a purified inactivated virus (PIV) edge gaps and areas of current scientific interest and
dengue vaccine candidate by inactivating with formalin each exploration.
of the dengue virus types [305–308]. A phase 1 trial of a There is no well-characterized and comprehensive animal
monovalent DENV-1 PIV adjuvanted with alum was com- model of dengue disease. However, significant resources
pleted in a small number of US flavivirus-naïve volunteers. have been allocated and development progress has been
The candidate had an acceptable safety profile with low to made [311, 312, 313, 314]. Two models currently being
moderate immunogenicity. GlaxoSmithKline Vaccines explored include the hu-HSC mouse model created by
(GSK) and the WRAIR are currently testing a tetravalent human hematopoietic stem cell (HSC) engraftment and the
372 S.J. Thomas et al.

BLT mouse model prepared by co-transplantation of the been difficult [322]. Newer methods of measuring neutral-
human fetal liver, thymus, and HSC [315]. Humanized izing antibodies have been proposed to increase throughput,
mouse models with a transplanted human immune system reduce human error, reduce assay-to-assay variation, and
have the ability to generate T cells, B cells, macrophages, increase the biologic and physiologic relevancy of the mea-
dendritic cells, and natural killer (NK) cells. Theoretically, surement [323–325].
these mice possess the immunologic “building blocks” Variation also permeates the measurement of cell-
required to generate the pro-inflammatory cascade of immu- mediated immune responses. It is generally accepted that
nologic events required to induce clinical outcomes such as components of the cellular immune system contribute to
plasma leakage and hemorrhage. Until further development both the disease severity and the immune profile which pro-
and optimization is achieved, mouse models will be absent tects against disease following homotypic reinfection [186,
from the “critical path” components of advanced drug or 326–333]. What is less clear is the sequence of immunologic
vaccine development programs. events which leads to one outcome (antiviral activity, asymp-
Following dengue virus exposure, nonhuman primates tomatic, or highly attenuated illness) versus another (pro-
develop infection as evidenced by measurable replicating inflammatory state, disease). How components of the
peripheral viremia and neutralizing antibody and cellular humoral and cellular immune systems interact qualitatively,
immune responses. Nonhuman primates do not consistently quantitatively, and kinetically during in vivo infection is
develop objective and measurable clinical or biochemical incompletely understood. Furthermore, the immunologic
markers of disease (i.e., elevated temperature, failure to feed, events that may determine the resulting clinical phenotype
lethargy, rash, depressed white blood cell and platelet count, likely occur prior to the onset of symptoms. As a result, when
evidence of plasma leakage, or hemorrhage) [316–318]. A and how to collect the appropriate biologic samples follow-
single study reported a hemorrhagic phenotype following ing infection remains unknown, and important immunologic
intravenous administration of a high-titer DENV-2 strain, but events are not measured.
these findings have not been reproduced [319]. Despite this, Key to the study of dengue immunopathogenesis is being
the nonhuman primate dengue virus infection and viremia able to link biologic samples (i.e., whole blood, peripheral
model has been a required and necessary component of den- blood mononuclear cells, etc.) collected before, during, and
gue vaccine development programs and the transition after dengue virus exposure and to associate these with vari-
between preclinical and clinical development phases. How ous clinical phenotypes observed following infection (i.e.,
informative the nonhuman primate model is as it relates to asymptomatic infection, dengue fever, severe dengue).
predicting human responses to dengue virus exposure or vac- Establishing a correlate and/or surrogate of protection is
cination with candidate dengue vaccines remains unclear. probably not possible without this approach. The complexity
The observation of low clinical efficacy against DENV-2 of this task is not trivial as it involves establishing prospec-
infection following vaccination despite protective efficacy in tive cohorts in dengue-endemic areas with sustained and
nonhuman primates underscores the need to rethink the util- relatively high annual clinical attack rates (1.5–2.0 %). To
ity of the model and the need to further optimize or explore complete comprehensive immunologic evaluations also
other options [249, 279]. requires a sample with a significant volume of biologic mate-
Identifying which cytokines, factors, and/or other cir- rial, a challenge in places where dengue disease is preferen-
culating immune mediators contribute to clinical disease tially experienced by toddlers and children. Prospective
or protection from the same is one challenge, and how to studies are performed over multiple years and are very costly.
best measure these is another. Neutralizing antibodies have Despite these challenges, numerous cohorts have been estab-
been associated with protection from other flaviviral diseases lished in Asia and Latin America and are making important
such as yellow fever and Japanese encephalitis [320]. It is contributions to our understanding of dengue [82, 115, 197,
therefore reasonable to assume that neutralizing antibodies 334–339].
play a similarly protective role in dengue. Measurement of Additional challenges to the dengue field include under-
neutralizing antibodies has classically been accomplished standing (1) how viral diversity over time and space may or
using a plaque reduction neutralization test (PRNT) [321]. may not impact disease severity or what diversity means for
Although very informative, properly performing and inter- developing a protective vaccine, (2) immune response matu-
preting PRNT results requires advanced skill and experi- ration following infection over time and the impact of wan-
ence. Variability among laboratories and technicians has ing immunity on both durable protection from disease and
been observed. Furthermore, the assay lacks robustness with vaccination policies once a vaccine is available, and (3) the
minimal modifications in assay reagents or testing condi- role of protective and non-protective heterotypic immunity
tions producing significant variability in results [105]. It [29, 340]. It is possible that the world will have a safe, effica-
is for these reasons direct comparison of immunogenicity cious, and effective dengue vaccine available before many of
readouts between vaccine candidates and developers has the above questions have been answered.
15 Flaviviruses : Dengue 373

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 Flaviviruses: Yellow Fever, Japanese
B, West Nile, and Others 16
Stephen J. Thomas, Luis J. Martinez, and Timothy P. Endy

1 Introduction Yellow fever virus (discussed in detail in this chapter) was

the first arbovirus identified in the 1800s as responsible for
Historically and currently the flaviviruses are important large epidemics of hemorrhagic fever in Africa and North,
human pathogens both as endemic viruses restricted to spe- Central, and South America. By 1960 scientists recognized
cific geographic areas and as emerging pathogens. Yellow serologically two distinct arboviruses: the group A arbovi-
fever, West Nile virus, and the dengue viruses (not discussed ruses now known as the family Togaviridae and the group B
in this chapter) represent previous, current, and future impor- arboviruses, renamed the family Flaviviridae [1]. Serologic
tant emerging pathogens that have produced large epidemics comparisons among the flaviviruses revealed cross-reac-
and human deaths. Why are these viruses such a threat to tivity within groups distinguishing the flaviviruses as mos-
human populations as emerging pathogens? Several impor- quito borne, tick borne, or nonvectored [2]. According to the
tant factors interplay to create these pathogens as agents International Committee on Taxonomy of Viruses, a sub-
of emerging diseases. As mostly arthropod-borne viruses group of the Division of Virology of the International Union
(arboviruses), they have complex life cycles involving both of Microbiology Societies, the Family Flaviviridae is com-
arthropods and vertebrate hosts with a life cycle involving all prised of three genera: Flavivirus, Hepacivirus, and Pestivirus
life stages of the arthropods (mosquitoes, ticks, and midges) (http://ictvonline.org/index.asp). Information on their isola-
and their reservoirs of vertebrates (birds or rodents) and ulti- tion, morphology, sensitivity to inactivation by chemicals,
mately higher vertebrates (humans) through the bite of the arthropod vectors, vertebrate hosts, laboratory propagation,
infected arthropod vector. The flaviviruses all contain ribo- serologic reactions, geographic distribution, clinical mani-
nucleic acid (RNA) as their genetic core and as such have festations, and epidemiology is found in the International
a high rate of mutations and thus adapt quickly to changes Catalogue of Arthropod-Borne Viruses, compiled by the
in vector competence and the environment. Climate change, American Committee on Arthropod-Borne Viruses (ACAV)
urbanization, and increasing ease of travel have created [3]. This exhaustive reference source has been used freely in
opportunities for the vector to spread and expand into human preparing the text that follows. Hepacivirus and Pestivirus
populations. The combination of these factors produces are discussed elsewhere in this textbook, and the focus of
a family of viruses that can change and emerge quickly as this chapter will be on the pathogenic viruses within the gen-
important human pathogens. era Flavivirus. The dengue viruses are discussed in a sepa-
rate chapter. Within the genus Flavivirus there are 53 species
of viruses and displayed in Table 16.1.
Despite their species variation, the flaviviruses have a
S.J. Thomas, MD • L.J. Martinez, MD remarkable genetic conservation throughout its genus [4].
Viral Diseases Branch,
The genome is a single positive-stranded RNA, 11 kilobases
Walter Reed Army Institute of Research,
503 Robert Grant Ave, Silver Spring, MD 20910-7500, USA in length, encoding the viral proteins from an open reading
e-mail: [email protected]; frame (ORF) of over 10,000 bases of approximately 3,434
[email protected] amino acids [4]. The proteins encoded from the ORF is 5′–C-
T.P. Endy, MD, MPH (*) prM (M)-E-NS I-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′.
Infectious Disease Division, Department of Medicine, The structural proteins C (capsid), M (membrane), and E
State University of New York, Upstate Medical University,
(envelope) comprise the outer coat of the flaviviruses and
725 Irving Avenue, E. Adams St., CPOB Suite 304,
Syracuse, NY 13210, USA the epitopes responsible for attachment to host cells and
e-mail: [email protected] cell entry. The nonstructural proteins include the highly

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 383

DOI 10.1007/978-1-4899-7448-8_16, © Springer Science+Business Media New York 2014
384 S.J. Thomas et al.

Table 16.1 Taxonomy of viruses of the family Flaviviridae, genera Flavivirus

Group (by vector) Abbreviation Geographic distribution Principal vector species Human disease
Tick-borne viruses
Mammalian tick-borne virus complex
Alkhurma hemorrhagic fever virus AHFV Egypt, Sudan Camel tick Hemorrhagic fever
Gadgets Gully virus GGYV Australia Ixodes uriae Unknown
Kadam virus KADV Uganda, Saudi Arabia Rhipicephalus pravus Unknown
Kyasanur Forest disease virus KFDV India, China Haemaphysalis spinigera Hemorrhagic fever
Langat virus LGTV Malaysia, Thailand, Siberia Ixodes granulatus Fever
Omsk hemorrhagic fever virus OHFV Russia and Central Asia Dermacentor pictus Hemorrhagic Fever
Powassan virus POWV Canada and Northern United States Ixodes spp. Encephalitis
Royal Farm virus RFV Afghanistan Argas spp. Unknown
Karshi virus KSIV Central Asia Ornithodoros papillipes Encephalitis
Tick-borne encephalitis virus TBEV Europe, Asia Ixodes spp. Encephalitis
European subtype Europe, Asia Ixodes spp. Encephalitis
Far Eastern subtype
Siberian subtype
Louping ill virus LIV United Kingdom Ixodes spp. Encephalitis
Irish subtype Ireland Ixodes spp. Encephalitis
British subtype United Kingdom Ixodes spp. Encephalitis
Spanish subtype Spain Ixodes spp. Encephalitis
Turkish subtype Turkey Ixodes spp. Encephalitis
Mosquito-borne viruses
Aroa virus complex
Aroa virus AROAV Venezuela Unknown Unknown
Bussuquara virus BSQV Brazil, Colombia, Panama Culex spp. Fever
Iguape virus IGUV Brazil Unknown Unknown
Naranjal virus NJLV Ecuador Culex spp. Unknown
Dengue virus complex
Dengue virus
Dengue virus type 1 DENV-1 Tropical and subtropical regions Aedes aegypti for all Fever, Hemorrhagic
Dengue virus type 2 DENV-2 of the world for all fever, encephalitis
Dengue virus type 3 DENV-3
Dengue virus type 4 DENV-4
Kedougou virus KEDV Senegal, Central Africa Aedes spp. Unknown
Japanese encephalitis virus complex
Cacipacore virus CPCV Brazil Unknown Unknown
Koutango virus KOUV Senegal, Central Africa Aedes spp. Unknown
Japanese encephalitis virus JEV Asia Culex spp. Encephalitis
Murray Valley encephalitis virus MVEV Australia Culex annulirostris Encephalitis
Alfuy virus ALFV Australia Unknown Unknown
St. Louis encephalitis virus SLEV North, Central, and South America Culex spp. Encephalitis
Usutu virus USUV Africa Mosquitoes Fever, rash
West Nile virus WNV Worldwide Culex spp. Encephalitis
Kunjin virus KUNV Australia, Asia Culex spp. Fever, rash
Yaounde virus YAOV Cameroon Culex spp. Unknown
Kokobera virus complex
Kokobera virus KOKV Australia Culex annulirostris Unknown
Stratford virus STRV Australia Aedes vigilax Unknown
Ntaya virus complex
Bagaza virus BAGV Africa Culex spp. Fever
Ilheus virus ILHV Central and South America Mosquitoes Fever
Rocio virus ROCV Brazil Mosquitoes Encephalitis
Israel turkey meningoencephalitis virus ITV Israel Unknown Unknown
Ntaya virus NTAV Africa Mosquitoes Fever
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 385

Table 16.1 (continued)

Group (by vector) Abbreviation Geographic distribution Principal vector species Human disease
Tembusu virus TMUV Malaysia, Thailand Culex spp. Unknown
Spondweni virus complex
Zika virus ZIKV Africa, Asia Aedes spp. Fever, rash
Spondweni virus SPOV Africa Aedes circumluteolus Unknown
Yellow fever virus complex
Banzi virus BANV Africa Culex spp. Fever
Bouboui virus BOUV Africa Unknown Unknown
Edge Hill virus EHV Australia Mosquitoes Unknown
Jugra virus JUGV Malaysia Mosquitoes Unknown
Saboya virus SABV Senegal Tatera kempi Unknown
Potiskum virus POTV Nigeria Unknown Unknown
Sepik virus SEPV New Guinea Mosquitoes Fever
Uganda S virus UGSV Africa Mosquitoes Unknown
Wesselsbron virus WESSV Africa, Asia Aedes spp. Fever
Yellow fever virus YFV Africa, South America Aedes aegypti Hemorrhagic fever
Viruses with no known arthropod vector
Entebbe bat virus complex
Entebbe bat virus ENTV Uganda Unknown Unknown
Sokoluk virus SOKV Kyrgyzstan Unknown Unknown
Yokose virus YOKV Japan Unknown Unknown
Modoc virus complex
Apoi virus APOIV Japan Unknown Unknown
Cowbone Ridge virus CRV USA Unknown Unknown
Jutiapa virus JUTV Guatemala Unknown Unknown
Modoc virus MODV USA Unknown Unknown
Sal Vieja virus SVV USA Unknown Unknown
San Perlita virus SPV USA Unknown Unknown
Rio Bravo virus complex
Bukalasa bat virus BBV Uganda Unknown Unknown
Carey Island virus CIV Malaysia Unknown Unknown
Dakar bat virus DBV Africa Unknown Fever
Montana myotis leukoencephalitis virus MMLV USA Unknown Unknown
Phnom Penh bat virus PPBV Cambodia Unknown Unknown
Batu Cave virus BCV Malaysia Unknown Unknown
Rio Bravo virus RBV USA, Mexico Unknown Fever
Adapted from Calisher et al. [1]

conserved proteins NS1, NS3, and NS5 and the four small animal host (i.e., they are zoonotic). The ecological specia-
hydrophobic proteins NS2A, NS2B, NS4A, and NS4B [4]. tions of these viruses are diverse. For example, the Japanese
Molecular and phylogenetic analysis of the genus encephalitis virus (JEV) group (e.g., Japanese encephalitis
Flavivirus reveals a family of viruses that has evolved rap- virus, West Nile virus (WNV), St. Louis encephalitis (SLE))
idly from a progenitor virus arising possibly in Africa several is maintained in a cycle of transmission between birds and
thousands of years ago. With evolution the flaviviruses have Culex mosquitoes. Mammalian hosts, humans, and horses
shown substantial ecological diversification with different are infected but are dead-end hosts as the viremia achieved is
lineages adapting to different vectors and modes of trans- too low for subsequent transmission to an insect vector. The
mission. They have also evolved unique strategies to evade tick-borne encephalitis group is transmitted among rodents
the host’s innate and adaptive immunity [5]. Figure 16.1 dis- by a tick vector; as with the JEV group, humans are a dead-
plays the phylogenetic tree of the genus Flavivirus show- end host. Yellow fever virus (YFV) is primarily maintained
ing the association of the groups of related viruses with in a sylvatic cycle involving nonhuman primates and Aedes
their invertebrate vectors, vertebrate hosts, and geographic mosquitoes; but it has shown the capacity to spread in urban
distribution. All human pathogenic flaviviruses are transmit- areas using Aedes aegypti mosquitoes and humans as its
ted by insect vectors, and all but the dengue viruses have an primary reservoir, resulting in extensive urban epidemics.
386 S.J. Thomas et al.

ALF
MVE
JE
USU
Old world
KOU
KUN
WN
YAO
CPC
AROA
IGU New world Culex spp and birds
BSQ
NJL
KOK
STR
BAG
ITV Old world
NTA
TMU
THCAr
ILH
ROC New world
SLE
DEN1
DEN2
DEN3
DEN4
SPO
ZIK
KED
Aedes spp and mammals
BAN
97 UGS
Mosquito Old world
JUG
POT
SAB
BOU
EH
YF
SEP
EB Vectors
100 SOK unknown
YOK
92 GGY
KFD
LGT
LI
NEG
WTBE TBE complex
RSSE ticks and
Sof mammals
FETBE Old world
Vs
OHF
Non- KSI
vectored 80 RF
precursor Tick
POW
KAD
MEA TYU complex
SRE ticks and
TYU seabirds
APOI
BC
PPB
Old world
CI
71 BB Bat
DB
RB Vectors
NKV unknown
MML
CR
MOD New world
SV Rodent
JUT
SP
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 387

Analysis of the genetic changes of the flaviviruses over an African virus unknown to the western world prior to the
time reveals a group of viruses that have evolved rapidly. 1600s, was introduced to the Western Hemisphere by the
Methods using coalescent theory and a maximum likelihood transportation of slaves from Africa with the first reported
(ML) demographic model reveal that the flaviviruses are outbreak of yellow fever in the Yucatan in 1648 [10]. The
growing at an exponential rate, with specific viruses such as transportation of the mosquito vector and infected pas-
the dengue viruses increasing rapidly in the recent past and sengers by ship and introduction of the virus into naive
Japanese encephalitis virus changing from constant popula- susceptible populations resulted over the next 200 years
tion size to exponential growth within the last century [6]. For in the transmission of the virus and illness to many large
St. Louis encephalitis virus (SLEV), a Bayesian coalescent urban populations with outbreaks occurring in the tropical
approach was used on the envelope gene sequence substitu- Americas, costal North America, and Europe. Yellow fever
tion rates and dates of divergence in the Americas [7]. The became known in the Hispanic world as La Vomito for the
mean substitution rate estimated for all SLEV was 4.1 × 10−4 black vomit that accompanies the hemorrhagic phase and
substitutions/site/year. The direction of the gene flow was yellow jack among the Europeans for the international
South to North between 158N and 308N latitude consistent signal flag for quarantine and its characteristic yellow and
with migratory bird patterns from their northern breeding black squares known by the same name. During the summer
grounds after having acquired infection while wintering in months, epidemics occurred in New York in 1668, Boston
the region of the Gulf of Mexico. This is an elegant example in 1691, and Charleston in 1699, as well as later in the cit-
of the role of the vector and the vertebrate host especially ies of the Gulf of Mexico and the Mississippi River. In 1793
migratory birds in influencing the gene flow and evolution a major yellow fever epidemic occurred in Philadelphia.
of the flaviviruses. The dengue viruses (DENVs) are also an Philadelphia at that time was the United States’ largest and
example of an emerged flavivirus and global health problem most cosmopolitan city. French refugees from a slave rebel-
that have changed dramatically over the past century. DENV lion in Haiti arrived on the banks of the Delaware River
has evolved to a molecular clock that is serotype and geno- which bounds the east side of the city bringing yellow
type specific [8]. Phylogenetic analysis and time analysis fever with them [11]. In July of that year, cases developed
suggest that dengue viruses serotypes separated within the among the working class living along the Delaware River
last 1,000 years, and the change of DENV from a sylvatic who suffered high fevers, yellowing of the skin and eyes,
cycle to sustained human transmission may have occurred hemorrhage, and death. By August 19 the epidemic poten-
between 125 and 320 years ago [9]. tial was recognized by Dr. Benjamin Rush, professor at
In this chapter we will review the characteristics of the the University of Pennsylvania, founder of the College of
flaviviruses, their modes of transmission, and their role in Physicians of Philadelphia, and signer of the Declaration
causing severe human clinical infection and emerging dis- of Independence. Dr. Rush later gave an accounting of
eases of potential epidemiologic significance. his clinical experience during this epidemic in a publica-
tion entitled, “An Account of the Bilious Remitting Yellow
Fever, as It Appeared in the City of Philadelphia, in the
2 Historical Background Year 1793.” The epidemic peaked in October of that year
with total deaths estimated to be 4,041–5,000 in a popula-
Yellow fever and its emergence as a deadly epidemic dis- tion of 45,000 (crude mortality of 9–11 %). Subsequently
ease is a model on how diseases emerge to become a large Philadelphia experienced outbreaks of yellow fever in
recurrent epidemic disease in urban populations. Originally 1797, 1798, 1799, 1802, 1803, and 1805.

Fig. 16.1 Phylogenetic tree showing the association of the groups of Langat, LI Louping ill, NEG Negishi, Sof Sofjin, FETBE far eastern
related viruses with their invertebrate vectors, vertebrate hosts, and geo- TBE, Vs Vasilchenko, OHF Omsk haemorrhage fever, KSI Karshi, RF
graphic distribution. ALF Alfuy, MVE Murray Valley encephalitis, JE Royal Farm, POW Powassan, KAD Kadam, MEA Meaban, SRE
Japanese encephalitis, USU Usutu, KOU Koutango, KUN Kunjin, WN Saumarez Reef, TYLI Tyuleniy, APOI Apoi, BC Batu Cave, PPB Phnom
West Nile, YAO Yaounde, CPC Cacipacore, ARO Aroa, IGU Iguape, Penh bat, CI Carey Island, BB Bukalasa bat, DB Dakar bat, RB Rio
NJL Naranjal, KOK Kokobera, STR Stratford, BAG Bagaza, IT Israel Bravo, MML Montana myotis leucoencephalitis, CR Cowbone Ridge,
Turkey meningoencephalomyelitis virus, TMU Tembusu, THCAr strain MOD Modoc, SV Sal Vieja, JUT Jutiapa, SP San Perlita, TBE tick-
of Tembusu, ILH Ilheus, ROC Rocio, SLE St. Louis encephalitis, DEN borne encephalitis, WTBE Western European TBE, RSSE Russian
dengue, SPO Spondweni, ZIK Zika forest, KED Kedougou, UGS spring and summer encephalitis, NKV refers to viruses with no known
Uganda S, JUG Jugra, POT Potiskum, SAB Saboya, BOU Bouboui, EH vector (Adapted from Ref. [2] with permission of the publisher)
Edge Hill, YF yellow fever, SEP Sepik, EB Entebbe bat, SOK Sokoluk, Reprinted with permission from [296]
YOK Yokose, GGY Gadgets Gully, KFD Kyasanur Forest disease, LGT
388 S.J. Thomas et al.

Yellow fever took its toll on deployed US soldiers in the Italy and was found to be highly effective. DDT became a key
tropical Americas, especially in Cuba. Army Surgeon-General component of the World Health Organization’s global malaria
George Miller Sternberg created the US Army Yellow Fever eradication campaign in 1955 [19]. This campaign resulted in
Commission in 1893 and directed Major Walter Reed to con- the elimination of both the malaria mosquito vector and Aedes
duct studies to establish its etiology. Together with his col- aegypti throughout South America and the virtual elimination
leagues and Carlos Finlay, a series of human clinical trials were of malaria, yellow fever, and dengue throughout the Americas.
performed in Havana, Cuba, during 1900–1901 to discover the The growing concerns of the environmental effects of DDT led
cause of yellow fever. Walter Reed, Jas Carroll, and Aristides to the end of the use of DDT as an effective mosquito control
Agramonte published in the JAMA in 1901 The Etiology of larvicide in 1969 [20]. The cessation of the DDT-based mos-
Yellow Fever: An Additional Note where they discussed the quito control programs in the Americas and the social disrup-
methods of their studies, results, analysis, and concluded the tion that resulted from World War II allowed the emergence
following: (1) the mosquito serves as the intermediate host for of dengue in Asia and its reintroduction and resurgence in the
the parasite of yellow fever; (2) yellow fever is transmitted to Americas.
the nonimmune individual by means of the bite of the mosquito By the 1950s, scientists of the Rockefeller Foundation, the
that has previously fed on the blood of those sick with this dis- US Army and Navy, the US Public Health Service, several
ease; (3) an interval of about 12 days or more after contamina- US universities, and many foreign governments recognized
tion appears to be necessary before the mosquito is capable of that arboviruses could be recovered in nature from appar-
conveying the infection; (4) yellow fever is not conveyed by ently healthy arthropods and wild vertebrate animals. This
fomites, and hence disinfection of articles of clothing, bedding, search in the natural cycles of arboviruses resulted in the
or merchandise, supposedly contaminated by contact with the discovery of over 500 different arboviruses with about 100
sick with this disease, is unnecessary; and (5) the spread of of them causing human disease. Unfortunately, the control
yellow fever can be most effectually controlled by measures of arbovirus infections has not kept pace with the discovery
directed to the destruction of mosquitoes and the protection of and spread of disease. Antiviral drugs for arboviruses are not
the sick against the bites of those Insects [12]. commercially available. Except for yellow fever, tick-borne
With this information, General William C. Gorgas engi- encephalitis, and Japanese encephalitis, there are no widely
neered the elimination of the mosquito initially from Havana used vaccines available for humans. Source reduction and
and then later from the environs of the Panama Canal control measures against the mosquito vector such as pesti-
construction site and subsequently yellow fever cases disap- cides and biological larvicides have neither been sustainable
peared [13–16]. In 1932, Soper and colleagues showed that nor effective in vector control.
there was a jungle cycle of yellow fever in the absence of
A. aegypti. This significant observation meant that yellow
fever could not be stopped just by controlling the mosqui- 3 Methodology Involved
toes in the cities. Theiler and Smith succeeded in cultivating in Epidemiologic Analysis
the Asibi strain of yellow fever virus, first in monkeys and
then in embryonated eggs, and attenuated it by passage [17]. 3.1 Sources of Mortality and Morbidity Data
In 1937, they announced their discovery of an attenuated
vaccine—the 17D strain [18]. This vaccine is used through- Mortality data are collected systematically but passively by
out the world today to prevent yellow fever. national governments for many arboviruses. Data are pub-
The discovery of the role of Aedes aegypti in the trans- lished in the Morbidity and Mortality Weekly Report of
mission and spread of yellow fever introduced the concept the US Centers for Disease Control and Prevention, in the
of mosquito control as an effective measure to disrupt yellow Weekly Epidemiological Report of the Pan American Health
fever transmission. The International Health Board and the Organization, and in the Weekly Epidemiological Report of
Rockefeller Foundation instituted mosquito control strate- the World Health Organization. The mortality data are grossly
gies including the use of a larvicidal, Paris green, throughout underreported but may serve as a comparative data base, since
the United States and Central and South America [19]. These underreporting may be uniform throughout much of the world.
techniques were applied to malaria control during the years The reporting of yellow fever mortality in Africa, for instance,
from 1924 to 1925 [19]. World War II created the Rockefeller was found to be about 10 % of the true figure [14, 21].
Foundation Health Commission in 1942 to support national The information flow to the World Health Organization
defense and malaria control for US forces. The need for lousi- is sometimes facilitated by informal networks of scientists
cides to combat typhus introduced a new insecticide developed and interested citizens. Nevertheless, the organization is
by the Swiss firm, Geigy, called dichlorodiphenyltrichloro- constrained from action until official reports are received.
ethane (DDT) [19]. Led by Fred Soper, the Rockefeller team This constraint often means a delay in control of a disease of
demonstrated the effectiveness of DDT as a lousicides and in regional or world importance.
disrupting typhus epidemics. DDT was soon used in aerial and The same sources supply morbidity data as supply mortal-
ground spraying for Allied Forces during a malaria outbreak in ity data. In the United States, of the arbovirus diseases, those
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 389

producing neuroinvasive and non-neuroinvasive illnesses are other hand, the survey is an excellent tool for determination
reported and include the California serogroup virus, Eastern of vaccination coverage and protection.
and Western equine encephalitis virus, and the flaviviruses One very important use of serosurveys is in the calcula-
Powassan virus, St. Louis encephalitis virus, and West Nile tion of the basic reproductive ratio, R naught (R0) [24], which
virus [22]. In addition, yellow fever is still reportable in the is defined as the number of new individuals in a susceptible
United States. Monath documented the time elapsed between population who are infected by a single individual during his/
onset of an epidemic of St. Louis encephalitis and its recog- her infectious period (see also Chaps. 1 and 5). The higher
nition [23]. That time varied between 2 and 8 weeks. In the the number, e.g., R0 >1, the more transmissible the infec-
Corpus Christi, Texas, epidemic of 1966, nearly 707 of the tion. For vector-borne diseases, in particular, R0 is important
cases had occurred before recognition. and complicated as it takes into account the susceptible and
infected vectors as well as the susceptible and infected ver-
tebrate hosts; R0 is thereby influenced by both the intrinsic
3.2 Serologic Surveys and extrinsic incubation periods. Serosurveys can calculate
the R0 as a measurement of the epidemic impact of a patho-
Serologic surveys entail the sampling of a defined popula- gen on a population but also as an estimate on the impact of
tion and measuring the amount of specific antibody to the interventions such as vector control on reducing disease in a
targeted protein which will indicate past or current infec- population. Furthermore, a concept integrally related to R0 is
tion. Because of the large sample size that is tested, high- the “critical vaccination threshold,” which is the number of
throughput assays such as enzyme-linked immunosorbent persons in a population needed to vaccinate in order to drive
assay (ELISA), complement fixation (CF), or hemagglutina- R0 to <1. Thus, determining R0 by serosurveys is important
tion inhibition (HAI or HI) are performed with confirmation not only in gauging the epidemic potential of a pathogen but
by more time-intensive assays such as the plaque reduction also in determining the interventions necessary to control an
neutralization titers (PRNT). The results give a point preva- epidemic. Age-stratified seroprevalence studies with cohorts
lence of past or current infection in different geographic including ages surrounding the age of peak incidence allows
areas and subpopulations or those at particular risk for infec- estimation of the force of infection over previous years based
tion. Distribution of the antibody in a population can give on different exposure rates across age groups and a calcula-
clues to the ecological conditions necessary for maintenance tion of R0 [25].
of the virus as well as the human behavior that might place
persons at risk for infection. Surveys of different age groups
will show if the virus is more prevalent as the population 3.3 Laboratory Methods
ages, typical of an endemically transmitted agent. Another
prevalence pattern in which antibody is present only in per- The laboratory is an all-important resource and key corner-
sons born before a certain year may indicate an epidemic in stone in the study of the epidemiology of arbovirus diseases
that year. Alternatively, a constant percentage of antibodies as well as understanding the pathogenesis of severe illness.
in each age group may indicate a recent introduction of the Diagnosis can rarely be made with certainty by clinical
virus in a naïve population. Broadly based serologic surveys examination and understanding the clinical course of symp-
of large populations can provide extensive information about toms, viremia, and antibody rise essential in utilizing the
virus distribution in different geographic areas, rural versus appropriate laboratory assays. Isolation of virus from arthro-
urban populations, different age and sex groups, and different pods, wild and domestic animals, and people is essential to
occupational types. Arbovirus serosurveys have limitations. determine the natural history of infection with these agents.
Cross-reactions occur among viruses of the same serogroup. Figure 16.2 demonstrates the typical flavivirus infection after
This is especially true of the HI test and ELISA with flavi- inoculation from a vector assuming an incubation period of 6
viruses. The neutralization test is more specific and should days. Viremia occurs prior to the first clinical symptoms with
be used where feasible. Surveys with HI or ELISA must be early symptoms, commonly fever, headache, myalgias, and
interpreted with caution unless one is certain that only one laboratory findings of leukopenia and thrombocytopenia.
flavivirus circulates in the region, or unless the results have Antibody rises late in the clinical illness with first IgM fol-
been confirmed by neutralization test with a portion of the lowed by IgG. IgM can persist for several months and disap-
negative and positive sera. pear, while IgG can persist for years after the infection. From
The serosurvey usually will not indicate when the infec- a laboratory perspective the utility of the assay is dependent
tion responsible for the antibody occurred. If the antibody is on the stage of infection. PCR-based assays and viral isola-
suspected to be of recent origin, the IgM antibody-capture tion are useful during the days of viremia in the early part of
ELISA is useful for detection of infections originating within the clinical illness, whereas antibody-based assays are useful
the prior 3–6 months. A serosurvey is not a reliable indicator during the latter part of the illness and in convalescent sera.
of natural infection in populations vaccinated for tick-borne Nonstructural protein 1 or NS1 is a highly conserved
encephalitis, yellow fever, or Japanese encephalitis. On the 48-kDa glycoprotein that is a requirement for flavivirus
390 S.J. Thomas et al.

Hemagglutination Inhibition
IgM and IgG ELISA
Plaque Reduction Neutralization test
Rapid tests
NS1 Levels

Virus isolation
Mosquito inoculation
Cell culture (C6/36, AP61)
Vero cells
Anti-flavivirus IgM

Molecular techniques Manifestations:

Blot hybridization
Polymerase chain reaction (PCR) Headache
Taqman Myalgias
NASBA Joint pains
Leukopenia
Thrombocytopenia

Fever Anti-
flavivirus
IgG
Viremia

0 2 4 6 8 10 12 14 16
Days after infection

Fig. 16.2 Typical flavivirus infection and the human host response by clinical course and appropriate diagnostic assay

RNA replication [26]. Interestingly NS1 exists in the cell as Details of the techniques of CF, HI, and virus neutral-
a homodimer associated with organelle membranes and is ization relating specifically to arboviruses are available in
actively transported to the mammalian cell surface where it current manuals. Fluorescent antibody (FA) techniques, anti-
is secreted as a soluble hexamer. Thus, NS1 can be detected gen-based ELISA, often coupled with monoclonal antibody,
in serum during flavivirus infections and has a half-life lon- and PCR are widely used for antibody, antigen, and RNA
ger than detectable RNA in serum [27]. NS1 ELISA-based detection, respectively. Such advances are greatly extending
assays have become available for dengue virus infections and the scope of field and laboratory investigations.
have the potential to be used for other flaviviruses [27]. The
advantage of these assays is that they can be used throughout
the early, middle, and late clinical course of infection with a 3.4 Genetic Sequencing
sensitivity and specificity equivalent to PCR.
Virus isolation is key in understanding the virus and its Molecular methods such as polymerase chain reaction
effects on the vector and human population, but isolation is (PCR) and the ability to detect and amplify small amounts
also the most challenging for laboratories. It requires serum of RNA or DNA to identify specific pathogens have revolu-
from the early part of clinical illness, −80 °C or colder tionized our diagnostic capabilities in identifying patients
freezers, and special laboratory techniques. Viral isolation who are acutely ill from flavivirus infections [28]. Reverse
requires inoculation of a small amount of serum into labo- transcriptase PCR (RT-PCR) methods are highly sensitive
ratory animals, arthropods like mosquitoes, or cell culture. and specific in detecting the infecting arbovirus during
Cell culture systems (vertebrate or insect cell cultures) are viremia [29, 30]. Real-time assays that utilize exonuclease
used for both virus isolation and neutralization titers. fluorogenic assays such as TaqMan® (Applied Biosystems,
In the study of material derived from patients, it is ideal to CA, USA) and SYBR® Green (Molecular Probes, Inc., OR,
have acute and convalescent sera available. As demonstrated USA) have significantly shortened the time of detection of
in Fig. 16.2, antibody titer rises rapidly from the acute to RNA and DNA in a specimen but also added the ability to
the convalescent serum, and a fourfold rise between the two quantify the amount of virus and correlate it to disease sever-
samples is indicative of an acute infection. Demonstration of ity [31]. Interest and need for point of care devices that can
IgM in ELISA permits a presumptive diagnosis with a single be utilized at a clinic with little expertise has prompted the
serum. development of new molecular platforms such as “isother-
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 391

mal” methodologies including nucleic acid sequence-based of individuals in any class is an integer with events occurring
amplification (NASBA), transcription-mediated amplifica- randomly with a given probability based on the associated
tion (TMA), and loop-mediated isothermal amplification deterministic model. The value of stochastic models is the
(LAMP) technologies [32]. This allows an isothermal reac- generation of different epidemics capturing the variabil-
tion and amplification to be performed at room temperature ity in the epidemic profile [35]. For example, a stochastic
with potential reduction in time to detection to a matter of metapopulation model with spatiotemporal transmissibility
minutes. scenarios was used to model the spread and transmission of
Sequencing of the arboviruses has transformed our abil- yellow fever [36]. This model was useful to understand and
ity to understand the genetics and epidemiology of these assess the risk of yellow fever in producing urban outbreaks
viruses as well as the relatedness of each virus as demon- and to identify locations at risk for yellow fever introduction
strated in Fig. 16.1. Genetic sequencing has rapidly evolved and subsequent transmission.
from a time-intensive process to technology for rapid
full-length automated sequencing. Partial and full-length
sequencing of the arboviruses has allowed phylogenetic 4 Biological Characteristics of the Virus
analysis and revealed a series of clades defined by their That Affect the Epidemiologic Pattern
epidemiology and disease associations [33]. Phylogenetic
analysis has identified mosquito-borne, tick-borne, and no- If you were to look at the world from the eyes of a flavi-
known-vector (NKV) virus clades, which have been further virus, you would find a bewildering array of environments
divided into clades associated to their principal vertebrate you were forced to adapt and propagate in. The vector inver-
host. Sequencing coupled with phylogenetic analysis is a tebrate environment is quite different from the vertebrate
powerful tool to understand the correlation between epi- environment in requiring the virus to propagate at a lower
demiology, disease, and geography. It furthers our under- body temperature and to utilize different cellular receptors
standing of the complex evolutionary relationships between for attachment and cellular entry. The virus upon infection
the virus, vector, and its vertebrate host. Metagenomics of its vector host from a blood meal will replicate, escape
takes this concept further. It is the coordinated study of from the midgut, evade vector host defenses, and replicate
multiple genomes—the population of genetic material in high concentration in the salivary glands. Environmental
and, as applied to viruses, the entire community of viruses factors including temperature and humidity affect vector lon-
within the host environment [34]. This approach utilizes gevity and viral replication and overwintering until the next
direct sequencing and bioinformatics to reconstruct the transmission season. Upon infecting their vertebrate host
viral population and as such reveals the genetic diversity after the bite of the infected vector, the flavivirus faces a new
and evolution of the virus and its vector hosts. Especially host environment with a different ambient temperature, new
powerful is the use of metagenomics when standard tech- receptors for host cell entry and replication, and new host
niques fail to identify a viral infection, such as in emerging innate and adaptive immunity. Flaviviruses are remarkably
diseases. For example, metagenomics was used to identify adept and successful at adapting to an array of environments
specific viruses from samples collected from an enterovi- to replicate and produce infection and ultimately epidemics
rus surveillance program in the Netherlands [34]. Samples in human populations. Ultimately the biological characteris-
were identified using conventional techniques including tics of the flavivirus that affect human epidemiology rely on
PCR that demonstrated cytopathic effects in cell culture its ability to be successful in the host vector. The capacity
without a virus. Metagenomics identified new viruses in all of the virus to replicate in the vector is influenced by many
the samples. factors including the environment, ecology, vector behavior,
and viral molecular factors. The interaction of these factors
is called the vectorial capacity. The extension of vectorial
3.5 Mathematical Modeling capacity to infect the host is known as vector competence—
the intrinsic ability of a vector to become infected and to
Mathematical modeling is a powerful technique used to subsequently transmit a pathogen to a susceptible host [37].
understand the interactions of the arbovirus, its vector, and The viral molecular factors involved in vectorial capacity
host infection that produce endemic transmission or explo- and the ability of the virus to be highly specific for its vector
sive epidemics in populations [35]. Mathematical models host are determined by how arboviruses exist as a collec-
have the potential to predict epidemics and, equally impor- tion of variable genomes within a host known as a mutant
tantly, to identify interventions by which to prevent or dimin- spectrum, mutant swarm, or quasispecies [37]. During a
ish the spread of the arbovirus through the use of vector replicative cycle, RNA viruses and its high mutation rate
reduction, personal protection methods, antivirals, or vac- will develop as a distribution of genetic variants which is
cines. Models have progressed from the basic susceptible, influenced by a balance between mutation and selection.
infected, and recovered SIR model to more advance model- Selection pressure will determine the most fit virus for
ing techniques using stochastic models in which the number the given environment with the least fit viruses going into
392 S.J. Thomas et al.

extinction. This Darwinian survival of the fittest is known 5 Epidemiology

as viral adaptability. Genetic bottlenecks, defined as survival
of a minority of one generation to become the majority of The epidemiology of flavivirus infections in humans as noted
the next generation, can occur in flaviviruses and have been in previous sections is a complex interplay between viral
well described for dengue virus [38]. During the course of evolution, selection pressures, vectorial capacity, and risk
an infection in a vector, host genetic bottlenecks can occur, of human infection from the vector and its spread in human
for example, when the virus leaves the midgut or replicates populations. In practical terms, many factors account for the
in the salivary glands allowing the selection of one mutant to dramatic changes in the epidemiology of the flaviviruses:
become the dominant genotype. Thus, bottlenecks diminish increasing travel and the speed of travel where an individual
viral diversity by selecting a subpopulation of viruses par- can go from rural areas of Africa or Asia to major metropoli-
ticularly adapted to that environment; as a result, frequent tan areas in less than 24 h, expanding trade and commerce,
bottlenecks enhance the evolution of phenotypic robustness population growth, increasing urbanization and population
of the virus [37]. detritus leading to increasing vector breeding areas, defores-
Viral fitness is inherently tied to vector fitness. A particular tation, and climate change [46]. Examples of this changing
virus strain that is selected for rapid killing of its vector host epidemiology are the spread of the dengue viruses through-
will have little success at survival to its next host, and an atten- out Asia, the Americas, and most recently Florida; resur-
uated virus that does not replicate well in its vector may not gence of yellow fever in South America and Africa; spread
propagate to the vertebrate host. Viral replication may disrupt of West Nile virus through North and Central America; and
the physiology of the vector host through disruption of diges- the spread of Japanese encephalitis in Southwest Asia [46].
tion, salivation, or changes in the vector midgut [39, 40]. Other
consequences to vector fitness from viral infection include
energy costs associated with immune activation, behavioral 5.1 Incidence and Prevalence
changes resulting in decreased feeding, and alterations in mat-
ing and propagation. A meta-analysis of studies across a range All infections with pathogenic flaviviruses discussed in
of mosquito–virus systems showed that arboviruses do reduce this chapter are acute and can be diagnosed either through
the survival of their mosquito vectors, but the overall impact molecular means during the time of infection or by serol-
on vector fitness varies by the virus taxonomic group and ogy consistent with acute infection, i.e., detection of IgM
mode of transmission [41]. Alphaviruses, for example, were antibody or a fourfold rise in antibody titer between acute
associated with the highest virulence levels in mosquitoes and and convalescent sera. Measurement of incidence requires
the least for the bunyaviruses. The conclusion was that virus documenting acute infection through the application of clini-
and vector fitness are interwoven and dependent on multiple cal criteria consistent with the flavivirus infection. All acute
selection factors that are evolving. The complexities of this flavivirus infections progress from a classic presentation of
relationship between virus and vector fitness coupled with the fever, chills, myalgias, and headache to a more flavivirus-
viral fitness required to propagate in a vertebrate host high- specific syndrome of an acute febrile illness, hemorrhagic
light the exquisite balance needed to maintain the life cycle fever, or encephalitis. These specific syndromes fall into a
and the disease process. This “trade-off” hypothesis suggests spectrum of overlapping clinical presentations, making it
that the replication in arthropod then vertebrate hosts shapes difficult to distinguish one flavivirus from another or from
virus evolution. It is when this trade-off results in an increase other pathogens based on clinical symptoms and signs alone.
in both vectorial capacity and vertebrate infection that human All infections display a biological gradation (“iceberg”) of
epidemics, global expansion, and emerging diseases may clinical responses and disease severity both at a cellular and
occur. An example of this is the emergence of chikungunya host response level (see Chap. 1). In the case of flaviviruses,
virus (CHIKV) infection in 2004 from the Indian Ocean to although unobserved (below the “waterline” of the iceberg),
India, attributed to a single amino acid mutation that enhanced subclinical infections are viremic and do contribute to the
vector capacity in a secondary vector Aedes albopictus [42]. transmission and infection of the vector and thus represent a
Another example is the emergence of a new lineage of West very important component of the epidemic. Further along the
Nile virus (WNV) in North America that was adapted for more gradient of infection are mild ambulatory illness, moderate
efficient transmission in Culex mosquitoes [43]. When vecto- illness requiring medical care, more severe illness requiring
rial capacity for an arbovirus is diminished or vector resis- hospitalization, and fulminant illness requiring intensive care
tance to virus infection is induced, virus transmission may with potentially fatal outcome. The shape of the gradient or
potentially be completely interrupted. The occurrence of this “iceberg” varies among the flaviviruses and depends on the
phenomenon may been realized recently with the observation preexisting immunity in the population, vectorial capacity,
that infection of Aedes aegypti by Wolbachia, an intracellular and environmental factors. The subclinical infections are not
bacterium, confers resistance of the mosquito to DENV infec- well studied due to the requirement for long-term prospective
tion [44]. These findings are now being used to infect natural studies of cohorts with the ability to diagnose asymptomatic
A. aegypti mosquitoes to diminish DENV transmission [45]. infections. The best documentation of subclinical infection
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 393

has come from the prospective cohort studies conducted in the most common mosquito-borne arboviral infection world-
Northern Thailand where at least 50 % of all DENV infec- wide (http://www.who.int/csr/disease/dengue/en/). Other sig-
tions were subclinical [47, 48]. In general, a large majority nificant flaviviruses that have vast global distributions include
of flavivirus infections occur below the waterline of detection West Nile virus, tick-borne encephalitis, and yellow fever.
and subclinical. The more severe forms of infection resulting Regional flavivirus threats that produce epidemic diseases
in death occur in less than 5 % of all infections. Understanding include Japanese encephalitis in Asia and West Nile virus and
the iceberg concept of infection is critical in thinking on the epi- St. Louis encephalitis in North and South America. All are
demiology and incidence of flavivirus infections. Surveillance endemic diseases that produce transmission and disease every
confined to hospitalized cases will detect only 10–20 % of all year with occasional epidemics.
cases. Extended to the ambulatory clinics, surveillance can
detect an additional 30 % of all infections. It is apparent that
even with detection of 50 % of acute cases, the incidence of 5.3 Temporal Distribution
infection is being grossly underestimated.
In contrast, seroprevalence studies (see Chap. 2) of fla- Temporal distribution is a consequence of overwintering
vivirus infection can define populations at risk, give a his- and seasonal breeding and is region specific. If birds are
torical account of the transmission, and provide an estimate the primary vertebrate host, temporal distribution is depen-
of the burden of infection in a population. A classic arbo- dent on their migratory patterns, nesting, and hatching of
virus seroprevalence study was the mapping of the world- young immunologically naïve chicks. In general, seasonal
wide distribution of yellow fever by Sawyer et al. in 1937 breeding of the vector occurs in spring, summer, and fall,
[49]. The virus neutralization test was performed using adult although this is affected by the climate with warmer years
white mice as test animals. Yellow fever virus was found having transmission during the winter months. Climate
to be more widely distributed in South America and Africa change and global warming will affect the temporal distri-
than had been earlier suspected and was absent from Europe, bution of the flavivirus by changing the abundance of the
Asia, and Australia. Neutralization tests measure durable vector. Global warming will increase the distribution of the
antibody that persists for years. High rates of antibody prev- vector as well as cause more rainfall and thus create more
alence could result from continued, widespread virus activ- potential areas for breeding in the case of mosquitoes. The
ity and/or from a large outbreak with a high attack rate. For natural weather pattern that occurs approximately every 5
example, yellow fever neutralization tests performed on sera years, La Niña/El Niño-Southern Oscillation, or ENSO,
collected from residents of Trinidad, West Indies, in 1953 can have dramatic effects on rainfall, flooding, and vector-
revealed no immunes under the age of 15 years and there- borne diseases including the flaviviruses [51, 52]. This
fore no apparent virus activity later than 1938. This situation periodic climate change occurs across the tropical Pacific
changed dramatically with the reappearance of yellow fever Ocean with variations in surface temperature (warming
on the island in epidemic form in 1954 [50]. known as El Niño and cooling as La Niña). In a recent
Incidence and prevalence studies provide two views on study, Murray Valley encephalitis virus (MVEV) in north-
flavivirus infection. The former can uncover newly occur- ern Australia and Papua New Guinea was monitored and
ring infections in the population and evidence of an out- correlated to the occurrence of rainfall [53]. Using multi-
break; the latter offer a historical perspective on previous satellite precipitation analysis, the authors found that
outbreaks and subpopulations that may be at risk. In most increases in monthly rainfall and monthly number of days
countries appropriate diagnostics are not available to diag- above average rainfall increased the risk of MVEV activ-
nose a specific flavivirus infection, and clinical criteria are ity at a time lag of 2–3 months. Clearly climate change
mostly used; as a result their population incidence is grossly and weather have an effect on the temporal distribution
underreported. of vector-borne infections with a particular impact on the
flaviviruses.

5.2 Epidemic Behavior and Geographic

Distribution 5.4 Age, Sex, and Other Demographic
Factors
Arbovirus infections are worldwide in distribution and may
occur whenever the appropriate mosquito or other arthropod Infections with arboviruses can occur at any age. The age
vectors abound in proximity to humans and a suitable amplify- distribution depends on the degree of exposure to the particu-
ing host. Table 16.1 displays both the human pathogenic flavi- lar transmitting arthropod relating to age, sex, occupation,
virus and those where illness hasn’t been documented, vector and recreational habits of the individual or group of individu-
if known and human illness if known. The flaviviruses occur als. In highly endemic areas where transmission occurs every
globally as both regional endemic diseases and epidemic dis- year, such as with Japanese encephalitis and dengue viruses,
eases that can spread worldwide. The dengue viruses are now flavivirus infection is seen primarily in children, with adults
394 S.J. Thomas et al.

protected by the antibody from previous infections [54, 55]. transovarially in arthropods, often referred to as “vertical
In areas where transmission is sporadic and the entire popu- transmission,” has been demonstrated (specific examples,
lation is at risk, extremes of age becomes a risk for severe tick-borne encephalitis in ticks, DENV in mosquitoes).
infection such as seen in WNV infection in adults over the Venereal transmission of SLE viruses in vector mosquitoes
age of 50 years [56]. has also been described. These mechanisms may be impor-
tant for survival of some arboviruses in nature, permitting
overwintering or survival over a protracted dry spell. Birds
5.5 Other Factors are important reservoir vertebrates for the viruses of JEV,
WEE, and SLE.
Genetic factors are known to influence disease severity There are a number of flaviviruses that cause human
from flavivirus infection and thus directly the epidemiol- infections where the principal vector species is not known
ogy of arbovirus infections. Nutrition may have an effect on (Table 16.1). These include the viruses Apoi, Dakar bat,
increasing disease severity in specific flavivirus infections Koutango, Modoc, and Rio Bravo. More research is needed
such as in dengue hemorrhagic fever though it is unknown if to establish the principal vector or, if not found, to gather
there is an effect with other infections [57]. Certainly malnu- details on their mode of transmission.
trition can suppress the immune response and affect disease There are two nonvector modes of transmission to
severity and thus the epidemiology. Genetic factors are very humans of the vector-borne flaviviruses that is essential
likely associated with disease severity. For example, in case– to know—transfusion mediated and sexual. During the
control gene association studies performed on cohorts of 2002 WNV epidemic in the United States, 23 patients were
DENV-infected patients in Asia and the Caribbean, specific confirmed to have acquired WNV through transfused con-
genes have shown associations with disease severity. These taminated blood products [61]. Of these the majority were
genes include the HLA molecules, the cell receptors for IgG immunocompromised due to transplantation or cancer.
(FcGII), vitamin D, and ICAM3 (DCSIGN or CD209) [58]. Sixteen donors with evidence of WNV viremia at donation
Also pathogen recognition molecules such as mannose- were linked to the 23 infected recipients. As a result of these
binding lectin (MBL) have associated with disease severity findings, the US blood supply is now screened for WNV.
as well as the blood cell-related antigens including ABO and Sexual transmission of another normally vector-borne fla-
human platelet antigens (HPA1 and HPA2) [58]. African vivirus was recently documented. Zika virus (ZIKV) is a
slaves were long known to be immune from the severity mosquito-transmitted flavivirus that has been isolated from
of yellow fever and were recruited to help stricken victims sick persons in Africa and Southeast Asia. Two American
during the Philadelphia 1793 yellow fever epidemic [11]. scientists working in Senegal became acutely ill with ZIKV
Clearly genetics that determine both acquired and innate [62]. One of the scientists upon returning to the United
immune responses contribute to disease severity or protec- States infected his wife who developed confirmed ZIKV
tion from flavivirus infections and can affect the epidemiol- symptomatic infection. Because she never traveled outside
ogy of these infections. of the United States, direct person-to-person transmission
was thought to be the mode of infection either through
saliva or infected semen.
6 Mechanism and Route of Transmission

By definition, the flaviviruses as arboviruses must be trans- 7 Pathogenesis and Immunity

mitted by arthropod vectors within which multiplication of
the virus is a necessary requirement. Non-arthropod-related Arbovirus infections are usually transmitted by the bite
transmission to humans can occur such as through goat, of the appropriate vector, and the skin is the normal por-
sheep, or cow milk in the case of TBEV and respiratory tal of entry. Even before the virus enters the host through
and alimentary tract with Omsk HF [59, 60]. The primary the skin, an important cofactor contributed by the vec-
vectors are mosquitoes and ticks. The duration of the nec- tor assists in shaping virus entry. Saliva from arthropods
essary period of virus multiplication within the arthropod (i.e., ticks and mosquitoes) has been shown to potenti-
host before it becomes infectious varies from virus to virus ate flavivirus infection and enhance in vertebrate hosts.
and vector to vector and is also directly temperature depen- Virus and arthropod saliva are delivered into the skin of
dent. For most viruses under average summer temperature the vertebrate host. Arthropod saliva contains a complex
conditions, the extrinsic incubation period falls in the 7- to mixture of bioactive molecules that are capable of alter-
14-day range. Once infected, vectors may remain infected ing homeostasis, immune response, and dendritic cells and
and able to transmit for many weeks or months. In the case may contribute to the ability of flaviviruses to establish
of ticks, this infection may be years. Transmission of virus an infection [63]. The potentiation of flavivirus infection
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 395

by arthropod saliva has been demonstrated for a number 8 Patterns of Host Response
of viruses including WNV and saliva from Culex tarsalis
which resulted in enhancement of early WNV infection in 8.1 Clinical Features
vertebrate hosts [64].
The first cells that typically come in contact with flavivi- Inapparent and subclinical human host responses predomi-
ruses are skin dendritic cells (DCs). These cells have been nate in most arbovirus infections. Clinical illness is fre-
shown to be important initial targets of infection that shape quently the exception rather than the rule. This varies from
the immune response for many of the pathogenic flaviviruses virus to virus. For example, infection with WNV, SLEV, and
including DENV, YFV, and tick-borne encephalitis virus JEV viruses results principally in mild and inapparent infec-
(TBEV) [50, 65–67]. TBEV targets DCs early in infection tions, whereas in infection with YFV, the host response is
and are major inducers of interferon (IFN) and the innate likely to be clinically apparent and often severe (Table 16.2);
immune response. TBEV modulates DCs and thus shapes the reasons for these differences are not known.
the innate response via INF antagonism. DCs become
infected and transport the virus to draining lymph nodes pro-
moting virus dissemination. After viral replication in target 8.2 Diagnosis
cells, the virus becomes widely disseminated throughout
the host. Viremia in the host is an important determinant for Cases of arbovirus infections are not likely to be diagnosed
additional infection in noninfected biting arthropods. The unless there is a high degree of clinical suspicion. Outbreaks
degree of viremia determines if the vertebrate host becomes of encephalitis in horses and dying off of birds may be an
a “dead-end host” where viremia with agents such as JEV early warning sign from some arboviruses. Recognition of
and WNV is minimal and no additional biting vectors are the arbovirus infection acquired by the traveler outside the
infected. In other flaviviruses, high and sustained viremia United States also depends on the alertness of the examin-
provides a source of additional vector infection, as is the ing physician. Rapid jet transport now permits exposed over-
case for DENV where humans are the primary reservoir for seas travelers to reach home and fall sick even within the
infection. short incubation period of such infections. The physician
The site of multiplication of most arboviruses remains must maintain a high degree of suspicion when seeing cen-
undetermined but is presumed to be in the vascular epithe- tral nervous system, influenza-like illnesses, or hemorrhage
lium and the reticuloendothelial cells on the lymph nodes, with fever occurring in travelers recently returned from areas
liver, spleen, and elsewhere. Liberation of virus from these endemic for arboviruses. Diagnosis will require specimens
organs constitutes the “systemic phase of viremia,” resulting (serum, cerebrospinal fluid) to be sent to specific laboratories
after 4–7 days in fever, chills, and aching joints. A number that have the capacity to perform the required assays such
of arbovirus infections have two phases—this early phase as the state laboratory or the Centers for Disease Control
and then a second phase with or without a few days of free- (CDC).
dom from symptoms. The second phase may be attended The laboratory diagnosis depends on the isolation of the
by encephalitis, joint involvement, rash, hemorrhage, and virus from the blood and/or a fourfold antibody rise in titer
involvement of the liver and kidneys. In most arbovirus or presence of specific IgM in sera taken during the acute
infections, only the first phase occurs, and the disease is mild and convalescent phases of illness. Often, the suspicion of
and “nonspecific.” In other instances, the early phase may be an arbovirus infection in individual cases arises too late for
missed, and only the severe manifestations occur. The early
phase is accompanied by leukopenia and the second phase Table 16.2 Patterns of host response to arbovirus infections in man
often by leukocytosis. Tissue injury may be the direct effect Response Examplesa
of viral multiplication in susceptible cells, as is the case with Asymptomatic infection WNV, DENV, JEV, SLEV
liver involvement in yellow fever. Mild febrile illness SLEV, YFV, JEV, DENV
Humoral antibodies regularly appear early in the course Influenza-like illness with aching DENV, ZIKV
of arbovirus infection and constitute the major basis of and joint pains
immunity. Such immunity may be lifelong. No infection Encephalitis, mild WNV, TBEV, SLEV
with yellow fever virus has been recorded in an individual Encephalitis, severe WNV, TBEV, SLEV
Jaundice, proteinuria YFV
who either had antibodies from an earlier infection or had
Rash, sometimes with hemorrhagic AHFV, DENV, KFDV
a history of yellow fever vaccination with development of manifestations
postvaccination antibody. The presence of antibodies in the Shock syndrome AHFV, DENV, KFDV
blood at the time of exposure to an infected arthropod vector a
Certain viruses have been selected for this list particularly to illustrate
provides a primary deterrent to reinfection with the homolo- the range of symptoms that may be seen in populations infected with a
gous virus. single virus
396 S.J. Thomas et al.

virus isolation or for demonstration of a rise in antibody titer. viruses are the Aroa virus, Japanese encephalitis virus,
Often only one acute serum specimen is sent without a con- Kokobera virus, Ntaya virus, Spondweni virus, and the yel-
valescent specimen. Under these circumstances, the presence low fever virus complexes. Under the viruses with no known
of a high antibody titer in a single serum may be significant if arthropod vector are the Entebbe bat virus complex, Modoc
the infection is an uncommon one in that region and particu- virus complex, and Rio Bravo virus complex. For each of the
larly if antibody surveys reveal a low antibody prevalence or viruses, the epidemiology, vector, human disease, diagnosis,
if prior surveys have demonstrated the absence of antibody and epidemic potential will be briefly covered.
in that community. The appropriate procedure in suspected
cases is to (1) notify the health department and seek back-
ground epidemiologic and clinical data and (2) send acute 10.1 Tick-Borne Viruses
and convalescent serum samples to the nearest public health
laboratory (usually a state laboratory), with a request for anti- 10.1.1 Mammalian Tick-Borne Virus Complex
body tests for arboviruses and other encephalitis-producing The mammalian tick-borne virus complex, a genetic and
viruses. Some state laboratories may not provide this testing, antigenically related group of viruses, share similar fea-
so a request for transshipment of sera to the CDC might be tures and have ticks as their vector and animal vertebrates
included. as a reservoir [69]. These viruses are pathogenic to humans
producing a range of diseases to include a mild febrile ill-
ness, severe hemorrhagic fever, and encephalitis and contain
9 Control and Prevention viruses that have emerged or emerging as global health prob-
lems. Some viruses, such as the agent of Kyasanur Forest
Major control methods include (1) control of the arthro- disease, remain localized to specific geographic areas and
pod vector, which may be by elimination of breeding sites, have the potential to emerge as regional health threats. The
termed source reduction, or modification of them by applica- mammalian tick-borne virus group includes Omsk hemor-
tion of insecticidal substances or by direct attack on the adult rhagic fever virus (OHFV), Langat virus (LGTV), Alkhurma
arthropods through residual insecticide treatment of adult hemorrhagic fever virus (AHFV), Kyasanur Forest disease
resting places; (2) avoidance of exposure to vector bites by virus (KFDV), Powassan virus (POWV), Royal Farm virus
screening of houses, by use of protective clothing, and by (RFV), Karshi virus (KSIV), Gadgets Gully virus (GGYV),
application of insect repellent sprays or creams when outside and Louping ill virus (LIV) [2].
in high-risk areas; and (3) immunization, a procedure widely
used only for yellow fever, TBEV, and Japanese encephalitis Gadgets Gully Virus (GGYV)
in endemic areas. GGYV was first isolated from Ixodes uriae on the penguin
Control of vectors through biological approaches ranges rockeries of Macquarie Island, Australia, in 1976 [70]. Six
from introduction of competing species such as the use of other strains were isolated from the same location in 1976
parasites lethal to the vector including protozoa, helminths, and 1977. To date GGYV has not been known to be a cause
bacteria, and viruses to introduction of genes deleterious to of human disease.
the vector population or influencing the vector behavior or
capacity to be infected with a pathogen [68]. Kyasanur Forest Disease Virus (KFDV)
KFVD, also known as monkey fever, is a disease of man
that is transmitted primarily by Haemaphysalis ticks in the
10 Characteristics of Selected tropical deciduous forests of the Karnataka State in South
Pathogenic Flaviviruses India [71]. Its subsequent discovery in the Yunnan Province,
China, in 1989, called Nanjianyin virus, is now known as
Selected flaviviruses are discussed in this section and a subtype of KFDV [72]. Results of a 1987–1990 seroepi-
focused on those that produce human infections and repre- demiologic investigation in the Yunnan Province demon-
sent emerged or emerging infection or have the potential to strated that residents of the Hengduan Mountain region
become global health problems. This section is organized had been infected with this virus [72]. This suggests that
by vector into tick-borne, mosquito-borne, or viruses with KFDV may have a wider geographic distribution, and migra-
no known vector (see Table 16.1). Under the tick-borne tory birds with attached infected ticks may be responsible.
flaviviruses are the mammalian tick-borne virus complex At-risk populations for KFDV include persons with recre-
(tick-borne encephalitis, Gadgets Gully virus, Kadam virus, ational or occupational exposure to the forests in Karnataka,
Kyasanur Forest disease, Alkhurma virus, Langat virus, India. Human disease may be accompanied by outbreaks
Omsk hemorrhagic fever, Powassan virus, Royal Farm of hemorrhagic disease in forest monkeys maintained in a
virus, and Louping ill virus). Under the mosquito-borne sylvatic cycle. After an incubation period of 3–8 days, the
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 397

symptoms of KFDV infection are fever, headache, severe Although wild-type LGTV has never been found to cause
muscle pain, cough, dehydration, and gastrointestinal symp- human disease, patients with terminal malignancies were
toms. After 1–2 weeks of symptoms, some patients recover inoculated with LGTV in a clinical study hoping the virus
while most patients experience severe hemorrhagic fevers might produce remission. The investigators also thought that
with a 2–10 % fatality [73]. Treatment is supportive care. A data from this study might also support the use of LGTV as
formalin-inactivated virus vaccine produced in chick embryo a live virus vaccine candidate against other tick-borne flavi-
fibroblasts is licensed and available in India [74–77]. viruses in healthy individuals. In this study, LGTV adminis-
tered subcutaneously was able to produce viremia, fever, and
Alkhurma Hemorrhagic Fever Virus (AHFV) leukopenia [84]. Further studies on this as a viable human
AHFV has been isolated several times since 1995 from vaccine are limited.
the blood of patients with severe hemorrhagic fever in
Saudi Arabia. It is associated with the camel tick. Among Louping Ill Virus (LIV)
16 patients, 4 had lethal outcome [78]. Sequence analysis Louping ill virus is a zoonotic disease of livestock on the
revealed the close genetic relationship of this virus to KFDV British Isles transmitted by the tick Ixodes ricinus. It is
[79]. AHFV may be an emerging infection and a risk to also a very rare cause of disease in humans for people who
travelers based on a report of tourists visiting southeastern work closely with sheep or the virus. There have been over
Egypt near the border of Sudan who were infected by AHFV 30 human cases of disease from LIV and several cases of
and developed hemorrhagic illness [80]. Like KFDV, AHFV laboratory-acquired infections [85, 86]. Four clinical syn-
may have its origin in Africa and in the case of AHFV trans- dromes can be seen in human disease to include influenza-
ported from Africa and other countries to Saudi Arabia by like illness, a biphasic encephalitis, a poliomyelitis-like
the transport of camels and other livestock carrying infected illness, and a hemorrhagic fever [86].
ticks [80].
Omsk Hemorrhagic Fever Virus (OHFV)
Karshi Virus (KSIV) OHFV is a significant cause of hemorrhagic fever and
KSIV has been isolated from Ornithodoros papillipes ticks encephalitis in Western Siberia. While the disease may
from Karshi, Uzbekistan. It has been reported in the Russian be transmitted by Dermacentor ticks, outbreaks related to
literature as a cause of encephalitis in humans though the direct human contact with the virus from muskrat trapping
exact numbers are not known (Dr. S. Khodjaev, personal and skinning may occur [78]. The most marked clinical sign
communication). The vector competence of human isolates of this disease is hemorrhage, but clinical symptoms also
of KSIV was tested in a variety of mosquitoes and ticks. include diffuse encephalitis which disappears during the
KSIV replicated in and was transmitted by all three species recovery period [78].
of Ornithodoros ticks tested (O. parkeri, O. sonrai, and O.
tartakovskyi) [81]. Experiments demonstrated that when Powassan Virus (POWV)
inoculated with Karshi virus, 90 % of Ornithodoros ticks POWV is a tick-transmitted virus of the tick-borne encepha-
transmitted this virus to suckling mice and transmission litis subgroup of flaviviruses. It is antigenically related to
continued for at least 1 year [81]. Female O. tartakovskyi TBEV discussed below. POWV is a North American virus
transmitted KSIV vertically to their progeny. This study sug- and originally isolated from the brain of a child who died
gests that Ornithodoros spp. are potential vectors and rodent in Ontario in 1959 [87]. Powassan virus has been found to
species a possible reservoir, with the tick responsible for the be widely distributed in small mammals in Canada and the
long-term maintenance of KSIV in the environment. northern states of the United States including New York,
Wisconsin, and Minnesota [88]. POWV was originally
Royal Farm Virus (RFV) thought to be a minor cause of human encephalitis with 27
RFV was first isolated from Argas hermanni nymphs from cases reported in Canada and the northeastern United States
Kabul, Afghanistan, by the US Naval Medical Research during 1958–1998 [89]. Over the past decade there has been
Unit in Cairo, Egypt [82]. Its group relation to Powassan an expansion of cases throughout Canada and the United
and Langat viruses was demonstrated by complement fixa- States with four Maine and Vermont residents with encepha-
tion (CF) and neutralization tests (N). By CF test it is closely litis found to be infected with POWV in 1998–2001. During
related to Powassan and by N test to Langat. RFV has never 1999–2005, there were nine cases of serologically confirmed
been found to be a cause of human disease. POWV disease: four from Maine, two from New York, and
one each from Michigan, Vermont, and Wisconsin [89,
Langat Virus (LGTV) 90]. The Michigan and Wisconsin cases represent the first
LGTV was first isolated in Malaysia and Thailand from pools reported from the north-central United States and suggest
of ticks of Ixodes granulatus and Haemaphysalis spp [83]. that this infection may be underreported in the United States.
398 S.J. Thomas et al.

POWV encephalitis is characterized as a classic encephali- cluded Aroa was a new group B virus most closely related to
tis with the acute onset of muscle weakness, confusion, and Bussuquara virus. Antigenic and biological properties cate-
neurologic signs. All patients described in this series recov- gorize AROAV closely with Israel turkey meningoencephali-
ered but most had long-term neurologic sequelae. POWV tis, Koutango, and Negishi viruses. AROAV grows in C6/36
is transmitted by hard ticks (Ixodidae spp.). Diagnosis can cells suggesting vector transmission potential [95]. There are
be accomplished by serology and PCR. There is no vaccine no reports of human infection or disease [3].
available for POWV or specific therapy for illness.
Bussuquara Virus (BSQV)
10.1.2 Tick-Borne Encephalitis BSQV was isolated in 1956 in Belem, Brazil, from a howler
Tick-borne encephalitis virus (TBEV) produces a fatal monkey (Alouatta belzebul). The virus has been isolated in
encephalitis in Europe and Asia [91]. The disease has been the Amazon from sentinel and wild Proechimys spp. rodents
known under several names, including Russian spring–sum- and Culex mosquitoes [96]. The virus was known to circulate
mer encephalitis and Far Eastern encephalitis. Clinically, in Brazil, Colombia, and Panama [97]. In vitro experiments
TBEV produces an acute febrile illness followed by enceph- demonstrate infection across a range of murine, chick, and
alitis with a high mortality rate. The virus is maintained in monkey cell lines [98]. Both Aedes and Culex mosquito spe-
an environmental cycle involving small mammals and birds cies demonstrate a potential for infection. Liver lesions simi-
in forested regions and transmitted by the ticks Ixodes rici- lar to those caused by YFV were noted in an infected howler
nus and I. persulcatus. Humans become infected by the bite monkey, Alouatta belzebul. There has been one human clini-
of infected ticks. Forest and construction workers, woods- cal case reported with fever, headache, profuse sweating, and
men, trappers, and farmers are at highest risk. TBEV is an arthralgia lasting 4 days [3, 99].
emerging infection spreading geographically and increasing
in mortality. From 1974 to 2003 there was a 400 % increase Iguape Virus (IGUV)
in morbidity in Europe and has spread to regions that were IGUV was isolated in 1979, from a sentinel mouse, in the
previously unaffected [92, 93]. TBEV is a notifiable disease rain forest of Iguape county, Sao Paulo state, Brazil [96,
in 16 European countries. There was an average of 8,755 100]. Serosurveys demonstrate wild birds may participate in
reported cases of TBEV per year in Europe and Russia the virus transmission cycle, as may rodents and marsupials.
between the years 1990 and 2007 as compared to 2,755 Defining virus reservoir–vector relationships requires further
cases per year between 1976 and 1989 [94]. The expansion study [100]. There have been no known human infections.
of TBEV may be caused by changes in the number and geo-
graphic range of the tick vector due to climate changes and Naranjal Virus (NJLV)
changes in land use. No specific therapy exists for TBEV. NJLV was first isolated from a hamster in 1976 in Guayaquil,
Prevention from infection is through control of tick popu- Ecuador. It is closely related to BSQV, but CF and neutral-
lations, protection against tick bite (repellents, protective izing antibody assays clearly demonstrate that it is distinct
clothing), and avoidance of tick habitats. An inactivated vac- [101]. Experimental infection of mice produces death and
cine is widely used in central Europe. Diagnosis is made by viremia over 5.6–8 days [3]. There are no reports of human
serology, virus isolation, and PCR. infection or disease.

10.1.3 Mosquito-Borne Viruses Japanese Encephalitis Virus Complex

The mosquito-borne viruses represent some of the most Cacipacore Virus (CPCV)
pathogenic viruses known to man. They have been histori- CPCV was first isolated from the Formicarius analis (black-
cally a cause of large epidemics and have become emerg- faced ant bird) in Oriximina, Para, Brazil, in 1997 [102].
ing or emerged pathogens. They produce a range of illnesses Serosurveys demonstrate evidence of infection in wild birds,
from a severe febrile illness to fatal hemorrhagic disease rodents, bats, and humans. Injection into mice produces
and encephalitis. As mosquito-borne viruses the majority death [102]. A 33-year-old farmworker in the Brazilian state
have an intermediate bird or rodent reservoir with humans of Rondônia, Brazil, was admitted to intensive care with
infected by the bite of infected mosquitoes. suspected leptospirosis and yellow fever; however, viral
sequencing identified CPCV as the cause of infection [103].
Aroa Virus Complex
Aroa Virus (AROAV) Koutango Virus (KOUV)
AROAV was initially isolated from a hamster (Cricetus KOUV was initially isolated in 1968 from a Tatera kempi
auratus) in the vicinity of the Aroa River, Venezuela, in the (gerbil) in the Koutango Village, Saboya region, Dakar,
early 1970s. Complement fixation (CF) experiments con- Senegal. KOUV is known to circulate in Senegal, Central
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 399

African Republic, Somalia, and Gabon [104, 105]. Natural JEV is a small (50 nm), enveloped virus containing a
host range includes rodents, ticks, mosquitoes, and a single 10.7-kb, single-stranded, positive-sense RNA genome. The
case of infection in man (laboratory accident). Mosquito viral envelope (E) protein serves as the cell receptor-binding
(Aedes aegypti) transmission and reinfection experiments protein and the fusion protein for virus attachment and entry
have been conducted as well as experimental transovarial into host target cells. Antibodies directed against E protein
transmission which suggest A. aegypti is a competent vector neutralize the virus and play an important role in protection
[3, 106]. [109, 125, 126]. The JEV was first isolated from the brain of
an encephalitis patient in Tokyo (1935) and was virologically
Japanese Encephalitis Virus (JEV) and serologically established as the prototype (Nakayama)
JEV is the leading cause of viral encephalitis in Asia [107]. strain [127]. Antigenic variation among JEV strains has been
JEV is a mosquito-borne zoonotic flavivirus transmitted pre- shown by serologic, virologic, and monoclonal antibody
dominantly between birds and mammals and to humans by analysis [127–129]. The virus causes cytopathic effect (CPE)
mosquitoes. The principal vector of JEV in northern Asia is in varied cell lines that include chick embryo, human epithe-
the rice paddy-breeding Culex tritaeniorhynchus summa- lial, mouse, Vero, LLC-MK2, and C6/36. A broad range of
rosus and in southern Asia C. tritaeniorhynchus and related hosts experience encephalitis and death following intracra-
C. vishnui group. Many species of wild birds function as nial or intraperitoneal administration of JEV to include mice,
reservoirs, supporting JEV circulation and transmission. hamsters, dogs, foxes, cats, and goats. Asymptomatic vire-
Domestic pigs may serve as amplifying hosts. Horses and mia is observed in monkeys, guinea pigs, rabbits, chickens,
humans are dead-end hosts because the virus circulates in pigs, and bats [3]. Culex tritaeniorhynchus, C. fuscocephal-
blood at low levels and for a short duration, thereby not sup- ues, and C. gelidus were experimentally infected feeding on
porting transmission [108, 109]. viremic pig and chicken and successfully transmitted infec-
Outbreaks of JEV occurred regularly in Japan, Korea, tion to chicken [130–133]. Transovarial transmission was
China, and Taiwan during the twentieth century. Seasonal demonstrated in Aedes albopictus and A. togoi [134].
outbreaks now involve portions of Vietnam, Thailand, Following infection there is a 5- to 7-day incubation period
Nepal, and India. The Philippines, Indonesia, and the north- and then a nonspecific viral prodrome. Early clinical symp-
ern tip of Queensland, Australia have witnessed smaller toms include lethargy, fever, headache, abdominal pain, nau-
outbreaks [108, 109]. Large-scale vaccination programs sea, and vomiting [135]. Nuchal rigidity, photophobia, altered
made it possible for Japan, Korea, and Taiwan to nearly consciousness, hyperexcitability, masked facies, muscle rigid-
eliminate JEV. More recent vaccine adopters (Thailand, Sri ity, cranial nerve palsies, tremulous eye movements, tremors,
Lanka, and Nepal) have also observed a reduced disease involuntary movement of the extremities, paresis, incoordi-
burden [108, 109]. Numerous JEV endemic regions remain nation, pathological reflexes, and meningeal (meningitis),
at risk despite the availability of safe and efficacious JEV parenchymal (encephalitis), or spinal cord (myelitis) signs
vaccines. may follow [136]. Sensory deficits are rare, and children (50–
In JEV endemic areas, the incidence of disease is greater 85 %) experience focal or general seizures more often than
in the young; attack rates in the 3- to 15-year age group are adults (10 %) and are associated with poor clinical outcome
five to ten times higher than in older persons; this is likely a [136]. In a recently published study of 1,282 adult patients
herd-immunity phenomenon [108, 109]. Numerous studies collected from 4 JEV epidemics in India (1978, 1980, 1988,
and epidemiologic observations document a weak protec- and 1989), altered sensorium occurred in 96 %, convulsions
tive effect of prior dengue virus infection on subsequent JEV in 86 %, and headache in 85 % [137]. Hyperkinetic move-
disease [110, 111]. The ratio of symptomatic JEV cases to ments were noted in 46 %, and most (83 %) were choreoath-
infections varies between 1:25 and 1:300 with the lower rates etoid in nature. Opsoclonus (20 %), gaze palsies (16 %), and
(1:200–1:300) observed in Asians indigenous to areas with pupillary changes (48 %) were observed, but cerebellar signs
enzootic JEV transmission, while higher rates were mea- were not. Dystonia and decerebrate rigidity were observed
sured in American military personnel [112–114]. The inci- in 43 and 6 %, respectively, of patients with 17 % having
dence of JEV for travelers from non-endemic countries to paralytic features and seizures in 30 %. Abnormal breathing
Asia is estimated at <1 case per million travelers. Expatriates patterns, pulmonary edema, and upper gastrointestinal hem-
and travelers who spend prolonged periods in JEV enzootic orrhage were observations of prognostic importance.
areas may share a similar or higher risk than the indigenous Elevated peripheral white blood cell counts and low
population [115–117]. Between 1973 and 2008, 55 cases of sodium may be observed. Cerebrospinal fluid (CSF) opening
JEV were reported in travelers from non-endemic countries. pressure, white blood cell content, and protein levels may be
Ten (18 %) of these were fatal and 44 % (N = 24) recovered normal or mildly elevated [136]. Electroencephalogram may
but experienced sequelae [115–124]. demonstrate diffuse delta wave activity and, rarely, seizure
400 S.J. Thomas et al.

patterns [136]. Imaging studies may demonstrate abnormal some resulting in hospitalization [144, 145]. The cause of the
findings in the white matter, thalamus, basal ganglia, cer- reactions is unclear, but the presence of murine neural pro-
ebellum, midbrain, pons, and/or spinal cord. teins, gelatin, and/or thimerosal in vaccine preparations has
If the patient improves, it will typically begin about 1 been implicated, but not proven. More serious was the case
week after symptom onset at the time of defervescence, in Japan of acute disseminated encephalomyelitis (ADEM)
but recovery of neurologic function may take weeks to temporally related to vaccination. ADEM has been reported
years. Seizure disorders, motor and cranial nerve paresis, as a severe drug reaction following administration of inacti-
and movement disorders may persist in up to one third of vated mouse brain vaccine in 5 × 10−4 to 1 × 10−6 administered
patients. Persistent behavioral and/or psychological abnor- doses [143]. No definite increased risk of ADEM temporally
malities occur in 45–75 % of survivors and are more severe associated with JEV vaccination has been proven.
in children [138]. JEV is managed with supportive care as To move away from mouse brain-derived vaccines, vero
there are no specific antivirals demonstrating clear benefit. cells were adopted for the development of numerous JEV
Fatality rates vary between 5 and 40 % and are often reflec- vaccine candidates [135]. Widespread distribution and
tive of medical care resources and capabilities. use in Asia have demonstrated an excellent safety profile,
Diagnosis by viral isolation is infrequent although feasi- immunogenicity, and efficacy [146]. Second-generation JEV
ble from brain tissue in fatal cases or CSF in about one third vaccines with improved safety profiles and lower dosage
of cases during the acute infection period; this portends a bad requirements present new options for immunization [135].
prognosis [139]. Serologic diagnosis is possible, demonstrat- The IC51 (IXIARO®, in Australia and New Zealand avail-
ing a fourfold rise in antibody titer using hemagglutinin inhi- able as JESPECT; Intercell AG, Vienna, Austria) vaccine is
bition, complement fixation, or neutralizing antibody assays a purified, formalin-inactivated, whole-virus JEV vaccine.
with appropriately timed acute and convalescent specimens. The product is approved in the United States (persons aged
Serum IgM antibodies appear early in the course of infec- ≥17 years), Europe, Canada, Switzerland, and Australia.
tion, persist for 3–6 months, and are relatively specific. The IC51 is manufactured by Intercell Biomedical (Livingston,
IgM-capture ELISA is especially well suited for diagnosis United Kingdom) and is distributed in the United States by
by detection of locally synthesized JEV antibody in the CSF Novartis vaccines (Cambridge, Massachusetts). The vac-
[140]. Earlier and more vigorous antibody responses corre- cine construct, developed at the Walter Reed Army Institute
late with improved survival. Serologic assays are plagued by of Research (Silver Spring, MD), is based on a SA14-14-2
cross-reactivity between JEV and other flaviviruses such as virus strain passaged in primary dog kidney (PDK) cells, cul-
dengue and West Nile virus. Newer serologic, reverse tran- tivated in Vero cells, and formulated with aluminum hydrox-
scriptase polymerase chain reaction and NS1 protein-based ide [147]. Numerous clinical trials have demonstrated IC51
assays tout improved sensitivity and specificity. safety and immunogenicity [148, 149]. A pooled, 6-month
Individuals can reduce exposure to vectors by use of mos- safety analysis of seven phase III trials included 3,558 sub-
quito repellant, wearing long-sleeved shirts and trousers, jects with at least one IC51 vaccination, 435 subjects with
avoiding outdoor activities in the evening, and sleeping under a JE-Vax® vaccination, and 657 with phosphate buffered
permethrin-impregnated mosquito nets or in screened or air- saline solution with 0.1 % Al(OH)3 (PBS + Alum) control
conditioned rooms [141]. Active immunization, in addition vaccination demonstrated a similar safety profile between
to personal protective measures, is the optimal strategy for the study arms with a superior local reactogenicity profile
preventing JEV. JEV vaccines have been available since the for IC51 compared to JE-Vax® [150]. Individuals previously
1950s [135]. Early vaccines were produced by inactivating given tick-borne encephalitis (TBE) vaccine and then vacci-
virus grown in mouse brain or in primary hamster kidney nated with IC51 demonstrated a 17 % higher incidence of at
(PHK) cells [135]. These vaccine constructs were used in least one adverse event in volunteers with previous TBE vac-
the United States and Europe (BIKEN; distributed as JE-Vax cination and slightly higher seroconversion rates and higher
by Sanofi Pasteur, Lyon, France) [142]. Seroconversion rates GMT in the IC51 group who had received TBE vaccination
(i.e., quantitative neutralizing antibody titers following vacci- (98 %, 470, compared to 92 %, 182) [151].
nation) and efficacy varied according to the population stud- Sanofi Pasteur has developed a JEV vaccine based on a
ied (indigenous vs. nonindigenous to JEV endemic area) and construct created at the St. Louis University Health Sciences
number of doses administered (one, two, or three doses) in Center, St. Louis, Missouri, and Acambis Inc., Cambridge,
the primary immunization series [135, 143]. Prolific vaccine Massachusetts [152]. JEV-CV is produced by inserting prM
use has contributed to significant decreases in JEV incidence and E genes from the SA14-14-2 JEV virus into the YFV
in Thailand, India, Korea, Taiwan, Vietnam, and areas of 17D viral strain “backbone” containing the YF nonstructural
Malaysia and Sri Lanka [135]. Since 1989, numerous cases genes [153]. The resulting chimeric RNA is electroporated
of moderate to severe hypersensitivity-type reactions tem- into Vero cells and replicated by the highly stable YFV RNA-
porally associated with JEV vaccination have been reported, dependent replicase. The safety and immunogenicity of a
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 401

single dose of JEV-CV at two concentrations (1 × 5.0 log- occur most often during summer (January to May) and follow-
10PFU versus 1 × 4.0 log10PFU) was comparable to licensed ing high rain periods, the ecology remains largely unknown
YFV-VAX (1 × 5.0 log10PFU) in a phase 1 trial. Low-level [161, 163]. Viral activity is monitored by trapping and test-
viremias were observed in both JEV-CV groups, and the ing mosquitoes or testing sentinel chicken flocks for serologic
frequency of all adverse events was similar in each group. seroconversion (http://medent.usyd.edu.au/arbovirus/index.
Overall, 96 % of subjects who received JEV-CV developed html, accessed 28 May 2012). Early indicators of MVEV cir-
neutralizing antibodies to one or more of the wild-type JEV culation trigger increased public health vigilance and education
strains tested (Beijing, P3, Nakayama), and all developed regarding personal protective measures.
antibodies to the homologous strain [154]. There is evi- Complement fixation and neutralizing and cross-
dence that JEV-CV induced sterilizing immunity. Safety and neutralizing antibody studies demonstrated MVEV was a
immune responses were evaluated in N = 10 JEV-CV (1 × 4.0– group B arbovirus in a subgroup containing Japanese enceph-
5.0 log10PFU) vaccine recipients who, about 9 months later, alitis, West Nile, St. Louis encephalitis, and Kunjin, Usutu,
were given a single dose of JE-Vax. No severe adverse events Kokobera, Stratford, and Alfuy viruses [3]. Nucleotide
were noted and seroconversion rates rose from 50 % pre-JE- sequencing associates the MVEV most closely with the
Vax to 90 % with a GMT greater than 1:150. Phase 3 stud- Japanese encephalitis virus. Phylogenetic studies suggest
ies assessed safety and immunogenicity comparing JEV-CV MVEV emerged in the Malay–Indonesian regions from an
and a mouse brain-derived (MBD)-JEV vaccine. The safety African progenitor virus, possibly Usutu. There appears to
profile of JEV-CV was superior to the MBD-JEV vaccine be persistent homogeneity of MVEV strains from wide-
and similar to placebo. The percent of the cohort serocon- spread areas throughout Australia different from those cir-
verting following a single dose of JEV-CV by PRNT with culating in New Guinea. The natural host range of MVEV is
the homologous strain was non-inferior to three doses of the broad including humans, chickens, species of water and land
MBD-JEV vaccine (99.1 % vs. 95.1 %) [155]. Additional birds, horses, dogs, foxes, and opossum. Experimental infec-
studies assessing immunization of toddlers and children, tion of mice, sheep, chick embryos, and hamsters resulted in
co-administration and sequential administration with yellow death [3]. Culex annulirostris and C. quinquefasciatus and
fever (YF) vaccine (YF-17D strain; Stamaril((R)), Sanofi Aedes occidentalis and A. vigilax demonstrated competence
Pasteur), and administration of a booster doses demonstrated to infect chicks following ingestion of an infected blood
excellent safety profiles and immunogenicity [156]. meal, and transovarial transmission following oral infection
was achieved with Aedes aegypti [49, 164].
Murray Valley Encephalitis Virus (MVEV) Approximately 1 of every 150–1,000 MVEV infec-
MVEV has caused numerous encephalitis epidemics tions results in clinical disease. Complete recovery is seen
(Australian X disease) in Australia since the early 1900s. The in approximately 40 %, long-term neurologic sequelae in
virus was first isolated in 1951 from the brain of a 19-year- 30–50 %, and death in 15–30 % [165]. Following an incuba-
old male who succumbed to infection [3]. Encephalitis out- tion period of 1–4 weeks, patients will experience 2–5 days
breaks in 1917, 1918, 1922, and 1925 were initially without prodrome of high fever and headache with anorexia, myal-
an etiologic agent but are now believed to have been MVE gia, nausea, vomiting, diarrhea, rash, or cough [159, 166].
[139]. Additional outbreaks occurred in 1956, 1971, 1974, Neurologic features include lethargy, irritability, and confu-
1978, 1981, and 1984, primarily in the Murray Valley of sion with or without seizures. The clinical spectrum of dis-
New South Wales and Victoria [157]. The 1974 outbreak ease can be broad ranging from fever and headache without
was unique in its geographic expansiveness, infecting peo- encephalitis to flaccid paralysis, cranial nerve and brainstem
ple in east central Queensland, Northern Territory, northern involvement, encephalitis with complete recovery, or death
and southeastern South Australia, and the Ord River Basin [161]. The disease appears to progress very rapidly in infants
of Western Australia [158]. Between 1978 and 1991 MVE [139].
was diagnosed in 26 patients, and 16 were in the Kimberley Magnetic resonance imaging (MRI) is the most sensitive
area of Western Australia [159]. During the 2007–2009 and specific imaging study available to support a diagnosis.
seasons, six human cases of MVEV were reported, at least T2-weighted images demonstrate bilateral hyperintensity of
two fatal, to the National Notifiable Diseases Surveillance the deep gray matter and may have findings mimicking her-
System (NNDSS) [160]. Sixteen people are believed to have pes simplex virus (HSV) encephalitis involving the temporal
contracted MVE in 2011 [161]. MVEV is endemic to Papau lobes, red nucleus, and cervical spinal cord.
New Guinea and Irian Jaya [162]. Laboratory confirmation of infection is achieved through
MVEV is a mosquito-borne flavivirus existing in an enzo- viral isolation, detection of MVEV RNA, a fourfold rise in
otic cycle involving primarily water birds [161]. Culex annu- IgG between acute and convalescent serum samples, or find-
lirostris is believed to be the principal vector, preferring to ing IgM in the serum of cerebral spinal fluid (CSF) (http://
breed in shallow, warm, fresh water. Although MVE outbreaks www.health.gov.au/internet/main/publishing.nsf/Content/
402 S.J. Thomas et al.

health-arbovirus-mve-guidelines.htm, accessed 29 May include primary chick embryo, hamster kidney, BHK-21,
2012). Cross-reactivity among antibody-based tests com- Vero, and LLC-MK2. Cell cultures from human, primate,
plicates distinguishing MVEV from other flaviviruses fol- rodent, swine, and avian cell cultures support replication
lowing infection or vaccination. Neutralization assays or an [224]. Natural host range includes wild vertebrate species
epitope-blocking ELISA may add diagnostic specificity. of birds as well as raccoons, opossums, and bats in North
There are no licensed vaccines or therapeutics to protect America and birds, rodents, nonhuman primates, and the
against or treat MVE. Trials of steroids, ribavirin, interferon three-toed sloth in tropical America. SLE varies in virulence
alpha-2a, and formulations of intravenous immunoglobulin for mice [139, 225, 226].
have been evaluated in the context of Japanese encephali- SLE virus has been responsible for thousands of deaths,
tis and West Nile virus infections but with little success more than 10,000 severe illnesses, and no fewer than a mil-
[167, 168]. Treatment is supportive and has reduced mortal- lion mild or subclinical infections [227, 228]. Only 1–2 % of
ity and morbidity. Passive immunization studies in animals SLE virus infections are symptomatic [210, 229, 230]. The
using gamma globulin from MVE and Japanese encephali- ratio of asymptomatic to symptomatic infection ranges from
tis virus survivors demonstrated prophylactic efficacy. The 800:1 in children to 85:1 in adults older than 60 years [200,
ability of a live chimeric Japanese encephalitis virus vaccine 231]. The infection incubation period ranges between 4 and
(ChimeriVax-JE) to cross-protect against MVEV in two 21 days. When the disease occurs, it may be classified into
encephalitis mouse models suggested a single dose elicited a three clinical syndromes: constitutional symptoms and head-
protective and durable immune response [169]. DNA-based ache (febrile headache), aseptic meningitis, and fatal enceph-
and Semliki Forest virus-vectored vaccines using the MVEV alitis [139, 200, 209]. Most cases start with fever, headache,
prM and E proteins as immunogens were investigated in a nausea, myalgias, and sometimes respiratory or abdominal
murine model. Both candidates induced durable, MVE- symptoms; most recover completely [231]. In those who
specific neutralizing antibody responses [170]. develop advanced disease, following the acute febrile pro-
drome, there may be an acute or subacute appearance of men-
St. Louis Encephalitis Virus (SLEV) ingeal and other neurologic signs and symptoms. Symptoms
The first recognized outbreak of SLE occurred in Paris, may include nuchal rigidity, disorientation, unsteady gait,
Illinois, in 1932 followed the next year by epidemics in St. vomiting, and diarrhea. Tremulousness involving the eyelids,
Louis and Kansas City, Missouri. The virus was first iso- tongue, lips, and extremities is observed. Cerebellar and cra-
lated from the brain of a fatal encephalitis case following nial nerve signs are common [231, 232]. Apathy, confusion,
inoculation in mice and rhesus monkeys [171]. Subsequent and disorientation leading to coma may occur. Nearly one
SLE outbreaks occurred in the Pacific coast states (1940s), quarter of patients experience urinary symptoms (frequency,
Florida (1959, 1961), Texas, Ohio-Mississippi Valley urgency, dysuria) [233]. The morality rate is approximately
(1970s), Colorado, California, Florida, Texas, and Arkansas 8 %, ranging from approximately 2 % in the young to 22 %
[139, 171–175]. Disease has been reported throughout the in the elderly who have comorbidities [139]. Advanced age
majority of the United States, southern Canada, Mexico, is the most significant risk for developing both symptomatic
and in limited areas of Central and South America [68, 173, disease and more severe encephalitis after infection [172].
175–209]. There is a diffuse endemicity over vast rural areas, People older than 60 years of age have the highest frequency
with low rates of seropositives in humans, but with occa- of encephalitis [139]. Almost 90 % of elderly SLE patients
sional epidemics of hundreds to more than a thousand cases. develop encephalitis [230]. Longer-term sequelae such as
The monitoring of sentinel flocks of birds for infection has asthenia, irritability, tremors, sleeplessness, depression,
proved useful as an early warning system [174]. memory loss, and headaches typically last less than 3 years
The virus is transmitted by a number of culicine mosqui- but may last longer. Persistent symptoms include gait and
toes, including Culex pipiens and C. quinquefasciatus in the speech disturbances, sensorimotor impairment, psychoneu-
East and urban areas of the West, C. tarsalis in rural areas of rotic complaints, and tremors [234–236].
the western states, and C. nigripalpus in Florida and southern Clinical laboratory findings are nondistinct. Peripheral
areas [210–214]. In rural areas of the West and Southwest, white blood cell count may be high and urine findings may
it is closely tied to C. tarsalis. Depending on the climate of include microscopic hematuria, proteinuria, and pyuria
the region, maintenance of the virus can occur by year-round [231]. Inappropriate antidiuretic hormone secretion and
horizontal transmission from bird to mosquito, overwinter hyponatremia may occur in a third of patients. Fewer than
survival of infected mosquitoes, or venereal or transovarian 200/mm3 white blood cells are typically seen in the cerebral
infection [215–221]. spinal fluid; polymorphonuclear cells predominate early
SLE virus is in the JEV–MVE–West Nile virus complex with an eventual shift toward a lymphocytic pleocytosis.
or subgroup [139, 222, 223]. The virus produces cytopathic Protein levels may be elevated to 1.5–2.0 times normal.
effect and plaques in numerous vertebrate cell lines that Electroencephalogram shows diffuse slowing and seizure
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 403

activity. Magnetic resonance imaging may show increased emerged in Vienna, Austria, in 2001 associated with avian,
signal in the substantia nigra, while radionuclide brain scans mainly blackbird, mortality. Infection has recently been
and computed tomography have been normal [139, 237]. noted in a variety of central European birds in Austria,
Diagnosis should be suspected if the patient presents Hungary, Switzerland, United Kingdom, Spain, Germany,
with a compatible clinical syndrome in the summer or and Italy [250, 251]. USUV was initially discovered to be
early fall, in an area known to have SLE virus circulation, clinically relevant in 1981 when a man in the Central African
with reports of similar cases with patients at the extremes Republic developed fever and rash following infection. In
of age. Although virus isolation has been documented 2004, a 10-year-old child in Burkina Faso developed fever
from the liver, spleen, lung, kidney, and brain, isolation is and jaundice following infection. In 2009, 2 immunosup-
very unusual from serum or CSF [238]. Polymerase chain pressed people (liver transplant, B-cell lymphoma) in Italy
reaction assays are being developed for diagnosis and sur- developed encephalitis [252, 253]. A recent serosurvey in
veillance [239–246]. Diagnosis most often is made using Northeastern Italy demonstrated 4 of 359 donors had IgG
serologic assays testing acute and convalescent serum sam- evidence of USUV infection [254].
ples or a cerebral spinal fluid sample [21]. IgM antibodies in
serum appear within the 4 days after infection, peak at 7–14 West Nile Virus (WNV)
days, and then decline and disappear by day 60 but may per- WNV was first isolated in a febrile woman in the West Nile
sist up to a year in a small percentage of patients [139, 231, District of Uganda in 1937 [255]. Since its discovery in West
247]. A fourfold change in serum antibody titer confirms Africa, outbreaks of WNV have occurred in many parts of
infection, and presence of IgM antibody in a single serum the world, and WNV is now endemic in Africa, Europe, the
is presumptive evidence. Hemagglutinin inhibition detects Middle East, West and Central Asia, Oceania, and North
group-reactive antigens and may be a useful screening test. America [256]. In the United States, the westward spread of
The presence of complement fixation antibodies in a single WNV across the continent from the initial cases in New York
serum sample is presumptive evidence of a recent infection, over the course of about 5 years was an epidemiologic phe-
but 20 % of patients with confirmed SLE virus infections do nomenon that was closely monitored [257]. WNV now has
not develop complement fixation antibodies [139]. Detection become the leading cause of neuroinvasive arboviral disease
of IgM antibodies in the cerebral spinal fluid by enzyme- in the United States [258].
linked immunosorbent assay (ELISA) provides a rapid and Wild birds are the primary vertebrate reservoirs of WNV
early diagnosis. in endemic regions [259] and WNV has been found to be
There is no licensed antiviral therapeutic to treat SLE. capable of infecting over 300 different species of birds [260].
The current care standard is supportive focusing on control- In many instances, WNV has spread across continents and
ling seizures, providing respiratory support, reducing cere- regions following bird migration patterns [261]. The virus is
bral edema, and managing metabolic derangements [231]. transmitted by mosquitoes usually of the Culex spp., though
There is no licensed vaccine to prevent SLE. Personal pro- various spp. of Aedes, Anopheles, and Ornithodoros ticks
tective measures and other vector avoidance practices are can be infected by WNV [3]. Mammals are less important
encouraged. Control in rural or an urban epidemics has been than birds in maintaining transmission cycles of the virus as
attempted by emergency application of mosquito control viremia is too low in most of the mammal species to reinfect
measures. Long-term control for urban and suburban locali- mosquitoes [262].
ties depends on good sanitation with respect to drainage and Nearly all humans are infected with WNV primarily
adequate disposal of waste water. In more specific rural areas by the bite of infected mosquitoes [257]. However, trans-
under irrigation, much can be accomplished through water mission of WNV to humans has also occurred through
management and directed application of insecticides to keep transplanted organs, and transfused blood, transplacen-
mosquito populations low. tal transmission [263], and transmission through breast
milk may also be likely [264]. Most infections with
Usutu Virus (USUV) WNV are asymptomatic. The typical incubation period is
The USUV is an avian virus discovered in South Africa 2–14 days, and a self-limited febrile illness without neu-
(1959) in a female mosquito (Culex neavei) [3, 248]. The rologic involvement, known as West Nile fever, may occur
virus has infectivity for numerous primary and continuous in approximately 20–30 % of infected cases. The symp-
animal species and human cell lines [3]. The life cycle is toms of West Nile fever may include malaise, eye pain,
complex, involving host bird species and Culex mosquitoes headache, myalgia, gastrointestinal discomfort, and rash.
as primary vectors and humans, horses, and other mam- A maculopapular rash occurs in about half of the persons
mals as incidental hosts. Geographic range initially includes with West Nile fever but is less commonly reported in per-
Senegal, Central African Republic, Nigeria, Uganda, sons with neuroinvasive disease. About 1 in 150 cases may
Burkina Faso, Cote d’Ivoire, and Morocco [249]. The virus progress to severe disease and develop encephalitis, men-
404 S.J. Thomas et al.

ingitis, or acute flaccid paralysis. Long-term complications New Mapoon Virus

(1 year or more after infection) are common in patients New Mapoon virus was originally isolated as part of viral
recovering from severe WNV infection. Currently, support- isolates obtained from mosquitoes in 1998 on Cape York
ive care is the only treatment available. Advanced age is Peninsula (located Far North Queensland, Australia) and
the most important predictor of death [265]. The mortality in 2000 on Saibai Island (one of the Torres Strait Islands
rate among neuroinvasive disease is approximately 10 %, in Australia, between the Australian mainland and the
with increased risk for patients with compromised immune island of New Guinea) and New Mapoon, Queensland [3].
systems and advanced age and with underlying conditions Viruses were characterized by partial genomic sequencing,
such as diabetes mellitus. monoclonal antibody-binding assays, and polyclonal cross-
Diagnosis of WNV is made primarily by the use of indi- neutralization tests. Two of these isolates were antigenically
rect antibody-capture ELISA or PCR. In most cases IgM will related to the KOKV complex but distinct. Sequencing dem-
be produced within 4–7 days and may persist for more than a onstrated a distinct novel virus named New Mapoon virus.
year [266]. ELISA for WNV may be performed on serum or
CSF. Other serologic tests such as neutralization assays gen- Ntaya Virus Complex
erally are less cross-reactive with other flaviviruses, but are Ilheus Virus (ILHV)
not widely available for clinical diagnosis. Antigen detection Ilheus virus (ILHV) was isolated from Aedes serratus and
assays are also being developed but are not widely used in Psorophora ferox mosquitoes near Ilheus, Bahia, Brazil, in
the clinical setting. PCR is widely available and is very spe- 1944 [269]. In the original study, Aedes aegypti was dem-
cific and sensitive. onstrated as a competent vector for this virus. After its dis-
There is no WNV vaccine available for use in humans, but covery, the virus was also isolated from a variety of other
a number of WNV vaccine candidates are in clinical trials. mosquito species and birds in Latin America. ILHV was
There are three WNV vaccines currently licensed for use in described as producing a febrile illness in humans in Central
horses; two are killed virus vaccines, and one is a chimeric and South America, but the burden of disease is not well
recombinant canarypox virus vaccine [262]. studied. In November 2005, a patient in Bolivia was reported
with ILHV who developed fever, malaise, asthenia, con-
Yaounde Virus (YAOV) junctival injection, vesicular rash, facial edema, arthralgia,
The original source of YAOV was a pool of Culex nebu- myalgias, bone pain, abdominal pain, headache, and earache
losis female mosquitoes collected in 1968 near Yaounde, [270].
Cameroon, Africa [3]. One month later the virus was iso-
lated via intracerebral inoculation in mice. Complement Ntaya Virus (NTAV)
fixation identified the virus as related to Usutu virus. Ntaya virus was originally isolated from mosquitoes in
Natural host range includes mosquitoes (Culex spp., Aedes, Ntaya, Uganda [3]. Isolation has been made from mosqui-
Eretmapodites), rodent, and bird. Injection in mice produced toes in Uganda, Cameroon, and the Central African Republic
death. There are no known human cases [3]. and may be endemic in Nigeria, Kenya, and the Zambia
[3]. Serologic surveys suggest that this virus may be wide-
Kokobera Virus Complex spread with antibodies to Ntaya virus in subjects in Tanzania,
Kokobera Virus (KOKV) Egypt, and the Far East. In a study of returning travelers from
KOKV is a mosquito-borne virus that was originally iso- Africa, three patients tested positive for Ntaya virus. Illness
lated from Culex annulirostris at the Kowanyama (Mitchell symptoms reported by the travelers included severe malaise,
River Mission) in northern Queensland in 1960 and named fever, headache, myalgia, and neurologic manifestations of
after a local Aboriginal tribe [267]. It is isolated throughout dizziness, numbness, and weakness of the left leg and arm.
Australia and Papua New Guinea [268]. The reservoir for In one traveler there was severe amblyopia and restriction of
the virus is thought to be kangaroos, wallabies, and horses. the peripheral visual fields.
Human infections with KOKV result in an acute polyarticu-
lar disease. Other Viruses Within the Ntaya Complex
Other viruses within this complex are primarily pathogens
Stratford Virus (STRV) of poultry causing large epidemics. These include Bagaza
STRV has a geographic distribution similar to KOKV from virus (BAGV), Israel turkey meningoencephalomyelitis
Culex species producing a febrile polyarticular illness. virus (ITV), and Tembusu virus (TMUV). All are spread by
Originally classified with KOKV as part of the JEV sero- a variety of mosquitoes primarily, Culex spp. These viruses
complex, serologic and sequencing of the virus demonstrated have the potential to produce human disease with serologic
that STRV is closely related to KOKV and its classification evidence of human infections though the burden of disease
into the KOKV complex [3]. within the human population is not known and thus are
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 405

briefly described. BAGV which is closely related to ITV has Asian lineage [278]. As noted earlier, ZIKV was documented
been described as producing a febrile illness in humans with to have been transmitted sexually from a scientist who was
one study demonstrating a possible cause of human encepha- infected in Senegal and, upon returning to the United States,
litis in India. In an outbreak investigation of a severe duck infected his spouse [62].
illness in China, a new virus related to TMUV and BAGV
was described named BYD virus [271]. Yellow Fever Virus Complex
Banzi Virus (BANV)
Spondweni Virus Complex BANV was first isolated in 1956 in a 9-year-old boy in
Spondweni Virus (SPOV) Ndumu, South Africa [279]. The clinical manifestations of
SPOV was first isolated in 1955 from mosquitoes, this virus was a nonspecific febrile illness. There have only
Taeniorhynchus uniformis collected in Tongaland, South been two acute cases of definitively diagnosed BANV infec-
Africa. The name was derived from the location they tions, but seroprevalence studies across sub-Saharan Africa
were collected, the district of Spondweni. A serosurvey of suggest BANV infections may be largely subclinical and
residents in Tongaland demonstrated evidence of infection underdiagnosed [3].
to SPOV. Human infection was demonstrated in laboratory
workers working with the virus characterized as a febrile Bouboui Virus (BOUV)
illness with generalized aches and pains, headache rigors, BOUV is a flavivirus that has been isolated in a baboon in
and vertigo [272]. SPOV has been isolated from humans in Senegal and in mosquitoes in Central Africa and Senegal.
Mozambique and Cameroon with serologic evidence of infec- While BOUV has never been isolated in humans, serop-
tion in humans in Angolal and Botswana [272]. The virus revalence studies (employing hemagglutination inhibition
has been isolated from several mosquito species including assays) involving over 4,000 patients in Central Africa have
Mansonia uniformis, Aedes circumluteolus, M. africana, A. found 15 patients with serum antibodies to this virus. The
cumminsii, Eretmapodites silvestris, A. fryeri, and A. fowleri clinical manifestations of this disease in humans are not
[272]. In 1982 a case of acute SPOV infection was reported known [3].
in a 40-year-old American man, who had worked in Bitou,
located in southeastern Upper Volta (Burkina Faso), near Wesselsbron Virus (WESSV)
the borders of Ghana and Togo [272]. Symptoms included WESSV is primarily an arboviral disease of sheep, cattle,
severe headache, dizziness, nausea, muscle aches, eye pain, and goats [280]. However, it has caused nine cases of human
and sensitivity to light. febrile disease in sub-Saharan Africa, sometimes with neuro-
logic complications to include disturbances in speech, hear-
Zika Virus (ZIKV) ing, and/or vision [281]. While WESSV is primarily located
ZIKV was originally isolated from a caged febrile rhesus mon- in Africa, it has been isolated in mosquitoes in Bang Phra,
key in the Zika Forest, Entebbe, Uganda [273]. Subsequent Thailand [3].
studies revealed Aedes africanus as the primary vector and a
serosurvey demonstrated evidence of human infection [274]. Yellow Fever Virus (YFV)
Human infection in a field worker and an experimental infec- YFV is the prototype member of the Flaviviridae family
tion of a volunteer demonstrated that ZIKV could produce a of viruses. The origin of YFV is speculated to have been
severe febrile illness associated with headache, myalgias, and Western Africa, although the disease was first distinguished
prostatitis symptoms [275]. Since its original isolation, ZIKV from other tropical febrile diseases by the Mayans and the
has been isolated from mosquitoes and a cause of human Spanish colonists in the Yucatan peninsula in 1648. During
infections throughout equatorial Africa, Indonesia, Malaysia, the eighteenth and nineteenth centuries, it was one of the
and Cambodia. Aedes aegypti was found to be the primary great plagues of the world occurring along the eastern US
vector in Southeast Asia [276]. In 2007, a large outbreak of seacoast, in Central America and South America, and in
ZIKV infection occurred in Yap Island, Micronesia [277]. Africa throughout the tropical area [164, 165, 167]. Major
In total there were 49 confirmed and 59 probable cases of epidemics occurred in many seaports of the United States,
ZIKV illness. Patients developed rash, fever, arthralgia, and and the 1905 outbreak in New Orleans was particularly
conjunctivitis. The authors estimated that 73 % of Yap resi- severe. The last US indigenous case occurred in 1911 and
dents 3 years of age or older had been recently infected with the last imported cases in 1924 [13, 214]. It never became
ZIKV. Aedes hensilli was the predominant mosquito species established in Europe above the range of the vector, A.
identified. Currently ZIKV is widely distributed outside of aegypti mosquitoes. YFV has never been reported from Asia
Africa with cases or serologic evidence of infection in India, and Australia, despite the endemic presence of A. aegypti in
Malaysia, the Philippines, Thailand, Vietnam, and Indonesia. tropical Asia [282]. The historical significance of YFV was
ZIKV was demonstrated to have evolved into an African and discussed in a previous section of this chapter.
406 S.J. Thomas et al.

The development of the YFV vaccine, Theiler’s attenu- series of epidemics of varying severity [285]. The period
ated live virus vaccine introduced in 1937, represents the first 1986–1990 represented an extraordinarily active period of
successful vaccine against an arbovirus and forms the basis YFV. The worldwide total of 17,728 cases and 4,710 deaths
for the present-day product [283]. YFV is maintained in the (i.e., a case fatality rate of 26.6 %) represents the greatest
environment in two cycles: an urban cycle involving human amount of YFV activity reported to the WHO for any 5-year
beings and A. aegypti mosquitoes and a sylvatic or jungle period since reporting began in 1948 [286, 287]. However, in
YFV cycle involving forest primates, principally monkeys, Africa, numerous studies have shown that only a small per-
and forest canopy mosquitoes, with human infections tangen- centage of African YFV cases are reported [287]. Due to the
tial to the transmission cycle [284, 285]. In 1901, eradication sylvatic cycle of jungle YFV, worldwide eradication is not
efforts directed toward A. aegypti mosquitoes were launched considered possible. Despite numerous studies, the question
under the direction of Dr. William Gorgas in Havana. These of maintenance of YFV in nature remains somewhat obscure.
eradication efforts, with concomitant reduction of YFV, were Although a continual vertebrate–vector maintenance cycle is
extended throughout Central and South America in the early possible in some environments, in other areas overwintering
1900s. The chain of urban A. aegypti-transmitted YFV was and maintenance probably occur via other mechanisms. The
successfully broken by the eradication program. The last laboratory and field studies that confirmed that YFV virus
endemic focus of A. aegypti-transmitted urban YFV was can be transovarially transmitted in many of its mosquito
in northeastern Brazil in 1934 [284]. The eradication of the vectors suggest that this mechanism might play an important
vector, and the concomitant reduction in urban YFV cases role in nature.
in the Americas, historically represents one of the most suc- Yellow fever patients have a characteristic but nondiag-
cessful public health campaigns against infectious diseases. nostic febrile disease with fever, headache, backache, nau-
Today, jungle YFV persists in the Western hemisphere sea, variable epistaxis, and a lack of correlation between
and is transmitted chiefly among monkeys, marmosets, and pulse and body temperature [289]. The clinical course is
possibly other forest-dwelling animals, commonly causing 2–4 days, followed by uneventful recovery or a remission
fatal infections. The vectors are mosquitoes of the forest can- period before development of severe yellow fever. Many
opy, chiefly of Haemagogus spp. and, to a lesser extent, A. tropical diseases, including a variety of arboviral infections,
leucocelaenus, Sabethes chloropterus, and possibly A. fulvus malaria, and relapsing tick fever, may present similar clinical
in Brazil [285]. For the last few decades in South America, syndromes, making a clinical diagnosis of suspected YFV
the vast majority of YFV cases were in males over 15 years difficult unless seen during a recognized epidemic. In a seri-
of age whose occupations increase their exposure to YFV- ous complication of the disease, the development of icterus
infected mosquitoes in endemic forest and jungle areas [286, occurs following a remission of the general manifestations.
287]. Up to 500 unvaccinated forest workers were infected in It develops as a yellowish tinge of the sclera, a very impor-
peak years. The majority of cases occur in the first 3 months tant diagnostic sign more easily seen in dark-skinned peo-
of the year in South America [287, 288]. A. aegypti has now ple, and only rarely becomes marked. Hemorrhagic signs
reinfested most of South and Central America and occu- and vomiting of blood characterize this more serious form
pies habitats just adjacent to the areas where endemic YFV of disease. They are more common preceding death, which
transmission occurs. A major threat is that this species could usually occurs within 9 days of onset. Mortality rates vary
transmit YFV in an urban cycle. widely and during epidemics reach from 20 to 80 % of the
In contrast to the endemic–sylvatic circulation of YFV in cases. Icteric YFV must be differentiated from infectious
the Western hemisphere in the late twentieth century, YFV in and serum hepatitis, leptospirosis, and poisoning.
Africa periodically explodes out of its sylvatic cycle to infect Because of these uncertain clinical criteria, laboratory
large numbers during major epidemics. During the decade diagnostic tests must be used. Isolation and identification
1980–1990, YFV reemerged as a major health problem in of YFV virus in blood samples or necropsy specimens or
Africa and, as mentioned above, threatens to reemerge in demonstration of specific antibody titer rises constitutes
South America. In Africa, two control strategies have been definitive diagnosis. While the virus generally grows well
attempted during the last 40 years. The first was routine in standard cell cultures or suckling mice, identification of
immunization programs, and the second was emergency YFV virus is now usually performed by PCR. Other rapid
control programs after the start of an outbreak. A routine, diagnostic assays have shown promise for demonstration
mandatory YFV immunization program was begun in the of YFV-specific antigen or IgG and IgM antibodies. Cross-
early 1940s in French West Africa, and the recurring pattern reactivity with other flaviviruses has been a historical sero-
of epidemics in West Africa was interrupted in those immu- logic problem. Demonstration of pathognomonic hepatic
nizing countries. This strategy was abandoned in 1960, and lesions in necropsy specimens is used when applicable,
the program switched to a post-outbreak, emergency immu- but needle biopsies of the liver have proved hazardous and
nization and control strategy. Since then, there has been a the procedure is not generally recommended. No specific
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 407

therapeutic regimen is available for YFV, and treatment is increased infectiousness, virulence, or transmissibility. To
chiefly supportive. Prevention is based both on protection control emerging viruses in general, and arthropod-borne
from exposure and on vaccination. The 17D YFV vaccine viruses in particular, there is a need for expanded (1) basic
was one of the earliest viral vaccines to be developed, and it and applied research that will help formulate coordinated
has proved to be one of the safest and most efficacious live strategies for anticipating, detecting, controlling, and pre-
attenuated vaccines [214, 290]. venting emergence or reemergence of viral diseases and (2)
Yellow fever in urban or jungle form is a continuing threat. basic and applied research on the virus, the infective process,
The two forms are nonetheless the same virus and the same and the host response to infection, which will be useful in the
disease, distinguished on epidemiologic grounds. Human development of vaccines and antiviral drugs.
beings can be protected by immunization with 17D YFV Additional unresolved problems are discussed from sev-
vaccine (not advised for infants under 1 year of age). A list of eral points of view, relating to the viruses, the vectors, the
the centers in the United States authorized to give the vacci- vertebrate hosts, and transmission cycles involving virus,
nation can be obtained from the Public Health Service (http:// vector, and host. The disease in the vertebrate host, which
wwwnc.cdc.gov/travel/yellow-fever-vaccination- clinics/ includes the host response to the pathogen, merits indepen-
search). dent consideration. Problems relating to the epidemiology
of each specific disease require a synthesis of many specific
Other Viruses in the Yellow Fever Virus Complex Not items. Finally, effective control exercised at the level of the
Pathogenic to Humans virus, the vector, or the vertebrate requires thorough under-
These viruses include Edge Hill virus (EHV) [3, 267], Jugra standing of the epidemiologic background. Specific exam-
virus (JUGV) [3], Saboya virus (SABV) [3], Sepik virus ples will help to illustrate problems.
(SEPV) [291], and Uganda S virus (UGSV) [3]. These
known viruses have been shown to cause illness in humans
but by potential exposure as indicated by positive antibody 11.1 The Viruses
titers.
Much progress has been made in recent years in cataloging
10.1.4 Viruses with No Known Arthropod Vector the several hundred described arboviruses and determining
A number of flaviviruses have no known arthropod vector the biochemical, growth, and morphological characteristics
and no documented naturally occurring infection in humans in intact vertebrates, in invertebrates, and in cell culture sys-
and includes Entebbe bat virus (ENTV) [3], Yokose virus tems of vertebrate and invertebrate cells. However, further
(YOKV) [3], Apoi virus (APOIV) [3], Cowbone Ridge virus work is needed to understand the evolution and emergence
(CRV) [292]. of epidemiologically relevant strains. In particular, new
Jutiapa virus (JUTV) [3], Modoc virus (MODV) [293], research is needed on the nucleic acid homology that may
Sal Vieja virus (SVV) [3], San Perlita virus (SPV) [3], and exist among the numerous members of a given arbovirus
Carey Island virus (CIV) [3]. grouping and on the mechanisms of recombination, reas-
sortment, and selection of new virus strains with important
virulence properties.
11 Unresolved Problems

The emergence of WNV in New York and its spread 11.2 The Vectors
throughout the Americas and the emergence of DENV as a
global health problem and recent outbreak in Florida have The factors that determine specific virus–vector associations
demonstrated the vulnerability of the United States to emerg- are still being elucidated. The role of the vector in genetic
ing viruses. Two studies by the Institute of Medicine deal- conservation of the arboviruses, as genetic bottlenecks, and
ing with emerging diseases warned that the threat posed by virus expansion into other vectors due to mutational events,
disease-causing microbes may be expected to continue and such as in the case of chikungunya virus, is not well under-
intensify in coming years [294, 295]. stood. Expanded research is needed before the principles
The natural life cycle of many arboviruses is multifac- governing virus–vector interactions are carefully delineated.
eted and, in addition to the virus, may include one or several There is a continuing need for taxonomic refinements
reservoir or amplifying hosts and often an arthropod vec- with respect to arthropods, such as the need for more infor-
tor. A change affecting the interaction of these fundamental mation on both Old World and New World mosquitoes of
elements might lead to the emergence or reemergence of a the genera Culex and Aedes. This need is generated by the
viral disease. In some instances, viruses might emerge as the increasing realization of their involvement with a large num-
result of selection of new genetic strains and variants with ber of arboviruses. The same remarks are pertinent for the
408 S.J. Thomas et al.

tick vectors. The need is equally great for more informa- Several of the tick-transmitted viruses apparently utilize
tion on the biology, feeding preferences, longevity, flight mechanisms of long persistence in ticks plus transovarial
range, and distribution of each arthropod species involved. transmission of virus to exist in an endemic form in defined
The genetic constitution of each vector species is basic to geographic areas. An excellent review of this topic is pre-
an understanding of what constitutes a vector, both physi- sented by Reeves [139] who discusses the epidemiologic
ologically and behaviorally, and will become increasingly problems of overwintering of arboviruses in northern coun-
important as control of vectors through genetic manipulation tries and possible transport via infected vectors on migrat-
is considered [118]. ing birds, with his discussion extending to the Old World
Unfortunately, in 1985, the Asian “tiger” mosquito, A. as well as the New World viruses. A related paper by Lord
albopictus, was introduced in old tires imported from Japan and Calisher [112] discusses the transport of arboviruses in
to Houston, Texas. It has now spread to infest over 22 states, infected migrating birds along the Atlantic coast flyway of
mostly in the South and Midwest, and has recently become the United States. Further research in these areas is vitally
established in several other countries. This mosquito is an needed to determine any point in the natural maintenance of
aggressive, opportunistic feeder with a wide host range that the virus where intervention might lead to control or eradi-
includes man. It adapts well to forest or urban settings and cation of the endemic disease.
it can vector many different arboviruses. In some areas, it Studies of transmission cycles are tied closely with sim-
already has replaced local mosquito species, and there is ulation of cycles by models with carefully defined param-
a concern it will transmit endemic viruses. Because of its eters. Such models may permit computer manipulation and
potential as a new vector of endemic or emerging arbovi- simulation of field conditions by varying the values applied
ruses in the United States and other countries, research on it to defined parameters, following which epidemic curves
should receive high priority. can be generated. Further work on models is needed, with
the hope of eventual prediction of emergence and spread of
disease.
11.3 The Vertebrate Hosts

For most of the arboviruses, the primary vertebrate host, i.e., 11.5 Disease in the Vertebrate Host
the host that serves as the basic unit for propagation of the
virus, is not man. For many of the arboviruses, the vertebrate Studies of the human response to arboviral infections are
hosts are not yet determined or are recognized on the most difficult, since the epidemics that provide numbers of cases
tenuous of evidence. Identification of the host(s) is a primary for study usually occur unpredictably in time and often far
need. Following this, an emphasis should be placed on elu- from modern facilities required for detailed clinical investi-
cidating a biological profile of the hosts, including the full gation. Classically, studies of infection in the vertebrate host
range of biological and ecological considerations, as well as (including man) have been part of research programs in the
the degree of host susceptibility to the virus. fields of pathology. However, animal models are available
for only a limited number of the arboviruses. The develop-
ment of these models will be crucial for the development
11.4 Transmission Cycles Involving Virus, modern vaccines and antiviral drugs.
Vector, and Vertebrate Recognition of disease in the vertebrate host, as well as
worldwide surveillance of disease, requires further develop-
The problem of virus persistence in nature is a particularly ment of simple diagnostic techniques and standardization of
baffling one. For example, there are many theories but few these assays using accepted reference reagents.
facts to explain how a given virus manages to overwinter
or survive past a long dry season, when vectors may practi-
cally disappear and vertebrate populations decline (or go into 11.6 Control
hibernation).
Current theories hypothesize that the virus persists in Virus vaccines are a highly cost-effective means of disease
vector populations that overwinter with some members control; yet for the arboviruses, only an attenuated yel-
harboring virus or the virus is in vertebrate populations low fever (17D) vaccine is in use on an international scale.
that overwinter and some infected individuals respond to The recent WHO recommendation that this vaccine be
a reactivation stimulus. In other cases, existing data point included in the Expanded Program of Immunization (EPI)
to an important role for transovarial transmission, permit- in YFV-endemic countries was a major step toward the con-
ting passage of virus to generation after generation of a trol of YFV in Africa [200, 201]. The threat of emergence
vector without the need for an intercalated vertebrate host. of YFV in South America has prompted some to call for
16 Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others 409

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 Hepatitis A Virus
17
Daniel Shouval

Abbreviation RIFIT Radioimmunofocus inhibition assay

RNA Ribonucleic acid
ACIP Advisory Committee on Immunization WHO World Health Organization
Practices
ALF Acute liver failure
ALT Alanine aminotransferase 1 Historical Background
Anti-HAV Antibodies to hepatitis A virus
BMI Body mass index Jaundice or icterus and other diseases of the liver were
CDC Centers for Disease Control (USA) described in the writings of a number of ancient societies
cDNA Complementary DNA including old Chinese, Greek, Roman, Babylonian, and
EIA Enzyme-linked immunoassay Talmud literature. Jaundice was generally considered
ELISA Enzyme-linked immunosorbent assay obstructive in origin, although large epidemics associated
EPI Expanded Program on Immunization with military campaigns were described as early as the sev-
GBD Global burden of disease enteenth century and continued through World War II [1–6].
GID Global incidence of disease Over the years and until discovery of the hepatitis A (HAV)
GMC Geometric mean concentration and hepatitis B (HBV) viruses, many synonyms were used to
HAV Hepatitis A virus describe viral hepatitis of undetermined origin, later referred
HBsAg Hepatitis B surface antigen to as HAV. These included among others the terms acute
HBV Hepatitis B virus catarrhal jaundice, epidemic catarrhal jaundice, epidemic
HCV Hepatitis C virus hepatitis, icterus epidemicus, infectious hepatitis, campaign
HIV Human immunodeficiency virus jaundice, and MS-1 hepatitis. From the 1940s through the
HLA Histocompatibility leukocyte antigen 1960s, extensive efforts to identify the agent(s) associated
IDU Intravenous drug use with viral hepatitis were unsuccessful [2, 6]. Epidemiologic
IEM Immune electron microscopy evidence supported two types of hepatitis viruses: infectious
IG Immunoglobulin hepatitis (later identified as HAV) and serum hepatitis (later
IV Intravenous identified as HBV). However, a clear distinction could not be
LAK Lymphokine-activated killer cells made between the two agents until transmission studies were
MSM Men who have sex with men conducted in primates [1, 7–11]. Studies, conducted between
NHANES National Health and Nutrition Examination 1940 and 1965, clarified clinical and epidemiologic distinc-
Survey tions and lack of cross-immunity between the two forms of
NK Cells natural killer cells viral hepatitis MS-1 and MS-2, later classified as HAV and
PBMC Peripheral blood mononuclear cells HBV, respectively. Clinical studies in the late 1940s and
onward established the efficacy of immune serum globulin
(IG) in preventing infectious hepatitis A [1, 9, 10, 12].
D. Shouval, MD The discovery of HBV in the early 1970s and development
Division of Medicine, of serological assays further differentiated these two forms of
Institute of Gastroenterology and Liver Diseases,
viral hepatitis. However, the critical advance occurred in 1973
Hadassah-Hebrew University Hospital,
Ein-Kerem, 12000, Jerusalem 91120, Israel when HAV particles were identified through immune electron
e-mail: [email protected] microscopy (IEM) in stool samples of patients with hepatitis

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 417

DOI 10.1007/978-1-4899-7448-8_17, © Springer Science+Business Media New York 2014
418 D. Shouval

A [13]. The development of serological assays to distinguish hepatitis in specific locations can be combined with reported
between acute and resolved HAV infection during the next age-specific prevalence data to estimate the overall morbid-
decade was pivotal in understanding the natural history, the ity resulting from hepatitis A [23, 24].
epidemiology, and the pathogenesis of HAV infection [4].
The discovery of the hepatitis E virus (HEV) and the develop-
ment of diagnostic assay for HEV enabled further distinction 2.3 Epidemiologic Studies
between HAV and HEV infections which share similar epide-
miology and clinical characteristics [14]. Finally, the identifi- Epidemiologic studies that used serological assays to differ-
cation and characterization of the HAV genome has added entiate acute from resolved HAV infections have greatly
power-full tool for the investigation of the phylogenesis and increased the understanding of this disease. Recently, nucleic
molecular epidemiology of HAV [15–18]. HAV was first acid variation within the region of the HAV genome that
grown in cell culture in 1979 [19], and vaccines developed codes for the capsid (surface) proteins has been used as a
from cell culture-derived attenuated virus have been shown to marker in epidemiologic studies [5, 17, 25].
effectively prevent hepatitis A [20, 21]. The spread of HAV infection can be assessed through
monitoring the overall and age-specific anti-HAV (IgG)
seroprevalence in selected populations which also enables
2 Methodology Involved indirect measurement of incidence rates. Overall prevalence
in Epidemiologic Analysis has been classified into high (≥90 % of population by age
10 years), intermediate (≥50 % by age 15 years with <90 %
2.1 Mortality by age 10 years), low (≥50 % by age 30 years with <50 % by
age 15), and very low (<50 % by age 30 years) [22].
HAV infection has a low case fatality rate, and mortality A new classification is emerging regarding endemicity of
from hepatitis A is a poor indicator of disease incidence. HAV worldwide based on reported incidence of serologically
Although viral hepatitis has been included in the International confirmed acute HAV cases through detection of anti-HAV
Classification of Diseases (ICD) for decades, neither sero- (IgM) antibodies [5, 22]. Accordingly, endemicity to HAV
logical testing nor appropriate coding to distinguish different may also be classified as very low, with an estimated inci-
viral etiologies was available until 1980. The ICD-coded dence of <5 cases/105; low, 5–15 cases/105; intermediate,
data on hepatitis A mortality has become more reliable in 15–150 cases/105; and high, >150 cases/105. Cross-sectional
developed countries where serological testing is widely studies of the prevalence of HAV infection in population sam-
available. Because accurate serological testing is not widely ples stratified by age, and in some cases by socioeconomic
available in developing countries, mortality statistics are status, have been completed in many parts of the world and
unreliable and remain poor indicators of disease incidence. provide the most accurate picture of HAV infection [23, 24,
26–28]. Serological testing of representative samples of acute
hepatitis cases in children and in adults has been used to
2.2 Morbidity define the proportion caused by HAV. In less-developed coun-
tries, these data can be combined with reported age-specific
Several factors have limited the usefulness of morbidity data in rates of acute hepatitis to estimate disease incidence. Studies
assessing the impact of HAV infection. In developed countries, to define the risk of HAV infection in certain groups or popu-
the reporting of hepatitis A and hepatitis B as different diseases lations have primarily been confined to persons traveling
only started in the late 1970s, and it was not until 1980 that a from countries with low HAV endemicity to regions with
diagnostic test for hepatitis A became available commercially. high rates of infection. Some data are available to define the
Furthermore, although viral hepatitis is a reportable disease in current risk of infection in men who have sex with men
almost all countries, underreporting is assumed to reach >80 % (MSM), drug users, the developmentally disabled, children
even in developed countries [5, 22]. In less-developed coun- attending day care, persons working in day-care settings, or
tries, serological testing is not widely available, and each type health-care workers [5]. Estimates on the national burden of
of viral hepatitis is not reported separately. Consequently, it is HAV-associated disease should include data on patients with
difficult to make strict comparisons of reported rates of hepati- fulminant hepatitis and those with HAV-induced liver failure
tis A from different geographic areas. referred for liver transplantation.
In spite of these well-recognized limitations, cyclical pat- Mechanisms or pathways of disease transmission have
terns of increased disease incidence and trends of disease been defined by serological testing of persons ill or exposed
over time are reasonable indicators of the epidemiology of in outbreaks and in some instances by analysis of genetic
hepatitis A. Data derived from studies using serological test- variation of HAV isolates [5, 17, 20]. Serological testing, or
ing to define the age-specific etiology of acute cases of viral HAV-RNA detection and sequencing, has improved our
17 Hepatitis A Virus 419

knowledge of the epidemiology of common-source out- 3 Biological Characteristics of the Virus

breaks (i.e., through contaminated strawberries, shellfish, or
lettuce), community-wide outbreaks, outbreaks associated HAV is a small (27–32 nm) non-enveloped RNA virus
with day-care centers and institutions for the developmen- belonging to the family Picornaviridae. The virus has icosa-
tally disabled, and outbreaks occurring in hospitals. hedral symmetry, has a buoyant density of 1.33 g/cc, and
contains a single-stranded positive-sense RNA of approxi-
mately 7,500 (Figs. 17.1 and 17.2). The virion is composed
2.4 Laboratory Methods of at least three major structural viral capsid polypeptides:
VP1, VP2, and VP3 (33,000–22,000 Da). Based on the
2.4.1 Isolation and Identification of the Virus nucleotide sequence, VP4, another viral capsid protein of
Primary virus isolation from clinical or environmental speci- approximately 2,500 Da, should be encoded but has not been
mens (i.e., feces, water, shellfish, or strawberries) has become identified in mature virions [20]. The genomic organization
possible using various primary or continuous cell lines. and replication of HAV appear similar to that of polio and
However, because of the slow growth characteristics of HAV other picornaviruses; viral RNA encodes a single large poly-
(up to 120 days), prolonged adaptation periods are required protein from which structural and nonstructural proteins are
before either infectious foci or HAV antigen can be detected subsequently cleaved [30, 40, 41].
[19, 29]. HAV generally does not produce a cytopathic effect When compared to other enteroviruses, HAV has essen-
in cell culture, and detection of virus requires special time- tially no nucleotide or amino acid homology, replicates more
consuming techniques such as the radioimmunofocus assay slowly in cell culture, and is more resistant to heat inactiva-
(RIFA), which uses radiolabeled antibody to detect infec- tion. Although HAV was initially classified in the genus
tious foci of HAV in fixed cells [29]. The inability to rapidly Enterovirus (Enterovirus 72), it has been reclassified in a
culture HAV has precluded the use of this method for the separate genus designated Hepatovirus [42].
diagnosis of infection or for detection of HAV in environ- HAV is stable in the environment, retaining infectivity in
mental samples. feces for at least 2 weeks and having only a 100-fold decline
Immune electron microscopy (IEM) was used extensively in infectivity over 4 weeks at room temperature [33, 34, 41,
in early studies to identify HAV [13] but has been supplanted 43, 44]. The virus resists extraction by nonionic detergents,
by more sensitive techniques. These include immunoassays chloroform, or ether and retains infectivity in pH 1.0 at 38 °C
to detect HAV antigen [30, 31] and detection of HAV-RNA for 90 min. HAV is more resistant than poliovirus to heat,
by polymerase chain reaction (PCR), the latter being the being only partially inactivated at 60 °C for 1 h [34]. When
method of choice for detection of low levels of HAV in clini- suspended in milk at 62.8 °C for 30 min, 0.1 % infectivity
cal and environmental samples [17, 25, 32–34]. However, remains, suggesting that pasteurization may not completely
the presence of HAV-RNA may not always equate with the inactivate HAV and temperatures of 85–95 °C for 1 min are
presence of infectious virus [35]. required to completely inactivate HAV in shellfish [34, 45,
46]. HAV is completely inactivated by formalin (0.02 % at
2.4.2 Serological and Immunologic 37 °C for 72 h) but appears to be relatively resistant to free
Diagnostic Methods chlorine, especially when the virus is associated with organic
In the 1980s, immunoassays to detect antibodies to HAV matter [34]. An outbreak of hepatitis A among swimmers sug-
structural antigens (anti-HAV) became available commer- gested that if a prescribed free chlorine level of 0.3–0.5 ppm
cially, including those that detected both IgG and IgM anti- existed in the swimming pool, it failed to inactivate HAV in
bodies (i.e., total anti-HAV often referred to as anti-HAV fecally contaminated water [47]. Only sodium hypochlorite,
(IgG)) and anti-HAV (IgM), which allow the differentiation 2 % glutaraldehyde, and quaternary ammonia compound
of past and current (within prior 6 months) infections [33, (QAC) with 23 % HC1 have been effective in reducing the
36]. To better evaluate the effectiveness of hepatitis A immu- titer of HAV by more than 104 on contaminated surfaces [48].
nization, quantitative assays for anti-HAV antibodies (IgG HAV exists as a single serotype, and monoclonal antibod-
and IgM) with increased sensitivity have been developed. ies that identify overlapping neutralization epitopes react
Cell culture assays that detect low levels of neutralizing anti- with virus isolates from all parts of the world [41]. The sin-
bodies have been used in the early phases of HAV vaccine gle immunodominant neutralization epitope appears to be
development [31, 37]. An experimental assay for detection of highly conformational and is composed of several sites
an immune response to nonstructural HAV proteins has raised located on VP1 and VP3 [34, 41, 49, 50]. There are seven
hopes to establish a method for differentiation between anti- known HAV genotypes, defined by sequence of the VP1/P2a
HAV (IgG) response to acute HAV infection and response to junction region of a global collection of viruses [17, 26].
immunization [38]. However, in contrast to HBV, no licensed Genotypes are defined by a sequence variability of ~15 % in
immunoassays are yet available for this purpose. these regions, while subgenotypes differ by 7.0–7.5 %. Four
420 D. Shouval

VP4 2A 3A3B
5’NTR 3’NTR
Vpg VP2 VP3 VP1 2B 2C 3Cpro 3Dpol AAAAAA
(3B)
IRES

VP4
2A
VP2 VP3 VP1

VP4 2A

VP2 VP3 VP1

Fig. 17.1 HAV genome organization. On top a scheme of the HAV tified cellular protease cleavage in VP1-2A is indicated by a red arrow.
genome is shown. The positive-strand (messenger-sense) RNA genome Structural proteins (P1) are indicated in blue; nonstructural proteins
contains a single open reading frame encoding a polyprotein that is (P2 and P3) are indicated in yellow and green, respectively (Reproduced
proteolytically processed by the viral protease 3Cpro. A yet-to-be-iden- by permission reference Cristina and Costa-Mattioli [39])

Fig. 17.2 HAV genome regions VP4 2A 3A3B

used for genetic analysis. The 5’NTR 3’NTR
thick red lines below the genome Vpg
scheme indicate the different VP2 VP3 VP1 2B 2C 3Cpro 3Dpol AAAAAA
(3B)
genomic regions used to IRES
characterize the different HAV
isolates (Reproduced by
permission reference Cristina and Full-length VP1 (900 nt.)
Costa-Mattioli [39])
VP3 VP1 2A

VP3 C terminal
VP1 N terminal VP1/2A junction
(206 nt.)
(171 nt.) (168 nt.)

genotypes, namely, I, II, III, and VII, were identified in required for detection of significant amounts of HAV antigen
infected humans, while genotypes IV, V, and VI have been in infected cells. The lack of a cytopathic effect allowed
found in infected nonhuman primates. Recently, the seven serial passage of virus that ultimately produced strains that
HAV genotypes have been re-classified into six genotypes replicate more rapidly and produce higher virus yields [34].
while genotype VII is now defined as a subgenotype of geno- HAV that has been adapted to grow efficiently in cell culture
type II [18]. The identification of the various HAV genotypes appears to have mutations in the 2B and 2C areas of the
and subgenotypes has significantly enhanced the ability to genome that codes for nonstructural proteins [34]. In addi-
investigate the molecular epidemiology of hepatitis A out- tion, cytopathic variants of HAV have been isolated, and
breaks and particularly its transmission routes [17, 25]. similar to polio, these variants produce plaques in cell cul-
Besides man, the host range of HAV includes nonhuman ture and have proven useful in laboratory studies [34, 55].
primates (marmosets, tamarins, owl monkeys, chimpanzees)
that have been infected experimentally and have served as
animal models of human HAV infection [51–53]. In addi- 4 Descriptive Epidemiology
tion, several species of Old World monkeys have been shown
to be infected with an HAV that is serologically similar to the 4.1 The Changing Epidemiology of HAV
human virus but genetically distinct and that does not appear and the Impact on Global Burden
to infect humans [7, 34, 54]. of Infection
In 1979, cell culture of HAV in fetal rhesus monkey kid-
ney cells was achieved only after the virus was passaged Prior to identification of its specific viral etiology and the devel-
multiple times in marmosets [19, 34]. Since then, HAV has opment of diagnostic tests, hepatitis A was presumed to be the
been cultivated directly from clinical or environmental sam- cause of most sporadic and epidemic hepatitis worldwide. Large
ples, but long adaptation periods (4–10 weeks) have been epidemics of hepatitis A occur in developing countries and
17 Hepatitis A Virus 421

Fig. 17.3 Epidemiologic risk sexual or house hold contact (7.8%)

groups for acute hepatitis A
virus infection obtained from
reported cases in the United
States in 2007. MSM men who international travel (17.5%)
have had sex with men, IVDA
intravenous drug abuse
(Modified from reference
Daniels et al. [61])
MSM (5.9%)
unknown
(67.7%) IVDA (1.2%)
child/employee day care center (3.8%)

suspected food or
waterborne outbreak (6.5%)

contact of daycare child/employee (4.6%)

other contact with an HAV patient (9.0%)

especially in countries in transition from high to intermediate cedures. Epidemiologic risk groups include populations of
endemicity of HAV. However, it appears that hepatitis E is also low socioeconomic status living under crowded conditions,
an important cause of epidemics of hepatitis that involved large household contacts of infected individuals, children visiting
numbers of people, especially in India and central Asia [3, 56]. day-care centers and kindergartens, international travelers
Data on the global incidence of disease (GID) and global from countries with low endemicity to areas with intermediate
burden of disease (GBD) of acute HAV infection is incom- or high endemicity, MSM, intravenous drug users, patients
plete, with an assumed underreporting rate of ≥80 % [28, with chronic liver disease, food handlers, caretakers of nonhu-
57–61]. The WHO has recently completed a preliminary sur- man primates, and patients with blood clotting disorders. The
vey for reassessment of the GBD of HAV disease. Estimates clinical expression of HAV infection is highly age dependent.
suggest an increase in the number of symptomatic acute Serological studies have confirmed that less than 10 % of
hepatitis A as well as subclinical cases globally from 177 infected children under age 6 will become jaundiced, whereas
million in 1990 to 212 million in 2005 and deaths due to in adults and children above age 5, infection may cause jaun-
hepatitis A to increase from 30,283 in 1990 to 35,245 in dice in 50–90 % of cases [65–68].
2005 [22, 23]. Increased numbers of cases were estimated to The increased incidence of HAV infection in adults has
occur in the age groups 2–14 years and >30 years. An epide- had an impact on the magnitude and severity of the disease
miologic shift from high to intermediate endemicity of HAV as recently reported from Korea [68, 69] and Brazil [70].
is now being recognized worldwide. As a result, more adults Furthermore, in countries in transition, pockets of intermedi-
in such areas of transition escape exposure to HAV in early ate endemicity may exist within the same areas of high ende-
childhood and become susceptible to infection during out- micity. Despite the observed low attack rates of clinical
breaks. This change in susceptibility to HAV infection is hepatitis, especially in areas of high but also in countries
paradoxically associated with an increase in disease inci- with intermediate endemicity in transition, HAV infection
dence rates in the presence of improved socioeconomic and has been identified as a leading cause of fulminant hepatic
sanitary conditions. An example for the potential conse- failure in a growing number of countries including Korea
quences of such a shift in endemicity was clearly demon- [71], Argentina [70, 72, 73], and Brazil [70].
strated during the massive 1988 outbreak of hepatitis A in The major risk groups for contracting HAV infection
Shanghai where >300,000 individuals contracted HAV reported by the US Centers for Disease Control and Prevention
infection within a short period [62, 63]. (CDC) for the year 2007 are shown in Fig. 17.3 [61].
HAV infection is mainly spread through the fecal–oral
route as well as through contaminated water and food. Shellfish
are able to ingest and concentrate HAV and as a result become 4.2 Prevalence and Incidence
a reservoir for spread of the virus [63, 64]. Transmission
occurs mainly through common-source outbreaks (i.e., food- The age-specific prevalence of anti-HAV (IgG) antibodies
and waterborne) as well as person-to-person contact. HAV is worldwide can be used to define several patterns and risks of
very rarely transmitted through blood products or medical pro- infection [23] (Fig. 17.4).
422 D. Shouval

Fig. 17.4 Global risk map of

HAV immunity in 2005. Age at
midpoint of population immunity
to HAV (Reproduced from
BioMed Central, reference [74])

Very high <5 years

High 5-14 years
Intermediate 15-34 years
Low >34 years

Many data on the global prevalence of HAV are outdated endemicity of infection is considered low or very low.
and recorded between the late 1970s and early 1990s prior to Until recently, in some US states, 30–60 % of US adults
major shifts in endemicity. Areas with a very high endemic- >40 years old had serological evidence for past HAV infec-
ity of infection primarily consist of less-developed and tion. In contrast, the prevalence of infection is less than
developing nations of Asia, Africa, South and Central 10 % in young adults and almost nil in children. In both
America, the Pacific Islands and certain populations in very-low- and low-endemicity areas, there is evidence that
southern regions of Eastern Europe [26]. In these countries a cohort effect accounts for the high prevalence of infec-
and until recently within ethnically defined populations or tion among older adults, indicating that HAV infection was
regions also in the United States, the prevalence of past HAV much more common in the past, especially among persons
infection in adults has reached 90 % or higher; almost all who were born prior to World War II [24]. The epidemiol-
older children had serological evidence of prior infection, ogy of HAV infection is rapidly changing in countries and
and most children became infected by the age of 10 years regions which introduced mass or universal vaccination
[24, 75, 76]. However, persons of upper socioeconomic sta- against HAV in babies and young children [5, 23, 81, 82].
tus residing in these regions have not become infected until Consequently, in such regions and countries, assessment
they were adolescents or young adults. of prevalence and incidence of HAV infection should be
In more developed counties in Western Europe (excluding divided into pre- and postvaccination periods. For exam-
northern regions) and some countries in Asia, the endemicity ple, in the United States, endemicity of HAV is shifting
of HAV infection was already intermediate to low in the pre- from intermediate to low and very low prevalence of infec-
vaccine era, and the prevalence of anti-HAV (IgG) varied tion in populations previously considered at risk [83] as
widely [24, 27]. During the 1980s in countries such as also shown in Israel [84] and some regions in Spain, Italy,
Greece, Italy, and Taiwan, prevalence of past HAV infection and Australia [5]. The impact of rising socioeconomic
in adults reached 80–90 %; while in children under age 10, conditions, improved sanitary conditions, and vaccination
the prevalence was only 20–30 %. The major increase in of children against hepatitis A is clearly shown in surveil-
prevalence of infection occurred between 10 and 19 years lance of incidence which has markedly dropped in age
[77–79]. In several Western European countries and the groups 5–39 years according to data obtained by the US
United States and prior to introduction of mass vaccination, Centers for Disease Control (CDC) [61, 83] as shown in
anti-HAV (IgG) prevalence in young adults varied from 30 to Fig. 17.5.
70 % but was less than 10 % in children under age 10, while
low socioeconomic status was associated with high rates
(endemicity) of past infection [80]. 4.3 Epidemic Behavior and Contagiousness
In some northern European countries, endemicity of
HAV infection is very low. In several regions in the United Rates of hepatitis A are not inherently stable, and periodic
States and in Japan, HAV infection is disappearing, and the widespread epidemics may occur in high, intermediate, and
17 Hepatitis A Virus 423

25 occur among adult contacts (parents, caretakers) of diapered

children in day-care settings [100, 101].
<5 yrs
20 5–14 yrs
15–24 yrs
25–39 yrs
15 4.4 Geographic Distribution
Incidence

≥40 yrs

10 As described previously, HAV infection occurs worldwide

with endemicity patterns defined by the socioeconomic
5
development of the individual nation and impact of vaccina-
tion campaigns. Published world maps on the prevalence of
HAV infection have been useful to convey public health
0
1990 1992 1994 1996 1998 2000 2002 2004 2006 messages as shown in Fig. 17.4. Yet most data on the global
Year prevalence of HAV infection are decades old. Consequently,
* Per 100,000 population. as the socioeconomic status of the population increases,
endemicity declines and the median age of infection
Fig. 17.5 US CDC report on decline of hepatitis A incidence between
increases, such maps require frequent updating. Recently, it
1990 and 2007 [85]
was suggested that world maps should also use the age at
midpoint of population susceptibility as a standard indicator
even low endemicity areas and produce cyclical patterns of for the level of HAV endemicity within the world regions
infection. However, over the past 20–40 years, nationwide [74]. In the least-developed countries, HAV transmission
epidemics of hepatitis A have gradually declined in most occurs exclusively during early childhood, almost the entire
countries with a low endemicity of infection, while epidemic population becomes infected before age 10 years, and clini-
cycles continue to occur in countries with either a high or cal disease is recognized only in older children. As hygiene/
intermediate endemicity in transition. Determinants of the sanitation standards improve, the age of infection shifts pro-
cyclical recurrence of epidemics include the age-specifc pro- gressively to include older children and young adults, par-
portion of the population susceptible to infection, the inter- ticularly in upper socioeconomic groups. This may result in
epidemic rate of HAV infection and the likelihood that HAV higher rates of clinical disease, and common-source out-
will be introduced into the susceptible population. Several breaks and/or nationwide epidemics appear if a substantial
patterns appear to produce these epidemic cycles: (1) high proportion of adults remain susceptible to infection. As
rates of asymptomatic infection in children under age 5 who sanitation and socioeconomic conditions improve further,
serve as a reservoir for spread of infection to susceptible the infection frequency in all age groups and overall rates of
older children, a pattern observed in American Indian popu- disease decrease; clinical disease is recognized in older chil-
lations [86]; (2) periodic introductions of HAV into largely dren and young adults, and localized outbreaks may occur.
susceptible isolated populations where epidemics occur due Finally, in some developed countries, infection rates decline
to living conditions that facilitate virus transmission and in all age groups, and cases of disease occur primarily as
where the high attack rate results in the disappearance of importations from areas having a high endemicity of
hepatitis A during interepidemic periods, a pattern observed infection.
in Alaskan Native villages and some Pacific Island popula-
tions [87, 88]; and (3) the periodic introduction of HAV
through vehicles such as food in populations where the stan- 4.5 Temporal Distribution
dard of living has improved and a large proportion of the
young adult population remains susceptible, a pattern Long-term follow-up data from Europe and the United States
observed in Shanghai, China, during the 1988 hepatitis A have shown a marked change in patterns of reported inci-
epidemic [63]. Smaller outbreaks continue to play a role in dence of disease in the past 50 years. Prior to and immedi-
disease transmission in the United States and elsewhere [89]. ately following World War II, hepatitis rates were high, and
Common-source outbreaks related to contaminated water, nationwide epidemics, presumably of hepatitis A, occurred
shellfish harvested from sewage-contaminated areas, or food at 6- to 10-year intervals. In addition, seasonal fluctuations
contaminated by an infected food handler have been recog- of the disease occurred, with peaks in the late summer and
nized for many years [34, 90–95]. Outbreaks among MEM early fall. Following World War II, disease rates began to
and drug users have been recognized in recent years, with decrease in northern Europe and epidemic cycles disap-
infections in the latter probably occurring from person-to- peared, as did seasonal variation in disease rates. In the
person spread or occasionally from common-source contam- United States, improvement in overall socioeconomic condi-
ination of illicit drugs, although HAV has rarely been tions, sanitation, and hygienic standards and introduction of
transmitted parenterally [96–99]. In addition, outbreaks may universal vaccination in states at risk have led to a reduction
424 D. Shouval

of epidemic cycles which gradually are waning. The reported cases occur primarily in children, as all adults are immune as
incidence of acute hepatitis A in the United States has a result of infection in childhood.
declined by 92 %, from 12.0 cases per 100,000 population in
1995 to 1.0 case per 100,000 population in 2007 [61]. In the
European Union (EU), though figures may vary among 4.7 Sex
countries, the overall incidence of hepatitis A has decreased
from 15.1 per 100,000 population in 1996 to 3.9 per 100,000 Until 2002, rates of acute hepatitis A had consistently been
in 2006 [89]. higher among males than among females at a ratio of almost
2:1; however, by 2007, overall rates had declined more among
males than among females, and the reported incidence in
4.6 Age males was 1.1 cases per 100,000 population, compared with
0.9 cases per 100,000 population among females [61].
In the United States and prior to introduction of HAV vac-
cination, children aged 5–14 years have had the highest
attack rates as shown in community-wide outbreaks affect- 4.8 Race
ing specific ethnic or low socioeconomic groups [86, 102,
103]. A bimodal distribution has been seen in day-care out- Hepatitis A occurs in all racial groups, and race per se is not
breaks, with children aged 5–9 years and adults 25–35 years believed to predispose to infection except when related to
most commonly affected [65]. In recent years, the overall socioeconomic status or country of origin [101]. Hepatitis A
incidence of hepatitis A declined among all age groups in rates in the United States have differed historically by race;
the United States, but the greatest decrease recorded the highest rates occurred among American Indian/Alaska
between 2001 and 2007 was among children <5 years of Natives and the lowest rates among Asians/Pacific Islanders.
age. However, asymptomatic infection is common among In 2007, rates among American Indian/Alaska Natives
young children, and symptomatic cases in children aged dropped from >60 cases per 100,000 population before 1996
<5 years represent only a limited proportion of infections to 0.5 cases per 100,000 population. Rates for Hispanics
that occur in this age group [61]. In 2007, rates were high- decreased 94 %, from the peak of 24.1 cases per 100,000
est for persons aged 25–39 years (1.3 cases per 100,000 population in 1997 to 1.4 cases per 100,000 population in
population) [61]. 2007 [61].
During 1999–2006 and following the introduction of uni-
versal vaccination in 17 US states, the overall seroprevalence
of anti-HAV (IgG) was 34.9 % as reported by the National 4.9 Occupation
Health and Nutrition Examination Survey (NHANES) [83].
During this period, US-born children living in vaccinating There does not appear to be an increased risk of HAV infec-
states had a higher seroprevalence (33.8 %) than children in tion associated with a particular occupational group, although
non-vaccinating states (11.0 %). Seroprevalence among chil- outbreaks of hepatitis A have occurred in persons working
dren increased from 8.0 % during 1988–1994 to 20.2 % dur- with certain nonhuman primates and sewage workers.
ing 1999–2006. For US-born children aged 6–19 years, the Employment in a day-care center is a reported source of
strongest factor associated with seroprevalence was resi- infection, but no studies have shown that this occupational
dence in vaccinating states. Among US-born adults aged group has an increased risk of infection compared to the gen-
>19 years, the overall age-adjusted seroprevalence of anti- eral population. Susceptible persons from low endemic
HAV (IgG) was 29.9 % during 1999–2006, which was not countries who work in or travel to countries with a high
significantly different from the seroprevalence during 1988– endemicity of HAV infection are at risk of infection, and this
1994 [83]. risk increases the longer they reside in the country [105,
Similar age-specific prevalence patterns occur in regions 106]. Outbreaks of hepatitis A in health-care workers are
which initiated mass vaccination campaigns in Europe and rare [107, 108]. Studies of health-care workers have shown
Australia [104]. In countries with a low or very low endemic- that they are not at increased risk of infection [109, 110].
ity of infection, the disease and the prevalence of anti-
HAV(IgG) are largely limited to adults, since the majority of
infections are imported. Seroprevalence rates vary consider- 4.10 Military and Other Settings
ably in various African, Asian, and South American coun-
tries where HAV endemicity is either high or in transition Hepatitis A among military personnel stationed in low ende-
[26]. In populations with a high endemicity of infection, micity countries does not appear to differ from that in the
17 Hepatitis A Virus 425

surrounding community. Outbreaks caused by contaminated included salads, sandwiches, glazed or iced pastries, and in
food, exposure to children in day-care centers, and sporadic the past some dairy products [34, 90, 121]. Outbreaks have
disease from person-to-person contact have been reported. been reported throughout the world when filter-feeding
However, prior to the availability of HAV vaccines, hepatitis bivalve mollusks (clams, oysters, mussels) harvested from
A posed a risk among troops deployed to parts of the world sewage-contaminated waters have been eaten with little or
where HAV infection is of high or intermediate endemicity. no cooking [34, 91, 122]. In addition, outbreaks have
However, in recent years, American troops sent to regions occurred from foods contaminated at the time of harvest or
with high or intermediate HAV endemicity are immunized processing that are subsequently served raw and have
prior to deployment. Consequently, the crude incidence of included lettuce, strawberries, and raspberries [34, 92, 93].
HAV in the US military is very low at 1.37/100,000 person- Nevertheless, during the 1990s, in the United States, disease
years, and the rate of hospitalization for HAV infection has traceable to contaminated food or water constituted less than
declined sharply [111]. 5 % of the overall disease burden [102].
Direct person-to-person transmission by the fecal–oral
route accounts for most infections in all parts of the world.
5 Mechanisms and Routes Susceptible household contacts have a 10–20 % risk of
of Transmission acquiring infection from a family member with acute illness
[123, 41]. Person-to-person transmission is the predominant
Studies in experimentally infected nonhuman primates and pathway in the day-care setting, in community-wide out-
in humans have shown that HAV is excreted in large quanti- breaks, and among homosexual men. The common denomi-
ties (up to 108 infectious units per ml) in feces during the late nator of close contact in settings with less than optimal
incubation period and first week of clinical illness. Some hygiene accounts for the ease of HAV transmission.
studies suggest that children may shed virus longer than Transmission in the day-care setting has received close
adults, but there is no evidence of chronic virus excretion study and provides a model for person-to-person disease
[112–114]. Fecal shedding of HAV has been reported in transmission [65]. In centers enrolling children in diapers,
patients with relapsing HAV infection [115]. Viremia occurs hepatitis A outbreaks may be common and involve transmis-
from the middle of the incubation period into the early clini- sion not only among young children but also among adult
cal illness; infectivity titers of serum are 103- to 106-fold contacts in the center, at home, or in the community. Infected
lower than those of feces. Virus is also present in saliva with children are rarely jaundiced, but they transmit HAV to adult
an infectivity that may be 104 lower than serum [114, 116]. contacts who are more likely to become symptomatic and
These physical data are firmly supported by epidemiologic comprise 70–80 % of recognized cases. Adult contacts of 1-
data that have implicated the fecal–oral pathway as the pre- to 2-year-old children are at highest risk of infection; risk of
dominant route of HAV transmission and occasionally dem- transmission decreases with increasing age of the child, and
onstrated blood-borne transmission. Ingestion of infectious contacts of older children (age 5–6 years) are not commonly
feces or vomits transferred from person to person or inges- affected. In centers not enrolling children in diapers, out-
tion of contaminated food or water is the major route of HAV breaks are uncommon, and spread following the introduction
transmission. Blood-borne transmission occurs rarely and of infection is limited. HAV spreads rapidly but silently
can lead to secondary outbreaks among health-care workers among mobile, fecally incontinent children in day care and
due to inapparent fecal–oral spread of the virus [117, 118]. subsequently to their contacts.
Transmission among MSM has been well documented; The day-care model indicates that asymptomatic HAV-
whether this occurs through sexual contact or simply by non- infected children are highly infectious and efficiently spread
sexual intimate contact is not certain [96, 119]. Finally, disease. Among persons without an apparent source for their
HAV-RNA was identified in breast milk of infected nursing HAV infection, almost 50 % have a child less than 5 years of
mothers but without evidence for HAV transmission to their age residing in the household. This suggests that much
babies [35]. community-acquired hepatitis A may be acquired from inap-
Common-source outbreaks have occurred from contami- parently infected young children.
nation of food and various sources of water, including Transmission of HAV by blood or blood products occurs
streams, individual wells, or community supplies [34, 41, infrequently and represents a very rare cause of posttrans-
120]. Food-borne outbreaks usually result from contamina- fusion hepatitis, i.e., in patients with blood coagulation dis-
tion by a food handler during the disease incubation period, order receiving multiple blood products [97, 117, 118].
and poor hygiene and diarrhea enhance the risk of transmis- Outbreaks of hepatitis A in hospitals have been traced to an
sion [90]. Implicated foods are usually handled extensively index case (often an infant) who acquired infection through
after cooking and/or are eaten uncooked. Such foods have transfusion. Transmission to hospital staff exposed to feces
426 D. Shouval

through breaks in infection control practices has occurred decline at the onset of clinical illness, and in most persons,
in neonatal intensive care units, with silent transmission it has decreased substantially 1 week later. A modest pro-
among infants and high attack rates among hospital staff portion (< 33 %) of those infected may continue to excrete
[117, 118]. virus during the second and third week of illness, and chil-
dren may excrete virus for longer periods than adults [112,
113, 118]. Between 5 and 20 % of cases may experience a
6 Pathogenesis and Immunity relapse of symptoms and ALT rise, 2–8 weeks after the ini-
tial illness, and virus excretion may occur during this time
6.1 The Viral Life Cycle [112, 115, 129]. Viremia is present from the middle of the
incubation period until early in the clinical illness and dur-
The virological, immunologic, and clinical events that occur ing relapse [129].
during HAV infection have been characterized in experimen- In tissue culture, HAV can replicate and be released with-
tally infected nonhuman primates and naturally infected out cell damage. No cellular injury occurs during the high
humans [124, 125]. The incubation period may range from rate of viral replication that occurs early in the infection. The
14 to 45 days, with a median of 28 days observed in common- mechanism of hepatic cell injury induced by HAV is not
source exposures. Studies in experimentally infected nonhu- fully understood, but available evidence suggests that it is
man primates suggest that the incubation period is lengthened immune mediated. The immune response to wild-type HAV
with the inoculation of a lower dose of virus or shortened infection involves the cellular, humoral, and innate limbs of
when inoculated parenterally but that the clinical severity of the immune system.
disease is not dependent on dose and route of inoculation
[114, 126]. Infection usually occurs through ingestion of
HAV-contaminated food or fluid when HAV penetrates the 6.2 Cellular Immune Response in Acute
gut mucosa reaching the liver through the portal circulation. Hepatitis A
HAV was demonstrated in intestinal crypts by immunofluo-
rescence but replication was not verified. HAV has a specific Infection with HAV leads to a cellular immune response
tropism for hepatocytes which are the primary site of replica- which is involved in the immunopathogenesis of HAV
tion [125, 127]. HAV entry into hepatocytes is mediated infection and the induction of hepatocyte injury [130–138].
most probably through a surrogate putative mucin-like gly- Despite the proven tropism of HAV for liver cells, the virus
coprotein receptor. Another hypothesis suggests that HAV is not cytopathic, and liver cell injury occurs through acti-
enters the liver cell as a virus–IgA complex through the vation of HAV multi-specific cytolytic T cells [137].
asialoglycoprotein receptor [128]. The virus replicates in the Inflammatory cell infiltrates isolated from liver biopsies of
liver and is then shed into the bile and feces and to a lesser patients with hepatitis A contain CD8-positive T cells which
degree into the bloodstream. Upon hepatocyte cell entry, can specifically lyse hepatitis A virus-infected target cells
host cell ribosomes bind to the viral uncoated RNA. HAV- in an histocompatibility leukocyte antigen (HLA) class
RNA is then translated into a major protein of 2,225 amino I-restricted manner [130]. Although there is only limited
acids. This large polyprotein is divided into three regions: information on involvement of the innate immune system
the P1 region encoding for the structural proteins VP1, VP2, in HAV infection, there is evidence that secretion of inter-
and VP3 and the P2 and P3 regions encoding for the nonstruc- feron gamma by activated T cells may facilitate the expres-
tural proteins involved in viral replication (Fig. 17.1). HAV- sion of HLA class 1 determinants on the surface of infected
RNA can be detected in body fluids and feces, using nucleic liver cells. Cytolytic T-cell epitopes residing on the struc-
acid amplification and sequencing techniques (Fig. 17.2). tural protein of HAV may be involved in cytolysis of HAV-
Such methods, mainly used by research laboratories, have infected hepatocytes [134, 135, 137]. Little is known about
been utilized for studies on the genetic organization of HAV the role of T-helper cells in mounting an immune response
infection [4, 17]. to HAV. One putative CD4 T-cell helper lymphocyte epitope
HAV antigen is found primarily in the cytoplasm of was identified on the VP3 102–121 sequence [132]. There
hepatocytes but can also be found in liver macrophages is also some evidence that nonspecific immune mecha-
(Kupffer cells). HAV appears in hepatocytes prior to detec- nisms, including natural killer cells (NK) and lymphokine-
tion in feces and prior to the onset of liver enzyme eleva- activated killer cells (LAK), are involved in the induction
tions. HAV is excreted via the biliary system into the feces of hepatocellular injury even before the initiation of cyto-
where it appears in high concentrations from 1 to 2 weeks toxic T lymphocyte injury [133]. Finally, impaired function
prior to onset of clinical illness. Virus excretion begins to of CD4+/CD25+ regulatory T cells has been linked to the
17 Hepatitis A Virus 427

frequent resolution of acute hepatitis A with spontaneous Clinical illness

recovery [136].

Response (relative titer)

Inoculation ALT
HAV
in stool IgG anti-HAV
6.3 Humoral Immune Response in Acute
Hepatitis A Viremia

HAV infection generates a humoral immune response

directed mainly against structural HAV proteins (Fig. 17.6). IgM anti-HAV
Diagnosis of acute hepatitis A is established through detec-
tion of anti-HAV (IgM) antibodies. Postinfection and post-
0 1 2 3 4 5 6 7 8 9 10 11 12 13
vaccination immunity is established through detection of Weeks after Exposure
total anti-HAV consisting mainly of IgG antibodies [17, 67,
139]. Presence of total anti-HAV antibodies in the absence of Fig. 17.6 Timeline of clinical and laboratory manifestations of acute
anti-HAV (IgM) antibodies signifies immunity against HAV hepatitis A. The sequence of events includes HAV viremia (green-blue)
and shedding of infectious HAV in feces (orange bar), followed by
and exclusion of acute HAV infection. Commercially avail-
increase in serum alanine aminotransferase (ALT) (green line) and the
able enzyme-linked immunoassays (EIA) are used for detec- appearance of anti-HAV (IgM) (gray) and total, mainly anti-HAV (IgG)
tion of anti-HAV (IgM) antibodies (directed against HAV antibody responses (blue dotted line). IgM antibody declines within 3
capsid proteins). EIAs are also used for detection of total months but can be detected in some patients as late as 6–12 months
postinfection as well as in relapsing patients by sensitive assays. Data
anti-HAV antibodies which consist mainly of anti-HAV
were obtained during the course of experimental infection in chimpan-
(IgG) antibodies and referred to as such in this chapter. Anti- zees inoculated IV with human strain HLD2 (Modified from references
HAV (IgM) assays utilize the principle of direct binding of Nainan et al. [17, 126])
anti-HAV (IgM) in the test sample to anti-human IgM-coated
matrix or particles. Qualitative competitive inhibition assays
for measurement of total anti-HAV (IgG) antibodies are used atic or subclinical [17, 142]. Immunity to HAV is established
in routine clinical practice. Experimental quantitative sensi- by convention once anti-HAV (IgG) antibodies rise to a titer
tive assays containing calibrators standardized against a above 10–20 mIU/ml, depending on the immunoassay used
WHO reference serum have been used in the assessment of for detection. However, the absolute lower limit of protective
anti-HAV (IgG) response to immunization. antibody level has not been determined [143].
Symptoms and signs of the acute infection usually occur Qualitative assays for total anti-HAV antibodies are used
within 2–4 weeks of exposure. IgM, IgG, and IgA anti-HAV for prevalence studies and may be used for assessment of
antibodies appear shortly before or during the onset of symp- immunity pending and following vaccination. However, this
toms [17, 126]. Anti-HAV (IgM) antibodies are detectable in method does not enable a distinction between immunity
symptomatic and asymptomatic patients alike. In symptom- generated by “natural,” wild-type HAV infection and
atic patients, anti-HAV (IgM) antibodies appear within vaccine-induced immunity. Such a differentiation may be
5–10 days before symptoms, or at the early phase of alanine possible in part through quantitative IgM and total anti-
aminotransferase (ALT) elevation, and persist for a period of HAV measurements using modified, more sensitive (and
about 4 months (range 30–420 days) (Fig. 17.6) [129, 140, experimental) immunoassays, since the humoral response to
141]. In patients with relapsing hepatitis A (3–20 % of immunization is generally weaker compared to the response
patients), anti-HAV (IgM), viremia, and shedding of HAV in to wild-type HAV infection [139, 144]. The inability to dis-
the feces may reappear intermittently for up to 6 months and tinguish clearly between these two situations led to an
occasionally even longer [33, 126, 129, 140]. False-positive attempt to develop an antibody assay against nonstructural
anti-HAV (IgM) may rarely be present >1 year postinfection proteins of the P2 and P3 regions of the HAV genome, for
or in patients with hyperglobulinemia [33, 67]. differentiation of the humoral immune response against rep-
Anti-HAV (IgG) antibodies are usually detectable at the licating virus from the response to a killed-inactivated HAV
onset of symptoms, and their titer rises slowly parallel to the vaccine [145, 146]. However, at present, these tests remain
decrease in titer of anti-HAV (IgM) antibodies (Fig. 17.6). a research tool only.
Anti-HAV (IgG) antibodies generated through natural infec- There are also other sensitive but also more time-
tion provide protection against rechallenge with HAV and consuming methods, compared to the commercially avail-
signify long-term immunity against hepatitis A apparently able assays for identification of neutralizing antibodies
for life, irrespective of whether the infection was symptom- against hepatitis A. These assays, which are mainly used as a
428 D. Shouval

research tool, include the radioimmunofocus inhibition assay indigestion, weight loss, emotional instability, and prolonged
(RIFIT), HAVARNA, and radioimmunoprecipitation assay indirect bilirubinemia. Acute HAV infection resolves sponta-
[139]. neously in >99 % of infected individuals. Relapsing hepatitis
The role of secretory immunity in hepatitis A remains A with subsequent complete resolution has been reported in
unclear. Anti-HAV (IgA) antibodies have been detected in 3–20 % of patients with clinical hepatitis A [129].
the saliva and feces of experimentally infected animals and Fulminant hepatitis A is rare, with a wide range of esti-
humans [145, 146]. mated rates as low as 1:10,000 or more in immunocompetent
individuals. About 70 % of fulminant hepatitis A patients
experience a relatively high spontaneous survival rate; the
7 Patterns of Host Response remaining patients either die or require emergency liver
and Clinical Outcome transplantation. In the United States, hepatitis A accounted
for 3 % of cases of fulminant hepatitis referred for liver
7.1 Clinical Features transplantation. The Acute Hepatic Failure Study group
reported in 2008 that only 29 % of 31 US patients with ful-
Acute HAV infection causes an acute necro-inflammatory minant hepatitis A underwent liver transplantation [59, 150].
process in the liver which normally resolves spontaneously. Recent reports from South America and Korea have raised
The clinical spectrum of acute disease is comparable to that concern that the current incidence of fulminant hepatitis A
of other types of viral hepatitis and ranges from few or no may be rising [59, 70, 71, 73]. Immunosuppressed patients
symptoms to fulminant hepatitis [147, 148]. However, in and patients with chronic liver disease such as chronic hepa-
contrast to HBV and hepatitis C virus (HCV) infection, HAV titis C and B are at an increased risk of developing severe or
infection does not lead to chronic liver disease. An important fulminant hepatitis [151]. Mortality in fulminant hepatitis is
feature of HAV infection includes the relationship of age to rare. Following the massive outbreak of hepatitis A in
clinical expression. Children under age 6 generally have Shanghai, there were 47 death among >300,000 infected
mild, often nonspecific, symptoms that may include nausea patients. Fatality in HBsAg + Chinese patients reached
and/or vomiting, malaise, diarrhea in 50–70 %, and fever or 0.05 % as compared to 0.015 % in non-HBV carriers [62]. In
dark urine in 30–50 % [65]. Among those under age 3, fewer Western countries, reports suggest a case fatality ratio in age
than 5 % become icteric, compared to 10 % of those aged groups <40 years of ~0.3–0.6 %. In age groups >50 years
4–6 years. Data are less complete for children aged and older, case fatality may rise to 1.8–5.4 % (these data
6–14 years but suggest that symptom patterns are compara- were obtained prior to introduction of liver transplantation
ble to those of adults. for HAV-associated liver failure) [11, 66, 81, 148, 152–154].
In adults, symptoms include malaise, fatigue, anorexia, Risk factors associated with development of fulminant HAV
vomiting, abdominal discomfort, diarrhea, pruritus, and less infection are poorly understood [155]. Familial clustering of
commonly fever, headaches, arthralgia, and myalgia [148]. fulminant HAV cases and putative sequence variations have
Five clinical patterns are recognized: (1) asymptomatic HAV been suggested as potential risk factors [156–158] but await
infection, often present in children under the age of 5 years; (2) confirmation.
symptomatic HAV infection with the appearance of dark urine Hepatitis surveillance in the United States has shown the
and sometimes clay-colored stools, often accompanied or fol- fatality rate among reported cases to be age dependent. The
lowed by jaundice; (3) cholestatic hepatitis characterized by highest rates were recorded among children under age five
pruritus, prolonged elevation of alkaline phosphates, gamma- (1.5 per 1,000 cases) and in persons over age 40 (27.0 per
glutamyl transpeptidase, bilirubinemia, and weight loss; (4) 1,000 cases), especially among those with underlying
relapsing hepatitis A infection manifested by reappearance of chronic liver disease [24].
the clinical, biochemical, and virologic markers of acute hepa-
titis A after initial resolution; and (5) fulminant hepatitis, which
frequently resolves spontaneously under conservative care but 7.2 Diagnosis of Acute Viral Hepatitis A
which occasionally may require liver transplantation.
Extrahepatic manifestations of acute hepatitis A may occur Diagnosis of hepatitis A requires demonstration of hepato-
and include skin involvement, vasculitis, pancreatitis, neuri- cellular liver injury by standard biochemical tests (e.g., ALT,
tis, encephalitis, carditis, glomerulonephritis, pneumonitis, AST, bilirubin) and the presence of anti-HAV (IgM) antibod-
hemolysis (especially in patients with glucose-6-phosphate ies. The intensity of hepatocellular injury (and clinical
dehydrogenase (G6PD) syndrome), cryoglobulinemia, and symptoms) is variable and ALT levels may rise to thousands
aplastic anemia [5, 41, 149]. Other post-icteric manifesta- of units accompanied by bilirubinemia and jaundice or
tions may occur in a minority of patients including prolonged remain very low, unicteric and undetected, especially in
fatigue, right upper quadrant discomfort, fat intolerance and young children. Historically, in situation of major outbreaks,
17 Hepatitis A Virus 429

documentation of hepatocellular injury and bilirubinemia efficacy of IG is well documented [143, 161, 162], but dura-
has been used to classify such events as HAV induced pro- tion of protection is limited to 12–20 weeks following intra-
vided one or more index cases were serologically positive for muscular injection of 0.02 and 0.06 ml/kg weight,
anti-HAV (IgM). Because of the higher frequency of nonspe- respectively. Preexposure prophylaxis is achieved within
cific symptoms or anicteric infection in young children, the hours of injection. Postexposure administration is ~85 %
index of suspicion should be high in particular circumstances effective when administered as close as possible to exposure
(i.e., those exposed in day-care centers), and testing for liver and no later than 14 days [143]. The mechanism of protec-
enzymes or anti-HAV (IgM) should be considered when an tion against hepatitis A conferred by IG is not fully estab-
adult contact of a child has hepatitis. Clinical symptoms are lished, but most probably involves neutralization of
inadequate to distinguish hepatitis types, and virus-specific circulating virus and possibly prevention of uptake of virus
serological testing must be done for all cases of presumed through the gut mucosa and hepatocytes.
viral hepatitis. Commercially available anti-HAV (IgM) tests Administration of IG is considered very safe, although
remain positive in virtually all cases tested within 2 months contraindicated in patients with IgA deficiency. Interference
of the disease, often remaining detectable for up to 6 months with live-attenuated vaccines such as measles, mumps,
after onset, and thus allow for the diagnosis based on a single rubella (MMR), and varicella requires special caution.
acute or early convalescent serum specimen. Testing for anti- Coadministration of IG with an active hepatitis A vaccine
HAV (IgG) will not differentiate acute from past infection, may blunt the initial quantitative anti-HAV (IgG) antibody
but is useful for identifying immunity as a result of prior response after the first vaccine dose [163, 164]. This effect,
infection or immunization. Measurement of plasma HAV- which is similar to the effect of passively transferred maternal
RNA levels by PCR is not used in routine clinical diagnosis anti-HAV (IgG) antibodies, is of minor significance, and such
but remains an important tool for investigation of the molec- vaccines respond well to a booster dose given 6 months after
ular epidemiology of HAV and used especially in situations the primary immunization. Finally, although administration
of outbreaks [15, 17]. of IG for pre- and postexposure short-term prophylaxis is
highly efficacious, the use of immunoglobulin worldwide is
now declining for a number of reasons: (a) nonspecific IG
8 Control and Prevention preparations increasingly fail to contain adequate amounts of
anti-HAV (IgG) [165–168]; (b) cost of specific HAV IG prep-
There are three measures for protection against HAV infec- arations is high [167, 169]; (c) duration of IG-mediated pro-
tion: (1) improvement in personal hygiene, sanitation, and tection against HAV infection lasts only several months as
socioeconomic status; (2) passive prophylaxis with immuno- compared to hepatitis A vaccines [143]; and (d) hepatitis A
globulin (IG); and (3) active immunization with vaccines have already been shown to induce very rapid pre- as
formaldehyde-inactivated or live-attenuated vaccines. well as postexposure protection against HAV following the
first out of the two recommended doses [20, 37, 162].

8.1 Personal Hygiene and Sanitation

8.3 Active Pre- and Postexposure
Personal hygiene and environmental sanitation to prevent Prophylaxis Against Hepatitis A
transmission through fecal contamination of food and water
or by personal contact have been the primary means to con- 8.3.1 Serological Measurement of Protection
trol hepatitis A in any setting. Improved sanitation is pre- A positive (qualitative) test for anti-HAV (IgG) antibodies
sumed to have lowered the incidence of infection and disease signifies immunity to hepatitis A. The lowest protective level
in developed countries. against challenge with HAV is unknown. The reported mini-
mal serum levels of anti-HAV (IgG) antibodies required for
protection against HAV in humans vary between 10 and
8.2 Passive Prophylaxis Against Hepatitis A 20 mIU/ml depending on the immunoassay used for detec-
with IG tion and regardless of whether these antibodies emerged fol-
lowing “natural” wild-type HAV infection or following
Since the late 1940s and until recently, administration of vaccination [31, 139]. Clinical experience suggests that pro-
human immunoglobulin (IG) prepared from pooled human tection against hepatitis A following passive immunization
plasma through ethanol fractionation [159] and containing with IG or active vaccination may still be present even in the
high concentration of anti-HAV (IgG) antibodies has been absence of detectable anti-HAV antibodies using standard
used as an efficient means for pre- and postexposure prophy- immunoassays [81]. Low levels of anti-HAV (IgM) antibod-
laxis against HAV infection [12, 143, 160]. The protective ies may be detectable by a conventional assay for a few
430 D. Shouval

weeks in ~20 % of recipients of HAV vaccines [37]. most probably lifelong immune memory and protection after
Therefore, anti-HAV (IgM) antibody assays cannot be used a complete vaccination has been predicted [104, 173, 182]
for reliable distinction between acute hepatitis A and anti- and already documented for at least 17 years [182, 183].
HAV response to vaccination. Consequently, booster doses are not recommended in vac-
cinees who completed a two-dose vaccination schedule [104,
182, 184].
8.4 Active Immunization The immunogenicity of inactivated hepatitis A vaccine is
blunted somewhat by preexisting antibodies, which occurs
All HAV vaccines contain HAV antigens derived from cell cul- when vaccine is coadministered with IG [185, 186] or when
tures of attenuated HAV strains adapted to grow in human and given to infants of previously infected HAV immune mothers
nonhuman mammalian cells. Viral attenuation is associated [187]. Yet, the clinical consequences of this phenomenon are
with a number of mutations generated through serial passage of most likely negligible in view of a recent report that vaccine-
wild-type HAV [4, 5, 17, 170]. Comparison of the nucleotide induced anti-HAV (IgG) seropositivity in children vacci-
sequence of complementary DNA (cDNA) cloned from wild- nated <2 years of age persists for at least 10 years regardless
type virus with attenuated HM-175/7 MK-5 HAV strain revealed of maternal anti-HAV (IgG) status [188].
a small number of nucleotide changes distributed throughout
the genome [170]. Most attenuated virus strains used for vac- 8.4.1 Preexposure Prophylaxis Through Active
cine production are grown in human diploid MRC-5 fibroblasts, Immunization
and the nucleotide and amino acid sequences of the virus are Inactivated hepatitis A vaccines have been licensed in
about 95 % identical among different strains. Cell culture- Europe, Asia, Australia, and the Americas. The high efficacy
derived HAV antigen is purified, inactivated by formaldehyde, of these vaccines in preventing hepatitis A in children has
and adsorbed to aluminum hydroxide for the following vac- been shown in two pivotal placebo-controlled clinical trials
cines: HAVRIX®, VAQTA®, and AVAXIM®). The HAV anti- conducted in Thailand and the United States demonstrating a
gen in EPAXAL® is formulated in influenza-reconstituted 99 % efficacy after three doses and 100 % after two doses of
virosomes. VAQTA®, HAVRIX®, EPAXAL®, HEALIVE®, aluminum hydroxide-formulated vaccines, respectively [18,
and the Chinese Lv-8 inactivated HAV vaccine are at present 19]. In a third study a single dose of an inactivated vaccine
preservative-free (Table 17.1). In addition to monovalent HAV formulated in virosomes was shown to provide complete
vaccines, formaldehyde-inactivated combination vaccines have protection against the disease [189]. Several factors have a
been developed in Europe against HAV and HBV or HAV and marginal impact on blunting of anti-HAV (IgG) antibody
typhoid [5, 174–179]. levels following immunization. These include overweight,
Two live-attenuated hepatitis A vaccines were developed older age, smoking, as well as passively transferred anti-
in China containing the H2 and LA-1 strains [172, 180, 181] HAV (IgG) antibodies from pregnant mothers to their new-
used in a single-dose immunization schedule. Both these borns. Lower sero-protection rates following vaccination
vaccines as well as two formaldehyde-inactivated vaccines, have been reported in human immunodeficiency virus-
administered in two doses, were tested in clinical trials and infected patients as well as solid organ and stem-cell trans-
integrated into the Chinese EPI in 2008 [172, 181] (http:// plant recipients. As observed with other vaccines, anti-HAV
www.who.int/wer/2010/wer8530/en/index.html). (IgG) levels were reported to be higher in females as com-
Formaldehyde-inactivated hepatitis A vaccines are highly pared to males, following a priming and booster dose of the
immunogenic and safe, providing rapid, protective immunity vaccine [5]. HAV vaccines have an excellent safety and toler-
to hepatitis A within 2–4 weeks after primary immunization. ability record and are interchangeable [5, 81].
A second dose is usually administered within 6–12 months As more information has become available on the extraor-
after the priming injection, but the interval may be extended dinary immunogenicity, effectiveness, and safety of hepatitis
to 18–36 months depending on vaccine type. Long-term, A vaccines, immunization strategies have shifted from vac-

Table 17.1 Attenuated hepatitis A virus strains used for production of formaldehyde-inactivated hepatitis A virus vaccines
Attenuated HAV strain Trade name Adjuvant HAV antigen dose/injection Manufactures Reference
Pediatric Adult
HM-175 HAVRIX® Alum hydroxide 720 EU 1440 EU GSK [21]
CR-326 VAQTA® Alum hydroxide 25 U 50 U MSD [20]
GBM AVAXIM® Alum hydroxide 80 U 160 U Aventis Pasteur [171] [171]
TZ84 HELIVE® Alum hydroxide 250 U 500U Sinovac Biotech Ltd [172]
RG-SB EPAXAL® Virosome 24 U 24 U Crucell/Berna Biotech [173]
17 Hepatitis A Virus 431

Total population Jewish population Non-Jewish population

Universal Immunization Universal Immunization Universal Immunization
Annual Incidence per 100000 Population

120 120 120

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0
1985 1990 1995 2000 1985 1990 1995 2000 1985 1990 1995 2000
Year Year Year

Total “Infectious Hepatitis”

Hepatitis A

Fig. 17.7 The impact of universal vaccination against hepatitis A of fied as “infectious hepatitis” (including A, B, C, and nonspecified).
18-month-old toddlers on the incidence rates in the Jewish, the Arab, Afterward, between 1993 and 2004, data include only serologically
and the overall population in Israel. Data obtained by passive and active confirmed hepatitis A cases (Reproduced by permission from reference
surveillance. Between1985 and 2004, acute hepatitis cases were classi- Dagan et al. [84])

cination of individuals belonging to specific risk groups to Similar projects were introduced in Puglia, Italy, in 1997
mass vaccination campaigns and then to universal vaccina- [191]; in Catalonia, Spain, in 1998 [192]; and in North
tion, which is however still restricted to a limited number of Queensland, Australia, in 1999 [193], leading to a 90–97 %
countries [61, 81, 82, 84, 104, 190]. decline in the reported incidence in these regions. The results
Following the licensure of HAV vaccines, four immuniza- of these highly successful vaccination projects in selected
tion strategies have been evaluated for preexposure prophy- geographic regions worldwide suggested that mass vaccina-
laxis. First, early efforts were aimed at immunization of tion of children in communities at risk is effective and will
individuals at increased risk, which included international lead to herd immunity even under moderate coverage.
travelers to areas of HAV endemicity, MSM, intravenous These early projects paved the way for the introduction of
drug users, patients with chronic liver disease, food handlers, the third immunization strategy, namely, universal vaccina-
day-care center staff, caretakers of nonhuman primates, and tion against HAV in selected countries with intermediate
patients with blood clotting disorders. This policy is still endemicity in transition. At present, 11 countries have
valid but has no or little public health impact on the herd embarked on the road toward universal HAV vaccination in
immunity at large. babies. In 1999, Israel, a country with intermediate HAV
The second strategy included regional mass vaccination endemicity in transition, became the first country to intro-
of pediatric populations at risk. Three demonstration projects duce universal HAV vaccination given in two doses to tod-
conducted in the United States in native Americans in dlers at 18 and 24 months of age. At a vaccination coverage
Alaska, in native American Indians, and in Butte County, of 90 and 85 % for the first and second dose, respectively, the
California, led to a 94–97 % reduction in incidence of symp- annual incidence of hepatitis A dropped sharply within
tomatic acute hepatitis [82]. Consequently, in 1999, the US 2–3 years of program initiation. An overall decline of 95 %
Advisory Committee on Immunization Practices (ACIP) in incidence was documented not only in babies but also in
issued a recommendation to introduce universal hepatitis A the unvaccinated adult population (Figs. 17.7 and 17.8) [84].
vaccination into routine childhood vaccination (two doses in Thus, immunization of ~3 % of the population annually led
children >2 years old and catch-up at the age of 10–12 years) to a marked decrease in attack rates of HAV infection in all
in 17 states in the United States with an annual incidence of age groups until the age of 44 years and a shift from a state
>20 cases/100,000. Postimmunization surveillance revealed of intermediate HAV endemicity to very low endemicity
that, despite variable first vaccine coverage of 50–80 %, a with an annual incidence of ~2.5 cases/100,000.
progressive decline in reported incidence of hepatitis A was In the United States, the public health objective of hepati-
observed from 21.1 cases/100,000 to 2.5 cases/100,000, tis A vaccination is the reduction of disease incidence and
which represents an 88 % drop (Fig. 17.5) [61, 82, 83]. possibly eradication of HAV infection through routine infant
432 D. Shouval

<1 Year 1-4 Years 5-9 Years

100 Universal Immunization 300 Universal Immunization 350 Universal Immunization

90
Annual Incidence per 100000 Population

Annual Incidence per 100000 Population

Annual Incidence per 100000 Population

Jewish 300
250
80 Non-Jewish

70 250
200
60
200
50 150
150
40
100
30 100
20
50
50
10

0 0 0

1993 1995 1997 1999 2001 2003 1993 1995 1997 1999 2001 2003 1993 1995 1997 1999 2001 2003
Year Year Year

10-14 Years 15-44 Years 45-64 Years

150 Universal Immunization 45 Universal Immunization 30 Universal Immunization

40
Annual Incidence per 100000 Population

Annual Incidence per 100000 Population

Annual Incidence per 100000 Population

125 25
35

100 30 20

25
75 15
20

50 15 10

10
25 5
5

0 0 0
1993 1995 1997 1999 2001 2003 1993 1995 1997 1999 2001 2003 1993 1995 1997 1999 2001 2003
Year Year Year

≥65 Years

90 Universal Immunization

80
Annual Incidence per 100000 Population

70

60

50

40

30

20

10

1993 1995 1997 1999 2001 2003

Year

Fig. 17.8 Annual age-specific incidences of reported hepatitis A disease among the Jewish and non-Jewish populations in Israel, 1993–2004.
Error bars indicate 95 % confidence intervals (Reproduced with permission from reference Dagan et al. [83])

immunization [187]. Major progress in this direction has In 2005, public health authorities in Argentina began an
already been achieved [83] (Fig. 17.5). experimental universal immunization program in 12-month-
The fourth strategy for preexposure prophylaxis includes old babies [196]. The original baseline incidence of HAV in
immunization with only a single dose of an inactivated HAV Argentina dropped from 70.5 to 173.8 cases/100,000
vaccine. The rationale for this strategy is based on the cumu- between 1995 and 2004 to ~10 cases/100,000 in all age
lative experience that anti-HAV (IgG) sero-protection rates groups within a few years, representing a >80 % decrease in
may reach 88 % in vaccinees within 2 weeks after a single incidence. These results also confirmed the experience
vaccine dose rising to 97–100 % at weeks 4–6 [37, 194, 195]. gained in mass and universal immunization programs
17 Hepatitis A Virus 433

elsewhere that effective immunization of toddlers will lead economic conditions in many countries and in regions within
to widespread herd immunity. It remains, however, to be individual countries has led to a shift in prevalence of HAV
seen if a single-dose immunization strategy will indeed pro- infection from high to intermediate and even low endemicity.
vide long-term protection against HAV or whether a booster Consequently, a growing population of adolescents and young
dose will be required after all [197]. adults becomes susceptible to HAV infection. Hepatitis A is a
The ultimate success of routine infant immunization to vaccine-preventable disease, and vaccination of babies is the
eliminate HAV transmission is dependent on the availability most efficient means for the control of the infection. Yet, at
of an affordable vaccine that potentially can be combined present only a few countries at risk have embarked on the route
with other antigens and provides long-term protection. Such toward implementation of universal vaccination. The demon-
a strategy would also be appropriate for countries with an strated effectiveness of hepatitis A immunization holds the
intermediate and high endemicity of infection where most potential for the eventual eradication of HAV infection.
persons become infected during childhood. Elimination of HAV transmission occurs when a critical level
of herd immunity is present in the population. As recently
8.4.2 Postexposure Prophylaxis stated in a 2012 WHO position paper on hepatitis A, “Countries
Evidence obtained in a number of clinical trials suggested should collect and review the information needed to estimate
that postexposure immunization against hepatitis A may also their national burden of hepatitis A” [22]. In addition to surveys
have similar effectiveness as IG, provided that immunization estimating age-specific prevalence of anti-HAV (IgG) antibod-
is started within 2 weeks of exposure. Support for this ies, this may require examining vital registration systems,
impression was initially obtained from the hepatitis A effi- acute disease surveillance, and health information systems
cacy trial conducted in the United States where the HAV vac- capturing fulminant hepatic failure cases and/or causes for
cine was administered during an HAV outbreak and no new liver transplantation. Economic evaluation, including cost-
cases of acute hepatitis were identified from day 17 onward effectiveness analyses of relevant immunization strategies,
after vaccination [20]. A similar experience was obtained in can serve as a useful additional element for decision making.
Slovakia [198], in Israel, and in Italy where a 79 % protective
efficacy of postexposure immunization was documented in Acknowledgments This chapter is an extensively revised and updated
Italian household contacts of acute hepatitis A cases [199]. version of the chapter printed in the 4th edition of this book authored by
HS Margolis, MJ Alter, and SC Hadler.
In Israel, as in Slovakia, prompt intervention with an active
vaccine in a community outbreak of hepatitis A led to effec-
tive control of an epidemic within 2 weeks of starting the
intervention, in contrast to the relatively poor performance of References
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167. Howell D, Barbara JA, Brennan M. Hepatitis A virus, blood 188. Sharapov UM, Bulkow LR, Negus SE, et al. Persistence of hepa-
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FE. Single dose inactivated hepatitis A vaccine: rationale and
 Hepatitis E Virus
18
Xiang-Jin Meng

1 Introduction 1978 Kashmir epidemic [1] or retrospectively in the earlier

outbreaks [2]. In addition, these outbreaks displayed unique
Although evidence for the existence of a new form of clinical features that were distinct from that of hepatitis A,
enterically transmitted non-A, non-B viral hepatitis came such as the high attack rate among young adults and high
from serological studies of waterborne epidemics of hepa- mortality rate in pregnant women [11]. This led to the recog-
titis in India in 1980 [1, 2], the identity of the virus was not nition of a new form of epidemic non-A, non-B, or enterically
known until 1990 when the genomic sequence of the hepa- transmitted non-A, non-B hepatitis [1, 2]. The term hepatitis
titis E virus (HEV) was determined [3]. The diseases caused E was designated only after the etiological agent responsible
by HEV are characterized by explosive outbreaks of acute for these outbreaks was identified in 1990 [3].
hepatitis in developing countries and sporadic and clus- In 1983, Mikhail Balayan (a Russian virologist) success-
tered cases in industrialized countries [4, 5]. The identifica- fully transmitted the disease to himself. He had experienced
tion of various animal strains of HEV has broadened the prior infection with the hepatitis A virus (HAV) and thus was
appreciation of the host range and diversity of the virus [6] immune to HAV, when he voluntarily ingested a suspension
and also provided unique homologous animal model sys- of the stool samples collected from non-A, non-B hepatitis
tems to study HEV replication and pathogenesis. Hepatitis patients [12]. About 36 days after ingestion of the stool sus-
E is now a recognized zoonotic disease, and pigs and likely pension, he developed severe acute hepatitis but acute diag-
other animal species are reservoirs for HEV [7]. A vaccine nostic markers for hepatitis A and B were not detected in his
has recently been licensed for use in China, although it is sera, thus indicating a new agent was responsible for the
not yet available in other countries [8]. Promising antiviral hepatitis. Immune electron microscopy identified 27–30 nm
agents have also been identified. viruslike particles in samples of his stool; they provided the
first direct evidence for the existence of a new enterically
transmitted viral hepatitis agent. Acute viral hepatitis was
2 Historical Background further transmitted to cynomolgus macaques by intravenous
inoculation of monkeys with the stool suspension collected
The epidemiological features of the large waterborne out- from the infected volunteer, and viruslike particles were
breaks of hepatitis in India in the 1950s and 1970s such as the visualized from the feces of the experimentally infected
1955–1956 Delhi, the 1975–1976 Ahmadabad, and the 1978 monkeys [12]. A nonhuman primate model was subsequently
Kashmir epidemics resembled hepatitis A in terms of the established for the study of the disease [13, 14].
mode of spread, incubation period, and clinical and biochem- In 1990, the agent responsible for the enterically transmit-
ical features [9]. In fact, the 1955–1956 Delhi epidemics were ted non-A, non-B hepatitis was successfully cloned and
initially reported as due to hepatitis A [10]. However, diag- sequenced and designated as hepatitis E virus (HEV) [3].
nostic markers of acute hepatitis A were not detected in the Subsequently, the development of diagnostic tests based on
recombinant HEV antigens or peptides enabled the study of
the HEV epidemiology [15]. In 1997, the first animal strain
X.-J. Meng, MD, PhD of HEV, swine hepatitis E virus (swine HEV), was discov-
Department of Biomedical Sciences and Pathobiology, ered and characterized [16], leading to the recognition of
Center for Molecular Medicine and Infectious Diseases,
hepatitis E as a zoonotic disease [6, 7, 17] and significantly
Virginia Polytechnic Institute and State University (Virginia Tech),
Blacksburg, VA 24061-0913, USA expanding our understanding of the natural history and ecol-
e-mail: [email protected] ogy of this pathogen [4–6, 18].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 439

DOI 10.1007/978-1-4899-7448-8_18, © Springer Science+Business Media New York 2014
440 X.-J. Meng

3 Methods for Epidemiologic Analysis 3.4 Laboratory Diagnosis

3.1 Mortality Immune electron microscopy is insensitive for the detection

of HEV particles and thus is of little value in the laboratory
Prior to the identification of the causative agent, the sources diagnosis of HEV infection [4, 35]. Specific serological
of mortality data were primarily studies of large waterborne assays to detect IgA, IgG, and IgM anti-HEV have been
outbreaks of acute hepatitis that were negative for hepatitis A developed [36, 37] and are commercially available in some
and B diagnostic markers [19]. With the development of sero- countries in Asia and Europe and recently in the United
logical and molecular diagnostic assays specific for HEV, the States. However, a considerable variation in the specificity
mortality data are now based on patients that are positive for and sensitivity of these assays has been reported [38, 39].
anti-HEV antibodies or HEV RNA [20, 21]. The mortality The available serological diagnostic assays primarily use the
associated with HEV infection is typically less than 1 % in recombinant ORF2 capsid protein expressed in bacterial or
the general population, but it can reach up to 25 % in infected baculovirus expression system as the antigen, although a
pregnant women especially during the third trimester [4, 5, mixture of both ORF2 and ORF3 proteins is also used as the
22]. Among the recognized hepatitis viruses, the high mortal- antigen in some assays. Some commercial serological assays
ity observed during pregnancy is unique to HEV. are approved for HEV diagnosis by regulatory agencies in
Asia and Europe, although none has been approved by the
FDA. When serum samples are collected during the acute
3.2 Morbidity stage of HEV infection, IgM anti-HEV is detectable in up to
90 % of the acute cases within 1–4 weeks after the onset of
The main sources of morbidity data have been investigations the disease [4, 15, 40, 41], but in <50 % of cases 3 months
of outbreaks and sporadic cases of acute viral hepatitis [19]. after the onset of the disease [15, 42]. A rising IgG anti-HEV
Due to the lack of accurate etiology-specific data for acute titer in samples collected 2–4 weeks apart after the onset of
viral hepatitis, the morbidity data for hepatitis E are not very the disease can also aid in the diagnosis of hepatitis E [4, 15,
reliable [23]. Investigations of acute viral hepatitis disease 40]. The duration of IgG anti-HEV persistence in infected
outbreaks using specific serological assays to detect IgG and individuals varies from <1 year in some pediatric patients to
IgM anti-HEV and PCR-based assays to detect HEV RNA 2–14 years in some adult patients [40, 41, 43].
help produce more accurate morbidity data regarding hepati- RT-PCR has been successfully used to detect HEV RNA
tis E. Unfortunately, many sporadic cases of acute viral hep- from outbreaks and sporadic cases of hepatitis E [44–48].
atitis are not routinely tested for hepatitis E and thus are not Real-time PCR assays have also been developed to detect
reported. Due to the lack of an FDA-approved diagnostic and quantify HEV genomes [49, 50]. By using a panel of
assay, underdiagnosis of hepatitis E in the United States and known HEV-containing plasma samples, the performance of
many other industrialized countries has further limited the 20 conventional or real-time RT-PCR assays from different
available morbidity data. laboratories worldwide was evaluated for the detection of
HEV RNA [51]. Significant variations in the sensitivity
(100–1,000-fold difference) and specificity of these PCR-
3.3 Serological Survey based assays were observed, indicating the need for a stan-
dardized PCR-based diagnostic assay for HEV. The existence
The seroprevalence of HEV infection has been primarily of at least four recognized and two putative genetically dis-
inferred from serological studies of archived samples and tinct genotypes of mammalian HEV [18, 52] further stresses
from reported outbreaks of the disease worldwide [24–27]. the need for a standardized universal RNA-based detection
The seroprevalence data from the retrospective studies assay for HEV.
should be interpreted with caution due to the potential sam-
pling bias of the convenient samples, the duration of IgG
anti-HEV response in infected individuals, and the variations 4 Biological Characteristics
of different serological assays. Age- and geography-matched
seroprevalence studies in special high-risk populations such 4.1 Physical and Biochemical Properties
as pig handlers, swine veterinarians, organ transplant recipi-
ents, and HIV-infected patents have also been reported [28– The buoyant density of HEV virion is 1.35–1.40 g cm−3 in
31]. The prevalence of HEV infection has been investigated CsCl and 1.29 g cm−3 in glycerol and potassium tartrate gra-
prospectively in the placebo groups in conjunction with dients. The HEV virion is sensitive to low-temperature stor-
vaccine clinical trials [32–34], which provided a more accu- age (between −70 °C and +8 °C) and iodinated disinfectants
rate estimate of HEV seroprevalence. [4, 18]. It is more heat labile than is HAV: HEV was about
18 Hepatitis E Virus 441

50 % inactivated at 56 ºC and almost totally inactivated 4.4 Genome Organization and Genotypes
(96 %) at 60 ºC for 1 h [53]. Liver suspensions containing
HEV remained infectious after incubating at 56 °C for 1 h, The HEV genome is organized into a short 5′ noncoding
although the virus is completely inactivated by boiling or region (NCR), three open reading frames (ORFs 1, 2, and 3),
stir-frying the HEV-contaminated livers for 5 min [54]. and a 3′ NCR [62]. ORF2 overlaps ORF3, but neither over-
laps with ORF1 [63]. A cap structure has been identified in
the 5′ end of the viral genome and may play a role in the
4.2 Morphology initiation of HEV replication [64]. A bicistronic subgenomic
mRNA encoding both ORF2 and ORF3 proteins has been
The HEV virions are nonenveloped icosahedral particles identified [65]. The 5′-NCR is only about 26 nt long and may
with a diameter of approximately 27–34 nm [52] (Fig. 18.1). play a role in the initiation of HEV replication. The 3′-NCR
The capsid is formed by capsomers consisting of homodi- contains a cis-reactive element. ORF1 encodes the nonstruc-
mers of a single capsid protein forming the virus shell. Each tural polyprotein, ORF2 encodes the major capsid protein,
capsid protein contains three linear domains forming distinct and ORF3 encodes a small phosphoprotein with a multifunc-
structural elements: S (continuous capsid), P1 (three-fold tional C-terminal region [62].
protrusions), and P2 (two-fold spikes). Each domain con- Currently HEV is the only species in the genus Hepevirus
tains a putative polysaccharide-binding site that may interact within the family Hepeviridae, and avian HEV from chick-
with cellular receptors [56, 57]. Native T = 3 capsid contains ens is classified as a floating species within the family [52].
flat dimers, with less curvature than those of T = 1 viruslike There exist at least 4 recognized and two putative genotypes
particles [58]. of mammalian HEV (Fig. 18.2): genotype 1 (Burmese-like
Asian strains), genotype 2 (a single Mexican strain and
some African strains), genotype 3 (strains from sporadic
4.3 Antigenic Properties human cases mostly from industrialized countries, and ani-
mal strains from pig, deer, rabbit and mongoose), and geno-
It is thought that there exists only a single serotype of HEV, type 4 (strains from sporadic human cases in Asia and swine
with antigenic cross-reactivity among strains in different strains) [52]. The strain of HEV recently identified from rats
genotypes [52]. Antibodies cross-reactive to human HEV in Germany [67] and the United States [68] appears to be a
strain capsid proteins have been reported in various animal new genotype, as does the recently identified HEV strain
species, but the viruses responsible for the antigenic cross- from wild boars in Japan [69]. The HEV strains from chick-
reactivity were genetically identified only in swine, chick- ens [55] and cutthroat trout [70] likely represent different
ens, deer, mongoose, rats, and rabbits [6, 18]. Common genera.
antigenic epitopes in the capsid protein between avian and
mammalian HEV strains and among different animal strains
of HEV have been identified [59–61]. 4.5 Gene Expression
and Replication of HEV

The mechanisms of HEV replication and gene expression

are largely unknown. The recent reports on the develop-
ment of improved cell culture systems for HEV may aid in
understanding the mechanisms of HEV replication in the
future [71, 72]. The ORF1 encodes a nonstructural poly-
protein that is important for viral replication, although it
remains unknown if the polyprotein functions as a single
protein or as individually cleaved smaller proteins [5, 52].
Functional activities such as methyltransferase [64, 73],
guanylyltransferase [73], deubiquitination [74, 75], NTPase
and 5′ to 3′ RNA duplex-unwinding activities [76], and
RNA 5′-triphosphatase [77] have been experimentally
demonstrated in the ORF1 protein. A proline-rich hyper-
variable region in the ORF1 protein is dispensable for viral
Fig. 18.1 An electron micrograph of a strain of hepatitis E virus par-
replication both in vitro and in vivo and is functionally
ticles (30–35 nm diameter). Negative staining, bar = 100 nm
(Reproduced with permission from Haqshenas et al. [55] the Society exchangeable between HEV genotypes [78, 79]. The ORF2
for General Microbiology) encodes the viral capsid protein that contains a typical
442 X.-J. Meng

Fig. 18.2 A phylogenetic tree of genotype 3 genotype 2

mammalian and avian strains of

V
hepatitis E viruses. The four

Ja

US

hHE
pa

pU S A
recognized genotypes of

1
ns

hH
mammalian Hepeviruses and the EV

EV
US2
sH

HE

EV

aH
three recognized genotypes of A

aH E V
V
US

SA
avian hepatitis E virus are Japa
n hHE

vU
indicated (Reproduced with V 67

aa
100 0
permission from Bilic et al. [66] 10

100
genotype 4
the Society for General
EV

100
Microbiology) 100 100
100 AaH
China hHEV 100
62 77
genotype 1
10
0

10
0
Ea

10

0
HE
V
genotype 2

EV
genotype 3

Bu
V
hH

HE

mr
o

a
ia h
ic

hH
ex

0.1
M

EV
Ind
genotype 1

signal peptide sequence and three potential glycosylation sequence and the stem-loop structure in the junction region
sites. Mutations within the glycosylation sites prevent the play important roles in HEV replication [90]. The mecha-
formation of infectious virus particles [80–82]. The ORF2 nisms of HEV assembly and release remain largely
capsid protein contains neutralizing epitopes and is the tar- unknown. The ORF3 protein is responsible for virion
get for vaccine development [82]. The ORF3 encodes a egress from infected cells [84, 91].
small cytoskeleton-associated phosphoprotein [83]. The N
terminus of ORF3 binds to HEV RNA and forms a complex
with the capsid protein, whereas the C terminus is multi- 4.6 Host Range and Cross-Species Infection
functional and may be involved in virion morphogenesis
and pathogenesis [62, 84]. Genetic identification of HEV strains from several ani-
HEV is thought to enter the host through the gastroin- mal species including pig [16], chicken [55], mongoose
testinal epithelial cells. HEV replication in small intestines [92], deer [93], rabbit [94, 95], rat [67, 68], and fish [70]
has been experimentally demonstrated [85, 86]. The cap- and the demonstration of cross-species infection by some
sid protein attaches to cell surface heparan sulfate proteo- animal strains of HEV [61, 96, 97] have significantly
glycans (HSPGs), specifically syndecans, in Huh7 human expanded the host range and genetic diversity of HEV
liver cells [87], although a specific cellular receptor for (Table 18.1). The strains of human HEV genotypes 1 and
HEV is unknown. The capsid protein is co-translationally 2 have a more limited host range and are restricted to
translocated across the ER membrane [88]. After uncoat- humans, whereas genotype 3 and 4 strains can infect
ing, viral genomic RNA is released to cytoplasm where across species barriers. Genotype 3 and 4 strains of swine
transcription, translation, and virus replication occur [62, HEV infected nonhuman primates [96, 97], and con-
89]. During HEV replication, an intermediate negative- versely genotypes 3 and 4 strains of human HEV infected
sense genomic RNA is produced to serve as the template pigs [98, 99]. Cross-species infection of HEV has also
for the production of positive-sense progeny viral genomic been reported in other animal species. Wistar rats were
RNA as well as the subgenomic RNA for the translation of reportedly infected by human HEV isolate [100]. Avian
ORF2 and ORF3 proteins [62, 89]. Negative-sense, repli- HEV from chickens infected turkeys but failed to infect
cative, viral RNA has been detected in the livers and gas- rhesus monkeys [101], suggesting that avian HEV may
trointestinal tissues of experimentally infected animals have a more limited host range and may not infect
[85, 86]. A conserved double stem-loop RNA structure humans. The HEV strains from rabbits infected pigs [61],
identified in the ORF1 and ORF2 junction region of HEV and genotypes 1 and 4 human HEV also reportedly
genome is critical for virus replication, and both the infected rabbits [102].
18 Hepatitis E Virus 443

Table 18.1 Animal reservoirs and host range of the hepatitis E virus (HEV)
Genotypes Natural hosts Epidemiological features in humans
Mammalian HEV
Genotype 1 Humans Epidemic outbreak
Genotype 2 Humans Epidemic outbreak
Genotype 3 Humans, domestic and wild pigs, deer, mongoose, rabbits Sporadic cases
Genotype 4 Humans, domestic and wild pigs, cattlea, sheepa Sporadic cases
Putative genotype 5 Rats Non-transmissible to humans
Putative genotype 6 Wild boars Unknown
Avian HEV
Genotype 1 Chickens (Australia, Korea) Non-transmissible to humans
Genotype 2 Chickens (USA, Canada) Non-transmissible to hum ans
Genotype 3 Chickens (Europe, China) Non-transmissible to humans
Fish HEV
Cutthroat trout virus Brown, Apache, and Gila trouts Non-transmissible to humans
a
Not independently confirmed

5 Descriptive Epidemiology the general populations in Egypt are positive for IgG anti-
HEV antibodies [20]. Interestingly, IgG anti-HEV preva-
5.1 Incidence and Prevalence lence in some industrialized countries is much higher than
expected: for example, in some regions of the United States,
The incidence of hepatitis E in the general population is up to 30 % of the blood donors were tested positive for IgG
unclear as there are few such studies to determine the disease anti-HEV antibodies [28, 30, 109].
incidence [103]. A prospective study of a cohort of 1,134
randomly selected individuals with 1,172 person-years of
follow-up in southern Bangladesh revealed that the overall 5.2 Epidemic Behavior
incidence of HEV infection was 64 per 1,000 person-years
over an 18-month period [104]. Based on surveys of clinical The sources of infection appear to be different for epidemics
diseases, the incidence of hepatitis E during outbreaks was and sporadic cases. HEV-contaminated drinking water is the
estimated at 1,400–1,650 per 100,000 population during main source for epidemics [4], which typically occur in small
large outbreaks in India and Nepal [19]. In 2005, a large out- towns and villages, refugee camps, as well as in cities of
break of 1,611 cases of hepatitis E was reported in Hyderabad, developing countries lacking modern sanitation [9, 19, 110].
India, with an attack rate of 40 per 100,000 population. A The outbreaks are often associated with inadequate water
significantly higher attack rate (203 per 100,000 population) treatment, contamination of water supplies with fecal materi-
was found in neighborhoods supplied by water lines that als, and poor sanitary conditions such as washing hands in a
crossed open sewage drains than in neighborhoods supplied group basin and storage of drinking water in large-mouthed
by non-crossing water pipes (38 in 100,000 population) vessels [105, 106, 111]. Person-to-person spread of hepatitis
[105]. Another large outbreak of 2,621 cases of acute hepati- E, although uncommon, does occur during an epidemic [112].
tis E was reported in refugee camps in Darfur, Sudan, with an The epidemic cycle is believed to be similar to hepatitis A
attack rate of 3.3 % and a case fatality rate of 1.7 % [106]. In with an occurrence of 7–10-year cycles [19]. It has been
industrialized countries, the incidence of sporadic or clus- reported that a second epidemic of hepatitis E occurred in a
tered cases of acute hepatitis E is difficult to determine but village that had experienced a prior outbreak 30 years earlier
appears to have been on the rise in recent years [107, 108]. [113], suggesting a loss of protective immunity over time in
Whether the increased incidence of sporadic cases of acute the community. Most outbreaks are associated with contami-
hepatitis E is due to increased awareness of the disease and nation of well water or leakage of untreated sewage into city
availability of diagnostic tests or due to actual increase in water treatment plants [19]. Attack rates are higher in villages
disease incidence remains debatable. The majority of the that rely on river water as compared to villages that rely on
sporadic or clustered cases are caused by zoonotic genotypes wells or ponds [114]. For villages relying on river water, the
3 and 4 strains of HEV [6, 18]. peak of epidemics is associated with the rise in the level of the
The prevalence of anti-HEV antibodies is very high in river during rainfall [19, 114]. HEV RNA has been detected
some developing countries: for example, more than 70 % of in sewage samples from an HEV-endemic region of India
444 X.-J. Meng

during a time when no HEV outbreak was being reported, 5.3 Geographic and Temporal Distribution
suggesting that HEV infection and fecal viral excretion may
be common in HEV-endemic regions throughout the year Seroepidemiological studies revealed that HEV infection is
even during nonepidemic periods. [115]. distributed worldwide [34, 117] (Fig. 18.3). Large explosive
The sources for sporadic cases of hepatitis E appear to be epidemics of hepatitis E occur primarily in the developing
contaminated animal meats, shellfish, and direct contacts countries of Asia, Africa, and Latin America [111, 118–122].
with infected animals [6, 18, 116]. In the United States, hav- Outbreaks are more common in geographic regions with hot
ing a pet in the home and consuming liver or other organ climates but are rare in temperate climates [34, 117].
meats more than once per month were significantly associ- Sporadic or clustered cases have been reported in developing
ated with increased odds of HEV seropositivity [109]. countries as well as in many industrialized countries [4]. As

Areas where >25% of

sporadic non-ABC hepatitis
is due to hepatitis E virus

Hepatitis E virus genotypes

1 1 and 4
2 1 and 2
3 3 and 4
4 1 and 3
2 and 3

Fig. 18.3 Worldwide prevalence of hepatitis E virus (HEV) (a) and the global geographic distribution of the different HEV genotypes (b)
(Reproduced with permission from Wedemeyer et al. [117] the American Gastroenterological Association)
18 Hepatitis E Virus 445

a fecal-orally transmitted disease primarily through contami- 5.5 Age, Sex, Race, and Other Factors
nated water or water supplies, the frequency of hepatitis E
outbreaks rises in seasons with monsoon rains and flooding. The highest attack rate of clinical disease is in young adults
A unique riverine ecology of HEV transmission has been of 20–29 years of age [15], although the seroprevalence of
reported in Southeast Asia [123]. Significantly higher preva- IgG anti-HEV is age dependent [130]. The seroprevalence of
lence of HEV infection has been associated with the use of IgG anti-HEV and the clinical attack rate are low in young
river water for drinking and cooking, personal washing, and children <15 years of age with the exception in epidemic set-
disposal of human excreta [123, 124]. In Indonesia, unusu- tings [4, 42, 130, 131]. In Pune, India, an HEV-endemic
ally dry weather led to decreased dilution of HEV in river region, HEV infection is rare in children and the prevalence
water and thus contributed to the risk of epidemic HEV does not peak (33–40 %) until early adulthood [130]. In
transmission. However, in Vietnam the risk of HEV infection Japan, an investigation of sera of 1,015 individuals collected
increased with river-flooding conditions and contamination in 1974, 1984, and 1994 showed that age-specific profiles of
[123, 124]. In the United States, individuals from tradition- IgG anti-HEV prevalence remained unchanged, with a peak
ally major swine states appear more likely to be seropositive at 40–49 years, suggesting ongoing silent HEV infection
for IgG anti-HEV than those from traditionally non-swine during the 20-year period studied [132] (Fig. 18.4). Age,
states: for example, subjects from Minnesota, a major swine socioeconomic status, and well water were significant inde-
state, are approximately five to six times more likely to be pendent variables for HEV infection in India [133]. In
seropositive for IgG anti-HEV than those from Alabama,
which is traditionally not a major swine state [30].
40 a Anti-HEV in men

5.4 Occupation 1974

30 1984
1994
Pigs are reservoirs for HEV [7], and therefore pig farmers,
Prevalence( % )

swine veterinarians, and other pig handlers in both developing 20

and industrialized countries have been shown to be at an
increased risk of HEV infection [28, 30]. An investigation of the
295 swine veterinarians from 8 US states with available age- 10
and geography-matched normal blood donors revealed that
swine veterinarians were 1.51 times more likely to be IgG anti-
HEV positive than normal blood donors [30]. Veterinarians who 0
reported having needle sticks while performing procedures on 40 b Anti-HEV in women
pigs were about 1.9 times more likely to be seropositive than
those who did not. Similarly, swine workers in North Carolina
30
had a 4.5-fold higher IgG anti-HEV prevalence rate (10.9 %)
than the control subjects (2.4 %) [125]. In Moldova, approxi-
Prevalence( % )

mately 51 % of pig farmers were positive for IgG anti-HEV, 20

whereas only 25 % of control subjects with no occupational
exposure to swine were seropositive. In Thailand, the preva-
lence of IgG anti-HEV in swine and poultry farmers was signifi- 10
cantly higher than that in government officers [126]. Human
populations with occupational exposure to wild animals have
0
also been found to have an increased risk of zoonotic HEV
0–9 10–19 20–29 30–39 40–49 50–59 >60
infection. For example, field workers from the Iowa Department
of Natural Resources had significantly higher seroprevalence of 1974 (23/21) (19/31) (12/38) (23/27) (39/12) (32/23) (37/12)

IgG anti-HEV than normal blood donors [127]. 1984 (21/26) (23/24) (16/23) (18/29) (27/24) (24/19) (15/35)
Sewage workers in India were found to have a signifi- 1994 (15/33) (11/39) (13/37) (8/42) (21/30) (23/27) (24/19)
cantly higher seroprevalence of IgG anti-HEV (56.5 %) than Age ranges (n: men/women)
the control subjects (19 %), and the IgG anti-HEV seroposi-
tivity significantly increased in sewage workers with >5 years Fig. 18.4 Age- and sex-specific prevalence of IgG anti-HEV at three
different times in an industrialized country. Numbers of men (a) /
in the occupation [128]. However, a cross-sectional study in
women (b) tested in each age group and year are indicated below in
Switzerland did not clearly identify sewage work as a high- parentheses (Reproduced with permission from Tanaka et al. [132]
risk occupation for HEV infection [129]. John Wiley and Sons)
446 X.-J. Meng

Pakistan, the attack rate was significantly higher in individu- 6.2 Foodborne Transmission
als 11–30 years of age (15.3 %) than in children <11 years of
age (1.4 %) [134]. In a large hepatitis E outbreak in 2004 in Sporadic and clustered cases of acute hepatitis E have been
refugee camps in Darfur, Sudan, the risk factors included age linked to the consumption of raw or undercooked pig livers
of 15–45 years (OR = 2.13) and drinking chlorinated surface and pork products. HEV RNA was detected in approximately
water (OR = 2.49) [106]. 2 % of the pig livers sold in local grocery stores in Japan [46]
No significant differences in IgG anti-HEV prevalence and 11 % in the United States [140]. The contaminating virus
between males and females have been observed [4, 42, 130, in commercial pig livers remains infectious [140]. The virus
131], although individuals with symptomatic HEV infec- sequences recovered from pig livers in grocery stores are
tion had male-to-female ratios ranging from 1:1 to 3:1 [19, closely related, or identical in some cases, to the viruses
132]. The higher rate of disease in men in certain outbreaks recovered from human hepatitis E patients [46]. Sporadic
may reflect the fact that men from rural areas worked out- cases of hepatitis E including a fulminant hepatic failure case
side the village whereas women stayed at home in local were linked to the consumption of raw or undercooked wild
villages [19, 132] (Fig. 18.4). A higher mortality of up to boar meats [141, 142]. In France, raw pig liver sausages
28 % was observed in infected pregnant females [22, 114, (figatelli) were the source for some sporadic cases of hepati-
135, 136]. Racial difference in HEV infection is not evi- tis E [44]. In Japan, a cluster of 4 cases of acute hepatitis E
dent. However, in a large seroepidemiological survey were linked to the consumption of raw deer meats in two
among US-born individuals, it was found that males, non- families [93, 143]. The viral nucleotide sequence recovered
Hispanic whites, and individuals residing in the Midwest from the leftover frozen deer meat was 99.7–100 % identical
and/or in metropolitan areas had the highest IgG anti-HEV to the viruses recovered from the four human patients [93,
seroprevalence [109]. 143, 144]. Additionally, cases of hepatitis E have been linked
to the consumption of shellfish [145, 146]. Taken together,
the available data provided compelling evidence of food-
6 Mechanisms and Routes borne transmission of HEV.
of Transmission

The main route of HEV transmission is fecal-oral. Under 6.3 Zoonotic Transmission
experimental conditions, however, infection of nonhuman
primates or pigs with HEV via oral inoculation proved to be The increased risk of HEV infection to persons in pig-
difficult [137], even though the animals could be readily handling occupations was noted above. In addition, potential
infected by HEV via the intravenous route of inoculation. transmissions of hepatitis E from a pet cat [147] and a pet pig
Chickens were successfully infected via the oral route of [148] to human owners have been reported. Zoonotic trans-
inoculation with an avian strain of HEV. Other routes such as mission of hepatitis E to individuals who consume infected
vertical, blood-borne, foodborne, and zoonotic transmissions pig and deer meats and pork products has also been docu-
have also been reported. mented [44, 93, 142].

6.1 Fecal-Oral Waterborne Transmission 6.4 Vertical Transmission

Contaminated water supplies are major sources of HEV There have been reports of HEV transmissions from the
infections. Historically, waterborne transmission is charac- mother to the fetus leading to premature birth, increased fetal
teristic of hepatitis E outbreaks in humans. Raw sewage loss and acute hepatitis in the newborns, and high neonatal
water contamination of drinking water and contaminated mortality [135, 149–151]. An investigation of 62 pregnant
well or river water used for washing and drinking purpose women with jaundice in the third trimester of pregnancy
are the main sources of HEV transmission in developing showed that vertical HEV transmission occurred in 33.3 %
countries. Zoonotically infected pigs and other animals of the cases [135]. In a study of mother-to-child HEV trans-
may excrete large amounts of HEV in feces [18]; thus, mission, 3/6 (50 %) of the cord blood samples tested were
HEV-containing animal manure and feces could contami- positive for HEV RNA [150]. Although HEV RNA has been
nate irrigation or coastal water with concomitant contami- detected in the colostrum of HEV-infected mothers, breast-
nation of produce or shellfish. Strains of HEV of both feeding appears to be safe for the infants, but transmission
human and swine origins have been detected in sewage may occur postpartum through close contact with infected
water [138, 139]. mothers [152].
18 Hepatitis E Virus 447

Although vertical HEV transmission has been reported in [4, 19]. In pigs experimentally infected with a genotype 3
patients, experimental evidence of vertical HEV transmis- human HEV [98], mildly-to-moderately enlarged hepatic
sion in animal models is still lacking. For example, infec- and mesenteric lymph nodes were observed from 7 to 55
tious HEV was detected in egg whites from eggs of chickens days postinoculation. Mild-to-moderate multifocal lympho-
experimentally infected with an avian strain of HEV, but evi- plasmacytic hepatitis and focal hepatocellular necrosis were
dence of complete vertical transmission is absent [153]. In observed. In chickens experimentally infected with an avian
another study, gilts (female pigs pregnant with their first lit- strain of HEV, gross lesions include subcapsular hemor-
ter) inoculated with a genotype 3 HEV became infected and rhages and slightly enlarged right intermediate lobe of the
shed virus in feces; however, vertical transmission was not livers. Foci of lymphocytic periphlebitis and phlebitis were
detected in the fetuses [154]. Similarly, pregnant rhesus the characteristic histological lesions in livers [166].
macaques experimentally infected with HEV failed to trans-
mit the virus to the offspring [155].
7.2 Immunity

6.5 Blood-Borne Transmission The immune response to HEV infection is characterized by a

transient appearance of IgM HEV antibodies followed by
Numerous reports have documented the transmissions of hepa- long-lasting IgG antibodies [4, 40] (Fig. 18.5). The HEV
titis E through blood transfusions in patients from both develop- capsid protein is immunogenic and induces neutralizing anti-
ing and industrialized countries [156–161]. A small proportion bodies and protective immunity. The capsid proteins of
of blood donors in Japan are viremic for HEV and thus pose a mammalian and avian HEV strains share common antigenic
potential risk for transfusion-associated hepatitis E [162, 163]. epitopes, and all HEV strains identified thus far appear to
Among the 41 blood donors with elevated ALT levels in Japan, belong to a single serotype. Protection against hepatitis E has
HEV RNA was detected in 8 serum samples (20 %) [164]. been demonstrated in passively or actively immunized cyno-
molgus monkeys [167]. It has also been demonstrated that
passive transfer of anti-HEV immunity from immunized
7 Pathogenesis and Immunity mothers to their offspring occurs both by transplacental and
lactation routes [168]. The characteristics of cell-mediated
7.1 Pathogenesis immune response against HEV infection are largely
unknown. The HEV-specific IFN-γ responses were found to
The pathogenesis of HEV is not well understood. After oral correlate strongly and significantly with the presence or
ingestion of the virus, HEV is believed to first replicate in the absence of IgG anti-HEV in experimentally infected chim-
gastrointestinal tract and then spread to its target organ, the panzees as well as in seroconverted human subjects [169].
liver, through viremia. Evidence of HEV replication in the
liver has been documented in nonhuman primates, pigs, and
chickens experimentally infected with human and swine 8 Patterns of Host Response
HEVs. After replication in the liver, the virus is released to
the gallbladder from hepatocytes and then is excreted in 8.1 Clinical Features
feces. In addition to livers, HEV replication has also been
identified in extrahepatic tissues including small intestines, The incubation period ranges from approximately 2 weeks to
colon, and hepatic and mesenteric lymph nodes [85, 86], 2 months. The clinical presentation of acute hepatitis E is
although the clinical and pathological significance of these often indistinguishable from other types of acute viral hepa-
extrahepatic sites of virus replication remains unknown. titis. Patients generally have jaundice, anorexia, dark urine,
Earlier studies in human volunteers provided some clues and hepatomegaly, and approximately 50 % of the patients
on the course of HEV infection in humans [12, 165]. Fecal also have abdominal pains and tenderness, nausea, vomiting,
virus shedding and viremia precedes the onset of clinical and and fever [4, 19]. Not all HEV-infected individuals develop
biochemical hepatitis, and virus shedding ends when the overt clinical disease, and in industrialized countries a sig-
level of serum liver enzyme returns to baseline [4, 117] nificant proportion of individuals are seropositive for HEV
(Fig. 18.5). Pregnancy increased the severity and mortality antibodies with no known history of hepatic disease [28, 30].
of the disease, although the mechanism of fulminant hepati- Hepatitis E is a dose-dependent disease: exposure to a higher
tis E during pregnancy remains unknown. Histological dose of virus may lead to manifestation of clinical symptoms
changes include focal necrosis with minimal infiltration and of hepatitis E, whereas patients exposed to a lower virus dose
moderate inflammation of Kupffer cells and leukocytes generally had only subclinical infections [4].
448 X.-J. Meng

Fig. 18.5 A diagram of a

serological, virological, and Acute self-limited infection (HEV)
clinical courses of acute and
chronic hepatitis E in humans.
+ + + + + +HEV-RNA (stool)
The detection and appearance of
viral RNA in blood and stool + + + +HEV-RNA (blood)
samples, serum IgG anti-HEV Anti-HEV lgG
antibodies, and levels of alanine
aminotransferase (ALT) levels
during the course of acute (a) or ALT
chronic (b) HEV infection are
schematically depicted
(Reproduced with permission
from Wedemeyer et al. [117] the
American Gastroenterological
Association)

Anti-HEV IgM

Weeks

b Chronic hepatitis E

+ + + + + + + + + +HEV-RNA (stool)

Anti-HEV lgG

ALT

Weeks

8.2 Hepatitis E and Pregnancy disease history, although the authors of the study concede
that the predominant HEV strain(s) in Egypt could be less
An important feature of HEV infection is the observed high virulent than those in South Asia [20]. In India, a large ret-
mortality during pregnancy, particularly in certain regions rospective study showed that HEV-related ALF was inde-
in India, where HEV infection causes severe hepatitis and pendent of the sex or the pregnancy status of the patients:
acute liver failure (ALF), leading to death in a significant case fatality was 51 % in HEV-related ALF in pregnant
proportion of infected pregnant women [22, 151]. For patients and 54.7 % in non-HEV-ALF in pregnant patients
example, it was reported that the case fatality among the [21]. Aggarwal has suggested that, in aggregate, pregnant
HEV-positive pregnant women was 27 % and that approxi- women appear to be at an increased risk of developing
mately two-thirds of the infected pregnant women had pre- HEV-associated ALF but that once ALF develops, the risk
term deliveries in India [135]. In Pakistan, the hepatitis E of death is similar in pregnant and nonpregnant women and
attack rate among the 162 pregnant females was 22 %, boys and men [151].
compared to only 11 % in nonpregnant females of child- Under experimental conditions, pregnant sows experi-
bearing age [134]. Other studies, however, question the mentally infected with a genotype 3 HEV at various stages
mortality data during pregnancy since the severity of hepa- of gestation did not develop clinical signs of hepatitis or
titis in pregnant women was found to be similar to that in elevation of liver enzymes [154]. Similarly, pregnant rhesus
nonpregnant women. For example, a study of 2,428 preg- monkeys experimentally infected with a genotype 1 HEV
nant women in Egypt did not link HEV exposure to liver did not develop more severe hepatitis than the nonpregnant
18 Hepatitis E Virus 449

monkeys [155]. The socioeconomic status, hormonal 9 Control and Prevention

changes and immunological status during pregnancy, and
the existence of coinfecting agents may explain the observed Protection of water system from fecal material contamina-
geographical difference of HEV-associated mortality during tion, practicing good hygiene, and avoiding drinking water
pregnancy. of unknown purity or consuming raw or undercooked animal
meats are important preventive measures [4–6, 18, 19, 188].
Washing hands thoroughly after handling infected animals is
8.3 Chronic Hepatitis E in also an effective measure to prevent zoonotic HEV transmis-
Immunocompromised Individuals sion [18].
A recombinant vaccine against HEV has recently been
Hepatitis E is generally regarded as a self-limiting acute viral licensed for use in China, although it is not yet available in
hepatitis that does not progress to a chronic infection [4]. other countries [8, 33]. Other experimental recombinant
However, recent studies revealed that chronic HEV infec- HEV vaccines based on the recombinant capsid protein of
tions do occur, more often than previously thought, espe- HEV also appear to be very promising [32]. The efficacies of
cially in immunocompromised individuals such as organ the recombinant HEV vaccines were 100 % for a bacteria-
transplant recipients and HIV-infected patients. For example, expressed recombinant vaccine [33] and 95.5 % for a
eight of the 14 HEV-infected patients who received organ baculovirus-expressed recombinant vaccine [32]. However,
transplants developed chronic hepatitis E with persistently the efficacies of these vaccines against the novel strains of
elevated aminotransferase levels, viremia, and chronic hepa- HEV including the emerging animal strains with zoonotic
titis lesions [170]. Numerous other studies also documented potential will need to be evaluated.
chronic hepatitis E in liver, kidney, heart, and kidney- Pegylated interferon and ribavirin have been shown to
pancreas transplant recipients [161, 170–177]. Additionally, clear HEV infection and improve liver histology in patients
chronic HEV infections have also been reported in individu- [173, 179, 189]. Ribavirin monotherapy inhibits HEV repli-
als with HIV infection [178–181]. Under experimental con- cation and induces a sustained virus inhibition in patients
ditions, prolonged viremia and fecal virus shedding were with chronic HEV infections [173, 177, 190, 191]. Pegylated-
also observed in a few animals experimentally infected by interferon-α-2a also induced a sustained virological response
HEV [99, 166, 182]. Taken together, these recent studies in an HEV-infected hemodialysis patient [189]. In addition,
indicated that acute HEV infection in immunocompromised in vitro treatment of A549 cells persistently infected with a
individuals can progress to a chronic infection and can rap- genotype 3 HEV with IFN-α for 72 h showed a dose-
idly progress into cirrhosis as well. dependent reduction in the levels of HEV RNA [192]. Other
experimental antivirals such as short hairpin RNA (shRNA)
[193], RNAi [194], and proteasome inhibitors such as
8.4 HEV Infection and Neurological MG132 [75] have also been shown to inhibit HEV replica-
Diseases tion in vitro and thus could serve as potential therapeutic
agents against HEV.
Increasing evidence indicates that neurologic disorders are
an emerging clinical manifestation of HEV infection. In a
kidney transplant patient chronically infected by HEV, 10 Unresolved Problems
peripheral nerve involvement with proximal muscular weak-
ness in the four limbs, with central nervous system involve- Hepatitis E is an important but extremely understudied dis-
ment and bilateral pyramidal syndrome, was reported [183, ease. The life cycle of HEV remains largely unknown in
184]. HEV RNA was detected in the serum and cerebrospi- large part due to the lack of a robust cell culture system for
nal fluid of the patient. In the United Kingdom and France, HEV. However, several recently identified cell lines that sup-
among the 126 patients with genotype 3 HEV infection, 7 port more efficient HEV replication should aid in future
patients (5.5 %) developed neurologic symptoms including studies of the HEV life cycle. Identification of a specific cel-
3 with inflammatory polyradiculopathy, 1 with Guillain- lular receptor for HEV will help establish a more efficient
Barré syndrome, 1 with bilateral brachial neuritis, 1 with cell culture system. The mechanisms of HEV transcription,
encephalitis, and 1 with ataxia/proximal myopathy [183]. In translation, and genome replication need to be delineated.
France, meningitis with diffuse neuralgic pain or polyra- One of the most pressing issues regarding HEV epidemiol-
diculoneuropathy was associated with HEV infection in 2 ogy is the elucidation of the now poorly understood ecology
adults [185]. Additionally, several cases of Guillain-Barré and natural history of this virus. The existence of IgG anti-
syndrome in different countries have been linked to HEV HEV in various animal species suggests that more animal
infection [186, 187]. species are likely infected by HEV. Unfortunately, the virus
450 X.-J. Meng

has been genetically identified only from a few animal spe- 11. Khuroo MS, Teli MR, Skidmore S, Sofi MA, Khuroo MI.
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171. Haagsma EB, van den Berg AP, Porte RJ, et al. Chronic hepatitis reports of ribavirin treatment for chronic hepatitis E. Ann Intern
E virus infection in liver transplant recipients. Liver Transpl. Med. 2010;153:85–9.
2008;14:547–53. 191. Gerolami R, Borentain P, Raissouni F, Motte A, Solas C, Colson
172. le Coutre P, Meisel H, Hofmann J, et al. Reactivation of hepatitis P. Treatment of severe acute hepatitis E by ribavirin. J Clin Virol.
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allogeneic stem cell transplantation. Gut. 2009;58:699–702. 192. Dong C, Zafrullah M, Mixson-Hayden T, et al. Suppression of
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tion. Gastroenterology. 2010;139:1612–8. 193. Kumar A, Panda SK, Durgapal H, Acharya SK, Rehman S, Kar
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 Influenza Viruses
19
John J. Treanor

1 Introduction in migratory waterfowl. Surveillance for these viruses in

birds, and their transmission to domestic poultry, swine,
More than 80 years after the recognition of influenza virus as other mammals, and ultimately man, has therefore become
the cause of the syndrome of influenza, or grippe, influenza an object of intense interest in recent years.
continues to be a major cause of acute respiratory illnesses. Two specific forms of influenza control approaches have
These illnesses rival acute gastroenteritis as causes of mor- been developed in the 80 years since the discovery of these
bidity and mortality throughout the world. Although the viruses, vaccines, and antiviral agents. In theory, developing
majority of cases of influenza are not identified by laboratory effective vaccines for influenza should be straightforward.
methods, a variety of direct and indirect methods have attrib- However, the same antigenic variation that circumvents nat-
uted a substantial proportion of the overall burden of acute ural immunity also has confounded efforts to develop effec-
respiratory illness directly to influenza. This burden includes tive influenza vaccines, and currently available live or
both deaths and hospitalizations, which occur most com- inactivated vaccines must be reformulated and readminis-
monly at the extremes of age and in persons with other tered annually to keep pace with these antigenic changes.
underlying heart or lung diseases and in pregnancy, as well Despite intense effort over many decades, a truly universal
as an enormous burden of transiently disabling illness in all influenza vaccine has never been successfully developed,
ages that result in substantial economic and productivity although recent progress in influenza immunology has
losses. increased optimism along these lines. Nevertheless, the cur-
Infection with influenza virus stimulates a coordinated rent vaccines do provide a substantial measure of protection
response of the innate, and cellular and humoral adaptive, and are important tools for reducing the overall disease bur-
immune response that leads to effective and long-lived resis- den of influenza.
tance to reinfection with the same strain of virus. However, The same high rate of evolution of influenza viruses has
influenza viruses uniquely subvert this immune response also complicated efforts to develop antiviral agents. Two
through rapid evolution of the viral surface glycoproteins viral proteins, the influenza A virus M2 protein and the neur-
resulting in antigenic changes that allow infection in the aminidase (NA) protein of influenza A and B viruses, are the
presence of immunity to prior strains. Relatively minor anti- targets of the current classes of antiviral agents, the
genic changes, traditionally referred to as “antigenic drift,” M2-inhibitors (amantadine and rimantadine) and
typically result in new viruses that cause the familiar sea- NA-inhibitors (zanamivir and oseltamivir), respectively.
sonal epidemics of acute influenza. In addition, influenza A Both classes of drug have been shown to reduce the duration
viruses occasionally and unpredictably undergo major of illness and viral shedding in acutely infected individuals
changes in antigenicity referred to as “antigenic shift” that and to reduce the rate of hospitalization and death when used
lead to worldwide, more severe epidemics, or pandemics of relatively early in the course of illness. However, the rapid
influenza. In many cases, it appears that pandemics viruses development of antiviral resistance has significantly impacted
emerge from the enormous gene pool of influenza A viruses the utility of these agents. At the moment, essentially all cur-
rent influenza A viruses are completely resistant to the M2
inhibitors, and there has been frequent development of resis-
tance to the NAI oseltamivir, particularly in the N1 neur-
J.J. Treanor, MD
aminidases. The use of multiple antiviral agents with
Department of Medicine, University of Rochester Medical Center,
601 Elmwood Ave, Box 689, Rochester, NY 14642, USA synergistic activities may represent a strategy to reduce the
e-mail: [email protected] emergence of resistance.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 455

DOI 10.1007/978-1-4899-7448-8_19, © Springer Science+Business Media New York 2014
456 J.J. Treanor

In this chapter, we will briefly review the history of influ- Finally, four antiviral agents in two classes have been
enza throughout. We will then describe the classification and approved for prevention and treatment of influenza. These
the basic virology of these agents and consider the data used include the so-called M2 inhibitors, amantadine in the mid-
to estimate their yearly impact on human health. We will 1960s, rimantadine in 1993, and the neuraminidase inhibi-
review the basics of the immune response to influenza and tors zanamivir and oseltamivir in 2000.
the characteristics of illness induced by these viruses and
then discuss the current approaches to prevention and
control. 3 Biological Characteristics

Influenza viruses are members of the Orthomyxoviridae

2 Historical Background family of viruses and are enveloped, single-stranded,
negative-sense RNA viruses. The viruses are further divided
The characteristics of influenza epidemics, such as the high into types A, B, and C based on substantial differences in
attack rates, explosive spread of disease, and characteristic proteins and genomic structure. Types A and B influenza are
cough and fever, have allowed identification of past influenza causes of seasonal epidemics of acute respiratory disease in
epidemic throughout history. Older studies identified proba- children, adults, and elders. Type C influenza is primarily a
ble influenza epidemics occurring at an average interval of minor cause of mild respiratory illness in children.
2.4 years between 1173 and 1875 [1]. The greatest pandemic Type A influenza viruses are further subdivided into sub-
in recorded history occurred in 1918–1919 when, during types based on antigenic and sequence differences in the two
three “waves” of influenza, 21 million deaths were recorded major surface glycoproteins: the hemagglutinin (HA) and
worldwide, among them 549,000 in the United States [2]. neuraminidase (NA). To date, a total of 16 distinct HA sub-
William Farr introduced the concept of “excess mortality” types have been identified, designated H1 to H16, and nine
in his vivid description of the London epidemic of 1847 [3]. distinct NA subtypes, N1 to N9. Recently, a new, unique
Frost [4] first used this concept in the United States to influenza virus was detected in yellow-shouldered bats in
describe the 1918 influenza epidemic, but it was Selwyn Guatemala [18]. Preliminary characterization of this virus
Collins who systematically used excess mortality as an index suggests that its HA and NA should be designated H17 and
for recognition of influenza epidemics [5, 6]. Collins esti- N10, although crystal structure suggests that the N10 neur-
mated baseline mortality by calculating weekly arithmetic aminidase does not actually have neuraminidase activity
means, and Serfling refined the baseline estimate by deriving [19].
a regressin function to describe seasonal variation in baseline While influenza A viruses of the H1N1, H2N2, and H3N2
mortality [7], the basic methodology which is still in use subtypes have caused substantial disease in humans, the nat-
today for assessing excess mortality. ural hosts of influenza A viruses are probably migratory
The first influenza virus was isolated from chickens with waterfowl, in which a genetically diverse ecology of all
fowl plague in 1901, but it was not recognized that this was known HA and NA subtypes have been found. These avian
an influenza A virus until 1955. Shope isolated the swine species thus are thought to act as a reservoir of genetic diver-
influenza virus in 1931 [8, 9]. Influenza A virus was first sity of influenza A viruses and as the source of emerging
transmitted from humans to ferrets and recognized as the pandemic influenza viruses (discussed below). Avian influ-
cause of influenza in 1933 [10]. Influenza B virus was iso- enza viruses are frequently transmitted from waterfowl to
lated by Francis in 1939 [11] and influenza C virus by Taylor domestic poultry, where they can cause widespread epidem-
in 1950 [12]. The discovery by Burnet in 1936 that influenza ics of severe or fatal disease. Transmission of influenza A
virus could be grown in embryonated hens’ eggs allowed viruses also commonly occurs to pigs, horses, mink and fer-
extensive study of the properties of the virus and the devel- rets, and marine mammals, in which the viruses can become
opment of inactivated vaccines [13]. Animal cell culture sys- established and maintained for years. Influenza in each of
tems for the growth of influenza viruses were developed in these populations is typically limited to certain subtypes,
the 1950s [14]. The phenomenon of hemagglutination, which with H1 and H3 viruses in pigs, H7 and H3 viruses in horses,
was discovered by Hirst in 1941, led to simple and inexpen- and H7 in marine mammals. Recently, influenza A has also
sive methods for the measurement of virus and specific anti- been described in felines and dogs.
body [15]. Influenza B viruses do not exhibit the same type of anti-
Evidence of the protective efficacy of inactivated vaccines genic and genetic variation in the HA and NA, and therefore
was developed in the 1940s [16]. The use of live vaccines for do not have subtypes. However, since 2001, two antigeni-
influenza was first suggested shortly after the virus was dis- cally lineages of influenza B viruses, termed the “Victoria”
covered [17], but the first live vaccine was not licensed in the lineage and the “Yamagata” lineage, have cocirculated in
United States until 2003, approximately 70 years later. humans [20]. In contrast to influenza A viruses, influenza B
19 Influenza Viruses 457

viruses appear to be limited to humans, although isolation of small amounts of M2 protein. Finally, the NA removes sialic
influenza B from seals has been described [21]. Possibly for acid from receptors on the cell surface or on the viral enve-
this reason, influenza B viruses have not been responsible for lope, allowing the progeny viruses to leave the infected cell.
pandemics of influenza.
An important feature of influenza viruses is that the
genome is segmented. Each gene segment is responsible for 4 Descriptive Epidemiology
the synthesis of one or more viral proteins. Both influenza A
and B viruses contain eight gene segments, although the spe- The epidemiology of influenza is characterized by seasonal
cific proteins assigned to each gene segment differ between epidemics of acute respiratory disease, punctuated, in the
these two types. The consequences of this are that when a case of influenza A, at random intervals by worldwide epi-
cell is infected with two different influenza viruses, the demics of varying levels of severity, referred to as pandem-
resulting progeny virus can in theory contain any combina- ics. Both of these events are felt to be driven by antigenic
tion of gene segments from the two parent viruses, meaning variation. In the case of seasonal epidemics of influenza, it is
that 256 possible combinations of genes can be derived from felt that population immunity drives the selection of muta-
reassortment event between two influenza viruses. However, tions in the immunologically critical HA and NA proteins
some combinations may not be compatible [22], and all that result in sufficient antigenic differences from previously
combinations are probably not equally likely. Genetic reas- circulating viruses to allow the selected antigenic variant to
sortment has been demonstrated to play an important role in efficiently replicate and infect individuals who have devel-
the generation of pandemic influenza A viruses and has also oped immunity to previous viruses of the same subtype.
been taken advantage of for the construction of attenuated Because these antigenic changes are relatively minor and are
live influenza vaccines. generally the result of a few mutations in critical antibody
Influenza viruses enter the cell by attachment of the viral epitopes, this process is frequently referred to as antigenic
hemagglutinin to sialic-acid-containing receptors on the cell drift.
membrane, followed by internalization of the virus into an In contrast, radical changes in the HA and NA result in
acidic endosome. In the acidic environment of the endo- the emergence of viruses that are able to spread rapidly in
some, the HA undergoes a conformational change that liber- populations with little or no effective immunity, resulting in
ates a fusion peptide and results in fusion of the viral envelope pandemics with high attack rates and, usually, high levels of
with the endosomal membrane. At the same time, the third morbidity and mortality (Fig. 19.1).
envelope protein, the M2 protein, acts as an ion channel Classically, pandemics involve the replacement of influ-
allowing H + ions to enter the virion from the endosome. enza A viruses of one subtype with influenza A viruses of a
This in turn allows the viral gene segments to leave the virion different subtype. For example, the most severe pandemic of
and enter the cytoplasm, a process known as uncoating. influenza in recently recorded history was the so-called
Viral gene segments are transported to the nucleus, where Spanish flu pandemic of 1918, due to a virus of the H1N1
the viral polymerase complex, comprised of the proteins subtype (the pandemic occurred before influenza virus was
PB1, PB2, and PA, directs the synthesis of the plus sense discovered and before the recognition of subtypes but was
messenger RNA as well as synthesis of negative-sense cop- classified as H1N1 retrospectively). Subsequent seasonal
ies that will serve as progeny genomic RNA. The polymerase epidemics of influenza were due to H1N1 viruses with vari-
proteins also may play a role in disruption of host cell protein ous degrees of antigenic change, most remarkably in 1947
synthesis. Because replication takes place in the nucleus, when a pseudopandemic was experienced due to an H1N1
some mRNA are spliced, giving rise in the case of influenza virus (A/Fort Monmouth/47) with enough antigenic change
A viruses to the M2 protein from the M gene segment, and from previous viruses that it was initially characterized as a
the NS2, or NEP (nuclear export protein) from the NS gene new influenza A virus, so-called A’ influenza [25]. H1N1
segment. Alternative start codons also give rise to a number viruses continued to cause seasonal epidemics in man until
of additional proteins from the polymerase genes of influ- 1957, when an influenza virus with an entirely new H and N
enza A viruses including PB1-F2 [23] and PA-X [24], which subtype, A/Japan/57 (H2N2), emerged to cause a second
may play roles in pathogenesis. pandemic of the twentieth century, the Asian flu. Initially,
Negative-sense daughter virion RNAs are encased in these viruses were referred to as strain A2, until the modern
nucleoprotein, associated with one copy of the polymerase typing system was developed and they were categorized as
complex and transported to the cytoplasm for assembly at H2N2 viruses. For reasons that are unclear, H1N1 viruses
the cell surface. Envelope proteins are glycosylated and ceased to circulate in man after the emergence of H2N2
transported to the cell surface. Virions bud from selected viruses.
lipid rafts at the cell surface, acquiring an envelope derived H2N2 viruses then underwent antigenic drift resulting in
from the cell membrane and decorated with HA, NA, and seasonal influenza until 1968 when these viruses were
458 J.J. Treanor

H3N2 H3N2
H2N2 (A2)
H1N1 (A0) H1N1 (A’)
H1N1 (sH1N1)
pH1N1

1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010

1918 1947 1957 1968 1977 2009

Fig. 19.1 Schematic diagram of the subtype circulation of influenza A between previous human and avian viruses (see text). In 1977 and 2009,
viruses in man. The pandemic of 1918 was caused by a virus of the novel H1N1 viruses which were antigenically related to previous H1N1
1918 subtype, which may have been introduced from an unknown ani- viruses (dotted lines) were introduced into the population
mal reservoir. Pandemics of 1957 and 1968 were reassortment events

replaced by the new subtype H3N2 viruses or the so-called from animal and human influenza viruses isolates have sug-
Hong Kong flu. This virus represented a slightly different cir- gested that the 1918 virus was introduced into humans from
cumstance in that while the HA was a new subtype, the NA such an animal population. In contrast, the 1957 and 1968
was retained from the previous H2N2 virus. Several studies pandemic influenza viruses were reassortant viruses that
have suggested that residual immunity to the retained N2 derived some genes from previously circulating human
component of the H3N2 viruses substantially ameliorated the viruses, while deriving the HA and sometimes NA genes
severity of this pandemic worldwide [26]. Again, for unclear from an avian influenza virus [28].
reasons, the emergence of H3N2 viruses coincided with the Because of the likely role of avian influenza viruses in the
disappearance of the previous H2N2 viruses, which have not generation of emerging pandemics, there has been intense
circulated in man since then. Studies of the serologic reactiv- interest in recent outbreaks involving transmission of avian
ity of banked sera (so-called seroarcheology) have suggested influenza viruses to man, with resulting disease. Most of
that an H3 virus, possibly of the H3N8 subtype, may have these transmission events have been quite limited, with small
also caused a pandemic in 1889–1891 [27]. numbers of persons affected, relatively mild disease, and
H1N1 viruses reemerged in 1977 and resulted in a rela- little or no evidence of person-to-person transmission. In
tively more mild pandemic referred to as the Russian flu (A/ most cases, virus has been transmitted to humans from
USSR/77). This virus was genetically and antigenically iden- infected domestic poultry, but cases have also occurred in
tical to influenza A viruses that had circulated in man in 1950, association with marine mammals and possibly wild birds.
and the mechanism that led to the preservation of this virus Subtype H7 viruses have been responsible for several small
over the subsequent 27 years has never been fully explained. outbreaks. Human infections with H7 AI viruses have generally
As expected, disease was largely restricted to younger per- been sporadic and mild in nature, with infected individuals pre-
sons born after 1957 who had not been previously exposed to senting with mild flu-like illness and/or conjunctivitis [29, 30].
H1N1 viruses, and the overall impact of this pandemic was Human cases have typically been associated with outbreaks in
much less severe than previous pandemics. In addition, the birds, although one case of human H7 infection was reported in
emergence of the H1N1 viruses did not result in the disap- a laboratory worker who was sneezed upon by a seal that was
pearance of the previous H3N2 virus. These H1N1 viruses infected with an H7N7 influenza virus [31]. The largest known
cocirculated with the H3N2 viruses until 2009, when a new cluster of H7N7 infections of humans occurred in 2003 in asso-
variant of H1N1 virus emerged from swine and replaced the ciation with an outbreak of highly pathogenic avian influenza in
previous H1N1, but not H3N2 viruses. commercial poultry farms in the Netherlands [32, 33]. While
almost all cases in this outbreak were mild or subclinical, there
was one confirmed fatal case in a 57-year-old otherwise healthy
4.1 Emergence of Pandemic Viruses veterinarian, who developed pneumonia [33].
from Birds A new outbreak of influenza illness due to viruses of the
H7N9 subtype has been recognized in western China since the
Extensive surveillance studies have identified influenza A spring of 2013 [34]. In contrast to previous H7 disease which
viruses of all 16 HA subtypes and all 9 NA subtypes in has been predominantly mild respiratory illness with conjunc-
migratory waterfowl. In these birds influenza A causes mild tivitis, cases in this outbreak have been more severe, with hos-
illness or may be shed asymptomatically at high levels and pitalizations and an approximately 20% case fatality rate [35].
for long duration in the feces. These birds may transmit Cases have been mostly recognized in older adults, for unknown
influenza to other animals, including domestic poultry, reasons, and fatalities have largely occurred in individuals with
horses, swine, and marine mammals, which may in turn underlying heart of lung disease , somewhat similar to seasonal
transmit these viruses to man. Comparisons of sequence data influenza [36]. Almost all cases have direct contact with poul-
19 Influenza Viruses 459

try, mostly in live bird markets. Because H7N9 viruses are not influenza A viruses from humans and from birds and have
highly lethal in poultry, outbreaks in markets are much harder always been considered to represent a potential “mixing ves-
to recognize and control, which may be contributing to the per- sel” in which reassortment between human and avian influ-
sistence of this outbreak in affected areas. enza viruses could occur. This concept was strengthened by
Subtype H9 viruses have rarely caused human disease. the recognition that the swine respiratory tract contains
H9N2 virus was isolated from two children in Hong Kong abundant receptors of both the α2-3 and α2-6 types favored
with mild febrile pharyngitis in 1999 [37]. Retrospective sero- by avian and human viruses, respectively [48].
logic cohort studies of individuals exposed to these two H9N2- In the late 1990s, one such reassortment event has been
infected children did not suggest person-to-person transmission recognized leading to a unique virus containing polymerase
[38]. Subsequently, H9N2 infection has been detected from genes of both swine and avian influenza virus origin. This
five individuals with typical influenza in China [39] and from unique combination of genes, referred to as the triple reas-
a child with a relatively severe influenza in Hong Kong. sortant cassette, apparently increased the frequency with
The greatest concern has been for H5N1 viruses, which which these viruses underwent reassortment with other vari-
were first recognized in humans in 1997 [40] and which have ants in swine populations. These viruses were also occa-
continued to cause substantial numbers of human cases since sionally transmitted to humans, typically in the context of
that time. From 2003 to October 1, 2012, a total of 608 state agricultural fairs. While most of the resulting disease
laboratory-confirmed human cases of H5N1 infection had was relatively mild, occasional severe disease and deaths
been reported to the WHO, of which 359 cases were fatal. were reported, primarily in pregnant women. However,
Cases have ranged in age from 3 months to 75 years with the person-to-person transmission was not observed.
median age being 20 years. Half of all cases have been in This situation changed in early 2009, when cases of swine-
people aged less than 20 years and 90 % of cases have been origin influenza A H1N1 viruses were first recognized in the
in those less than 40 years of age. The median duration from United States and rapidly spread throughout the world [49]. The
onset of illness to hospitalization has been 4 days (range of virus responsible for this pandemic was determined to be a qua-
0–18 days). The case fatality rates have been the highest for druple reassortant virus derived from the triple reassortant virus
those in the 10–19-year age group, lowest for people 50 or by the addition of M and NA genes from a swine virus of
older, and in between for children aged <10 years [41]. Eurasian lineage (Fig. 19.2). The exact circumstances that led to
Most cases have had close contact with ill poultry in the this event remain mysterious, but it appears that the M1 protein
week before the onset of illness. Activities like plucking and derived from the Eurasian M gene conferred on these viruses an
preparing diseased birds, playing with birds, especially enhanced ability to transmit from person to person [50].
asymptomatically infected ducks, and handling fighting The age distribution of cases in the resulting pandemic dis-
cocks are risk factors for infection [42]. Other apparent modes played the relative sparing of older adults that has been
of acquisition have included eating undercooked poultry or observed in previous pandemics and might be explained by the
drinking raw duck blood or exposure to contaminated water exposure of older adults to antigenically similar viruses in their
[43]. However, instances of person-to-person transmission childhood [51]. Thus, the bulk of the disease occurred in ado-
have been rare. Fifteen family clusters of infection involving lescents and young adults. As a result, the estimated number of
≥2 family members were documented between January 2004 excess deaths due to the pandemic in 2009 was estimated to be
and July of 2005 [44], with the largest cluster identified thus only 12,000 in the United States, which, while substantial, was
far involving seven confirmed cases in family members of a considerably less than often experienced during seasonal influ-
woman who died of an acute respiratory illness [45]. In addi- enza, especially in years predominated by H3N2 viruses.
tion, there is one well-documented transmission of virus from However, as many of these deaths occurred in young people,
an ill child in Thailand to her mother [46]. the impact on years of life lost was much greater and overall
more representative of the impact of the pandemic.
Since the emergence of the pH1N1 viruses, influenza sur-
4.2 Emergence of Pandemic Viruses veillance activities have increased their focus on domestic
from Swine swine, and a new potential pandemic threat in the form of
quadruple reassortant viruses with the same internal genes as
Domestic swine have also been recognized as a potential the pH1N1 but containing variant H3 HA genes has been
source of pandemic influenza viruses in man. Genomic data recognized [52]. Antigenically, these viruses resemble
have suggested that influenza A (H1N1) viruses were intro- human H3 viruses that circulated in the early 1970s [53].
duced into swine populations at around the same time that Approximately 300 cases of human disease have been identi-
H1N1 viruses emerged in man in 1918 [47]. Since that time, fied, almost all as the result of transmission from swine to
these H1N1 viruses, or classic swine viruses, continued to be humans during agricultural fairs. As expected, almost all of
maintained in domestic swine where they caused minor ill- these cases have occurred in children. There has been little
nesses and underwent relatively little antigenic evolution. evidence of person-to-person spread, but there remains a
During this time, swine were also occasionally infected with need for continued vigilance regarding this possibility.
460 J.J. Treanor

North American
Eurasian Pandemic H1N1 virus- "A(H1N1)pdm09"
SWine H1N1 triple reassortant virus
swine
virus (es)

PB2– Avian, North American PB2– Avian, North American

PB1– Human, Seasonal H3N2 PB1– Human, Seasonal H3N2
PA– Avian, North American PA– Avian, North American
H1 HA– Swine, North American (~1918)
HA– Swine, North American
NP– Swine, North American NP–Swine, North American
N1 NA– Swine, North American (~1918) NA– Swine, Eurasian
M (M2)– Swine, North American M (M2)– Swine, Eurasian
NS (NEP)– Swine, North American NS (NEP)– Swine, North American

PB2– Avian, North American PB2 -Avian, North American

PB1–Human, Seasonal H3N2 PB1 -Human, Seasonal H3N2
PA–Avian, North American PA-Avian, North American
H3 HA–Swine, North American (~1998) H3 HA- Swine, North American (~1998)
NP–Swine, North American NP-Swine, North American
N2 NA–Swine, North American (~1998) N2 NA- Swine, North American (~1998)
M (M2)–Swine, North American M (M2) - Swine, Eurasian
NS (NEP)–Swine, North NS (NEP)- Swine, North American
American

SWine H3N2 triple gene reassortant viruses

(1998-2011) H3N2 variant innuenza A- "A(H3N2)v"

Fig. 19.2 Current swine-origin viruses are the result of a complex series of reassortment events. Both current pH1N1 viruses and H3N2v viruses
derive the M gene from a Eurasian swine lineage

4.3 Seasonal Influenza 4.3.1 Disease Impact

Influenza epidemics are regularly associated with excess
Influenza epidemics during the interpandemic period gener- morbidity and mortality, usually expressed in the form of
ally display a marked seasonal periodicity in regions with excess rates of pneumonia- and influenza-associated (P&I)
temperate climates, with the majority of disease activity hospitalizations and deaths during epidemics. In order to
occurring between November and April in the Northern estimate the disease burden of influenza, observed P&I
Hemisphere and between May and September in the Southern events during periods of influenza epidemic activity are com-
Hemisphere (Fig. 19.3). pared with an expected seasonal baseline derived from a
Seasonal periodicity is also observed in tropical climates, time-series regression model, and the excess event rate
with increased activity during periods of low absolute humid- attributable to influenza is calculated. Because not all
ity, although influenza can occur throughout the year and influenza-related deaths are manifested as pneumonia, P&I
seasonal fluctuations are not as marked [54]. The reasons for mortality statistics may underestimate the true impact of
these seasonal changes are not entirely clear but might be the influenza on the population [57]. Although less precise, sea-
result of more favorable environmental conditions for virus sonal excess all-cause mortality is probably a more accurate
survival [55]. Studies in a model of transmission of influenza reflection of the total burden of influenza.
in guinea pigs have also supported a role for conditions of From 1979 to 1991, these methodologies have led to esti-
cold temperature and dry humidity in facilitating transmis- mated rates of influenza-associated hospitalizations ranging
sion [56]. Colder temperatures are also associated with from 55,000 to 431,000 annually, with an overall average of
behavioral changes that may increase transmission, such as 226,000 hospitalizations attributable to influenza [58].
indoor crowding or school attendance. Estimates of influenza-associated deaths have increased in
19 Influenza Viruses 461

Number of specimens positive for influenza by subtype

12000

10000

Number of specimens
8000

Northern Hemisphere 6000

4000

2000

0
45
46
47
48
49
50
51
52

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
2
3
4
5
6
7
8
9

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
2011 2012
Weeks

B (Lineage not determined) A (Not subtyped) A(H1)

B (Victoria lineage) A(H3) A(H5)
B (Yamagata lineage) A(H1N1)pdm09

1400

1200

1000
Number of specimens

Southern Hemisphere 800

600

400

200

0
45
46
47
48
49
50
51
52

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
2
3
4
5
6
7
8
9

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
2011 2012

Fig. 19.3 Representative example of the global circulation of influenza in the northern and southern hemisphere. In temperate climates, influenza
peak activity occurs in months with cooler, drier climate conditions

recent decades, possibly because of the increasing numbers respiratory secretions [61]. Influenza also results in more
of older, at-risk members of the population. Recent estimates severe disease and significant mortality in individuals with
suggest an average of approximately 8,000 excess P&I- human immunodeficiency virus (HIV) infection [62, 63].
associated deaths annually, but much higher numbers of all- The increased risk of influenza during pregnancy was dra-
cause mortality associated with influenza, with an average of matically demonstrated during the 2009 pandemic [64].
36,000 deaths annually with a range as high as 51,000 deaths Previous studies had identified an increased risk of hospital-
in the United States [59]. Generally, the level of excess mor- ization associated with influenza epidemics during preg-
tality is highest in years when influenza A (H3N2) viruses nancy, especially in the second and third trimester and in the
predominate [60] and lower in years with predominant H1N1 immediate postpartum period [65]. Several groups at
activity, possibly reflecting age-related susceptibility to these increased risk for severe influenza-related disease and deaths
two subtypes. were recognized during the 2009 H1N1 pandemic. Women
Excess morbidity and mortality are particularly high in in each stage of pregnancy, or in the immediate post partum
those with certain high-risk medical conditions, including period, were clearly over represented among those admitted
adults and children with cardiovascular and pulmonary con- to hospitals and ICUs. This observation was perhaps not sur-
ditions such as asthma, or those requiring regular medical prising as pregnancy has long been recognized as a risk fac-
care because of chronic metabolic disease, renal dysfunc- tor for influenza mortality during previous pandemics and to
tion, hemoglobinopathies, or immunodeficiency, and in indi- a lesser extent during seasonal influenza as well. The
viduals with neurologic conditions that compromise handling mechanism(s) by which pregnancy enhances the risk of
462 J.J. Treanor

120 sought was from 1.1 to 3.6 per year, depending on severity
Visits/100 of the outbreak and age of the patient. In children, outpatient
100
ARD Hospitalizations/10,000 visits are 10–250 times more common than hospitalizations
80 P&I Mortality/100,000 [72]. It is worth noting that direct medical costs of illness
account for only about 20 % of the total expenses of a case
Rate

60
of influenza, with a major proportion (30–50 %) of the eco-
40 nomic impact due to loss of productivity [74]. In one study,
influenza in schoolchildren resulted in 37 missed school
20
days by children and 20 days of missed work by parents, per
0 100 children [75]. Influenza is also associated with decreased
<5 5–9 10–14 15–19 20–24 25–34 35–44 45–54 55–64 >64
job performance in working adults [76] and reduced levels
Age group
of independent functioning in older adults [77]. Data from
Fig. 19.4 Age-related annual incidence of acute illness visits, hospital- the Tecumseh Community Health Study have been used to
izations, and deaths during the interpandemic era in Houston, Texas estimate that influenza is responsible for 13.8–16.0 million
(Data from Couch et al. [66]) excess respiratory illnesses per year in the United States
among individuals less than 20 years of age and for 4.1–4.5
influenza is not clear but might include the increased cardio- million excess illnesses in older individuals [78].
vascular demands of pregnancy as well as hormonally medi-
ated changes to the innate and adaptive immune response.
Obesity also emerged as a risk factor for influenza mor- 5 Mechanisms and Routes
bidity and mortality that had not been recognized in previous of Transmission
seasonal epidemics or pandemics. While compromise to
respiratory mechanics as a direct result of extreme obesity Influenza viruses are transmitted from person to person via
undoubtedly plays a role, there is also evidence to support a the respiratory route. Three potential modes of transmission
detrimental role of adipose tissue in the inflammatory have been suggested [79]. Coughing and sneezing could gen-
response that might also enhance the influenza disease pro- erate small particle aerosols (<10 um mass diameter) which
cess. As a result of these observations, obesity is not recog- can remain suspended in air for many hours and could trans-
nized as an important factor for targeting influenza vaccine. mit infection to individuals at a substantial difference. Larger
Influenza is usually associated with a U-shaped epidemic particles or droplets will typically fall to the ground within
curve (Fig. 19.4). Attack rates are generally highest in the 3 m of the infected person and would be expected to infect
young, whereas mortality is generally highest among older individuals in direct contact. Finally, viral particles could
adults [66, 67], in part because the prevalence of high-risk land on surfaces, where influenza viruses remain infectious
conditions is greater in this group. Influenza is also recog- [80, 81] and could infect others through indirect contact.
nized as an important health problem in young children. There is substantial evidence for all three modes of transmis-
Rates of influenza-related hospitalizations are particularly sion in experimental studies and epidemiologic observations,
high in healthy children under 2 years of age, where rates but the relative roles of each mode of transmission are uncer-
approach those of older children with high-risk conditions tain and remain controversial, with obvious implications for
[68–70]. In addition, a high rate of secondary complications, infection control practices and for potential interventions to
particularly otitis media and pneumonia, occurs in children mitigate pandemics.
with influenza infection [71]. Outpatient clinic visit rates for Small particle aerosols are generated by infected humans,
laboratory-documented influenza have been observed at and influenza genome can be detected in these small parti-
50–95 and emergency room visit rates of 6–27 per thousand cles by polymerase chain reaction techniques [82, 83]. It has
person years in children under five [72]. While rare, influenza- not been proven that these aerosols contain significant
related deaths occur each year in previously healthy children amounts of infectious virus, but experimental studies in
[61]. Notably, many of these deaths occur in children who humans have shown that vary small amounts (~5 infectious
were not recognized to have high-risk conditions prior to their particles) may be sufficient to infect humans by the aerosol
illness. route [84, 85]. Aerosol transmission has also been demon-
Much of the impact of influenza is related to the malaise strated in animal models in which infected and exposed fer-
and consequent disability that it produces, even in young, rets or guinea pigs are separated by several meters, with
healthy individuals. It has been estimated that a typical case transmission occurring in the direction of airflow [56, 86].
of influenza, on average, is associated with 5–6 days of Airborne transmission has also been implicated in multi-
restricted activity, 3–4 days of bed disability, and about 3 ple observations of outbreaks where an airborne route of
days lost from work or school [73]. The average number of transmission appears to be the most plausible explanation for
medical visits for cases in which medical attention was the characteristics of the outbreak. The most often cited such
19 Influenza Viruses 463

outbreak occurred in a commercial airliner that was delayed (IgA), by nonspecific mucoproteins to which virus may
for approximately 4½ h with a poorly functioning ventilation attach, or by the mechanical action of the mucociliary appa-
system. The risk of transmission of influenza A from the ratus. After adsorption, virus replication begins, leading to
index case to other passengers was related to the amount of cell death by inhibiting cellular protein synthesis and dis-
time passengers spent on the aircraft and not on their seating rupting other cellular functions and by inducing apoptosis.
proximity to the index case. Since most of the passengers did Virus release continues for several hours before cell death
not have direct contact with the index case, airborne transmis- ensues. Released virus then may initiate infection in adjacent
sion appears to be likely [87]. In a well-investigated hospital and nearby cells, so within a few replication cycles, a large
outbreak, nosocomial cases occurred significantly less fre- number of cells in the respiratory tract are releasing virus
quently in a hospital ward where the air was treated with UV and dying as a result of the virus replication. The time
light than in an otherwise similar ward without UV light treat- between the incubation period and the onset of illness and
ment [88]. In an outbreak in a long-term care facility, there virus shedding varies from 18 to 72 h depending in part on
appeared to be an association between the risk of nosocomial the inoculum dose [94].
influenza and the air handling systems in several wards [89]. Quantitation of virus in respiratory tract specimens from
Additional observations consistent with airborne trans- otherwise healthy young adults reveals a characteristic pat-
mission were made during an early study of zanamivir pro- tern (Fig. 19.5). Virus is first detected just before the onset of
phylaxis of influenza in families. In this study [90], subjects illness (within 24 h), rapidly rises to a peak of 3.0–7.0 log10
who received short-term prophylaxis with inhaled zanamivir TCID50/mL, remains elevated for 24–48 h, and then rapidly
were protected compared to placebo recipients, but recipi- decreases to low titers [95]. Usually, virus is no longer
ents of zanamivir administered by nasal spray were not. In detectable after 5–10 days of virus shedding. However,
addition, the combination of nasal and inhaled zanamivir because of the relative lack of immunity in the young, more
was no better than inhaled zanamivir alone. prolonged shedding of higher titers of virus is expected in
In most of these outbreaks, there are alternative explana- children.
tions for the observations that could at least partially explain fections of healthy adults, the severity of illness corre-
the epidemic behavior without requiring aerosol transmis- lates well with the quantities of virus shed, suggesting that a
sion [91], and the real role of aerosol transmission remains major mechanism in the production of illness is cell death
controversial. If aerosol transmission plays a dominant role resulting from viral replication. Although the clinical mani-
in influenza, then health-care workers would need to wear festations of influenza are dominated by systemic symp-
filtering face masks, and patients would require negative toms, viral replication is limited to the respiratory tract.
pressure isolation, to prevent nosocomial transmission of Instead, systemic symptoms are probably due to the release
influenza. This has prompted several studies that have of potent cytokines, such as type I interferons, tumor necro-
attempted to evaluate the role of facemasks in infection pre- sis factor, and interleukins (ILs), by infected cells and
vention in hospitals. In one large, randomized trial, nursing responding lymphocytes [95]. In fact it has been suggested
staff who were randomly assigned to wear N-95 respirators that an overly vigorous cytokine response to infection may
had the same rate of influenza as staff assigned to wear sim- contribute to the high fatality rate seen with H5N1 influenza
ple surgical masks while caring for patients with influenza [96, 97].
[92]. This study suggests that airborne transmission does not Bronchoscopy of individuals with typical, uncomplicated
play a major role at least in nosocomial influenza, although it acute influenza has revealed diffuse inflammation of the lar-
has been pointed out that cases in the N-95 group could have ynx, trachea, and bronchi, with mucosal injection and edema
been acquired outside the hospital and that compliance with [98, 99]. Biopsy in these cases has revealed a range of histo-
these masks is frequently poor. In contrast, hand hygiene and logic findings, from vacuolization of columnar cells with cell
simple surgical masks were reported to be modestly effective loss to extensive desquamation of the ciliated columnar epi-
in the prevention of influenza transmission in households thelium down to the basal layer of cells [99]. Individual cells
[93] suggesting that in this setting, droplet spread was the show shrinkage, pyknotic nuclei, and a loss of cilia. Viral
predominant modality. antigen can be demonstrated in epithelial cells [100].
Generally, the tissue response becomes more prominent as
one moves distally in the airway [99]. Epithelial damage is
6 Pathogenesis and Immunity accompanied by cellular infiltrates primarily composed of
lymphocytes and histiocytes. Histologic findings on autopsy
6.1 Pathogenesis in more severe cases show extensive necrotizing tracheo-
bronchitis, with ulceration and sloughing of the bronchial
Once virus is deposited on the respiratory tract epithelium, it mucosa [101], extensive hemorrhage, hyaline membrane for-
can attach to and penetrate columnar epithelial cells if not mation, and a paucity of PMN infiltration. Patients with sec-
prevented from doing so by specific secretory antibody ondary bacterial pneumonia have the changes characteristic
464 J.J. Treanor

Clinical and virologic response Nasal cytokine response

Virus titer IL-6

IFN-α
Score
TNF-α
Mucus IL-8

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Day postinoculation with A/Texas/91 (H1N1) virus

Fig. 19.5 Time course of influenza in healthy adults who were experimentally infected with influenza A/Texas/91. Symptoms are temporally
correlated with the peak of virus shedding and production of a variety of cytokines (Data adapted from Hayden et al. [95])

of bacterial pneumonia in addition to the tracheobronchial Submucosal hyperemia, focal hemorrhage, edema, and cel-
findings of influenza. Recovery is associated with rapid lular infiltrate are present. The alveolar spaces contain vary-
regeneration of the epithelial cell layer and with ing numbers of neutrophils and mononuclear cells admixed
pseudometaplasia. with fibrin and edema fluid. The alveolar capillaries may be
Abnormalities of pulmonary function are frequently dem- markedly hyperemic with intra-alveolar hemorrhage.
onstrated in otherwise healthy, nonasthmatic young adults Acellular hyaline membranes line many of the alveolar
with uncomplicated (nonpneumonic) acute influenza. ducts and alveoli [109]. Pathologic findings seen by biopsy
Demonstrated defects include diminished forced flow rates, of lung in nonfatal cases are similar to those described in
increased total pulmonary resistance, and decreased density- fatal cases [110].
dependent forced flow rates consistent with generalized Bacterial superinfection is a well-recognized complica-
increased resistance in airways less than 2 mm in diameter tion of influenza that may account for a substantial propor-
[102, 103], as well as increased responses to bronchoprovo- tion of morbidity and mortality, especially in adults. For
cation [102]. In addition, abnormalities of carbon monoxide example, the frequency of pneumococcal hospitalizations
diffusing capacity [104] and increases in the alveolar-arterial has been shown to increase in association with influenza epi-
oxygen gradient [105] have been seen. Of note, pulmonary demics [111]. Consequently, the spectrum of disease and
function defects can persist for weeks after clinical recovery. pathophysiology of bacterial superinfection has been studied
Influenza in asthmatics [106] or in patients with chronic intensively, and a number of factors have been identified in
obstructive disease [107] may result in acute declines in viral respiratory disease that could play a role in increasing
forced expiratory vital capacity (FVC) or forced expiratory the risk of bacterial infection. Uncomplicated influenza is
volume in 1 s (FEV1). Individuals with acute influenza may associated with significant abnormalities in ciliary clearance
be more susceptible to bronchoconstriction from air pollut- mechanisms [112]. In addition, increased adherence of bac-
ants such as nitrates [108]. teria to virus-infected epithelial cells has been demonstrated
Primary viral pneumonia is an uncommon but frequently [113]. The disruption of the normal epithelial cell barrier to
severe complication of acute influenza. In this situation, infection and loss of mucociliary clearance undoubtedly
virus infection reaches the lung either by contiguous spread enhance bacterial pathogenesis. In addition, influenza infec-
from the upper respiratory tract or by inhalation. The tra- tion may upregulate certain cell surface receptors involved in
chea and bronchi contain bloody fluid, and the mucosa is bacterial adherence [114]. Alterations in PMNs and mono-
hyperemic [109]. Tracheitis, bronchitis, and bronchiolitis nuclear cells may also contribute to enhanced bacterial
are seen, with loss of normal ciliated epithelial cells. infection.
19 Influenza Viruses 465

6.2 Immunity function which is directly related to binding to cellular recep-

tors. Thus, antibody that can block hemagglutination, or so-
Epidemiologic and experimental observations in humans called hemagglutination-inhibiting antibody (HAI), has been
have shown that infection with influenza virus results in studied intensively and is generally accepted as a surrogate
long-lived resistance to reinfection with the homologous for virus neutralization and protection against infection.
virus. In addition, variable degrees of cross-protection within Serum antibody to the HA has been demonstrated to have a
a subtype have been observed [115]. Infection induces both protective role against influenza infection and disease in both
systemic and local antibody, as well as cellular immune animal models as well as in experimental infection of humans
responses, each of which plays a role in recovery from infec- and in epidemiologic observations. An increased risk of
tion and resistance to reinfection (Fig. 19.6) [116]. laboratory-documented influenza among those with the low-
est titers of preexposure HAI or neutralizing antibody is a
6.2.1 Systemic Antibody Responses consistent finding of most but not all studies. However, there
Infection with influenza virus results in the development of is considerable uncertainty about the actual level of HA anti-
antibody to the influenza virus envelope glycoproteins HA body that is the best predictor of protection, with estimates
and NA, as well as to the structural M and NP proteins. ranging from HAI titers of 1:8 to 1:160 or higher [118].
Some individuals may develop antibody to the M2 protein Given the substantial variation from laboratory to laboratory
as well [117]. The antibody response is more rapid after in the estimation of the HAI titer on the same set of samples
reinfection. Peak antibody responses after primary infec- [119], the inability to use an absolute value for protection is
tion are seen at approximately 4–7 weeks and decline not unexpected. In addition, the amounts of antibody needed
slowly thereafter. Antibody titers can sometimes be to mediate protection could vary by population, degree of
detected years after exposure, for example, persons born exposure, age, and specific influenza type or subtype,
before 1968 frequently have detectable titers of antibody although this has not been analyzed comprehensively.
against H2 viruses. The mechanisms that allow such persis- B cells secreting HA-specific antibody that binds to the
tent antibody are not known. stem region rather than the head of the HA have been detected
As implied by the name, the HA protein of influenza is in the blood of individuals experiencing infections with
defined by the ability to hemagglutinate red blood cells, a novel influenza viruses such as pH1N1 or H5N1 [120, 121].

URI URI
LRI
Fever

Virus

Serum Ab
Titer

Mucosal Ab

CD8+ T cells

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 1 2 3 4 5 6 7 8

Time (days)

Primary infection Reinfection

Fig. 19.6 Immune response to influenza infection and reinfection (Adapted from Subbarao et al. [116])
466 J.J. Treanor

These antibodies exhibit neutralizing activity in some assays 6.2.3 Cellular Immune Responses
but do not inhibit hemagglutination. Stalk-binding antibody The induction of cellular immune response to influenza virus
can be detected in serum as well [122]. Because the stalk infection has been studied intensively in murine models, and
region is well conserved, these antibodies can be cross- such studies suggest that B cell, CD4+ T cell, and CD8+ T
neutralizing among HA subtypes and have increased interest cell responses all can play a role in protection against disease
in the potential creation of a universal influenza vaccine. and recovery from infection. A large number of HLA class
In contrast to anti-HA antibody, anti-NA antibody does I-restricted (CD8+ T cell) and HLA class II-restricted (CD4+
not neutralize virus infectivity but instead reduces efficient T cell) epitopes have been described, and in situations where
release of virus from infected cells, resulting in decreased those epitopes are on relatively well-conserved influenza
plaque size in in vitro assays [123] and in reductions in the proteins such as the polymerase, NP, and M proteins, the cel-
magnitude of virus shedding in infected animals [124, 125]. lular responses are cross-reactive between subtypes, but not
Observations on the relative protection of those with anti-N2 between types A and B.
antibody during the A/Hong Kong/68 (H3N2) pandemic [26, Cellular immune responses to influenza vaccination and
126], as well as experimental challenge studies in humans infection have not been studied as extensively in humans, but
[127], have shown that anti-NA antibody can also be protec- B cell (memory B cell and antibody-secreting cell), CD4+ T
tive against disease and results in decreased virus shedding cell, and CD8+ T cell responses in peripheral blood have
and severity of illness but that it is infection permissive [128]. been described after infection or vaccination [136]. It can be
Antibody to other influenza viral proteins has also been difficult to capture the peak of the response and detectable
evaluated for potential protection. Antibody to M2 reduces increases in antigen-specific cells may only be seen on a few
plaque size in vitro, and passive transfer studies in mice days after exposure. Generally, the peak cellular response
have also suggested that antibody to the M2 protein of occurs somewhere between 5 and 14 days depending on the
influenza A viruses may be partially protective if present status of the subject and the nature of the response. As seen
in large enough amounts [129]. The mechanism of protec- in murine models, a major component of the cellular response
tion in vivo is related to mediation of antibody-dependent is directed at conserved peptides to which the subject has
cytotoxicity [130]. Antibody to internal viral proteins such already been exposed during previous infections or
as M or NP is also cross-reactive among type A viruses, vaccinations.
but they are non-neutralizing. Studies in mice have sug- Antibody-secreting cells (ASC) appear in blood and ton-
gested that such non-neutralizing but cross-reactive anti- sils as early as 2 days after vaccination and are detected in
body may mediate protection under some circumstances the blood of adults and older children more frequently than
[131]. The mechanism by which antibody to viral proteins in young children after immunization [137]. An increase in
that are not exposed on the surface can mediate protection cytotoxic T lymphocytes, directed primarily at the con-
is unclear. served internal proteins, has been shown in healthy adults
with a peak at 14 and 21 days after vaccination and return to
6.2.2 Mucosal Antibody Responses baseline at 6 months. An increase in HA-specific CD8+ T
Both natural viral infection and live attenuated vaccines cells on day 7 after vaccination has also been detected by
have been found to induce significant mucosal antibody tetramer staining in adults receiving inactivated influenza
responses. Nasal HA-specific IgG is predominantly IgG1, vaccine [138].
and its levels correlate well with serum levels of HA-specific An important role of the cellular immune response in
IgG1, suggesting that nasal IgG originates by passive diffu- recovery from influenza infection in humans is strongly
sion from the systemic compartment [132]. Nasal supported by the observation of prolonged illness and viral
HA-specific IgA is predominantly polymeric and IgA1, shedding in individuals who are lymphopenic as a result of
suggesting local synthesis. Studies in mice and ferrets have disease or chemotherapy. However, it has been difficult to
emphasized the importance of local IgA antibody in resis- develop specific markers of T cell immunity as correlates of
tance to infection, particularly in protection of the upper protective immunity. Activated T cells, in the form of gran-
respiratory tract. Studies in humans have also suggested zyme B-positive T cells, have been associated with protec-
that the resistance to reinfection induced by virus infection tion in older subjects [139]. In the human challenge model,
is mediated predominantly by local HA-specific IgA, early studies identified the early induction of virus-specific
whereas that induced by parenteral immunization with cytotoxic T cells as measured by cytochrome release assays
inactivated virus depends also on systemic IgG [133]. as correlated with reductions in the duration and level of virus
Importantly, either mucosal or systemic antibody alone can replication in adults [140]. In a subsequent study done many
be protective if present in high enough concentrations, and years later by the same group, prechallenge CD4+ T cells,
optimal protection occurs when both serum and nasal anti- but not CD8+ T cells, correlated with relatively lower lev-
bodies are present [134, 135]. els of viral shedding and symptoms following experimental
19 Influenza Viruses 467

infection [141]. In a large study of the efficacy of live atten- complications include myositis and myoglobinuria [149],
uated vaccine in children, it was shown that higher levels myocarditis and pericarditis [150, 151], toxic shock syn-
of influenza-specific T cells assayed by gamma-interferon drome [152, 153], Guillain-Barré syndrome and transverse
ELISPOT was associated with a decreased risk of PCR- myelitis [154], and Reye’s syndrome particularly in children
documented influenza [142]. The development of more who have been given aspirin to treat fever.
sophisticated markers that can specifically identify reactive
cells in peripheral blood will help to define the role of cel-
lular immunity in protection, but the field remains limited 8 Control and Prevention
by the lack of convenient access to compartments other than
peripheral blood in humans. 8.1 Antiviral Drugs

Two classes of antiviral drugs have been used clinically to

7 Patterns of Host Response treat and prevent influenza. The adamantanes amantadine
and rimantadine are related primary symmetrical amines
Typical uncomplicated influenza often begins with an abrupt whose mechanism of action involves inhibition of the M2
onset of symptoms after an incubation period of 1–2 days. ion channel activity of susceptible viruses. The function of
Many patients can pinpoint the hour of onset. Initially, sys- the M2 ion channel in viral replication is to acidify the inte-
temic symptoms predominate, including feverishness, chilli- rior of the virion, disrupting the interaction between the
ness or frank shaking chills, headaches, myalgia, malaise, matrix and nucleoproteins and allowing the ribonucleopro-
and anorexia. Usually, myalgia or headache is the most trou- teins to be transported to the nucleus, where replication
blesome symptom, and the severity is related to the height of occurs [155]. Similar ion channels have been described for
the fever. Respiratory symptoms, particularly cough and sore influenza B and C viruses; however, at clinically achievable
throat, are usually also present at the onset of illness. The levels, these drugs are active against only influenza A.
predominance of systemic symptoms is a major feature dis- Amantadine and rimantadine are effective in the therapy
tinguishing influenza from other viral upper respiratory of both experimentally induced and naturally occurring
infections. Older adults may simply present with high fever, influenza A, with more rapid decrease in fever, more rapid
lassitude, and confusion without the characteristic respira- improvement in symptoms, and decreased shedding of virus
tory complaints, which may not occur at all. Generally there [156, 157]. Rimantadine has also been shown to be effective
is a wide range of symptomatology in healthy adults, ranging in the treatment of influenza A in children [158].
from classic influenza to mild illness or asymptomatic Drug resistance has been a factor in limiting the use of
infection. these antiviral agents. Resistant viruses emerge frequently in
Two manifestations of pneumonia associated with influ- treated individuals, particularly children, in whom subpopu-
enza are well recognized: primary influenza viral pneumonia lations of resistant virus can be detected following treatment
and secondary bacterial infection. The syndrome of primary in virtually all cases [159]. Resistance is the result of single
influenza viral pneumonia was first well documented in the point mutations in the membrane-spanning region of the M2
1957–1958 pandemic, predominantly among persons with protein, and it confers complete cross-resistance between
cardiovascular disease, especially rheumatic heart disease amantadine and rimantadine [160]. Resistant virus can be
with mitral stenosis, and to a lesser extent in others with transmitted to, and can cause disease in, susceptible contacts.
chronic cardiovascular and pulmonary disorders [109]. The Prolonged shedding of resistant viruses may occur in immu-
illness begins with a typical onset of influenza, followed by a nocompromised patients, particularly children, and may con-
rapid progression of fever, cough, dyspnea, and cyanosis. tinue even after therapy is terminated [161], consistent with
Secondary bacterial pneumonia classically presents after a the relative fitness of these resistant viruses. While previ-
brief period of improvement [143, 144], although most ously rare, a rapid increase in the prevalence of de novo
patients do not fit this classic pattern. Bacteria frequently resistance to M2 inhibitors was noted in 2005, and essen-
associated with influenza include Streptococcus pneumoniae tially all H3N2 viruses are now resistant to these agents [162,
or Haemophilus influenzae and, notably, an increased fre- 163]. Although previously circulating seasonal H1N1 viruses
quency of Staphylococcus aureus. An increased frequency of remained sensitive to these agents, the emerging pH1N1
community-acquired, methicillin-resistant S. aureus has viruses are also completely resistant to the M2 inhibitors,
been seen in children and adults following influenza out- which now lack activity against all strains of influenza virus
breaks [145]. currently circulating.
In addition to pneumonia, other pulmonary complications The neuraminidase inhibitors act by inhibiting the func-
of influenza include croup [146] and exacerbations of chronic tioning of the influenza virus neuraminidase, which is criti-
bronchitis or asthma [147, 148]. Recognized non-pulmonary cal in allowing newly formed viruses to egress from the cell
468 J.J. Treanor

and spread to other cells [164]. The two licensed inhibitors, virus, so-called trivalent influenza vaccine. As mentioned
zanamivir and oseltamivir, have shown very similar results in above, two antigenically distinct lineages of influenza B
clinical trials. Both drugs reduce the duration of symptoms viruses have cocirculated in humans since 2004, and quadri-
and enhance the return to normal activities when used within valent formulations of vaccine are currently being
the first 36 h of symptoms in otherwise uncomplicated influ- considered.
enza in healthy adults [165–168]. Both drugs have also been
shown to be effective in reducing the duration and severity of 8.2.1 Safety
influenza in children [169, 170]. IIV are chemically inactivated and have been administered
Antiviral resistance to these agents has also been detected either as the so-called “whole-virus” preparations or as
both in treated and untreated individuals. Mutations within detergent-disrupted and partially purified “split product” or
the catalytic framework of the NA that abolish binding of the “subunit” vaccines. Hundreds of millions of doses of IIV are
drugs have been described [171, 172]. The specific muta- administered each year, and the safety of these vaccines has
tions conferring resistance are dependent on the specific NA, been repeatedly confirmed. For example, no increase in clin-
that is, common resistance mutations in N1 (e.g., H274Y) ically important medically attended events has been noted
are different than the ones seen in the N2 (e.g., R292K or among over 251,000 children <18 years of age who were
E119V) or influenza B (e.g., D198N). In addition, depending enrolled in one of the five health maintenance organizations
on the location of the mutation, these viruses may be specifi- within the Vaccine Safety Datalink, the largest published
cally resistant to only one inhibitor [173]. Resistance muta- post-licensure population-based study of vaccine safety
tions in the NA may be associated with altered characteristics [183]. The most common adverse events reported following
of the enzyme with significantly reduced activity [174, 175]. immunization with IIV are tenderness and/or pain at the
Some resistant viruses appear to have reduced fitness, injection site. Most injection site reactions are mild and
with reduced levels of replication, attenuation in animals, rarely interfere with daily activities. Systemic reactions fol-
and reduced ability to be transmitted from animal to animal lowing immunization of adults with inactivated vaccine are
[176–179]. Drug resistant viruses were also isolated very uncommon. In placebo-controlled clinical trials in younger
infrequently from oseltamivir-treated individuals in clinical and elderly adults, rates of systemic reactions were similar
trials, being seen in less than 2 % of treated adults and among groups given inactivated vaccine or placebo [184,
detected in 5.6 % of children [169]. However, subsequent 185]. However, whole-virus IIV are associated with fever in
studies have demonstrated that resistant viruses can be children [186] and are not recommended in this age group.
detected in up to 18 % of treated children when using sensi- Recently, an increased frequency of fever and febrile sei-
tive PCR techniques to pick up minor subpopulations [180]. zures was observed among young children given one specific
Beginning in 2006, spontaneously resistant H1N1 viruses IIV during the 2010 influenza season in Australia [187]. The
carrying the H274Y mutation began to be detected in viruses reasons for this unexpected reactogenicity are unclear, but
from individuals who did not have a history of exposure to preliminary studies have suggested that this vaccine prepara-
oseltamivir [181]. By 2008, all H1N1 viruses isolated in the tion stimulated unusually high cytokine responses in in vitro
United States were resistant to oseltamivir. The mechanism assays. In addition, concomitant immunization of young
that led to the selection of seasonal H1N1 clades resistant to children with IIV and pneumococcal conjugate vaccine
oseltamivir as the predominant circulating H1N1 viruses is (PCV) was shown to be associated with an increased risk of
unclear. However, the emerging pH1N1 virus has remained developing febrile seizures.
largely susceptible to oseltamivir. Immediate hypersensitivity reactions (hives, wheezing,
angioedema, or anaphylactic shock) following inactivated
vaccine can also occur, and vaccine is considered contraindi-
8.2 Vaccines cated for persons who experienced a previous anaphylactic
reaction following vaccine. Clinical protocols have been pro-
Two types of influenza vaccines are currently licensed and posed to administer inactivated vaccine to persons who are at
are used in various countries, inactivated influenza vaccines high risk for severe or complicated influenza who also have
(IIV) and live attenuated influenza vaccines (LAIV). Shortly a history of immediate hypersensitivity to eggs, if the benefit
after these vaccines were introduced, it was recognized that of immunization is judged to outweigh the risk [188].
their efficacy might depend on the antigenic match between Several unusual syndromes have been associated with
the strain(s) contained within the vaccine and the circulating IIV. The Guillain-Barré syndrome (GBS), an acute inflam-
viruses [182], and since that time, the vaccines have been matory demyelinating polyneuropathy, was associated with
continuously reformatted to keep pace with the ongoing evo- the 1976 swine influenza vaccination campaign, with an
lution of influenza viruses. Since 1977, influenza vaccines increased risk of approximately 1 per 100,000 vaccinees.
have contained a representative A/H3N2, A/H1N1, and B More recent studies have suggested a statistically significant
19 Influenza Viruses 469

but very slight increased relative risk of GBS within 7 weeks a wide variety of nonrandomized cohort and retrospective
of influenza vaccination [189]. The oculorespiratory syn- study designs which assess vaccine effectiveness. Endpoints
drome (ORS) is a syndrome of red eyes, facial edema, and/or evaluated in these studies have included both laboratory-
respiratory symptoms such as coughing, wheezing, sore confirmed influenza and non-laboratory-confirmed respira-
throat, hoarseness, difficulty breathing, or chest tightness tory illnesses. In this regard, it has been recognized that
that develop within 2–24 h after vaccination, associated with studies that utilize a serologic definition of influenza infection
a specific influenza vaccine used in Canada, but not else- may overestimate the efficacy of influenza vaccine, since it
where [190]. The specific mechanism underlying this phe- will be harder to demonstrate postvaccination to post-season
nomenon is unknown. antibody increases in the vaccinated group [194].
Although not studied as extensively, LAIV also appear to Randomized studies of IIV efficacy against laboratory-
be quite well tolerated. Nasal symptoms (runny nose, nasal confirmed influenza have mostly been conducted in healthy
congestion, or coryza) and sore throat have been the most adults. These studies have shown a wide range of efficacy,
frequently identified adverse symptoms following LAIV. from approximately 40–80 %, with lower levels of efficacy
Children under 8 have had slightly increased rates of low- typically seen in years with apparent antigenic mismatch. A
grade fever, runny nose, and abdominal symptoms in the 7 recent meta-analysis of 8 randomized, controlled trials in
days following vaccination compared to placebo recipients. healthy adults during 2004–2008 estimated the pooled effi-
However, when considering all the pediatric studies in aggre- cacy of IIV against culture-confirmed influenza to be 59 %
gate, no consistent symptom was significantly more common (95 % CI 51–67) among those aged 18 through 64 years
in LAIV compared to placebo recipients. In older children, [195]. The role of antigenic mismatch in the efficacy
11 to <16 years of age, sore throat was observed slightly observed in these trials is unclear, and some studies in young
more frequently in LAIV recipients than IIV recipients. adults have demonstrated high levels of efficacy (76 %)
In larger studies, wheezing has been associated with despite a degree of antigenic mismatch. Recent studies using
LAIV in young children, although occurring at low rates. In virus culture and/or PCR endpoints have demonstrated simi-
the largest trial, medically significant wheezing within 42 lar levels of efficacy for both egg-grown and cell culture-
days of vaccination was reported in 3.8 % of children grown IIVs [196].
<2 years old after receipt of LAIV compared to 2.1 % in Relatively few placebo-controlled trials of the efficacy of
those who received IIV [191]. Wheezing generally occurs in LAIV have been conducted in adults. In the human challenge
the youngest, previously unvaccinated children following the model, cold-adapted and inactivated influenza vaccines were
first dose of vaccine. Because of this observation, LAIV is of approximately equal efficacy in the prevention of experi-
currently approved for use in the United States for children mentally induced influenza A (H1N1), A (H3N2), and B.
≥2 years old who do not have a history of asthma. The combined efficacy in preventing laboratory-documented
LAIV can be recovered from nasal secretions of about influenza illness due to the three wild-type influenza strains
half of adult recipients, although generally shedding of was 85 % for LAIV [197]. In a randomized, controlled study
LAIV by adults is of low titer and short duration [192]. in healthy persons aged 1 through 64 years, of whom most of
Although young children shed much higher levels of vaccine the participants were adults, the efficacy of a pre-licensure,
virus, no transmission of LAIV from vaccine recipients to bivalent preparation of LAIV for preventing culture-
susceptible contacts was detected in studies of young chil- confirmed influenza A illness in adults was 85 % (95 % CI
dren involved in day-care-like settings where LAIV and pla- 70–92 %) for H1N1 and 58 % (95 % CI 29–75 %) for H3N2
cebo recipients played together for up to 8 h a day for [198]. LAIV was also evaluated in a large study against clini-
7–10 days after vaccination. In the largest study, 197 chil- cal endpoints performed in 4,561 healthy working adults
dren between 8 and 36 months of age were randomized to [199]. In this study, the effectiveness of LAIV-T in prevent-
receive LAIV or placebo, and vaccine virus shedding was ing severe febrile respiratory illness of any cause during the
assessed for 21 days after vaccination. Although 80 % of influenza season was 29 %.
LAIV recipients shed at least one vaccine strain, for a mean Relatively few recent prospective trials have assessed IIV
of 7.6 days, clear evidence of transmission was detected in efficacy in children. In one randomized, controlled trial in
only one placebo recipient [193]. healthy children aged 6 through 23 months, vaccine efficacy
was 66 % (95 % CI 34–82) in the first year, but efficacy could
8.2.2 Efficacy and Effectiveness not be assessed in the second year due to a very low influenza
The ability of influenza vaccines to prevent influenza has attack rate (efficacy −7 %: 95 % CI −247–67) [200].
been assessed in numerous clinical studies which vary Immunization of asthmatic children has also been shown to
greatly in design, populations, and endpoints. These studies reduce the incidence of influenza. More recently, the efficacy
have included prospective, randomized controlled studies, in of IIV against PCR-confirmed influenza was assessed in a
which case they are referred to as efficacy studies, as well as randomized, placebo-controlled trial in healthy children
470 J.J. Treanor

between the ages of 6 and 72 months [201]. The efficacy of meeting a particular case definition are tested for influenza
IIV against all influenza strains was 43 % compared with the using a highly sensitive and specific diagnostic test, and the
placebo group. vaccination exposure of test-positive cases and test-negative
LAIV was demonstrated to be efficacious in the preven- controls is determined [207]. Large surveillance networks
tion of influenza in a pivotal 2-year, randomized placebo- for this purpose have been established in Canada, the United
controlled trial conducted in 1,314 children aged 15 to States, Europe, and Australia for purposes of making interim
<72 months [202]. The efficacy against culture-confirmed and end-of-season estimates of vaccine effectiveness.
influenza illness in the first year of this trial was 95 % (95 % Studies using this design have shown variable results with
C.I. 88–97 %) for influenza A/H3N2 and 91 % (95 % C.I. estimates generally ranging from as low as 20 %, or in some
79–96 %) for influenza B. In the second year of the trial, the cases, no effectiveness, to as high as 70 % [195]. While the
H3 component of the vaccine (A/Wuhan/93) was not a close various networks vary in their study design and the specific
match with the predominant H3 virus that season, A/ selection criteria for subject inclusion, a few overall general-
Sydney/95. However, the efficacy of LAIV against this vari- izations can be stated. Failure to detect VE has typically
ant was 86 % (95 % C.I. 75–92 %) [203]. Overall, the effi- occurred in studies with very low prevalence of influenza in
cacy of LAIV to prevent any influenza illness during the the study population, or in years with substantial antigenic
2-year period of surveillance in this field study was 92 % mismatch between the vaccine and circulating strains, most
(95 % C.I. 88–94 %). The overall efficacy of LAIV against often involving influenza B lineage mismatch. The relation-
culture-confirmed influenza among children 6 to <36 months ship of antigenic mismatch with vaccine effectiveness for
who were attending day care was recently shown to be 85 influenza A/H1N1 and A/H3N2 viruses is not as consistent,
and 89 % in the first and second year of the study, respec- but even in situations of antigenically matched viruses, VE
tively [204]. Significant protection against flu-associated remains in the 50–60 % range [208]. In some cases, viruses
acute otitis media also was demonstrated (>90 % in both recovered from subjects in studies with low vaccine effec-
years). Studies done in Asia have reached similar conclu- tiveness have been shown to have substantial changes on a
sions, with an efficacy of LAIV compared to placebo of HA sequence level despite appearing well matched by tradi-
between 64 and 84 % over multiple seasons, depending on tional HAI tests [209].
the antigenic match with the vaccine [205]. Most studies have not enrolled enough subjects in a single
Although annual vaccination of elders and other high-risk season to make age-specific estimates of VE. However, there
persons has been recommended for many years, there are is a trend towards decreased VE in elderly, not surprising
very few randomized trials demonstrating efficacy in these given their diminished immune response to vaccination.
groups, in part because the existing vaccine recommenda- After accumulating cases over several seasons, it was
tions make it difficult to do studies using a placebo group. In recently possible to use the same test-negative case–control
the most commonly referenced study, TIV was 52 % (95 % design to demonstrate VE of approximately 60 % against
CI 29–67) efficacious in preventing serologically docu- influenza-related hospitalization in a population of
mented influenza illness in a population of adults 60 years of community-dwelling older adults [210].
age and older [184]. When the groups were further stratified While the use of a study design in which testing is per-
by age, efficacy estimates against serologically documented formed without knowledge of vaccination status may elimi-
influenza illness were 57 % (95 % CI 33–72 %) in those 60 nate some biases related to health-care access and
through 69 years old but only 23 % (95 % CI −51–61 %) in health-seeking behavior, the results are influenced by the
those ≥70 years old. accuracy of the diagnostic testing, since errors in assignment
In a recently reported randomized, double-blind, placebo- to the case or control group will bias VE towards nil.
controlled clinical trial of LAIV among community-dwelling Recently, in a study done in children, it was demonstrated
ambulatory adults ≥65 years old, the overall efficacy of that using the test-negative case–control approach, estimates
LAIV against viruses that were antigenically similar to the of VE were substantially higher when children with docu-
vaccine was 42 % [206], similar to the protection seen with mented infections with viruses other than influenza were
inactivated vaccine. However, LAIV is not currently licensed used as a control group, rather than using all children who
for use in individuals over 49 years of age. were test negative for influenza [211].
Monitoring influenza vaccine efficacy on an annual basis A larger body of data exists from nonrandomized or obser-
by conducting randomized placebo-controlled studies would vational studies of vaccine effectiveness. These studies have
clearly be a very difficult undertaking and is probably not suggested that influenza vaccination can reduce pneumonia
possible in children, elders, and other high-risk groups. and influenza (P&I) hospitalizations and death among the
Therefore, a number of observational study designs have elderly regardless of whether they have other conditions that
been used for this purpose. Many recent studies have utilized place them at high risk for complications following influ-
a test-negative, case–control design, in which individuals enza [212]. While post-licensure observational studies are
19 Influenza Viruses 471

important tools for monitoring vaccine effectiveness, such effects of vaccine are suboptimal, particularly in some groups
studies relating to the elderly are particularly challenging to at high risk for influenza-related complications including
perform and interpret. Frailty selection bias (a higher baseline young children and elders. Thus, there has been significant
risk of hospitalization and death among unvaccinated vs. vac- interest in the potential use of adjuvants to enhance the pro-
cinated subjects) and nonspecific endpoints may overestimate tective effects of vaccination. Although influenza vaccines
vaccine effectiveness in cohort studies [213]. adsorb well to aluminum salts, these compounds have not
Infants less than 6 months of age are at substantial risk for been effective adjuvants for influenza, for unknown reasons.
influenza-related morbidity but are too young to receive Early studies suggested that water in oil emulsions using
influenza vaccine. One strategy to protect vulnerable infants mineral oil could very substantially improve antibody
is maternal immunization, with protection mediated by both responses in military recruits allowing the use of lower doses
transfer of maternal antibody and reduced potential for con- [219]. However, the use of these adjuvants was restricted by
tact with an influenza-infected mother. In a randomized substantial local side effects including the development of
study of maternal immunization, infants born to mothers sterile abscesses at the site of administration; [220] the use of
immunized with influenza vaccine had substantially lower peanut oil appeared to reduce the observed toxicity [221].
rates of laboratory-documented influenza in the first 6 Subsequently, oil-in-water emulsions based on the metab-
months of life than did infants born to mothers immunized olizable oil squalene have been shown to substantially
with pneumococcal vaccine [214]. Similarly, in a retrospec- improve immune responses with an excellent safety profile.
tive case–control study, the frequency of influenza immuni- While there is relatively little effect on the immune response
zation was substantially lower in the mothers of infants to seasonal vaccines in healthy adults [222], the oil-in-water
hospitalized with PCR-confirmed influenza than in mothers emulsion MF59 results in an approximately 50 % increase in
infants hospitalized who were PCR negative, with an esti- antibody titers in older adults [223], and MF59 adjuvanted
mated protective effect of 92 % [215]. seasonal IIV has been licensed for use in elders in Italy for
There has been considerable interest in potential strate- several years. Recently, a large randomized trial in young
gies to protect such individuals indirectly by preventing ill- children compared MF59-adjuvanted IIV with unadjuvanted
ness and viral transmission within highly susceptible IIV over two seasons. Absolute efficacy against all influenza
populations that probably play a role on community trans- strains was 86 % in the group given IIV-MF59 and 43 % in
mission, such as schoolchildren. Several studies have sug- the group given unadjuvanted IIVs when compared with the
gested that this may be possible. During the 1968 pandemic, placebo group [201].
it was observed that the incidence of respiratory illness dur- Oil-in-water emulsions have demonstrated especially
ing the period of influenza A circulation was among unvac- striking improvements in the antibody responses to candi-
cinated adults was substantially lower in a community in date H5 pandemic vaccines. These studies have shown higher
which schoolchildren were immunized than in a comparison titers of antibody against the vaccine virus, as well as against
community with no school immunization. Influenza B was antigenic variants, the development of B cells that recognize
not contained in the vaccine, and during a subsequent influ- a larger variety of HA epitopes, and broadened and more vig-
enza B epidemic, there was no difference in adjusted respira- orous CD4 T cell responses [224, 225].
tory illness rates in adults in the two communities [216]. In a
recent study, closed agricultural communities of Hutterites 8.2.4 Comparisons of Inactivated and Live
were randomized to vaccination of schoolchildren with influ- Influenza Vaccines
enza vaccine or with hepatitis A vaccine as a control. In the While relatively few randomized direct comparisons of the
subsequent influenza A epidemic, the rate of laboratory- efficacy of live and inactivated vaccines have been per-
documented influenza A in unvaccinated adults residing in formed, the available studies are consistent with the observed
school-vaccinated communities was reduced by 61 % (95 % effects of age and prior influenza experience on immunoge-
CI 8–83 %) compared to adults in unvaccinated communities nicity. When these vaccines have been compared in young
[217]. Observations in Japan, where it appeared that substan- children 12 months through 59 months of age, LAIV has
tial fluctuations in overall influenza-related mortality (occur- shown consistently superior protection, with an approxi-
ring mostly in the elderly) were directly related to the rate of mately 50 % greater protective efficacy than inactivated vac-
school-aged influenza vaccination, also support a potential cine [191, 226]. Studies conducted in healthy young children
role for school vaccination in protection of elders [218]. have generally concluded that LAIV may be more effica-
cious than IIV, including both against viruses which are well
8.2.3 Adjuvants matched antigenically to the vaccine virus and those which
For many years, licensed inactivated influenza vaccines have are antigenically drifted [191].
been administered without adjuvants. However, it is clear In contrast to young children, studies that have directly
that the immune response to vaccination and the protective compared the vaccines in adults have suggested that the
472 J.J. Treanor

vaccines have similar efficacy or that IIV vaccine is slightly use of antiviral strategies, but the basic pathogenesis of influ-
more efficacious than live vaccine. In one 3-armed study, the enza creates an extremely narrow time window for antiviral
efficacy of LAIV compared to placebo for prevention of intervention in most cases, further complicating antiviral
laboratory-confirmed influenza in healthy adults was 57 %, therapy. Novel approaches that focus on modifying the host
while the efficacy of the IIV was 77 %, but the difference or the host innate immune response may offer one alternative
between the two vaccines was not statistically significant that might overcome some of these problems.
[227]. In a subsequent season in the same population, the Although it should be recognized that the cumulative dis-
absolute efficacies of IIV and LAIV were 68 and 36 %, ease burden of seasonal influenza is generally larger than that
respectively [228]. Similar results have been reported from a of pandemic influenza, the threat of a severe influenza pan-
retrospective study evaluating the effectiveness of IIV and demic represents another unresolved issue in the field.
LAIV against medical visits for pneumonia- and influenza- Extensive efforts have been devoted to comprehensive sur-
related diagnoses in the US military [229]. Generally, rates veillance for novel influenza viruses and potential pandemic
of such visits were lower in recipients of IIV, except for per- threats in a wide variety of domestic and wild animal popula-
sonnel who had not been vaccinated in previous years, in tions, and our understanding of the ecology of influenza is
which there was not apparent difference in the effectiveness expanding rapidly. However, our ability to actually predict
of the two types of vaccines. In a recent effectiveness study the emergence of any specific pandemic remains extremely
that included both LAIV and IIV recipients, there was no limited.
clear-cut difference in effectiveness between the two vac-
cines [208].

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2011;365:1406–16. 221. Weibel RE, McLean A, Woodhour AF, Fredman A, Heilleman
202. Belshe RB, Mendelman PM, Treanor J, et al. The efficacy of live MR. Ten-year follow-up study for safety of adjuvant 65 influenza
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203. Belshe RB, Gruber WC, Mendelman PM, et al. Efficacy of vac- safety, tolerability, and immunogenicity of a MF59-adjuvanted
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influenza virus vaccine against a variant (A/Sydney) not contained adults. Vaccine. 2003;21:4234–7.
in the vaccine. J Pediatr. 2000;136:168–75. 223. Podda A. The adjuvanted influenza vaccines with novel adjuvants:
204. Vesikari T, Fleming DM, Aristegui JF, et al. Safety, efficacy, and experience with the MF59-adjuvanted vaccine. Vaccine.
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dren attending day care. Pediatrics. 2006;118:2298–312. reactive antibodies after boosting long-lived human memory B
doi:10.1542/peds.2006-0725. cells primed by an MF59 adjuvanted prepandemic vaccine. Proc
205. Tam JS, Capeding MR, Lum LC, et al. Efficacy and safety of a live Natl Acad Sci U S A. 2009;106:7962–7.
attenuated, cold-adapted influenza vaccine, trivalent against 225. Khurana S, Verma N, Yewdell JW, et al. MF59 adjuvant enhances
culture-confirmed influenza in young children in Asia. Pediatr diversity and affinity of antibody-mediated immune response to
Infect Dis J. 2007;26:619–28. pandemic influenza vaccines. Sci Transl Med. 2011;3:85ra48.
206. DeVilliers PJT, Steele AD, Hiemstra LA, et al. Efficacy and safety 226. Ashkenazi S, Vertruyen A, Aristegui J, et al. Superior relative
of a live attenuated influenza vaccine in adults 60 years of age and efficacy of live attenuated influenza vaccine compared with
older. Vaccine. 2010;28:228–34. inactivated influenza vaccine in young children with recurrent
207. Orenstein EW, de Serres G, Haber MJ, et al. Methodologic issues respiratory tract infections. Pediatr Infect Dis J. 2006;25:
regarding the use of three observational study designs to assess 870–9.
478 J.J. Treanor

227. Ohmit SE, Victor JC, Rotthoff JR, et al. Prevention of antigeni- Osterholm MT, Kelley NS, Sommer A, Belongia E. Influenza vaccine
cally drifted influenza by inactivated and live attenuated vaccines. efficacy and effectiveness: a new look at the evidence. Lancet Infect
N Engl J Med. 2006;355:2513–22. Dis. 2012;12:36–44. This thought-provoking review and meta-
228. Monto AS, Ohmit SE, Petrie JG, et al. Comparative efficacy of analysis of published studies of influenza vaccine efficacy and
inactivated and live attenuated influenza vaccines. N Engl J Med. effectiveness provides a thorough review of the challenges of influ-
2009;361:1260–7. enza vaccine.
229. Wang Z, Tobler S, Roayaei J, Eick A. Live attenuated or inacti- Poehling KA, Edwards KM, Weinberg GA, Szilagyi PG, Staat MA,
vated influenza vaccines and medical encounters for respiratory Iwane MK, Bridges CB, Grijalva CG, Zhu Y, Bernstein DI, Herrara
illnesses among US military personnel. JAMA. 2009;301: G, Erdman D, Hall CB, Seither R, Griffin MR. The underrecognized
945–53. burden of influenza in young children. N Engl J Med. 2006;355:31–
40. This clearly established the important role that influenza played
in young children and was one of the studies that encouraged cur-
rent recommendations for the vaccination of children.
Suggested Reading Thompson WW, Shay DK, Weintraub E, Brammer L, Bridges CB, Cox
NJ, Fukuda K. Influenza-associated hospitalizations in the United
Hayden FG, Fritz R, Lobo MC, Alvord W, Strober W, Straus SE. Local States. JAMA. 2004;292:1333–40.
and systemic cytokine responses during experimental human influ- Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ,
enza A virus infection. Relation to symptom formation and host Fukuda K. Mortality associated with influenza and respiratory syncytial
defense. J Clin Invest. 1998;101:643–9. Comprehensive description virus in the United States. JAMA. 2003;289:179–86. These two papers
of the clinical and innate immune response to influenza infection in comprehensively describe the methodology and the most complete esti-
the challenge model. mates of the overall disease burden of influenza in the United States.
 Noroviruses, Sapoviruses,
and Astroviruses 20
Ben A. Lopman, Jan Vinjé, and Roger I. Glass

1 Introduction where despite careful questioning, their illnesses cannot be

linked to an outbreak or to other common cases or
Acute gastroenteritis (AGE) is among the most common dis- exposures.
eases of humankind affecting people of all ages and particu- Noroviruses and sapoviruses belong to two separate gen-
larly those at the extremes of life: children and the elderly. In era of the family Caliciviridae [5]. These single-stranded
developing countries, AGE remains one of the most impor- RNA nonenveloped viruses are highly infectious agents
tant causes of death in children [1] and has been associated transmitted through a variety of routes including person-to-
with a cycle of malnutrition and problems of impaired neuro- person; contact with fecally contaminated food, water, or
cognitive development [2]. In developed countries, the dis- environmental surfaces; or possibly via airborne droplets. In
ease is an important cause of doctor visits and hospitalization the United States, noroviruses are now recognized to be the
of children, and a less frequent but persistent problem for most common cause of outbreaks of AGE, the most com-
adults and the elderly, all at appreciable medical cost [3, 4]. mon cause of sporadic AGE across all ages, and the most
Prior to 1970, an etiology could be specified for fewer than common cause of foodborne disease [6]. They are also an
15 % of episodes of AGE, but since that time, more than 50 important cause of healthcare-associated infections in both
different infectious agents and toxins have been identified to long-term and acute care settings. In addition to account-
cause the disease. This wealth of new information has chal- ing for the high burden of AGE, noroviruses are often
lenged investigators and public health professionals to detected in people without the occurrence of symptoms,
understand the relative importance of each agent, assess its particularly among people in resource-poor settings [7].
contribution to human health, and consider prospects for pre- Immunity is complex and incompletely understood but gen-
vention and control. Control has focused on understanding erally regarded to be short-lived [8]. The sapoviruses, which
routes of transmission of each pathogen and determining initially were thought to cause AGE in children, are now
whether public health interventions could interrupt their recognized to cause outbreaks in people of all age groups
spread or investigating whether natural immunity to repeat including the elderly [9]. Astroviruses are non-enveloped,
infection might provide an opportunity to consider preven- single-stranded RNA viruses in the family Astroviridae.
tion with vaccines. Many of the infectious agents of AGE Although astroviruses have been detected in all age groups,
have been discovered in epidemics where a common illness most infections are in children <2 years of age and tend to
has led to the identification of a single point source of infec- be relatively mild, rarely requiring hospitalization [10].
tion. However, patients with these infections also present Over the past two decades, this new appreciation of the
individually as “sporadic cases” in clinics and hospitals, major burden of norovirus and sapovirus AGE has been
brought about by laboratory advances in the molecular detec-
tion of these viruses and their genetic characterization. In the
B.A. Lopman, PhD (*) • J. Vinjé, PhD 1990s, the prototype Norwalk virus and its close relative,
Division of Viral Diseases, National Center for Immunization Southampton virus, were cloned and sequenced [11, 12],
and Respiratory Diseases, Centers for Disease Control
and Prevention, 1600 Clifton Road NE, Atlanta 30333, GA, USA
e-mail: [email protected]
R.I. Glass, MD
Fogarty International Center, National Institute of Health, The findings and conclusions in this report are those of the authors and
31 Center Drive, MSC 2220, Bethesda, MD, USA do not necessarily represent the views of the Centers for Disease
e-mail: [email protected] Control and Prevention.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 479

DOI 10.1007/978-1-4899-7448-8_20, © Springer Science+Business Media New York 2014
480 B.A. Lopman et al.

paving the way for the development of new molecular diag- linked to an outbreak of AGE at a school in Norwalk, Ohio,
nostic tests based upon RT-PCR detection and sequencing of which had been investigated by CDC epidemiologists 4 years
virus directly from fecal specimens [13–15]. Furthermore, earlier [18]. The discovery of the Norwalk virus opened a
expression of the capsid proteins meant that serologic assays golden era for electron microscopists to discover new viral
could be performed with antigens that could be replenished in pathogens in fecal specimens and use IEM to characterize
the laboratory [16] rather than relying on immunoassays that the patient’s immune response in acute and convalescent
required fecal specimens for antigen and paired sera collected sera. A stream of other enteric viruses were then discovered
from human volunteers. Genetic factors associated with resis- – rotaviruses, astroviruses, “classic caliciviruses,” as well as
tance to infections and acquired immunity both play a role in antigenically distinct variants of Norwalk virus, named
susceptibility to infection and disease. With this in mind, and according to the location where first identified (e.g., the
because of the great difficulty in controlling most outbreaks Hawaii agent, the Snow Mountain agent, the Sapporo virus,
through routine public health interventions, vaccines for nor- the Taunton agent) [19–21]. Other viruses were seen in fecal
ovirus are currently a priority for development. specimens in the absence of a corresponding serologic
Unfortunately, there are currently few specific interven- response, a sine qua non of a true infection – parvoviruses,
tions to prevent the spread of these viruses. Management of reoviruses, enteroviruses, and picobirnaviruses. Thus began
outbreaks is based on identifying, where possible, the means the journey to discover the relevance of each of these viruses
of spread, be it from person to person or from fecally con- to cause disease and to public health. Viruses found in stool
taminated food and water, and then intervening with control were first classified by their morphologic appearance by EM.
measures to interrupt that spread. Treatment is supportive Noroviruses were first classified simply as “small round-
and based on oral rehydration solution (ORS) or intravenous structured viruses” (SRSVs) to distinguish them from small
fluid replacement in cases of severe dehydration. Recent round viruses (SRVs) without a distinct surface structure
advances have led not only to major changes in our recogni- such as parvovirus and enterovirus. Differentiating between
tion of the importance of these viruses in human health but variant SRSVs required IEM using human paired sera speci-
also novel approaches to their control through public health mens, but few research groups had the human reagents or
measures and the potential future use of vaccines. skills needed to distinguish one SRSV from another.
Following their discovery, the Norwalk-like viruses began
to be linked to the majority of outbreaks of AGE where
another bacterial or parasitic pathogen could not be found
2 Historical Background [22]. The frequency of detection depended initially upon the
skill of the microscopist. Because no animal model for noro-
The history of noroviruses and our understanding of the virus infection was available, a long series of human volun-
viruses that cause AGE track directly with the development teer challenge studies was conducted to assess immunity to
of modern diagnostic methods to detect and characterize infection [23, 24] and cross-protection between strains [25]
these viruses. Each advance in diagnostics has furthered our and to develop novel immunoassays using fecal specimens
understanding of the epidemiology of the virus and expanded and paired acute and convalescent sera from human volun-
our appreciation of the role that these viruses play in human teers. Virus was often not seen in fecal specimens from
health. Consequently, over the past 40 years, noroviruses patients directly implicated in outbreaks. Therefore, sero-
have emerged from being an obscure and rarely detected logic assays, both radioimmunoassay (RIA) and an enzyme
viral cause of outbreaks of AGE to become the most com- immunoassay (EIA), were developed using human reagents
mon cause of these outbreaks and of sporadic episodes of (stool for antigen and paired sera) to monitor the immune
AGE in developed countries and a common pathogen in responses of those involved in outbreaks [26, 27]. At CDC,
developing countries. In the 1940s, investigators suspected for more than a decade, these assays became the diagnostic
viruses to be possible causes of AGE because volunteers tests of choice [28]. The application of serology greatly
administered bacteria-free stool filtrates from patients suffer- increased the number of outbreaks of AGE that were attrib-
ing from AGE developed the disease [17]. Unfortunately, uted to the Norwalk virus. Of note, however, was that some
they were unable to isolate a pathogen. Most episodes and people with documented exposure to the contaminated food
outbreaks of AGE, one of the most common illnesses of or water did not become ill or seroconvert, suggesting a role
humans, went without an etiologic diagnosis and were for innate or acquired immunity. Also, some people involved
defined with descriptive terms – diarrhea of weaning, travel- in outbreaks of norovirus did not seroconvert to stool filtrates
ers’ diarrhea, winter vomiting disease, or idiopathic diarrhea. from Norwalk volunteers; however, they did respond to
The first major breakthrough occurred in 1972 when human reagents developed from volunteers challenged with
Kapikian discovered a virus-like particle by immune elec- an inoculum from patients infected with antigenically dis-
tron microscopy (IEM) in the fecal specimen of victims tinct Norwalk-like viruses such as the Snow Mountain agent
20 Noroviruses, Sapoviruses, and Astroviruses 481

or the Hawaii agent suggesting serotype differences in sus- grouped into at least six different genogroups of which
ceptibility and immunity to disease [25]. A laboratory diag- viruses from GI and GII viruses being the most common [6,
nosis of norovirus was rarely sought in patients routinely 40]. GII viruses have caused the majority of all norovirus
hospitalized for diarrhea because no simple diagnostic test outbreaks over the past decade worldwide [41].
was commercially available, and few had a history of expo- Traditionally, public health laboratories studied noroviruses
sure in an outbreak. Noroviruses remained almost exclu- almost exclusively in the context of outbreaks reported to them.
sively agents of outbreaks of AGE and were not associated However, AGE is responsible for both mild disease leading to
with sporadic illness leading to hospitalization [28]. doctor and clinic visits and severe disease leading to hospital-
All this changed in the early 1990s when the Norwalk izations and some deaths [3, 42, 43]. That few of these patients
and Southampton viruses were cloned and sequenced [11, are directly linked to an outbreak suggested a role for norovi-
12, 29], an advance that revolutionized the field. These dis- ruses as a cause of sporadic AGE. A national cohort study in
coveries led immediately to the development of molecular England was the first to widely apply RT-PCR for noroviruses
assays that were more sensitive, specific, and reproducible to study patients with AGE in the community [44–46].
than electron microscopy and that used chemical reagents Norovirus was identified as the most common cause of disease
that could be produced or purchased rather than biological among adults and second only to rotavirus as the causative
reagents obtained from human volunteers. Molecular char- agent among children. A study of the etiology of AGE in adults
acterization immediately placed the Norwalk virus and the hospitalized or treated in the emergency room in three sites in
Norwalk “family of viruses,” i.e., the other SRSVs, into a the United States yielded similar results: noroviruses were the
separate genus, Norovirus, in the family Caliciviridae. The most common cause of diarrhea in US adults [47]. With the
Sapporo virus and related viruses with typical calicivirus advent and widespread use of rotavirus vaccine in the United
morphology were grouped in their own genus Sapovirus [5]. States, norovirus has become the most common cause of medi-
Knowledge of the sequence meant that individual strains cally attended gastroenteritis [48]. Application of molecular
could be identified and traced within and between outbreaks diagnostics have now demonstrated noroviruses and sapovi-
allowing for the identification of common source exposures ruses to be common in children and adults as well and a major
[30, 31]. Furthermore, strains previously characterized sim- cause of AGE in low- and middle-income countries [49].
ply by the location where they had been found, or studied for In a parallel trajectory, astroviruses were first discovered
antigenic differences by IEM, could now be sequenced and in 1975 through electron microscopic examination of stools
classified into genogroups and genotypic clusters [32]. When from an outbreak of pediatric diarrhea [50, 51]. The modern
reverse transcription polymerase chain reaction (RT-PCR) standard for laboratory diagnosis is real-time RT-PCR [52,
was applied to examine fecal specimens from a large col- 53]. In the pediatric population, astroviruses are consistently
lection of outbreaks of AGE in the United States, Europe, associated with acute diarrhea, though at substantially lower
Australia, and Japan, which had gone without an etiologic levels than the noroviruses [54].
diagnosis, more than 80 % could be attributed to a norovirus,
making it the most common cause of outbreaks of AGE [28,
33, 34]. Knowledge of sequence allowed new studies of the 3 Epidemiology
molecular epidemiology of the virus and facilitated tracing
transmission of a single strain to a common source, such as Noroviruses are now recognized to be one of the most com-
fecally contaminated shellfish or foods prepared by a single mon pathogens causing AGE in a wide range of settings,
sick food handler [30, 35, 36]. Similarly, outbreaks caused ages, and risk groups. In the United States, they are the most
by foods irrigated with sewage (e.g., raspberries transported common cause of AGE in the community, the most common
in a worldwide distribution chain) had a mixture of viruses cause of outbreaks of AGE, and the most common cause of
representing multiple sources of contamination [37, 38]. foodborne disease outbreaks [3, 55, 56]. Following the intro-
The cloning of a number of HuCVs cleared the way for duction of routine rotavirus immunization in the U.S., noro-
their single capsid protein to be expressed in different vec- viruses are now the most common cause of pediatric
tors [11, 12, 29]. When the Norwalk virus capsid gene was gastroenteritis requiring medical care [48]. They also cause
introduced into a baculovirus expression system, it formed the great majority of outbreaks of AGE in institutional set-
viruslike particles (VLPs) that were indistinguishable by EM tings such as nursing homes, hospitals, and chronic care
from natural virus [11]. VLPs could be used as antigens in facilities in industrialized countries [57]. Sapoviruses are a
immunoassays to monitor the immune response to specific less common cause of AGE in all age groups and in a wide
infections, to differentiate between strains of virus, and to variety of settings as well. For norovirus, sapovirus, and
consider as potential vaccines [39]. They also provided tar- astrovirus, our more complete appreciation of the burden of
gets to study the structure of related viruses and consider disease has developed over the last decade, largely due to the
novel targets for drugs. Noroviruses can genetically be availability of more sensitive and specific diagnostic tests.
482 B.A. Lopman et al.

3.1 Methods for Epidemiological Analysis was clear that the virus was highly infectious with primary
and secondary attack rates of 50 and 30 %, respectively [18,
3.1.1 Epidemics and Outbreak Investigation 67]. The incubation period was determined to be short at
Data from the United States and Europe have demonstrated <48 h, and occurrence in both students and teachers implied
that norovirus is responsible for approximately 50 % of all a lack of protective immunity with age. Other outbreak
reported AGE outbreaks (range 36–59 %) [58]. These gener- investigations from the 1980s demonstrated that norovirus
ally occur throughout the year, although there is a seasonal could be transmitted by drinking water [10], as well as recre-
pattern of increased activity during the winter months ational water use [68]. Transmission by contaminated oys-
(Fig. 20.1) [58]. Outbreaks occur in various settings; they are ters was demonstrated to be an important route of
propagated by nosocomial transmission in hospitals and transmission, as were a range of other foodborne exposures
nursing homes, foodborne spread in restaurants and aboard (e.g., to leafy greens, raspberries, and foods prepared by ill
cruise ships, and waterborne outbreaks, all affecting people food handlers) [69–72]. The role of norovirus in healthcare
of all ages. Although initial reviews of norovirus outbreaks facilities, including nursing homes, also becomes apparent
in the United States implicated contaminated food as the from these early investigations [73]. Outbreak investigation
main vehicle of infection [59], newer reports suggest that the led to the observation that norovirus can also be transmitted
majority involve person-to-person transmission [55, 60–62]. directly by apparent airborne or droplet spread [74–77], not
Moreover, given the high infectivity and environmental sta- only directly from person to person. More recent investiga-
bility of noroviruses, transmission during outbreaks may tions have provided compelling evidence of environmentally
involve multiple routes [63], and contaminated fomites, sur- mediated transmission [78, 79] and even by unusual fomites
faces, and droplets may also act to perpetuate outbreaks such as reusable shopping bags [80, 81]. An other outbreak
[64–66]. started from a contaminated food product, subsequently
HuCV infections often come to the attention of public spread by person to person among players on opposing foot-
health authorities in the form of outbreaks, and these events ball teams, and went on to attack the roommates of the play-
present an opportunity to learn about transmission of the ers several days later; it demonstrated the potential for
virus. In the United States, thousands of outbreaks have been multiple transmission routes in a single outbreak [82]. While
identified and investigated over the past four decades, and a astroviruses primarily cause sporadic disease, outbreaks
few have provided critical clues to understand transmission. have been reported in a range of settings and may be a fairly
The original Norwalk virus outbreak occurred in an elemen- common cause of nosocomial gastroenteritis in children’s
tary school and provided important initial observations. It hospitals [83].

500

400
Reported Outbreaks

300

200

100

0
Fig. 20.1 Suspected and
January-08

January-09

January-10
January-07

July-07

July-08

July-09

confirmed norovirus outbreaks

reported by 30 US states,
January 2007–April 2010
(Adapted from Yen et al. [55])
20 Noroviruses, Sapoviruses, and Astroviruses 483

a Europe b United States

Hospital
Other 5%
14% Other
Hospital 22%
33%
Food Outlet
6%

School/nursery
9% Food Outlet
8%
Long-term care facility
61%
Long-term care facility School/nursery
38% 4%

Fig. 20.2 Setting of reported outbreaks of norovirus AGE in (a) Europe, 2004 [33], and the (b) United States, 2006–2008 [89]. In both settings,
the majority of outbreaks occur in healthcare settings; in Europe many more outbreaks are reported from acute care hospitals

Periodically, the number of norovirus outbreaks has Outbreaks of sapovirus-associated AGE have been
increased at the same time that new genogroup II genotype 4 reported in settings similar to those of noroviruses – schools
(GII.4) strains have emerged, likely because these new vari- [95], child care facilities, [96] hospitals [97], long-term
ants escape immunity in the population [84, 85]. These care facilities, and occasional foodborne outbreaks [98,
emergent GII.4 strains rapidly replace circulating strains and 99]. The broad age range of individuals affected in these
can sometimes cause unusually severe seasons, as occurred outbreaks demonstrates that sapovirus infection is not
in 2002/2003 and 2006/2007 [41, 86, 87]. GII.4 variants pre- restricted to young children as was previously thought.
dominate across all settings, though the role of GI and other Although the molecular epidemiology of sapovirus is less
GII genotypes appears to be greater in settings that involve well studied than norovirus, strains belonging to four dif-
foodborne or waterborne transmission compared to the GII.4 ferent sapovirus genogroups (GI, GII, GIV, GV) have been
viruses [88]. observed to infect humans. Two of these viruses, GI.2 and
A major difference in the reported epidemiology of noro- GIV, have been most commonly associated with outbreaks
virus between the United States and most other high-income in Europe, Asia, and North America primarily affecting
countries lies in the frequency of outbreaks in acute care older adults [9, 100, 101].
(hospital) settings. In Europe approximately one-third of Similar to the norovirus and sapovirus, astrovirus out-
reported outbreaks occur in hospitals compared with 4 % in breaks may occur in a range of settings including long-term
the United States (Fig. 20.2) [90]. It is not known whether and acute care facilities, schools, and child care centers
this difference in reported outbreaks represents a real differ- [102]. However, such outbreaks appear to be much less com-
ence in the epidemiology of outbreaks, an underreporting of mon than those caused by norovirus.
hospital outbreaks in the United States, or differences in Few studies have quantified the direct healthcare or soci-
infection control practices in hospitals. etal costs due to the noroviruses, but given how common
Recently, more sophisticated statistical and modeling stud- these infections are, the direct healthcare costs and the indi-
ies have used data from traditional outbreak investigations to rect costs to society are likely to be substantial. Most studies
examine issues of virus transmission. Transmission models to date have only quantified the cost of outbreaks, as opposed
have been fit to demonstrate the important role that vomiting to endemic disease, for which the cost must be much greater.
plays in the spread of norovirus [91]. By using methods that For example, an outbreak in a single 946-bed US hospital
reconstruct the most likely transmission trees [92], Sukhrie cost an estimated $650,000 [103]. During the 2002–2003
and colleagues found that in healthcare settings, people with season, the cost to the English National Health Service of
symptoms are far more likely to transmit infection than those nosocomial AGE outbreaks was estimated at $184 million
who remain asymptomatic, and patients are more important to [57]. Norovirus foodborne disease in the United States costs
virus transmission than staff [93, 94]. an estimated $2 billion annually [4].
484 B.A. Lopman et al.

3.2 Surveillance understanding patterns of disease when national surveillance

systems are incomplete or do not exist [114, 115].
There are some major limitations to what can be learned
from individual outbreak investigations since published 3.2.1 Etiologic Studies and Endemic Disease
reports likely represent a biased sample of events in terms of Globally, norovirus is estimated to account for 12 % (95 %
disease severity [104, 105] and provide limited insight into CI 9–15 %) of community- or clinic-based AGE cases and
the efficacy of control measures and the full burden of dis- 11 % (95 % CI 8–14 %) of emergency department- or
ease [106]. In most countries, AGE from norovirus is not a hospital-based cases [49]. These proportions are similar in
notifiable cause of disease; surveillance has focused on the developing and developed country populations [49]. In the
recognition of outbreaks, many of which would not be United States, norovirus causes an estimated 21 million
reported in the peer-reviewed literature. What constitutes an cases of AGE [3], 1.7 million outpatient visits [116], 400,000
outbreak can be hard to define; as a result, the data collected emergency care visits, 70,000 hospitalizations [42], and 800
by any given surveillance system may in part reflect the defi- deaths annually across all age groups (Fig. 20.3) [43]. The
nitions used, the size of the outbreaks reported, and the focus burden of hospitalizations and death surges by approxi-
of the surveillance effort. For example, in the United States, mately 50 % in those years when novel GII.4 variant strains
outbreaks linked to food historically were prioritized for emerge [86, 87, 117]. Although symptomatic norovirus
reporting, whereas in England and Wales, surveillance tar- infections are usually mild and self-limiting in otherwise
geted outbreaks occurring in National Health Service hospi- healthy adults, they may be fatal among the elderly [118] and
tals, thereby emphasizing nosocomial spread [107]. When immunocompromised persons [119]. Excess mortality asso-
more broad-based assessments have been conducted, the ciated with norovirus has been documented in a number of
profile of outbreaks in the United States has been consistent countries as well [120, 121].
with those from other industrialized countries where a major- Etiologic studies of norovirus can be grouped into two
ity occur in healthcare settings and are spread by person-to- categories: (1) community-based cohort/case–control stud-
person transmission [55]. In response to this surveillance ies or (2) healthcare facility-based studies, with the former
gap, CDC has recently developed the National Outbreak being much less common. Community-based cohort studies
Reporting System (NORS) as an integrated national surveil- are expensive and logistically complicated to conduct, but
lance system for all enteric disease outbreaks [108]. when conducted, have established norovirus to be the most
Launched in February 2009, NORS now provides the frame- common cause of AGE in the community [46, 54, 122]. In
work through which all outbreaks of enteric disease, regard- England and the Netherlands, these studies have estimated
less of transmission mode, may be reported from state and the incidence of norovirus in the general population to be 4.1
local health departments to the CDC. CDC also coordinates and 4.6 cases per 100 person-years [46, 54, 122], with
a norovirus outbreak surveillance network known as regional studies providing generally consistent results [116,
CaliciNet, also launched in 2009 [109]. State and local pub- 123]. This means that with a life expectancy of ~80 years, a
lic health and food regulatory agency laboratories upload person will experience an average of three to five episodes in
sequences of norovirus outbreaks to allow rapid comparison their lifetime. Incidence is approximately five times higher
to potentially link outbreaks with a common (e.g., food) in children under the age of 5 years [46]. The incidence of
source as well as to identify emerging norovirus strains (e.g., norovirus at the general practitioner (primary care) level has
GII.4 Sydney in late 2012). been estimated at 0.49 per 100 person-years in England and
Large reviews of surveillance datasets have been pub- 0.62 in northwest Germany, suggesting about one in ten who
lished from a number of countries including England and are ill with AGE seeks medical care [46, 54, 123].
Wales, [61, 110] some European countries (as part of the Community-based studies of the incidence of sapoviruses
Foodborne Viruses in Europe group) [33, 90], Australia, and have been less common. In England and Wales, the inci-
New Zealand [111, 112]. These broad-based surveillance dence of sapovirus-associated illness in the community was
systems have consistently demonstrated that the majority of estimated at 2.6 cases per 100 person-years [54]. Estimates
reported outbreaks occur in healthcare settings (including from a study of a Healthcare Management Organization pop-
nursing homes or hospitals) and are predominantly spread ulation in the state of Georgia yielded similar results, 9 and 1
from person to person while at the same time identifying cases per 1,000 population, respectively [116]. A prospective
an important role for food in disease transmission. Systems study of Finnish children <2 years of age reported sapovi-
dedicated to the surveillance of outbreaks in healthcare set- ruses in 9 % of sporadic gastrointestinal illness compared
tings have shown a large burden of disease, in both acute with rotavirus in 29 % and norovirus in 20 % [124].
and long-term care settings, as well as a high degree of As noted earlier, astroviruses are also comparatively less
severe disease and economic burden [112, 113]. Regional common than norovirus. In the England and Wales study,
or state-wide surveillance reports have also been useful for incidence across the age range (at 0.5 per 100 person-years)
20 Noroviruses, Sapoviruses, and Astroviruses 485

Annual estimate Lifetime risk

Deaths 570–800 1 in ~5,000–7,000

Hospitalizations 56,000–71,000 1 in ~50–70

Emergency department 414,000 1 in ~9

visits

Outpatient 1.7–1.9 million 1 in ~2

visits

Cases 19–21 million ~5

Fig. 20.3 Annual cases, outpatient visits, emergency room consultations, hospitalizations, and deaths from norovirus annually in the United
States. Lifetime risk estimate based on a life expectancy of 80 years and US population of 300 million [125]

was about one-tenth and one-fifth of the incidence of norovi- young children and the elderly residing in long-term care
rus and sapovirus, respectively, in 2008/2009, while in a facilities (LTCFs) or hospitals [105, 107]. In nearly all
recent study in the United States, the classical human astro- instances of norovirus-associated death reported in the lit-
viruses caused about one-fourth of the norovirus cases [54, erature, the decedents had other serious underlying heath
126]. Astroviruses are usually detected in <10 % of young conditions, such as immunosuppression [130–133]. Data
children treated for gastroenteritis in outpatient clinics or in from passive surveillance systems and outbreak investiga-
hospitals [127, 128]. tions provide estimates of norovirus case hospitalization in
The only nationally comprehensive estimate of the burden the range of 0.1–5 hospitalizations per 1,000 cases [61,
of disease due to human caliciviruses and the associated dis- 107, 110, 112, 134]. A large systematic review of published
ability-adjusted life years (DALYs) exists for the Netherlands. outbreak reports estimated norovirus-associated hospital-
This assessment included the incidence of cases in the com- ization and mortality rates at 7 and 0.7 per 1,000 cases,
munity that did not seek healthcare, those visiting a general respectively [105]. However, severity may be overestimated
practitioner, hospitalizations and deaths that were derived when based on published outbreak reports since the larger
from cohort studies, and surveillance data and literature. In and more severe outbreaks are more likely to be investi-
total, these yielded an estimate of 1,622/100,000 (95 % CI gated and reported.
966–2,650) disability-adjusted life years in a population of It has been challenging to isolate the risk factors leading
16.5 million [129]. This burden is similar to that of disease to hospitalization and death because the mode of transmis-
due to Salmonella spp. in the same population. While sion, population affected, and genotype causing the out-
endemic disease-related costs have not been comprehen- breaks are highly correlated. Specifically, GII.4 outbreaks
sively assessed, norovirus-associated hospitalizations spe- are predominant in healthcare facility outbreaks, which
cifically have been estimated at nearly $500 million annually affect vulnerable individuals (i.e., the elderly) and are typi-
in the United States [42]. cally spread from person to person [135, 136]. A review of
over 800 outbreaks has highlighted that hospitalizations and
3.2.2 Severe Disease Outcomes deaths were much more likely to occur in healthcare out-
Although norovirus AGE is typically a mild self-resolving breaks and, somewhat surprisingly, in GII.4 virus-associated
illness in healthy adults, it can lead to severe dehydration, outbreaks, independent of those factors that could be ana-
hospitalization, and, in rare cases, death. These severe out- lyzed. This suggests that in addition to increased vulnerabil-
comes are more common in vulnerable populations such as ity of certain population groups, there is increased severity
486 B.A. Lopman et al.

associated with GII.4 viruses. GII strains are shed at higher Because humans are the only known reservoir for human
levels [137], may be more likely to induce vomiting [138], norovirus, sapoviruses, or astroviruses, in a sense, all trans-
and cause more severe disease in children; [139] this geno- mission is ultimately person to person. From that perspec-
group thus appears to demonstrate a consistent pattern of tive, foodborne, waterborne, and environmental transmission
higher virulence. The relatively high hospitalization rates in are “special cases” of person-to-person spread.
long-term care facilities and mortality in all healthcare set-
tings underscore the vulnerability of populations affected by
outbreaks in these settings. 4.1 Direct Person-to-Person Spread

3.2.3 Risk Factors Direct person-to-person transmission is believed to be the

Consistently, the strongest risk factors for community dis- primary mode of spread in most outbreaks [55, 61] and in
ease are proxies for contact with an infectious person. For sporadic disease [141, 146]. The proportion of outbreak
both young children and older children/adults, reporting a spread primarily by person-to-person transmission is highest
symptomatic household member, especially a child, is a in settings with close contacts. Direct person-to-person
strong predictor of disease [122, 123, 140, 141]. It appears transmission is reported as the primary route in >90 % of
that young children frequently bring infection into the house- norovirus outbreaks in hospitals, long-term care facilities,
hold, and older children/adults acquire many of their infec- and schools [61]. GII and, specifically, GII.4 viruses are
tions within the household [141, 142]. Foreign travel is also more commonly associated with person-to-person transmis-
a risk factor; [141, 143] the increased risk may be attribut- sion [62] or found in settings where person-to-person trans-
able to changes in behavior while traveling or exposure to a mission is common [135, 136]. Furthermore, there is a strong
different spectrum of norovirus strains. Although outbreak wintertime seasonality of person-to-person outbreaks [55,
investigations frequently attribute norovirus AGE to contam- 147–149], a pattern not clearly seen for norovirus spread by
ination of food during preparation by a range of mechanisms other routes of transmission [61]. However, when new strains
and in a range of settings, food-related risk factors have not of GII.4 emerge by escaping population immunity, aberrant
shown consistent associations with disease in community- seasonal patterns may occur, [41, 87, 150, 151] highlighting
based studies [122, 141]. In fact, consumption of raw fruits the importance of host factors (i.e., population immunity) in
and vegetables, often considered potential vehicles for trans- transmission [152]. The most consistently identified risk
mission, is generally associated with a protective effect factors for transmission are all related to the exposure of a
[141]. Other potential factors such as recreational water symptomatic contact (see Sect. 3.2.3). Although infection in
exposure and animal contact have also been associated with asymptomatic individuals is common, the importance of
reduced risk [122, 123, 141]. Taken together, the unexpected these shedders in person-to-person spread is unclear.
relationships observed in these studies suggest that these However, based on current evidence, it appears that disease
putative risk factors are correlated with other lifestyle factors symptoms, especially vomiting, are fundamental to disease
that may actually be protective against norovirus or that fre- transmission [91, 93, 94, 153].
quent exposure results in immunity. The foods consistently
associated with disease are oyster and other shellfish har-
vested from areas where the seabeds are contaminated with 4.2 Foodborne Disease
sewage [141, 144]. However, in most populations, consump-
tion of these products is not common, and this exposure Norovirus is the most common cause of foodborne disease
likely accounts for only a small fraction of disease. outbreaks in the United States [154, 155], accounting for
>50 % of foodborne disease outbreaks with known causes
reported to CDC during 2006–2008 [154, 155]. Foodborne
4 Mechanisms and Routes transmission most frequently occurs by contamination from
of Transmission infected food handlers during preparation and service. Only
a small dose of virus is needed to cause infection, and thus
Norovirus is extremely contagious, with an estimated infec- infected food handlers can contaminate large quantities of
tious dose as low as 18 viral particles [145]. In contrast, product, especially when they put their hands into large-
approximately five billion infectious doses are contained in volume liquids (e.g., salad dressing), which allow for mix-
each gram of feces during peak shedding. Humans are the ing. In one example, an estimated 3,000 cases of AGE were
only known reservoir for human norovirus infections. traced to icing prepared by an ill baker and put on a variety
Transmission occurs via fecal-oral and vomit-oral pathways of bakery products [71]. Unlike with direct person-to-person
by four general routes: direct person-to-person, foodborne, transmission, the role of both pre- and post-symptomatic
waterborne, or through environmental fomites (Fig. 20.4). shedding has been clearly linked with onward transmission
20 Noroviruses, Sapoviruses, and Astroviruses 487

Environment
Disinfection

Infected person Uninfected person

Host factors related to transmissibility Host factors related to susceptibility
-Symptomatic/asymptomatic infection -Acquired immunity
-Presence of vomiting Direct -Genetic susceptibility
-Behavior Person-to-person -FUT2 secretor status
-Hand hygiene -Behavior
-Social distancing -Hand hygiene
-Food handling

Food

Water

Fig. 20.4 Routes of transmission of norovirus from infected to unin- routes and to illustrate that all pathways require shedding of virus
fected people (Reproduced with permission from Current Opinion in from infectious hosts. Different control measures may be targeted at
Virology [7]). Norovirus transmission can occur via a range of trans- each arrow; here, the role of environmental disinfection is high-
mission routes. Characteristics and behaviors of the infected host and lighted. Certain practices (such as hand hygiene) may reduce trans-
potential susceptible individuals may mitigate the risk of transmis- mission through all pathways, while targeted interventions (such as
sion. This simple schematic is not meant to depict all the intricacies of exclusion of ill food handlers from work) may reduce transmission
each pathway but rather to highlight the interaction of the various through specific pathways

[156–158]. Foodborne astrovirus outbreaks have been asymptomatic and prolonged [164, 166, 167]), small infec-
reported, [159] though this route of transmission, relative to tious dose, [145] and widespread contamination by vomitus
person-to-person spread, is not well defined. [65, 77, 91, 164, 165, 167–169]. The viruses are relatively
Food can become contaminated with norovirus at any stable in the environment, [149, 167, 170] resistant to disin-
point along the farm-to-fork continuum, including produc- fection, [171, 172] and can contaminate a range of fomites
tion, processing, and preparation. Thus, a variety of products [65, 165, 168, 173, 174]. The most convincing evidence of
have been implicated in outbreak investigations, especially environmental transmission comes from outbreaks where
those foods irrigated with or grown in fecally contaminated groups in a common setting with no known direct contact
water and eaten raw, such as leafy vegetables, fruits, raspber- have been sequentially affected [175]. Such transmission has
ries, and shellfish [31, 108, 154, 160–163]. Because gross occurred aboard airplanes and cruise ships, [78] [63] in a
sewage contamination will contain a collection of viruses concert hall, [176] and from the use of a reusable grocery
circulating in the community, multiple norovirus genotypes bag [80]. In all these examples, the environment or fomites
are often detected in such outbreaks. likely became contaminated by airborne transmission fol-
lowing an episode of vomiting. Widespread contamination
of environments during outbreaks has been documented,
4.3 Environmental Transmission particularly in hospital settings where virus has been detected
on surfaces of many different objects – switches, televisions,
Many factors may facilitate environmental transmission of cellular phones, public phones, water taps, toilet light
norovirus. They include a large human reservoir, [3, 46, 116, switches, microwave ovens, keyboards, bed frames, and
122] high levels of shedding [164, 165] (which can be chairs [165, 168]. The role of this contamination is nevertheless
488 B.A. Lopman et al.

unclear because noroviruses are hardy: outbreak strains have icosahedral symmetry (where T is triangulation number),
been detected on environmental surfaces during non- [193] using two distinct dimer types to form the higher-order
outbreak periods, and the converse has also been observed structure [194]. Noroviruses cannot be classified by sero-
[173, 177]. Although the highest levels of contamination types, since it cannot be grown in cell culture and neutralized
probably occur on surfaces directly contaminated by vomitus with antisera, but can be divided into at least five genogroups
or feces, virus has been detected on mantle pieces and light (G), designated GI–GV, based on amino acid identity in the
fittings, located above 1.5 m in a hotel affected by an out- major structural protein (VP1) [195]. Viruses from at least
break [65]. one additional genogroup have been recognized in dogs [40,
196]. The strains that infect humans are found in genogroups
GI, GII, and GIV, whereas the strains infecting cows and
4.4 Waterborne Transmission mice are found in GIII and GV, respectively (Fig. 20.5a).
Although interspecies transmission of noroviruses has not
Sewage-contaminated water is also a recognized route of been documented, strains that infect pigs are found in GII
transmission [178–180]. Norwalk virus can remain infec- [197], and a GIV norovirus was discovered recently as a
tious in groundwater for at least 2 months and can be detected cause of diarrhea in dogs [196], suggesting the potential for
for over 3 years [170, 181]. Drinking water or ice may zoonotic transmission. On the basis of phylogenetic analysis
become contaminated with norovirus and result in outbreaks of the complete VP1, noroviruses can be further classified
in food service settings. The same contaminated water can into genotypes, with at least nine genotypes belonging to GI
cause disease directly through drinking and when used in and 21 genotypes belonging to GII (Fig. 20.5a). At least
food preparation [182]. Recreational and drinking water can since 2001, GII.4 viruses have caused the majority of viral
become contaminated from septic tank leakage and sewage AGE outbreaks worldwide [41, 198]. Recent studies have
or from breakdowns in municipal treatment plants, resulting demonstrated that these viruses evolve over time through
in large community outbreaks [183–185] [186]. However, serial changes in the VP1 sequence, which allow evasion of
outbreaks have even been reported from wells built in com- immunity in the human population [117]. Sapoviruses are
pliance with regulations when groundwater becomes con- divided into at least seven different phylogenetic clusters,
taminated by septic systems or percolation of sewage through four (GI, GII, GIV, and GV) of which include viruses that
unusual geologic formations [187]. For reasons that are not infect humans, while GIII, GVI, and GVII have only been
clear, most waterborne outbreaks are associated with GI nor- found in swine [199]. Detection of additional sapovirus
oviruses [88, 188]. However, in contrast to these epidemio- genogroups in mink and recently in bats illustrates the
logical observations, some laboratory data suggest that GII enormous genetic variability and host range of viruses within
viruses are more stable in water as well as on surfaces [181, the Sapovirus genus (Fig. 20.5b).
189]. Waterborne transmission of astrovirus has also been Astroviruses, first discovered in 1975 [200, 201], are
documented [190]. Because they often result from gross con- nonenveloped, single-stranded RNA viruses in the family
tamination, waterborne outbreaks are also more commonly Astroviridae. Astroviruses are 28 nm in diameter with a
associated with mixed infections with multiple noroviruses smooth edge and may have a characteristic 5- or 6-pointed
or even multiple pathogens [88]. starlike appearance in the center (Greek, astron = star).
Since then eight serotypes of human astrovirus (classical
human astroviruses) have been identified and character-
ized. Serotype 1 is the most common, though multiple sero-
5 Biological Characteristics types can co-circulate during the same season. Greater
serotype diversity may be found in developing countries
Noroviruses are a group of nonenveloped, single-stranded [202, 203]. In addition, viruses belonging to two other phy-
RNA viruses with an icosahedral symmetry classified into logenetic clades (MLB and VA) have been detected in
the genus Norovirus of the family Caliciviridae. Other gen- human stools, some of which has been directly linked to
era within this virus family include Sapovirus, which also AGE in children [204, 205].
causes AGE (AGE) in humans, as well as Lagovirus,
Vesivirus, and Nebovirus, which are not pathogenic for
humans [5]. Upon approval by the calicivirus study group of
International Committee on the Taxonomy of Viruses 6 Pathogenesis
(ICTV), novel caliciviruses detected in rhesus monkeys and
swine may be accepted as additional genera [191, 192]. By The primary replication site for noro- and sapoviruses has
structural analysis, the Norwalk VLP capsid is formed with not been established. It is likely that these viruses replicate
180 capsid molecules organized into 90 dimers with a T =3 in the upper intestinal tract because volunteers who develop
20 Noroviruses, Sapoviruses, and Astroviruses 489

gastrointestinal illness following oral administration of brush border enzymes (trehalase and alkaline phosphatase)
virus have histopathologic lesions on biopsies from the are significantly decreased compared with baseline and
jejunum [19, 206]. Pigs infected with porcine sapovirus convalescent-phase values [206]. It has been proposed that
demonstrate blunting and shortening of the villi of the abnormal gastric motor function is responsible for the nau-
proximal small intestine [207]. Interestingly, the same sea and vomiting associated with noroviruses [208], but the
characteristic jejunal lesion has also been observed in vol- precise mechanism responsible for illness is not known.
unteers who were fed Norwalk or Hawaii virus but did not Astrovirus infection appears to be restricted to villous
become ill [208–210]. Increased mononuclear infiltrates in enterocytes and the exposed epithelium. Histological dam-
the lamina propria and villous blunting in intestinal biop- age and inflammation is generally mild [212], and the spe-
sies of pediatric patients compared with uninfected con- cific mechanism by which astrovirus causes diarrhea is not
trols have been reported [211]. Levels of small intestinal known [102, 213].

GV.1
Genetic classification of noroviruses

GV:Murine
GII: Human and Swine

GII.18 GII.19
GII.2
GII.5
GII.6 GII.11
GII.10
GII.16 GII.3
GII.15
GII.1 GI.6
GII.12
GII.19 GI.1
GII.13
GII.17 GI.2
GI.4
GI.5
GII.8 GI.3
GI.9
GII.9
GII.14
GII.7 GI.8

GI.7 GI: Human

GII.20
GII.4

GIII: Bovine
GVI.2 GVI.1
GIV.1
GIV.2 GIII.2 GIII.1
GVI: Canine GIII.3
0.1
GIV: Human and Lion
Updated July 2012

Fig. 20.5 Genetic classification of (a) noroviruses and (b) sapovi- different genogroups (GI–VII) with several additional viruses (mink
ruses based on phylogenetic analysis of sequences of the major cap- sapovirus and bat sapovirus) that are tentative new genogroups.
sid protein VP1. Noroviruses can be grouped into six different Sapoviruses in GI (5 genotypes and 1 unassigned), GII (3 genotypes
genogroups (GI–VI) of which GI (9 genotypes), GII (18 of the 21 and 2 unassigned), GIV (1 genotype), and GV (1 genotype) can infect
genotypes), and GIV (1 of the 2 genotypes) can infect humans. humans. The scale bar of 0.1 reflects the number of amino substitu-
Strains belonging to GIII, GV, and GVI infect bovine, murine, and tions per site (Data analysis and graphs were developed by Everardo
canine species. (b) Sapoviruses can be grouped into at least seven Vega, CDC)
490 B.A. Lopman et al.

Genetic classification of sapovirus GVI.1

GVI: Porcine

Mink sapovirus

GV: Human/Porcine/Pinniped

UA
UA GV.1

GIII: Porcine
GI: Human

GI.2 GI.4
GI.5

GI.3 Bat sapovirus

UA
GI.1
GII.3
UA
GII: Human
UA GII.1 GII.2

GVII: Porcine
Canine sapovirus
GVII.1 0.1
GIV: Human
Updated July 2012

Fig. 20.5 (continued)

7 Patterns of Host Response demonstrated that homologous antibody protection might

last anywhere from 8 weeks to 6 months [23, 216]. Because
Protective immunity to norovirus appears to be different preexisting antibodies among challenged volunteers did not
from other enteric viral pathogens and is incompletely under- confer immunity in all individuals and because some persons
stood. Although seroepidemiological studies have shown an remained uninfected despite significant exposure, both
antibody prevalence to norovirus of >80 % by age 20, adults innate host factors and acquired immunity have been hypoth-
consistently demonstrate a high degree of susceptibility to esized to contribute to the susceptibility to infection [23].
both naturally occurring and experimentally administered However, the infectious dose of virus given to volunteers in
noroviruses. In a classic study from the 1970s, 50 % of adult these challenge studies was several thousand-fold greater
volunteers infected with Norwalk virus consistently devel- than the small dose of virus capable of causing human ill-
oped illness following challenge [25, 214]. In human chal- ness, and thus immunity to a lower, more natural challenge
lenge studies, infected volunteers were susceptible to dose might be greater and more cross-protective. A mathe-
reinfection with the same strain as well as to infection with matical modeling study suggests the duration of protective
heterologous strains [23, 25, 215, 216]. In addition, individu- immunity may be on the order of 4 to 8 years [217].
als with preexisting antibodies were not protected from Histo-blood group antigens (HBGAs), including H type,
infection unless repeated exposure to the same strain ABO blood group, and Lewis antigens, are a diverse family
occurred within a short period. Two of these studies of carbohydrates expressed on mucosal surfaces. Several
20 Noroviruses, Sapoviruses, and Astroviruses 491

studies indicate that they act as putative receptors or co- serum IFN-γ and IL-2, but not IL-6 or IL-10, on day two after
receptors for noroviruses. Although there is no evidence that challenge [224]. Interestingly, in an in vitro study using a
binding to HBGA is mediating viral entry, there is a strong Norwalk virus replicon-bearing cells, IFN-α efficiently
correlation between polymorphic expression of HBGA and cleared the NV replicon in a dose-dependent manner at com-
human susceptibility to norovirus infection [8]. Genetic parable levels to hepatitis C virus, indicating a potential thera-
resistance to norovirus infections has been associated with peutic application of IFN to norovirus infection [230].
mutations in the FUT2 (or “secretor”) gene, which encodes Because diarrheal disease caused by astrovirus is largely
an α1,2-fucosyltransferase; in the homozygous state, null restricted to children, immunity is believed to be long last-
mutant alleles (FUT2−/−) lead to the absence of the α1,2- ing; however, little is known regarding the specific immune
linked fucose residue on the H-type carbohydrate structures, responses that result in immunity to astroviruses. CD4+ T
which characterize the so-called non-secretor phenotype cells may be involved in the anti-astrovirus response [231]
[218]. The most frequent null allele (se428) is characterized and animal models point to a possible role for the innate
by the 428G > A nonsense mutation, and homozygous non- immune system [232].
secretors (FUT2−/−) represent up to 20 % of the European
population [219]. Results from two landmark studies of
human volunteers who were challenged with Norwalk virus 8 Control and Prevention
demonstrated that non-secretors were not infected by the
virus and none of the volunteers showed an increase in anti- Efforts to prevent norovirus and sapovirus disease are
Norwalk virus antibodies or had detectable RNA in feces, directed at interrupting the person-to-person transmission
while secretors (FUT2+/+ or FUT2+/−) excreted the virus cycle, even in the case where contaminated food or water can
and developed a strong antibody response [8, 220]. In addi- be identified. Most gastroenteritis viruses are transmitted via
tion, both volunteer studies demonstrated that FUT2-positive the ingestion of infectious fecal (or, less commonly, vomitus)
individuals of the B blood group type were less likely to be material. Therefore, standard sanitation and hygienic precau-
infected than A or O individuals but, if infected, more likely tions are key. These include frequent hand hygiene, environ-
to remain asymptomatic. Thus, alleles at the FUT2 locus mental disinfection, proper disposal of fecal or vomit-soiled
determine sensitivity or resistance to the Norwalk virus materials, and limited contact with ill persons. Even when
strain, and the polymorphism at the ABO locus modulates these precautions are put firmly in place, our ability to con-
sensitivity within the secretor-positive group. HBGAs are trol outbreaks remains limited [233].
differentially expressed in humans, and several conserved
amino acids of the P2 domain of VP1 are important for
HBGA binding [221]. The expression of HBGAs has been 8.1 Hand Hygiene
shown to be associated with strain-specific susceptibility to
norovirus infection [8, 222–224]. However, specific binding The single most important method to prevent norovirus infec-
profiles are not genotype or genogroup exclusive [225] sug- tion and control transmission is appropriate hand hygiene
gesting a host-pathogen coevolution driven by carbohydrate- [234, 235]. Washing with soap and water is the preferred
protein interactions. For GII.4 viruses, single amino acid method to prevent norovirus transmission, with alcohol-based
replacements seem to drastically alter the binding capacity hand sanitizers useful only as an adjunct when hands are not
of the VLP [226]. Overall no single norovirus strain seems to visibly soiled [236]. Plain soap and water reduces the number
be able to cover the whole spectrum of human HBGAs diver- of microbes on hands via mechanical removal of loosely
sity, although collectively, they are likely to be able to infect adherent microorganism [234]. In finger pad studies, soap
nearly everyone. The only notable exception is the subgroup and water used for 20 s have been shown to reduce norovirus
of individuals who have a rare genotype, either FUT2−/− or by 0.67–1.20 log10 by RT-PCR assay [235]. The use of alco-
FUT3−/− [218], who may be completely resistant. hol-based hand sanitizers remains controversial, due to both
Recent studies have reported that innate immunity plays inconclusive in vitro finger pad studies [171, 235, 237] and
an important role in the control of murine norovirus infection, epidemiological studies where higher rates of infection have
but little is known about cell-mediated immune responses been detected during outbreaks in long-term care facilities
against noroviruses [227, 228]. A study using oral immuniza- that use alcohol-based hand sanitizers [238], though the rea-
tion of human volunteers with Norwalk viruslike particles sons for association in this one study are debated [239].
showed an increase in interferon-γ (IFN) in the absence of Surrogate viruses such as murine norovirus (MNV) or
IL-4 production, suggesting a dominant Th1 pattern of cyto- feline calicivirus (FCV) and porcine sapovirus are typically
kine production [229]. This dominant Th1 response was con- used since norovirus cannot be cultured, so its infectivity
firmed in a study of 15 volunteers infected with Snow cannot be directly assessed. Additionally, detection and
Mountain virus, who experienced significant increases in quantification of viral RNA is not necessarily a reliable
492 B.A. Lopman et al.

means of estimating the effectiveness of hand sanitizers 8.3 Food Handling

against human norovirus [171]. Studies on disinfectant and
hand sanitizers using MNV and FCV have given contradic- Food may also be potentially contaminated with enteric
tory results [237, 240]. The sensitivity of FCV to low pH and viruses during production if growing or irrigation waters are
the relatively high susceptibility of MNV to alcohols suggest contaminated with human feces; thus, shellfish should be
that disinfectants that are effective against both surrogate adequately cooked and fresh produce washed thoroughly
viruses may be more likely to be effective against human before consumption [108].
norovirus [171].

8.4 Environmental Decontamination

8.2 Exclusion and Isolation
Chemical disinfection is a central approach inactivate noro-
Considering the highly infectious nature of norovirus, virus [246, 247]. The US Environmental Protection Agency
exclusion and isolation of infected individuals are often the maintains a list of approved products for norovirus disinfec-
most practical means of interrupting transmission of virus tion (http://www.epa.gov/oppad001/list_g_norovirus.pdf)
and limiting contamination of the environment. This is true based on their efficacy against FCV. Notably, FCV exhibits
in settings where people reside or congregate such as long- different physiochemical properties than human norovirus
term care facilities, acute care hospitals, cruise ships, and and therefore might not reflect a similar disinfection effi-
college dormitories as well as in the case of infected food cacy profile. Largely due to the uncertainty from in vitro
handlers. studies, CDC recommends chlorine bleach solution at a
Unfortunately, empirical evidence for the effectiveness of concentration of 1,000–5,000 ppm (5–25 tablespoons
exclusion and isolation strategies is limited; [241] these household bleach [5.25 %] per gallon of water) for disinfec-
strategies are based on general infection control principles tion of hard, nonporous, environmental surfaces whenever
rather than direct evidence. The principle underpinning iso- feasible [108, 172]. In healthcare settings, cleaning products
lation is to minimize contact with persons during the most and disinfectants used should be EPA registered and have
infectious periods of their illness. This includes the acute label claims for use in healthcare settings [108]. Hand
phase of illness, a period following recovery while the per- hygiene (discussed above) is also a key part of the environ-
son is still shedding virus at high levels, and, in some situa- mental transmission cycle since contaminated hands can
tions in healthcare facilities, exclusion of exposed and transfer virus to touched surfaces, and hands may be a vehi-
potentially incubating individuals. Isolation of well persons cle for transferring virus from contaminated surfaces back
(i.e., quarantine) may be useful during outbreaks in long- to humans [175].
term care facilities and hospitals to help break the cycle of
transmission and prevent additional cases.
In healthcare facilities, ill patients may be cohorted 8.5 Vaccination and Treatment
together in an isolatable unit, with the same dedicated
nursing staff providing care only for infected individuals No specific treatment exists for most AGE viruses, so treat-
[242]. Ill patients should not generally be transferred to ment is supportive and includes therapy for dehydration and
unaffected units in the facility – except in the case of med- electrolyte imbalances. First-line treatment should be oral
ical necessity and after consultation with infection control rehydration solutions, while severe dehydration or shock
staff. Analogously, passengers with AGE on cruise ships may warrant intravenous fluid therapy. Antiemetics, antimo-
may be asked to remain isolated in their cabins during tility agents, and antibiotics are generally not recommended
their illness and for a period of 24–48 h after recovery. To [248]. Certain compounds with antiviral properties have
minimize the risk of spread from incubating or asymp- shown promise in laboratory studies, but their value in the
tomatically infected patients and staff in healthcare facili- clinic remains uncertain given the short and acute infection
ties, such individuals should not be transferred to or work caused by caliciviruses.
in unaffected areas, typically for 48 h after exposure. In No vaccines for noroviruses are currently available, but a
certain situations, units in a healthcare facility may be number of norovirus vaccines are at various stages of devel-
closed to new admissions to prevent the introduction of opment. The product furthest developed is based on recom-
new susceptible patients, though guidelines differ on this binant virus-like particles (VLPs) produced by the expression
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facilities as well as infected food handlers should be VP1. An intranasally delivered formulation was shown to be
excluded during their illness and for 24–48 h following safe and immunogenic in phase 1 and 2 trials [249]. In a
resolution of symptoms [108]. challenge study, where participant were vaccinated and
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 Orthopoxviruses: Variola, Vaccinia,
Cowpox, and Monkeypox 21
Brett W. Petersen, Kevin L. Karem, and Inger K. Damon

1 Introduction of a single, linear, covalently closed double-stranded DNA

molecule with inverted terminal repetitions (ITRs) at each
1.1 Biological Characteristics of the two ends. The genomes of most orthopoxviruses are
~200 kbp in length and contain a guanine plus cytosine con-
Poxviruses are a family of large, complex DNA viruses tent of ~36 % [1]. The complete genomic DNA sequences of
characterized by their unique ability to replicate entirely many orthopoxviruses, including representative strains all of
within the cytoplasm of infected cells [1]. The Poxviridae
family is divided into the subfamilies Chordopoxvirinae
and Entomopoxvirinae which infect vertebrate and insect
hosts, respectively. Orthopoxviruses are members of the
Chordopoxvirinae subfamily along with seven other genera:
Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus,
Suipoxvirus, Molluscopoxvirus, and Yatapoxvirus. Four species
of orthopoxviruses are known to infect humans: variola (small-
pox), vaccinia (smallpox vaccine), cowpox, and monkeypox.
Due to their large size compared to other animal viruses,
poxviruses were the first viruses to be seen with a microscope
[1, 2]. With the increased resolution of electron microscopy,
orthopoxvirus virions appear brick shaped with slightly
rounded edges [1, 3]. The mature virion (MV) is the most
basic infectious form of poxvirus and consists of a dumbbell-
shaped core surrounded by a single lipid membrane bilayer
[1, 4] (Fig. 21.1).
The orthopoxvirus genome is located in the viral core
within a nucleoprotein complex (nucleosome) [4]. It consists

B.W. Petersen, MD, MPH • I.K. Damon, MD, PhD (*)

Poxvirus and Rabies Branch, Division of High-Consequence
Pathogens and Pathology, National Center for Emerging
and Zoonotic Infectious Diseases, Centers for Disease Control
and Prevention, 1600 Clifton Rd NE, Mailstop G-43,
Atlanta, 30329-4018, GA, USA
e-mail: [email protected]; [email protected]
K.L. Karem, PhD
Laboratory Reference and Research Branch,
Division of Sexually Transmitted Disease and Prevention,
National Center for HIV/AIDS, Fig. 21.1 Electron micrograph of smallpox virus virions depicting the
Viral Hepatitis, STD and TB Prevention, dumbbell-shaped viral core (From the US CDC Public Health Image
1600 Clifton Rd NE, MS AIZ, Library [PHIL] Image ID 1849. Credit: CDC/Dr. Fred Murphy; Sylvia
Atlanta, 30329-4018 GA, USA Whitfield. This image is in the public domain and thus free of any copy-
e-mail: [email protected] right restrictions)

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 501

DOI 10.1007/978-1-4899-7448-8_21, © Springer Science+Business Media New York 2014
502 B.W. Petersen et al.

the viruses described in this chapter, have been determined development of immunity against orthopoxviruses [18].
and described previously [5–8]. To thwart these host responses, poxviruses have developed
During virion morphogenesis, a minority of MV undergo numerous mechanisms to evade the immune system [19, 20].
further processing with the addition of a second outer lipid Many of these disrupt the innate immune system by targeting
membrane bilayer to form an extracellular enveloped virion mediators of inflammation such as interferons, tumor necro-
(EV). Both MV and EV are infectious particles but are sis factors, interleukins, complement, and chemokines [19,
thought to have different mechanisms of binding and enter- 21–27]. Further studies using DNA microarrays to evaluate
ing cells. MV binding to cell glycosaminoglycans or laminin gene expression profiles in response to infection with variola,
is mediated by four viral proteins associated with the single vaccinia, and monkeypox viruses confirmed the absence of
MV membrane [9]. While the binding protein(s) of EV has interferon and tumor necrosis factor responses and other fac-
yet to be identified, it is believed that the second outer mem- tors with critical roles in the activation of the innate immune
brane is disrupted to expose the MV membrane prior to entry response [28, 29]. This complex interplay between the host
[9, 10]. Subsequently, membrane fusion is initiated by 11–12 response and viral immune evasion mechanisms has made it
transmembrane proteins on the MV surface, a process that difficult to establish a direct correlate of immunity despite
can occur directly with the cell plasma membrane or with technological advances [4]. Historically, the presence of a
the membrane of an endocytic vesicle [9, 10]. When fusion “take,” i.e., demonstrable lesion or scar at the administration
occurs at the plasma membrane, the viral entry and fusion site of smallpox vaccine, was used as evidence of successful
proteins now embedded in the cell membrane also mediate vaccination against smallpox and protection against disease.
the formation of syncytia with adjacent cells and cell to cell However, this practice can mistake a bacterial superinfection
spread of virus [10]. Once inside the cell, viral replication of the inoculation site or vaccination with bacillus Calmette-
commences and occurs completely within the cytoplasm. For Guérin (BCG) vaccine with protection [4]. Furthermore, an
this reason, poxvirus virions contain a nearly complete RNA alternate correlate of protection would be useful for more
polymerase system that enables primary transcription of viral recently developed highly attenuated smallpox vaccines that
genes without the use of the cell’s nuclear machinery [1]. This do not produce visible lesions. Continued research is needed
early gene expression provides nonstructural proteins needed to better elucidate the patterns of host response and corre-
for DNA replication as well as gene expression of intermedi- lates of immunity for orthopoxvirus infections.
ate and late genes. The intermediate and late genes encode
structural proteins required for virion assembly in addition to
early transcription factors that are packed together with RNA 2 Smallpox
polymerase in assembling virions [1]. In total, orthopoxvi-
rus genomes contain approximately 200 genes of which half 2.1 Historical Background
encode proteins that are ultimately packaged into virions [4].
Smallpox is one of the oldest described human diseases.
Examinations of several mummies from ancient Egypt,
1.2 Patterns of Host Response including that of the pharaoh Ramses V, suggest that small-
pox may have occurred in humans over 3,000 years ago [2].
At present, there is an incomplete understanding of the pat- Early written records containing detailed descriptions of
terns of host response that occur in human orthopoxvirus smallpox also exist including those of Ko Hung from China
infections. Of the four orthopoxviruses that infect humans, in 340 AD, Ahrun from Alexandria in 622 AD, and the
vaccinia virus has been the best studied as a model for under- Persian scholar Al-Razi in 910 AD [2]. Periodic epidemics
standing poxvirus biology. Vaccinia virus has also been of smallpox likely plagued primitive civilizations until their
exploited as a vaccine due to the ability of orthopoxviruses populations became sufficiently dense to establish endemic-
to induce cross-reactive antibodies that protect against infec- ity [2]. By the mid-eighteenth century, smallpox had become
tion from other orthopoxvirus species. Neutralizing antibod- endemic on all continents with the exception of Australia [2].
ies to orthopoxviruses generally appear within the first week Throughout the centuries, smallpox caused untold human
of illness and can remain present beyond 20 years [11, 12]. suffering and loss of life. Even as late as the 1950s, over
In contrast, hemagglutination-inhibition and complement- 150 years after Edward Jenner introduced vaccination, small-
fixation antibodies become detectable 16–18 days after pox was estimated to produce 50 million cases worldwide
infection with levels decreasing after 1 year [2, 13]. The each year [30]. Subsequently, a global eradication campaign
development of such antibodies has been associated with was initiated by the World Health Organization (WHO) in
protection in studies of both animals and humans [14–17]. 1959 to rid the world of this ancient scourge. The success of
However, in addition to humoral responses, cell-mediated these efforts culminated in the identification of the last known
and T-cell responses also appear to be important in the case of naturally occurring smallpox in Somalia in 1977.
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 503

In 1980 the WHO’s General Assembly officially declared 2.3 Descriptive Epidemiology
smallpox eradicated, and recommendations for routine vac-
cination against smallpox were discontinued. All known The host range of variola virus was restricted to humans,
stockpiles of variola virus were collected and consolidated and there are no known animal or insect reservoirs [2].
and are currently stored in two locations at the Centers for However, recent genetic analyses suggest that the disease
Disease Control and Prevention (CDC) in Atlanta, Georgia, may have emerged from a rodent-borne variola-like ancestor
United States, and the State Research Center of Virology and in Africa [45, 46]. Among humans, smallpox cases occurred
Biotechnology VECTOR in Novosibirsk, Russia. Following in all ages, genders, races, and ethnicities. Indeed, smallpox
eradication, smallpox was allegedly developed as a bio- has been described as a disease of “princes and peasants”
logical weapon by the Soviet Union and produced in large due to its indiscriminate nature in inflicting disease upon
quantities [31]. This revelation, along with other intelligence all people [47]. Two principal forms of disease were distin-
concerns, has led to the belief that unauthorized stocks of guished epidemiologically: variola major and the less severe
smallpox may exist and could be used as a biological weapon variola minor (including a biologically distinct virus type
by terrorists or rogue nations. Though the likelihood of this alastrim). Outbreaks of variola major produced case fatality
happening is believed to be low, efforts to be prepared, utiliz- rates up to 30 %, while those for variola minor were gener-
ing a smallpox response research and development program, ally less than 1 % [2, 4]. Population studies including both
for the possible reemergence of smallpox into human circu- vaccinated and unvaccinated subjects revealed that case fatal-
lation are ongoing [32, 33]. ity rates were highest in the very young and older age groups
[40, 48]. Pregnant women also had extraordinarily high case
fatality rates from smallpox (34.3 % overall and nearly 70 %
2.2 Methods for Epidemiologic Analysis for unvaccinated pregnant women) and were particularly
susceptible to hemorrhagic smallpox [2, 49–51]. Secondary
Given the eradication of the disease, all epidemiologic data attack rates of 30–80 % among unvaccinated contacts within
on smallpox is historical in nature. However, the widespread households were similar for both variola major and minor [4].
nature of the disease prior to eradication provided numer- The age distribution during outbreaks of smallpox depended
ous cases for study. Among the largest and most systematic largely on the immunization or disease-convalescent status of
clinical and epidemiologic analyses of smallpox in hospi- the population affected. Children generally had the highest
talized patients were those done by Thomas F. Ricketts, A. attack rates in smallpox outbreaks in most areas including the
Ramachandra Rao, James P. Marsden, and Cyril W. Dixon United States, Europe, and Asia as most adults had acquired
[2, 34]. The work of Ricketts, Rao, and Dixon was based immunity through either vaccination or previous smallpox
on personal examination of hundreds of patients with small- infection [52]. In contrast, the age distribution of cases was
pox hospitalized in London, England; Madras, India; and generally proportional to the age distribution of the population
Tripolitania (modern-day Libya), respectively [35–37]. in rural areas where vaccination was less common and adults
Similarly, Marsden’s experience in the London smallpox were more likely to be vaccine naïve and susceptible to dis-
hospitals provided insights into the epidemiology of the ease [40, 52]. Smallpox outbreaks exhibited a seasonal pattern
milder form of smallpox variola minor [2, 38]. Further stud- with the highest incidence of cases occurring during the winter
ies evaluating the epidemiology of smallpox in nonhospital and early spring [40, 53]. This observation was consistent with
settings were also undertaken during the global eradica- the findings that other orthopoxviruses (i.e., vaccinia) survived
tion campaign [39, 40]. Currently, smallpox surveillance is better under conditions of low temperature and humidity [54].
focused on strengthening and maintaining the capacity to
identify the disease in the event that human infection reoc-
curs. Smallpox is a nationally notifiable disease in the United 2.4 Mechanisms and Routes
States, and the CDC has developed a clinical algorithm to of Transmission
evaluate the risk of smallpox in patients with acute vesicu-
lar or pustular rash illnesses, to assist in developing a differ- The most common route of transmission of smallpox was
ential diagnosis, and to help inform the need for diagnostic from person to person primarily via respiratory-salivary
testing [41, 42]. To increase capacity for diagnostic testing, droplets expelled from the oropharynx of infected individu-
the Laboratory Response Network (LRN) was established by als. Transmission generally occurred following direct face-
the CDC with collaboration from multiple partners includ- to-face contact, though rare occurrences involving airborne
ing the Association of Public Health Laboratories and the dissemination over longer distances have been documented
Federal Bureau of Investigation [43, 44]. The LRN provides [55]. Common features linked to these occurrences included
laboratory diagnostic testing for smallpox in addition to primary cases with extensive rash and cough, low humidity,
other potential biological warfare agents. and hospital ventilation favoring the formation of air currents
504 B.W. Petersen et al.

[55]. Transmission of smallpox also occurred through direct

contact with active rash lesions or other sloughed exudative
materials, though this route of transmission was uncommon
[2]. Smallpox was also rarely transmitted via contaminated
fomites, e.g., soiled clothing or bed linens [2, 34]. Lastly,
smallpox infection by direct inoculation occurred among
nursing mothers, postmortem workers, and fabric workers
(particularly those working with lace) or more commonly
through variolation – a technique for immunizing patients by
purposefully infecting them with material from the pustules of
smallpox patients with mild smallpox disease [2]. Variolation
was practiced widely before the advent of vaccination.
Peak viral shedding is seen during the rash phase of ill-
ness; consequently, almost all transmission occurred during
the time when rash is present [56, 57]. As such, the major-
ity of smallpox transmission occurred within households,
hospitals, and other health-care settings. Large outbreaks in
schools were uncommon, and transmission on trains, planes,
or buses was also rare [58, 59]. Despite containing signifi-
cant amounts of virus, scabs were not found to be particu-
larly infectious presumably due to the encasement of viral
particles within the protein-fibrin matrix [2].

2.5 Pathogenesis and Immunity

Infection with smallpox began with entry of the virus through

the respiratory mucosa or alveoli. The clinical course of ordi-
nary smallpox then began with an asymptomatic incubation
period generally lasting 10–14 days (range of 7–17 days).
Illness onset generally occurred suddenly with fever and
malaise. Temperatures rose to 38.5–40.5 °C and were vari-
ably accompanied by headache, backache, abdominal pain,
vomiting, and pharyngitis [2]. These prodromal symptoms Fig. 21.2 Smallpox lesions on the arm and palm (From the US CDC
PHIL Image ID 2007. Credit: World Health Organization; Diagnosis of
lasted approximately 2–3 days before the first appearance of Smallpox Slide Series. This image is in the public domain and thus free
lesions [2]. The rash developed in a centrifugal pattern with of any copyright restrictions)
lesions appearing first and in highest numbers on the oro-
pharynx, face, and extremities before spreading to the trunk.
Those on the oropharynx quickly ulcerated as a result of the
lack of stratum corneum, releasing high titers of virus into
the saliva [58, 60]. The presence of lesions on the palms and
soles of the hands and feet was also characteristic (Fig. 21.2).
Lesions progressed from macules to papules to vesicles over
the course of 4–5 days. Vesicles were often umbilicated and
evolved to pustules within a day or two (Fig. 21.3).
Pustules were described as round, tense, firm to the touch,
and deep seated within the dermis. At any given time, lesions
exhibited the same stage of development in any one area of
the body [4]. Lesions typically began to crust on the eighth
or ninth day of rash with scab formation and sloughing
around day 14 post rash onset. Resolving skin lesions left
pitted scars which were particularly evident on the face due Fig. 21.3 Umbilicated smallpox lesions on the trunk (From the US
to the destruction of the sebaceous glands, shrinking granu- CDC PHIL Image ID 284. Credit: CDC/James Hicks. This image is in
lation tissue, and fibrosis [2]. In fatal cases, death usually the public domain and thus free of any copyright restrictions)
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 505

occurred during the second week of illness. The exact cause A similar system for subdividing smallpox into catego-
of death in smallpox remains unclear, and the pathogenesis ries with prognostic value was described by Dixon [34,
may have differed depending on whether the clinical mani- 37]. He defined a total of nine types of smallpox, each
festations were hemorrhagic, flat, or “malignant” as opposed with associated mortality estimates. Fulminating smallpox
to the ordinary type [34]. In some cases reviewed retrospec- (purpura variolosa) was the first and most deadly type with
tively, mortality was attributed to some combination of renal a mortality rate of nearly ~100 %. This type of smallpox
failure, shock secondary to volume depletion, and respira- was characterized by a hyperacute course leading to death
tory compromise induced by cytopathic effects of the virus within 4–5 days after onset of symptoms. Illness began
[61]. Toxemia associated with viral antigens in plasma and with fever, prostration, and anxiety accompanied by early
immune complexes of antigen and antibody have also been hemorrhages, particularly of the mucous membranes. The
proposed to be contributing factors [2, 58]. absence of any vesicular eruption was a distinct clinical
During the eradication of smallpox, the WHO estab- feature making differentiation from other acute hemor-
lished four main clinical types of disease based on disease rhagic catastrophes difficult. The second and third types of
presentation and rash burden: ordinary, modified, flat, and smallpox were termed malignant confluent and malignant
hemorrhagic [62]. The most common type was ordinary semiconfluent. Malignant smallpox typically began with a
smallpox which accounted for approximately 85 % of cases high initial fever during the first 3–4 days of illness that
in outbreaks of variola major. The clinical presentation of defervesced before reappearing during the fourth to twelfth
ordinary smallpox followed the description above and, as days of illness. The rash of malignant smallpox was soft,
classified by WHO for epidemiologic description, was fur- velvety, hot, and tender with a slow evolution. Vesicular
ther subdivided based on the density of rash lesions on the lesions were rare though hemorrhaging could occur late in
face and body. In ordinary confluent, the rash was conflu- the skin as well as the mucosa. The prognosis depended
ent on the face and arms, whereas ordinary semiconfluent largely on the distribution of the rash with 70 % mortality
demonstrated a confluent rash on the face only. Ordinary in patients with confluent rash on the face and arms (malig-
discrete was characterized by areas of normal skin between nant confluent) as opposed to 25 % mortality in patients
pustules on the face and body. These subdivisions had with confluent rash on the face only (malignant semicon-
prognostic value as higher rash burden portended a higher fluent). Benign confluent and benign semiconfluent were
likelihood of death; confluent, semiconfluent, and discrete the fourth and fifth types of smallpox described by Dixon.
ordinary smallpox had case fatality rates of 62, 37, and 9 %, These types were distinguished primarily by the distinc-
respectively, in unvaccinated individuals [2, 36]. Modified tive rash of ordinary smallpox that followed a centrifugal
smallpox was similar to ordinary smallpox apart from being distribution and progressed through the macular, papular,
“modified” by previous vaccination to produce disease with vesicular, and pustular stages. As with malignant small-
a milder prodrome, fewer lesions, and an accelerated clini- pox, the rash of benign semiconfluent extended to the face
cal course. This type accounted for 5–7 % of cases and was only compared to the face and arms in patients with benign
not fatal [2, 62]. In contrast, flat smallpox occurred at a sim- confluent and conferred a mortality benefit (10 vs. 20 %,
ilar frequency but was usually fatal in both vaccinated and respectively). Lastly, Dixon described four types of less
unvaccinated individuals [2, 36]. The lesions of flat smallpox severe smallpox that differed primarily in the number of
were slow to mature and generally remained as soft, velvety lesions; the discrete type exhibited over 100 (but no areas
vesicles without pustulation [30]. Most cases of flat small- of confluence), 20–100 in the mild type, less than 20 in
pox were seen in children and may have been associated the abortive type (often without pustulation), and no lesions
with deficient cellular immune responses though no stud- in variola sine eruptione. Death was uncommonly encoun-
ies were undertaken to confirm this [2]. Lastly, hemorrhagic tered in these types of smallpox with only discrete small-
smallpox was a rare (<1 % of cases) but deadly form involv- pox manifesting a 2 % mortality rate.
ing extensive bleeding into the skin and mucous membranes Variola minor infections produced disease that was clini-
that almost invariably led to death within a week of disease cally identical to the ordinary or modified types of smallpox
onset [4]. This type occurred mostly in adults (particularly with typical prodrome and rash [38]. Prior to eradication,
pregnant women) with equal frequencies in vaccinated and variola major and minor could only be distinguished in out-
unvaccinated individuals [2]. Variola infection without the break settings by the difference in case fatality rates, whereas
development of rash was thought to occur rarely in previ- virologic differentiation is now possible for certain biologi-
ously vaccinated individuals exposed to smallpox cases and cally discrete viruses causing minor disease (i.e., alastrim)
was referred to as variola sine eruptione. Patients typically [2, 58].
presented with sudden onset of fever, headache, and some- Survivors of smallpox were thought to be protected for
times backache that resolved within 48 h and showed evi- life [2, 13]. In contrast, complete protection is not lifelong
dence of acute infection by high or rising antibody titers and following smallpox vaccination. Vaccine-induced immunity
occasionally viral isolation [2]. is most effective in the first 1–3 years following vaccination
506 B.W. Petersen et al.

when preexposure vaccine efficacy may approach 100 %, [70]. The second study actively surveyed physicians in ten
and substantial protection may endure for up to 15–20 years states to solicit reports of complications of smallpox vaccina-
[4, 63, 64]. Postexposure vaccination was also effective in tion [71]. Not surprisingly, the adverse event rates calculated
preventing and/or ameliorating disease when given to con- using the active surveillance from the ten statewide surveys
tacts of patients with smallpox. Such postexposure prophy- were higher than those calculated from the passive national
laxis appeared to be most effective when given as soon as surveillance [70, 71]. More recently during 2002–2004,
possible following exposure and particularly in previously adverse events were monitored, while the United States
vaccinated individuals; vaccination beyond 3 days after Department of Defense (DOD) and Department of Health
exposure appeared to be less effective based on interpreta- and Human Services (DHHS) increased efforts to vacci-
tions of data from the eradication era [65–68]. nate service members and civilians to enhance prepared-
ness against the possibility of a bioterrorism event involving
smallpox. In contrast to those persons vaccinated during the
3 Vaccinia and Cowpox 1968 studies, these populations involved adults that were
highly screened for predisposing conditions associated with
3.1 Historical Background higher rates of adverse events (e.g., eczema, immunodefi-
ciency, or therapeutic immunosuppression). As such, overall
The disease known as “cowpox” was so named because of rates of adverse events were much lower in both the DOD
the pustular lesions it produces on the teats of milking cows. and DHHS programs when compared to historical rates [72–
During the 1700 s, rural Europeans observed that milkmaids 74]. One exception was the identification of myopericarditis
(and others exposed to cows) were rarely afflicted with the and cardiac ischemic events at higher rates than anticipated
scars of smallpox, and it was hypothesized that exposure to based on historical data [73, 75].
cowpox induced resistance to infection with smallpox. In With respect to cowpox, the most detailed epidemiologic
1798, Edward Jenner published the first scientific evidence analysis is based on a series of 54 human cases investi-
that cowpox could be used prophylactically to produce gated between 1969 and 1993 [76]. Limited epidemiologic
immunity against challenge with smallpox. This concept information has also been gleaned from outbreaks and case
of preventing disease through inoculation was later termed reports of naturally acquired as well as laboratory-associated
“vaccination” by Louis Pasteur using the Latin word for cow cowpox infections [77–85].
(vacca) to honor the work of Jenner. Initially, poxviruses
taken from or grown on a wide range of species including
cows, horses, goats, sheep, pigs, and buffaloes were used [2]. 3.3 Descriptive Epidemiology
However, smallpox vaccination quickly gained acceptance,
and by the twentieth century, almost all smallpox vaccines The vast majority of cases of vaccinia virus infection and
contained vaccinia virus. Recent genotypic analysis suggests its complications are associated with vaccination. In the
that vaccine strains of vaccinia virus are most closely related United States, select military service members still receive
to cowpox viruses from continental Europe [69]. Regardless routine vaccination against smallpox using vaccinia virus
of its precise origin, the use of vaccinia virus in combination [72]. Serious infections with vaccinia virus have been seen
with surveillance and monitoring led to the eradication of among vaccinees as well as contacts of vaccinees [74, 86,
smallpox in 1980. 87]. Vaccination with vaccinia virus is also recommended
for laboratory workers who work directly with non-highly
attenuated strains of vaccinia virus or other orthopoxviruses
3.2 Methods for Epidemiologic Analysis that infect humans [88]. Vaccinia virus is commonly used in
laboratory research, and multiple exposures and infections
Early interest in the epidemiology of vaccinia stemmed pri- have been documented in laboratory workers [89].
marily from its use as a routine childhood vaccine for the In addition to its use in laboratory research, vaccinia
prevention of smallpox. In particular, considerable attention viruses can also be genetically altered to produce recombi-
has been paid to describing the adverse events associated nant vaccines. One such vaccine is a recombinant vaccinia
with vaccinia when used as a smallpox vaccine. Two studies virus containing a rabies virus glycoprotein that is dis-
conducted by the Centers for Disease Control in 1968 were tributed in baits to control rabies in wildlife. Two cases of
among the most comprehensive to evaluate the incidence of human vaccinia virus infection have been reported in dog
adverse events following childhood smallpox vaccination owners following exposures to baits retrieved by their dogs
with vaccinia virus. The first study relied on passive report- [90, 91]. Surveillance for further human exposure to oral
ing of patients with suspected complications of smallpox rabies vaccine is ongoing and provides additional data for
vaccination to seven separate national surveillance systems epidemiologic analysis [92].
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 507

Natural infection with vaccinia virus is rare but does occur.

Evidence suggests that a vaccinia virus related to a Brazilian
smallpox vaccine strain has become established in cattle in
Brazil and shows signs of spread within the country [93–95].
Human vaccinia virus infections among farmworkers exposed
to cattle in Brazil have been reported [94, 96]. In addition,
outbreaks of buffalopox virus, a subspecies of vaccinia virus
found in milking buffalo and cattle, have been reported to
cause vaccinia-like lesions on animals’ teats and milkers’
hands in India, Egypt, Bangladesh, and Indonesia [97, 98].
Human cowpox virus infection is classically described as
a zoonotic infection associated with occupational exposure
to cattle. However, multiple other species have been impli-
cated as sources of human infection including rats, pet cats,
and zoo and circus elephants [76]. Two cases of laboratory-
acquired human infection with cowpox virus have also been
documented [77, 78].

3.4 Mechanisms and Routes

of Transmission

Both vaccinia virus and cowpox virus are most commonly

transmitted to humans through contact of skin with puri-
fied virus, active lesions, or materials from lesion exudates.
Intentional infection with vaccinia virus for the purpose of
immunization is achieved by inoculating virus through scari-
fication of the skin using a bifurcated needle. Inadvertent
inoculation (to others or to a distant site of the same patient)
also occurs as vaccinia virus and cowpox virus lesions have
the capacity to produce infectious material until a scab has Fig. 21.4 Progressive vaccinia in a 7-year-old male with microcephaly
formed and detached to reveal an underlying intact layer of and cerebral palsy after receiving smallpox vaccine (From the US CDC
PHIL Image ID 14250. Credit: CDC/Dr. Cocke. This image is in the
skin. public domain and thus free of any copyright restrictions)

3.5 Pathogenesis and Immunity typically occurs as a single lesion on the hand, finger, or face
but can present with multiple lesions secondary to multiple
Human infections with vaccinia and cowpox virus produce primary inoculations and autoinoculation and rarely through
similar clinical syndromes. Both produce an ulcerative skin lymphatic or hematogenous spread of virus [76]. Most vac-
lesion that progresses through a well-characterized sequence cinia and cowpox infections are self-limiting and resolve
of events. The site of inoculation develops a papule which within 6–8 weeks though recovery can take up to 12 weeks
becomes vesicular after 3–5 days. Subsequently, the vesicles in some cases. However, severe systemic illness can be seen
become pustular and lesions reach their maximum size at in individuals with immunosuppression or immunodefi-
day 8–10. The lesions then dry forming a hard black crust ciency [70, 71, 79, 86].
which typically separates at day 14–21 leaving a perma- A number of rare and sometimes fatal complications can
nent scar. During the vesicular and pustular stages, lesions result from vaccinia virus infection following vaccination
are often painful with surrounding erythema and edema. or naturally acquired infections. Progressive vaccinia, also
Infection is usually also associated with systemic symptoms previously referred to as vaccinia necrosum or vaccinia gan-
including fever, lymphadenitis, fatigue, and malaise com- grenosum, can occur in persons with severe cell-mediated
monly described as influenza-like. Vaccination with vaccinia immunodeficiency [99]. The disease is characterized by
virus is typically performed on the upper deltoid; however, uncontrolled spread of infection from the site of inocula-
secondary autoinoculation or contact transmission can lead tion resulting in persistent enlargement of the primary lesion
to multiple lesions in other anatomical locations. Cowpox often associated with necrosis (Fig. 21.4).
508 B.W. Petersen et al.

Postvaccinial encephalomyelitis (PVEM) is a rare but

important complication that occurs following primary vac-
cination with vaccinia virus. Symptoms of encephalitis or
myelitis including fever, headache, vomiting, and malaise
typically develop abruptly 10–13 days following vaccina-
tion [2]. Decreased consciousness, seizures, and coma
frequently ensue [105]. One case with a similar clinical syn-
drome has also been reported following infection with cow-
pox virus [106]. Unlike progressive vaccinia and eczema
vaccinatum, no known predisposing factors for PVEM
are known [2]. The pathophysiology of PVEM is poorly
understood with evidence of direct viral infection in some
cases, while others display a demyelinating process more
closely resembling an acute disseminated encephalomyeli-
Fig. 21.5 Eczema vaccinatum in an 8-month-old male infected with tis (ADEM) [105, 107, 108]. Reported rates of PVEM can
vaccinia by a sibling recently vaccinated against smallpox (From the
US CDC PHIL Image ID 3311. Credit: CDC/Arthur E. Kaye. This
vary widely based at least partially on differences between
image is in the public domain and thus free of any copyright vaccine virus strains. The New York City Board of Health
restrictions) (NYCBOH) strain, a strain with lower incidence of PVEM
compared to those used in other countries, produced a rate
Subsequent dissemination to other parts of the body can of 3–12 cases per million primary vaccinees in the United
sometimes follow. A vaccination site that continues to show States during 1968 [2, 70, 71]. The fatality rate of PVEM is
progression without signs of healing 15 days after vaccina- estimated to be 25 % [109].
tion should prompt consideration of the diagnosis [100]. A syndrome similar to PVEM is also rarely seen in infants
Progressive vaccinia is generally lethal in those with a com- less than 2 years of age. Termed postvaccinial encephalopa-
plete lack of cellular immunity (e.g., infants with primary thy (PVE), cases develop the same symptoms as PVEM
immunodeficiencies), though survival has been documented but typically present earlier following vaccination (usually
in adults with acquired immunodeficiencies [86, 99]. The within 6–10 days) [110]. The development of hemiplegia and
incidence of progressive vaccinia was approximately one aphasia can also be distinguishing features of PVE [110].
case per million recipients of routine smallpox vaccination Generalized vaccinia is a vesicular rash that develops
in the United States during 1968 [70, 71]. after vaccination due to dissemination of virus from the site
Eczema vaccinatum is a potential complication of vac- of inoculation. Lesions typically appear 6–9 days after vacci-
cinia virus infection that can occur in individuals with atopic nation and follow a similar progression as the primary lesion
dermatitis (eczema) regardless of the current status of dis- only more rapid [2]. The rash can be profuse and cover the
ease activity. Patients typically present with firm, deep- entire body but does not follow a centrifugal pattern of dis-
seated vesicles or pustules 3–14 days following exposure to tribution as seen with smallpox [2]. Generalized vaccinia is
vaccinia virus [101]. The lesions can be locally or widely typically self-limiting, and serious cases are only seen when
distributed, often at the site of a previous eczematous lesions, underlying immunodeficiency is present [100]. While gen-
and generally demonstrate the same stage of development eralized vaccinia is thought to result from viremic spread
(Fig. 21.5). of the virus, isolation of virus from lesions is uncommon
Lesions may be accompanied by systemic symptoms [111, 112]. During 1968, generalized vaccinia was reported
including fever, malaise, and lymphadenopathy. Bacterial in 23–242 per million primary smallpox vaccinations per-
superinfections and supraglottic edema leading to air- formed in the United States [70, 71].
way compromise are potentially fatal complications [101]. Infections with vaccinia virus can pose a risk to the
Underlying defects in the immune system of atopic indi- unborn fetus. Although an analysis of pregnancy outcomes
vidual’s skin render them particularly vulnerable to infec- among 376 women vaccinated against smallpox did not
tion with vaccinia virus and subsequent local spread or wider reveal higher-than-expected rates of pregnancy loss, pre-
dissemination [102, 103]. This susceptibility persists even in term birth, or birth defects, approximately 50 cases of fetal
the absence of active disease as one case series found that vaccinia have been documented previously [113, 114]. This
two thirds of eczema vaccinatum patients had no active or complication often results in death of the fetus or neonate
obvious eczema at the time of vaccination [104]. Eczema and occurs at an estimated rate of 1/10,000 to 1/100,000 in
vaccinatum occurred at a rate of 10–39 cases per million pri- vaccinated pregnant women [114]. For this reason, vacci-
mary smallpox vaccinations and 1–3 cases per million small- nation with vaccinia virus is generally avoided in pregnant
pox revaccinations in the United States during 1968 [70, 71]. women.
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 509

Cardiac complications have been associated with small- Central and West Africa providing the first opportunities for
pox vaccination, particularly among civilian and military per- epidemiologic analyses [125, 126]. Following the eradica-
sonnel vaccinated in the United States since 2002 [73, 74]. tion of smallpox, the WHO sponsored enhanced surveillance
The observed rate of myopericarditis among this vaccinated efforts for monkeypox in the DRC during 1980–1986 which
population has been higher than the calculated background detected 350 cases with the assistance of a monetary reward
incidence [75, 115, 116]. Clinical studies suggest that myo- for reporting [126]. Between 1987 and 1995, surveillance
pericarditis may occur in 1 in 175 adults who receive primary efforts were greatly reduced and few cases were reported. A
vaccination against smallpox [117]. However, most cases large outbreak in Zaire in 1996 led to further studies over the
appear to be mild and self-limited with few documented cases ensuing 2 years that were partially hampered by civil unrest
of dilated cardiomyopathy [115, 118]. The pathophysiology [126]. The first appearance of human monkeypox outside of
of myopericarditis associated with smallpox vaccine remains the African continent occurred in the United States in 2003
unclear. However, the absence of direct viral invasion seen and provided new insights into the epidemiology of the dis-
by histopathologic examination of myocardial tissue from ease outside of the geographic locations and populations typ-
vaccinees with myocarditis suggests this may be an immune- ically affected. Human monkeypox continues to occur in the
mediated phenomenon [115, 119]. While temporally associ- DRC, and efforts to improve prevention and control through
ated cardiac ischemia and myocardial infarction have been enhanced surveillance remain ongoing.
observed among smallpox vaccinees, the incidence of these
complications does not exceed the expected background
rates, and there is currently no evidence to suggest a causal 4.3 Descriptive Epidemiology
association with vaccination [115, 119].
With the exception of isolated outbreaks in the United States
and Sudan, human monkeypox is limited geographically to
4 Monkeypox Central and West Africa. Males and females are affected
equally. Younger age groups are disproportionately affected;
4.1 Historical Background 90 % of cases identified in the DRC during 1980–1985
were less than 15 years old [127]. As with smallpox, the
Monkeypox was so named due to its identification in 1958 vaccination status of an affected population may influence
during an outbreak of a pox-like illness in captive monkeys the age distribution of monkeypox cases in an outbreak.
in Denmark [2, 120]. Initially, this gave rise to concerns However, more recent investigations of outbreaks occur-
that smallpox eradication would not be possible if monkeys ring 20–30 years after the cessation of smallpox vaccination
served as a reservoir for variola virus. However, subsequent have found that the incidence and attack rates for monkey-
laboratory analyses revealed the monkeypox virus to be a pox remain highest among those 14 years and younger [124,
distinct species within the Orthopoxvirus genus. The first 128]. Furthermore, recent data suggest that the incidence of
case of human monkeypox was identified in the Democratic monkeypox is increasing in rural Africa due to the grow-
Republic of the Congo (DRC) in 1970 [121, 122]. Further ing proportion of unvaccinated individuals in the population,
human cases were confirmed when nine samples from Zaire, waning immunity in vaccinated individuals, decreasing num-
Nigeria, Cote d’Ivoire, Sierra Leone, and Liberia tested bers of smallpox survivors, and human encroachment into
positive for monkeypox virus as part of the efforts of the areas inhabited by animal reservoirs [124, 128–130]. Most
Commission to Certify Smallpox Eradication in West Africa reported deaths occur in unvaccinated children under the age
and the Congo Basin sponsored by the WHO [123]. Since of 14 years. While the overall case fatality rate in Africa is
that time, sporadic epizootics continue to occur primarily in typically ~10 % (range 3.7–11.3 %), rates as high as 14.9 %
Central Africa with evidence suggesting the incidence has have been seen among the youngest age groups (0–4 years)
increased since the cessation of smallpox vaccination [124]. [2, 127, 128]. In contrast, no deaths were observed in 37
The emergence of human monkeypox cases in the United confirmed monkeypox cases in North America [131, 132].
States in 2003 and in South Sudan in 2005 further demon- Differences in the level of medical care administered, route
strates the potential for outbreaks in locales discrete and/or of infection, and underlying host factors are all potential con-
distant from the historic geographic range of the virus. tributors to this disparity in lethality. In addition, genetically
distinct isolates of monkeypox virus have been identified
from West Africa and the Congo Basin [130, 133–135]. The
4.2 Methods for Epidemiologic Analysis West African clade has shown decreased virulence compared
to the Congo Basin clade in animal models of monkeys,
In the decade following the discovery of human monkey- mice, and prairie dogs; the West African monkeypox virus
pox infection, 54 cases were identified and investigated in identified in North America may also have contributed to the
510 B.W. Petersen et al.

absence of fatalities and relatively mild disease observed in spread from person to person, monkeypox does not exhibit
the 2003 outbreak [134, 136–138]. Serologic surveys of per- sustained transmission within human populations. The sec-
sons without vaccination scars were performed in the 1980s ondary attack rate of 9.3 % among unvaccinated household
in Central and Western Africa to estimate the prevalence of contacts documented in African outbreaks is far lower than
monkeypox. These studies found that 1,583/10,300 (15.4 %) that observed for smallpox [123, 145]. Moreover, the longest
sera tested demonstrated orthopoxvirus antibodies by hem- documented chain of uninterrupted human-to-human trans-
agglutination inhibition or immunofluorescence assays mission lasted six generations [146]. Secondary transmission
[125]. Of these positive sera samples, 420 were tested using may also be influenced by the clade of virus involved; the lon-
a more monkeypox-specific serologic assay (the radioimmu- gest chain of interhuman transmission occurred in the Congo
noassay adsorption test), 73 of which gave positive results Basin, whereas no human-to-human transmission of West
[125]. Prevalence rates did not vary significantly between African monkeypox virus has been documented [139, 147].
the African regions surveyed, providing further epidemio-
logic evidence for a difference in virulence between West
African and Congo Basin monkeypox virus clades when one 4.5 Pathogenesis and Immunity
considers that fewer than 10 % of monkeypox cases and no
fatalities occurred outside of the Congo Basin [125, 134]. The pathogenesis and clinical presentation of monkey-
Initial studies through 1979 evaluating seasonality reported pox resembles that of ordinary smallpox in many respects.
that most cases occurred during the dry season (December, Following exposure, patients enter an asymptomatic incuba-
January, and February), whereas surveillance involving the tion period lasting approximately 12 days (range 7–21 days)
use of monetary incentives to report cases during 1981–1986 [2, 123, 125, 128]. The subsequent prodromal phase is usu-
showed peak incidence in June, July, and August [125, 139]. ally heralded by fever and often accompanied by headache,
The complete host range of monkeypox remains uncer- prostration, back pain, myalgia, and malaise. In contrast to
tain. Humans and monkeys appear to be incidental hosts, and smallpox, up to 90 % of patients with monkeypox also dis-
growing evidence suggests that one or more species of squir- play prominent lymph node enlargement during this phase
rels or rodents serve as the animal reservoir for monkeypox [2]. The lymphadenopathy is most often generalized but can
virus [140–142]. However, monkeypox virus has only been sometimes be localized to the cervical or inguinal regions
isolated from one animal infected in the wild – a squirrel and present as 1–4 cm firm, tender, and occasionally painful
(Funisciurus anerythrus) with skin eruptions captured in lymph nodes [2, 125]. As with smallpox, the rash of mon-
1985 near a village where human monkeypox was previously keypox progresses from macules to papules to vesicles to
identified [140]. More recently, prairie dogs (Cynomys spe- pustules before umbilicating, crusting, and separating to
cies) housed with rodents from West Africa were implicated leave a pitted scar. The rash lesions are typically found in the
as vectors in the North America outbreak in 2003 [132, 143]. same stage of development and are distributed peripherally
This discovery has led to the establishment of prairie dogs as including the palms and soles [2, 125] (Fig. 21.6).
an animal model for monkeypox infection [138]. In total, the illness generally lasts 2–4 weeks. Potential
complications include secondary bacterial infections of the
skin and respiratory tract, severe dehydration secondary
4.4 Mechanisms and Routes to vomiting and diarrhea, and keratitis and corneal lesions
of Transmission which can lead to blindness [123, 125]. The severity of
disease is correlated not only with the source of infection
As a primarily zoonotic infection, monkeypox virus is most (Congo Basin vs. West Africa) but also with the mechanism
commonly transmitted to humans through direct contact of transmission. Monkeypox cases reporting a bite or scratch
with the blood, bodily fluids, or lesion exudates of infected exposure were more likely to experience systemic signs or
animals. The source and types of animal exposures reported symptoms of disease and undergo hospitalization than those
by primary contacts prior to rash onset are varied. Case con- with only noninvasive exposures [148].
trol studies have implicated hunting, skinning, trapping, Recent vaccination against smallpox (within 3–19 years)
eating, cooking, or playing with carcasses of nonhuman pri- is estimated to provide 85 % protection against monkey-
mates, terrestrial rodents, antelopes, gazelles, and tree squir- pox based on comparisons of attack rates between vacci-
rels [123, 125]. These studies, however, were confounded by nated and unvaccinated contacts of index cases in Africa [2,
multiple animal exposures in both controls and cases. 123, 145]. The monkeypox outbreak in North America in
Secondary human-to-human transmission occurs though 2003 provided some evidence that vaccine-induced immu-
inhalation of virus-containing respiratory droplets or direct nity may be maintained for decades and confer a reduced
contact with lesion exudates from an infected person. risk of infection [63, 124]. However, remote vaccination
Additionally, vertical transmission has been reported in one (>30 years) did not provide complete protection against
case of congenital monkeypox [144]. Despite the ability to monkeypox [131, 143, 149].
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 511

Fig. 21.6 Images of the vesiculo

pustular rash of monkeypox.
A centrifugal distribution, and
lesions on the palms and soles,
are characteristic of the
generalized rash

5 Prevention, Control, and Treatment during the eradication campaign such as Dryvax that were
typically propagated in the skin of livestock animals. Second-
The cross-protective immunity provided by smallpox vaccine generation smallpox vaccines are manufactured using mod-
makes vaccination an effective method of prevention for all ern cell culture techniques but are fully replicative live virus
orthopoxvirus infections. Vaccine technology has advanced vaccines believed to have essentially equivalent efficacy and
significantly from the first-generation smallpox vaccines used safety profiles to the first-generation vaccines. ACAM2000®,
512 B.W. Petersen et al.

a clonal derivative of the NYCBOH/Dryvax smallpox vac- has demonstrated anti-orthopoxvirus activity in vitro and
cine, is the prototypical second-generation smallpox vaccine protection against mortality in mouse models of vaccinia
that is currently the principal vaccine stockpiled by the United and cowpox [154, 156, 157]. However, cidofovir has low
States for use in an emergency [117]. Third-generation small- oral bioavailability and must be administered intravenously.
pox vaccines are live virus vaccines that have been attenuated The development of CMX001, a lipid conjugate of cidofovir,
to improve the safety profile and decrease the risk of adverse provides several potential advantages over cidofovir includ-
events in high-risk populations. IMVAMUNE® is one such ing increased oral bioavailability allowing oral administra-
vaccine attenuated through multiple passages in chicken tion, decreased toxicity, and enhanced antiviral activity due
embryo fibroblasts such that it is unable to complete a full to greater cellular uptake [158]. Initial safety and pharmaco-
replication cycle in mammalian cells [150]. Lastly, fourth- kinetic studies in humans have been successful in achieving
generation vaccines remain in development and include pro- predicted therapeutic plasma concentrations with no dose-
tein subunit, DNA, and recombinant vaccines [151]. limiting adverse events reported [158, 159].
The global eradication campaign implemented a strategy ST-246 is another orally bioavailable drug with antivi-
of ring vaccination in combination with rapid case identifi- ral activity specific for poxviruses. It was discovered using
cation and isolation to successfully achieve its goals. These a high throughput screening approach that assessed com-
concepts have been incorporated into modern plans for pounds for their ability to inhibit virus-induced cytopathic
responding to the use of smallpox as an agent of bioterrorism effects [158, 160]. ST-246 acts as an inhibitor of extracel-
[152]. A similar approach would be unlikely to succeed in lular virus formation through by targeting a major enve-
eradicating monkeypox, however, owing to the ability of the lope protein critical to this process [160]. Animal studies
virus to propagate within an animal reservoir. Furthermore, in mice, rabbits, and nonhuman primates have shown
implementing a vaccination program to control monkeypox ST-246 to be efficacious in preventing death following
would be challenging due to the relatively low incidence challenge with lethal doses of vaccinia virus, rabbitpox
in remote areas that are difficult to access, risks of adverse virus, monkeypox virus, and variola virus [160–162]. This
events, and associated costs [153]. As such, most programs protective effect was seen even when treatment was begun
to prevent human infection with monkeypox currently focus up to 3 days after the viral challenge was administered in
on reducing the risk of exposure to animals that can trans- most cases [158]. Clinical evaluation of ST-246 in humans
mit the virus, controlling the trade of animals to prevent the has been limited to safety and pharmacokinetic studies and
spread of virus outside of Africa, and avoiding close physical rare instances of investigational and compassionate use in
contact to limit human-to-human transmission after an out- treating orthopoxvirus infections [86, 87, 90, 163, 164].
break has already occurred. Similarly, vaccinia and cowpox Notably, the development of resistance to ST-246 during
infections can be prevented by avoiding exposures to the ani- the course of treatment of one case of progressive vaccinia
mal vectors. Following smallpox vaccination, proper care of has been documented [164]. The clinical significance of
the vaccination site is also critical in preventing inadvertent this finding remains unclear, however, as the patient did
transmission of vaccinia virus. ultimately recover. ST-246 has recently entered the US
Limited options are available for treating orthopoxvirus Strategic National Stockpile and is intended to be used in
infections after the onset of clinical signs and symptoms. the treatment of smallpox disease in the event of an out-
Vaccinia immunoglobulin is the only product currently break. This would likely occur under an investigational
licensed by the US Food and Drug Administration (FDA) new drug and/or emergency use authorization regula-
for the treatment of an orthopoxvirus infection (i.e., vac- tory mechanism given its current status as an unlicensed
cinia). The licensed indications include eczema vaccinatum, product.
progressive vaccinia, severe generalized vaccinia, vaccinia New options for the treatment of orthopoxvirus infections
infections in persons with skin conditions (e.g., eczema, continue to be sought. For example, a nineteenth-century
burns, impetigo, varicella zoster, or poison ivy), and aberrant therapy for smallpox involving a botanical preparation of the
vaccinia infections in areas that would constitute a special carnivorous plant Sarracenia purpurea has recently dem-
hazard such as the eyes or mouth [110]. onstrated antipoxvirus activity against vaccinia virus, mon-
Though no FDA-approved antivirals are currently keypox virus, and variola virus [165]. However, establishing
licensed for treatment of orthopoxvirus infections, several the efficacy of antiviral therapy in treating human orthopox-
drugs with antiviral activity against these viruses have been virus infections remains difficult due to the low incidence
identified and are in clinical development [154, 155]. One of cases requiring treatment. In particular, evaluation of the
such drug is cidofovir, a nucleotide analog with broad anti- potential synergistic effects of combination antiviral therapy
viral activity against a number of DNA viruses. It is thought suggested by animal studies is not possible with the limited
to inhibit viral DNA polymerases as well as the proofreading human data available [166]. While the FDA has the ability
activity of the poxvirus exonuclease [154, 156]. Cidofovir to approve drugs or license biological products on the basis
21 Orthopoxviruses: Variola, Vaccinia, Cowpox, and Monkeypox 513

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 Paramyxoviruses: Henipaviruses
22
Stephen P. Luby and Christopher C. Broder

1 Introduction close contact with the index mare during the final stages of
her fatal illness. The horse trainer, whose hands and arms
The henipaviruses are species of the genus Henipavirus, fam- had abrasions, attempted to force feed the mare by placing
ily Paramyxoviridae, and subfamily Paramyxovirnae. The his bare hands with food into the sick mare’s mouth. Both the
natural reservoir of the three known henipaviruses, Hendra stable hand and the horse trainer became ill 5–6 days after
virus, Nipah virus and Cedar virus, are old world fruit bats of the death of the mare with fever, myalgia, headaches, leth-
the genus Pteropus (order Chiroptera, suborder, Mega- argy, and vertigo. The stable hand remained lethargic for sev-
chiroptera, family Pteropodidae). Human infection with eral weeks but eventually recovered. The horse trainer
either Hendra or Nipah virus causes a widespread vasculitis developed progressive respiratory failure and died. His
that commonly progresses to a fatal encephalitis or pneumo- autopsy findings were consistent with interstitial pneumonia,
nia. People who recover from acute infection with either of with focal alveolitis and syncytial formation [2]. An identi-
these henipaviruses are at risk of recrudescent infection. cal virus, which was ultimately named Hendra virus, was
grown from samples from both the affected horses and the
affected people [3].
2 Historical Background Nipah virus was first recognized in a large human enceph-
alitis outbreak in peninsular Malaysia [4–6]. The initial cases
Hendra virus, previously referred to as equine morbillivirus, were identified among pig farmers who lived near the city of
was first identified in an outbreak in September 1994 in Hendra, Ipoh within the state of Perak in late September 1998.
a suburb of Brisbane, Queensland, Australia [1, 2]. Nineteen Patients presented with fever and headache; many progressed
horses residing in or immediately next to the outbreak stable to unconsciousness and death [7]. By December 1998, larger
developed illness; 14 horses died. At autopsy the lungs were clusters of similar cases were reported within the Port
heavy and edematous with hemorrhage and frothy secretions in Dickson District of Negeri Sembilan, 300 km south of Ipoh
the airways. Histopathological investigations identified inter- [8]. In March 1999, a novel paramyxovirus was isolated
stitial pneumonia with focal necrotizing alveolitis and syncytial from the cerebrospinal fluid of an affected patient from
giant cells within the vascular endothelium [3]. Sungai Nipah village [9]. Immunostaining demonstrated the
Two employees at the stable, a 40-year-old male stable virus within the pathological lesions confirming it as the
hand and a 49-year-old male horse trainer, had particularly cause of the outbreak [4]. Ultimately, the Malaysian Ministry
of Health reported 283 cases with 109 (39 %) fatalities [6].
Cedar virus, is a henipavirus isolated from urine of Pteropus
S.P. Luby, MD (*) bats in Australia and reported in 2012 [10]. Although Cedar
Center for Innovation in Global Health, Woods Institute
virus readily infects guinea pigs and ferrets in the laboratory,
of the Environment, Stanford University,
Yang and Yamazaki Environment and Energy Building, 473, unlike Hendra or Nipah virus it does not cause serious illness
Via Ortega, Stanford, CA 94305, USA in these animals [10]. Human infection with Cedar virus has
e-mail: [email protected] not been described.
C.C. Broder, PhD RNA consistent with a novel henipavirus was identified
Emerging Infectious Diseases Graduate Program, from feces of Eidolon helvum, the African Straw-colored
Department of Microbiology and Immunology,
fruit bat in Ghana in 2008 [11]. Subsequent sampling of 6 bat
Uniformed Services University of the Health Sciences,
4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA species across 5 African countries have identified a diversity
e-mail: [email protected] of henipavirus like RNA sequences, and the full genome of

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 519

DOI 10.1007/978-1-4899-7448-8_22, © Springer Science+Business Media New York 2014
520 S.P. Luby and C.C. Broder

an apparently novel Henipavirus was characterized [12]. spective individual level diagnostics are only conducted at the
Human infection with African henipaviruses has not been three facilities that have identified the most cases of Nipah
described. virus during the 3 months, January through March, when the
most cases have been identified. To efficiently identify out-
breaks of Nipah virus in Bangladesh over a broader geograph-
ical area and wider seasonality, a complementary surveillance
3 Methodology Used in Epidemiologic activity focuses on identifying clusters of patients with
Analysis encephalitis, that is, at least two people who live within a
30-minute walk of each other and who within 2 weeks of each
3.1 Wild Animal Studies other developed symptoms of meningoencephalitis including
fever, new seizures, or mental status changes [22].
The known henipaviruses are not part of the normal human
microbiota. Their ecological niche is within old world
fruit bats of the family Pteropodidae [13, 14]. Closely 3.4 Laboratory Diagnosis
related henipaviruses have been identified among different
Pteropodidae. Nipah virus infects P. vampyrus, P. hypomela- 3.4.1 Virus Detection
nus, P. lylei, and P. giganteus [15–17]. Hendra virus infects The most definitive confirmation of henipavirus infection is
all four Pteropus species in Australia [18]. RNA fragments recovery of the virus from a patient sample. Both Hendra and
suggestive of closely related henipaviruses were collected Nipah virus grow readily on Vero cell culture [3, 4].
from feces of Eidolon helvum, a fruit bat from family Henipavirus has been successfully cultured from human
Pteropodidae that lives in Africa [19]. These infections of cerebrospinal fluid, respiratory secretions, and urine [9, 23,
genetically related Pteropodidae with genetically related 24]. Both Nipah and Hendra virus can be identified through
henipaviruses suggests that these viruses coevolved with polymerase chain reaction (PCR) using either nested or real-
their Pteropodidae hosts [19, 20]. time platforms. Different laboratories have used different
Although not part of the lifecycle of the henipaviruses, primers. PCR has been used successfully on respiratory
humans and many other mammals are susceptible to infec- secretions, cerebrospinal fluid, blood, and urine [23, 24–28].
tion, infections that can have catastrophic consequences. Two research groups have developed a recombinant vesicu-
When a human infection with henipavirus is recognized, an lar stomatitis virus that expresses the F and G protein of
underlying question is how this bat virus got into a human. Nipah virus (pseudotyped virus) [29–31]. This pseudovirus-
based assay can be used safely in a biosafety level II labora-
tory, has high analytical sensitivity, and provides an
3.2 Outbreak Investigations alternative more rapid assessment of virus neutralization.

Most of what is known of the epidemiology of human infec- 3.4.2 Measurement of Virus-Specific
tion with henipaviruses comes from outbreak investigations. Antibodies
Investigators have conducted in-depth case studies to Although there are no commercially available tests for sero-
describe unique or illustrative situations. This has been par- logical diagnosis of henipavirus in humans, several labora-
ticularly important for Hendra virus, because so few human tories have developed enzyme-linked immunosorbent assays
infections have been recognized. Outbreak investigators (ELISA) to detect antibody against Nipah and/or Hendra
have conducted case–control studies and evaluated cohorts virus. To secure henipavirus antigen for the ELISA, some
of people who were potentially exposed to henipavirus to laboratories grow henipavirus-infected cells in tissue culture
assess risk factors for transmission. and then use various procedures to purify viral materials that
To complement outbreak investigations, scientists have are then used to detect antibody in samples [32–34]. Since
conducted serological surveys to explore the frequency of most countries consider Nipah and Hendra virus as biosafety
unrecognized infection and the risk to groups who were not level IV pathogens, only laboratories with biosafety level
implicated in outbreak investigations. IV facilities can produce reagents, and the disease occurs in
places where there are few such laboratories. Alternatively,
some laboratories have developed recombinant henipavi-
3.3 Surveillance rus antigens [35– 38]. These recombinant-based ELISAs do
not require a biosafety level IV laboratory to develop, but
Recurring Nipah virus outbreaks in Bangladesh have charac- their test performance has not been evaluated against a wide
terized the population at risk sufficiently to permit prospective diversity of samples. Using either viral culture-derived or
surveillance for the virus in hospitals which have repeatedly recombinant henipavirus antigens, laboratories have devel-
identified cases [21]. Nipah virus represents less than 2 % of oped indirect ELISA assays to detect Henipavirus-specific
all cases of encephalitis presenting to these hospitals, so pro- IgG antibodies and capture ELISA to detect Henipavirus-
22 Paramyxoviruses: Henipaviruses 521

specific IgM antibodies [32, 35]. These ELISA assays pro- 4 Biological Characteristics
vide an efficient approach to detect antibody and to screen a
high volume of samples, for example, in a surveillance sys- 4.1 Morphology and Morphogenesis
tem. However, since the system depends on antibody detec-
tion, its diagnostic sensitivity early in illness is suboptimal. Henipavirus particles are enveloped and pleomorphic, rang-
Among patients tested within the first 4 days of illness, 30 % ing in size from 40 to 1,900 nm and can vary from spherical
of those ultimately confirmed as infected with Nipah virus to filamentous forms when imaged by electron microscopy
were anti-Nipah IgM antibody negative by ELISA [33]. [3, 40, 41] (Fig. 22.1).
The envelope carries surface projections composed of the
3.4.3 Clinical Diagnosis of Henipavirus viral transmembrane anchored fusion (F) and attachment (G)
Infection glycoproteins. Hendra virus possesses a double-fringed
During outbreaks in Bangladesh, many people die from appearance of spikes, whereas Nipah virus appears to have a
apparent Nipah virus infection before samples can be col- single fringe of projections [41]. Like other paramyxoviruses,
lected or before they develop IgM antibodies [39]. During the henipavirus genomes are an unsegmented, single-strand,
such outbreaks, clinical assessment is sometimes sufficient negative-sense RNA [42, 43]. At the time of their discovery,
for diagnosis. When previously healthy people develop char- the genomes of Hendra and Nipah virus were the largest
acteristic symptoms of Nipah virus infection – fever with among all members of the Paramyxoviridae family, one factor
new onset seizures or mental status change – and when they that justified their separation into their own genus, Henipavirus
are linked to a laboratory confirmed case, either because they [44]. Henipavirus genomes conform to the “rule of six,” i.e.,
shared a common exposure that was implicated in the trans- the total number of nucleotides is evenly divisible by six [45],
mission of Nipah or because of exposure to the respiratory an important feature in the way the nucleocapsid (N) protein
secretions of a confirmed case, they are generally classified interacts with the viral genomic RNA [43]. The RNA genome
as probable cases [39]. The risk of misclassification is small, in association with the N protein is referred to as the ribonu-
because the rapid onset of a fatal illness with symptoms of cleoprotein core that has a characteristic herringbone appear-
meningoencephalitis is uncommon. It is unlikely that such ance by electron microscopy. The ribonucleoprotein core is
an uncommon event would occur from a different etiology contained within a lipid bilayer (envelope) that is derived from
during a Nipah outbreak. the infected host cell during virus assembly and budding.

a b

Fig. 22.1 Henipavirus particles visualized by electron microscopy. (a) profiles of transversely sectioned nucleocapsids. Arrowhead indicates a
Transmission electron micrograph of Hendra virus negative stained released virus particle with internal nucleocapsids, the membrane is
with 2 % phosphotungstic acid. Arrow indicates the nucleocapsids disrupted. Scale bar represents 200 nm (Images courtesy of the AAHL
being released. Scale bar represents 200 nm. (b) Transmission electron Biosecurity Microscopy Facility, Australian Animal Health Laboratory
micrograph of Nipah virus-infected Vero cells in thin section. Arrow (AAHL) Livestock Industries CSIRO, Australia)
indicates virus budding from the plasma membrane with underlying
522 S.P. Luby and C.C. Broder

The relative gene order of the henipaviruses is conserved as problems (www.outbreak.gov.au). All confirmed human
compared to other paramyxoviruses with the N gene first, fol- Hendra virus infections occurred among people who had
lowed by P (phosphoprotein), M (matrix), F, G, and L (large/ close contact with a sick horse in Queensland, Australia.
polymerase) genes (in a 3′–5′ order) [42]. The N, P, and L Serological studies among people who cared for ill horses,
proteins form a complex that replicates the viral RNA; poly- among healthcare providers who cared for Hendra virus-
merase activity resides within L [43, 45]. The henipavirus P infected humans [23, 56], and among Australians with close
gene is also the largest among the paramyxoviruses and contact with Pteropus bats have identified no evidence of
encodes the V and W proteins through a transcriptional editing asymptomatic infection [57]. Apparently, human infection
mechanism that adds untemplated G nucleotides. An alterna- with Hendra virus occurs rarely.
tive start site within the P gene encodes the C protein [42]. The
henipavirus M protein, which underlies the viral membrane,
organizes viral proteins during virion budding from the host 5.2 Nipah
cell; the Nipah virus M protein can independently bud from
expressing cells forming virus-like particles (VLPs) [46, 47]. 5.2.1 Malaysia/Singapore
Finally, the G and F envelope glycoproteins project from the In the large Malaysian Nipah outbreak in 1998, cases
surface of the virion as well as on infected cells and are essen- occurred almost exclusively among people who worked on
tial for the binding and entry steps of virus infection (reviewed pig farms. Males and persons of Chinese ethnicity were more
in [48]). The G glycoprotein attaches the virion to host cell via likely to be pig farmers in Malaysia and were more likely to
receptors and the F glycoprotein fuses the viral membrane be infected with Nipah virus [58]. Abattoir workers in
with the host cell membrane (reviewed in [49]). The G and F Singapore who handled pigs imported from Malaysia during
glycoproteins are also the principal antigens to which virtually the Malaysian Nipah outbreak also became infected [59].
all henipavirus-neutralizing antibodies are directed and are the Among asymptomatic persons who lived or worked on pig
major components or targets of well-advanced vaccine and farms where cases of human Nipah infection were identified,
antiviral strategies (reviewed in [50]). 11 % had antibody to Nipah virus [58]. The human outbreak
of Nipah virus infection ceased after widespread deployment
of personal protective equipment to people contacting sick
4.2 Physical Properties pigs, restriction on livestock movements, and culling over
900,000 pigs [60]. Malaysian authorities confirmed 265
As enveloped paramyxoviruses, Hendra and Nipah virus cases of human Nipah virus infection and 105 deaths during
would be expected to be more labile to environmental factors the outbreak [4]. Authorities in Singapore confirmed 11
as compared to non-enveloped virions; however, there is some cases and 1 death [5]. Since the outbreak ended in 2013, no
evidence of persistence. A patient with confirmed Nipah virus human or porcine Nipah virus infections have been reported
infection in Malaysia who neither entered nor went near pig from Malaysia or Singapore.
farms prior to his illness worked repairing pig cages [51]. His
illness suggests that pig secretions or excretions remain infec- 5.2.2 Nipah Bangladesh/India
tious at least for hours and perhaps for days. Nipah virus spill- The epidemiology of Nipah virus in Bangladesh/India
over events in Bangladesh have been attributed to contaminated infection has been quite different from Malaysia. In 2001
food [52, 53]. In the laboratory, henipavirus survived greater in Siliguri, India, 47 patients with encephalitis were
than 4 days at 22 °C in pH-neutral fruit bat urine and survived linked in a web of person-to-person transmission among
in various fruit juices from several hours to 4 days depending patients and healthcare workers who had contact with
on temperature and pH [54]. human Nipah cases [26]. Over 70 % of people infected
with Nipah virus in Bangladesh and India have died
[26, 52]. In Bangladesh, all recognized Nipah outbreaks
have occurred in rural communities. Both adults and chil-
5 Descriptive Epidemiology dren have been infected, including many children age
5–15 years [52]. Nipah cases have been remarkably clus-
5.1 Hendra tered in both space and time [61]. All but one of the rec-
ognized human infections with Nipah virus in Bangladesh
All four Pteropus species native to Australia commonly have have occurred in the west, central, and northwest regions.
serum antibody against Hendra virus [18] and three of the Cases have occurred disproportionately in close proxim-
four have been confirmed to shed Hendra virus, at least occa- ity to major rivers. The two reported outbreaks from India
sionally, in their urine [55]. From 1994 to 2012, only seven have occurred within 50 km of the border with Bangladesh
human infections with Hendra virus have been recognized; 4 [26, 62], adjacent to areas where human cases have been
(57 %) died and 1 reportedly has serious, ongoing health recognized in Bangladesh (Fig. 22.2).
22 Paramyxoviruses: Henipaviruses 523

Fig. 22.2 Location of Nipah

outbreaks in Bangladesh/India 2001
2001–2012

2011
2007
India

2012

2001

Bangladesh

2005

India 2007 2003 2008

2007
2004
2010
20082004

100 kilometers Bay of Bengal

Myanmar

Surveillance is particularly intense in these areas had IgG antibody against Nipah virus suggesting prior
where outbreaks have repeatedly been identified, but the infection [64].
Government of Bangladesh maintains national surveillance
for outbreaks and investigates clusters of various sorts of
illness throughout the country. All of the outbreaks which 6 Mechanism and Route
have been confirmed to be Nipah through March 2012 have of Transmission
occurred in this more restricted region. From 2001 to 2014,
human Nipah cases were identified every year except in 2002 6.1 Hendra
and 2006. Primary cases of human Nipah infection are those
apparently acquired through contact with contaminated non- Four species of Pteropus bats, P. alecto, P. conspicillatus, P.
human secretion/excretions. Among 121 primary cases in poliocephalus, and P. scapulatus, live in Queensland,
Bangladesh, all have been identified between December and Australia [55]. Because of changes in their native habitat,
April with 79 (65 %) identified in January. Pteropus bats in Queensland now frequently occupy trees in
Sero-surveys of communities affected by outbreaks in urban communities [65]. Hendra virus has been recovered
Bangladesh [63] have identified a much lower proportion of from urine collected underneath bat roosts of each of these
asymptomatic infections than sero-surveys in Malaysia dur- species [55]. Longitudinal collection of bat roost urine speci-
ing the outbreak. A study of 104 healthcare workers who mens suggests that bat colonies shed Hendra virus intermit-
cared for Nipah patients identified two nursing students who tently without a regular seasonal pattern [55]. Hendra virus
524 S.P. Luby and C.C. Broder

has also been recovered from uterine fluid [13]. Through with heavy exposure to respiratory secretions and without
January 2012, 67 equine infections with Hendra virus have wearing personal protective equipment. Other people with
been recognized [66], though the precise pathways that the close contact with these same horses did not develop Hendra
virus moved from Pteropus bats to horses are unknown. virus infection. These observations suggest that Hendra virus
The first recognized Hendra virus infection occurred in a is not easily transmitted from horse to human. It apparently
pregnant mare that became ill while staying in an open pad- requires a horse that is an unusually efficient transmitter and
dock and was moved to a stable in the Brisbane suburb of a person with a high exposure to infectious secretions.
Hendra and died 2 days later [2]. Between 8 and 11 days
after the mare’s death, 18 other horses residing in or near the
stable became ill. Among 18 horses with clinical illness, 14 6.2 Nipah
died, 12 horses from the Hendra stable, one horse staying in
the paddock adjoining the stable, and one horse living on a 6.2.1 Malaysia
neighboring property that had very close contact with horses There are two Pteropus species native to Malaysia, Pteropus
from the Hendra stable. The index mare was the apparent hypomelanus which is smaller and prefers island habitats
source of all the subsequently infected horses since they and Pteropus vampyrus. Both species commonly have anti-
developed illness within one incubation period (8–11 days bodies against Nipah virus [15], and their colonies intermit-
after the death of the index mare). Whether the virus was tently shed Nipah virus in their urine [68, 69].
transmitted directly from horse to horse or whether transmis- The precise mechanism of transmission of Nipah virus
sion was facilitated by the activities of people caring for the from Pteropus bats to pigs in Malaysia is not confirmed,
horses is unknown. The absence of a successive wave of though the index farm was surrounded by native P. vampy-
infection among horses and the rarity of horse-to-horse rus habitat, and both the index farm and other pig produc-
transmission in subsequent investigations suggest that ers commonly raised mangoes, a food attractive to P.
Hendra virus super spreader horses are exceptional. vampyrus, on the same property where they were raising
In addition to the two employees at the Hendra stable who pigs. Mathematical modeling suggests that multiple spill-
had particularly close contact with the index mare during the overs from bats into the pig population would be necessary
final stages of her fatal illness, other exposures in Queensland to create a dynamic population with sufficient susceptible
that resulted in horse-to-human Hendra virus transmission pigs to sustain Nipah virus transmission within pigs for
included a farm worker who cared for two sick horses, one months [70].
with acute respiratory distress, and the other with a rapid Unlike the severe illness seen in Hendra virus-infected
onset of neurological symptoms. After both horses died, the horses, most pigs infected with Nipah virus had mild illness.
farm worker assisted a veterinary surgeon during the nec- Among three pigs infected with Nipah virus through experi-
ropsy of the two horses [25]. Throughout caring for the mental oral inoculation or sharing a cage with an inoculated
horses and the necropsy, the farm worker never wore gloves, pig, all developed asymptomatic infections [71]. On farms
mask, or protective eyewear. the case fatality among adult-infected pigs ranged from <1 to
A veterinarian developed Hendra infection after conduct- 5 % [72]. A minority of Nipah virus-infected pigs developed
ing a limited autopsy on a 10-year-old horse that died of a more severe illness with fever, agitation, trembling, and
rapidly progressive respiratory illness with large amounts of twitching accompanied by rapid labored respirations,
bloodstained frothy nasal secretions [67]. While not wearing increased drooling, and a nonproductive loud barking cough
gloves or other personal protective equipment, the veterinar- [72]. Pathological examination of severely affected pigs
ian reached deep into the carcass to examine internal organs showed extensive involvement of the lungs with a giant cell
and became heavily contaminated with the horse’s body flu- pneumonia with multinucleated syncytial cells containing
ids. The two autopsy assistants and an adult family member Nipah virus antigen in the lungs and epithelial cells lining
who held the dying animal’s head and were exposed to frothy the upper airways [4]. Nipah virus was recovered from respi-
bloody nasal secretions did not become ill and were sero- ratory secretions of infected pigs, and Nipah virus antigen
negative for Hendra virus infection [67]. was detected in renal tubular epithelial cells [4, 71].
A 33-year-old male veterinarian and a 21-year-old female Humans infected with Nipah virus in Malaysia were more
veterinary nurse became infected with Hendra virus during likely than controls to have direct contact with pigs that
an outbreak that affected five horses in a veterinary practice appeared sick and to have close contact with pigs through
in Thornlands, Queensland [23]. Both the veterinarian and feeding pigs, processing baby pigs, handling dead pigs, and
the nurse performed nasal cavity lavage on a horse during the assisting in breeding, birthing, or medicating pigs [58].
3 days before the horse developed symptoms of what was Abattoir workers in Singapore who developed Nipah virus
later confirmed to be Hendra virus infection. infection were more likely than controls to be exposed to pig
Each of the recognized human cases of Hendra virus had urine or feces from pigs that had been imported from
intimate contact with a Hendra virus-infected horse, usually Malaysia during the Malaysian Nipah virus outbreak [5].
22 Paramyxoviruses: Henipaviruses 525

The isolation of Nipah virus from pigs’ lungs and respira-

tory secretions and the observation that human cases of
Nipah virus infection had more direct contact with pigs than
controls suggests that Nipah virus was transmitted from
infected pigs to humans through contaminated saliva and
possibly urine.
All human Nipah virus infections in the outbreak in
Malaysia/Singapore in 1998–1999 may have been linked to a
single event of Nipah virus transmission from an infected bat
to an immunologically primed pig population, leading to a
sustained porcine epidemic which in turn led to a human epi-
demic. The genomic sequences from Malaysian Nipah virus
isolates from pigs and people were nearly identical [4, 73].
There was little evidence of person-to-person transmis-
sion of Nipah virus in Malaysia. Although asymptomatic
infections were identified among members of the households
Fig. 22.3 Infrared photograph showing a Pteropus bat licking date
of cases [58], these infections may have resulted from shared palm sap from the shaved part of the tree
exposures to infected pig secretions or excretions. A cohort
study enrolled 363 healthcare workers from three hospitals
that cared for Nipah patients [74]. Healthcare workers lick the sap stream [83] (Fig. 22.3). Infrared cameras placed
reported skin (n = 89) or mucus membrane (n = 39) exposure in the seven trees that were the source of fresh date palm sap
to body fluids of Nipah virus-infected patients and needle- drunk by the human Nipah virus cases in the Manikganj/
stick injuries (n = 12). None reported any serious illness, Rajbari outbreak in 2008 identified an average of four P.
encephalitis, or hospital admission. None of the initial 363 giganteus bat visits where the bat licked the sap stream per
serum samples had detectable Nipah virus IgG or IgM anti- tree per night [21].
body by ELISA. Among 293 serum samples collected Nipah virus has not been isolated from a domestic animal
30 days later, three (1 %) were positive for Nipah virus IgG in Bangladesh, but outbreak investigations have linked
antibody, though none had detectable IgM and all three were human Nipah virus cases to apparent domestic animal infec-
negative for anti-Nipah virus-neutralizing antibodies. tions. The index case in the 2001 outbreak in Meherpur
District developed illness on April 20, the latest post-winter
6.2.2 Bangladesh/India onset of any confirmed Nipah virus outbreak in Bangladesh,
Pteropus giganteus is the single Pteropus species currently past the end of the date palm sap season in most communi-
living in Bangladesh. These bats commonly have antibody to ties. Nipah virus cases in Meherpur were eight times more
Nipah virus [17, 75] and urine collected from P. giganteus likely to report contact with a sick cow than controls [17]. In
occasionally contains detectable Nipah virus RNA [76]. the Naogaon outbreak in 2003, Nipah virus cases were six
Drinking raw date palm sap is the most common pathway times more likely than controls to report contact with a pig
of Nipah virus transmission from Pteropus bats to people herd that visited the community 2 weeks before the human
identified in outbreak investigations in Bangladesh [21, 53, outbreak [84]. In 2004, a child developed Nipah virus infec-
77, 78]. Human Nipah virus outbreaks in Bangladesh and tion 2 weeks after playing with two goats that developed an
India coincide with the cool dry date palm sap harvesting illness that began with fever and progressed to difficulty
season [52]. At the beginning of the season, the bark is walking, frothing at the mouth, and death [61].
shaved off of one side of the tree (Phoenix sylvestris) and a Person-to-person transmission of Nipah virus has been
small hollow bamboo tap is placed at the base [79]. In the frequently recognized in Bangladesh/India. The two largest
late afternoon, the date palm sap harvester scrapes the area recognized outbreaks caused by person-to-person transmis-
where the bark was removed so the sap can flow freely and sion include 47 linked cases in the Siliguri, India, 2001 out-
ties a 2–4 l clay pot underneath the tap. During the night, the break mentioned above [26] and a person-to-person chain of
sap rises to the top of the tree, and some sap oozes out from transmission that infected 33 persons with Nipah virus in
where the bark is denuded, flows through the tap, and drips Faridpur, Bangladesh, in 2004 [27]. Nipah virus RNA has
into the clay pot. At daybreak, sap collectors gather the clay been frequently identified in the saliva of Nipah virus patients
pots and often sell some of their sap to village residents who [24, 85]. In Faridpur, Nipah virus cases were more likely than
drink it fresh in the morning. controls to report touching a Nipah virus-infected patient who
Pteropus bats occasionally shed Nipah virus in their saliva later died [27]. Similarly, Nipah virus-infected cases in
[80–82]. Infrared photography confirms that P. giganteus Thakurgaon in 2007 were more likely than uninfected con-
bats commonly visit date palm trees during collection and trols to have been in the same room when the index case was
526 S.P. Luby and C.C. Broder

coughing [75]. Across all recognized outbreaks in Bangladesh Less is known about the memory responses or cell-
from 2001 to 2007, Nipah virus patients who had difficulty mediated immune response to henipavirus infection though
breathing during their illness were more likely to transmit both are likely present. In Nipah virus challenge studies in
Nipah virus than Nipah virus patients who did not have diffi- African green monkeys when animals survive a severe infec-
culty breathing (12 vs. 0 %, p = 0.03) [52]. tion and are rechallenged with very high doses of virus, they
remain completely protected from subsequent infection and
disease (TW Geisbert and CC Broder 2008, unpublished).
7 Pathogenesis and Immunity There are a few published reports on cytokine responses
to henipavirus infection. In vitro infection of endothelial
7.1 Cellular Tropism and Host Range cells with Nipah virus induced increases in the secreted
inflammatory chemokines, monocyte chemotactic protein-1
A remarkable feature of the henipaviruses that separate them (MCP-1), interleukin-8 (IL-8), and interferon-inducible pro-
from all other paramyxoviruses is their exceptionally broad tein 10 (IP-10) or C-X-C motif chemokine 10 (CXCL10),
species tropism and ability to cause fatal disease in multiple and infected cell culture supernatants could induce mono-
vertebrate hosts including humans, monkeys, pigs, horses, cyte and T-lymphocyte chemotaxis [111]. These pro-
cats, dogs, ferrets, hamsters, and guinea pigs, which, in addi- inflammatory chemokines produced by Nipah virus-infected
tion to their bat reservoir hosts, spans 6 mammalian orders endothelial cells in vitro are consistent with the prominent
[81, 86–98]. The henipaviruses use host cell membrane pro- vasculitis observed in infections in vivo. In the hamster
teins as entry receptors and bind to ephrin-B2 and ephrin-B3 model of henipavirus infection, the development of neuro-
using their G glycoproteins [99–102]. Human ephrin-B2 and logical disease was correlated with disruption of the blood–
ephrin-B3 are members of a large family of surface expressed brain barrier (BBB) and upregulation of tumor necrosis alpha
glycoprotein ligands that bind to Eph receptor tyrosine (TNF-á) and interleukin 1 â (IL-1â) and IP-10 (CXCL10),
kinases and both the ephrins and Eph receptors mediate bidi- and these inflammatory mediators are likely important in
rectional cell-cell signaling within the nervous, skeletal, and henipavirus pathogenesis [112]. Nipah virus-infected ham-
vascular systems [103, 104]. The ephrin-B2 and ephrin-B3 sters expressed IP-10 (CXCL10) mRNA in various organs
molecules are highly sequence-conserved proteins including with kinetics that followed the replication of virus. Elevated
among those hosts susceptible to henipavirus infection; the levels of CXCL10 were also identified in brain tissue of
human, horse, pig, cat, dog, and flying fox have amino acid patients with fatal Nipah virus infection from the Malaysian
identities of 95–96 % for ephrin-B2 and 95–98 % for ephrin- outbreak [113].
B3 [105]. Ephrin-B2 expression is prominent in arteries,
arterioles, and capillaries in multiple organs and tissues
including arterial smooth muscle and human bronchiolar 7.3 Antagonism of the Host Interferon
epithelial cells [106], while ephrin-B3 is found predomi- Response
nantly in the nervous system and the vasculature (reviewed
in [107, 108]). The identification of ephrin-B2 and ephrin- Products of the P gene inhibit both double-stranded RNA
B3 as entry receptors for Hendra virus and Nipah virus helps signaling and interferon signaling and the P, V, W, and C
explain both the broad species tropism that the henipaviruses proteins can all antagonize the host interferon response, fea-
possess and the observed distribution of viral antigen within tures that are thought to contribute to viral pathogenicity.
infected animals and people (reviewed in [89, 109]). The details of these mechanisms have been reviewed else-
where [114–116]. The W protein is the most potent inter-
feron antagonist and P protein is the least [117]. However, a
7.2 Immune Response recent study conducted using live virus infection indicated
that interferon signaling remains functional during henipavi-
Henipavirus infection in people and other susceptible ani- rus infection of human cell lines while interferon production
mals induces strong humoral responses. In humans with was inhibited [118]. Further, henipavirus infection of bat
clinical Nipah virus encephalitis, anti-Nipah virus antibod- cells does not induce interferon expression and interferon
ies were detected in sera (71 %) and cerebral spinal fluid signaling is inhibited; these observations suggest that the
(CSF) (31 %) [7]; levels of virus-specific IgM antibodies control of virus infection and lack of disease in these natural
were higher than IgG antibodies in CSF and serum [5, 9, hosts is mediated by mechanisms other than the interferon
110]. Seroconversion of IgG against Hendra virus was response [119]. In addition, recombinant Nipah virus indi-
observed in two human cases of Hendra virus infection in vidually lacking either the V, C, or W proteins antagonized
2008 following an influenza-like illness that progressed to the interferon response, and wild-type virus and in vivo
encephalitis [23]. Nipah virus lacking either the V or C protein were
22 Paramyxoviruses: Henipaviruses 527

significantly less pathogenic. The roles of these proteins in [14, 81] also produced no clinical disease or gross pathologi-
pathogenicity thus appear to be independent of their inter- cal findings even with high doses (50,000 TCID50) of virus
feron-antagonist activity [120]. inoculation; however, bats do seroconvert [81, 128, 129].
Transplacental transmission of Hendra virus in bats can also
occur [129] but fetal tissues show no pathology. Virus shed-
8 Patterns of Host Response ding from experimentally infected bats also occurs but is rare
and has only been observed from urine [14, 81].
8.1 Henipavirus Infection in People
8.2.2 Infection in Spillover Hosts
The initial site of henipavirus replication is ill-defined but is Evaluating henipavirus pathogenesis among various animal
likely within the respiratory system followed by apparent species has been critical for understanding their biology, for
hematogenous systemic spread [110]. Established infection is modeling human disease, and as platforms for testing vac-
characterized by a widespread vasculitis and endothelial cell cines and therapeutics. A major limitation to in vivo henipa-
tropism resulting in multinucleated syncytial cells. There is virus infection studies is the requirement for BSL-4
prominent parenchymal cell infection and pathogenesis of containment which impacts the size and scope of animal
many, if not most, major organs with the brain, lung, heart, challenge studies. These difficulties were compounded by
kidney, and spleen significantly involved [9, 110, 121]. the early observation that henipaviruses did not cause a
Clinically, severe infection in humans presents as a severe pathogenic infection in mice, rats, and rabbits [95, 96].
respiratory disease, encephalitis, or a combination of both [2, Several animal modeling platforms of henipavirus infection
7, 39]. The widespread endothelial cell infection and resulting accurately reflect the pathogenic processes seen in either
vasculitis, thrombosis, and parenchymal cell infection in many naturally infected humans or in economically important live-
organ systems, particularly in the CNS and respiratory system, stock (horses and pigs).
contribute to fatal human henipavirus infection [42, 89, 110]. All cases of natural Hendra virus spillover infections in
Human henipavirus infection can also result in a clini- Australia have been in horses, while Nipah virus infection
cally quiescent period following a recovery from an acute had first occurred in pigs in Malaysia, although dogs, cats,
infection, which can later recrudesce as encephalitis. For and horses were also infected (reviewed in [130]). Hendra or
example, the second recognized fatal case of human Hendra Nipah virus infection in horses results in more severe disease
virus infection occurred in an individual who experienced than either virus causes in pigs (reviewed in [131]). Natural
relapsed encephalitis 13 months after infection [25]. Hendra virus infection in horses is often severe and experi-
Similarly, among cases of human Nipah virus infection in the mental infections are essentially uniformly fatal. The incuba-
Malaysian outbreak, neurological disease frequently pre- tion period is 8–11 days and the animals initially become
sented >10 weeks following recovery from acute encephali- anorexic, depressed, with general uneasiness and ataxia, and
tis [122] and relapsed encephalitis has presented from several become febrile with sweating. A severe and fatal respiratory
months to 11 years following infection [123–125]. An exam- disease will often rapidly progress. Neurological disease can
ination of the first two fatal human cases of Hendra virus also present but is less frequent [128, 132]. Infection is wide-
infection, one from an acute respiratory disease and another spread with an endothelial cell tropism with syncytia [89],
from relapsed encephalitis, demonstrated that Hendra virus and virus can be recovered from a number of internal organs,
can cause both acute encephalitis without clinical signs or including lung, and from saliva and urine [128, 133–135].
relapsed encephalitis similar to Nipah virus [121]. Relapsed Experimental Nipah virus infection of horses has not been
encephalitis is presumably caused by the recrudescence of reported but a naturally infected horse had viral antigen and
virus replication that is restricted to the central nervous sys- nonsuppurative meningitis [89].
tem (CNS) [121, 123]. In natural and experimental Nipah virus infection of pigs,
the respiratory system is a primary target organ of virus repli-
cation and pathology, with viral antigen present in the respira-
8.2 Natural and Experimental Henipavirus tory epithelium and syncytia in small blood and lymphatic
Infections of Animals vessels [71, 89]. The involvement of the CNS appears less
common, with meningitis or meningoencephalitis more com-
8.2.1 Infection in Pteropus Bats mon than encephalitis [71]. Nipah virus infection of Landrace
No clinical disease caused by Hendra virus [13, 126] or piglets resulted in a mild clinical disease with fever and respi-
Nipah virus [69, 127] infection has been reported in natu- ratory signs, but neurological disease could also appear [93].
rally infected fruit bats. Experimental infections with Hendra Virus replication is seen in the respiratory system, the lym-
virus in P. poliocephalus and P. alecto [14, 128, 129] and phoid tissues, and the CNS and was greatest in the respiratory
with Nipah virus in P. poliocephalus and P. vampyrus system but syncytia were less frequent. Virus was recovered
528 S.P. Luby and C.C. Broder

from the respiratory, lymphatic, and nervous systems, and Henipavirus infection of African green monkeys results in a
virus shedding was observed in nasal, pharyngeal, and ocular uniformly lethal disease with low dose challenge by intratra-
fluids. Experimental Hendra virus infection of pigs also cheal inoculation within 7–10 days. Virus infection yields
resulted in a respiratory disease with possible CNS involve- widespread vasculitis and endothelial and arterial smooth
ment. Hendra virus appeared to cause a more severe disease in muscle cell syncytia formation in virtually all organs and tis-
pigs in comparison to Nipah virus, and Hendra virus shedding sues [87, 92]. Subjects develop severe respiratory disease;
was noted in nasal, oral, rectal, and ocular fluids [97]. lungs show significant congestion, hemorrhage, polymerized
Cats were discovered to be susceptible to natural Nipah fibrin, and viral antigen [87]. Monkeys infected with either
virus infection and disease in the Malaysian outbreak [89], Nipah or Hendra virus also exhibit neurological disease,
and cats were later shown to be highly susceptible to infec- along with vascular and parenchymal lesions in the brain
tion and disease by both Hendra and Nipah virus. Hendra including infection of neurons and significant involvement
virus disease in cats is similar to that seen in horses, reveal- of the brainstem.
ing widespread vasculitis and parenchymal lesions in the
lungs and other tissues and organs [89, 133]. Experimental
Nipah virus infection in the cat is similar to Hendra virus 9 Control and Prevention
infection but with more extensive inflammation of the respi-
ratory epithelium [71, 89, 91]. The systemic vasculitis seen 9.1 Preventing Henipavirus Outbreaks
in the cat model is consistent with the resulting pathology of
Nipah virus infection in humans. In utero transmission of Preventing henipavirus transmission from bats to domestic
Nipah virus in cats has also been demonstrated with evidence animals or humans depends on adopting regular practices
of extensive viral replication in many tissues of a pregnant that reduce transmission risk. A high index of suspicion for
adult cat and in fetal tissues [136]. infection in intermediate hosts including horses in
Queensland and pigs in Southeast Asia can identify poten-
8.2.3 Hamsters and Ferrets tially high-risk situations when increased attention to infec-
The golden hamster is the only small animal model of henipa- tion control can be lifesaving. Regular surveillance for
virus infection that accurately reflects human pathology and henipavirus infection among domestic animals can better
was first described with Nipah virus challenge experiments characterize the magnitude of risk and help to focus preven-
[96] and later with Hendra virus [88]. Intranasally infected tive efforts.
hamsters die 9–15 days later, displaying progressive signs of Healthcare providers should maintain a high index of sus-
neurologic disease and breathing difficulties; viral genome is picion for human infection with henipavirus among people
detectable in multiple organ systems and severe pathology is who are at high risk for infection including veterinary health-
most evident in the brain [96]. Hendra virus infection of ham- care providers in Queensland, people who raise pigs in
sters also yields pathology that resembles acute human Nipah Southeast Asia, and people in South Asia who either drink
virus cases [88], including widespread endothelial infection raw date palm sap or who recently provided care for persons
and vasculitis and parenchymal lesions, especially in the CNS. with fever and mental status changes. Early recognition of
Henipavirus infection in hamsters with higher doses of either human cases can identify high-risk situations and help to
virus manifests greater respiratory disease, while lower doses prevent additional infections.
yield greater neurological disease [112].
Ferrets are also an excellent model of henipavirus infection 9.1.1 Preventing Hendra Virus Spillover
and disease [86, 137, 138]. Six to ten days after low dose oral- The first step to prevent human infection with Hendra virus
nasal challenge with Nipah virus, ferrets develop severe respi- is to prevent Hendra virus infection in horses. Although the
ratory and neurological disease with widespread vasculitis and precise pathway the virus moves from Pteropus bats to
parenchymal lesions including the CNS neurons [86, 137]. horses is not defined, through 2009 all infected horses were
Hendra virus-infected ferrets, with doses as low as 50 TCID50, stabled in open paddocks [139]. Prudent steps to minimize
rapidly progressed with severe disease between 6 and 9 days horse exposure to Pteropus saliva, urine, and birth products
following infection [138]. Clinical signs and Hendra virus- include placing horse feed and water containers under shelter
mediated disease were essentially identical to those observed and removing horses from open paddocks when nearby flow-
with Nipah virus infection. Overall, the ferret model repro- ering and fruiting trees are attracting flying foxes [140]. Sick
duces all the hallmarks seen in henipavirus-infected people. horses should be isolated from other horses [140].
Next, all persons who work with sick horses in Queensland
8.2.4 Nonhuman Primates and the surrounding area should recognize that a sick horse
The African green monkey accurately reproduces human may be shedding Hendra virus. The very high levels of expo-
Hendra virus and Nipah virus-mediated disease [87, 92]. sure to respiratory secretions and other body fluids among
22 Paramyxoviruses: Henipaviruses 529

human cases of Hendra virus infection, while most atten- Chloroquine, an antimalarial drug first demonstrated to block
dants were uninfected, suggest that modest steps to improve the proteolytic processing of Hendra virus-F [145], was later
infection control, if consistently applied, could prevent trans- shown to inhibit henipavirus infection in vitro [146]. Both
mission. People who have contact with horses in areas where ribavirin and chloroquine have been evaluated in vivo in ani-
Hendra virus infections have been recognized should mini- mal challenge models. Ribavirin only delayed Nipah virus
mize their contact with equine blood and body fluids espe- disease and death and had no benefit against Hendra virus
cially when a horse develops a rapidly progressive febrile infection in hamsters [147, 148]. Ribavirin therapy also only
illness characterized by respiratory distress and frothy nasal delayed Hendra virus disease and pathogenesis by 1 or
discharge. Horse handlers should regularly wash their hands 2 days in African green monkeys [92]. Chloroquine treat-
with soap and isolate sick horses from other horses and peo- ment, alone or in combination with ribavirin, had no thera-
ple [141]. If Hendra virus infection is suspected, horse han- peutic benefit in ferrets challenged with Nipah virus or in
dlers and veterinary personnel should wear personal hamsters challenged with either Nipah virus or Hendra virus
protective equipment to protect their clothing, skin, and face [137, 147, 148]. Ribavirin is still considered a possible treat-
from contact with horse blood or body fluids. ment for henipavirus infection, though there is little addi-
tional evidence of benefit. In 2008, two Hendra virus-infected
9.1.2 Preventing Nipah Virus Transmission patients showed no discernible benefit after treatment with
People who raise pigs in areas where Pteropus bats live ribavirin [23], and combined treatment with chloroquine and
should remove trees that are attractive to Pteropus bats from ribavirin was unsuccessful in one Hendra virus-infected per-
the immediate area where pigs are being raised. son in 2009 [149].
In Bangladesh, the government is encouraging people to PolyIC12U, a strong inducer of interferon-α and
avoid drink fresh date palm sap, because of the risk of Nipah interferon-β production and a specific activator of the toll-
virus. Researchers have been exploring deploying barrier like receptor 3 pathway (reviewed in [150]), blocked Nipah
skirts around date palm sap trees during collection to prevent virus replication in cell culture and was tested in vivo against
physical access of bats to the sap [79, 83]. These have been Nipah virus infection in the hamster model [148]. Continuous
quite effective in excluding the bats, but only a small minor- administration of polyIC12U for 10 days beginning at the
ity of date palm sap collectors are using barrier skirts. time of Nipah virus challenge prevented lethal disease in five
Combined efforts to discourage fresh sap consumption, or, if of six animals. Further studies using TLR3 agonists such as
people insist on drinking fresh sap, to ensure that such sap PolyIC12U as immunotherapeutic agents appear warranted.
was collected from a tree protected by a date palm skirt, are Heptad peptide-based membrane fusion inhibitors corre-
currently being evaluated. sponding to either of the heptad repeat (HR) domains of a
Preventing person-to-person henipavirus transmission is paramyxovirus F glycoprotein can potently block virus
part of a broader effort to reduce transmission of dangerous infection in vitro (reviewed in [151]). HR-2 peptides have
saliva-transmitted pathogens in low-income countries. In been used more often and act by blocking the formation of
high-income countries, healthcare providers are professionals, the 6-helix bundle structure in F which is required for virus
trained on procedures to reduce risk of infection, and provided and host cell membrane merger. A 36 amino acid HR-2-
with disposable personal protective equipment to reduce their based F glycoprotein sequence (Nipah virus-FC2) [152] was
risk. In low-income countries, where families are the primary the first one tested and it is analogous to the approved HIV-
care providers [142, 143], they have no training in infection 1-specific drug enfuvirtide (Fuzeon™). An HR-2 peptide
control, and they neither have the access to nor can afford derived from the human parainfluenza virus type-3 (hPIV3)
infection control supplies. An active area of research is to F glycoprotein has also proved to be a potent Hendra virus
identify practical strategies for low-resource settings that can fusion inhibitor [153]. A sequence-optimized and cholesterol-
reduce the risk of saliva-transmitted infections. tagged hPIV3-based HR-2-derived peptide was tested in the
Nipah virus hamster model [154] and showed it could pene-
trate the CNS and exhibit some effective therapeutic activity
9.2 Limiting Human Disease against Nipah virus. Peptide fusion inhibitors as henipavirus
therapeutics are actively being investigated.
9.2.1 Antiviral Strategies
Ribavirin, a ribonucleoside analog with broad antiviral activ- 9.2.2 Passive Immunization Strategies
ity, was used in the Nipah virus outbreak in Malaysia. The The henipavirus envelope glycoproteins are the major targets
morbidity and mortality among treated patients were signifi- of virus-neutralizing antibodies (reviewed in [155]). Challenge
cantly lower than was observed among untreated patients studies carried out in hamsters demonstrated that protec-
earlier in the outbreak [144], though it is unclear how much tive passive immunotherapy with either Nipah virus G- or
of this improved survival can be attributed to ribavirin. F-specific polyclonal antiserums or mouse monoclonal anti-
530 S.P. Luby and C.C. Broder

bodies (mAbs) was possible [88, 156, 157]. Using recombinant noted in a Nipah virus challenge and m102.4 postexposure
antibody techniques, henipavirus-neutralizing human mAbs therapy study conducted in African green monkeys (TW
specific for the G glycoprotein were isolated from a naïve Geisbert and CC Broder 2014, submitted).
human phage-displayed antibody library [158]. One mAb, During the 2010 Hendra virus spillover occurrence in
m102, exhibited strong cross-reactive neutralization activity Queensland, Australia, two individuals considered to be at
against both Hendra virus and Nipah virus and was affinity high risk of Hendra virus infection were offered m102.4
maturated yielding mAb m102.4 and converted to IgG1 for- therapy on a compassionate use basis even though no human
mat for production in a CHO-K1 cell line. The m102.4 mAb safety testing had been carried out and it was not recom-
has exceptionally potent neutralizing activity and with 50 % mended for use in humans. There were no adverse reactions
inhibitory concentrations below 0.04 against Nipah virus and resulting from infusion of the mAb and both individuals
0.6 μg/ml against Hendra virus [159]. The epitope recognized remain well. In July 2012, a third asymptomatic individual
by m102.4 maps to the ephrin receptor binding site [160], and in Australia also received a 20 mg/kg dose of m102.4 fol-
it neutralizes all known isolates of Hendra virus and Nipah lowing high-risk Hendra virus exposure, also with no
virus [86]. adverse reaction [162].
The efficacy of m102.4 treatment in vivo has been exam-
ined in both the ferret and African green monkey models of 9.2.3 Active Immunization Strategies
Hendra virus and Nipah virus infection. In a pre- and postex- Active immunization strategies against Nipah virus infection
posure Nipah virus challenge study in the ferret, animals was first examined in hamsters with an attenuated vaccinia
were given a single 50 mg dose (~25 mg/kg) at 24 h before virus strain (NYVAC) using recombinants prepared with
(pre-) or 10 h following (post-) challenge by intravenous either the Nipah virus F and G glycoprotein genes [156]. The
infusion. Virus was administered oronasally as a 5,000 vaccine demonstrated complete protection from Nipah virus-
TCID50 dose of Nipah virus (tenfold the minimal infectious mediated disease but with evidence of Nipah virus replica-
dose – 50 %). Control ferrets became febrile with severe tion and an anamnestic antibody response in challenged
clinical illness and were euthanized by day 8, while one sub- animals. Recombinant canarypox-based vaccine (ALVAC)
ject in the pre-group and all in the post-group were febrile candidates, encoding Nipah virus F and G glycoprotein
with depression, but by day 10 fevers began to abate. At genes, for use in pigs were tested by immunization of
13 days postinfection, 2/3 animals in the pre-group pro- 4-week-old pigs twice within 2 weeks [163]. Each ALVAC
gressed with disease and were euthanized, whereas all other vector was tested alone and in combination, and piglets were
ferrets (3/3 in the post-group and 1/3 pre-group) were free of challenged intranasally with Nipah virus. All pigs were pro-
any disease signs and remained well until study end point. tected from Nipah virus-mediated disease by either vector
Control subjects revealed severe systemic pathology, and the alone or in combination. The individual ALVAC-F- and
two pre-group-treated animals which experienced a delayed ALVAC-G-vaccinated animals had only low levels of detect-
disease course had markedly reduced pathology, but there able virus shedding by nucleic acid analysis but no infectious
was no pathology seen in any of the surviving ferrets [86]. virus could be recovered, while the combined vaccination
Similar findings have been obtained in a Hendra virus chal- with ALVAC-F/G appeared superior with neither infectious
lenge study in ferrets followed by m102.4 postexposure ther- virus nor Nipah virus RNA being detected. These data sug-
apy (J Pallister and CC Broder 2012, unpublished). gest that an ALVAC-G or combination vector formulation
The m102.4 human mAb has also recently been examined could serve as a protective vaccine against Nipah virus dis-
in the African green monkey henipavirus challenge model ease in pigs but the combination of vectors would be needed
[161] as postexposure passive immunotherapy. Fourteen to assure the prevention of virus shedding [163].
monkeys were challenged intratracheally with Hendra virus, A subunit vaccine strategy for henipaviruses has been
and 12 animals were infused twice with a 100 mg dose extensively explored using a soluble oligomeric form of the
(~20 mg/kg) of m102.4 beginning at 10, 24, or 72 h postex- G glycoprotein (sG) [164] and Hendra virus-sG is now com-
posure with the second infusion ~48 h later. All 12 animals mercially deployed as a livestock vaccine [165]. Hendra
that received m102.4 survived infection, whereas the virus-sG, produced by recombinant expression in mamma-
untreated control subjects succumbed to severe systemic dis- lian cell culture and properly N-linked glycosylated [166],
ease by day 8. Animals in a 72 h treatment group, where retains its oligomeric form and ability to bind ephrin recep-
mAb therapy began 3 days after lethal challenge, exhibited tors [99]. Hendra virus-sG elicits potent cross-reactive neu-
neurological signs but recovered by day 16. At study end, tralizing antibody responses in a variety of animals and
there was no evidence of Hendra virus-specific pathology in presents more cross-reactive epitopes (anti-Nipah virus G
any of the m102.4-treated animals. Further, no infectious antibodies) as compared to sG from Nipah virus [167].
Hendra virus could be recovered from any of the m102.4- Henipavirus sG vaccination was first tested in the Nipah
treated animals. Essentially identical findings have been virus cat model with both Hendra virus-sG and Nipah virus-
22 Paramyxoviruses: Henipaviruses 531

sG administered as three 100 μg doses 3 weeks apart. Both 10 Unresolved Problems

immunogens induced a completely protective humoral
immune response against lethal subcutaneous Nipah virus The contribution of strain differences within henipaviruses
challenge [91]. A follow-up study examined lower levels of to observed epidemiological differences is an open question.
pre-challenge neutralizing antibody titers together with a Are some strains of henipavirus more likely to infect humans,
high-dose oronasal challenge of Nipah virus [168]. Two more likely to cause a primary respiratory disease and more
doses of either 50, 25, or 5 μg of Hendra virus-sG formu- likely to cause person-to-person transmission than other
lated spaced by 3 weeks were tested. All vaccinated animals strains? If strain differences confer different infectious com-
were completely protected from a high-dose oronasal chal- petencies, what are the genetic characteristics that underlie
lenge of Nipah virus (50,000 TCID50). Hendra virus-sG these competencies? We do not yet have enough strains of
immunization has also been tested in the ferret model [138]. henipavirus, paired with detailed epidemiological informa-
Ferrets were immunized twice with a 20-day interval with tion to resolve these questions, but continued careful out-
either a 100, 20, or 4 μg dose of Hendra virus-sG. Animals break investigation and collection of additional isolates may
were challenged oronasally with a 5,000 TCID50 dose (ten- provide additional insight. Collecting additional strains may
fold the minimal infectious dose 50 %), and all vaccinated also clarify how much genetic variation occurs within the
ferrets remained free of any signs of fever or clinical dis- henipavirus genome. Ultimately, a better understanding of
ease. Postmortem examination of vaccinated animals genetic diversity and the relationship between genomic dif-
showed all subjects completely normal except one of four ferences and epidemiology would permit a sound assessment
animals in the lowest dose group. There was no evidence of of the risk of spillover of a henipavirus strain capable of sus-
virus or viral genome in any tissues or samples from ani- tained person-to-person transmission.
mals in the 100 and 20 μg vaccine groups. Only a very low Studies in non-Pteropus bats from China and Ghana [171,
level of genome was detected in the nasal washes in one 172], pigs in Ghana [173], and cattle and goats in Bangladesh
animal in the 4 μg group. No virus was recovered from any [174] have identified antibodies against henipavirus, but the
vaccinated animal. serum neutralizes neither Nipah nor Hendra virus-infected
Recently, Hendra virus-sG vaccination of African green cells. These serological results as well as the PCR fragments
monkeys followed by intratracheal Nipah virus challenge of henipavirus identified in feces of E. helvum suggest the
[169] or Hendra virus challenge (Geisbert and Broder, existence of other henipaviruses. Isolating the specific
unpublished) has demonstrated complete protection from viruses and exploring their ecology and the impact of such
henipavirus infection and disease with no recoverable virus infections on animals including humans would broaden our
or evidence of virus shedding. The application of Hendra understanding of the diversity and risk of henipaviruses.
virus-sG as an equine vaccine against Hendra virus infection While human infections with Hendra and Nipah virus
has been tested as a strategy to prevent both infection and have a remarkably high case fatality rate, human infection is
virus shedding [170]. Several vaccination and Hendra virus rare. Australian animal handlers regularly care for sick
challenge studies have been carried out in Australia; at the horses without complications. Bangladeshi villagers have
high-containment biological safety level 4 (BSL-4) facilities been enjoying raw date palm sap for generations with only a
of the Animal Health Laboratories (AAHL), Commonwealth very small proportion of people enjoying this local delicacy
Scientific and Industrial Research Organisation (CSIRO), in suffering any adverse effect. This rarity of adverse outcomes
Geelong. The Hendra virus-sG was used to immunize horses means that most persons at risk for henipavirus infections
(2 doses) and both a high and a low dose of Hendra virus-sG never observe a connection between their own behavior and
antigen were examined. Horses were challenged with a high a health outcome. This represents a substantial barrier to
oronasal dose of Hendra virus (2 × 106 TCID50), and to date, adopting and maintaining behaviors to reduce their risk.
all vaccinated horses have remained clinically disease-free This small number of human infections also means that
with no evidence of virus replication or virus shedding fol- with limited budgets and competing priorities, preven-
lowing virus challenge [165]. Development of the equine tion efforts have to be extremely low cost in order to be
vaccine against Hendra virus was a collaborative effort cost-effective. Although the m102.4 mAb has been remark-
among investigators of the US and Australian Government ably effective in animal studies and is presently in preclinical
laboratories and private industry. On November 1, 2012, the development stages in both the United States and Australia,
Hendra virus horse vaccine called Equivac HeV® was there have been insufficient financial commitments from
released for use in Australia, and it is the first vaccine either government or private industry to support further
licensed and commercially deployed for public use against a developmental costs including human safety testing trials.
BSL-4 agent. Preclinical development of Hendra virus-sG in Because of the high economic value of competition horses,
vaccine formulations that could potentially be suitable for the high mortality associated with Hendra virus infection, and
use in humans is in progress. the subsequent risk to people living in a high-income country,
532 S.P. Luby and C.C. Broder

there is a potential private market among horse owners for an 2. Selvey LA, Wells RM, McCormack JG, et al. Infection of humans
effective livestock vaccine against Hendra virus. Indeed, and horses by a newly described morbillivirus. Med J Aust. 1995;
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would require substantial additional investment. A human malian paramyxoviruses. Nature communications. 2012;3:796.
henipavirus virus vaccine is unlikely to be developed by private 13. Halpin K, Young PL, Field HE, Mackenzie JS. Isolation of Hendra
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Acknowledgments The opinions or assertions contained herein are the reemergence, Bangladesh. Emerg Infect Dis. 2004;10:2082–7.
private ones of the author(s) and are not to be construed as official or 18. Young PL, Halpin K, Selleck PW, et al. Serologic evidence for the
reflecting the views of the Department of Defense, the Uniformed presence in Pteropus bats of a paramyxovirus related to equine
Services University of Health Sciences, and the Centers for Disease morbillivirus. Emerg Infect Dis. 1996;2:239–40.
Control and Prevention. C.C.B. and S.P.L. are supported in part by the 19. Drexler JF, Corman VM, Gloza-Rausch F, et al. Henipavirus RNA
Department of Health and Human Services, National Institutes of in African bats. PLoS One. 2009;4:e6367.
Health, grants AI054715, AI077995, and 07-015-0712-52200. The 20. Halpin K, Hyatt AD, Plowright RK, et al. Emerging viruses: coming
authors thank Sandy Crameri and Dr. Alex Hyatt for generously provid- in on a wrinkled wing and a prayer. Clin Infect Dis. 2007;44:711–7.
ing the electron micrograph images of Hendra virus and Nipah virus 21. Rahman MA, Hossain MJ, Sultana S, et al. Date palm sap linked
and Salah Uddin Khan for providing the image of the Pteropus bat lick- to Nipah virus outbreak in Bangladesh, 2008. Vector Borne
ing date palm sap. Zoonotic Dis. 2012;12:65–72.
22. Naser AM, Hossain MJ, Sazzad HM, et al. Cluster surveillance for
Competing Interests C.C.B is a US federal employee and is coinven- detecting encephalitis outbreaks in Bangladesh. In: International
tor on patents relating to human monoclonal antibodies against Hendra conference on emerging infectious diseases. Atlanta: American
and Nipah viruses and soluble forms of Hendra and Nipah envelope Society of Microbiology; 2012.
glycoproteins and vaccines; assignees are the United States of America 23. Playford EG, McCall B, Smith G, et al. Human Hendra virus
as represented by the Department of Health and Human Services encephalitis associated with equine outbreak, Australia, 2008.
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 Paramyxoviruses: Measles
23
William J. Moss and Diane E. Griffin

1 Introduction 2 Historical Background

Measles is one of the most important infectious diseases of Measles virus is closely related to rinderpest virus, a recently
humans and has caused millions of deaths since its emergence eradicated Morbillivirus pathogen of cattle, and measles
as a zoonotic disease thousands of years ago. For infectious likely evolved as a zoonotic infection in communities where
disease epidemiologists, measles has served as an exemplary cattle and humans lived in close proximity. Measles virus is
model of an acute infectious disease conferring lifelong believed to have become established as a human pathogen
immunity to reinfection. Kenneth Maxcy, the second chair of approximately 5,000–10,000 years ago when communities
the Department of Epidemiology at the Johns Hopkins achieved sufficient population size in Middle Eastern river
University School of Public Health, wrote in 1948, “The sim- valley civilizations to maintain virus transmission [2]. The
plest of all infectious diseases is measles” [1]. Despite the earliest extant descriptions of measles are those of Abu
apparent simplicity, much has been learned about measles in Becr, also known as Rhazes, who wrote a treatise on mea-
the 60 years since Maxcy’s chapter on the epidemiology of sles and smallpox in the tenth century. Interestingly, he con-
infectious diseases. Detailed investigations of the virology, sidered measles to be the more severe of the two diseases.
immunology, epidemiology, and transmission dynamics have Linguistic evidence suggests that measles was recognized as
shown measles to be a much more complex disease than a distinct disease between 450 and 650 AD. In the mid-­
Maxcy’s statement suggests. This ignorance, however, has eighteenth century, Francis Home attempted to prevent
not impeded global measles control. Remarkable progress measles through inoculation of blood obtained from the skin
has been made in reducing measles morbidity and mortality of an afflicted individual, analogous to Jenner’s approach to
through measles vaccination programs. This achievement preventing smallpox.
attests to the enormous public health significance of measles The first to explicitly document the contagious nature
vaccines. However, this progress is threatened by failures to of measles was the Danish physician Peter Panum during
maintain high levels of measles vaccine coverage and popula- an outbreak of measles on the sparsely populated Faroe
tion immunity. Strategies for the global eradication of mea- Islands in 1846. Through careful documentation of clini-
sles are currently being considered. cal cases, Panum provided accurate measurement of the
incubation period for measles and evidence of the long-
term protective immunity conferred by measles [3].
Specifically, Panum observed that adults who acquired
W.J. Moss, MD, MPH (*) measles during the prior outbreak six decades earlier were
Departments of Epidemiology, International Health and the protected from disease, despite absence of exposure to
W. Harry Feinstone Department of Molecular Microbiology
and Immunology, Bloomberg School of Public Health, Johns
measles between epidemics.
Hopkins University, Room E6547, 615 North Wolfe Street, Attenuated and killed measles vaccines were introduced
Baltimore, MD 21205, USA in the 1960s after successful isolation and growth of measles
e-mail: [email protected] virus in tissue culture by John Enders [4] and further attenu-
D.E. Griffin, MD, PhD ation of measles vaccine virus by Maurice Hilleman. As a
W. Harry Feinstone Department of Molecular Microbiology consequence of high levels of measles vaccine coverage, the
and Immunology, Bloomberg School of Public Health,
Johns Hopkins University, 615 North Wolfe Street,
WHO Region of the Americas interrupted indigenous trans-
Suite- E5132, Baltimore, MD 21205, USA mission of measles virus in 2002. The Measles Initiative
e-mail: [email protected] (now the Measles and Rubella Initiative) was founded in

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 537

DOI 10.1007/978-1-4899-7448-8_23, © Springer Science+Business Media New York 2014
538 W.J. Moss and D.E. Griffin

2001 as a partnership committed to reducing global measles of the N protein, with up to 12 % variability between wild-
mortality and has been instrumental in providing technical type viruses. The World Health Organization recognizes
and financial support for measles control activities. eight clades of measles virus (designated A through H) and
24 subclades or genotypes (Fig. 23.1) [9]. New genotypes
may be identified with enhanced surveillance and molecu-
3 Methods for Epidemiologic Analysis lar characterization. As measles control efforts intensify,
molecular surveillance of circulating measles virus strains
3.1  linical and Laboratory Diagnosis of
C can be used to document interruption of measles virus
Measles transmission and identify the source and transmission path-
ways of measles virus outbreaks [10, 11]. Six genotypes
Epidemiologic studies of measles require accurate diag- have not been reported since at least 2000, and an addi-
nosis and valid case series. The characteristic clinical fea- tional five genotypes have not been reported since 2006,
tures of measles are of sufficient sensitivity and specificity despite enhanced surveillance efforts [9], and may be inac-
to have high predictive value in regions where measles is tive. A global database for measles virus genotypes was
endemic and allow for epidemiologic analyses of measles developed by the World Health Organization LabNet to
case series over many decades. However, laboratory con- collate information generated from enhanced measles virus
firmation is necessary, particularly where measles virus surveillance activities (http://www.who-measles.org/
transmission rates are low or in immunocompromised Public/Web_Front/main.php).
persons who may not develop the characteristic clinical
manifestations. Infection with rubella virus, parvovirus
B19, human herpes virus 6, and dengue viruses may 3.3 Modeling Measles Virus Transmission
mimic measles. Detection of IgM antibodies to measles Dynamics
virus by enzyme immunoassay (EIA) is the most com-
monly used method to diagnose measles [5]. Alternatively, Because of characteristic epidemic cycles and historical
seroconversion using IgG-specific EIA or virus neutral- case series, measles has served as a model for understand-
ization assays can be used to diagnose measles based on ing the transmission dynamics of an acute infectious dis-
testing serum or plasma obtained during acute and conva- ease and the impact of vaccination strategies. Mathematic
lescent phases. Serologic surveys to detect measles virus- modeling of measles dynamics was first attempted
specific IgG antibodies using blood or oral fluid samples 100 years ago by Sir William Hamer [12]. The basic prin-
provide age-specific data on population immunity and the ciple underlying early models was the law of mass action,
average age of infection, from which the force of infec- in which the periodicity of measles incidence was described
tion can be derived [6]. as a function of the changing number of infectious and sus-
Measles virus can be isolated in tissue culture from white ceptible persons as they contact one another. The number
blood cells, respiratory tract secretions, and urine, although of susceptible persons, in turn, was determined by the
the ability to isolate measles virus diminishes quickly introduction of new susceptibles through birth and the
after rash onset. Amplification and detection of measles removal of susceptibles through the acquisition of protec-
virus RNA by reverse transcription polymerase chain reac- tive immunity following infection. However, the epidemic
tion (RT-PCR) from blood, urine, and nasal discharge is cycles generated by these simple deterministic models
highly sensitive in detecting measles virus RNA and allows approached a steady state, losing their periodicity. The
sequencing of the measles virus genome for molecular introduction of stochastic (chance) and seasonal processes
­epidemiologic studies. resulted in models of measles epidemics that closely resem-
bled observed data. Further development of these models
allowed for predictions of the impact of vaccination strate-
3.2 Molecular Epidemiology gies on various epidemiologic characteristics of measles
virus transmission, including the increase in the average
Although measles virus is considered to be monotypic, age of infection and the interepidemic period [13]. More
variability within the genome is sufficient to allow for sophisticated models of measles virus transmission dynam-
molecular epidemiologic investigations. Genetic character- ics have investigated the role of nonlinear dynamics in tem-
ization of wild-type measles viruses is typically based on poral and spatial patterns of measles incidence [14],
sequence analysis of the genes coding for the nucleocapsid traveling waves of infection originating in large cities and
(N), hemagglutinin (H), and phospho (P) proteins [7, 8]. spreading to small towns [15], seasonality [16], and the loss
One of the most variable regions of the measles virus of spatial synchrony in measles epidemics following the
genome is the 450-nucleotide sequence at the carboxy-terminal introduction of measles vaccination [17].
23  Paramyxoviruses: Measles 539

Distribution of measles genotypes, 2011. Dat as of 6 February 2012

Incidence:
West Africa in set West Europe Genotypes:
(per 100,000)

B3 <0.1

D4 ≥0.1 - <1

D8 ≥1 -< 5
D9 ≥5

D11 No data reported

G3
Chart proportional to
H1 number of genotypes
5
1

0 2500 5000 Kilometers

Fig. 23.1  Distribution of measles virus genotypes, 2011 (Source: World Health Organization http://www.who.int/immunization_monitoring/
diseases/measles_monthlydata/en/index1.html)

4 Biologic Characteristics host cell. The genome encodes eight p­ roteins, two of which
(V and C) are nonstructural proteins expressed from the P
Measles virus is a spherical, nonsegmented, single-stranded, gene. Of the six structural proteins, P, large protein (L),
negative-sense RNA virus and a member of the Morbillivirus and N form the nucleocapsid housing the viral RNA. The
genus in the family of Paramyxoviridae. Although RNA H, F, and matrix (M) proteins, together with lipids from
viruses have high mutation rates, measles virus is considered the host cell membrane, form the viral envelope. This rela-
to be an antigenically monotypic virus, meaning that the sur- tively simple combination of proteins and ribonucleic acid
face proteins responsible for inducing protective immunity has evolved to be one of the most highly infectious, directly
have retained their antigenic structure across time and space. transmitted agents known and the cause of millions of
The public health significance is that measles vaccines devel- human deaths.
oped decades ago from a single measles virus strain remain In terms of understanding the epidemiology and control
protective worldwide. Measles virus is killed by ultraviolet of measles, the two surface proteins F and H are most impor-
light and heat, and attenuated measles vaccine viruses retain tant. The H protein interacts with F to mediate fusion of the
these characteristics, necessitating a cold chain for transport- viral envelope with the host cell membrane [18]. The primary
ing and storing measles vaccines. function of the H protein is to bind to the host cellular recep-
The measles virus RNA genome consists of approxi- tors for measles virus. The H protein elicits strong immune
mately 16,000 nucleotides and is encapsalated and responses, and the lifelong immunity that follows infection
enclosed in a lipid-­containing envelope derived from the is due primarily to neutralizing antibodies against H.
 540 W.J. Moss and D.E. Griffin

Three cellular receptors have been identified: CD46, CD150 ress in reducing measles incidence and mortality has been,
(SLAMF1), and nectin-4. CD46 is a complement regulatory and continues to be, made in resource-­poor countries as a
molecule expressed on all nucleated cells in humans. SLAMF1, ­consequence of increasing measles vaccine coverage, provision
an acronym for signaling lymphocyte activation molecule fam- of a second dose of measles vaccine through supplementary
ily member 1, is expressed on activated T and B lymphocytes immunization activities (SIA), and efforts by the World Health
and antigen-presenting cells. Nectin-4, the most recently iden- Organization, the United Nations Children’s Fund (UNICEF),
tified receptor, is an adherens junction protein found on epithe- and their partners to target endemic countries for accelerated
lial cells [19, 20]. The distribution of these host proteins and sustained measles mortality reduction. Specifically, this
determines the tissue tropism and cell types infected by mea- targeted strategy aimed to achieve greater than 90 % measles
sles virus. Wild-type measles virus enters cells primarily vaccination coverage in every district and ensure that all chil-
through the cellular receptor SLAMF1, and most vaccine dren received a second dose of measles vaccine. Provision of
strains can use CD46 as a receptor, although wild-type measles vitamin A as a part of polio and measles SIAs has probably
virus may use both CD46 and SLAMF1 as receptors during contributed further to the reduction in measles mortality.
acute infection [21]. Nectin-4 permits measles virus to enter the In 2003, the World Health Assembly endorsed a r­ esolution
basolateral surface of respiratory epithelial cells. that urged member countries to reduce the number of deaths
attributed to measles by 50 % compared with 1999 estimates
by the end of 2005. This global public health target was met,
5 Descriptive Epidemiology with estimated measles mortality reduced by 60 % from an
estimated 873,000 deaths in 1999 (uncertainty bounds
5.1 Disease Burden 634,000–1,140,000) to 345,000 deaths in 2005 (uncertainty
bounds 247,000–458,000) [22]. Further reductions in global
The disease burden due to measles has decreased as a result measles mortality were achieved by 2008, during which there
of several factors. Measles mortality declined in developed were an estimated 164,000 deaths due to measles (uncertainty
countries in association with economic development, improved bounds 115,000 and 222,000 deaths) (Fig. 23.2) [23]. Using a
nutritional status, and supportive care, particularly antibiotic refined model to estimate measles mortality, the number of
therapy for secondary bacterial pneumonia. Remarkable prog- deaths attributed to measles declined 74 % from an estimated

Estimated measles deaths with vaccination Estimated measles deaths in absence of vaccination
95% CI of estimated measles deaths Deaths averted by measles vaccination
with vaccination
1250

1000
Estimated deaths (1000s)

750

500

250

0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Year

Fig. 23.2  Global estimated measles nortality and measles deaths averted (Source: World Health Organization [23])
23  Paramyxoviruses: Measles 541

Fig. 23.3  Epidemic cycles of Measles noftifications in England and Wales, 1950−79
measles cases in England and
35
Wales by week, 1950–1979.
The arrow indicates the 30
beginning of the national
measles vaccination program 25
in 1968 (Source: Fine and
Clarkson [26]) 20

15

10

Number of Measles Cases Notified (in Thousands) 5

0
1950 1952 1954 1955 1958
40

35

30

25
Beginning of
20 national measles
vaccination program
15
10

0
1960 1962 1964 1966 1968
15

10

0
1970 1972 1974 1976 1978
Years

535,000 in 2000 (95 % confidence intervals 347,200–976,400) populations where periodic introduction of measles virus
to an estimated 139,000 deaths in 2010 (71,200–447,800) results in widespread but self-limited outbreaks.
[24]. Prior to this effort, an estimated 30 million cases of mea-
sles occurred each year, with more than one million deaths.
5.3 Temporal Patterns and Seasonality

5.2 Geographic Distribution Measles incidence has a typical temporal pattern characterized
by yearly seasonal epidemics superimposed upon longer epi-
Measles is a global disease but was absent from the Americas demic cycles of 2–5 years or more (Fig. 23.3). These epidemic
prior to contact with Europeans. Measles, in combination with cycles have been well documented over many decades in differ-
smallpox, likely was responsible for large numbers of deaths ent geographic locations and have been characterized analyti-
of Native Americans, facilitating European conquest [25]. cally in both simple and sophisticated mathematic models. In
Progress in measles elimination efforts has resulted in the temperate climates, annual measles outbreaks typically occur in
interruption of measles virus transmission in large geographic the late winter and early spring. These annual outbreaks are
regions, including the Americas. Because of its mode of trans- likely the result of social networks facilitating transmission
mission and high infectivity, measles virus is most readily (e.g., congregation of children at school) and environmental fac-
maintained in densely populated urban settings. Migration of tors favoring the viability and transmission of measles virus
infected persons to rural areas results in outbreaks in suscep- [26]. In the tropics, measles outbreaks have variable relation-
tible rural populations too small to sustain measles virus trans- ships with rainy seasons, which combined with high birth rates
mission. An extreme example occurs in isolated island result in highly irregular, large measles outbreaks [16].
542 W.J. Moss and D.E. Griffin

Measles cases continue to occur during the interepidemic 5.7 Average Age of Infection
period in large populations but at low incidence. The longer
cycles occurring every several years result from the accumu- The average age of measles virus infection depends upon the
lation of susceptible persons over successive birth cohorts rate of contact with infected persons, the rate of decline of pro-
and the subsequent decline in the number of susceptibles fol- tective maternal antibodies, and the vaccine coverage rate.
lowing an outbreak. In the absence of a vaccination program, Young infants in the first months of life are protected against
these longer epidemic cycles tend to occur every 2–4 years. measles by maternally acquired IgG antibodies. An active
Measles vaccination programs that achieve coverage rates in transport mechanism in the placenta is responsible for the
excess of 80 % extend the interepidemic period to 4–8 years transfer of IgG antibodies from the maternal circulation to the
by reducing the number of susceptibles. fetus starting at about 28 weeks’ gestation and continuing until
birth [31]. Three factors determine the degree and duration of
protection in the newborn: (1) the level of maternal anti-mea-
5.4 Reservoir sles virus antibodies, (2) the efficiency of placental transfer,
and (3) the rate of catabolism in the child. Although providing
Humans are the only reservoir for measles virus, a fact impor- passive immunity to young infants, maternally acquired anti-
tant for the potential eradication of measles. Nonhuman ­primates bodies can interfere with the immune responses to the attenu-
may be infected with measles virus and develop an illness simi- ated measles vaccine by inhibiting replication of vaccine virus.
lar to measles in humans, with rash, coryza, and conjunctivitis. In general, maternally acquired antibodies are no longer pres-
However, populations of wild monkeys are not of sufficient size ent in the majority of children by 9 months of age, the time of
to maintain measles virus transmission [2, 27]. routine measles vaccination in many countries [32]. The half-
life of anti-measles antibodies was estimated to be 48 days in
the United States and Finland, but it is shorter in developing
5.5 Incubation Period countries. Women with vaccine-induced immunity tend to have
lower anti-­measles virus antibody concentrations than women
The incubation period for measles, the time from infection to with naturally acquired immunity, and their children may be
clinical disease, is approximately 10 days to the onset of susceptible to measles at an earlier age [33]. Infants born to
fever and 14 days to the onset of rash, with a median of HIV-infected women may have lower levels of protective
12.5 days (95 % confidence intervals 11.8–13.3 days) [28]. maternal antibodies independent of their HIV infection status
The incubation period may be shorter in infants or following and also may be susceptible to measles at a younger age [34].
exposure to a large inoculum of virus, and it may be longer In densely populated urban settings with low vaccination
in adults. During this seemingly quiescent period, the virus is coverage rates, measles is a disease of young children. The
replicating and infecting target tissues. The incubation period cumulative distribution can reach 50 % by 1 year of age, with a
is best measured during outbreaks in which the time of expo- significant proportion of children acquiring measles virus
sure to the index case can be precisely determined. infection before 9 months, the age of routine vaccination. As
measles vaccine coverage increases, or population density
decreases, the age distribution shifts toward older children. In
5.6 Infectious Period such situations, measles cases predominate in school-age chil-
dren. Infants and younger children, although susceptible if not
The infectious period is more difficult to measure than the protected by immunization, are not exposed to measles virus at
incubation period as it requires careful observation of the con- a rate sufficient to cause a large disease burden in this age
tacts of exposed persons prior to the onset of rash. Generally, group. As vaccination coverage increases further, the age dis-
persons with measles are infectious for several days before tribution of cases may be shifted into adolescence and young
and after the onset of rash, when titers of measles virus in the adults, necessitating targeted measles vaccination programs for
blood and body fluids are highest. The fact that measles virus these older age groups.
is contagious prior to the onset of recognizable disease hinders
the effectiveness of quarantine measures. Detection of measles
virus in body fluids by a variety of means, including identifica- 5.8 Infectivity
tion of multinucleated giant cells in nasal secretions or the use
of RT-PCR, suggests the potential for prolonged infectious Measles virus is one of the most highly contagious, directly
periods in persons ­immunocompromised by severe malnutri- transmitted infectious agents, and outbreaks can occur in
tion or human immunodeficiency virus (HIV) infection populations in which fewer than 10 % of persons are suscep-
[29,  30]. However, whether detection of measles virus by tible. Chains of transmission commonly occur among house-
these methods indicates prolonged contagiousness is unclear. hold contacts, school-age children, and healthcare workers.
23  Paramyxoviruses: Measles 543

The contagiousness of measles virus is best expressed by the 5.10 Critical Community Size
basic reproductive number Ro, which represents the mean
number of secondary cases that arise if an infectious case is To provide a sufficient number of new susceptibles through
introduced into a completely susceptible population [35]. Ro births to maintain measles virus transmission, a population
can be empirically measured, although the introduction of size of several hundred thousand persons with 5,000–10,000
measles virus into a completely susceptible population is births per year is required [2]. Measles virus is believed to
rare. In the 1951 measles epidemic in Greenland, the index have become established in human populations about 5,000–
case attended a community dance during the infectious 10,000 years ago when human populations achieved suffi-
period resulting in a Ro of 2000 [36]. Ro also may be esti- cient size in the Middle Eastern river valley civilizations to
mated from the average age of infection (A), the life expec- maintain virus transmission. The critical community size is a
tancy (L), and the duration of protection from maternally key parameter for the persistence of measles virus transmis-
acquired antibodies (M) using the following equation: sion in insular populations [2, 39].
L-M
Ro =
A-M 6  echanisms and Routes
M
The average age of infection can be inferred from age-­ of Transmission
specific seroprevalence data in the absence of vaccination.
The estimated Ro for measles virus is 12–18 [13], in contrast Measles virus is transmitted primarily by respiratory drop-
to only 5–7 for smallpox virus and polioviruses. The high lets small enough to traverse several feet but too large to
infectivity of measles virus implies that a high level of popu- remain suspended in the air for long periods of time. The
lation immunity is required to interrupt measles virus symptoms induced during the prodrome, particularly sneez-
transmission. ing and coughing, enhance transmission. Measles virus also
may be transmitted by the airborne route, suspended on
small particles for a prolonged time [40]. Direct contact with
5.9 Herd Immunity Threshold infected secretions can transmit measles virus, but the virus
does not survive long on fomites as it is quickly killed by
Interruption of measles virus transmission does not require heat and ultraviolet radiation.
that all persons be immunized and protected. If a sufficient
proportion of the population is immune, the probability that
an unprotected person will encounter an infectious individ- 7 Pathogenesis and Immunity
ual is reduced to almost zero. Protection of unvaccinated per-
sons by a reduction in the risk of exposure is referred to as 7.1 Pathogenesis of Measles Virus Infection
herd immunity [37, 38], and the level of population immu-
nity necessary to interrupt transmission is known as the herd Respiratory droplets from infected persons serve as vehicles
immunity threshold (H). The herd immunity threshold can of transmission by delivering infectious virus to respiratory
be derived using analytical models of infectious disease tract mucosa of susceptible hosts. During the incubation
dynamics from the equation period, measles virus replicates and spreads within the
infected host. In the standard model of measles virus patho-
H =1-1/Ro
genesis, viral replication occurs initially in epithelial cells in
where Ro is the basic reproductive number [13]. A number of the upper respiratory tract and the virus spreads to local lym-
assumptions are made in deriving this formula, including the phatic tissue (Fig. 23.4). Replication in local lymph nodes is
unrealistic assumption of homogenous mixing of the popula- followed by viremia and the dissemination of measles virus
tion (i.e., an individual has an equal chance of coming into to many organs, including lymph nodes, skin, kidney, gastro-
contact with any other individual). Nevertheless, this simple intestinal tract, and liver, where the virus replicates in epithe-
equation provides a means of assessing the level of popula- lial and endothelial cells as well as in lymphocytes,
tion immunity required to interrupt transmission based upon monocytes, and macrophages. In a rhesus macaque model,
a measure of infectivity (Ro). For measles, with a Ro of 12–18, the predominant cell types infected by measles virus were
the herd immunity threshold is 93–95 %. This does not rep- CD150+ cells and dendritic cells [41]. Recently, an additional
resent the level of vaccine coverage but the proportion of the model of measles virus pathogenesis was proposed in which
population protected against measles. This level of popula- measles virus enters respiratory epithelial cells from infected
tion immunity cannot be achieved with a single dose of mea- lymphocytes and monocytes through the basolateral surface
sles vaccine, for which the primary vaccine failure rate is [42, 43]. Virus then buds from the apical surface, allowing
about 15 % when administered at 9 months of age. for respiratory transmission.
544 W.J. Moss and D.E. Griffin

Fig. 23.4  Basic pathogenesis a

of measles virus infection. (a) Virus replication
Virus infection starts in the
respiratory tract and then Skin
spreads to infect multiple organs Liver
including lymphoid tissue, liver, Thymus
lungs, and skin. Virus clearance
begins with the onset of rash. Lung
Clearance of infectious virus is Lymphatic tissue
complete 20 days after infection
Spleen
but viral RNA persists at
Blood
multiple sites. (b) Clinical signs
and symptoms begin about 10 Local lymph nodes
days after infection with Respiratory tract
prodromal symptoms of fever,
conjunctivitis and appearance of b Clinical symptoms
Koplik’s spots followed by the
maculopapular rash that lasts
3–5 days. (c) The rash is a
manifestation of the adaptive
immune response with Conjunctivitis Rash
infiltration of CD4+ and CD8+
Cough
T cells into sites of virus
replication and initiation of virus
Fever
clearance. There is a rapid
activation, expansion, and then Koplik’s
contraction of virus-specific spots
CD8+ T cells. The CD4+ T cell
response appears at the same
time, but activation is prolonged.
Measles-virus specific IgM c Immune responses
appears with the rash and is
commonly used to confirm the
CD8T cells
diagnosis of measles. This is
followed by the sustained
synthesis of measles-virus- lgG
specific IgG. Immune suppres-
sion is evident during acute
disease and for many weeks
after recovery. (d) Cytokines and CD4T cells lgM
chemokines that are produced
during infection in sufficient
quantities to be found in Immune suppression
increased concentrations in
plasma are of several distinct d
types. Shortly after infection, Cytokine responses
the chemokine IL-8 is increased.
During rash, IFN-γ and IL-2 are
produced by activated type 1 IFN-γ
IL-2 IL-4
CD4+ T cells and by CD8+ T
IL-8 IL-10
cells. After resolution of the
IL-13
rash, type 2 and regulatory
CD4+ T cells produce IL-4,
IL-10, and IL-13. Dashed line
viral RNA, IFN interferon, IL
interleukin.

0 5 10 15 20
Days after infection

Although measles virus infection is clinically inapparent these processes can be detected. During the incubation period,
during the incubation period, the virus is actively replicating the number of circulating lymphocytes is reduced (lymphope-
and the host immune responses are developing. Evidence of nia). Measles virus can be isolated from the ­nasopharynx and
23  Paramyxoviruses: Measles 545

blood during the later part of the incubation period and in the The duration of protective immunity following wild-type
several-day prodromal period prior to the onset of rash when measles virus infection is generally thought to be life long
levels of viremia are highest. The prodrome ends with the [52]. The immunologic mechanisms involved in sustaining
appearance of the measles rash. The rash results from measles high levels of neutralizing antibody to measles virus are not
virus-specific cellular immune responses and marks the begin- completely understood, although general principles of
ning of viral clearance from blood and tissue. Clearance of immunologic memory probably govern this process.
infectious virus from the blood and other tissues occurs within Immunologic memory to measles virus includes both contin-
the first week after the appearance of the rash, although mea- ued production of measles virus-specific antibodies by long-­
sles virus RNA can be detected in body fluids of some children lived plasma cells in the bone marrow and the circulation of
for at least 3 months using a PCR-based assay [29, 44]. measles virus-specific CD4+ and CD8+ T lymphocytes [53].
Although immune protection is best correlated with the lev-
els of anti-measles virus antibodies, long-lasting cellular
7.2 Immune Responses to Measles Virus immunity almost certainly plays an important role in protec-
tion from infection and disease.
Host immune responses to measles virus are essential for Measles vaccines also induce both humoral and cellular
viral clearance, clinical recovery, and the establishment of immune responses. Antibodies first appear between 12 and
long-term immunity. Early nonspecific (innate) immune 15 days after vaccination and peak at 21–28 days. IgM anti-
responses occur during the prodromal phase of the illness. bodies appear transiently in blood, IgA antibodies are pre-
These innate immune responses contribute to the control of dominant in mucosal secretions, and IgG antibodies persist
measles virus replication before the onset of more specific in blood for years. Vaccination also induces measles
(adaptive) immune responses [45]. The adaptive immune ­virus-­specific T lymphocytes. Although both humoral and
responses consist of measles virus-specific humoral (anti- cellular responses can be induced by measles vaccine, they
body) and cellular responses. The protective efficacy of anti- are of lower magnitude and shorter duration compared to
bodies to measles virus is illustrated by the immunity those following wild-type measles virus infection.
conferred to infants from passively acquired maternal anti-
bodies and the protection of exposed, susceptible individuals
following administration of anti-measles virus immune glob- 7.3 Immune Suppression
ulin [46]. The first measles virus-specific antibodies produced
after infection are of the IgM subtype (Fig. 23.4). The IgM The intense immune responses induced by measles virus
antibody response is typically absent following re-­exposure infection are paradoxically associated with depressed
or revaccination and serves as a marker of primary infection. responses to unrelated (non-measles virus) antigens, lasting
IgA antibodies to measles virus are found in mucosal secre- for several weeks to months beyond resolution of the acute
tions. The most abundant and most rapidly produced antibod- illness [54]. This state of immune suppression enhances sus-
ies are against the N protein, and the absence of antibodies to ceptibility to secondary bacterial and viral infections causing
N protein is the most accurate indicator of seronegativity to pneumonia and diarrhea and is responsible for much of the
measles virus. Although not as abundant, antibodies to H and morbidity and mortality attributed to measles. Delayed-type
F proteins are responsible for virus neutralization and are suf- hypersensitivity (DTH) responses to recall antigens, such as
ficient to provide protection against measles. tuberculin, are suppressed [55], and cellular and humoral
Evidence for the importance of cellular immunity to mea- responses to new antigens are impaired following measles
sles virus clearance is demonstrated by the ability of children virus infection [56]. Reactivation of tuberculosis and remis-
with agammaglobulinemia (congenital inability to produce sion of autoimmune diseases have been described after mea-
antibodies) to fully recover from measles, whereas children sles and are attributed to this state of immune suppression.
with severe defects in T-lymphocyte function often develop Abnormalities of both the innate and adaptive immune
severe or fatal progressive disease [47]. CD4+ and CD8+ T responses have been described following measles virus
lymphocytes are activated in response to measles virus infec- infection. Transient lymphopenia with a reduction in CD4+
tion (Fig. 23.4) [48]. CD4+ T cells secrete cytokines capable and CD8+ T lymphocytes occurs in children following
of modulating the humoral and cellular immune responses measles virus infection. Functional abnormalities of
(Fig. 23.4) [49]. The initial predominant Th1 response (char- immune cells have also been detected, including decreased
acterized by IFN-γ) is essential for viral clearance, and the lymphocyte proliferative responses [57]. Dendritic cells,
later Th2 response (characterized by IL-4) promotes the major antigen-­ presenting cells, mature poorly, lose the
development of measles virus-specific antibodies. CD8+ T ability to stimulate proliferative responses in lymphocytes,
cells are cytotoxic and important for control and clearance of and undergo cell death when infected with measles virus in
infectious virus [50, 51]. vitro [58]. The dominant Th2 response in children recover-
546 W.J. Moss and D.E. Griffin

ing from measles can inhibit Th1 responses and increase Measles
susceptibility to intracellular pathogens [54]. Engagement First day Third day
of CD46 on monocytes suppresses IL-12 production, a of rash of rash
cytokine important for Th1 responses [59]. Engagement of
CD46 and CD3 on CD4+ T cells induces production of
high levels of IL-10 and transforming growth factor Confluent
(TGF)-β, an immunomodulatory and immunosuppressive Koplik’s spots maculopapules
on buccal mucosa
cytokine profile characteristic of regulatory T cells [60].
The role of these cytokines in the immune suppression fol-
lowing measles is supported by in vivo evidence of ele-
vated levels of IL-10 in the plasma of children after Rash
measles virus infection [61]. discrete

8 Patterns of Host Response

8.1 Clinical Disease and Complications

Clinically apparent measles begins with a prodrome charac-

terized by fever, cough, coryza (runny nose), and conjuncti-
vitis (Fig. 23.4). Koplik’s spots, small white lesions on the
buccal mucosa inside the mouth, may be visible during the
Discrete
prodrome and allow the astute clinician to diagnose measles maculopapules
prior to the onset of rash. The prodromal symptoms intensify
several days before the onset of rash. The characteristic ery-
thematous and maculopapular rash appears first on the face
and behind the ears and then spreads in a centrifugal fashion
to the trunk and extremities (Fig. 23.5). The rash lasts for
3–4 days and fades in the same manner as it appeared.
Malnourished children may develop a deeply pigmented rash
that desquamates or peels during recovery [62].
Fig. 23.5  Development and distribution of measles rash (Source:
In uncomplicated measles, clinical recovery begins soon Perry and Halsey [69]. Krugman S. St. Louis: Mosby, 1958; Infectious
after appearance of the rash. Unfortunately, complications Diseases of Children)
occur in up to 40 % of measles cases, and the risk of com-
plication is increased by extremes of age and malnutrition
[62]. Complications of measles have been described in almost Because the rash of measles is a consequence of the
every organ system. The respiratory tract is a frequent site of cellular immune response, persons with impaired cellular
complication, with pneumonia accounting for most measles-­ immunity, such as those with the acquired immunodefi-
associated deaths [63]. Pneumonia is caused by secondary viral ciency syndrome (AIDS), may not develop the character-
or bacterial infections or by measles virus itself. Pathologically, istic measles rash. These persons have a high case fatality
measles virus infection of the lung is characterized by multi- and may develop a giant cell pneumonitis caused by mea-
nucleated giant cells that form when measles virus proteins on sles virus. T-lymphocyte defects due to causes other than
the surface of infected cells facilitate cell fusion. Other respi- HIV ­ infection, such as cancer chemotherapy, also are
ratory complications include laryngotracheobronchitis (croup) associated with increased severity of measles.
and otitis media (ear infection). Mouth ulcers, or stomatitis, Rare but serious complications of measles involve the cen-
may hinder children from eating or drinking. Many children tral nervous system. Post-measles encephalomyelitis compli-
with measles develop diarrhea, which further contributes to cates approximately 1 in 1,000 cases, mainly older children
malnutrition. Eye disease (keratoconjunctivitis) is common and adults. Encephalomyelitis occurs within 2 weeks of
after measles, particularly in children with vitamin A defi- the onset of rash and is characterized by fever, seizures, and a
ciency, and was a frequent cause of blindness. variety of neurologic abnormalities. The ­finding of ­perivenular
23  Paramyxoviruses: Measles 547

demyelination, the induction of immune responses to myelin are at particular risk of death following measles. Measles, in
basic protein, and the absence of measles virus in the brain turn, can exacerbate malnutrition by decreasing intake (par-
suggest that post-measles encephalomyelitis is an autoim- ticularly in children with mouth ulcers), increasing metabolic
mune disorder triggered by measles virus infection. Other demands, and enhancing gastrointestinal loss of nutrients as a
central nervous system complications that occur months consequence of a protein-losing enteropathy [66]. Measles in
to years after acute infection are measles inclusion body persons with vitamin A deficiency leads to severe keratitis,
encephalitis (MIBE) and subacute sclerosing panencephalitis corneal scarring, and blindness [67].
(SSPE). In contrast to post-measles encephalomyelitis, MIBE
and SSPE are caused by persistent measles virus infection.
MIBE is a rare but fatal complication that affects individuals 8.4 Sex Differences
with defective cellular immunity and typically occurs months
after infection. SSPE is a slowly progressive disease charac- Interestingly, some data suggest that measles mortality may
terized by seizures, progressive deterioration of cognitive and be higher in girls. Among persons of different ages and
motor functions, followed by death that occurs 5–15 years across different regions, measles mortality in girls was esti-
after measles virus infection. It most often occurs in persons mated to be 5 % higher than in boys [68]. Although older
infected with measles virus before 2 years of age. historical data and recent surveillance data from the United
States do not support this conclusion [69], if true, the higher
mortality in girls is in contrast to most infectious diseases in
8.2 Measles Mortality and Case Fatality which disease severity and mortality is highest in males.
Supporting the hypothesis of biologic differences in the
The measles case fatality proportion is highest at extremes of response to measles virus was the observation that girls were
age. Vaccinated children, should they develop disease after more likely than boys to have delayed mortality following
exposure, have less severe disease and significantly lower receipt of high-titer measles vaccine [70]. The underlying
mortality rates. Vaccination programs, by increasing the mechanisms are likely differences in immune responses to
average age of infection, shift the burden of disease out of measles virus between girls and boys, although no cogent
the age group with the highest case fatality (infancy), further biological theory has been developed [71].
reducing measles mortality.
Measles case fatality proportions vary widely, depend-
ing upon the average age of infection, nutritional status of 8.5 Host Genetics
the population, measles vaccine coverage, and access to
health care [64]. In developed countries, such as the All persons without preexisting protective immunity are
United States, fewer than 1 in 1,000 children with measles believed to be susceptible to infection with measles virus,
die. In endemic areas in sub-Saharan Africa, the measles and geographic differences in disease severity and mortality
case fatality proportion may be 5 %. Measles is a major are almost certainly due to environmental and nutritional
cause of child deaths in refugee camps and in internally factors rather than genetic differences. Nevertheless, genetic
displaced populations. Measles case fatality proportions factors (e.g., genes regulating cytokine production) may
in children in complex emergencies have been as high as explain some of the differences in response to measles virus
20–30 %. During a famine in Ethiopia, measles alone or between individuals. The host genetics underlying immune
in combination with wasting accounted for 22 % of 159 responses to measles vaccine have been more extensively
deaths among children younger than 5 years of age and studied and suggest that polymorphisms in human leukocyte
17 % of 72 deaths among children aged 5–14 years [65]. antigen (HLA) genes are associated with differences in anti-
body responses [72].

8.3 Nutritional Status

9 Control and Prevention
Measles and malnutrition have important bidirectional inter-
actions. Measles is more severe in malnourished children, 9.1 Measles Vaccines
although separating the independent effects of nutritional and
socioeconomic factors is often difficult. Children with severe The best means of preventing measles is active immuniza-
malnutrition, such as those with marasmus or k­washiorkor, tion with measles vaccine [73]. The first attenuated measles
548 W.J. Moss and D.E. Griffin

vaccine was developed by passage of the Edmonston strain pared to the unvaccinated group (VE = 1 − RR). A number of
of measles virus, isolated by John Enders, in chick embryo field methods and study designs can be used to measure
fibroblasts to produce the Edmonston B virus [74]. Licensed measles vaccine efficacy [81, 82]. For example, measles vac-
in 1963 in the United States, this vaccine was protective but cine efficacy can be estimated by measuring the proportion
also induced fever and rash in many vaccinated children. of measles cases occurring in vaccinated persons and the
Further passage of the Edmonston B virus produced the proportion of the population that is vaccinated. Measles vac-
more attenuated Schwarz vaccine that was licensed in 1965 cine efficacy (VE) then can be calculated using the following
and currently serves as the standard measles vaccine in much equation:
of the world. The Moraten strain (meaning “more attenuated
PPV − PCV
Enders” and licensed in 1968) was developed by Maurice VE =
Hilleman and is used in the United States. Attenuated mea- PPV − ( PCV × PPV )

sles vaccine strains have mutations that distinguish them
from wild-type viruses [75] and exhibit decreased tropism where PPV is the proportion of the population that is vacci-
for lymphocytes [76]. nated against measles and PCV is the proportion of measles
The recommended age of first vaccination varies from 6 to cases that are vaccinated.
15 months and is a balance between the optimum age for sero- Immunologic markers of protective immunity are com-
conversion and the probability of acquiring measles before monly used to assess measles vaccines. Measurement of anti-
that age [77]. The proportions of children who develop protec- bodies to measles virus by the plaque reduction neutralization
tive levels of antibody after measles vaccination are approxi- assay is best correlated with protection from infection and
mately 85 % at 9 months of age and 95 % at 12 months of age remains the gold standard for determination of protective
[78]. Two doses of measles vaccine are necessary to achieve antibody titers. Neutralizing antibody levels of 120 mIU/mL
sufficiently high levels of population immunity to interrupt (or 200 mIU/mL depending upon the WHO reference serum
transmission [35]. The first dose is typically administered used) are considered protective following vaccination [83].
through the primary healthcare system. The World Health
Organization recommends that the first dose of measles vac-
cine be administered at 9 months of age [73], although coun- 9.3 Optimal Age of Vaccination
tries in which the risk of measles is low frequently provide the
first dose at 12–15 months of age. Two strategies to administer The optimal age of measles vaccination is determined by con-
the second dose of measles vaccine are through the primary sideration of the age-dependent increase in seroconversion
healthcare system or mass immunization campaigns called following measles vaccination and the average age of infec-
supplementary immunization activities, an approach first tion. In regions of intense measles virus transmission, the
developed by the Pan American Health Organization (PAHO) average age of infection is low and the optimal strategy is to
for South and Central America and modeled after polio eradi- vaccinate against measles as young as possible. However, both
cation strategies [79]. These campaigns are used to deliver maternally acquired antibodies and immunologic immaturity
other health interventions, including insecticide-treated bed reduce the protective efficacy of measles vaccination in early
nets for prevention of malaria, vitamin A, antihelminthic infancy [84]. In many parts of the world, 9 months is consid-
drugs, and other vaccines, such as rubella vaccine. ered the optimal age of measles vaccination and is the age
The duration of vaccine-induced immunity is at least sev- recommended by the Expanded Programme on Immunization
eral decades if not longer [52]. Secondary vaccine failure (EPI). Most countries following the EPI schedule administer
rates have been estimated to be approximately 5 % at measles vaccine alone, although more countries are introduc-
10–15 years after immunization, but are probably lower when ing combined measles and rubella vaccines as rubella control
vaccination is given after 12 months of age [80]. Decreasing programs expand. In communities with intense measles virus
antibody concentrations do not necessarily imply a complete transmission, a significant proportion of children may acquire
loss of protective immunity, as a secondary immune response measles before 9 months of age. Under some circumstances,
usually develops after re-exposure to measles virus, with a provision of an early dose of measles vaccine at 6 months of
rapid rise in antibody titers without overt clinical disease. age (e.g., in outbreaks or to HIV-infected children) is appropri-
ate. In contrast, in regions that have achieved measles control
or elimination, and where the risk of measles in infants is low,
9.2 Measures of Protection the age of measles vaccination is increased to ensure that a
higher proportion of children develop protective immunity.
Measles vaccine efficacy under study conditions, or effec- For example, in the United States, the first dose of measles
tiveness under field conditions, is measured as 1 minus a vaccine is administered at 12–15 months of age, as a com-
measure of the relative risk in the vaccinated group com- bined measles, mumps, and rubella (MMR) vaccine.
23  Paramyxoviruses: Measles 549

9.4  urveillance and Outbreak

S Several candidate vaccines with some of these charac-
Investigation teristics are undergoing development and testing. Naked
cDNA vaccines are thermostable and inexpensive and could
Disease surveillance is an important component of measles theoretically elicit antibody responses in the presence of pas-
control programs, providing estimates of measles incidence sively acquired maternal antibody. DNA vaccines encoding
and mortality, the effectiveness of the control program, and either or both the measles H and F proteins are safe, immu-
information to support targeted interventions [85]. Case-­ nogenic, and protective against measles challenge in naive,
based surveillance with laboratory confirmation of sus- juvenile rhesus macaques [91]. A different construct, con-
pected cases should be the goal. Not all suspected cases in taining H, F, and N genes and an IL-2 molecular adjuvant,
an outbreak need to be laboratory confirmed if they can be provided protection to infant macaques in the presence of
epidemiologically linked to a confirmed case. As the inci- neutralizing antibody [92, 93]. Alternative techniques for
dence of measles declines, other viral causes of fever and administering measles virus genes, such as alphavirus [86],
rash may be mistaken for measles. Many surveillance pro- parainfluenza virus [94], or enteric bacterial [95] vectors, are
grams test specimens for rubella virus-specific IgM antibod- also under investigation. Oral immunization strategies have
ies. Although confirmation typically requires detection of been developed using plant-based expression of the measles
anti-measles virus IgM antibodies in plasma or serum, less virus H protein in tobacco [96], and dry powder attenuated
invasive specimen collection methods, including oral fluid measles vaccine delivered by inhalation was shown to induce
swabs and dried blood spots collected on filter paper, can protective immunity in rhesus macaques [97].
also be used [5].

10.3 Measles Eradication

10 Unresolved Problems In the WHO Region of the Americas, intensive vaccination

and surveillance efforts interrupted endemic measles virus
10.1 Measles Virus Persistence transmission [98], and all five remaining WHO regions set
measles elimination targets of or before 2020. Progress in
Recent observations of measles pathogenesis in a rhesus reducing measles incidence and mortality in sub-­Saharan
macaque model, in conjunction with observations of pro- Africa [99] led to the proposal to eliminate measles in the
longed detection of measles virus RNA in children [29, 44], WHO African region by 2020 [100].
suggest that measles virus RNA persists in peripheral blood The elimination of measles in large geographic areas, such
mononuclear cells for up to 4 months after infection and is as the Americas, suggests that global eradication is feasible
associated with a biphasic T-cell response with peaks at with current vaccination strategies. Potential barriers to eradi-
7–25 days and 90–110 days [86]. These observations suggest cation include the following: (1) lack of political will, (2) dif-
measles may not be as acute a viral infection as once under- ficulties of measles control in densely populated urban
stood. Persistent measles virus replication could explain the environments, (3) the HIV epidemic, (4) waning immunity
prolonged state of immune suppression and the live-long and the potential transmission from subclinical cases, (5)
immunity following infection [87]. transmission among susceptible adults, (6) the risk of unsafe
injections, and (7) unfounded fears of disease caused by mea-
sles vaccine [101, 102]. Whether the threat from bioterrorism
10.2 Ideal Measles Vaccine precludes stopping measles vaccination after eradication is a
topic of debate, but, at the least, a single-dose rather than a
The ideal measles vaccine would be inexpensive, safe, heat-­ two-dose measles vaccination strategy could be adopted.
stable, and immunogenic in neonates or very young infants
and administered as a single dose without needle or syringe
[88]. The age at vaccination would ideally coincide with
other vaccines in the EPI schedule to maximize compliance
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 Paramyxoviruses: Mumps
24
Steven A. Rubin

1 Introduction Although in modern times mumps is generally thought of

as a disease of childhood, historically it was considered to be
Mumps is an acute contagious disease caused by a non- an illness that affected armies during times of mobilization.
segmented negative strain RNA virus in the paramyxovirus Mumps was a major cause of morbidity among Confederate
family. Infection with the mumps virus typically results in soldiers during the American Civil War [2] and was the lead-
unilateral or bilateral parotitis, frequently accompanied by ing cause of days lost from active duty by US troops in
more significant complications, such as meningitis, pancre- France during World War I [3, 4]. The annual hospital admis-
atitis, or orchitis. Symptoms usually resolve within 2 weeks sion rate for mumps among US soldiers at that time was 55.8
of onset; serious complications are rare. Approximately per 1,000, exceeded only by influenza and gonorrhea [5].
one-third of infections are unrecognized or present with The highest reported rate of mumps in the US Army (75.5 in
nonspecific or primarily respiratory symptoms. Prior to 1,000) occurred in 1918 and declined markedly to 6.9 in
implementation of national mumps immunization programs, 1000 in 1943. Although mumps incidence was greatly
more than 90 % of most populations had serologic evidence reduced during World War II relative to preceding wars, it
of exposure to the virus by adolescence. Following wide- remained a major problem, having a frequency three times
spread use of mumps-containing vaccine, disease incidence higher than that of the next most common condition, measles
declined sharply, particularly in countries with a two-dose [4]. Mumps outbreaks continued to occur in military settings
vaccine program. However, over the past few years there has until the implementation of routine MMR immunization of
been resurgence in mumps cases globally, even in highly military recruits [6].
vaccinated populations. A viral etiology of the disease was suggested in several
experiments conducted in animals in the early part of the
1900s [7–9], but it was not until 1934 that mumps was deter-
2 Historical Background mined to be caused by a virus present in the saliva of infected
patients [10, 11]. In these studies, Johnson and Goodpasture
Mumps was first described by Hippocrates in the fifth cen- injected filtered, bacteria-free material from the saliva of
tury as an illness accompanied by swelling around one or patients with epidemic parotitis into the Stenson’s ducts
both ears and, in some cases, painful swelling of one or both of rhesus monkeys and produced nonsuppurative parotitis.
testes. Central nervous system (CNS) involvement was first A diluted emulsion from the parotids of these monkeys
described in the literature by Hamilton in 1790 [1]. The ety- caused parotitis in susceptible children but not in immune
mological origin of the term mumps is unclear but may stem controls. Ten years following this landmark study, Habel
from the English noun, mump, meaning lump, in reference to [12] and Enders [13, 14] cultivated the virus in developing
the distinctive facial swelling that accompanies the disease, chick embryos, leading to the development of an inactivated
or, alternatively, the name may derive from the mumbling vaccine that was tested in humans a few years later [15]. The
speech of patients with parotitis. first live attenuated mumps virus vaccines were used in the
late 1950s in the Soviet Union [16] and in the early 1960s in
the United States (US) [17].
S.A. Rubin, PhD
Division of Viral Products, Center for Biologics Evaluation
and Research, Food and Drug Administration,
N29A 2C20, HFM-460, Bethesda, MD 20892, USA This chapter is a revised version of Chapter 17. Harry A. Feldman, Viral
e-mail: [email protected] Infections in Humans, 3rd edition. Plenum Press, 1989.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 553

DOI 10.1007/978-1-4899-7448-8_24, © Springer Science+Business Media New York 2014
554 S.A. Rubin

3 The Agent 4 Methodology Involved

in Epidemiologic Analysis
Mumps virus is a member of the genus Paramyxovirus in the
family Paramyxoviridae, genus Rubulavirus, which includes 4.1 Sources of Data
human parainfluenza viruses 2, 4a, and 4b; parainfluenza
virus 5 (formerly simian virus 5); Mapuera virus; porcine The epidemiologic study of mumps is limited by the frequency
rubulavirus (La Piedad Michoacan Mexico virus); simian of asymptomatic infection, the lack of serological testing in
virus 41; and tentative genus members Menangle virus and cases of parotitis with other etiologies misdiagnosed as
Tioman virus. The virus is readily inactivated by heat, forma- mumps, and the failure to report cases. Large outbreaks, as
lin, ultraviolet light, and other agents. well as specific studies, have provided information on inci-
The enveloped mumps virion is polymorphic and con- dence and complications. The experiences during World War
tains a negative-sense RNA genome of 15,384 nucleotides I and World War II have contributed greatly to our under-
encoding seven genes that generally parallel those of the standing of the epidemiology of the disease [3, 4, 45, 46].
other paramyxoviruses. These include the nucleocapsid (N) Mumps was first made a nationally reportable disease in 1922
gene, V gene, matrix (M) gene, fusion (F) gene, small hydro- but was removed from the list of reportable diseases in 1950.
phobic (SH) gene, hemagglutinin-neuraminidase (HN) gene, Mumps was reinstated as a notifiable disease in 1968 follow-
and the large (L) gene [18]. Each gene encodes a single pro- ing the licensure of a live attenuated mumps virus vaccine.
tein, with the exception of the V gene, a faithful transcript of The number of cases of mumps reported each week is pub-
which produces the nonstructural V protein, whereas co- lished in the Morbidity and Mortality Weekly Reports by the
transcriptional addition of non-templated guanine residues Centers for Disease Control and Prevention (CDC), and inci-
produces the phosphoprotein (P) and I protein [19, 20]. The dence data (derived from the National Notifiable Diseases
N protein coats the viral RNA to form the ribonucleocapsid, Surveillance System) are summarized annually [47].
which complexes with the P and L proteins, which together
provide the polymerase activity responsible for transcription,
replication, methylation, capping, and polyadenylation 4.2 Serological Surveys
[21–24]. The M, F, SH, and HN proteins are all associated
with the viral envelope. The M protein is involved in viral Over the years, the seroprevalence of mumps immunity has
assembly, presumably by linking the N proteins of the ribo- been assessed in a number of serological surveys using vari-
nucleocapsid with the cytoplasmic tails of the HN and the F ous methods available at the times the studies were con-
proteins that span the host cell-derived viral envelope [25]. ducted [6, 48–61].
The HN protein is responsible for cellular attachment during
the initial infection process as well as release of nascent viri-
ons from the cell surface postinfection [26, 27]. The HN and 4.3 Laboratory Methods
F proteins work cooperatively to facilitate fusion of the
virion membrane with the host cell membrane and introduce The diagnosis of mumps can be confirmed by isolation of the
the viral nucleocapsid into the host cell. These two viral pro- virus, by detection of viral RNA, or, with some limitations,
teins also induce cell-to-cell fusion following infection [21, by serological testing for virus-specific antibodies. The latter
28]. The V and SH proteins are accessory proteins, acting as also forms the basis for assessing susceptibility to mumps
antagonists of the host antiviral response, with the former infection and responses to vaccination.
interfering with the interferon response [29–31] and the lat-
ter interfering with the TNF-α-mediated apoptotic signaling 4.3.1 Virus Isolation and RNA Detection
pathway [32]. Studies of the role of the I protein in virus The virus can be recovered from most body fluids and secre-
replication and infection are lacking. tions with variable degrees of success. The highest success
The HN and F glycoproteins, being exposed on the virion rates have been achieved with buccal swabs obtained from
surface, are the main targets of the humoral immune response. the area proximal to the Stensen’s duct. The virus can also
Only antibodies directed to these two proteins have been be readily detected in saliva and, in cases of mumps menin-
shown to neutralize virus infectivity and to confer protection gitis, from the cerebrospinal fluid (CSF). Rates of success
[33–36]. Although mumps virus is serologically monotypic with other clinical specimens, such as urine and semen, are
[37], antigenic differences among the various strains of lower. Regardless of the source material, virus detection
mumps virus have been demonstrated in laboratory studies rates dramatically decline after the first week of symptoms.
[38–42], and there is some evidence that such differences Despite the apparent high frequency of viremia during
may allow for breakthrough infections in persons previously mumps (based on the evidence of wide dissemination of the
immunized, particularly upon waning of antibody levels [40, virus), mumps virus or viral RNA has rarely been identified
41, 43, 44]. in blood or serum [62, 63], possibly related to the presence
24 Paramyxoviruses: Mumps 555

of anti-mumps virus antibodies, which begin to appear at in virus-specific IgG can be demonstrated using paired acute
the time of symptoms [64]. and convalescent serum samples, results of serological test-
For virus isolation, a number of cell lines can be used, the ing must be carefully considered. Before the widespread use
most permissive of which are Vero and CaCo-2 [65]. Cultures of vaccine, enzyme immunoassay (EIA) detection of virus-
typically display syncytia formation within 5 days of incuba- specific antibody was reliable for confirming mumps, but in
tion with mumps virus, followed by lysis; however, reliance the present era where nearly everyone has a history of mumps
on cytopathic effects alone is not a reliable means of con- vaccination, both cases and non-cases would be expected to
firming the presence of mumps virus since some mumps be IgM negative and IgG positive. Thus, a negative IgM
strains are non-cytopathic, and many of the viruses in the result no longer rules out mumps, and a positive IgG result
differential diagnosis of mumps cause cellular pathology no longer confirms mumps. It should be noted that some
indistinguishable from that induced by mumps virus. Instead, EIAs are highly sensitive, and although IgM is not produced
confirmation by PCR amplification of viral RNA is often in high amounts in the anamnestic immune response, it is
required. Because of this need, and the ability to directly nonetheless present. Thus, the detection of IgM in a person
assay clinical material by PCR without an intervening in with mumps with a history of vaccination is not an evidence
vitro culture step, virus isolation by culture is often unneces- of primary vaccine failure [86].
sary [44, 66–68]. The success of virus isolation or PCR- While the EIA remains the most commonly used serol-
based detection may be influenced by vaccination status. ogy test, it is worth noting that detection of IgG by EIA
While mumps virus RNA has been detected in >80 % of does not imply the presence of protective antibody in that
clinically diagnosed cases in children with a history of 0 or 1 both virus-neutralizing (protective) and non-neutralizing
dose of mumps-containing vaccine [44, 69, 70], only about antibodies are detected by this method. Seroconversion can
30 % of mumps cases involving children with a history of be demonstrated by EIA even in the absence of demonstra-
two doses of vaccine may test positive [66, 71]. While the ble neutralizing antibody [87]. For the purpose of assess-
reason for this phenomenon has not been established, it is ing protective immunity, only assays that measure levels of
likely due to an attenuated course of virus growth and spread virus-neutralizing antibody, such as the plaque reduction
in fully vaccinated individuals. neutralization assay or the microneutralization assay, are
Data from PCR-based detection of the viral genome is also relevant. Other once prevalent techniques such as comple-
used for molecular epidemiological purposes. Such analyses ment fixation (CF), radioimmunoassay (RIA), hemagglu-
are usually based on the sequence of the 316-nucleotide-long tination inhibition (HI), immunofluorescence assay (IFA),
SH gene, the most variable in the genome and therefore the and hemolysis-in-gel (HIG) are no longer used due to their
genomic region used for genotype classification. Presently, time-intensive nature, relative insensitivity, and/or prob-
12 different mumps virus genotypes have been assigned lems with cross-reactions with other paramyxoviruses [77,
based on the SH gene sequence, designated A–N (excluding 87–92].
E and M, which have been merged with genotypes C and K, Mumps antibody can also be detected in the CSF of
respectively) [72–74]. patients with mumps meningitis. Antibody in CSF is detect-
able within a few days of onset of illness and peaks within 2
4.3.2 Serological Tests weeks. Elevated CSF–serum IgG antibody ratios from sam-
The humoral response to mumps infection follows a course ples collected on the same day indicate mumps infection
typical of that to respiratory viruses. Salivary IgA develops because mumps antibodies are uncommon in the CSF in the
5–7 days after infection and is followed by virus-specific presence of other infections [93–96].
serum IgA and IgM, detectable within a few days of symp- Skin tests are unreliable and should not be used to assess
tom appearance. Levels of IgA and IgM peak 1–2 weeks immunity to mumps [97, 98].
later and decline over the next several weeks [43, 64, 75–78].
During this time, low-avidity serum IgG is produced [64,
77, 79]. Upon a second exposure to mumps virus, a high- 5 Descriptive Epidemiology
avidity, virus-neutralizing IgG is produced, whereas IgM
and IgA antibodies are often Undetectable [43]. Although 5.1 Incidence and Prevalence
virus-specific IgG can persist for many years to a lifetime,
it appears that in the absence of periodic natural boosting, Within a decade after the December 1967 licensure of mumps
antibody levels and antibody effectiveness decline over time vaccine in the United States, the number of reported cases
[37, 80–85], possible resulting in the susceptibility of per- declined by nearly 90 % from 152,209 in 1968 when mumps
sons previously immune. was reinstituted as a reportable disease to 16,817 cases in
Historically, serological testing has been the gold stan- 1978 (Fig. 24.1). Mumps previously had been a report-
dard for a laboratory confirmation of mumps; however, in the able disease between 1922 and 1950. Following a period
present era of high vaccine coverage, unless a significant rise of a record low incidence in the early 1980s, a resurgence
556 S.A. Rubin

Fig. 24.1 Reported cases per Vaccine licensed

100,000 population per year. United
States, 1968–2011. (Data from
Centers for Disease Control and
100
Prevention)
Mumps Cases, by Year, United States, 1986-2011
90
14000

80 12000

10000

Reported Cases
70
8000

Rates per 100,000

6000
60
4000
50 2000

0
40
86 989 992 995 998 001 004 007 010
19 1 1 1 1 2 2 2 2

30 Year

20

10

0
68 71 74 77 80 83 86 89 92 95 98 01 04 07 10
19 19 19 19 19 19 19 19 19 19 19 20 20 20 20

Year

of mumps occurred in the United States between 1986 and disease and the associated lack of need to seek medical care
1987, which resulted in an almost fivefold increase in the in many instances. A serological survey conducted in Florida
annual incidence rate per 100,000 population (1985, 1.1; [55] highlighted an infrequent number of physician contacts
1987, 5.2) [99]. This resurgence was attributed primarily and the failure of physicians to report cases; only 27 % of the
to the low vaccination coverage of adolescents and young 126 cases identified were seen by a physician and only 6 %
adults who were born between 1967 and 1977 [100, 101]. were reported. The estimated annual incidence rate based on
Although the vaccine was licensed in 1967, its use was lim- this study was ten times the rate based on national surveil-
ited until 1977 when it was recommended for routine use lance figures. Low reporting efficiency was identified in sev-
in young children. By 2003, the number of cases of mumps eral other studies [3, 106, 107]. In addition, the high rate of
reported to the CDC reached an all-time low of 231 cases, subclinical mumps infections, estimated to approach 50 % in
representing a 99.9 % decrease in the number of cases from several studies [108, 109], contributes to a gross underesti-
the prevaccine era. Mumps incidence remained at historic mation of the true incidence of infections. On the other hand,
lows until 2006 when the United States experienced its larg- cases reported as mumps based on the presence of parotitis
est mumps epidemic in 19 years with 6,584 cases reported, without laboratory confirmation may not be mumps, since
mostly in the Midwest (Fig. 24.1, inset) [102–104]. Unlike there are other causes of parotitis. However, this is not a
the 1986–1987 mumps resurgence, the mumps outbreaks concern for cases of parotitis reported during an outbreak or
in 2006 were not due to unvaccinated cohorts, as nearly for cases epidemiologically linked, since only mumps virus
all cases occurred in persons with a history of vaccination. causes outbreaks of parotitis.
The number of reported cases precipitously declined in the
2 years that followed but then spiked again in 2009–2010
with focal outbreaks in New York and New Jersey [105]. 5.2 Survey Data
Provisional data for 2011 indicates a return to pre-outbreak
levels. Numerous population surveys have been carried out using
The number of cases of mumps reported each year is gen- questionnaires and interviews, skin tests, and serological
erally considered to be far less than what actually occurs. tests to assess patterns of susceptibility and immunity in
Underreporting results in part from the mild nature of the communities.
24 Paramyxoviruses: Mumps 557

In the 1960s, Harris and coworkers [110, 111] used ques- countries and in the United States was similar (20–25 %). In
tionnaires to study the incidence of mumps among health the older age groups, significantly higher rates of seroposi-
professionals, university faculty members, and households. tivity were found among US children compared to children
During this time, which was before the wide use of mumps in the Middle American countries. Among children 4–6 years
vaccine, cases were most likely among pediatricians and old, 70 % of children in the United States had mumps anti-
least likely among the general university faculty. Among body compared to approximately 55 % of children in the
households, the peak incidence of mumps was found to Middle American countries (p < 0.05).
occur at 7 years of age, and 74 % of the study population Variation in the average age of infection has been found in
reported mumps before 10 years of age. other serosurveys from other parts of the world. In St. Lucia,
An early study by Henle et al. [52] in 1951 showed that 70 % of unvaccinated children were found to be seropositive
70–80 % of persons with a history of mumps had positive by 4 years of age [117]. In contrast, a majority of children from
findings on the CF test and skin tests. Although these tests the Netherlands [118] and from Scotland [119] remained sus-
are now known to be insensitive or unreliable, fewer than 2 % ceptible at this age. In the Netherlands, most children develop
of those who tested positive subsequently acquired mumps. mumps between 4 and 6 years of age, and by 14 years of age,
Early serological surveys were conducted in military pop- 90 % are seropositive. Before vaccination campaigns were ini-
ulations. Serosurveys in the prevaccine era from 1951 to 1962 tiated in Spain, 53 % of 3–5-year-old and 61 % of 6–7-year-
using the CF or HI tests showed that 46–76 % of army recruits old children were immune [120]. The lack of immunity was
in the United States were immune to mumps [48, 56, 57]. associated with rural residency, low socioeconomic status, and
Later serosurveys [49, 58, 59] conducted more than two lack of school attendance or siblings. In the United Kingdom,
decades after the licensure of mumps vaccine and using the before routine mumps vaccination was initiated in 1988, most
more sensitive EIA found that 84–88 % of military recruits cases were in children 6–7 years of age [121]. Subsequent to
were immune. In contrast to World War I data that showed the routine vaccination program, the average age of infection
increased susceptibility among men from rural areas [112], has decreased, perhaps because of increased contacts among
no significant urban–rural differences were found in the later preschool age children [122]. Serological data from England
serosurveys conducted in 1962 and in 1989 [48, 49]. Kelley and Wales from 1990 showed that 70 % of 1–2-year-olds and
and coworkers [49] found that black, non-Hispanic recruits 73 % of 3–4-year-olds were immune [123].
were more likely to be seropositive than those from other Data from the 1999–2004 US National Health and
racial or ethnic groups. Nutrition Examination Survey (NHANES) involving partici-
Serosurveys also have been conducted in civilian popula- pants aged 6–49 years [60] revealed an overall age-adjusted
tions of all ages. Retrospective tests on sera in the World mumps seroprevalence of 90.0 %, with a lower confidence
Health Organization (WHO) Serum Bank at Yale University limit of 88.8 %, well below the 92 % level estimated to be
showed a wide variation in the proportion of seropositive required for herd immunity [124, 125]. Seropositivity was
findings according to geographic area in the prevaccine era higher (93.4 %) among participants in the earliest birth
[50]. Among children 5–9 years of age, mumps antibody was cohort (1949–1956) and lowest (85.7 %) among those born
not found in any of the children from Alaska and in only from 1967 through 1976. Seroprevalence among persons
15 % of those from Tahiti. In contrast, antibody was detected born from 1977 to 1986 and from 1987 to 1998 (the young-
in more than half of 5–9-year-olds from New Haven, est cohort in the study) was 90.1 and 90.3 %, respectively.
Connecticut (52 %), the Cape Verde Islands (55 %), the Seroprevalence was higher among US-born non-Hispanic
Bahamas (74 %), and Iceland (79 %). Among persons of all blacks (96.4 %) and non-US-born Mexican Americans
ages residing in remote islands off Alaska, only 7 % of resi- (93.7 %). Sex, education, and birthplace were also found
dents of St. Paul Island [113] and 12 % of those from St. to be independent predictors in some cohorts. As discussed
George Island [114] were seropositive when assessed in the in Sect. 9, seroprevalence in young children is likely higher
late 1960s. Similarly, mumps antibody was found in an aver- now than indicated by the 1999–2004 NHANES, given that
age of only 33 % of members of isolated Indian tribes in the median coverage with two doses of MMR vaccine among
South America [115]. In comparison, mumps antibody was kindergartners during the 2011–2012 school year in the
found in 90 % of the residents of New Haven and 88 % of United States was estimated to be approximately 95 % [126].
medical students tested in another seroprevalence study
using the neutralization test [97].
Kenny et al. [116] measured mumps-neutralizing anti- 5.3 Epidemic Behavior and Contagiousness
body in sera collected between 1970 and 1973 in children
1–15 years old in the Dominican Republic, Honduras, the Mumps outbreaks typically occur wherever children and
Republic of Panama, and the United States. The proportion young adults aggregate, as in schools, military barracks, and
of seropositive 1–3-year-old children in the Middle American other institutions. Mumps is less contagious than measles or
558 S.A. Rubin

chickenpox. Hope-Simpson [127] demonstrated the infec- United States, mumps outbreaks typically occur when a
tiousness of mumps by assessing the incidence of cases sufficient number of susceptible persons accumulate to sup-
resulting from every exposure of a susceptible person in the port an outbreak. It follows that the incidence is higher in
home and concluded that the susceptible–exposure attack more densely populated areas. Recent data has shown that
rate for mumps was 31.1 % compared to 75.6 % for measles mumps outbreaks can even occur within highly vaccinated
and 61.0 % for chickenpox. The higher rate of subclinical populations, particularly among groups of individuals in
infection with mumps may account for an underestimation close contact, such as university campus environments.
of the true infectiousness of the virus; however, the higher Investigations of three different mumps outbreaks in the
average age at infection observed for mumps [128] supports United States occurring in 2006, all on college/university
the less efficient transmission of the virus. campuses, revealed that 96–100 % of cases occurred in per-
Outbreaks in virgin populations have been used to esti- sons with a history of vaccination during childhood, most
mate the contagiousness of mumps. In three isolated island with two doses [66, 130, 131]. Mumps outbreaks among
groups off Alaska, where no outbreak of mumps had occurred high two-dose vaccinated populations were also identified in
for more than 50 years and approximately 90 % of persons close-contact environments in other countries [132–134].
tested were seronegative [113, 114, 129], clinical mumps There is ample evidence of waning of mumps immunity
occurred in 35–65 % of the population and an additional postvaccination [81, 83–85, 135–140], and this is likely a
20–24 % had subclinical infections following the introduc- significant contributory factor in these outbreaks.
tion of mumps virus. Nucleotide sequencing of clinical isolates (see Sect. 4.3.1)
indicates that virus genotypes D and G predominately circu-
late in the Western hemisphere, whereas genotypes F, C, and
5.4 Geographic Distribution I predominately circulate in the Asia-Pacific region and gen-
otypes B, H, J, and K in the Southern hemisphere (Fig. 24.2).
Mumps occurs worldwide with the exception of isolated Co-circulation of multiple genotypes within a region, or even
island groups and remote, sparsely populated areas. In the within an outbreak, does occur. Some have suggested that

West Europe Legend

B
C
D
F
G Pie slice size proprotional to the
number of years each genotype
H was reported 2005-2011.
I
5
J 1
K 0 2,500 5000 Kilometers

Fig. 24.2 Distribution of reported mumps genotypes, 2005–2011. (Source: courtesy of WHO, with permission http://www.who.int/immuniza-
tion_monitoring/diseases/mumps/en/index.html, accessed September 11, 2012)
24 Paramyxoviruses: Mumps 559

certain virus genotypes are more virulent than others [141– of cases were reported among older children and adoles-
143]; however, this has not been adequately examined and cents aged 10–19 years and among young adults [147–149].
remains to be demonstrated. Between 1988 and 1993, persons 15 years of age and older
have accounted for more than one-third of reported cases with
known age compared to an estimated 8 % in this age group
5.5 Temporal Distribution during the 5 years following licensure [150, 151]. By 1992, the
highest age-specific incidence shifted back to the 5–9-years-
In the North Temperate Zone, mumps occurs more frequently old age group, remaining in this age group until the multistate
in winter and spring than at other times of the year. This pat- mumps outbreak in 2006 when the age-specific incidence
tern has not changed since the wide use of vaccine and the shifted again to older age groups [103, 152] before returning to
associated dramatic reduction in incidence. Seasonal differ- the younger age group after the outbreak [153, 154]. This pat-
ences are not apparent in tropical areas. Epidemiologic tern again repeated itself during the recent mumps outbreaks in
reports suggest an interepidemic period for mumps of New York and New Jersey in 2009–2010 [105]. Mumps inci-
approximately 3 years [121, 122, 144]. dence by age group since 1990 is presented in Fig. 24.3.
The 1986–1987 resurgence reflected underimmunization
of the cohort born between 1967 and 1977, a period of time
5.6 Age Distribution when mumps vaccine was not administered routinely to chil-
dren and the risk for exposure to mumps was decreasing
In the prevaccine era, the largest proportion of cases occurred [146]. More recent outbreaks, which have occurred in highly
in children 5–9 years of age. Although mumps vaccine was vaccinated populations, are thought to be due to waning
licensed in the United States in 1967, it was not until 1977 that immunity (see Sect. 5.4).
the Advisory Committee on Immunization Practices (ACIP)
recommended routine vaccination of all children 1 year of age
or older [145]. In the years following, the average age of cases 5.7 Gender Distribution
began to increase, reflecting protection in younger popula-
tions. This shift to older age groups was first observed in 1982 During non-outbreak years when only sporadic cases are
[146]. A more marked shift occurred during a resurgence of reported, a trend toward higher attack rates is apparent
mumps in 1986–1987 when a disproportionately high number among males than females (Fig. 24.4), likely a consequence

4
≥40
Cases per 100,000

3 25-39
18-24
)
rs

2 10-17
ea
(y

5-9
p
ou

1 1-4
gr
e

<1
<1
Ag

0
1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

Year

Fig. 24.3 Annual mumps incidence by age group, 1990–2011, as reported to the CDC. Figure courtesy of Albert Barskey, National Center for
Immunization and Respiratory Diseases, CDC
560 S.A. Rubin

Fig. 24.4 Mumps incidence by 3.00

gender, as reported to the CDC, *
1995–2010, from Summary of 2.50 males
Notifiable Diseases (http://www. females
cdc.gov/mmwr/mmwr_nd/index.
html). * Denotes years during 2.00
which large focal outbreaks

Rate (per 100,000 population)

significantly contributed to the 1.50
total number of reported cases
*
1.00 *
0.50

0.25

0.00
95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10
19 19 19 19 19 20 20 20 20 20 20 20 20 20 20 20

of mumps in postpubertal males being frequently compli- rates among black persons ranged from 1.2 to 8.2 times those
cated by orchitis, and, thus, cases among males are more of other racial groups during the 4-year period of 1990–1993
likely to be seen by physicians and to be reported. However, [151]. Since that time, the trend has changed with somewhat
during mumps outbreaks, several investigations found higher higher rates of infection among whites (Fig. 24.5). In non-
attack rates in females [131, 139, 155–157]. This was par- outbreak years, incidence rates among other racial/ethnic
ticularly apparent during the multistate outbreak of mumps populations have been significantly higher than among blacks
in the United States in 2006. It has been postulated that and whites. However, caution must be used when drawing
higher attack rates among females may be a reflection of conclusions from these data as different racial/ethnic popula-
gender differences in social behaviors facilitating increased tions might have different patterns of access to health care,
transmission or exposure, but this is not clear [131, 155]. In regional differences in reporting compliance may exist, and
contrast, during the 2009–2010 mumps outbreaks in the many outbreaks may target racially/ethnically concentrated
United States, attack rates were much higher among males, populations due to circumstances unrelated to race/ethnicity.
but this was a consequence of the outbreak setting, all male Persons engaged in occupations that involve contact with
schools within orthodox Jewish communities in New York persons in age groups or settings associated with the highest
and New Jersey [105]. Fewer than 3 % of cases associated incidence of mumps are likely to have frequent exposures to
with the 2009–2010 mumps outbreaks occurred among per- the mumps virus. A large mail survey conducted in the late
sons outside this community. 1960s when mumps was primarily a disease of young chil-
Factoring out gender-specific manifestations (e.g., orchi- dren showed that the risk for acquiring mumps from an occu-
tis, oophoritis, mastitis), rates of complications are similar in pational exposure was highest among practicing pediatricians
males and females, with the exception of neurological and lowest among university staff [110]. In this study, the
manifestations, which appear with a 3:1 or greater male– risk of mumps among teachers was related inversely to the
female ratio [117, 158–160]. age of their students. With recent outbreaks mostly occurring
on university campuses, it would stand to reason that occu-
pational exposure is now higher among university staff than
5.8 Race and Occupation among practicing pediatricians; however, this has not been
examined.
Historically, incidence rates among the black population
tended to be higher than among the white population. This
was first shown in the military and has been demonstrated 5.9 Occurrence in Different Settings
subsequently in studies of civilian outbreaks [161, 162].
Based on data from 28 states in which race and ethnicity Any setting in which a pool of susceptible persons are in
were reported for at least one-half of the cases, incidence close contact facilitates the transmission of the mumps virus.
24 Paramyxoviruses: Mumps 561

Fig. 24.5 Mumps incidence by

race/ethnicity, as reported to the 2.0 American Indian/Alaskan Native *
CDC, 1999–2010
Asian/Pacific Islander
Hispanic
1.5
Black

Rate (per 100,000 population)

White
1.0

0.5

0.4
*
0.3
*
0.2

0.1

0.0
99 00 01 02 03 04 05 06 07 08 09 10
19 20 20 20 20 20 20 20 20 20 20 20

5.9.1 Families and Schools [49, 58, 59] (see Sect. 5.2). Mumps outbreaks in military
Families and schools have had an important role in the trans- settings continued into the postvaccine era [163, 164] but
mission of mumps [106]. Children exposed to mumps at appeared to have tapered off by the mid-1990s, coinciding
school introduce the virus into the household where it may with the US military’s practice of routinely administering
spread to susceptible family members. Prior to routine vac- MMR vaccine to all recruits without regard to prior vaccina-
cination of young children, the younger children in a family tion status [6, 165]. The exception to this was the Air Force,
usually acquired mumps at an earlier age than their oldest which targeted MMR vaccination to recruits lacking sero-
sibling. During that time period, most outbreaks occurred in logic evidence of immunity to measles or rubella [49, 166].
school settings, with peak incidence in children during the
first 5 years of school attendance. In the present era of 5.9.3 Hospitals
decades of high vaccine coverage among young children, Mumps is a relatively uncommon cause of nosocomial dis-
peak incidence has shifted to older age groups, presumably ease. Sporadic cases resulting from transmission in hospitals
due to waning immunity in those individuals. Indeed, the have been documented, including transmission from asymp-
time since the last vaccination has been linked to declining tomatic staff [98, 167–169]. Data from a study in an area
levels of mumps virus-specific antibodies [81, 83, 135, 136], with widespread outbreaks of mumps suggested that the
decreased vaccine effectiveness [85, 137, 138], and an introduction of mumps into hospitals by employees or by
increased odds of being a case [84, 139, 140]. patients is likely when local incidence is high [170]. In a
retrospective cohort study conducted at a hospital in Chicago,
5.9.2 Military in a state reporting 795 cases of mumps during the 2006 mul-
Historically, epidemiologic data were derived largely from tistate mumps outbreak, nine mumps cases were found to
studies of military populations (see Sect. 2). The seasonal- have resulted in 339 exposures, 98 % of whom were hospital
ity of mumps was recognized by the high proportion (70 %) staff [171]. The cost to the institution, which experienced
of cases occurring during winter and spring. The incidence ongoing transmission for 4 weeks, was calculated at $29,199
was higher among blacks than among whites. Mumps was per mumps case.
associated with high rates of hospitalization and days lost
from duty. Mortality was low, although deaths resulting
from secondary infections such as pneumonia were more 5.10 Other Factors
common during World War I than in later times when effec-
tive treatments for these complications became available. Socioeconomic status has not been an important risk factor
Seroprevalence studies carried out among military recruits for mumps, except among impoverished populations where
in the prevaccine era found larger proportions of susceptible overcrowded living conditions facilitate the transmission of
individuals [48, 56, 57] than were found in later serosurveys mumps virus as well as other infective agents.
562 S.A. Rubin

6 Mechanisms and Routes lase levels may be elevated as a result of inflammation and
of Transmission tissue damage in the parotid gland [191]. Viral excretion in
saliva clears with the appearance of mumps-specific secretory
Humans remain the only known reservoir of mumps virus. IgA antibody about 5 days after onset [109, 173, 174].
Although a virus with over 90 % similarity to mumps virus Involvement of the CNS is common and may occur
at the amino acid level was recently identified in bats [172], whether or not parotitis is present [192]. More than one-half
the only established route of transmission of mumps is of clinical mumps infections involve the CNS as measured
person-to-person. In 1948, Henle and colleagues transmit- by pleocytosis of the CSF [193, 194], although only a frac-
ted mumps to mumps-naïve children by following both oral tion of these cases present with overt CNS symptoms. The
and nasal routes of inoculation, suggesting that infection virus can be recovered from the CSF early in the course of
is spread through droplets containing mumps virus infect- meningitis [192, 195]. Experimental infection in hamsters
ing the upper respiratory tract [109]. The spread of mumps suggests that mumps virus enters the CSF through the cho-
among persons in close contact also suggests this mode of roid plexus, permitting distribution of the virus through the
transmission. Mumps virus may be present in the saliva of ventricular pathways and the subarachnoid space [189].
infected individuals for up to 7 days before onset of clinical Based on experimental infection studies, virus-infected cho-
illness [173] and 8–9 days following onset; however, it is roidal and ependymal epithelia become inflamed and slough
usually present from 2 to 3 days before and 4–5 days after off into the CSF, a postulated mechanism for obstructive
onset of symptoms [174]. The virus also is present in the hydrocephalus and aqueductal stenosis commonly observed
saliva of persons with inapparent infections [109]. Outbreak in laboratory animals infected intracerebrally [196–198].
investigations performed during the prevaccine era demon- Notably, hydrocephalus is a complication of mumps in
strate that most transmissions occur before symptom appear- humans, although relatively uncommon [199, 200]. While
ance or during the first few days of symptom onset [106]. encephalitis can also develop, it too is rare, occurring in less
Transmission of certain vaccine strains of the virus has also than 0.5 % of clinical cases [158, 159, 201]. Encephalitis is
been demonstrated, but is an uncommon event [175–181]. probably a result of viral penetration of the brain paren-
In addition to horizontal transmission, data from animal chyma by spread from contiguous ependymal cells that line
studies and from case reports of humans provide evidence the ventricular cavities of the brain [18]. Viral entry into neu-
that mumps virus can also be transmitted transplacentally rons allows the virus to spread widely along neuronal path-
[182–186]. ways. In addition to the evidence for viral persistence from
laboratory studies of cell cultures [202–205] and from ani-
mal models [189, 206], cellular responses and oligoclonal
7 Pathogenesis and Immunity humoral responses have been shown to persist within the
CNS of patients, implying continued antigenic stimulation
During the incubation period of 16–18 days (range, 12–25 and suggesting the persistence of the virus [18, 207–209].
days) following exposure, it is believed by inference from The association of such persistence with late-occurring pro-
related viruses that the mumps virus multiplies in the upper gressive CNS disease has been suggested [210].
respiratory mucosa and spreads to the regional lymph nodes, Mumps epididymo-orchitis is also common, occurring in
resulting in transient viremia. Despite presumed viremia, the up to 30 % of cases in postpubescent males. Epididymo-
virus has only been infrequently isolated from the blood orchitis is believed to occur by direct invasion of testicular
[63], possibly due to the coincident development of humoral cells by the virus, as evidenced by the recovery of the virus
antibody. Both through experimental infection of animals, from the semen and testis during orchitis [211, 212], although
laboratory studies on human samples, and in vitro studies, an indirect immune-mediated mechanism also has been pos-
viremia appears to be predominately cell associated, with tulated [213]. The pathology in cases of epididymo-orchitis
activated T lymphocytes being the most likely infected cell involves both Leydig and germ cells and can lead to altered
type [63, 187–189]. Cell-free viremia has been suggested by levels of hormones, including decreased testosterone pro-
the isolation of virus from clarified human serum on one duction [214–216]. Hypofertility can result but is uncom-
occasion [63], but this has not been subsequently reported. mon. Sterility is exceptionally rare.
The occurrence of viremia during mumps explains the Renal involvement occurs frequently and is mild. One
wide dissemination of the virus, as indicated by multiple group of investigators detected viruria in 80 % of specimens
organ involvement. Parotitis or other salivary gland involve- collected within the first 5 days [217], and in another study
ment is evident in nearly all clinical cases, a function of the of young servicemen, viruria persisted in some patients for
clinical case definition being based on salivary gland swell- as long as 25 days following onset [218]. All of these men
ing. Viral replication in the parotid gland produces periductal had some abnormal renal function tests; however, none had
interstitial edema and local inflammation, primarily involving generalized edema or hypertension, and all had negative cul-
lymphocytes and macrophages [190]. Serum and urine amy- tures and normal renal function at the end of the study.
24 Paramyxoviruses: Mumps 563

Involvement of the pancreas is characteristic of experi- in seropositive individuals [227] and are largely dependent
mental mumps, and human pancreatic beta cell cultures are on T lymphocytes with IgG receptors [228].
permissive to the virus [219]. Pancreatic involvement in
patients with mumps is generally limited to epigastric pain;
however, extensive damage can occur, resulting in hemor- 8 Patterns of Host Response
rhagic pancreatitis, but this has rarely been reported [220].
Although uncommon, transient or permanent deafness 8.1 Common Clinical Features
is one of the distinctive features in mumps, and mumps
virus is the most frequent cause of acquired unilateral sen- Mumps virus infections may be asymptomatic or associated
sorineural hearing loss in children. The pathogenesis of only with nonspecific or respiratory symptoms in more than
deafness in mumps is unclear but likely involves retro- one-half of persons infected [108, 229]. Inapparent infection
grade penetration of the virus from cervical lymph nodes may be more common in adults than in children, and paroti-
into the perilymphatic fluid, resulting in infection of the tis may be more common in children. Among persons with
cochlea and causing damage to the organ of Corti and the clinically apparent disease, host responses vary, depending
tectorial membrane [221–225]. No pathogenic link has on which organs may be affected. The major clinical mani-
been demonstrated between deafness in mumps and other festations of mumps and their frequency are presented in
complications. Table 24.1, gleaned from a review of data from 47 mumps
Humoral immunity is most likely the primary mode of outbreaks involving at least 20 cases.
protection. Studies of infections by related viruses demon- Swelling and tenderness of the salivary glands are com-
strate cellular responses to also be important, particularly in mon, primarily the parotid glands; however, sublingual and
the recovery and long-term protection from disease; how- submandibular glands also may be involved. Parotitis may
ever, the significance of cell-mediated immune responses in be unilateral or, more commonly, bilateral, peaking on about
mumps infections is unclear. In persons with severely com- the third day of illness and resolving within 10 days. A pro-
promised T cell responses, the course of mumps was found dromal period may precede the parotitis by several days with
to not differ from that seen in healthy individuals [226], sug- nonspecific symptoms, including fever, malaise, myalgia,
gesting a limited, if any, role of cellular immune responses in headache, and anorexia. In some patients other glandular
mumps symptoms and duration. Similarly, neither severe tissues may be infected, causing epididymo-orchitis [230],
symptoms of mumps nor a protracted course of illness has oophoritis, mastitis [231], pancreatitis [220], or thyroid-
been reported in persons with AIDS. Despite this, in vitro itis [232]. Pleocytosis of the CSF is common [193, 194],
lymphocyte proliferative responses to mumps antigen occur but only in a fraction of such cases is aseptic meningitis

Table 24.1 Major clinical

Frequency (%)
manifestations of mumps
Manifestation Mean Range 90 % confidence interval Number of studies
Glandular
Parotitis or other salivary gland swelling 98 83–100 96,99 49
Epididymo-orchitis 1 13 1–39 11,16 46
Oophoritis 4 <1–17 1,7 9
2
Mastitis 10 <1–31 2,18 6
Pancreatitis 4 <1–27 1,6 22
Neurologic
Meningitis 3 5 <1–33 3,7 35
Encephalitis 0.7 <1–2 0.1,1 8
Other
Myocarditis 4 6 1–15 2,10 5
Nephritis 0.4 <1–1 0,1 2
Deafness (transient or permanent) 2 <1–7 <1,4 9
Data sourced from Vaccine, 6th ed., Rubin and Plotkin, Mumps Vaccine, p.420, Copyright Elsevier 2013, with
permission
1
Male patients 12 years of age or older
2
Female patients 12 years of age or older
3
Includes symptoms described as severe headache and nuchal rigidity
4
Based on electrocardiogram abnormalities
564 S.A. Rubin

Table 24.2 Incidence of infection and clinical disease in a virgin soil 8.2 Involvement of the Central Nervous
outbreak of mumps in 561 residents of St. Lawrence Island, Bering Sea System
Feature Number Percent
Mumps infectionsa 460b 82 Mumps virus is as an important cause of aseptic meningitis
Males All infections 300b 53 and encephalitis [235–241]. The high frequency of involve-
Clinical infection 205 68 ment of the CNS in mumps infection demonstrated by
Asymptomatic infection 95 32 numerous studies conducted over the past 50 years has led to
Females All infections 261b 47 CNS involvement frequently being considered part of the
Clinical infection 158 61
natural history of the disease.
Asymptomatic infection 103 40
Many studies of CNS involvement have categorized asep-
Both sexes All infections 561 100
tic meningitis and encephalitis together under the term
Clinical infection 363b 65
“meningoencephalitis” rendering it difficult to differentiate
Asymptomatic infection 198b 35
Total infection ratec 85
between these two CNS complications that have markedly
Clinical mumps Any manifestation 363b 65 different clinical courses and prognoses. Variability in inci-
Salivary gland swelling 344 95 dence rates also reflects the case definition used, the skill of
Stiffness or neck 40 11 the observers, the age distribution of the population, whether
Scrotal swellingd 52d 25 cases are hospitalized or are diagnosed as outpatients, and
Swelling of breastse 24 15 the frequency of the use of lumbar puncture.
Data derived from Philip et al. [129] CSF pleocytosis most likely occurs in 40–65 % of patients
a
Based on serological data with mumps, but symptoms of meningeal irritation are evident
b
Of total population of 561 in fewer than 30 % of closely followed cases [193, 242].
c
An additional 3 % had disease without antibodies
d
Of cases in males
Reported rates of meningitis diagnosed during outbreaks range
e
Of cases in females widely from less than 1 % to approximately 20 % (Table 24.1).
The CSF profile consists of normal opening pressure,
apredominance of lymphocytes with cell counts commonly
clinically apparent, much less so for encephalitis. Transient between 200 and 600/mm3, elevated protein in up to 70 % of
renal abnormalities also are common, but they are seldom patients, and moderately low glucose concentrations in up to
accompanied by clinical manifestations and, thus, are rarely 30 % of patients [159, 195, 243–247]. Aseptic meningitis
reported [218]. Mumps virus can cross the placenta and associated with mumps is a benign disease that resolves
infect the fetus [185], and mumps has been associated with spontaneously and is not associated with long-term morbid-
spontaneous abortions and intrauterine fetal death [233] but ity. There is no correlation between the level of CSF
not congenital malformations [234]. pleocytosis and the severity of the illness or outcome [159,
An investigation of mumps in a seronegative population of 246, 248].
Eskimos living on St. Lawrence Island in the Bering Sea in Encephalitis complicating mumps is much less frequent
the 1950s provided rates of clinical findings in serologically than aseptic meningitis, probably occurring in 0.1 % of cases
confirmed cases (Table 24.2) [129]. Mumps infection was [159, 201]. Mumps had been considered the most common
confirmed in 82 % of the 561 residents. Orchitis and mastitis cause of viral encephalitis until the early 1980s when the inci-
were age related with sharp increases in frequency at puberty. dence of mumps was greatly reduced and other viruses such as
Orchitis was bilateral in 37 % of cases. There were a few herpes simplex, enteroviruses, arboviruses, and varicella-
women who had lower abdominal pain suggesting oophoritis. zoster virus became the leading cause of encephalitis. Although
In a few cases there were symptoms of thyroiditis. Delirium, mumps encephalitis may be a severe disease, most patients
vomiting, and high fever were associated with stiff neck in completely recover [195, 243, 249, 250]. Postencephalitis
some of the patients, but all recovered. One of four deaths ataxia, behavioral changes, and electroencephalographic
reported during the outbreak occurred 2 days following the abnormalities, should they occur, typically resolve within a
onset of parotitis in an infant; however, the cause of death few weeks [195]. Rarely, permanent neurological sequelae can
was not determined. There were three spontaneous abortions result from mumps encephalitis, including behavioral disor-
among eight women with clinical mumps in the first trimes- ders, seizure disorders, cranial nerve palsies, muscle weak-
ter of pregnancy and one abortion among 12 women with ness, ataxia, chronic headaches, aqueductal stenosis, and
inapparent infections. No stillbirths or miscarriages occurred hydrocephalus [159, 192, 196, 200, 244, 245, 248, 251, 252].
among women who acquired infection after the first trimester, Myelitis or polyneuritis also may follow mumps encephalitis
and there were no congenital malformations in infants born to [253–256]. Overall, the mortality rate associated with mumps
mothers with mumps during pregnancy. encephalitis is less than 2 % and is higher in adults than in
24 Paramyxoviruses: Mumps 565

children. Neuropathologic findings at autopsy are variable, common. Follow-up of men who had orchitis during the
and many reports come from unconfirmed cases. In most care- large outbreaks of mumps that occurred during World War I
fully examined cases, there is evidence of both cellular destruc- and World War II did not show an association with impo-
tion, suggesting a direct effect of the virus, and demyelinization, tence or sterility. Although testicular atrophy occurs in
suggesting an autoimmune process [257]. approximately one-third of the cases of mumps orchitis
Among children with mumps, CNS disease is three- to [282], an effect on quality or quantity of sperm is infrequent
fourfold more common in males than in females [159, 195, [282, 283], and sterility rarely occurs [211, 215]. A small
242–247]. increased risk of testicular cancer following mumps orchitis
Sensorineural deafness is an important sequelae of mumps has been reported [230, 284, 285], but the significance of this
and may occur in the absence of meningitis or encephalitis observation is unknown.
[258, 259]. Deafness usually has a sudden onset, is unilateral
in about 80 % of cases, and is usually thought to be perma-
nent [260–263], although one prospective study of 398 ser- 8.5 Mumps and Diabetes
vicemen being treated for mumps at a military hospital found
hearing loss due to mumps to be transient in most cases Pancreatic involvement in mumps has been linked to diabe-
[259]. The incidence of hearing loss associated with mumps tes mellitus, but a causal relationship remains to be proved
has been estimated to range from 0.05 per 1,000 cases [260] [227, 286, 287]. Notably, pancreatic damage has not been
to over 40 per 1,000 cases [259], which may be an underes- documented in case reports of diabetes in children following
timate since inapparent mumps can cause deafness [264]. mumps infection [288–295]. It has been speculated that
The virus may cause direct damage to the cochlea and to mumps and diabetes have a related periodicity based on the
cochlear neurons [223, 265, 266]. Mumps virus has been iso- occurrence of outbreaks of diabetes months or years follow-
lated from the perilymph of a patient with mumps and sud- ing outbreaks of mumps [286]. These studies were uncon-
den unilateral deafness [221]. trolled and relied on the clinical diagnosis of mumps without
supporting laboratory tests. In one study, mumps accounted
for 8–22 % of the 30 % of cases of insulin-dependent diabe-
8.3 Involvement of the Heart tes mellitus in which a viral infection may have been the pre-
cipitating event [296]. Another study found a seasonal
Acute myocarditis may result from various viral infections, relationship of diabetes with coxsackievirus but not with
including mumps [267–274]. Among persons hospitalized other viruses [297]. In a subsequent study, the association of
for mumps, 3 % of children and 7 % of adults show tran- mumps and coxsackievirus with diabetes could not be con-
sient electrocardiographic abnormalities involving the ST firmed [298]. Less than 1 % of cases of juvenile-onset diabe-
segment or atrioventricular conduction defects [267, 268]. tes followed recent infections with mumps virus in a sample
Electrocardiographic abnormalities suggestive of myocardi- of 1,663 diabetic patients in England [299]. A study in preg-
tis have been detected in 1–15 % of clinical cases, typically nant women found no association between the presence of
between days 5 and 10 of the illness [275], but clinically mumps, coxsackie B or respiratory syncytial virus antibod-
apparent cardiac complications are rare. When it does ies, and diabetes [300]. Other investigators also have failed
occur, myocarditis is usually self-limited; however, it may to find a relationship between antibodies to mumps or other
be catastrophic [267, 268, 271, 276]. Mumps has also been viruses and diabetes mellitus [301–303].
implicated in cases of endocardial fibroelastosis. Some inves-
tigators have suggested that maternal intrauterine infection
may result in endocardial fibroelastosis based on positive 8.6 Other Complications
mumps skin test results; however, the data is inconclusive
[273, 277–280]. Nonetheless, mumps virus RNA has been A variety of other acute and delayed complications have
detected in endomyocardial biopsies or autopsy samples been reported following mumps infection. Arthropathy is a
from children with endocardial fibroelastosis [274, 281]. rare complication that occurs predominantly in young men.
Gordon and Lauter [304] reviewed the literature on mumps
arthritis from the first description in 1850 through the early
8.4 Orchitis and Sterility 1980s. They noted that a total of 32 well-documented cases
of mumps arthritis had been reported in the literature since
Mumps may be complicated by orchitis in over one-third of 1924 when 6 cases (0.44 %) were reported during an out-
postpubertal males (Table 24.1). In most cases, testicular break of 1,334 cases of mumps in Paris. A retrospective
swelling and pain is unilateral, but bilateral orchitis is survey of 2,482 patients with mumps in England and Wales
566 S.A. Rubin

failed to identify any cases with arthritis. Arthropathy fol- United States or most other countries [322, 323]. Thus, vac-
lowing mumps may be manifest as arthralgia without signs cination remains the only practical control measure.
of inflammation; as polyarticular arthritis, which is often The first mumps vaccines were formalin-inactivated virus
migratory; or as monoarticular arthritis of the knee, hip, or preparations, produced within a year of the 1945 publica-
ankle. The arthropathy may last from 2 days to 6 months tions of Habel [12] and Levens and Enders [324] reporting
and is self-limited without sequelae. Mumps virus can repli- the propagation of mumps virus in embryonated eggs. These
cate in human joint tissue in vitro [305, 306], and one study products were found to be safe and effective and were widely
reported detection of mumps virus RNA in the synovial fluid used [15, 325–329], although the duration of protection was
in 3 of 42 patients with early rheumatoid arthritis [307]; relatively short-lived. In the United States, inactivated
however, there are no reports of isolation of the virus from mumps vaccine was used from 1950 to 1978.
affected joints of patients. The observation that vaccines using killed antigens did
Viruria is a common finding in patients with mumps not produce long-lasting protection led to the development
[308]. In one study all 20 patients with laboratory-confirmed of live attenuated mumps virus vaccines in the 1950s in the
mumps had abnormal renal function at some time during the Soviet Union [16] and in the 1960s in the United States [17].
acute infection, but all had normal renal function and nega- This effort was stimulated by the earlier work of Habel [12,
tive urine cultures within 24 days [218]. Clinically apparent 330] and Enders and Levens [14] demonstrating that live
nephritis is uncommon and generally benign [309–312]; virus that had undergone continuous passage in chick embryo
however, fatal cases have occurred [309, 313]. Nephritis may produced inapparent infections, but good immune responses
result from direct infection of the kidney or immune com- in monkeys. At least ten different live attenuated mumps vac-
plex glomerulonephritis [313]. cine strains have been used in various countries around the
Ocular manifestations of mumps also have been reported, world [331].
including involvement of the lacrimal gland, which affected A live attenuated mumps virus vaccine using the Jeryl
20 % of soldiers in a 1903 epidemic, and optic neuritis, Lynn strain of mumps virus (named after the child from
which may be associated with involvement of other sites in whom the strain was first isolated) was developed in 1965
the CNS [314]. For unknown reasons, such complications of and licensed in the United States in 1967 [17, 332–334]. This
mumps are now rarely reported. Keratitis, iritis, conjunctivi- remains the only live mumps virus strain used in the United
tis, scleritis, endonitis, corneal endotheliitis, and central reti- States and is the most widely distributed vaccine globally.
nal vein occlusion have been reported rarely. Extensive prelicensure field trials were carried out by Stokes
Although the persistence of mumps virus has been sug- et al. [332] in Philadelphia and by others in several field stud-
gested as a factor in neurological and muscular disorders of ies [333–337]. Overall seroconversion rate was 97 % among
unknown cause, there is no compelling evidence for a causal children and 93 % among adults. Efficacy was approximately
association [315–317]. 95 % based on a follow-up period of 20 months. Vaccine-
induced antibody persists for decades [338–341], although
there is ample evidence of declining antibody titers with time
9 Control and Prevention postvaccination [81, 82, 135]. Whether such declines in
virus-specific antibody levels can lead to breakthrough infec-
Given the high rate of asymptomatic infections and that tions is a matter of controversy [80, 84, 85, 130, 140].
transmissions often occur before symptom appearance or Mumps vaccine administered after exposure does not
during the first few days of symptom onset [106], case isola- appear to provide clinical protection or alter the severity of
tion is of limited value in preventing the spread of disease. the disease [336]. However, evidence from observational
Where indicated, the recommended period of patient isola- studies suggests that mass vaccination or “ring vaccination”
tion is 5 days following parotitis onset [318], a guideline during a mumps outbreak may help contain the disease and
based on recent data showing that fewer than 15 % of terminate the outbreak [161, 342]. This approach, although
patients continue to shed virus beyond day 4 of symptom unlikely to prevent infection in those already exposed, will
onset [319]. act to prevent infection in susceptible contacts.
Standard immune globulin or gamma globulin preparations Mumps vaccine is often administered in combination
have been found to be of little value for postexposure prophy- with measles, mumps, and rubella (MMR) vaccine or in
laxis [114, 320], although some success has been achieved combination with measles, mumps, rubella, and varicella
with mumps virus-specific immune globulin preparations, but (MMRV) vaccine. In the United States, mumps vaccine is no
only when administered early after the outbreak [320, 321]. longer available in monovalent formulation. Incorporation of
Nonetheless, the value of mumps-specific immune globulin mumps vaccine into multivalent formulations with other
to public health was considered to be not high enough to war- viruses was found not to affect mumps vaccine safety or
rant routine use and the product is no longer available in the immunogenicity [331, 343–348].
24 Paramyxoviruses: Mumps 567

In 1967, when the live attenuated mumps virus vaccine [355]. Inadvertent vaccination during pregnancy should not
was licensed in the United States, mumps control was not a be considered an indication for termination of pregnancy.
high priority in the public health community, as the disease Because live mumps vaccine is produced in chick embryo
was considered primarily a mild inconsequential disease of cell culture, persons with a history of anaphylactic reactions
childhood. At that time, the Advisory Committee on to eggs should be vaccinated with caution according to estab-
Immunization Practices (ACIP) recommended that the newly lished protocols [356]; however, egg allergy is not a contra-
licensed mumps vaccine be considered for use in children indication for vaccination. The vaccine also contains trace
approaching puberty, for adolescents, and for adults who amounts of neomycin; thus, persons with anaphylactic
have not had mumps, but was not recommended for routine reactions to neomycin should not receive the vaccine. Mumps
use in younger individuals due to uncertainties over the dura- vaccine should not be administered to immunodeficient per-
tion of immunity [349]. The specific concern was that pro- sons; however, when vaccination against measles is indicated
tection against mumps in young children might be fleeting, for symptomatic HIV-infected children, MMR vaccine can
resulting in an increased incidence of postpubertal suscepti- be used. Children who are infected with HIV but are asymp-
bility. A series of progressively stronger statements from the tomatic should be vaccinated.
ACIP on mumps control followed [350, 351], with routine The benefit-to-cost ratio for mumps vaccination is high,
childhood immunization recommended in 1977. In 1989 the with an estimated savings of $7–$14 per dollar spent on
ACIP recommended that all children receive two doses of mumps vaccination programs [357, 358]. Zhou and col-
measles vaccine [352]. Because measles vaccine is generally leagues calculated a savings of over $1.5 billion annually by
given to children in the United States as MMR, the effect of vaccinating against mumps in the United States, equating to
implementation of this recommendation has been that most a saving of $3,614 per case prevented per year based on 2001
children receive two doses of mumps vaccine. data [359].
In general, people who can be considered immune to School immunization laws beginning during the late
mumps (1) have documentation of vaccination with live 1970s have had a dramatic effect on outbreaks and the over-
mumps virus vaccine on or after their first birthday, (2) have all incidence of mumps. In 1977 when the ACIP recom-
laboratory evidence of mumps immunity, (3) have documen- mended that children routinely be vaccinated against mumps
tation of physician-diagnosed mumps, or (4) were born [350], only five states had immunization laws. By 1993, this
before 1957. During mumps outbreaks, vaccination with number steadily increased to 43, including the District of
mumps-containing vaccine should be considered for people Columbia (DC). Presently, all states but Iowa require vacci-
born before 1957. nation with mumps-containing vaccines for entrance into
In 2009 the ACIP revised their recommendations for primary school [360]. The dramatic reduction in reported
evidence of mumps immunity for healthcare personnel to cases of mumps in the United States since 1991 reflects
include documented administration of two doses of vaccine increasing implementation of school immunization laws
containing live mumps vaccine or laboratory evidence of requiring two doses of MMR.
immunity or laboratory confirmation of disease. For unvac- In 1985, states without immunization laws had twice the
cinated personnel born before 1957 who lack laboratory incidence rates as states with comprehensive laws requiring
evidence of mumps immunity or laboratory confirmation proof of immunity against mumps [100]. During the resur-
of mumps, healthcare facilities should consider vaccinating gence of mumps in 1986, the reported incidence of mumps in
with two doses of mumps-containing vaccine at the appropri- 15 states without any requirement for mumps vaccination
ate interval [353]. There is no increased risk associated with was 12-fold higher than that observed in states that had a
vaccinating immune persons; thus, those who are unsure of comprehensive requirement [100]. An outbreak in New
their histories of mumps disease or vaccination should be Jersey in 1983, in which students in the sixth grade were
vaccinated. nearly seven times more likely to develop mumps than those
Adverse effects of vaccination with the Jeryl Lynn vac- in lower grades, reflected the partial school law that covered
cine, such as parotitis and low-grade fever, are infrequent students from kindergarten through grade 5 [361, 362].
and minor. Mild allergic reactions of short duration such as Although children can be exempted from immunization
rash, pruritus, and purpura occur infrequently. Postvaccination laws for various reasons, including religious, personal, or
aseptic meningitis has been reported in association with sev- moral beliefs, the end effect of immunization laws is that
eral mumps vaccine strains used outside the United States, presently nearly all children entering kindergarten have
but not for the Jeryl Lynn strain [354]. received at least one dose of mumps-containing vaccine.
Mumps vaccine is contraindicated for pregnant women By 1996, ≥90 % of 19–35-month-old children had received
because of a theoretical risk to the fetus. Mumps vaccine at least one dose of MMR [363–365]. For the 2011–2012
virus has been shown to infect the placenta, but there is no school year in the United States, median coverage with two
evidence that it causes congenital malformations in humans doses of MMR among kindergartners was 94.8 % based on
568 S.A. Rubin

47 reporting states and the DC [126]. Among 49 report- gence in the United States in 2006, despite belonging to the
ing states and the DC, exemption rates ranged from <0.1 same age group and residing in barracks across the United
to 7.0 %, with a median rate of 1.5 % [126]. Based on the States, paralleling the high density, close-contact environ-
2011 National Immunization Survey (NIS), among children ment of university campuses. The likely reason for sparing of
19–35 months of age (born January 2008–May 2010), cover- the military may lie with the military’s practice in 1991 of
age with one or more doses of MMR vaccine was estimated routine administration of MMR to recruits without regard to
at 91.6 % [366], which is above the national Healthy People prior vaccination status [6, 165]. This was later changed by
2020 target of 90 %. some branches to targeted MMR vaccination of those lacking
Over 250 million doses of mumps-containing vaccine documentation of two doses of MMR or lacking serologic
have been distributed in the United States since licensure in evidence of immunity to measles, mumps, or rubella.
1967. During this time, the incidence of mumps has been While it is clear that the neutralizing antibody is required
reduced by 99.8 %, with only 363 cases reported in 2011 for protection [327, 375, 376], the amount required is not
compared to over 200,000 annually in the prevaccine era. known. Neutralizing antibody titers in the range of 1:4–1:8
were found to be associated with protection in studies con-
ducted during the prevaccine era [51, 98, 376, 377]; however,
10 Unresolved Problems it does not appear that vaccine-induced antibody of this con-
centration is adequate [378]. More work is needed in this
Despite the remarkable success of mumps vaccination pro- area to firmly establish a correlate of protection, if one exists.
grams in reducing disease incidence, sporadic and some- Particular attention should be given to evaluating the role of
times large mumps outbreaks continue to occur, even in cellular immunity in disease prevention. While the presence
settings of high two-dose vaccine coverage. In 2006, the or absence of mumps virus-specific cell-mediated responses
United States experienced its largest mumps outbreak in 19 correlates with the presence or absence of virus-specific anti-
years, with 6,584 cases reported, representing a 20-fold body, the magnitudes of the two types of immune responses
increase in the disease incidence in recent years. Disease do not correlate [379, 380], and, in some instances, cell-
incidence returned to the baseline the following year but then mediated immune responses have been detected in seronega-
spiked again in 2009–2010. Whereas outbreaks in previous tive persons [381, 382]. The precise role of cell-mediated
decades predominantly affected young, often unvaccinated immune responses in protection needs to be investigated.
children in primary and secondary schools, more recent out- In this era of routine use of more sophisticated epidemio-
breaks involve young adults on college and university cam- logical tools, such as viral gene amplification and nucleotide
puses, the overwhelming majority of whom have a history of sequencing, it has been observed that virus isolates invariably
mumps vaccination [66, 132, 138, 367–373]. Thus, rather cluster into genotype groupings distinct from those of the
than a failure to vaccinate, contemporary outbreaks appear to vaccine strains used, leading to the speculation by some that
be a consequence of vaccine failure, with the preponderance the recent rash of mumps outbreaks are due to the evolution
of evidence pointing toward waning immunity [81, 83–85, of virus strains capable of escaping vaccine-induced immu-
135, 137–140]. Indeed, vaccine effectiveness during recent nity. While it was demonstrated that serum from recent vac-
outbreaks is significantly lower than that reported under con- cinees possessed strong neutralizing activity against a wide
trolled clinical trials. In contrast to the prelicensure studies array of mumps virus strains [37], in light of the evidence of
demonstrating a single dose of vaccine to be ≥95 % effective waning immunity, the persistence of such broad virus-neu-
in preventing mumps [333, 334], more recent data from out- tralizing activity is not known and needs to be established.
break investigations have estimated vaccine efficacy to be
less than 70 %, even for two doses of vaccine [84, 138, 369,
374]. These data support the growing body of evidence of References
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 Paramyxoviruses: Parainfluenza
Viruses 25
Janet A. Englund and Anne Moscona

1 Introduction clinical manifestations, but serious illnesses occur less fre-

quently. Infections with parainfluenza virus type 4 (HPIV4)
Acute respiratory infection is now the leading cause of are detected less frequently.
mortality in young children under 5 years of age, account- With the use of sensitive molecular detection methods, the
ing for nearly one-fifth (20 %) of childhood deaths world- frequency and importance of HPIV infections in the commu-
wide and killing two to three million children each year [1]. nity and hospital settings are becoming more appreciated. A
Human parainfluenza viruses (HPIVs) types 1, 2, and 3, recent survey study estimated that HPIV accounted for 7 %
human metapneumovirus [2], and respiratory syncytial virus of all hospitalizations for fever and/or acute respiratory ill-
(RSV) cause the majority of childhood lower respiratory ness in children under 5 years in the United States or 23,000
tract disease in the United States. The parainfluenza viruses hospitalizations every year in the United States that can be
types 1 and 3 belong to the Respirovirus genus within the attributed to HPIV [7]. Half of these result from HPIV3
Paramyxovirinae subfamily of the Paramyxoviridae family infection, with most of the remainder caused by HPIV1. The
of negative-stranded RNA viruses, while parainfluenza virus effective use of corticosteroids and nebulized epinephrine for
type 2 belongs to the Rubulavirus genus. HPIV1-associated croup has decreased the need for hospi-
Human parainfluenza viruses (HPIVs) cause lower respi- talization for croup, and this development helps explain the
ratory tract diseases including bronchitis, bronchiolitis, and decreased fraction of severe cases caused by HPIV1 [8].
pneumonia in infants, children, and immunocompromised Despite the impact of these viruses on illness and hospi-
individuals [3] and are responsible for up to 30–40 % of all talization of young infants worldwide, no specific treatment
acute respiratory tract infections in infants and children. In or prevention strategies are yet available, with the exception
adults, the impact of these respiratory viruses may be seri- of the symptomatic benefit derived from corticosteroids for
ous as well. For example, in adult hematopoietic stem cell HPIV1-associated croup [9]. Development of antiviral drugs
transplant patients, respiratory viruses cause about two- and vaccines for these viruses has been hindered by gaps
thirds of the respiratory illnesses, with high mortality [4, 5]. in fundamental knowledge about the pathogens and about
HPIV3 accounts for the vast majority of HPIV infections the human immune response to respiratory viruses. Recent
following transplantation, causing pneumonia with a 35 % discoveries provide a basis for optimism that effective treat-
acute mortality rate and a 75 % mortality rate at 6 months ments and vaccines may be available in the near future.
[4, 6]. Parainfluenza virus type 1 (HPIV1) is the princi-
pal cause of croup (laryngotracheobronchitis) in children.
Parainfluenza virus type 2 (HPIV2) resembles HPIV1 in its 2 Historical Background

Chanock [10] first reported the isolation of a virus from

J.A. Englund, MD (*)
Department of Pediatric Infectious Diseases, human sources in Cincinnati in 1956 from children
Seattle Children’s Hospital, University of Washington, with croup, a virus originally designated as the “croup-
4800 Sand Point Way NE, Seattle, WA 98105, USA associated” (CA) virus. Two additional strains were identi-
e-mail: [email protected]
fied by their ability to adsorb guinea pig erythrocytes onto
A. Moscona, MD† (*) infected rhesus monkey kidney cells in culture in 1958 [11].
Departments of Pediatrics and of Microbiology and Immunology,
These two viruses, first designated “HA-1” and “HA-2,” for
Weill Cornell Medical Centers,
505 East 70th Street, New York, NY 10021, USA hemadsorption 1 and 2, shared many biological properties
e-mail: [email protected] with CA virus while being antigenically distinct, and they

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 579

DOI 10.1007/978-1-4899-7448-8_25, © Springer Science+Business Media New York 2014
580 J.A. Englund and A. Moscona

Hemagglutinin-neuramindase The trimeric glycoprotein (F) directly mediates fusion of

tetramer (HN) virus and cell membranes during viral entry. The mature
Fusion protein (F)
Matrix protein (shown as a trimer) form of the F glycoprotein is generated by enzyme cleav-
(M) age, which results in two disulfide-linked subunits, F1 and
Fusion peptide
F2 [24], and requires an activation step in order to medi-
Lipid ate fusion; activation is provided by the HN upon receptor
bilayer engagement [25–28]. The viruses are relatively antigeni-
cally stable; although subgroups of the specific types may
Large RNA polymerase have recognizable antigenic differences at certain epitopes,
complex (L) these differences are stable and nonprogressive. Conserved
epitopes—particularly on the fusion proteins—generate
antibodies that neutralize across these subgroups [29, 30].
HPIV3 viruses remain infectious in an aerosol for periods
greater than 1 h [31]. While there are no reported sys-
Phosphoprotein tematic studies of the stability of other HPIVs in aerosol,
(P) HPIV1 has been isolated from air samples collected near
Nucleocapsid protein
infected children [32].
(NP) A biological feature characteristic of the HPIVs is their
ability to replicate in the respiratory epithelium without
deeper invasion or immediate host cell death [26]. In vitro
models of HPIV3 infection in the human airway epithe-
lium indicate that infection occurs exclusively in ciliated
epithelial cells and utilizes sialic acid residues for initiat-
Fig. 25.1 A schematic diagram of the parainfluenza virion. L large ing infection [33]. The virus buds from the host cell mem-
RNA polymerase protein, M matrix protein, NP nucleocapsid protein, P brane without lysing the cell, permitting continued release
phosphoprotein [26]
of infectious particles from a single cell [22]. HPIV3
virus may infect the mucosa of infants in the presence of
were later reclassified as HPIVs: HPIV1 (HA-2), HPIV2 maternally derived circulating antibodies [34–37] and also
(CA), and HPIV3 (HA-1) [12]. HPIV4 was first isolated frequently reinfects older children despite circulating anti-
in 1960 [13], with two subtypes of this virus now recog- bodies [38]. In fact, multiple reinfection is the hallmark of
nized: HPIV4a and HPIV4b. During the same period, these these viruses. Excretion of virus may be prolonged up to
viruses were compared with isolates obtained from animals. 1 month or longer, even with the second or third infection
The Sendai virus [14] recovered from rodents was found to [39–41]. Immunocompromised patients may shed HPIV for
share antigens with HPIV1 and is classified as a subtype of many months, often with no or minimal intermittent symp-
this strain [15, 16]. In 1959, a hemadsorbing virus [17] anti- toms [42]. Epidemiologic evidence suggests that reinfected
genically similar to HPIV3 virus [18] was recovered from children are infectious for their contacts [38]. The incubation
cattle with “shipping fever.” A simian virus, SV5 [19], has period is approximately 3–6 days, and the virus spreads rap-
been shown to be related to HPIV2 virus [20, 21] and is now idly to a high percentage of persons in closed populations,
called HPIV5. indicating a high degree of infectiousness. In studies carried
out in the 1950s, adult volunteers with preexisting antibody
were infected with as little as 1,500 median tissue culture
3 Biological Characteristics infectious doses (TCID50) and more than half of the volun-
teers infected with HPIV developed signs and symptoms of
HPIVs are RNA-containing viruses with a spike-covered an upper respiratory illness [43].
lipoprotein envelope [22]. Figure 25.1 shows a schematic
diagram of a representative virus. The genome consists
of single-stranded, non-segmented, negative-sense ribo- 4 Diagnostic and Laboratory Methods
nucleic acid. The spikes are formed by oligomers of two
glycoproteins, the hemagglutinin-neuraminidase (HN) and 4.1 Culture
the fusion protein (F). The tetrameric HN is responsible
for the receptor-binding (hemagglutinating) and recep- Classical methods of HPIV isolation in tissue culture have
tor-cleaving (neuraminidase) activities [23], as well as been utilized in most epidemiologic studies. However, direct
for activating the membrane fusion process during entry. identification of HPIV from cell culture usually requires fur-
25 Paramyxoviruses: Parainfluenza Viruses 581

ther evaluation because many viruses do not cause a direct and other respiratory viruses can be reliably reported to the
cytopathic effect. Infection is therefore detected by a second- clinician within 1 h [71]. Nonetheless, as for all rapid diag-
ary step, for example, HPIV-type-specific labeled monoclo- nostic assays available at the present time, confirmation by
nal or polyclonal antibodies [35, 44] or specific molecular other means may still be important, especially in the face
detection using amplification strategies. The epidemiology of a negative result in an ill child. Simple, effective assay
of HPIV4 has been obscured due to difficulties in viral detec- kits (whether antigen or nucleic acid based) should allow
tion; HPIV4 does not cause cytopathic effect in many cell for simple screening of children and are likely to be used
lines and detection of this virus was frequently missed using more commonly in the future as antiviral therapies become
standard culture techniques. available. Identification of the etiologic agent, even if no
specific therapy is available, is key to containing respira-
tory virus outbreaks and avoiding transmission to vulnerable
4.2 Molecular Identification individuals.
The MassTag polymerase chain reaction method pro-
Molecular methods have been used to detect virus and to vides a paradigm for new detection strategies for early
examine questions of antigenic diversity among the HPIVs recognition and containment of a wide range of respira-
[29, 45–50]. Batteries of monoclonal antibodies directed tory pathogens and exemplifies new molecular techniques
toward epitopes of the surface glycoproteins were used for that are sensitive and specific and potentially adaptable to
antigenic characterization, and gene sequencing later defined diverse laboratory settings. Briese et al. have described
these epitopes. Reverse transcriptase-polymerase chain reac- the development of a MassTag polymerase chain reaction
tion (RT-PCR)-based sequencing assays have been adapted (PCR) for differential diagnosis of respiratory disease [72].
to epidemiologic studies of nosocomial infections [51–58]. In this multiplex assay, the pathogen gene targets are coded
The development of more sensitive, specific, and rapid diag- by a library of 64 distinct mass tags. The microbial RNA
nostic assays for respiratory viruses is critical for two major or DNA is amplified by multiplex RT-PCR using up to 64
reasons. Diagnosis is becoming increasingly important to primers. Each primer is labeled with a different molecu-
clinical management of patients and nosocomial disease in lar weight tag, attached to the primer by a photo-cleavable
the health-care setting, and there is an urgent need for evalu- link. After amplification, the mass tags are released from
ation of the global distribution of disease. the amplified material by UV irradiation, the identity of the
The significant progress in applying molecular biology tag determined by mass spectrometry, and the identity of
advances to respiratory virus diagnosis has led to several the organism determined by the presence of its two spe-
new clinically useful strategies [59–67]. For the practitioner, cific tags, one from each primer. The technology has been
guidelines and clear data about the situations in which spe- successfully applied to respiratory disease [72], where
cific kinds of assays may be appropriate are needed. In the multiplex primer sets were designed to identify up to 22
clinical setting, rapid identification of HPIV has been most respiratory pathogens in a single reaction. Such technology
frequently performed in clinical specimens using antigen could be of use in the public health setting for identification
detection with commercially available kits for rapid screen- of outbreaks and global surveillance.
ing that have sensitivities and specificities of 80–90 %.
Antigen detection has been the most widely used type of
rapid test for HPIV until the early 2010s. These tests are per- 4.3 Serology
formed directly in NP secretions, generally using fluorescent-
conjugated antibody (DFA) and less frequently employing Serological studies have been the main source of data
an enzyme-linked immunosorbent assay [60]. Most recently, delineating the importance of the parainfluenza viruses as
molecular detection assays using multiplex quantitative etiologic agents of respiratory disease. Such studies have
reverse transcription-PCR-enzyme hybridization have been demonstrated the ubiquity of viral infection with HPIV and
optimized to identify a panel of respiratory viruses and dif- the similarity of age at acquisition of antibody throughout
ferentiate between HPIV1, HPIV2, and HPIV3 [68–70]. the world [73–80]. Many community studies have utilized
Several FDA-licensed molecular-based diagnostic multiplex both serological and viral isolation data but have relied
RT-PCR assays for the detection of HPIV and other clinically most heavily on serological data to determine the frequency
important respiratory viruses are available, and the sensitiv- of infection within the population [81–86]. Vaccine studies
ity, specificity, and positive and negative predictive values also rely on serology as indicators of vaccine infectivity
of these methods are excellent. Relatively simple RT-PCR [74]. However, the use of newer, more sensitive technolo-
kits such as those produced by Luminex Technology, Idaho gies such as RT-PCR techniques and MassTag polymerase
Technology, or 3 M are currently being used in many medi- chain reaction method for surveillance in the future will be
cal center settings; sensitive and specific detection of HPIV important.
582 J.A. Englund and A. Moscona

5 Epidemiology mortality from HPIV3 after transplantation include graft-

versus-host disease, steroid use, the presence of copatho-
5.1 Morbidity and Mortality Data gens [121–123], and infection with HPIV3 within the first
100 days after transplant [123]. The lack of any effective
Official morbidity reports do not include acute respiratory therapy is especially troubling in these vulnerable groups
infections; furthermore, laboratory diagnosis is required to (addressed in detail below).
establish the diagnosis of an HPIV infection. Morbidity data, The role of HPIVs as etiologic agents of respiratory ill-
therefore, are based on prospective research studies of respi- nesses in adults has also been investigated [81, 85, 124–129].
ratory disease conducted around the world. Standardized These studies have reported sampling of patients with acute
techniques have been employed to provide some estimate of respiratory disease in hospitals, military services, university
the impact of HPIV as causes of acute respiratory disease in student health services, and industrial medical facilities,
diverse populations, with older studies relying on some com- generally relying on culture and serology. More recently,
bination of viral culture and serology and more recent stud- respiratory disease in hospitalized patients with underlying
ies mainly relying on molecular detection. Most studies have respiratory conditions has been prospectively evaluated uti-
been cross-sectional studies of the etiology of either illnesses lizing a combination of culture, serology, and RT-PCR tech-
of hospitalized patients or epidemics in closed populations niques [130, 131]. In the past, challenge experiments with
and provide a limited view of the total worldwide morbid- HPIV have been conducted in volunteer studies in the United
ity resulting from these viruses. Prospective investigations States [132–135] and Great Britain [43, 135, 136].
of the etiology of acute respiratory disease conducted over Evidence of infections with paramyxoviruses has been
extended periods of time in large populations merit special demonstrated throughout the world [77–79, 137], includ-
mention because of the unique perspective provided. Seven ing tropical climates [80, 138–140] and isolated commu-
studies carried out in the latter half of the twentieth century nities [75, 141], with the notable exception of the absence
in Washington, DC [34, 87]; Chapel Hill, North Carolina [36, of HPIV1 and HPIV2 antibody in children in very remote
88, 89]; Tecumseh, Michigan [86, 90]; Seattle, Washington Indian tribes in South America [142]. In developing coun-
[82]; Houston, Texas [39, 91, 92]; Tucson, Arizona [93, 94]; tries, HPIVs are second only to RSV as the most commonly
and Great Britain [95, 96] differed in methods of patient identified respiratory pathogens in children [138, 140, 143–
selection, types of illnesses surveyed, and ages of subjects, 150]. As in Europe and North America, HPIV3 is the most
but as a group, they documented respiratory illness over an common HPIV isolated from severely ill infants, but types 1
extended time period in widely diverse geographic environ- and 2 have also been found [146, 150, 151].
ments and socioeconomic groups. Limited mortality data are available and consist chiefly of
Other studies have focused on the occurrence of respi- case reports [152–154], fatal pneumonia in severely immu-
ratory illness in specialized population groups. Children nocompromised patients [106, 121, 155–157], and case
in hospital or outpatient settings have been studied exten- series in children with hematologic malignancy or HSCT
sively both in North America and Great Britain [7, 35, [9]. Most deaths caused by HPIV are likely related to HPIV3
97–104]. These patients include those with underlying virus infections in young infants, but even in this age group,
conditions that put them at high risk for serious conse- the etiology of fatal cases is not documented sufficiently to
quences of respiratory infections [45, 105–107]. Studies of allow a reasonable estimate of the mortality rate in the gen-
uncomplicated respiratory disease in institutionalized chil- eral population. This problem is magnified in developing
dren [38, 108–110] day-care groups [41, 111] and school countries, where mortality attributed to acute respiratory dis-
children [112] have contributed information about the spec- ease is many times greater than in Europe or North America
trum of disease caused by these agents. Additional fam- [1, 158]. Although the exact impact of HPIV in more recent
ily studies addressing the importance of HPIVs have been studies in Africa has had varying results [159, 160], avail-
described using more classical techniques [113, 114]. The able data suggest that HPIV infections contribute to excess
use of PCR detection in children in the family and day-care mortality [143, 145, 148, 149].
setting [111, 115] has contributed to the epidemiology by
documenting the presence of prolonged shedding, multiple
infections and reinfections, and asymptomatic disease. The 5.2 Incidence and Prevalence Data
role of HPIV has also been studied specifically in children
with bronchiolitis [116], asthmatic children [117, 118], 5.2.1 Serological Surveys
patients with chronic bronchitis [103], newborn nurseries HPIVs infect most persons during childhood. Serological
[119], and children with hematologic malignancies [120]. surveys show that 90–100 % of children have antibodies to
HPIVs, especially HPIV3, are a serious problem for chil- HPIV3 by 5 years of age [75, 80, 84]. Antibodies to HPIV1
dren with immune system compromise, including stem and HPIV2 are not as universal as antibodies to HPIV3 or
cell transplant (HSCT) patients. Known risks for increased RSV, although 74 % of children over 5 years of age have
25 Paramyxoviruses: Parainfluenza Viruses 583

Table 25.1 Frequency of infection with parainfluenza virus type 3 (para 3) among children studied from birth, Houston Family Study,
1975–1980

Number of Number with infection with HPIV3

Age (months) child-years Primary Reinfection Total (rate)a LRDb (rate)
0–12 121 75 6 81 (66.9) 14 (11.6)
13–24 90 30 31 61 (67.8) 9 (10.0)
25–36 63 2 21 23 (36.5) 2 (3.2)
37–48 39 0 13 13 (33.3) 1 (2.6)
49–60 24 0 4 4 (16.7) 0
Totals 337 107 75 182 (54.0) 26 (7.7)
a
Per 100 child-years
b
Lower respiratory tract disease

been shown to possess antibody against HPIV1 and 59 % In Houston, 121 children were followed from birth for
against HPIV2 [161]. Similar data have been obtained in infection with HPIV3 [92] (Table 25.1). About two-thirds
serological surveys in a wide variety of geographic areas were infected during each of the first 2 years of life. The
[75–77, 80, 84, 140, 141] but not in remote South American risk of illness was at least 30 per 100 children per year. Most
tribes [142]. Serological studies of natives of Tristan da lower respiratory tract illnesses accompanied primary infec-
Cunha who moved to the United Kingdom demonstrated the tion with a risk of 13 per 100 child-years. Over 90 % were
rapidity with which an isolated population acquires antibod- infected at least once by 24 months of age, and almost 40 %
ies to these viruses when they move to a heavily populated had been infected twice. Similarly, 21 of 62 (34 %) infants
area [162]. in Chapel Hill followed longitudinally through the first 2
years of life developed respiratory illnesses associated with
5.2.2 Association of HPIVs with Illnesses HPIV3 virus infection and 6 (9.7 per 100 children) had lower
HPIV viruses were isolated from about 3 % of persons of all respiratory tract involvement. Four of fifteen were reinfected
ages with acute respiratory disease presenting for medical during the second year, yet 66 % of the total had neutralizing
care in Houston over a 5-year period [92]. During this time, antibodies at the end of 2 years, indicating that mild or inap-
30 % of infections were type 1 and 10 % were type 2. Over parent infections occurred at a rate similar to that of children
40 % of persons with HPIV1 and HPIV2 infections occurred presenting with illness. These two studies with intensive fol-
among persons over 5 years of age, an older population than low-up show that lower respiratory tract involvement due to
in those infected with HPIV3. Studies of lower respiratory HPIV3 is common in toddlers.
disease have shown the highest isolation rates of HPIV1 and In contrast, seroepidemiological studies in school-age
HPIV2 viruses to be in children between 4 months and 5 children [112] and young adults [81, 85, 126] have revealed
years of age [34, 36]. The rate for lower respiratory disease a relatively low rate of HPIV seroconversions, although
associated with HPIV1 virus infection in the Chapel Hill infections by these agents are well documented in adults.
pediatric practice was 17 per 1,000 children per year for chil- Prospective studies in long-term care facilities have shown
dren under 4 years of age [36, 88, 89]. Infants followed in relatively similar rates of HPIV1, HPIV2, and HPIV3 infec-
the Tucson study had lower rates in the first 6 months of life, tion determined by prospective serological surveys compared
but thereafter had rates comparable to those reported from to rates of infection with RSV, HMPV, and influenza [130].
Chapel Hill [94]. Lower respiratory disease after 2 years HPIV3 virus infections have been detected in all studies
of age was relatively uncommon [37, 128]. Rates of HPIV of hospitalized children with acute lower respiratory disease
infection, diagnosed by PCR methods, were demonstrated in [34, 87, 101, 104, 163], and this virus is now recognized
12 % of day-care attendees with respiratory illness, but only to be second only to RSV as a cause of hospitalization for
a minority (4/37, 11 %) were due to types 1 and 2 [52, 111] bronchiolitis and pneumonia in infants [7, 35–37, 89, 95].
(Table 25.1). Henrickson et al. assessed the disease burden in Wisconsin
Few studies of open populations have had sufficiently over 2 years (1996–1998) using multiplex PCR detection.
sized population denominators to allow calculation of attack The authors estimated that approximately 545,000 hospital-
rates. In Chapel Hill, studies of children with lower respi- izations of children under 18 years old for lower respiratory
ratory disease presenting to a pediatric practice from 1964 tract disease occurred each year; the most common viruses
to 1975 showed that 15 children per 1,000 per year were detected each year in hospitalized children were RSV (A and
infected with HPIV3 each year for the first 3 years of life [36, B, 117,000), HPIVs (HPIV1 and HPIV2, 48, 000; HPIV3,
88, 89]. The rates for lower respiratory disease in Tucson 18,000), and influenza (A and B, 39,000). The HPIVs were
infants were similar in the first 6 months but fell below those detected much more frequently in immunocompromised
in Chapel Hill during the last half of the first year [94]. children than in previously healthy children (33 % vs. 16 %).
584 J.A. Englund and A. Moscona

A recent New Vaccine Surveillance Network study virus was endemic [34]. Beginning in 1962–1974, sharp epi-
enrolled children younger than 5 years of age in the United demics of HPIV1 virus occurred every 2 years in the autumn
States who were hospitalized with febrile or acute respiratory of even-numbered years [34, 87]. A similar pattern was noted
illnesses in the 2000s and found that HPIV accounted for in Great Britain between 1962 and 1977 [95, 96]. Epidemics
almost 7 % of hospitalizations for fever and/or acute respira- of HPIV1 virus occurring in the fall of even-numbered years
tory illness (ARI) in these young children. The authors esti- after 1962 have been described in other reports as well [89,
mated that HPIV3 is responsible for half of the 23,000 HPIV 97–99, 101]. After 1970, a 3-year lapse occurred before
hospitalizations yearly [7]. In this study, nasal turbinate/ resumption of biannual epidemics [169]. Since 1973, HPIV1
throat swabs were tested for the pediatric respiratory viruses epidemics have occurred in the autumn of odd-numbered
HPIV, RSV, and influenza virus, with culture and reverse years in Houston, accompanied by HPIV2 [92]. In other lon-
transcription-polymerase chain reaction. Over the course of gitudinal studies, including those in Tecumseh [86], HPIV1
4 years (2000–2004), 2,798 children were enrolled, and 191 virus occurred in the endemic pattern until 1971 but thereaf-
HPIVs were identified from 189 children (6.8 % of enrolled: ter seen in autumn and early winter [90]. The evidence sug-
73 HPIV type 1, 23 HPIV type 2, and 95 HPIV type 3), com- gests that HPIV1 has varied patterns of occurrence but the
pared with 521 RSV and 159 influenza viruses. Mean HPIV predominant pattern in temperate zones is biannual epidem-
hospitalization rates were 3.01, 1.73, 1.53, 0.39, and 1.02 per ics in the fall [168].
1,000 children per year for ages 0–5, 6–11, 12–23, 24–59, HPIV2 appears to be nearly as ubiquitous as HPIV1 based
and 0–59 months, respectively. The investigators concluded on serological surveys, but is not associated as frequently
that the pediatric HPIV inpatient burden is substantial [7]. with severe clinical disease. Because many laboratories have
depended on the detection of viruses from more seriously
5.2.3 HPIV4 Virus Infections ill or hospitalized patients with lower respiratory disease to
HPIV4 virus has not been reported as frequently as the other document the presence of the virus in the community, less
HPIVs, but serological surveys indicate that infection may be information on the occurrence of HPIV2 is available; this is
common [164–166]. Most of these infections are considered changing with the availability of rapid molecular diagnostic
to be asymptomatic, but isolation of the virus has likely been methods for respiratory viruses. In the past surveillance stud-
missed in studies relying on culture techniques [164]. More ies of young children with minor respiratory illness, HPIV2
recent studies in hospitalized children [167] or studies of chil- has had a distinct epidemic pattern with high attack rates in
dren attending day care [52] have documented variations in small, defined populations [41, 109, 110]. HPIV2 appears
infection over time, with rates of symptomatic disease similar to occur in a sporadic epidemic pattern, often disappearing
to that and sometimes exceeding that of other HPIV types. from the community for fairly long periods of time. Several
studies in the United States have noted HPIV2 epidem-
ics to occur in the fall of the odd-numbered years [87, 89,
5.3 Epidemic Behavior 90, 92, 170]; however, in Great Britain [95] and in the first
Tecumseh study [86], HPIV2 occurred in well-defined but
HPIV can be isolated in any month of the year in both tem- somewhat erratic epidemics, and in the Seattle surveillance
perate [34, 89] and tropical climates [138–140]. Although studies [82], it was rarely identified.
HPIV3 circulation peaks generally in the spring, HPIV2 gen-
erally peaks in the fall, and HPIV1 seems to peak in the fall 5.3.2 HPIV3
of only odd-numbered years [168] (Table 25.2). Infections with HPIV3 virus have usually been described as
endemic in nature, occurring throughout the year [87, 89].
5.3.1 HPIV1 and HPIV2 Small outbreaks have been noted, but no predictable peri-
The longest continuous observation of the epidemic behavior odicity in their occurrence was noted until after a sharp out-
of HPIV1 has been carried out at the Children’s Hospital, break occurred in Houston in 1977 (Fig. 25.2) [92]. Since
Washington, District of Columbia. From 1957 until 1961, the that time, HPIV3 infections have tended to occur in the

Table 25.2 Clinical and epidemiologic manifestations of various HPIV types

Human serotype Major clinical syndrome Peak age (months) Sex predominance Periodicitya
HPIV1 Croup 6–24 Male Epidemic (fall)
HPIV2 Croup 6–24 Male Epidemic (fall)
HPIV3 Pneumonia and bronchiolitis 0–6 None Endemic
HPIV4 URI Unknown Unknown Endemic
a
Peak season in parentheses
25 Paramyxoviruses: Parainfluenza Viruses 585

Fig. 25.2 Schematic Sialic acid-containing

illustration of the parainfluenza Genome (–)
receptions
virus life cycle. RER, rough
HPIV
endoplasmic reticulum [26] Host cell Virion
Binding

Transcription
of mRNA

RER Antigenome(–)
Replication

Uncoating

Translation
M L NP P Fusion

Genome(–)

Golgi
apparatus

Budding Release

Glycosylation of
HN and F completed
Transfer to membrane

spring or during the months when HPIV1 and HPIV2 were ding was documented for up to 16 days, with a median of
not prevalent [168]. Although other major respiratory viruses 12 days. In addition, HPIV4 was detected in 4/127 (3 %)
generally cause relatively discrete epidemics, outbreaks of asymptomatic enrollment swabs, with no asymptomatic
HPIV3 have occurred concurrently with outbreaks of other shedding documented for either HPIV1 or HPIV2, and
viruses [89]. 3/127 (2 %) for HPIV3.
Other recent studies using molecular techniques to detect
5.3.3 HPIV4 HPIV4 have been primarily based on hospitalized children,
HPIV4 has been assumed to cause relatively mild disease with a potential bias toward children with more severe ill-
and generally has not been included in rapid antigen detec- ness. The proportion of HPIV4 as a cause of HPIV infection
tion panels or diagnostic tests offered in pediatric centers. in these hospital studies ranges from 1.2 to 9.1 %, with a
The addition of PCR techniques has increased detection median of 3.5 % [58, 167, 171, 172], compared to the 10 %
rates of this virus. Recently, HPIV4 infection was specifi- rate documented in the day-care study. The relatively higher
cally investigated in 225 young children attending day care rate of HPIV4 infection in the outpatient study suggests that
in Fort Lewis, WA, using RT-PCR techniques [52]. HPIV HPIV4 may not be as likely to cause severe infection.
was detected in 87/523 (17 %) of illnesses, with HPIV4
present in 10 % of these HPIV illnesses, the second most
common type following HPIV3 (85 % of HPIV-positive 5.4 Geographic Distribution
illnesses). Marked differences in the proportion of HPIV4
infections between the early and later years of the 3-year HPIVs are found throughout the world and cause illness
study period were seen (22 % the first 2 years vs. 2 % the in young children wherever they have been identified.
last year). No differences in the demographic profiles of There is remarkable similarity of serological and isola-
children infected with HPIV4 versus other HPIV types tion or PCR data obtained from tropical [80, 138, 173],
were detected, including age, sex, race, or duration of time temperate [38, 78, 84], and arctic [141, 174] climates.
spent in day care, and no seasonal pattern was apparent However, antibody to HPIV1 was found in very few Tiriyo
for HPIV4 infection. Only one of 9 HPIV4 infections was Indians in South America and none under age 20; in the
associated with croup; the remaining cases all had uncom- Xikrin tribe, there was no antibody in persons under age
plicated upper respiratory tract infections. HPIV4 shed- 17 years. HPIV2 antibody was present in Xikrin of all ages
586 J.A. Englund and A. Moscona

but almost entirely absent in other tribes. This suggests almost 15 to 7 per 1,000 children per year for children under
that fresh introductions of virus are needed in very remote 3 years of age [88].
tribes in which the population base is too small to sustain After the first 3 years of life, the incidence of lower
the infection [142]. respiratory illnesses associated with HPIV3 infections
falls off considerably, although studies indicate that rein-
fections are common in older children and adults. These
5.5 Age Distribution infections are not generally associated with evidence of
lower tract involvement [38, 81, 113, 125, 126]. Studies of
5.5.1 HPIV1 and HPIV2 HPIV3 infection in adults hospitalized with lower respira-
Data from studies of lower respiratory disease show few cases tory tract infection indicate an infection rate of approxi-
of severe disease in infants under 4 months of age [35, 36, mately 3 % based on serology alone [177]. In a prospective
82, 95, 104, 138], with the most severe disease documented study in adults with underlying respiratory disease, and
in premature infants or in newborn intensive care units [175, using diagnostic techniques based on culture, PCR, and
176]. After 4 months of age, the number of cases of croup serology, HPIV3 was the most common respiratory virus
and other lower respiratory diseases increases dramatically after influenza detected in adults over the age of 45 [83].
[36], until approximately age 6 years. Lower respiratory HPIV infections are being increasingly recognized, like
illnesses in persons infected by HPIV1 and HPIV2 viruses RSV, as an etiology of serious respiratory disease in
are unusual in adolescents [89] and adults [81, 128], although elderly adults. Like RSV, HPIV3 causes outbreaks in geri-
they have been reported [129, 177]. atric facilities and can cause severe disease in older indi-
Studies of milder illness have shown a similar age distri- viduals [179–181].
bution. Infection and minor clinical illness occur more fre-
quently in younger children than in adolescents and adults
[86, 113, 178]. Infections do occur in older persons [81, 85, 5.6 Gender Differences in Infection
86, 90, 95, 114, 126], most of which presumably represent
recurrent infections in persons with antibody. Studies con- Boys have been long recognized to have a greater frequency
ducted in Tecumseh [86, 90] showed serological evidence of of croup [182]. The Chapel Hill pediatric practice studies
frequent infections in adults in age groups likely to include demonstrated infection rates for HPIV1 virus of 1.8 lower
parents of school children. In family studies, infection in respiratory illnesses per 100 boys compared to 1.1 per 100
adults occurred concurrently with illness in their children, girls [36]. This sex difference disappeared after age 6 years.
but attack rates in adults were distinctly lower than those in A similar predominance of serious illness in young males
younger family members [113, 178]. Rates of hospitaliza- has been noted in other studies of lower respiratory disease
tion for HPIV1 and HPIV2 in adults with lower respiratory [183]. Rates of infection in young boys and girls seem to be
tract disease in the United States appear to represent only 0.2 the same, but clinical manifestations of infection are more
and 2.5 % of adults hospitalized for lower respiratory tract severe in boys [89]. Insufficient data are available to ana-
disease [177]. lyze HPIV2. HPIV3 caused identical rates of lower respira-
tory illness in males and females for children observed in
5.5.2 HPIV3 Chapel Hill [89] and in the Fort Lewis day-care study [111];
Initial infections with HPIV3 virus occur early in life. however, in the Houston longitudinal study, boys were more
Among children followed from birth in the Houston Family likely to have lower respiratory tract involvement with pri-
Study, 62 % were infected during the first year of life, and by mary infection [92].
the end of the second year, 92 % had been infected at least
once and 36 % had one or more reinfections [92]. Similar to
RSV infections, HPIV3 virus infections often occur in the 5.7 Special Settings
very first months of life, despite the fact that these infants
may still possess circulating antibodies derived from their Nosocomial infections with respiratory viruses occur readily
mothers [34–37, 95]. Infants born with the highest liters of if susceptible individuals are not isolated from those with
maternal antibody to HPIV3 may be spared during the first HPIV shedding or respiratory disease. HPIV infections are
months of life. The risk of lower respiratory disease is greater detected commonly in studies of hospital-acquired infections
for primary infection during the second year than for primary in children on pediatric wards [163, 184–186] and neonatal
infection during the first year [92]. In studies of lower respi- units [119, 175, 176, 187].
ratory disease in the Chapel Hill pediatric group practice, Increasing reports of outbreaks and severe disease
the age-specific attack rate for HPIV3 disease paralleled that associated with HPIVs are being reported in immunocom-
of RSV [36]. The average annual attack rate ranged from promised hosts [45, 105, 106, 121, 155–157, 188–190].
25 Paramyxoviruses: Parainfluenza Viruses 587

Hematopoietic stem cell marrow transplant units and commonly associated with an economically important dis-
hematology/oncology wards are particularly susceptible to ease of cattle usually called “shipping fever” [198], but there
nosocomial infections due to cohorts of vulnerable patients is no evidence of spread between cattle and man.
who may shed viruses for prolonged periods of time, even
asymptomatically [9, 42, 54, 191, 192]. Bronchoalveolar
lavage fluid from these patients may contain high viral 7 Pathogenesis and Immunity
loads [193], and patients may be hospitalized for prolonged
periods with increased exposure to viruses and other patho- 7.1 Pathogenesis
gens. Lung transplant recipients are also at high risk for
serious morbidity and mortality from HPIVs [106], and The viral surface glycoproteins play a key role in the first
outbreaks in lung transplant centers have been reported steps of infection by HPIV. Infection of host cells is initiated
[194]. Symptom-based isolation, institution of good hand- by attachment of the virus to the host cell through interac-
washing, and viral surveillance of patients with sensitive tion of the receptor-binding glycoprotein, hemagglutinin-
and rapid methods of detecting infection are necessary to neuraminidase (HN) with a sialic acid-containing receptor
institute control measures and recognize potential candi- molecule on the host cell surface, as diagrammed in the
dates for experimental antiviral therapy. schematic viral life cycle shown in Fig. 25.2. Upon interac-
tion with receptor, the receptor-binding glycoprotein triggers
or activates the viral fusion protein (F) to its fusion-ready
6 Mechanisms and Route state, promoting fusion of the virus into the cell and initia-
of Transmission tion of infection [25]. The fusion glycoprotein F is synthe-
sized as a single polypeptide chain that forms a trimer before
Transmission of HPIV is by direct person-to-person contact being cleaved by host cell proteases to yield a membrane-
or large-droplet spread. These viruses do not persist long in distal and membrane-anchored subunit. The new N-terminal
the environment, but HPIV1 has been recovered from air region of the membrane-anchored subunit, termed the fusion
samples collected in the vicinity of infected patients [32]; peptide, contains the hydrophobic residues that insert into
from 1 to 10 % of HPIV3 virus particles in aerosol may be target membranes during fusion at neutral pH (reviewed in
viable after 1 h [31]. Adult volunteers who have had prior [199]). Infection also may result in fusion between cells,
natural infection have been reinfected experimentally by which involves the interaction of receptor-binding proteins
inoculation of the upper airway with a coarse aerosol or nasal and fusion proteins expressed on the surface of an infected
drops or both [43, 132]. The high rate of infection early in cell with the membrane of an adjacent uninfected cell. The
life coupled with the high frequency of reinfection suggests role of the HPIV HN molecule in promotion of fusion during
that the virus spreads readily, that reinfected persons may be viral entry has been elucidated in recent years [25, 27, 200–
infectious, and that a relatively small inoculum may result 202]. Unlike influenza viruses, in which separate surface
in infection. glycoproteins are responsible for the neuraminidase (NA)
Outbreaks of HPIV1 and HPIV3 in symptomatic and receptor-binding (HA) activities, the HPIV HN is a dual-
and asymptomatic healthy young adults stationed at the function molecule, carrying out both receptor binding during
Amundsen-Scott South Pole Station in 1978 after 10 and 29 entry and receptor cleavage during budding and release of
weeks of total isolation present an interesting addition to the virus. The third function of HN, once receptor engaged, is
story of HPIV shedding and viral transmission [195, 196]. to trigger or activate F to undergo its final conformational
Both HPIV1 and HPIV3 were recovered from frozen throat alteration that permits fusion [25]. One bifunctional active
swabs of personnel overwintering at this station, and serolog- site on HN possesses both binding and neuraminidase activi-
ical responses during midwinter outbreaks of HPIV infection ties, while a recently identified second site on HN possesses
when no new personnel had arrived for over 8 weeks suggest binding and F-protein-triggering activities [27, 203]. Finally,
that persistent infection accompanied by prolonged shedding recent evidence suggests that HN interacts with F prior to
of HPIV occurs in healthy persons and may lead to outbreaks engaging receptor and exerts a stabilizing effect on F that
of respiratory illness. prevents F from being activated before it is in a suitable posi-
There is little evidence of animal reservoirs for human tion with respect to the target cell [204].
disease and no evidence of vector spread. Sendai virus is a After fusion of the viral envelope with the plasma mem-
rodent strain of HPIV1 virus, but there is no evidence that brane of the cell, the viral genetic material is released into
it is related to disease in humans [22]. HPIV5 (formerly the cytoplasm, as shown in Fig. 25.2. The nucleocapsid con-
called SV5), a simian virus related to HPIV2 virus, has been tains the genome RNA in tight association with the viral
reported to cause human infections but only on very rare NP protein, and this RNA/protein complex is the template
occasions [20, 197]. Bovine HPIV3 virus is one of the agents for both transcription and genome RNA replication. The
588 J.A. Englund and A. Moscona

first step in the viral growth process, termed primary tran- virus infection [218]. For the paramyxoviruses including the
scription to denote transcription directly from the infecting HPIVs, evasion of this response is mediated by the C and
nucleocapsid template, initiates at the 3’ end of the genome. V proteins. The V proteins are involved in a wide range of
Each of the mRNAs is transcribed and present in infected mechanisms for evading the immune response, including
cells, at a level correlated to its relative location on the prevention of apoptosis [219, 220], cell cycle alterations
negative-sense viral genome. For five of the six genes of [221], inhibition of double-stranded RNA signaling [219,
the HPIVs, transcription generally leads to generation of 222], and prevention of IFN biosynthesis [219, 220, 222].
a single mRNA species that encodes a single protein mol- The strategies whereby different V proteins inhibit STAT
ecule. However, the P gene of HPIVs encodes a number of proteins have been found to be highly diverse. For example,
proteins, and in several members of the family, more than HPIV type 2 V protein targets STAT2 for degradation [223,
one mRNA is synthesized and the mRNA also encodes a 224], while mumps virus V protein can target both STAT1
number of smaller protein products in an alternate reading [225] and STAT3 [226] and can also interact with cellular
frame from the P protein. These “C” proteins, as they are RACK1, potentially modifying IFN receptor activity [227].
commonly called, are a nested set of carboxy-coterminal
proteins, with each protein encoded in the same reading
frame but differing by its start site on the mRNA. The func- 7.2 Immunity
tions of the C proteins are known to include key roles in
evasion of the host immune response [205]. HPIVs replicate in the epithelium of the upper respiratory
Unlike transcription, replication of the viral RNA genome tract and spread to the lower respiratory tract within 3 days.
depends on ongoing protein synthesis and therefore is linked The disease manifestations result from inflammatory obstruc-
to prior transcription of the viral genome. The second stage tion of the airway. Epithelial cells of the small airways may
of genome RNA replication occurs when the newly repli- become infected with resultant necrosis and inflammatory
cated and encapsidated full-length plus-stranded RNA infiltrates. The interplay between virus-induced cell damage,
(antigenome)-containing nucleocapsids are copied into beneficial immune responses, and inflammatory responses
negative-stranded RNA-containing progeny nucleocapsids that contribute to disease for HPIV has not been well studied
identical to the nucleocapsid that is packaged into progeny as it has for RSV; however, it is likely that in many cases, dis-
viral particles. These progeny viral nucleocapsids are tem- ease severity is increased and the pathology of clinical dis-
plates for transcription. ease is actually caused by the inflammatory response rather
After replication, as shown in Fig. 25.2, new viruses are than by the cytopathic effects of the virus. This concept is
formed by budding of newly replicated and assembled nucleo- highlighted by the fact that virus titers in the infected host
capsids containing the viral RNA genome along with the P and are generally waning by the time disease symptoms become
L proteins through areas of the plasma membrane that contain apparent [228] and that virus titer does not correlate with the
the viral surface glycoproteins and the M (matrix) protein. severity of lower respiratory disease. The pathologic changes
In polarized epithelial cells, the viruses bud from the apical that are present in children that have died with parainflu-
surface of the cell. It is thought that the non-glycosylated M enza infection suggest exaggerated inflammation [152, 229]
protein binds to the nucleocapsid, and also likely interacts rather than simply tissue destruction by virus.
with the cytoplasmic tails of the transmembrane glycopro- HPIV infections can induce both humoral and cellular
teins, and thereby mediates the alignment of the nucleocapsid immune responses in infected humans, including innate
and the areas of the plasma membrane containing viral gly- responses, local and systemic IgG and IgG responses, and
coproteins in preparation for budding [206–211]. It is likely T-cell responses. HPIV primary infection does not confer
that entry, fusion, and budding involve the participation of permanent immunity; however, although reinfection occurs,
the cellular actin cytoskeleton as well as microtubule func- immunity is usually sufficient to restrict virus replication
tion [212–217]. Host cellular proteins that are involved in cell from the lower respiratory tract and prevent severe disease.
mobility may be important for these steps; one possible com- Mucosal IgA levels correlate with protection from replica-
ponent of the entry/fusion mechanisms involves interaction tion of HPIVs in humans [134, 135] and seem to provide the
between paramyxovirus F proteins and Rho A [215, 216]. best correlate for protection, at least in adults. Cell-mediated
Host cells have developed a variety of defense mecha- immunity figures importantly in prevention of disease: for
nisms in the face of viral infection. In some of these, an example, HPIV3 infection of T-cell-deficient infants can
interferon (IFN)-induced antiviral state is created; some cause a fatal giant-cell pneumonia [134, 135], and in bone
cell types undergo apoptosis, which limits viral replica- marrow transplant recipients, HPIV pneumonia has a 30 %
tion. Many viruses have been found to possess strategies mortality [106]. However, neutralizing antibodies against
for evading these host immune responses, particularly the the HN and F proteins of HPIV are critical for long-term
innate immune response that plays a key role in controlling protection [230].
25 Paramyxoviruses: Parainfluenza Viruses 589

8 Patterns of Host Response only 20 % of the time [38], but this is likely higher than
that in the family setting.
8.1 Clinical Manifestations Evidence that HPIV infections may predispose to bacte-
rial infections is accumulating [232–235]. Both otitis media
Primary infections with HPIVs are usually symptomatic, and bacterial tracheitis in children have been associated with
with clinical manifestations ranging from an afebrile upper these infections [236], with otitis media documented in about
respiratory illness to severe, life-threatening lower respira- 1/3 of children diagnosed with acute onset of HPIV infection
tory disease. The most characteristic and clinically impor- [237], rates similar to that of otitis media following influenza
tant syndrome associated with infections with HPIV1 and or rhinovirus infection. An outbreak of invasive pneumococ-
HPIV2 is croup or laryngotracheobronchitis. HPIV1 was cal disease in a chimpanzee colony followed infections with
isolated from 20 % of patients with croup in studies in HPIV3 [238]. Animals with HPIV3 upper respiratory illness
the Chapel Hill pediatric practice [36, 169], but HPIV2 were 5.7 times more likely to develop invasive pneumococ-
virus is much less frequently associated with croup than cal infection despite the fact that most of the animals had
is HPIV1 [34, 89, 169]. Studies in ambulatory populations received pneumococcal vaccine.
suggest that most initial infections with HPIV1 virus result
in febrile upper respiratory illness, whereas initial infec-
tions with HPIV2 virus result in less severe upper respira- 8.2 Clinical Syndromes
tory illness with many illnesses presenting without fever
[41, 109]. Reinfection with HPIV results in similar respi- 8.2.1 Pediatric Disease
ratory symptoms from those caused by other viruses [92, Clinical diagnosis of the etiology of sporadic episodes of
113]. The clinical manifestations of infections with HPIV acute respiratory disease is difficult because many viral respi-
viruses may differ in developing countries [147], with more ratory diseases have similar signs and symptoms. Infections
cases of lower respiratory disease noted, although differ- with HPIV may be suspected when characteristic clini-
ences in surveillance methods could account for some of cal manifestations occur in known epidemiologic patterns.
these features. The association of HPIV with croup, well The determination of HPIV outbreaks in the community is
described in developed countries, does not appear to be best ascertained using sensitive laboratory methods such as
as common in developing countries [138, 146, 151, 231], RT-PCR (see Sect. 4.2).
where HPIV1 and HPIV2 infections are typically associ- In developing countries, HPIV1 and HPIV2 infections
ated with pneumonia and tracheobronchitis [138, 146, 151, appear to be less consistently associated with croup. In
231] (Table 25.2). an HPIV1 epidemic in Trinidad, pneumonia was the most
The clinical manifestations of infections with HPIV3 common lower respiratory tract diagnosis [138]. In such
are varied. In the Chapel Hill studies, the clinical diagno- a setting, the clinical disease pattern will be less useful in
sis varied with age [89]. Infants under 1 year of age were identifying the occurrence of an HPIV1 epidemic. HPIV3
likely to present with bronchiolitis or pneumonia, whereas virus infections are much less predictable, but this etiology
children from 6 to 18 months might often have croup. should be considered for infants less than 6 months of age
Older children were usually diagnosed as having tracheo- with bronchiolitis or pneumonia occurring at times other
bronchitis. In general, no consistent clinical presentation than during RSV epidemics. Although medically attended
of HPIV3 virus infection was found. The severe manifesta- lower respiratory illnesses associated with HPIV3 virus have
tions of HPIV3 infection, usually pneumonia, in develop- had an even sex distribution [36], longitudinal studies have
ing countries are similar to those in developed countries. found the incidence of both croup and lower tract involve-
Figure 25.3 shows representative radiographic evidence of ment more common in boys [92].
the pulmonary disease associated with HPIV3 in an immu-
nocompromised child. 8.2.2 Disease in the Immunocompromised
Prospective studies of children indicate that primary Host
infection with HPIV3 is usually symptomatic but often The severe impact of HPIV infection in immunocompro-
mild [41, 92]. In children followed for the first 2 years mised patients was first recognized in children with under-
of life, about one-third of primary infections involved the lying immunodeficiencies, in whom giant-cell pneumonia
lower respiratory tract, but only 5 % of primary infec- has been documented at autopsy [156, 239]. Persistent
tions resulted in lower respiratory illnesses for which respiratory tract disease and shedding have been observed
families sought medical. Frequency of reinfection—both in these children (Fig. 25.3) as well as HIV-infected patients
symptomatic and asymptomatic—decreased with age. [240]. In adult hematopoietic stem cell transplant patients,
Reinfection was symptomatic among young children at respiratory viruses cause about two-thirds of the respiratory
the Washington, DC, children’s home, Junior Village, illnesses, with high mortality [5]. HPIV3 accounts for up to
590 J.A. Englund and A. Moscona

and may be less with nonmyeloablative conditioning [9,

121, 243]. Radiographic findings can vary from mild focal
infiltrates to diffuse interstitial and alveolar interstitial infil-
trates (Figs. 25.3 and 25.4) [106]. Risk of death following
the development of lower tract disease can range up to 45 %
[244]. Dissemination of disease to the pancreas, brain, peri-
cardium, and myocardium has been reported [156, 245],
but the most common serious adverse sequelae associated
with HPIV infection in stem cell transplant patients and
lung transplant recipients are significant declines in pulmo-
nary function posttransplant [194, 246], even in stem cell
transplant recipients who had only upper respiratory tract
HPIV infection.

9 Control and Prevention Based

Fig. 25.3 Chest radiograph of an 8-month-old child with severe com-
bined immunodeficiency infected with HPIV2 obtained 3 weeks fol- on Epidemiologic Data
lowing a hematopoietic stem cell transplant. The child was infected
with HPIV2 pretransplant but unable to clear the virus due to the immu- 9.1 Vaccines
nodeficiency and did well clinically until 3 weeks posttransplant, when
he had respiratory distress requiring oxygen support. Chest radiograph
is remarkable for right perihilar opacities associated with mild perihilar The development of a vaccine for the HPIVs, as for RSV,
peribronchial cuffing, suggestive of viral lower tract infection. The has been difficult because of the need to induce an immune
child was treated with the experimental drug DAS181 (Ansun response in young infants, who have both an immature immune
Biopharma, San Diego, CA), under an emergency IND, cleared the system and maternal antibodies that may interfere with the
HPIV, and was discharged home
development of an adequate immune response. An inactivated
HPIV1, HPIV2, and HPIV3 vaccine used in infants in the late
90 % of these cases [121], causing pneumonia (Fig. 25.4) 1960s was immunogenic but did not offer protection from
with a 35 % acute mortality rate and a 75 % mortality rate infection [247, 248]. Since that time, it has been challeng-
at 6 months posttransplant [121]. High rates of pneumo- ing to identify vaccine viruses that are adequately attenuated
nia have been described in patients undergoing stem cell for the vulnerable seronegative infant, yet also immunogenic.
transplantation [106]; solid organ transplantation with The development of reverse genetic systems has allowed for
kidney, heart, or lung [194, 241, 242]; and patients with specific engineering of attenuated yet immunogenic vaccine
hematologic malignancies undergoing chemotherapy, par- viruses. Experimental vaccines are now under evaluation, with
ticularly induction chemotherapy [243]. The largest reports a reasonable expectation that a vaccine for HPIV3, and per-
of HPIV infection have been reported in hematologic stem haps also HPIV1, will be feasible [230].
cell transplant recipients [121], where HPIV infection was Two different strategies of interest are currently under
diagnosed in 253/3,577 transplant recipients (7.1 %), with active development by NIAID and industrial partners for
78 % of the infections community acquired. In this study HPIV3 vaccines: candidates based on live-attenuated bovine
based on patients hospitalized in the 1990s, diagnosis was parainfluenza type 3 (BPIV3) strains and those based on a
confirmed mainly using direct immunofluorescence testing cold-adapted live-attenuated strain. Studies of both bovine-
and culture; rates of HPIV infection using more sensitive derived vaccines and live-attenuated human HPIV3 vaccines
methods at the same institution in the early 2000s were derived from well-characterized passages of human-derived
noted to be higher—up to 13 % during the first year post- vaccine strains have shown good evidence of safety, infectiv-
transplant [42]. Most infections in the transplant patients ity, and immunogenicity in children and infants [249–257].
begin with typical symptoms of an upper respiratory tract In addition, new live chimeric candidate vaccines based
infection with a low-grade fever, although asymptomatic on a chimeric construct expressing the HPIV3 F (fusion)
infections or intermittent asymptomatic shedding have and HN (hemagglutinin-neuraminidase) proteins and the
been noted [42]. Sinusitis may be present in up to 40 % of RSV F proteins from the bovine HPIV3 viral genome have
patients [243]. Progression from upper to respiratory tract been utilized in human trials in seropositive and seronega-
disease is variable but common, ranging in various reports tive children and young infants [258, 259], suggesting that
from 18 to 77 %. Risk of progression in stem cell transplant a combined HPIV3/RSV vaccine could be feasible [252,
recipients is associated with steroid use and lymphopenia 260]. Recombinant HPIV3 vaccines have now passed phase
25 Paramyxoviruses: Parainfluenza Viruses 591

a b

Fig. 25.4 Chest radiographic (a) and computed tomography findings beginning on day 7 posttransplant. He required intubation on day 18
(b, c) of a neutropenic adult male hematopoietic stem cell transplant posttransplant and, despite treatment with ribavirin, IVIG, and steroids,
recipient with HPIV3 infection diagnosed on day 11 posttransplant. No died soon afterward. (a) Chest radiograph, day 11 posttransplant. (b)
other pathogens other than HPIV3 were identified despite two bron- Computed tomography of chest, day 11 post stem cell transplant. (c)
choalveolar lavage procedures and PCR testing for multiple bacterial, Pulmonary consolidations and pleural effusions shortly before death,
fungal, and viral pathogens. His history was significant for sinusitis pre- day 18 posttransplant
transplant, with fever and upper respiratory tract symptoms documented

I evaluation and are undergoing testing in proof-of-concept (Sendai virus) is being evaluated in a similar strategy to the
trials [230]. bovine virus strategy for HPIV3. Approaches to HPIV2
The approach farthest along for HPIV1 vaccines is an also include specifically attenuated engineered viruses.
engineered live-attenuated virus, with a series of attenuat- Attenuated HPIV1 and HPIV2 vaccines are both at the pedi-
ing mutations in several genes. In addition, a murine HPIV1 atric phase I stage of clinical trials [230].
592 J.A. Englund and A. Moscona

9.2 Antivirals transient intermediate to draw the viral and cell membranes
together, and a conserved coiled-coil motif required for this
Therapy for the treatment of HPIV infections has relied on step can be specifically targeted by fusion-inhibitory pep-
nonspecific symptomatic or anti-inflammatory treatments. tides that bind to specific sequences on the F protein. Such
For example, symptomatic benefit for the treatment of moder- peptides inhibit viral fusion and entry in a dominant-negative
ate to severe HPIV1-associated croup has been demonstrated manner by binding to F’s transient intermediate, preventing
with short courses of glucocorticoids, such as dexametha- F from advancing to the next step in fusion. Recently, it has
sone, shown to provide clinical and economical benefits, been shown that membrane targeting of fusion-inhibitory
although ineffective for HPIV2- or HPIV3-associated respi- peptides by use of a lipid tag allows efficient inhibition of
ratory disease [261, 262]. There is no specific antiviral ther- the transient intermediate of fusion and also increases serum
apy available for the treatment of HPIV infections. While half-life and tissue biodistribution of the peptides, making
ribavirin has been discussed as a potential HPIV antiviral them promising candidates for development as HPIV antivi-
based on in vitro data, it has no proven benefit, although rals [277–279].
there are anecdotal reports of benefit and lack of benefit in Finally, it is possible to subvert the normal process
nonrandomized case series [121, 263–265]. Ribavirin ther- whereby HN activates F and cause HN to activate the F pro-
apy is not recommended [266] as published reports do not tein’s fusion mechanism before it reaches the target host cell,
suggest a link between ribavirin use in cases of HPIV infec- thus incapacitating F before it can mediate viral entry; com-
tion and improved outcome. pounds are under development that will act by this mech-
The potential benefit of antiviral treatment, especially anism [280]. As with other rapidly evolving RNA viruses
for very young infants and immunocompromised hosts, is (influenza, HIV, etc.), it may be important to use several anti-
increasingly recognized [26, 244]. Several features of the viral agents simultaneously to attack different viral targets,
viral life cycle make HPIV vulnerable to attack. HPIVs to avoid the inevitable development of resistance to single
enter their target cell by binding to a receptor molecule and antivirals. It will therefore be critical to develop several anti-
then fusing their viral envelope with the cell membrane to viral strategies in parallel.
enter the cytoplasm. Binding, fusion, and each step in cell
entry are critical stages which could be targeted to prevent
HPIV infection and disease [26, 267, 268]. One potential 10 Unresolved Problems
strategy for HPIV and other sialic-binding viruses targets
the removal of lung epithelial sialic acid receptor for HPIV, The clinical spectrum of illness at a young age, the failure of
thereby preventing viral entry. A new recombinant sialidase, serum antibody to adequately protect against disease, and the
first developed as an antiviral agent for influenza, functions ubiquity of infections make the HPIVs serious and challeng-
by cleaving sialic acids from the host cell surface, thereby ing foes. Better understanding of the pathogenesis of disease
inactivating the host cell receptor recognized by HPIV [269]. and of the protective immune response will facilitate the
Reports of successful use of this agent in several adult and development of effective antivirals and vaccines. The deter-
pediatric transplant recipients under compassionate use have minants of protective immunity need to be defined for natu-
been reported [270, 271, 272]. The molecule is an engineered ral infection, so that the natural response can be duplicated
neuraminidase molecule (DAS 181; Fludase) [268] and has by active immunization. The use of attenuated live virus
not entered clinical trials at this time for HPIV but shows vaccines and particularly live virus vaccines built on the
promise in proof-of-concept treatment of immunocompro- backbone of HPIV requires further investigation as a poten-
mised humans [273], and trials in specific populations are tially beneficial and economical approach to immunization
underway. of young children. In addition, immunization of women of
The HN molecule, in addition to binding to receptors, con- childbearing age should be considered as a means of protect-
tains neuraminidase (receptor-cleaving) activity and cleaves ing infants by passively acquired antibody during the first
the sialic acid moieties of cellular receptors, allowing new months of life.
virions to be released from the host cell surface and infection As the number of immunocompromised patients increases
to spread. While neuraminidase inhibition is unlikely to be as due to advances in transplantation and antiviral HIV therapy
effective for parainfluenza viruses as it has been for influenza and new methods of monoclonal antibody immunosuppres-
virus as an antiviral strategy [26, 274, 275], specific inhibi- sion and treatment for a wide variety of diseases, the impact
tion of this activity could prevent virion entry into additional of respiratory viruses in general and HPIV in particular is
uninfected cells [276]. becoming more well known. The need for safe and effective
The fusion protein undergoes a series of structural transi- antivirals for the prophylaxis and treatment of HPIV infec-
tions as it mediates viral entry, and each of these steps is tions in immunocompromised patients should be a high pri-
a potential target for antiviral development. F must form a ority, and the need to avoid resistance by potentially using
25 Paramyxoviruses: Parainfluenza Viruses 593

several simultaneous antiviral strategies is important to any 11. Chanock RM, Parrott RH, Cook K, et al. Newly recognized myxo-
antiviral approach designed to combat RNA viruses. viruses from children with respiratory disease. N Engl J Med.
1958;258:207–13.
A major unsolved problem is the substantial excess mor- 12. Andrewes CH, Bang FB, Chanock RM, Zhdanov VM. Para-
tality from acute respiratory disease in developing countries. influenza viruses 1, 2, and 3: suggested names for recently described
Acute respiratory disease remains one of the leading causes of myxoviruses. Virology. 1959;8:129–30.
death in children under 5 years of age in the developing world; 13. Johnson KM, Chanock RM, Cook MK, Huebner RJ. Studies of
a new human hemadsorption virus. I. Isolation, properties and
available information suggests that HPIVs contribute signifi- characterization. Am J Hyg. 1960;71:81–92.
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 Paramyxoviruses: Respiratory
Syncytial Virus and Human 26
Metapneumovirus

James E. Crowe Jr. and John V. Williams

1 Human Respiratory Syncytial Virus common viral agent of serious pediatric respiratory tract dis-
ease worldwide. In most areas of the world, RSV is the most
1.1 Introduction common cause of pneumonia and bronchiolitis in infants less
than 1 year of age. RSV causes severe disease in young
Human respiratory syncytial virus (RSV) and human infants, but disease is not restricted to the early life period.
metapneumovirus (MPV) are members of the family The virus can cause severe lower respiratory tract illness in
Paramyxoviridae of the Mononegavirales order, com- large numbers of elderly subjects and also in subjects who
prising the nonsegmented negative-strand RNA viruses. are severely immunocompromised such as hematopoietic
Paramyxoviridae has two subfamilies: Paramyxovirinae, stem cell transplant recipients [3–7].
which includes the parainfluenza viruses 1–4 and measles
and mumps viruses, and Pneumovirinae, which includes RSV
and MPV. Pneumovirinae has two genera: Pneumovirus, 1.3 Epidemiologic Analysis
which includes human RSV, bovine respiratory syncytial
virus, and pneumonia virus of mice, and Metapneumovirus, 1.3.1 Mortality Data
which includes human MPV and avian metapneumovirus, Mortality in infants and children caused by RSV is uncom-
sometimes called avian pneumovirus. mon in developed countries with modern critical care units.
Estimates of the mortality rate are about 0.3 % of hospital-
ized children or less. Mortality has been dropping over the
1.2 Historical Background last several decades, and by the late 1990s the estimated
number of deaths in the USA was several hundred children a
RSV was isolated first in 1956 from an ill chimpanzee in a year or less [8, 9]. Large epidemiologic studies report that
laboratory setting that had an illness similar to the common the US mortality in children may be only about 100 cases a
cold. A virus causing a similar cytopathic effect in cultured year. Interestingly, while many providers think of RSV infec-
cells was recovered from infants with respiratory illness tion as principally a pediatric illness, there are over 17,000
shortly after, and studies of human antibodies in the serum of deaths per year in the elderly, making them the highest risk
infants and children indicated that infection was common population for death [9]. In less developed countries, how-
early in life [1, 2]. Now it is known that RSV is the most ever, infant deaths due to RSV infection may be more
common.
J.E. Crowe Jr., MD Infants with underlying illness are at highest risk among
Vanderbilt in Vaccine Center, young children for morbidity and mortality from RSV infec-
Departments of Pediatrics, Pathology, Microbiology
tion. Infants with chronic lung disease requiring supplemen-
and Immunology, Vanderbilt Vaccine Center,
Vanderbilt University Medical Center, tal oxygen following treatment for prematurity, due to
11475 Medical Research Building IV – 2213 Garland Ave., bronchopulmonary dysplasia, are perhaps at the highest risk
Nashville, TN 37232-0417, USA for prolonged, severe, or fatal illness due to RSV [10]. Infants
e-mail: [email protected]
with severe congenital pulmonary or cardiac disease have
J.V. Williams, MD (*) been reported to be at risk of death in 3–4 % of cases when
Division of Infectious Diseases,
hospitalized, although this rate is likely decreasing in the
Department of Pediatrics, Vanderbilt University Medical Center,
D-7235 MCN, Nashville, TN 37232-2581, USA USA [11]. Both children and adults with primary immuno-
e-mail: [email protected] deficiency or medically induced immunosuppression are at

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 601

DOI 10.1007/978-1-4899-7448-8_26, © Springer Science+Business Media New York 2014
602 J.E. Crowe Jr. and J.V. Williams

high risk of mortality due to RSV infection. The most best functional marker of infection, but sensitivity is much
severely immunocompromised, and thus those at highest risk higher in antigen binding assays using individually purified
of mortality, are hematopoietic stem cell transplant patients RSV F or G proteins [22].
of any age [12]. In some settings, the mortality rate of RSV
infection in hematopoietic stem cell transplant patients with 1.3.4 Laboratory Methods
severe immunosuppression verges on 100 % [12]. Isolation of the virus in cell culture provides a definitive test
for diagnosis of active infection. Various methods of obtain-
1.3.2 Morbidity Data ing respiratory virus secretions for testing have been com-
Hospitalization rates of infants for RSV disease vary with pared. Most studies suggested that aspiration or gentle
the setting, probably due to variations in exposure, genetics, flushing of nasal secretions using a solution like saline is
socioeconomic, and other risk factors and due to the local best, though some types of nasal swab have given reasonable
practice style of medical providers. Many developed coun- results. The virus is more thermolabile than most, and thus
tries report hospitalization rates of about 1 in 100–200 samples should be transported on wet ice to the diagnostic
infected infants during the first year of life [13, 14]. Studies laboratory and processed rapidly. Prolonged times of trans-
of RSV disease in developed countries suggest that of those port to remote reference laboratories reduce the effectiveness
infants hospitalized, about 9 % require mechanical ventila- of isolation. After inoculation onto susceptible cell culture
tion [15, 16]. There are certain populations at extraordinarily substrates, highly trained staff can recognize cytopathology
high risk of hospitalization with RSV, for example, Alaskan in the cell monolayers by visual inspection and conventional
native infants younger than 1 year have been reported to have bright-field microscopy after about 3–7 days of incubation.
a hospitalization rate of 53–249 per 1,000 infants [16]. Low Detection may be more efficient when using shell vial cul-
socioeconomic status is a risk factor for higher rate of hospi- tures and immunofluorescence [23]. Various cell lines have
talization in most areas. been used for RSV detection, such as HEp-2 epithelial cells,
RSV also is one of the most common viral causes of serious MRC-5 fibroblasts, and rhesus monkey kidney cells, but the
lower respiratory tract illness in the elderly, especially in insti- R-Mix commercial mix of human and mink lung cells may
tutionalized subjects [17]. Exacerbations of chronic obstruc- perform better for detection of RSV [24].
tive pulmonary disease (COPD) are frequently associated with Culture is expensive and requires highly trained staff and
RSV infection [18, 19]. The elderly do not exhibit a remark- therefore is not usually available at the point of care, which is
ably diminished level of antibodies to RSV [20]. Decreased often an outpatient clinic or emergency department.
levels of T cell memory in the elderly and specifically in Therefore, rapid diagnostic methods were developed for the
patients with (COPD) may contribute to the increased suscep- detection of viral proteins or RNA in respiratory secretions.
tibility to RSV infection in these populations [21]. Many think RSV antigen tests mostly rely on direct immunofluorescent
of influenza virus as the principal viral respiratory pathogen in assays (DFA) on exfoliated cells in secretions or enzyme
this population, but in a hospitalized cohort, influenza A virus immunoassay (EIA). Nucleic acid detection assays based on
and RSV infection resulted in similar mortality, lengths of RT-PCR are now widely available for RSV, often in a multi-
stay, and rates of use of intensive care [17]. RSV infection plexed panel for detection of multiple respiratory virus patho-
accounted for over 10 % of hospitalizations for pneumonia. gens. These tests are typically more sensitive than any of the
virus isolation or protein-based detection assays discussed
1.3.3 Serological Surveys above. Enhanced sensitivity is especially helpful when test-
Seroepidemiology studies suggest that virtually all children ing adults, who often shed virus in very low titers. Positive
are infected in the first 2 years of life, and early infection is RT-PCR tests need to be interpreted in the context of the clin-
especially common in infants attending group day care. ical scenario, since the tests can remain positive for prolonged
Serological methods are helpful in epidemiology and vac- periods of time after infection, well beyond the period during
cine studies, but serologies are not often used for diagnosis which infectious virus can be isolated. Since children may
in clinical settings. Because of the transfer of maternal RSV- experience symptomatic respiratory infections every few
specific antibodies across the placenta, and the high preva- weeks during the winter, caution must be used in interpreta-
lence of early infection, it is unusual to find infants who are tion of positive PCR tests, especially when multiple viruses
RSV seronegative. In older children and adults, a fourfold are detected simultaneously in a sample. Some instances of
rise in serum antibodies is often used as evidence of RSV multiple PCR test positivity likely represent residual RNA
infection, but asymptomatic infections in which viral shed- from a previous virus infection and a second RNA type repre-
ding is low in titer often are not accompanied by serum anti- senting live virus from the active current infection.
body rises. Serological tests in infants are even less sensitive, RSV typically is propagated in monolayer cell cultures of
because young infants may not exhibit a large or durable rise continuous cell lines of human epithelial cell origin, such as
in antibodies. Neutralizing antibody tests are considered the HEp-2 cells. Monkey kidney cells of various types are also
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 603

NS1 N P M M2 G F SH L
3′ 5′ Respiratory syncytial virus
NS2

N P M F M2 SH G L
3′ 5′ Metapneumovirus

Fig. 26.1 Schematic of the genomic organization of respiratory syncytial virus and human metapneumovirus. Gene segments drawn roughly to
scale

used commonly for propagation in the laboratory. In fact, the large RNA-dependent RNA polymerase (L). It is suspected
virus replicates to some extent in most cell lines of mamma- that the M protein helps the particle to form by bridging the
lian origin. In non-polarized epithelial cells, the virus often nucleocapsid and the lipid envelope with its incorporated
causes a typical cytopathic effect in which multinucleated surface proteins. The surface of the particle incorporates
giant cells form due to cell-cell fusion, termed cell syncytia. three integral membrane surface glycoproteins, the highly
This in vitro effect is the origin of the virus name, although glycosylated RSV G protein suspected to be the attachment
it is not clear that RSV causes syncytia in vivo. In polarized protein, the fusion protein F, and the small hydrophobic pro-
epithelial cells in culture, the virus assembles and buds from tein (SH). RSV F (a Type I integral membrane protein) and
the apical surface of cells, mimicking to some extent the bud- RSV G (a Type II integral membrane protein) form oligo-
ding of virus into the lumen of the airway. mers on the surface of the particles, which appear like small
spikes with globular heads when seen in electron micro-
scopic (EM) images by negative stain. The morphology of
1.4 Biological Characteristics particles in EM images or in fluorescent microscopy images
labeled by conjugated antibodies suggests that the virion par-
The virus has a negative-sense single-stranded RNA genome ticles are filamentous. However, spherical particles, fila-
with 10 genes encoding 11 proteins. Figure 26.1 compares ments, and more pleomorphic forms have been observed;
the genomes of RSV and MPV, which are similar in many therefore, it is uncertain what the morphology of infectious
respects. particles in vivo during natural infection is.
The replication proteins are common to both of the viruses, The F protein is critical for entry into cells, by breaching
as are the matrix (M) protein and the surface fusion (F) and the barrier of the cell lipid bilayer. It is thought that binding of
glycosylated attachment (G) glycoproteins. The gene order virion particles to susceptible cells at physiologic pH triggers
differs slightly, and RSV possesses two nonstructural (NS) a conformational change of the F protein from a metastable
genes NS1 and NS2 that are not present in MPV. The func- pre-fusion state [25] to an altered post-fusion conformation
tions of these genes are not fully defined, but involve interac- [26, 27] in which the hydrophobic fusion peptide in the pro-
tions with the host response machinery, especially interferons. tein is exposed and extended to insert into the host cell mem-
The presence of these host response-modifying genes may brane. This membrane insertion event accomplishes a fusion
explain in part why RSV appears to cause severe disease more of the viral and host membranes, allowing delivery of the
commonly than MPV. Many of the gene sequences exhibit nucleocapsid to the cytoplasm where viral replication occurs.
some clear global sequence relatedness; however, the extent This event is termed “fusion from without” when the particle
of the relatedness of many of the sequences of corresponding enters a cell. An alternative fusion event (“fusion from
proteins is relatively low. Based on sequence homology, it is within”) occurs when newly expressed F protein on the sur-
not expected that there is a significant amount of immuno- face of infected cells causes fusion of an infected cell to an
logic cross-reactivity in responses to the two viruses. adjacent cell in culture causing “syncytia.” It is not certain
The RSV virion buds from airway epithelial cells, incor- whether this latter form of fusion (cell-cell fusion) occurs dur-
porating host cell membrane as the lipid bilayer that forms ing natural infection and contributes to pathogenesis or if the
the envelope of the particle. Since the virus is enveloped, formation of syncytia is a tissue culture phenomenon only.
chemicals that disrupt lipid bilayers (detergents) inactivate
the virus, leading to the strong recommendations for health-
care provider hand washing following patient contact. The 1.5 Descriptive Epidemiology
genome is a single strand of RNA, which forms a helical
complex with the nucleoprotein (N). The final nucleocapsid Although there are many animal forms of RSV, there is no
structure likely is formed by the complete set of replication known animal reservoir of human RSV; close contact with
proteins, which also include the phosphoprotein (P) and the infected humans is the only source of RSV infection.
604 J.E. Crowe Jr. and J.V. Williams

1.5.1 Incidence and Prevalence Data glycoproteins showed that the F proteins are 50 % related
Early prospective studies showed that approximately half of antigenically and the G proteins are 1–7 % related [44].
infants in the USA are infected during their first year of life; Consistent with this level of antigenic relatedness, F protein
most were infected after two winter epidemics [28]. About a expressed by a vector immunization was equally protective
quarter of infants exhibit signs and symptoms of lower respi- in small animals against challenge with the homologous or
ratory tract disease (wheezing and/or pneumonia) during a heterologous subgroup virus, whereas the G protein was
primary RSV infection [14, 28–31]. RSV is the most com- 13-fold less effective against the heterologous subgroup
mon virus associated with hospitalization for respiratory ill- virus [45]. Thus, the F protein is responsible for most of the
ness and in fact is one of the most common of all causes of observed cross-subgroup neutralization and protection. In
infant hospitalization. For example, RSV caused 13 hospital- some communities a pattern of infection with viruses of
izations per 1,000 US children younger than 1 year in one alternating subgroups has been described, but this is not a
large study [32]. During winter RSV epidemics, over 75 % universal phenomenon. RSV subgroup B virus is more dif-
of the children hospitalized for acute lower respiratory tract ficult to isolate and propagate in culture, so subgroup B
infection are infected with RSV [33, 34]. The rate of very viruses are less commonly associated with severe disease in
severe disease in hospitalized infants is high, with about some studies. However, clearly viruses of both subgroups
2–5 % of hospitalized infants requiring mechanical ventila- can cause severe disease leading to hospitalization [46–50].
tion for respiratory failure [35].
1.5.3 Risk of Serious Lower Respiratory
1.5.2 Risk of Infection and Reinfection Infection (LRI) During Infancy
Although primary infection of infants is probably most effi- Infants exhibit the highest risk of severe lower respiratory
cient, RSV can infect subjects of any age [28, 36, 37]. Some tract disease. This elevated risk is explained by a myriad of
adult infections are asymptomatic, and most are limited physiologic, immunologic, and other factors. First, the high-
symptoms related to infection of the upper respiratory tract, est point of resistance in the airways is the bronchioles, and
such as the common cold [28, 36, 38]. Since otherwise the resistance of airways is inversely proportional to the air-
healthy adults all possess measurable RSV serum antibodies way radius to the fourth power (resistance ~ 1/radius4, from
and RSV-specific T cells, it is clear that prior exposure and Poiseuille’s law). The bronchioles of infants are narrow, and
induction of immune responses may not prevent infection. inflammation and secretion in the bronchioles leads to turbu-
However, disease severity is usually reduced after one or sev- lent airflow, and further reduction of the airway lumen size.
eral infections early in life, thus immune effectors such as These physical factors lead to the clinical signs of wheezing
serum neutralizing antibodies do prevent severe disease dur- (a sign of outflow obstruction) and air retention. Increased
ing reinfections. Unlike influenza virus, RSV does not respiratory rate can compensate to some extent for respira-
exhibit a progressive antigenic drift. Although RSV anti- tory compromise, but prolonged tachypnea can lead to
genic diversity is observed in field strains, diversity of the fatigue and eventual respiratory failure. Also, during primary
antigenic proteins is not required for RSV to cause reinfec- infection infants do not possess active immunity to infection,
tion [39–41]. Most experts believe that serum neutralizing which allow this efficient virus to replicate in the airway to
antibodies protect against severe lower respiratory tract dis- titers as high as 107 infectious particles per mL of secretion.
ease but do not result in sterilizing immunity against infec- Mothers pass RSV antibodies to infants across the placenta
tion at the respiratory mucosa. Thus, healthy adults show during the third trimester, but premature infants do not obtain
signs and symptoms of the common cold in about half of maternal antibodies prior to 32 weeks’ gestation. Also, the
cases of natural or experimental infections, even though they airways of premature infants are smaller than those of term
have experienced numerous previous RSV infections [39]. infants. Another factor leading to respiratory difficulty is
There is a single serotype of RSV, but two antigenic sub- dehydration. Obstruction of the nasal passages with thick
groups of RSV, with about 25 % antigenic relatedness, have secretions can impede the feeding of infants, who are obli-
been defined using immune sera; the subgroups are desig- gate nose breathers.
nated A and B. Antigenic dimorphism for RSV had been
noted in an early study [42] and subsequently was delineated 1.5.4 Role of RSV Infections in Adults
using MAbs, which identified extensive differences in the G RSV also is an important cause of serious infection in elderly
protein and less extensive differences in F and other proteins adults; in fact RSV appears to cause substantially more fatal-
[43]. The two subgroups exhibit a three- to fourfold recipro- ities in elderly adults than in children [17, 51]. As above, a
cal difference in neutralization by polyclonal convalescent large study of the US population showed that RSV was asso-
serum. Analysis of glycoprotein-specific responses in ciated with approximately 17,000 all-cause deaths per year
experimental rodent models or human infants by enzyme- among persons of all ages in the USA [9], with most of those
linked immunosorbent assay (ELISA) with purified F and G deaths in the elderly.
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 605

1.5.5 Role of RSV in Infections with Underlying contact precautions, which are universally recommended for
Cardiopulmonary and Other Medical RSV patients. A high level of compliance with precautions is
Diseases difficult to achieve in busy care settings but is needed to pre-
Virtually any serious medical or genetic condition is associ- vent transmission by healthcare providers. The use of the
ated with some increased risk of severe diseases [32]. Certain prophylactic monoclonal antibody palivizumab has been
particular categories of subjects are at highest risk for severe studied to interrupt an outbreak in a neonatal intensive care
RSV disease, including infants with congenital heart or unit setting [89], but currently palivizumab use is not recom-
chronic lung disease, immunodeficient subjects of any age mended for this purpose.
and the elderly [9, 10, 17, 33, 52–57]. These latter subjects
are thought to have reduced competency of RSV-specific T 1.5.8 Role of RSV in Otitis Media
cells. Prematurity increases risk to a small extent, but more Bacterial otitis media is a common complication of RSV
importantly chronic lung diseases are important factors [10, upper respiratory tract infection. In fact, RSV infection is
33, 56, 58–62]. Infants who are born prematurely and then probably the most common precipitating factor associated
suffer persistent chronic lung disease, especially those need- with otitis media. RSV antigens and nucleic acids have been
ing oxygen supplementation, are at very high risk of hospi- reported in middle ear fluids [90, 91]. The disease is pre-
talization with RSV. Children with cystic fibrosis are at high dominantly due, however, to Eustachian tube dysfunction,
risk of severe disease [63, 64]. In children younger than 2 resulting in bacterial stasis in the middle ear and subsequent
years with cystic fibrosis, the consequence of RSV infection otitis media.
may be prolonged respiratory morbidity [65]. Children with
congenital heart disease also are at increased risk [33, 56, 58, 1.5.9 Epidemic Behavior
62, 66, 67]. RSV regularly occurs in annual epidemics. The US National
Respiratory and Enteric Virus Surveillance System
1.5.6 Role of RSV Infections in Patients with (NREVSS) considers that the RSV season starts when the
Immunodeficiency first of two consecutive weeks during which the mean per-
RSV is a common cause of severe respiratory disease in centage of specimens testing positive for RSV antigen is
immunocompromised patients, including lower respiratory ≥10 %. The RSV season is considered to have ended in a
disease [68]. Mortality rates in some populations of immu- community when the mean percentage of positive specimens
nocompromised patients verges on 50 % [7, 69]. Infants with is ≤10 % in reference laboratories for two consecutive
congenital severe combined immunodeficiency are at special weeks.
risk [57, 70], but any acquired immunosuppressive condition
such as cancer or transplantation puts patients at risk, espe- 1.5.10 Geographic Distribution
cially when T cell function is compromised [68, 71, 72]. RSV infection occurs in infants and adults worldwide, in
RSV infection can cause severe lung disease in recipients of yearly epidemics. The virus has been isolated in every area
lung transplants, sometimes with a long-term outcome of of the world in which surveillance studies have been con-
obliterative bronchiolitis [73–75]. Children with HIV infec- ducted. The principal season varies depending on the climate
tion shed RSV for extended periods, but disease is not espe- and region, but infection is ubiquitous. Virtually all children
cially severe in HIV-infected children prior to onset of AIDS in the world are infected within the first few years of life.
[76–78]. Interestingly, although most immunocompromised
subjects appear to be at risk because of T cell problems, 1.5.11 Temporal Distribution
infants with phagocytic cell defects including those with Epidemics occur in the winter and early spring in the USA.
interferon-γ receptor deficiency or chronic granulomatous Onset of the annual epidemic varies by region of the country
disease also are at risk of severe RSV disease. and by year but typically begins in the USA in October or
November and lasts through late spring. Within a region, the
1.5.7 Role of RSV in Nosocomial Infection timing of RSV season changes slightly each year. Florida
Transmission of RSV in the hospital setting can lead to seri- often has the earliest onset of RSV epidemic and the longest
ous disease, especially in critical care units with neonates or lasting season in the USA. Near the equator, infections may
other high-risk infants [79–86]. Nosocomial outbreaks in be more common during rainy season.
inpatient transplantation facilities are sometimes severe and
unit outbreaks can be difficult to terminate because trans- 1.5.12 Occurrence in Different Settings
plant patients can shed RSV for many months [57, 58, 69, During community outbreaks of RSV, the venues with the
84, 87, 88]. Theoretically, transmission in inpatient health- highest level of young infants and children exhibit the high-
care settings should be preventable through strict compliance est rates of transmission of virus, especially large families
with infection control practices, especially hand washing and and day-care settings with large numbers of children per
606 J.E. Crowe Jr. and J.V. Williams

room. Hospital infection control practices must be used to illness. The incubation period is not known definitely but is
prevent RSV spread, using measures including careful hand about 3–6 days and likely varies according to the intensity of
hygiene, contact precautions for patient isolation, gowns and exposure and the amount of virus in the inoculum. The
gloves [92, 93], and, when direct coughing occurs, face- period of viral shedding is many days; infants can shed infec-
masks and goggles [94, 95]. RSV rapid diagnostic testing tious virus for weeks [98, 99]. RSV can survive on hard sur-
can be used in hospital infection control practice to identify faces for greater than 24 h.
RSV-infected patients during the admission process [96, 97].
RSV is shed for prolonged periods. It has been reported that
92–100 % of hospitalized children are still shedding infec- 1.7 Pathogenesis and Immunity
tious virus after 7 days [98, 99].
Infection occurs by inoculation of the nasal or conjunctival
1.5.13 Environmental Risk Factors mucosa, often by self-inoculation with infected secretions
Exposure to tobacco smoke and poor nutrition increase the from a close contact. Adult volunteers were infected experi-
incidence of disease [100–102]. Low socioeconomic status mentally if virus was inoculated onto the conjunctival sac or
increases the risk of severe disease for uncertain reasons. into the nose, but not following introduction into the mouth
Lower income populations exhibit a five- to tenfold increased [112]. The incubation period from time of inoculation to
risk of hospitalization in many studies [14, 32, 103]. onset of illness for RSV is likely about 4–5 days [113]. Virus
replication initiates in the nasopharynx and rapidly can reach
1.5.14 Other Factors concentrations of over a million particles per mL in the upper
Breast-feeding may confer some protective benefit against airway in children. Adults, who are partially immune, typi-
RSV disease, but the extent of the benefit of breast-feeding cally shed much lower amounts of virus. Virus spreads
has been controversial. The results of epidemiologic studies quickly to the lower respiratory tract, often causing symptoms
on benefit have been conflicting, although recent studies sug- within days of onset of upper respiratory symptoms. Virus
gest that breast-feeding may have a strong protective effect may spread from cell to cell, but also it is likely that small
only in girls [104]. The sex of the infant modulates the sever- aspirations of upper respiratory secretions with virus inocu-
ity of RSV infection for additional reasons that are not fully late the lower tract.
understood. Even though the same high proportion of both Higher titers of virus in respiratory secretions usually are
male and female infants become infected, males have a associated with increased severity of disease, in prospective
higher incidence of RSV lower respiratory tract disease than studies of natural infection [114] or of clinical vaccine trials
girls [30, 32, 105–108]. Ethnic and genetic factors appear to [115]. RSV infection is an acute infection and virus shedding
play a role. Alaska and other Native American children are at usually resolves within days to weeks. RT-PCR tests for
increased risk of severe respiratory disease during RSV virus nucleic acid may remain positive for prolonged peri-
infection [109]. ods. Some animal studies suggest that a negligible amount of
infectious virus may persist in airways far after apparent
resolution of shedding, as evidenced by the recovery of low
1.6 Mechanisms and Routes amounts of infectious virus during immunosuppression sev-
of Transmission eral months after infection [116].
The virus infects respiratory epithelial cells in the lung
Direct contact with secretions from an infected human, usu- and airway. It is not clear whether RSV also replicates pro-
ally by fomites (contaminated objects, including hands), ductively in macrophages and dendritic cells in the airway or
allows transmission of virus. In some cases, infected subjects not. RSV protein antigens have been detected in circulating
may inoculate contacts at short distance by coughing via mononuclear cells [117], and viral genomic RNA and mRNA
large-particle droplets. However, the virus is not spread effi- have been detected by RT-PCR in blood cells [118], but this
ciently by small-particle aerosols [110, 111]. The nasal and probably represents cells from the airway recirculating, as
conjunctival mucus membranes are probably the most com- infectious virus in the blood (viremia) is not detected. RSV
mon portals of entry [112]. RSV is one of the most infectious replicates in the cells at the luminal surface of the respiratory
viruses spread by contact, and transmission is very efficient epithelium and virus both enters and is shed from the apical
even among subjects who possess partial immunity due to surface of infected epithelial cells [119, 120]. Studies of
prior infection. Spread among family members and day-care polarized, differentiated respiratory epithelial cells in vitro
contacts is especially common. The infectious doses for show that RSV infection preferentially infects ciliated cells
humans are probably only a few infectious particles per mL at the luminal face [121], but it is not clear if infection is
of respiratory secretions, but infants routinely shed at least a restricted to such cells in humans. Histopathology studies of
millionfold higher concentration of virus during the peak of infected humans are very limited, but show RSV antigens in
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 607

the superficial cells of the airway in a patchy distribution, antibody levels [15]. Most infants and children in whom
with antigen-positive cells and debris in the airway lumen. maternal antibodies have declined to a low level make their
Pathology caused by RSV infection during infant bron- own serum and secretory antibodies to both the F and G sur-
chiolitis includes necrosis and proliferation of the bronchio- face glycoproteins in response to RSV infection [126].
lar epithelium and destruction of ciliated epithelial cells Antibody responses in neonates are particularly low in qual-
[120, 122]. There is an influx of a large variety of immune ity and magnitude due to immunologic immaturity and the
cell types including neutrophils, lymphocytes, and macro- suppressive effect of passively acquired maternal antibodies
phages. The respiratory tissues become edematous. Mucous [126, 127]. Antibody-mediated immune suppression by pas-
secretion, sloughing of dead cell debris, and the influx of sive antibodies primarily affects humoral rather than cell-
apparent inflammatory cells obstruct the lumen of the narrow mediated immunity [128, 129]. High levels of serum
bronchioles and alveoli. The small diameter of infant bron- antibodies do not appear to provide solid immunity against
chioles is easily obstructed in the presence of dead cells and disease in the upper respiratory tract. Mucosal secretory IgA
edema of the airway tissues [123]. Virus-infected cells can be appears to contribute to local protection against reinfection
identified in the epithelium of the bronchi, bronchioles, and in the airway, although potent protective IgA responses are
alveoli [120]. It is surprising, given the extent of disease, that likely relatively short lived. In human infants, the decrease in
RSV antigen staining is usually patchy or focal, and even in virus shedding in nasal secretions was associated with the
some cases of fatal RSV bronchiolitis, antigen is present appearance of RSV-specific IgA antibodies [130].
only in small amounts [119, 120]. The number of cases that T Cells. T cells clearly play a major role in resolution of
have been collected is limited, however, and there is virtually active infection. RSV-specific T cells with cytolytic activity,
no histopathology from milder cases. thought to be CD8+ T cells, have been detected in peripheral
RSV upper respiratory infection is complicated frequently blood mononuclear cells from infants with RSV disease
by otitis media caused by bacteria. It is unusual to observe [131]. Immunodeficient children, especially those with T
frank bacterial pneumonia or sepsis as a complication of cell defects, often fail to clear RSV infection and can shed
RSV infection, in contrast to some other respiratory viruses virus for many months [57]. Adults with leukemia or hema-
like influenza. Although some clinicians may empirically use topoietic stem cell transplant also have a very high incidence
antibiotics during infant pneumonia, there is no evidence that of prolonged RSV infection leading to severe disease and
antibiotic therapy alters the course of RSV bronchiolitis or sometimes death.
pneumonia. Antimicrobial therapy should not be used in
most cases of RSV bronchiolitis or pneumonia because of
the lack of benefit and risk of selection of antibiotic-resistant 1.8 Patterns of Host Response
colonizing organisms. Nevertheless, there is some sugges-
tion that bacterial-viral interactions may affect the overall 1.8.1 Symptoms
rate of disease in the population. It is interesting that annual RSV infection usually causes upper respiratory tract symp-
RSV and influenza virus epidemics correlate directly with toms during primary infection in otherwise healthy term
the time of peak incidence of invasive pneumococcal disease infants; asymptomatic primary infection is not common.
in many population studies [124]. A double-blind, random- There is often profuse rhinorrhea, and the upper tract disease
ized, placebo-controlled trial of pneumococcal conjugate is very often complicated by otitis media. Symptoms of
vaccine showed a vaccine-attributable reduction in rates of lower respiratory tract involvement occur in about a third of
childhood viral pneumonia requiring hospitalization, caused primary cases [28]. The principal diagnoses are bronchiolitis
by any of seven respiratory viruses, including RSV [125]. (manifested by tachypnea and wheezing) and pneumonia.
The immune mechanisms responsible for resolution of These entities are probably not discrete processes but more
infection and protection against reinfection by RSV are not likely represent a continuum of disease involving increasing
fully defined. tissue distribution. The typical illness starts with nasal con-
Antibodies. Most experts agree that high levels of serum gestion, followed in a few days by cough. The infection is
neutralizing antibodies are associated with relative protec- sometimes associated with fever, which is usually low grade.
tion against severe lower respiratory tract disease in other- After several days of upper tract symptoms, infants may
wise healthy subjects. This idea is supported strongly by the wheeze. Many infants suffer mild wheezing that resolves,
observation that prophylaxis of high-risk infants with a neu- but some cases progress with tachypnea, diffuse inspiratory
tralizing monoclonal antibody prevents about half of hospi- crackles, and expiratory wheezes. Most children recover in
talizations in that group of patients. Many population studies 1–2 weeks with supportive care and observation. If expira-
suggest that infants born with high levels of transplacental tory obstruction becomes severe, however, hyper-expansion
RSV-neutralizing maternal antibodies develop milder illness of the chest occurs due to air retention, and the compliant
or illness at an older age than infants with low maternal nature of the infant chest wall leads to intercostal and
608 J.E. Crowe Jr. and J.V. Williams

subcostal retractions during tachypnea. With prolonged healthy adults, especially in those with frequent exposure to
tachypnea, fatigue may occur with poor oxygenation and small children [80]. In the elderly, particularly those with
CO2 retention (measured by pulse oximeter or arterial blood underlying medical diseases, severe pneumonia may occur,
gas measurement), markers of respiratory failure. Intubation leading to hospitalization or even death.
and mechanical ventilation is used in this setting to manage
the respiratory failure. 1.8.2 Diagnosis
Infection during the first day or weeks of life may just be Astute clinicians can often make a presumptive diagnosis of
characterized by temperature instability or fever, irritability, infection based on the clinical signs of wheezing or pneumo-
and lethargy even in the absence of overt respiratory signs or nia in an infant during a local epidemic. Laboratory testing
symptoms. In very young infants, especially those born pre- of nasal or lower airway secretions by antigen test (ELISA)
maturely, apneic spells may occur in response to RSV infec- or nucleic acid detection (by RT-PCR) provides rapid diag-
tion. Apnea may be the first reported evidence of infection in nosis of the presence of virus in many cases. The gold stan-
some cases, and apneic spells may recur during the acute dard for diagnosis is isolation of the virus in cell culture, but
infection. These events thankfully are usually self-limited this test is typically only available in referral laboratories
and rarely cause neurologic damage. Apneic events are an because of the need for extensive equipment and a high level
indication for hospitalization and careful medical supervi- of technical expertise.
sion with respiratory monitoring. Because of the association
with apnea, some have considered whether RSV is associ-
ated with sudden infant death syndrome (SIDS). Although 1.9 Control and Prevention
RSV has been detected in the lung tissues of some cases of
SIDS, there is no statistically significant association between 1.9.1 Treatment
RSV and SIDS. The reported cases likely reflect a temporal Medical Treatment
association caused by the high prevalence of RSV in this age Primary treatment is supportive care, which includes oral or
group, the prolonged pattern of shedding, and the simultane- intravenous hydration; monitoring of respiratory status,
ous peak incidence of both RSV infection and SIDS in win- especially of oxygen saturation during tachypnea; use of
ter months [132]. supplemental oxygen; removal of secretions from the upper
It is not clear whether infection with RSV causes pro- airway; and, in the case of respiratory failure, intubation and
longed abnormal pulmonary function during childhood or mechanical ventilation. Advances in support care in pediatric
whether children with underlying predisposition to lower critical care units have caused a major decrease in morbidity
respiratory tract disease of all causes manifest their suscepti- and mortality from RSV in the developed world. Infants hos-
bility first to RSV because of the young age of RSV infec- pitalized for RSV disease should be monitored for apnea.
tion. Certainly measureable pulmonary function Investigators have studied nitric oxide [138, 139] mixtures of
abnormalities are common after RSV lower respiratory tract helium and oxygen [140, 141] and surfactant treatment [142]
disease, and these findings may persist for a decade or more in clinical experimental studies in the support of infants with
[133]. Recurrent wheezing is common during subsequent severe RSV disease. Nitric oxide treatment does not appear
viral infections after severe RSV bronchiolitis or pneumonia, to mediate a bronchodilator effect during RSV infection
with an incidence of 10–50 % [134]. A large case-control [139].
study of 200 children hospitalized for bronchiolitis or pneu-
monia in which RSV was the most common cause found that Antivirals
7 years later there was a strong predisposition in these sub- Therapy of RSV disease by any antiviral agent is challenging
jects toward decreased pulmonary function, recurrent cough, because it is a rapid acute infection, and by the time the onset
wheezing, and bronchitis [135]. Seminal prospective studies of disease is recognized, it may be too late to alter the course
by Martinez et al. involved measurement of pulmonary func- of disease by reducing viral load. The guanosine (ribonucleic
tion in infants at birth and then found a strong correlation acid) analog ribavirin is a nucleoside inhibitor that inhibits
between prior lower pulmonary function and the develop- viral RNA synthesis and viral mRNA capping. The drug has
ment of wheezing during RSV infection [136]. This correla- in vitro antiviral activity against RSV, and aerosolized ribavi-
tion persisted in children during the first 3 years of life [136]. rin therapy has been associated with a small but statistically
Even some individuals who do not typically exhibit recurrent significant increase in oxygen saturation during the acute
wheezing have postexercise or pharmacologically induced infection in several small studies. Decreases in mechanical
bronchial reactivity [137], which may be responsive in part ventilation and duration of RSV-associated hospitalization
to bronchodilators [134]. have not been proven (reviewed in [143]). Ribavirin was
Symptomatic upper respiratory tract RSV infections man- approved in 1986 in the USA for treatment of RSV infec-
ifested by common cold symptoms are common in otherwise tion [144]. Clinically, the drug usually is administered as a
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 609

small-particle aerosol using a tent, mask, or mechanical ven- beta-agonist therapy for treatment of bronchiolitis suggest
tilator, delivered for 6–18 h daily for a period of 3–7 days. that they offer little benefit [148, 149]. Alpha-adrenergic
The drug now is not recommended for routine use because receptor stimulation results may decrease interstitial and
follow-up studies have not shown a major benefit. The drug mucosal edema [150], and use of nebulized epinephrine
may be considered for use in select patients with docu- (with combined alpha- and beta-adrenergic activity) has
mented, potentially life-threatening RSV infection. Over a been studied with conflicting results [151, 152]. Alpha-
dozen other experimental small molecule inhibitors of RSV agonist stimulation of the sympathetic nervous system is
fusion to cells have been described and tested in preclinical expected to reduce capillary leakage by constricting precap-
studies for inhibition of RSV, but none have progressed in illary arterioles, reducing hydrostatic pressure and conse-
development to date. A third approach employs short inter- quently bronchial mucosal edema [150]. Racemic
fering RNAs (siRNAs), taking advantage of an ancient host epinephrine treatment relieves some respiratory distress but
cell regulatory system. Single-stranded and double-stranded does not affect length of stay [153]. The usefulness of such
RNA molecules that exhibit RSV-specific small interfering agents in the management of RSV bronchiolitis is not clear.
RNAs have been developed for treatment, which cause RNA
interference activity against RSV, destroying the correspond- 1.9.2 Prevention and Immunoprophylaxis
ing RSV RNA. These novel compounds have shown promis- The most effective mode of prevention is to avoid contact
ing results in preclinical studies [145] and have been tested with infected subjects. In the hospital setting, careful adher-
in small clinical trials. Human immune globulin with a high ence to infection control practices is important for the
titer of RSV antibodies and the RSV monoclonal antibody protection of high-risk patients from RSV infection. Careful
palivizumab have been tested as therapy of acute RSV dis- hand washing may reduce transmission in family and day-
ease, but they were not effective for treatment of established care settings. Pharmacologic intervention is indicated to pre-
disease. vent hospitalization for the highest risk infants, however.
Passive RSV immunoprophylaxis with antibodies has proven
Anti-inflammatories a costly but relatively effective intervention. Parenteral infu-
Anti-inflammatory strategies have been investigated. No sion of RSV-neutralizing antibodies into experimental ani-
benefit of corticosteroid therapy on disease severity or length mals was shown early on to confer substantial resistance in
of hospital stay has been demonstrated, despite studies in the respiratory tract to a subsequent RSV virus challenge
over a dozen randomized clinical trials of outpatients or hos- [154]. Significant reductions in RSV-associated hospitaliza-
pitalized infants with RSV bronchiolitis. Since the drug is of tions and disease severity in high-risk human infants were
no benefit on its own [146] and may prolong virus shedding, first accomplished with prophylactic administration of
it is not recommended. In the future, a possibility might be to human immunoglobulin with high RSV-neutralizing activity
combine an effective antiviral treatment with an anti- given by the intravenous route (RSV-IVIG; FDA licensed in
inflammatory agent [147]. 1996) [155, 156]. Monthly intravenous infusions during the
RSV season reduced the frequency of pediatric hospitaliza-
Antibiotics tion and duration of stay by approximately 55 % and
Intravenous antimicrobial therapy is not appropriate in hos- decreased the number of days spent in intensive care by
pitalized infants with RSV bronchiolitis or pneumonia unless 97 %. The use of RSV-IVIG was superseded by the use of a
there is clear evidence of secondary bacterial infection. Otitis monoclonal antibody (MAb) that was developed subse-
media occurs very often in infants with RSV bronchiolitis; quently that could be given by intramuscular route, and pro-
oral antimicrobial agents can be used for therapy of otitis duction of the former has been discontinued. Several MAbs
media if necessary. were developed for immunoprophylaxis against RSV. The
most successful of these was based on murine MAb 1129
Bronchodilators [157], which is specific to the F protein and efficiently neu-
It is intuitive to think of using beta-adrenergic agents, com- tralizes viruses of both RSV subgroups A and B. This MAb
monly used for the treatment of asthma, to treat the wheezing was humanized by recombinant methods by transferring its
associated with RSV infection. These agents are not usually variable regions onto a human IgG1 backbone, resulting in a
recommended for routine care of first-time wheezing associ- recombinant antibody now named palivizumab [158]. This
ated with RSV bronchiolitis. Short-term improvements in MAb is 50–100-fold more effective for in vitro neutraliza-
oxygenation and clinical scores can be achieved by these tion on a per weight basis than was RSV-IVIG, and thus the
therapies, but it has not been established that their use results total amount of immunoglobulin administered could be
in improvements in duration or severity of illness or disease reduced to an amount that could be given IM. Palivizumab
outcomes. Studies in this area have been conflicting, but sys- (trade name Synagis) was licensed in 1998 for RSV prophy-
tematic reviews of randomized clinical trials of nebulized laxis of high-risk infants, following studies demonstrating its
610 J.E. Crowe Jr. and J.V. Williams

safety and efficacy [159–162]. Palivizumab is administered candidates such as emulsion vaccines [171] and nanoparticle
monthly through the RSV season and has been widely used protein preparations [172, 173].
in high-risk patients with prematurity, chronic lung disease, Live-attenuated vaccines represent an attractive strategy
and hemodynamically significant heart disease [163]. More for preventing RSV, since live infection induces a balanced
potent derivatives of this recombinant antibody have now immune response that is not associated with enhanced dis-
been developed [164]; however, the lead candidate from ease on subsequent natural infection. Many live-attenuated
these affinity maturation efforts exhibited increased side RSV vaccine candidates have been developed over several
effects in a large efficacy study. decades. It has proven difficult to identify a candidate that is
satisfactorily attenuated while remaining satisfactorily
1.9.3 Vaccines immunogenic in the youngest infants. Clinical trials of a
Prevention of severe disease probably will best be accom- safe, live-attenuated RSV vaccine for intranasal administra-
plished by development and use of an effective vaccine. tion have shown restriction of viral replication in infants fol-
Vaccine development for RSV has proven exceptionally dif- lowing administration of a second dose and have been
ficult, however. First, young infants are a difficult population encouraging [174], and additional attenuated vaccine candi-
to immunize. Obstacles to immunization at this early age dates are being developed [175].
include immunologic immaturity and immunosuppression
by maternal antibodies, as already noted [165]. Also, severe
adverse events occurred in early RSV vaccine trials. A 1.10 Unresolved Problems
formalin-inactivated RSV vaccine candidate (FI-RSV) was
developed and evaluated in infants and children in the 1960s Despite over 50 years of research on RSV, many challenges
[166, 167]. This vaccine suspension was made by mixing and questions remain. There are many unanswered funda-
concentrated, inactivated virus with alum adjuvant and was mental questions about the biology of the organism and the
delivered by the intramuscular (IM) route. This inoculation pathogenesis of disease. Why does reinfection occur through-
did not protect against infection or disease, but rather during out life? What are the definitive mechanisms of immunity in
subsequent natural infection vaccinees experienced more humans? Is there a genetic basis for susceptibility to severe
frequent and severe disease. Most FI-RSV vaccinees (80 %) disease? Does severe RSV disease cause asthma? What
required hospitalization during subsequent natural infection, drives the seasonality of RSV?
compared to 5 % in the control group. Autopsies of two fatal- The mortality in infants has been greatly reduced in the
ities showed evidence of RSV replication and pulmonary USA through advances in critical care, but little RSV-specific
inflammation [167]. This event put a chilling effect on RSV intervention is available. Currently, there is little to offer for
vaccine development efforts. Therefore, RSV protein vac- therapy except for supportive care. Prophylaxis of high-risk
cines have been problematic for use in infants given their infant with a MAb prevents some hospitalizations but is
possible potential for disease enhancement, together with the expensive and is not always effective. There are no licensed
poor immunogenicity in this population. vaccines. Given the disaster of early FI-RSV trials, it is not
However, an RSV protein vaccine might be useful in clear that a non-replicating vaccine can be proven safe
boosting immunity in RSV-experienced older children and enough in preclinical models to absolutely assure that
adults who are at increased risk of severe RSV disease due enhanced disease will not occur. On the other hand, the
to underlying disease or advanced age. Protein vaccines for explosion of new technologies for generation of recombinant
RSV have been evaluated clinically for use in such RSV- RSV strains, the determination of pre- and post-fusion anti-
experienced individuals, in whom they appear to be safe. gen structures, and new tools for the detailed study of the
One experimental subunit vaccine consisted of purified F molecular and genetic basis of human immune responses
protein (PFP) isolated from RSV-infected cell culture. This suggest that much progress will be made in the RSV research
purified protein vaccine candidate was evaluated in adults, in field in the coming years.
older children with and without underlying medical diseases,
and in the elderly [168]. The PFP vaccine candidate was well
tolerated and moderately immunogenic in these settings. A 2 Human Metapneumovirus
large multicenter study in children 1–12 years of age with cys-
tic fibrosis did not provide evidence of significant protection 2.1 Introduction
against RSV infection [169]. PFP also has been evaluated for
maternal immunization in the third trimester of pregnancy. Human metapneumovirus (HMPV, MPV) is a paramyxovi-
In the single study to date, the increase in antibody titers rus isolated in 2001 from Dutch children with acute respira-
was only minimal [170]. Maternal immunization studies are tory infection (ARI). Epidemiologic studies have since
being pursued currently with newer non-replicating vaccine confirmed that MPV is a leading cause of upper and lower
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 611

respiratory infection in children and adults worldwide. MPV extremely restricted or no replication during experimental
causes many hospitalizations annually in young children and infection of chickens or turkeys and thus is a true human
presumably deaths in developing nations. Children with pathogen [176]. Human poultry workers exhibit serological
comorbid conditions including prematurity, immune com- evidence of asymptomatic infection with AMPV, providing
promise, and chronic cardiopulmonary disease are at higher evidence for the feasibility of an original trans-species trans-
risk for severe disease. However, the majority of MPV- mission event [181].
associated hospitalizations occur in otherwise healthy infants While recently identified, MPV is not truly a new virus.
and children. Conversely, while MPV is associated with Studies using archived sera collected in the 1950s revealed a
severe lower respiratory infection in adults at rates similar to 100 % seroprevalence in humans greater than 5 years old
those of influenza virus and RSV, almost all of the MPV [176]. Nasal washes collected prospectively during the 1970s
infections in this population are in patients with comorbidi- from children with ARI had detectable MPV RNA upon ret-
ties. MPV causes predictable annual outbreaks with late rospective testing by RT-PCR 25 years later [182]. The spe-
winter-early spring predominance in temperate regions. cialized cell culture requirements, slow growth, and limited
Substantial progress has been made in defining the biology, CPE of MPV likely prevented the earlier discovery of this
pathogenesis, and immunology of the virus. Small animal common respiratory pathogen.
and nonhuman primate models of MPV have been developed
and used to elucidate mechanisms of immunopathogenesis
and test candidate vaccines. A variety of vaccine approaches 2.3 Methods for Epidemiologic Analysis
against MPV are under study, including recombinant sub-
unit, vectored, and live-attenuated vaccines. Human and 2.3.1 Sources of Mortality Data
murine monoclonal antibodies have been generated that Limited mortality data are available and consist of sporadic
exhibit potent in vivo efficacy in rodent models. A subset of case reports, case series, or the identification of fatal cases of
integrins with a binding site for a natural ligand that contains MPV infection in research studies. MPV is not a reportable
an Arg-Gly-Glu (RGD) motif (RGD-binding integrins) has infection and there is no specific ICD-9 diagnostic code.
been identified as receptors that mediate MPV attachment Thus, an accurate estimate of the mortality associated with
and entry via an RGD motif in the MPV fusion protein. MPV is not feasible. However, lower respiratory infections
Remarkable scientific progress has been made during the are a leading cause of death in children worldwide, primarily
decade since the discovery of MPV. in developing nations. MPV is a common cause of severe
lower respiratory infection and thus likely accounts for a
substantial number of deaths globally.
2.2 Historical Background
2.3.2 Sources of Morbidity Data
MPV was discovered by investigators in the Netherlands A substantial body of literature has accumulated in the last
who cultivated specimens from children with respiratory decade describing the epidemiology, disease burden, and
infection on a variety of cell types [176]. A hitherto unknown clinical features of MPV. Many groups have used standard
virus produced cytopathic effects (CPE) in tertiary monkey techniques to provide some estimate of the burden of MPV
kidney cells, but could not be identified by antibody staining in diverse populations. Most reports have been cross-
or RT-PCR for common viruses. Electron microscopy of sectional studies of selected populations, usually based on
infected cells revealed pleomorphic enveloped particles with convenience samples of patients with acute respiratory ill-
surface projections suggesting protein spikes, while bio- ness. These studies are thus limited by potential selection
chemical experiments showed that the virus contained a lipid bias, narrow time periods, and incomplete demographic or
envelope and did not hemagglutinate avian or mammalian clinical data and often lack controls. Nonetheless, the broad
red blood cells. Elegant randomly primed RT-PCR experi- application of these studies to sizable global populations has
ments yielded multiple fragments of genome, which illuminated the frequency of MPV infection. Many of these
sequence analysis identified as related to avian metapneumo- studies have focused on special populations, such as patients
virus (AMPV). with asthma, immune compromise, or chronic obstructive
AMPV (formerly turkey rhinotracheitis virus), identified pulmonary disease (COPD), and thus offer valuable infor-
in 1979, is an important global pathogen of poultry including mation about MPV among these persons.
turkeys and chickens [177]. There are four serotypes of A number of prospective, well-designed studies in adults
AMPV (A–D), and MPV is most closely related genetically and children have been published and offer the best estimates
to AMPV-C [178]. Phylogenetic analysis shows that MPV of the population-based incidence of MPV infection. Some
likely diverged from AMPV-C ~200 years ago and thus MPV of these used preexisting prospective data and samples
is of zoonotic origin [179, 180]. However, MPV exhibits collected prior to the discovery of MPV for retrospective
612 J.E. Crowe Jr. and J.V. Williams

analysis. Classical methods including active day care and described [188–195]. Many early assays were based on lim-
clinic surveillance, as well as newer approaches such as ited sequence data and subsequently were found to be subop-
home surveillance with parent-collected swabs, have been timal for detecting multiple diverse strains [195]. Both
used. These studies have been built upon the foundations of individual and multiplexed RT-PCR assays offer more sensi-
seminal longitudinal studies conducted to investigate other tive detection of MPV than culture; however, multiplex
viruses including influenza, parainfluenza viruses, and RSV. assays sometimes balance decreased sensitivity for a single
Taken together, these reports provide a broad survey of MPV agent with the convenience of detecting multiple viruses
epidemiology across diverse geographic environments, simultaneously [196]. Another limitation of molecular detec-
socioeconomic populations, and high-risk groups. tion for all viruses is the ability to detect low levels of viral
nucleic acid in the absence of infectious virus. It has become
2.3.3 Serological Surveys common to detect more than one virus in a single specimen,
Seroprevalence studies using diverse methods have been per- and the interpretation of these data is far from clear.
formed in different populations. Most have used enzyme- Community respiratory viral infections are frequent in child-
linked immunosorbent assay (ELISA) techniques against hood, and the likelihood of detecting viral genome prior to
whole virus or purified proteins to detect IgM or IgG. A few the onset of illness or for prolonged periods after illness res-
have used immunofluorescent detection of MPV-specific olution complicates the assignment of causation to one of
antibodies or measured serum virus-neutralizing antibodies. several co-detected viruses.
These data have mainly been useful in determining the ubiq-
uity of infection with MPV. Some studies have measured
acute and convalescent sera to diagnose MPV infection, 2.4 Biological Characteristics
while others have attempted to establish a serum neutralizing
titer that correlates with susceptibility to infection. Inherent MPV is an enveloped pleomorphic virus ranging in size from
limitations of serological surveys include the potential for 150 to 600 nm, containing a single-stranded negative-sense
cross-reactive antibodies and the lack of standardized RNA genome [178]. Complete genomic sequences of numer-
reagents for MPV. ous MPV strains have been published [178, 180, 197]. The
genome comprises eight separate open reading frames
2.3.4 Laboratory Methods encoding nine distinct proteins (Fig. 26.1). MPV genes are
Most studies have used RT-PCR to detect MPV due to the analogous to those of RSV (though NS1 and NS2 are absent
difficulty in cultivating the virus. The original isolation of in MPV), but the organization of genes differs. AMPV and
MPV in tertiary monkey kidney cells was possible because MPV have been taxonomically classified in the separate
the investigator had access to a monkey kidney cell source Metapneumovirus genus based on the gene order.
that was free of the endogenous simian foamy virus Phylogenetic analysis of MPV genes consistently identifies
(A.D.M.E. Osterhaus, personal communication). Primary four genetic clades, two major groups designated A and B,
monkey kidney cells commercially available in the USA all each with two minor groups designated A1, A2, B1, and B2
contain SFV, and even the addition of anti-SFV antisera can- [180, 198–203]. One group has suggested further sublin-
not prevent the growth of this endogenous virus prior to the eages based on partial F sequence diversity [204], but there
slow emergence of MPV. Further, the fusion protein of MPV is no evidence that this further genetic distinction is of any
requires cleavage by exogenous trypsin for robust in vitro antigenic or immunologic importance.
growth. Trypsin is added by most clinical virology laborato- The two major surface proteins are the fusion (F) and
ries only to cultures of Madin-Darby canine kidney (MDCK) attachment (G), with a third integral membrane short hydro-
cells for the isolation of influenza virus; however, MDCK phobic (SH) protein. F is the target of neutralizing antibodies
cells are very poorly permissive for MPV even in the pres- in animal models, F-only vaccines induce protection in ani-
ence of trypsin. Finally, primary isolation of MPV often mals, and F-specific monoclonal antibodies provide passive
requires one or more passages prior to visible CPE and few protection [205–213]. In contrast, G-specific antibodies do
laboratories routinely follow this procedure. Unlike RSV, not neutralize virus and G-only vaccines induce neither neu-
MPV is not particularly labile to freeze-thaw cycles [183] tralizing antibodies nor protection [206, 209, 214]. Thus, it
and thus can be retrospectively isolated from PCR-positive appears that MPV is unique among human paramyxoviruses
specimens. Fluorescent antibody staining of patient speci- in that the attachment protein does not contribute to protec-
mens or shell vial cultures can facilitate more rapid identifi- tive antibodies. Further, the G protein exhibits a high degree
cation [184–187]. of genetic variability between subgroups, with as low as
Thus, molecular diagnostic techniques have been used for 29 % amino acid identity between the major A and B sub-
virtually all studies of MPV epidemiology. A number of sen- groups and a minimum 60 % identity within subgroups [202,
sitive and specific real-time RT-PCR assays have been 215–218]. The selective pressure for this diversity is unclear.
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 613

In contrast to G, the F protein is conserved, with a mini- 2.5.2 Risk of Infection and Reinfection
mum 94 % amino acid identity between A and B subgroups The serological data show that MPV infection is nearly ubiq-
and a minimum 98 % identity within subgroups [179, 202, uitous during the first years of life. Further, reinfection
203]. Presumably there are functional constraints on the occurs throughout life. In children, primary MPV infection
diversity of F, since the mutation rate of MPV is high, similar is associated commonly with lower respiratory illness, while
to other RNA viruses. The major question regarding the reinfection is associated with upper tract disease [182, 238].
diversity between major or minor subgroups is whether it
contributes to antigenic variation or escape in human 2.5.3 Risk of Serious Lower Respiratory
populations. Infection (LRI) During Infancy
Cross-neutralization against heterologous virus from the Most epidemiologic studies of MPV in children show that
A and B lineages was tested using experimental infection of the virus is the second leading cause of lower respiratory
ferrets [202]. This study found relative neutralization of infection after RSV. The prevalence of MPV in studies of
homologous to heterologous virus ranging from 12 to 96-fold children with LRI is 5–25 %. A 25-year prospective study of
difference, thus providing some evidence for antigenic sero- otherwise healthy children <5 years old detected MPV in
types. However, subsequent experiments using hamsters, 12 % of children with LRI; several children experienced
African green monkeys, chimpanzees, and rhesus macaques recurrent infection [182]. A 2-years multicenter study of
found that the A and B groups were 64–99 % related anti- inpatient and outpatient Japanese children with ARI identified
genically [219]. Infected animals developed neutralizing MPV in 57/637 (8.9 %) [239]. A 5-years observational study
antibodies that were highly effective against heterologous of otherwise healthy Korean children <5 years old found
virus, and previously infected primates were protected MPV in 24/515 (4.7 %), similar to the rates for influenza and
against challenge with heterologous virus. Cynomolgus parainfluenza virus type 3 (PIV-3) [240]. A very large obser-
macaques infected with A or B subgroup viruses or with can- vational study in Queensland, Australia, tested specimens
didate vaccines exhibited only a 6–16-fold difference in neu- obtained from patients of all ages with LRI from 2001 to
tralizing titer against homologous and heterologous viruses 2004; MPV was detected in 707/10,025 (7.1 %). Ninety-two
[220, 221]. Taken together, these data show that while MPV percent of patients with MPV were <5 years old, and MPV
F exhibits some antigenic diversity, the virus does not have was the second most common virus after RSV in these chil-
truly distinct serotypes. The potential implications for human dren [241]. A South African group tested specimens from
epidemiology are discussed further below. children hospitalized with ARI who were subjects in a pro-
spective pneumococcal vaccine trial; MPV was present in
126/1,409 (8.9 %) and was the most common virus after
2.5 Descriptive Epidemiology RSV. The estimated incidence of MPV-associated hospital-
ization in HIV-negative children was 29/1,000; a number of
2.5.1 Incidence and Prevalence Data children had repeat infections [242]. A prospective study of
Numerous studies document the fact that MPV infection is hospitalized children in Hong Kong over a 13-month period
ubiquitous and that reinfection is common. Serosurveys test- found MPV in 32/587 (5.5 %); the estimated incidence of
ing large sample collections in Canada, China, Croatia, MPV-associated hospitalization was 4.4 per 1,000 children
Germany, Israel, Japan, the Netherlands, Taiwan, Thailand, <6 years old [243]. A Chinese group conducted a prospective
the USA, and Uruguay show that 95–100 % of children have 2-years study of children hospitalized with ARI and identi-
antibodies against MPV by the age of 5 years [222–231]. In fied MPV in 227/878 (25.9 %), with most <6 years old [244].
many of these studies, 50–75 % of children are seropositive A large, population-based, prospective surveillance study
by age 2 years, suggesting that most acquire primary MPV conducted in three US cities over 6 years found that the inci-
infection early. Most identify a decrease in serum MPV anti- dence of hospitalization for MPV in children <5 years old
body titer from birth to 6–12 months, presumably due to the was 1 per 1,000, lower than the rate of RSV-associated hos-
expected decline of maternally derived antibodies. Studies in pitalization in the same cohort (3/1,000) but similar to the
Japan and India that compared MPV and RSV titers in the rates for influenza (0.9/1,000) and PIV-3 (0.5/1,000) [245].
same cohort found that after the expected nadir during early
infancy, RSV titers began increasing at an earlier age than 2.5.4 Role of MPV Infections in Adults
MPV [232, 233]. This finding is interesting in light of epide- Respiratory disease is among the leading causes for hospital-
miologic data suggesting that primary MPV infection peaks ization of adults in the USA, and “influenza and pneumonia”
between 6 and 12 months of life compared with the peak of ranks among the top 10 causes of deaths annually. Although
RSV at 2–3 months (discussed below). Longitudinal studies the data are limited, MPV appears to be associated with a
in adults and children have documented reinfection by a four- substantial burden of ARI in adults, primarily those with
fold rise in serum antibody titer [222, 230, 231, 234–237]. comorbidities. A prospective study in Rochester, NY,
614 J.E. Crowe Jr. and J.V. Williams

enrolled four cohorts during four winters: healthy adults children with MPV bronchiolitis during infancy compared to
>65, high-risk adults >65 with comorbidities, healthy adults infants with acute gastroenteritis found that MPV infection
19–40 years old, and adults hospitalized for acute cardiopul- early in life was significantly associated with a later diagno-
monary illness [246, 247]. Overall, MPV infection was sis of asthma and recurrent wheezing at 5 years of age [258].
detected in 8.5 % of ARI in the cohort. The rate of MPV
infection was highest in young adults at 13 %, though many 2.5.6 Role of MPV in Infections with Underlying
of these were asymptomatic and detected only serologically. Cardiopulmonary Disease
Of note, this group had a mean age of 33, was predominantly MPV causes severe disease in children with comorbid con-
female, and had daily exposure to children. Of the hospital- ditions such as cardiac and pulmonary disease or prematu-
ized patients, the incidence of MPV annually ranged from rity [259–263]. A prospective 1-year study of hospitalized
4.4 to 13.2 %. More than 85 % of the hospitalized MPV- children found that 34 % of patients with MPV had a history
infected subjects had underlying conditions, chiefly cardio- of prematurity, chronic lung disease, complex congenital
pulmonary disease or diabetes mellitus. There were six heart disease, or immunodeficiency [264]. Vicente et al.
deaths in this study, all with comorbidities; one had concom- detected MPV by RT-PCR in 6 % of adults >64 years old
itant bacteremia with Streptococcus pneumoniae. with acute exacerbations of COPD; none had other patho-
Interestingly, the incidence of MPV infection was similar to gens identified by culture or PCR [265]. A Canadian study
the annual average infection rate for RSV (5.5 %) and influ- found that 4 % of hospitalized adults with community-
enza A (2.4 %) in these cohorts during the same study period. acquired pneumonia or COPD exacerbations tested positive
A prospective, population-based study in Nashville, TN, for MPV, all with comorbid conditions; one also had influ-
recruited adults hospitalized for ARI at several county hospi- enza A and S. pneumoniae [266]. RSV was present in 9 %
tals over three winters [248]. Of 508 subjects, 23 (4.5 %) had and influenza A in 6 % of the cohort. MPV was detected in
MPV, 33 (6.5 %) influenza, and 31 (6.1 %) RSV. Notably, 12 % of adults hospitalized for COPD exacerbation during
MPV-infected subjects were significantly older than one winter in Connecticut, none with other viruses co-
influenza-infected subjects (mean 76 vs. 60 years) and had detected; RSV was present in 8 % and influenza A in 4 % of
higher rates of chronic cardiopulmonary disease (78 % vs. the entire cohort [267].
52 %). The overall population-based rates of hospitalization
for the three viruses were similar, at 1/1,000 for MPV, 2.5.7 Role of MPV Infections in Patients
1.5/1,000 RSV, and 1.2/1,000 for influenza. However, for with Immunodeficiency
subjects ≥65 years, hospitalization rates were much higher Severe and fatal MPV disease can occur among immuno-
for MPV and RSV at 2.2/1,000 for MPV and 2.5/1,000 for compromised individuals, including solid organ and stem
RSV compared to 1.2/1,000 for influenza, likely reflecting cell transplant recipients, HIV-infected persons, and chemo-
the use of influenza vaccine for older adults. A prospective therapy patients [268–275]. MPV is associated with morbid-
study of community-acquired pneumonia in Canada found ity and mortality in adults with hematologic malignancies
MPV in 4 % of hospitalized cases during an 18-month and stem cell transplant; [270, 276] MPV was detected in
period, all with underlying conditions [249]. A Dutch group bronchoalveolar lavage specimens from 5/163 (3 %) epi-
detected MPV in 2 % of bronchoalveolar lavage specimens sodes of acute respiratory infection in stem cell transplant
from intensive care unit patients. All were >50 years old with recipients, and four died [270]. HIV-positive South African
comorbid conditions and 83 % died [250]. Similarly, a retro- children with MPV were significantly more likely to receive
spective study in North Ireland found MPV in only 0.8 % of a diagnosis of pneumonia and experience longer hospitaliza-
residual respiratory specimens from adults, but 33 % of these tion, lower mean oxygen saturation, and bacteremia; further,
died [251]. Together, these data show that MPV is an impor- HIV-positive children were fivefold more likely to be infected
tant cause of acute respiratory disease in adults, primarily with MPV than HIV-negative children [242].
older adults or those with underlying comorbid conditions.
2.5.8 Role of MPV in Nosocomial Infection
2.5.5 Role of MPV Infections in Patients with MPV has been implicated in several hospital and institu-
Asthma tional outbreaks leading to mortality [277, 278]. Kim and
MPV is associated with acute asthma exacerbations [252– colleagues [279] report the transmission of MPV to pediatric
254]. MPV was detected in 10 of 132 hospitalized Finnish hematology-oncology patients during a nosocomial out-
children with acute wheezing [255]. Similarly, MPV was break. The incubation period was between 7 and 9 days.
isolated from 7 % of adults hospitalized for acute asthma Standard, but not droplet, precautions were used. In labora-
exacerbation, only one of whom tested positive 3 months tory studies, infectious virus persists on metal and nonporous
later [256]. Premature infants who developed MPV bronchi- surfaces for up to 8 h [183]. Due to the significant morbidity
olitis within the first year of life had decreased lung function and mortality of MPV in high-risk children, isolation precau-
at 1 year of age [257]. A prospective, case-control study of tions are important.
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 615

2.5.9 Role of MPV in Different Clinical seasonality have been described in some European studies
Syndromes [298, 299]. Annual rates of MPV-associated ARI are lower
MPV is associated with both upper and lower respiratory than RSV and comparable to parainfluenza virus types 1–3
tract disease [182, 280–282]. Rhinorrhea and cough are the combined and influenza [240, 241, 243, 282, 294, 300] although
most frequent symptoms, while hoarseness, laryngitis, sore MPV does on occasion surpass RSV in incidence [293].
throat, and croup are less common [238–241, 243, 282]. A
large prospective study of children with URI detected MPV 2.5.13 Occurrence in Different Settings
in 5 % of patients, similar to RSV, influenza, and PIV but less Multiple outbreaks have been reported in nursing homes and
frequent than adenovirus and rhinovirus. In these children other long-term care facilities (LTCF). MPV was the only
with URI associated with MPV, fever was present in 54 %, pathogen identified in an outbreak of ARI that occurred over
coryza in 82 %, cough in 66 %, pharyngitis in 44 %, hoarse- a 6-weeks period at a Quebec LTCF, with 6 PCR-confirmed
ness in 8 %, and conjunctivitis in 3 % [280]. MPV is associ- and 96 epidemiologically linked probable cases. There were
ated with acute otitis media and has been detected in nasal three deaths among the confirmed and nine deaths among the
secretions and middle ear fluid [182, 280, 283–285]. probable cases [278]. A California study described an out-
Signs and symptoms of LRI with MPV include cough, break of ARI in 26/148 (18 %) residents and, importantly, 13
wheezing, and rhonchi. A large Chinese study of children staff of a LTCF; MPV was confirmed in 5 residents, 2 of
with acute respiratory infections found that wheezing was whom were hospitalized, and no other viruses were detected
more common in children with MPV than with RSV [282]. In in any case [301].
that study, children with MPV were diagnosed with pneumo-
nia significantly more than children with RSV, 47 % versus 2.5.14 Environmental Risk Factors
31 % (p = 0.002). Conversely, a larger percentage of children Attendance in out-of-home day care, breast-feeding, and
with RSV were diagnosed with bronchiolitis compared to passive smoke exposure are not significantly associated with
MPV, 62 % versus 42 %, respectively (p < 0.001). Other stud- MPV infection [245]. MPV infection is not associated with
ies also note the trend toward a higher percentage of children lower socioeconomic status.
with MPV and pneumonia compared to RSV [182, 238, 245]
although this is not always statistically significant [243]. 2.5.15 Other Factors
MPV has been associated rarely with neurologic compli- Viral coinfection has been suggested with MPV; dual infec-
cations, including febrile seizures and altered mental status, tion with RSV and MPV increased the risk of PICU admis-
but there is no conclusive evidence for direct neural infec- sion compared to RSV alone in one small study [302].
tion. One case report describes a patient who died and had However, this finding was refuted by subsequent larger
MPV isolated by RT-PCR from brain and lung tissues [286]. reports [303–305]. Other studies demonstrate no significant
MPV was detected by RT-PCR in nasal specimens from 4 of difference in children with coinfections, including adenovi-
1,570 persons with encephalitis of unknown etiology [287]. rus, bocavirus, coronavirus, influenza virus, parainfluenza
Several other reports describe the detection of MPV in a viruses, or RSV [240, 241, 282, 293, 306, 307].
respiratory specimen from patients with encephalitis [286, MPV has four distinct genetic lineages or subgroups: A1,
288–290]. Only one case reports detection of MPV in cere- A2, B2, and B2 [202, 203, 218]. The predominant subtype
brospinal fluid [291]. varies by year and location [241, 280, 293, 297, 308]. In
Italy, over a 3-years period, all four subtypes were identified
2.5.10 Epidemic Behavior each season but the predominant subtype changed. In 2001–
The incidence of viral infection varies between countries and 2002, A1 accounted for 59 % of all strains, the following
years, but MPV circulates in every year [176, 182, 239–241, year B1 and B2 were present equally, and in 2003–2004,
243, 282, 292–296]. 72 % of strains were A2 [308]. Similar variation was observed
over 20 years in a US study, with multiple subgroups present
2.5.11 Geographic Distribution in most seasons (Fig. 26.3) [280].
Epidemiologic studies have verified the presence of MPV It is unclear whether viruses from different subgroups dif-
worldwide. fer in virulence. A study in Spain reported that children with
group A infection more frequently had pneumonia and
2.5.12 Temporal Distribution higher disease severity [309], while in Canada, group B was
MPV is present during all months in temperate regions, associated with more severe disease in hospitalized patients
although predominant in late winter-early spring, often fol- [261]. Patients with group B strains in a French study were
lowing the peak of RSV (Fig. 26.2) [176, 182, 191, 239, 241, more likely to have abnormal chest radiographs but did not
246, 264, 280, 282, 294, 297]. have significant differences in oxygen saturation, hospital-
In subtropical climates such as Hong Kong, a spring- izations, or clinical severity scores [310]. Other studies have
summer season similar to RSV occurs [243]. Biannual peaks of found no major distinctions in disease severity [239, 241,
616 J.E. Crowe Jr. and J.V. Williams

20 2001/02 2003/04 2005/06 2000/01 2002/03 2004/05 2006/07 35

percentage HMPV of all ARI cases

30
number of HMPV cases 15
25

20
10
15

10
5
5

0 0

Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

50 total tested 12
RSV
40

number of HMPV cases

HMPV
total/number of RSV cases

8
30

20
4

10

0 0
10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10

10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10

2000/01 2000/02 2002/03 2003/04 2004/05 2005/06 2006/07

Fig. 26.2 Number of infants admitted to hospital with acute respira- where >10 % of specimens test positive by PCR and the last week of
tory illness and the seasonality of RSV and MPV in Austria from 2000 >10 % positive preceding 2 consecutive weeks of <10 % positive. Gray
to 2007 (top panel). The weeks of onset and end of RSV and MPV indicates total tested; yellow, RSV; red, HMPV (Used with permission
activity (bottom panel) are defined as the first of 2 consecutive weeks from Aberle et al. [298])

10
B2
8 B1
A2
No. of isolates

6 A1

0
82
83
84
85

86
87
88
89
90
91

92
93
94
95
96
97
98

99
00
01
19
19
19
19

19
19
19
19
19
19

19
19
19
19
19
19
19

19
20
20

Fig. 26.3 Rates, by year, of each genetic lineage of human metapneumovirus. Data are from specimens collected from children with acute respira-
tory infection between 1982 and 2001 in the Vanderbilt Vaccine Clinic (Used with permission from Williams et al. [280])
26 Paramyxoviruses: Respiratory Syncytial Virus and Human Metapneumovirus 617

311], laboratory abnormalities [228], or symptoms [240] limited cross-protection between subgroups, since reinfec-
between subgroups. Group A viruses replicate more effi- tions occur in children and adults [246, 248] with genetically
ciently in animal models, suggesting some meaningful bio- different strains [182, 239, 240, 268, 320] as well as strains
logical differences between groups [219, 220]. from the same subgroup [280]. Early protection against rein-
fection following primary infection was confirmed in
macaques; however, when challenged 12 weeks later, virus
2.6 Mechanisms and Routes replication was detectable despite the presence of serum
of Transmission antibodies [220]. Eleven months later, antibody levels had
waned still further, and all macaques challenged with heter-
MPV is an enveloped virus and thus inactivated by soap, dis- ologous virus and two of three animals reinfected with
infectants, or alcohols. Spread is thought to occur by direct homologous virus had no evidence of protection [220]. A
or close contact with contaminated secretions. Infectious prospective study in humans noted that baseline MPV anti-
virus can persist at room temperature, especially nonporous bodies were lower in patients who subsequently became
surfaces, for up to 8 h [183]. Contact precautions are recom- infected versus those who did not become infected [321].
mended as for RSV with meticulous hand hygiene. Cohorting Thus, cross-protection and duration of antibody responses
of patients and caregivers should be considered during out- are important issues for vaccine development.
breaks in care facilities.

2.8 Patterns of Host Response

2.7 Pathogenesis and Immunity
2.8.1 Symptoms
Human data are limited; studies in rodents and nonhuman MPV causes both upper and lower respiratory tract signs and
primates reveal mild erosive and inflammatory changes in symptoms clinically indistinguishable from disease associ-
the mucosa and submucosa of the airways, with viral replica- ated with RSV and other respiratory viruses [182, 239, 300,
tion observed in ciliated epithelial cells in the respiratory 322, 323]. Fever is present in most cases, especially children
tract [312–314]. Inflammatory infiltrates with a lymphocytic [239–241, 243, 264, 280, 293]. Transient maculopapular
and monocytic predominance are present in perivascular and rash has been described in a minority of patients [241, 243,
peribronchial areas. Sumino and colleagues [315] reviewed 293], and vomiting or diarrhea are described with low fre-
the lung pathology of five adults with MPV infection. quency [182, 239, 243]. Laboratory abnormalities are
Histopathology in three patients demonstrated acute, orga- uncommon, although one study identified 27 % of patients
nizing lung injury with diffuse alveolar membrane formation with elevated ALT and AST values [293]. White blood cell
and the presence of smudge cells. The fourth patient had no count and C-reactive protein were not significantly different
evidence of lower respiratory tract infection, and the fifth between RSV and MPV [238, 282].
patient had nonspecific acute and chronic inflammation. A MPV is rarely detected in asymptomatic persons [182,
similar study in children revealed chronic inflammatory 191, 324–326], though in otherwise healthy young adults,
changes of the airways with intra-alveolar macrophages MPV infection can be subclinical [246]. The duration of
[316]. A major limitation of reports of human pathology is shedding in healthy individuals is approximately 7–14 days
that patients had been mechanically ventilated prior to death, [239, 327].
making it difficult to distinguish virus-induced pathology
from barotrauma and nonspecific inflammation. 2.8.2 Diagnosis
MPV lacks genes present in other paramyxoviruses that Detection in cell culture requires prolonged incubation and
inhibit interferon responses; nevertheless, MPV is capable is both insensitive and often impractical. Shell vial culture
of blocking type I interferon responses by an unknown offers increased sensitivity over traditional culture [184,
mechanism [317, 318]. MPV and other respiratory viruses 328]. IFA has demonstrated a sensitivity of 73 % and speci-
induce pulmonary CD8+ T cells that fail to secrete IFNγ or ficity of 97 % with RT-PCR as the gold standard [329]. DFA
exhibit cytotoxic degranulation in response to viral peptides; has shown similar results [330]. Commercial antibody kits
these impaired CD8 T cells resemble exhausted CD8 T cells for immunofluorescent detection of MPV are available.
induced by chronic infections such as HIV and hepatitis C RT-PCR is most commonly used for detection of the virus in
virus [319]. epidemiologic studies and is becoming more common in
Humans develop neutralizing antibodies to MPV, and clinical laboratories [176, 320, 331, 332]. Real-time RT-PCR
passive antibodies alone can protect in animal models. targeting the conserved N gene has high sensitivity for detec-
However, immunity wanes over time and likely provides tion of all four subgroups [189, 195].
618 J.E. Crowe Jr. and J.V. Williams

2.9 Control and Prevention bined with the transient nature of mucosal IgA means that
reinfection is possible throughout life. However, serum IgG
2.9.1 Treatment appears to offer protection against severe lower respiratory
The primary therapy for MPV infections is supportive care, involvement, and thus a vaccine would likely ameliorate the
including oral or intravenous hydration, monitoring of respi- most severe cases. The best candidate vaccine is not yet
ratory status and oxygen saturation, supplemental oxygen, clear. Further, MPV has as yet unidentified mechanisms to
and mechanical ventilation for frank respiratory failure. subvert host immunity. Elucidation of these pathways might
There are no licensed antivirals for MPV. Reports of pharma- help guide vaccine development and uncover novel host tar-
cologic treatment of MPV are limited to severely ill or gets for immunomodulation.
immunocompromised patients. Ribavirin, an antiviral agent
used in severe RSV infection, reduced inflammation and
viral replication in mice with MPV infection [333]. References
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 Parvoviruses
27
Kevin E. Brown

1 General Introduction 2 Parvovirus B19 (B19V)

Parvum is Latin for small, and Parvoviridae are among Parvovirus B19 (B19V) was discovered in 1975 [6] and,
the smallest known DNA-containing viruses that infect until the discovery of the human bocaviruses, was the only
mammalian cells. The virions are nonenveloped par- human pathogenic parvovirus. Unlike many virus infections,
ticles about 22 nm in diameter with icosahedral symme- the clinical manifestations of infection with parvovirus B19
try. The Parvoviridae are divided into two subfamilies, vary widely with the immunologic and hematologic status of
Parvovirinae and Densovirinae, on the basis of their the host. In individuals with underlying hemolytic disorders,
ability to infect vertebrate or invertebrate cells, respec- B19V is the primary cause of aplastic crisis. In immunocom-
tively. The Parvovirinae are currently further subdivided promised patients, persistent parvovirus B19 viremia may
into eight genera on the basis of their transcription map, manifest as pure red cell aplasia and chronic anemia, and in
their ability to replicate efficiently either autonomously the fetus, where the immune response is immature infection,
or with helper virus, and their sequence homology. The it may lead to fetal death in utero or hydrops fetalis. The
five genera are Protoparvovirus (formerly Parvovirus), major disease manifestation in normal immunocompetent
Dependoparvovirus, Erythroparvovirus, Bocaparvovirus, individuals is erythema infectiosum (EI), also called fifth
Amdoparvovirus, Aveparvovirus, Copiparvovirus and disease or “slapped-cheek” disease. Generally, this is an
Tetraparvovirus [1]. innocuous rash disease of childhood, but in adults, it may
At least four different parvoviruses are known to infect also be associated with an acute symmetrical polyarthropa-
humans. Parvovirus B19 (B19V) is the best characterized thy, which can mimic acute rheumatoid arthritis.
and is classified as the type member of the
Erythroparvovirus genus. The other human viruses are the
human adeno-associated viruses (Dependoparvoviruses), 2.1 Historical Background
human bocaparvoviruses (Bocaparvovirus), and human
Parv4, a member of the newly recognised Tetreparvovirus 2.1.1 The Virus
genus [2–4]. Parvovirus B19 was discovered by Yvonne Cossart and co-
The human dependoparvoviruses are nonpathogenic and workers [6] in England. They were evaluating tests for hepa-
as such are being utilized as vectors for gene therapy [5]. titis B surface antigen (HBsAg) using panels of serum
They will not be discussed further in this chapter. samples. One serum (coded 19 in panel B) gave anomalous
results, positive in counterimmune electrophoresis (CIE)
with human antisera but negative in the more specific radio-
immunoassay (RIA) and reverse passive hemagglutination
(RPHA) tests that used hyperimmune animal sera. When the
precipitin line from the CIE was excised, electron micros-
copy (EM) showed the presence of 23-nm particles resem-
bling parvovirus. There was no reactivity with antisera to
K.E. Brown, MD adeno-associated viruses or to rat parvovirus, and the virus
Virus Reference Department, Public Health England,
was originally labeled “serum parvovirus-like particle”
Microbiology Services, 61 Colindale Avenue,
London NW9 5EQ, UK (SPLV). Approximately 30 % of adults had antibody to the
e-mail: [email protected] new virus detectable by CIE.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 629

DOI 10.1007/978-1-4899-7448-8_27, © Springer Science+Business Media New York 2014
630 K.E. Brown

The first clinically significant illness associated with In 1983, following an outbreak of fifth disease in London,
B19V infection was hypoplastic crisis in patients with sickle- England, 31 of 31 children or adolescents who had been
cell anemia [7]. Sera from such patients contained B19V affected had anti-B19V-specific immunoglobulin M (IgM)
antigen, detectable by CIE or EM at the time of crisis, and antibody in their serum detectable by RIA [13]. Similar
convalescent sera lacked virus but showed evidence of anti- results were obtained in other epidemics of fifth disease
body seroconversion. worldwide, and parvovirus B19 is now known to be the etio-
In 1985, the virus was officially recognized as a member of logic agent for EI [14–16].
the Parvoviridae, and the International Committee on Taxonomy
of Viruses (ICTV) recommended the name B19V to prevent
confusion with other viruses (i.e., human papillomavirus). It is 2.2 Methodology Involved
classified as a member of the Erythrovirus genus with the name in Epidemiologic Analysis
parvovirus B19 (official abbreviation, B19V) [1].
2.2.1 Sources of Mortality Data
2.1.2 Transient Aplastic Crisis Parvovirus B19 is a common infection and death due to
Transient aplastic crisis (TAC) was the first clinical illness B19V must be rare. However, life-threatening B19V infec-
associated with B19V infection. The term “aplastic crisis” tion can occur in patients with underlying hemolytic disease.
was coined by Owren [8] to describe the abrupt onset of In one study in Jamaica, of 308 children with homozygous
severe anemia with absent reticulocytes in patients with sickle-cell disease followed from birth to 15 years of age,
hereditary spherocytosis. (Hemolytic crises in hereditary 114 (37 %) became infected with B19V and four deaths were
spherocytosis patients are associated with increased bone attributable to the infection [17]. The prevalence of B19V as
marrow turnover and reticulocyte production.) Also, in con- a cause of chronic anemia and its contribution to excess mor-
trast to hemolytic crises, aplastic crisis occurred as a single tality in immunocompromised patients is still unknown,
episode in the patient’s life. In TAC cases, there was a com- although deaths have been reported [18–21]. Case reports of
mon history of a preceding prodromal illness and the occur- myocarditis following parvovirus B19 infection have also
rence of epidemics in large kindreds of hereditary been described [22–24]. Finally, it has also been suggested
spherocytosis suggested an infectious etiology. that B19V infection contributes to the mortality of malaria in
When stored sera from (over 800) children admitted to a tropical countries [25–28].
London hospital were examined for B19V antigen by CIE, a
precipitin line was found in a child with sickle-cell disease 2.2.2 Sources of Morbidity Data
suffering a hypoplastic crisis. Five other patients presenting Parvovirus B19 infection is not a notifiable disease in any
with similar symptoms were also investigated, and all had country, and official morbidity reports in the United States
evidence of recent infection with B19V (either antigenemia do not include EI or parvovirus B19 infection. Morbidity
or seroconversion). All were Jamaican immigrants with data are therefore based on studies of outbreaks of EI (with
sickle-cell disease presenting with aplastic crisis. There was and without serological confirmation) or on case reports of
a reduced hematocrit and deficient red cell production in confirmed infections. In England, laboratory-confirmed par-
their bone marrow [7]. Retrospective studies of sera from vovirus B19 infections are reported to the Public Health
Jamaican sickle-cell patients showed that 86 % of TACs England. The majority of B19V infections are not reported
were associated with recent parvovirus infection [9]. since laboratory investigation is rarely performed for fifth
disease. Similarly, there are no formal reporting systems or
2.1.3 Erythema Infectiosum for documenting the incidence of parvovirus B19 infections
This exanthematous rash illness of childhood was probably in pregnancy.
first described by Robert Willan in 1799 and subsequently
illustrated in his 1808 textbook [10]. The disease was redis- 2.2.3 Serological Surveys
covered in Germany, and in 1899, Sticker gave it the name Serological surveys were originally performed using the rel-
erythema infectiosum. Six years later, Cheinisse classified it atively insensitive CIE [6, 29, 30], now superseded by more
as the “fifth rash disease” of the six classical exanthemata of sensitive enzyme immunoassays (ELISA). Due to the inabil-
childhood [11]. Subsequently, many outbreaks of fifth dis- ity to grow B19V in standard cell culture systems, early
ease were documented in the medical literature but the cause serology assays were based on the use of synthetic peptides
remained a mystery. Often the epidemiologic data suggested [29] or fusion proteins in Escherichia coli [31] as antigen.
“a common source exposure to a highly effective transmit- However, the epitopes presented by these products do not
ter,” and an atypical rubella virus or echovirus was thought to accurately reproduce the epitopes of the native capsids, and
be responsible [12]. However, neither virus could be repro- the sensitivity and specificity were disappointing. The
ducibly isolated from fifth disease patients. expression of B19V capsid proteins as virion-like particles
27 Parvoviruses 631

using transfected B19V genome into CHO cells [32], COS-7 limited, consisting of a single strand of DNA of 5,596 nucle-
cells [33], or the use of yeast [34] or baculovirus [35, 36] otides. The genome has two large open reading frames
expression systems appears to have overcome these prob- (ORF) with the left ORF encoding the nonstructural protein
lems, with results based on these antigens showing good cor- and the right ORF encoding the two capsid proteins, VP1
relation with assays based on native virus. The antigens are and VP2, by alternative splicing. Parvovirus B19 particles do
not only relatively easy to mass produce; they are also non- not appear to contain lipids or carbohydrates. The virion
infectious and therefore without hazard to laboratory work- itself has phospholipase activity [38, 39], which is probably
ers. They are widely used in seroprevalence studies. critical for viral entry into cells.
As a consequence of their lack of an envelope and limited
2.2.4 Laboratory Methods DNA content, parvoviruses are extremely stable to physical
Virus Isolation and Detection inactivation. Depending on the environment, heat treatment at
In the absence of suitable methods for isolating virus from 60 °C does not inactivate the virus [40, 41], and in clinical
clinical specimens, documenting the presence of virus relies studies heat-treated clotting factors (56 °C for 60 min or even,
on the detection of viral DNA by molecular biology tech- 80 °C for 72 h) did not prevent transmission [42]. B19V is
niques. B19V DNA can be detected in serum at the time of resistant to lipid solvent treatment (ether, chloroform) but can
transient aplastic crisis by hybridization or more commonly be inactivated by formalin, β-propiolactone, and oxidizing
by quantitative polymerase chain reaction (PCR). Due to the agents. Gamma irradiation will also inactivate B19V, with
exquisite sensitivity of PCR, B19V DNA can then remain 1.4 mR producing a 10 log10 reduction in infectivity [43].
detectable for months or even years at low levels even fol-
lowing complete recovery, and thus quantitative PCR is 2.3.2 Morphology
required to distinguish recent infection from previous infec- The parvovirus B19 virion is an icosahedron consisting of 60
tion with the virus. copies of the capsid proteins. Most of the capsid protein is
In situ hybridization can be used to identify B19V DNA VP2, with 5 % or less of the larger VP1 protein [32]. VP2
within specific cells. Assays for B19V antigen based on capsid proteins self-assemble in the absence of B19V DNA,
monoclonal antibodies are relatively insensitive (<106 virus and in these systems, protein expression leads to formation
particles/ml). Electron microscopy (EM) can be used to of recombinant virus-like particles. VP1 is not required for
detect B19V in serum with the same sensitivity as antigen capsid formation [35, 44].
assays. Within cells, EM cannot always distinguish intracel- The atomic structure of both infectious B19V and B19V
lular virus from ribosomes. VP2 empty capsids has now been resolved to <0.75 nm [45–47].
The virion surface has a major depression encompassing the
Serological Assays for B19V-Specific Seroprevalence fivefold axis, similar to the canyon structure found in RNA-
ELISA-based assays using virus-like particles are both sen- containing icosahedral viruses. In B19V capsids, there is
sitive and specific. Although parvovirus B19V IgG can be also a hollow cylindrical structure about the fivefold axes
detected by both capture assay and indirect assay, an indirect that appears to penetrate to the inside of the virion. The
format is generally used for seroprevalence studies. Antibody structural distribution of VP1 in the B19V capsid structure is
to virus is usually present by the seventh day of illness still unknown. VP1 capsomers alone will not self-assemble
(aplastic crisis) or day after the onset of rash, and probably is to form icosahedral capsids; rather, they form smaller and
lifelong thereafter, although some waning of antibody has irregular structures. In native virions, the VP1 unique region
been suggested [37]. appears to be on the outside of the capsid, and it has been
proposed that it may extend through the fivefold axis cylin-
der to the outside of the virion [48].
2.3 Biological Characteristics of B19V
2.3.3 B19V Strain Variation
2.3.1 Physical Properties Parvovirus B19 is now recognized to have three different
Parvovirus B19 has the typical features of a member of the genotypes, with ~10 % variability at the DNA level between
Parvoviridae. On EM, the particles are nonenveloped, genotypes [49–51]. Most of the B19V identified is genotype
15–28 nm in diameter, and show icosahedral symmetry. 1 [49], the original B19V genotype, which is distributed
Often both “empty” and “full” capsids are visible. Mature worldwide. Genotype 3 seems to be the predominant B19V
infectious parvovirus particles have a molecular weight of genotype in Ghana, representing more than 90 % of the
5.6 × 106, a buoyant density in cesium chloride gradients of sequences identified [52]. Genotype 2 has been primarily
1.41 g/ml, and a sedimentation coefficient of 110S. identified in tissues of older patients (born before 1973), sug-
The DNA in infectious particles made up 19–37 % of the gesting that it may have circulated more frequently prior to
total mass of the capsid. The genome size is extremely the 1970s [53]. However, blood samples or donations
632 K.E. Brown

250

all other cases

200
cases in women aged 17-44yrs
Total laboratory reports

150

100

50

0
01 -0
4
-0
7
-1
0
-0
1
-0
4
-0
7
-1
0
-0
1
-0
4
-0
7
-1
0
-0
1
-0
4
-0
7
-1
0 1 4 7
-1
0
-0
1
-0
4
07- 7 7 7 8 8 8 8 9 9 9 9 0 0 0 0 1-0 1-0 1-0 1 2 2
20 2 00 200 2 00 200 2 00 200 2 00 2 00 200 2 00 200 2 01 2 01 201 2 01 2 01 2 01 2 01 201 2 01 2 01

Year-month

Fig. 27.1 Laboratory-confirmed cases of parvovirus B19 infection 2007–2012 in England and Wales, showing the seasonal variability (© Crown
copyright. Reproduced with permission of Public Health England [83])

containing high titer genotype 2 are occasionally identified insensitive assay that would detect high titer viremia (indicat-
[54, 55]. The observation of both genotype 2 and 3 sequences ing acute infection) was used between 1:20,000 and 1:40,000
in blood and tissues from many different parts of the world units of blood during epidemic seasons were positive [72].
[56–58] indicates a more widespread distribution than With more sensitive PCR assays then ~1 % of all donations
originally assumed. have low-level parvovirus B19 DNA detectable [73].
Despite the differences in the DNA sequences, the capsid Due to the risk of adverse outcome in pregnancy, a num-
protein sequence is conserved between the different geno- ber of studies have attempted to calculate the incidence of
types, and there is evidence for both serological and cross- infection in women of childbearing age [74–77]. This varies
neutralization [59]. There is no evidence that the different between countries but is estimated for women with no known
genotypes show any differences in virological or disease exposure to be between 0.6 % [63] and 2.4 % [78], with most
characteristics [60, 61]. estimates around 1 % [63, 79, 80].

2.4.2 Epidemic Behavior and Contagiousness

2.4 Descriptive Epidemiology Infections in temperate climates are more common in late
winter, spring, and early summer months [81–83]. Rates of
2.4.1 Prevalence and Incidence infection may also increase every 3–5 years (Fig. 27.1), and
Parvovirus B19 is a common infection in humans, and this is reflected by corresponding increases in the major clin-
although there is some variation in different countries [62], by ical manifestations of B19V infection, TACs, and EI [84].
age 15, approximately 50 % of children have detectable IgG The virus can be readily transmitted by close contact. In
[63]. Infection also occurs in adult life, and more than 80 % studies in Europe, the highest force of infection (FOI) is in
of the elderly have detectable antibody [64]. Significantly children aged 7–9, with an overall R0 (basic reproduction
higher seroprevalence is found in some parts of Africa [65] number) of between 1.5 and 3 [85]. The secondary attack
and Papua New Guinea [66], with >80 % of 10-year-olds hav- (seroconversion) rate has been calculated in various settings.
ing detectable antibody. In contrast, some parts of Asia [67– In one study the secondary attack rate from symptomatic
69] and some isolated communities in other parts of the world TAC or EI patients to susceptible (IgG negative) household
have a much lower seroprevalence [70, 71]. contacts was approximately 50 % [84]. In school outbreaks,
The prevalence of parvovirus B19 DNA in blood samples serological studies are generally not available, but 10–60 %
depends on the sensitivity of the assay used. When a relatively of students may develop a rash disease consistent with B19V
27 Parvoviruses 633

infection [75–77]. The highest secondary attack rates and 2.6 Pathogenesis and Immunity
also seroprevalence and annual seroconversion rates even in
the absence of known outbreaks of infection are observed 2.6.1 Pathogenesis
among workers such as day-care providers and school per- Parvovirus B19, like all autonomous parvoviruses, is depen-
sonnel who have close contact with affected children [64, 86]. dent on mitotically active cells for its own replication.
In one modeling study using seroprevalence data from five Parvovirus B19 also has a very narrow target cell range and
European countries, the highest risks of transmission were can only be efficiently propagated in human erythroid pro-
within households and schools, and where there was close genitor cells. For erythroid progenitors from bone marrow,
physical contact on a daily basis and for at least 1 h [85]. susceptibility to parvovirus B19 increases with differentia-
Although nosocomial transmission in hospital situations tion; the pluripotent stem cell appears to be spared and the
has been described [87–89], especially in patients with per- main target cells are CD36 positive erythroid colony-forming
sistent disease, this is unusual, and health-care workers do cells (CFU-E) and erythroblasts [94, 95]. In erythroid pro-
not have a significantly higher seroprevalence than age- genitors, the virus is cytotoxic, producing a cytopathic effect
matched controls [64]. However, patients with TAC or per- with characteristic light [96] and electron microscopic [97,
sistent infection should be considered infectious and 98] changes. Infected cultures are characterized by the pres-
appropriate precautions taken to limit transmission. ence of giant pronormoblasts or “lantern cells,” which are
early erythroid cells, 25–32 μm in diameter, with cytoplas-
mic vacuolization, immature chromatin, and large eosino-
2.5 Mechanism and Routes of Transmission philic nuclear inclusion bodies. On EM, virus particles are
seen in the nucleus and lining cytoplasmic membranes of
Parvovirus B19 DNA has been found in the respiratory infected erythroid progenitors, and infected cells show mar-
secretions of patients at the time of viremia [16, 84] suggest- ginated chromatin, pseudopod formation, and cytoplasmic
ing that B19V infection is generally spread by a respiratory vacuolation, typical of cells undergoing apoptosis [99]. The
route of transmission. In contrast to other respiratory viruses, light microscopic findings are also seen in the bone marrow
however, no site of replication for B19V has been found in of infected patients [100].
the nasopharynx. There is little evidence of virus excretion in The basis of the erythroid specificity of parvovirus B19
feces or urine [90]. may be explained by the tissue distribution of the virus cel-
The virus can be found in serum, and infection also can lular receptor, globoside, also known as blood group P anti-
be transmitted by blood and blood products. Although ~1 % gen [101]. P antigen is found on erythroblasts and
of blood donations have low-level B19V DNA detectable megakaryocytes. (It is also present on endothelial cells,
[73], reports of transmission of B19V infection by individ- which may be targets of viral infection involved in the patho-
ual units of blood or platelets are rare. In a linked donor- genesis of transplacental transmission, possibly vasculitis
recipient study, transmission of infection only occurred and the rash of fifth disease, and on fetal myocardial cells
with components that contained B19V DNA at concentra- [102].) Rare individuals who do not have P antigen on their
tions greater than 106 IU/mL (estimated to occur in 1:6,000 cells are resistant to B19V infection, and their bone marrow
donations) [91]. cannot be infected with B19V in vitro [103].However, ery-
Without prior screening for parvovirus B19, pooled prod- throid specificity may also be modulated by specific ery-
ucts could contain high concentrations of parvovirus B19. throid transcription factors [104], and other cofactors
Parvoviruses, including B19V, are very heat resistant and at including a hypoxic environment [105, 106].
high virus concentration can withstand the usual heat treat- Studies in normal volunteers have shown that B19V infec-
ment (80 °C for 72 h) used to destroy infectivity. In addition, tion leads to an acute but self-limited (4–8 days) cessation of
solvent–detergent methods, which only inactivate lipid- red cell production and a corresponding decline in hemoglo-
enveloped viruses, are ineffective. Parvovirus B19 infection bin level [90, 107]. In patients with normal erythroid turn-
has been transmitted by steam- or dry-heated factor VIII or over, this short interruption of red cell production does not
IX [42, 92] and by solvent–detergent-treated factor VIII lead to anemia; but in patients with high red cell turnover, due
[92]. Hemophiliacs who received heat-treated factor VIII to hemolysis, blood loss, and so forth, the interruption can
alone had lower prevalence of B19V antibody and lower precipitate an “aplastic crisis.” The crisis resolves as virus is
rates of seroconversion compared to those receiving nonheat- “cleared” from the bone marrow by the immune response. In
treated factor [93]. patients who are immunocompromised, the infection may
Recommendations in Europe and America now require persist and produce chronic pure red cell aplasia.
all plasma pools for fractionation to be screened for high lev- The infected fetus may suffer severe effects because red
els of parvovirus DNA to try to minimize the transmission of blood cell turnover is high and the immune response deficient.
parvovirus B19 by blood products. During the second trimester, there is a great increase in red cell
634 K.E. Brown

a B19V IgG
12

B19V IgM / IgG

Arbitrary scale
B19V IgM
B19V DNA
(IU/mL)

B19V DNA
3

0
7 14 21 28 2 3 4
Days Months
Fever
Rash
Drop in reticulocytes

a
B19V DNA
12

B19V IgM / IgG

Arbitrary scale
B19V DNA
(IU/mL)

3 B19V IgG

B19V IgM
0
7 14 21 28 2 3 4
Days Months
Time

Drop in reticulocytes

Fig. 27.2 Schematic diagram of the clinical and virological events fol- immunosuppressed patient prior to treatment with intravenous immu-
lowing B19 parvovirus infection. (a) Virology and immune response in noglobulin (Data from Anderson et al. [90], Potter et al. [107] and the
immunocompetent individual. (b) Virology and immune response in case presented by Kurtman et al. [18])

mass. Parvovirus particles can be detected by EM within the studies, the rash and joint symptoms appeared when viremia
hematopoietic tissues of liver and thymus [108], and B19V was no longer detectable and at the time of development of a
DNA and capsid antigen have been detected in the myocar- detectable immune response [90]. Similar findings have been
dium of infected fetuses [109]. In addition, there is evidence reported in chronically infected individuals treated with
that the fetus may develop myocarditis [110, 111], compound- immunoglobulin therapy [112].
ing the severe anemia and secondary cardiac failure. By the
third trimester, a more effective fetal immune response to the 2.6.2 Immune Response to B19V Infection
virus may account for the decrease in fetal loss. Both virus-specific IgM and IgG antibodies are made follow-
The pathogenesis of the rash in EI and polyarthropathy is ing experimental [90] and natural [113] B19V infection
almost certainly immune complex mediated. In volunteer (Fig. 27.2). Following intranasal inoculation of volunteers,
27 Parvoviruses 635

virus can first be detected at days 5–6 and levels peak at days Perhaps surprisingly, prolonged activation of the CD8 response
8–9. IgM antibody to virus appears about 10–12 days after is seen even after apparent acute infection and resolution of
experimental inoculation; IgG antibody appears in normal symptoms [124], presumably in keeping with the low levels of
volunteers about 2 weeks after inoculation. B19V DNA that are detectable in serum and tissues for months
There is a similar time course in natural infections. In or even years following infection [128, 129].
patients with TAC, 108–1014 genome copies/ml of virus DNA
may circulate [114, 115]. IgM antibody may be present in
patients with TAC at the time of reticulocyte nadir and dur- 2.7 Patterns of Host Response
ing the subsequent 10 days; IgG may not be present at the
time of reticulocyte depression but appears rapidly with 2.7.1 Asymptomatic Infection
recovery. High levels of B19V DNA are not detectable in Most people with B19V specific antibody have no recollec-
patients with clinical fifth disease (the manifestations are tion of any specific symptoms. In one epidemiologic study of
secondary to immune complex formation), although B19V a school outbreak, B19V caused asymptomatic infection in
DNA can still be detected by PCR [116]. approximately 25 % of adults [77]. In household contacts of
IgM antibody may be found in serum samples for several patients with aplastic crises or EI due to B19V, 32 % (17/52)
months after exposure [117]. IgG persists for life and levels reported no symptoms [84]. There were more asymptomatic
rise with reexposure [90]. Transient aplastic crisis due to par- infections in those with darker skin (69 %) compared to
vovirus B19 infection does not occur more than once in the whites (17 %), but the numbers studied were small and the
life of a sickle-cell patient [17]. Measurable IgA antibodies clinical presentation of the index infection was different in
specific to B19V may play a role in protection against infec- the two groups (TAC for the black contacts and EI for the
tion by the nasopharyngeal route [118]. white contacts). Parvovirus B19 may go unrecognized in
In immunocompetent individuals, the early antibody those with darker skin color who do not have an underlying
response is to the major capsid protein VP2, but as the hemolytic anemia, since the rash is particularly difficult to
immune response matures, reactivity to the minor capsid see in these circumstances.
protein, VP1, dominates [119, 120]. Sera from patients with
persistent B19V infection typically have antibody to VP2 but 2.7.2 Erythema Infectiosum
not to VP1 [121]. Recovery from infection correlates with As indicated earlier, EI, otherwise known as fifth disease or
the appearance of circulating specific antivirus antibody. slapped-cheek disease, is the major manifestation of B19V
The importance of the anti-VP1 response in developing infection and was well characterized clinically before the
neutralizing antibody has been confirmed in animal experi- discovery of B19V [130, 131]. The nonspecific prodromal
ments using recombinant capsid. Rabbits immunized with cap- illness often goes unrecognized and may be associated with
sids containing only VP2 produced a strong antibody response, symptoms of fever, coryza, headache, and mild gastrointesti-
but the sera had low neutralization titers. In contrast, rabbits nal symptoms, such as nausea and diarrhea. Two to five days
immunized with capsids containing VP1 produced antibody later, the classic slapped-cheek rash appears a fiery red erup-
with neutralizing titers comparable to those produced in tion on the cheek, accompanied by relative circumoral pallor.
humans following acute B19V infection [122, 123]. One to four days after the slapped-cheek rash, the second-
Persistent B19V infection is the result of failure to pro- stage rash may appear—an erythematous maculopapular
duce effective neutralizing antibodies by the immunosup- exanthem on the trunk and limbs. As this eruption fades, it
pressed host. Perhaps because of the limited number of takes on a typical lacy appearance. There may be great varia-
epitopes presented to the immune system by parvovirus B19, tion in the dermatological appearance, and rarely it may
the congenital immunodeficiency states associated with per- present as papulo-purpuric glove and sock syndrome
sistent infection may be clinically subtle, with susceptibility (PPGSS) [132–136]. The classic slapped-cheek rash is more
largely restricted to parvovirus, although multiple immune common in children than adults, and the second-stage erup-
system defects are apparent once directed testing of T- and tion may vary from a very faint erythema that is easily missed
B-cell function is performed. Administration of commercial to a florid exanthema and may be transient or recurrent over
immunoglobulins can cure or ameliorate persistent parvovi- 1–3 weeks. The rash may be accompanied by pruritus, which
rus infection in immunodeficient patients [112]. can be the dominant symptom and may be especially promi-
The role of the cellular immune response in limiting parvo- nent on the soles of the feet [77, 137].
virus B19 infection has been studied less intensively. Using a
combination of recombinant capsids and peptides from the 2.7.3 Polyarthropathy Syndrome
nonstructural and capsid proteins, it is now clear that B19V In children, B19V infection is usually mild and of short dura-
infection induces both a profound CD8 [124, 125] and CD4 tion. However, in adults and especially in women, there may
[126, 127] response, both of which are required for viral control. be arthropathy in approximately 50 % of patients [77]. The
636 K.E. Brown

joints can be painful, often with accompanying swelling and the worsening anemia. Congestive heart failure and severe
stiffness. The distribution is usually symmetrical; mainly the bone marrow necrosis may develop [151, 152] and the illness
small joints of hands and feet are involved. Joint symptoms can be fatal [17]. Aplastic crisis can be the first presentation
last 1–3 weeks, although in 20 % of affected women, arthral- of an underlying hemolytic disease in a well-compensated
gia or frank arthritis may persist or recur for more than 2 patient [153].
months, even to 2 years. In the absence of a history of rash, TAC and B19V infection in hematologically normal
the symptoms may be mistaken for acute rheumatoid arthri- patients are often associated with changes in the other blood
tis, especially as B19V infection can be associated with tran- lineages. There may be varying degrees of neutropenia,
sient rheumatoid factor production [138–140]. In one study thrombocytopenia, and transient pancytopenia [145]. Some
of patients attending an “early synovitis” clinic in England, cases of idiopathic thrombocytopenia purpura [154] and
19 of 153 (12 %) had evidence of recent infection with B19V Henoch–Schönlein purpura [155, 156] have been linked to
[140]. Parvovirus B19 infection should be considered as part parvovirus B19 infection. Less often, agranulocytosis has
of the differential diagnosis in any patient presenting with been described following B19V infection [157, 158].
acute arthritis. Community-acquired aplastic crisis is almost always due
It has been postulated that B19V is involved in the initia- to parvovirus B19 [159] and should be the presumptive diag-
tion and perpetuation of rheumatoid arthritis leading to joint nosis in any patient with anemia due to abrupt cessation of
lesions [141], but these results have not been reproducible by erythropoiesis as documented by reduced reticulocytes and
other groups. In contrast, parvoviral B19V DNA is fre- bone marrow appearance. In contrast to patients with EI,
quently found in synovial tissue of patients with rheumatoid TAC patients are often viremic at the time of presentation,
arthritis, chronic arthropathy, and control subjects. In one with concentrations of virus as high as 1014 genome copies/
carefully performed controlled study, although B19V DNA ml, and the diagnosis is readily made by detection of B19V
was detected in synovial tissue of 28 % of individuals with DNA in the serum. As B19V DNA levels fall in serum,
chronic arthritis, it was also found in 48 % of nonarthropathy B19V-specific IgM becomes detectable.
controls [142], indicating that PCR-detectable DNA may TAC is readily treated by blood transfusion. It is a unique
persist in synovial tissues for months or years. In addition, in event in the patient’s life, and following the acute infection
one study with long-term follow-up, none of the 54 patients immunity is lifelong.
with B19V-associated arthralgia reported persistence of joint
swelling or restricted motion, and no evidence of inflamma- 2.7.5 Infection During Pregnancy
tory joint disease was found [143]. Therefore, it seems and Congenital Infection
unlikely that B19V plays a role in classic erosive rheumatoid B19V infection in pregnancy may be associated with miscar-
arthritis. The association of B19V and juvenile rheumatic riage or nonimmune hydrops fetalis [160]. Although nonim-
disease is more convincing [144], but whether it is the cause mune hydrops fetalis (NIHF) is rare (1 per 3,000 births) and
of the disease or one of many potential triggers is less clear. in approximately 18 % of cases the etiology is unknown, 7 %
can be ascribed to an infectious cause, generally parvovirus
2.7.4 Aplastic Crisis B19 [161]. In one study of 63 fetal deaths due to NIHF, 8
Transient aplastic crisis is the abrupt cessation of erythropoi- were due to parvovirus B19 infection [162]. In cases where
esis characterized by reticulocytopenia, absent erythroid pre- pathological studies were undertaken, the fetuses showed evi-
cursors in the bone marrow, and precipitous worsening of dence of leukoerythroblastic reaction in the liver and large,
anemia. TAC was the first clinical illness associated with pale cells with eosinophilic inclusion bodies and peripheral
B19V infection [7]. TAC due to B19V has been described in condensation or margination of the nuclear chromatin.
a wide range of patients with underlying hemolytic disor- Parvovirus B19 DNA could be detected by DNA hybridiza-
ders, such as hereditary spherocytosis, thalassemia, and red tion [163] and in situ hybridization [164], and parvovirus par-
cell enzymopathies such as pyruvate kinase deficiency and ticles could be seen by electron microscopy [108].
autoimmune hemolytic anemia [145]. TAC can also occur Even in the absence of treatment, an adverse fetal out-
under conditions of erythroid “stress,” such as hemorrhage, come is not typical after maternal B19V infection. In a pro-
iron deficiency anemia, and following kidney, bone marrow, spective British study of more than 400 women with
or liver transplantation [146–149]. Acute anemia has been serologically confirmed B19V during pregnancy, the excess
described in hematologically normal persons [150], and a rate of fetal loss was confined to the first 20 weeks of preg-
drop in red cell count (and reticulocytes) was seen in healthy nancy and averaged only 9 % [165]. No abnormalities were
volunteers [90]. found at birth in the surviving infants, even when there was
Although suffering from an ultimately self-limiting dis- evidence of intrauterine infection by the presence of B19V
ease, patients with aplastic crisis can be severely ill. Symptoms IgM in the umbilical cord blood, and there were no long-
may include dyspnea, lassitude, and even confusion due to term sequelae attributable to B19 infection in the 129
27 Parvoviruses 637

children observed for more than 7 years. The findings in immunosuppression may allow the host to produce antibody
studies in other countries were similar [166–170]. and resolve the virus infection.
Many cases of parvovirus B19-induced hydrops fetalis are Virus-associated hemophagocytic syndrome (VAHS) is
now treated with intrauterine blood transfusion. In one study characterized by histiocytic hyperplasia, marked hemo-
of follow-up of 20 children, no long-term sequelae were phagocytosis, and cytopenia in association with a systemic
observed [171]. However, in a second study of 24 transfused viral illness [181]. In contrast to malignant histiocytosis,
infants, 5/16 infants that were followed had delayed psycho- VAHS is usually a benign, self-limiting illness in which his-
motor development [172]. Neither study included controls, tiocytic proliferation is reversible. Hemophagocytosis is not
and it is not clear whether the developmental abnormalities uncommon and occurs in the setting of a wide range of infec-
were a direct effect of the virus or due to the treatment inter- tions, not only viral but also bacterial, rickettsial, fungal, and
vention. Even in the absence of intrauterine transfusion rare parasitic [182]. However, in many patients, there is underly-
case reports of congenital ocular and neurological abnormali- ing immunosuppression, usually iatrogenic, so that the role
ties after maternal B19V infection have been reported. of the incriminated pathogen as an etiologic agent or coinci-
Rare cases of congenital anemia after a history of mater- dental opportunistic infection remains unclear.
nal B19V exposure have been reported [173]. In these cases, Parvovirus B19 infection has been detected in 15 cases of
the virus load is generally low and the anemia does not hemophagocytosis syndrome among children and adults
respond to immunoglobulin therapy. The B19V infection [183]. The majority of patients were previously healthy, but
may mimic Diamond–Blackfan anemia [174, 175], and the four patients were immunosuppressed by drug therapies. In
role of in utero B19V infection in inducing constitutional all but one case, there was a favorable outcome (one immu-
bone marrow failure such as that in Diamond–Blackfan ane- nosuppressed patient died of fulminant aspergillosis).
mia is still not clear. Further studies are required to determine whether parvovirus
B19 is a major cause of VAHS as well as the rate of VAHS in
2.7.6 Chronic Bone Marrow Failure otherwise uncomplicated parvovirus B19 infection.
Persistent B19 infection resulting in pure red cell aplasia has
been reported in patients with a wide variety of immunosup- 2.7.7 Vasculitis
pressive conditions, ranging from congenital immunodefi- The role of parvovirus B19 in vasculitis remains unclear.
ciency, acquired immunodeficiency syndrome (AIDS), and Several case reports have described positive B19V serology
lymphoproliferative disorders to transplantation [176]. The in patients with vasculitis and/or polyarteritis nodosum
stereotypical presentation is with persistent anemia rather [184–187]; however, in each individual report, it was uncer-
than the immune-mediated symptoms of rash or arthropathy. tain as to whether the association was coincidental or caus-
The patients have absent or low levels of B19V-specific anti- ative. More recently, parvovirus B19 infection has been
body and persistent or recurrent parvoviremia as detected by associated with acute systemic necrotizing vasculitis [188].
high levels (>109 IU/mL) of B19V DNA in the serum. Bone Recent infection with parvovirus B19 was indicated in three
marrow examination often shows the presence of scattered patients by the presence of both B19V IgM in the serum and
giant pronormoblasts. Often there is a pure red cell aplasia, B19V DNA in serum and tissues. Treatment with intrave-
but other lineages may also be affected. nous immunoglobulin led to clearing of the virus and resolu-
The prevalence of B19-induced anemia in human immuno- tion of the patients’ symptoms.
deficiency virus (HIV)-seropositive patients is probably higher A similar report linked parvovirus B19 to Kawasaki dis-
than recognized. In one early study of 50 patients with AIDS, ease, a multisystem vasculitis of early childhood. In a study
no patients with B19V viremia were identified. In a larger from Italy, parvoviral B19 DNA and/or IgM antibodies were
cohort study, B19V DNA was found in only 1 of 191 (0.5 %) found in 10 of 15 patients with Kawasaki disease compared
HIV-seropositive men who have sex with men. However, to 0 of 36 control children [189, 190]. The authors do not
B19V DNA was found in 5 of 30 (17 %) of the transfusion- report on treatment of their cases, but immunoglobulin ther-
dependent HIV seropositives, and when a hematocrit of less apy is known to be beneficial in Kawasaki disease. However,
than 20 was used as a criterion, 4 of 13 (31 %) had detectable other studies have not shown a relationship between
B19V DNA [177]. In contrast to the earlier studies, the mar- Kawasaki disease and parvoviral infection [189, 191, 192].
row morphology need not be suggestive of pure red cell apla-
sia, and giant pronormoblasts may not be present. 2.7.8 Myocarditis
Administration of immunoglobulin can be beneficial There have been several case reports of myocarditis associ-
and ameliorative even if not curative [178]. Temporary ces- ated with B19V infection in both children and adults [193,
sation of maintenance chemotherapy also led to resolution 194]. Many of the case reports attributed the syndrome to
of the anemia, and in two cases, reinstitution did not lead to B19V as the cause based simply on the detection of B19V
recurrence [179, 180] suggesting that decreasing the level of DNA genome; but given the known persistence of B19V
638 K.E. Brown

DNA in tissues, this may be erroneous. The putative role of convalescent-phase antisera [121] and commercial immuno-
B19V in the pathogenesis of myocarditis nevertheless war- globulin preparations [201, 202] contain neutralizing anti-
rants further investigation, particularly because P antigen is bodies to parvovirus, as assessed in vitro using erythroid
found on fetal myocardial cells and B19V appears to cause colony systems or tissue culture. In addition, commercial
myocarditis in the fetus [110, 111]. immunoglobulin from normal donors can cure or ameliorate
Less convincing is the evidence for parvovirus B19 as a cause persistent B19V infection in immunosuppressed human
of cardiomyopathy [195]. Many parvovirus-associated cases are patients [18, 112].
based simply on the detection of B19V DNA in cardiac tissue. Prospects for a B19V vaccine should be good, as
However, when control studies have been performed, B19V baculovirus-produced B19V capsids induce neutralizing anti-
DNA is also found in control cardiac tissue [196–198]. bodies in experimental animals [203], even without adjuvant.
The presence of VP1 protein in the capsid immunogen
appears critical for the production of antibodies that neutral-
2.8 Diagnosis of B19V Infections ize virus activity in vitro, and capsids with supranormal VP1
content are even more efficient in inducing neutralizing activ-
The detection of B19V viremia is based on quantitative PCR. ity in immunized animals [123]. Candidate vaccines have
High titer parvovirus B19 DNA (>109 IU/ml) can be detected been produced, but the most recent study was halted due to
in serum at the time of TAC (Fig. 27.2). In immunocompe- unexplained cutaneous reactions in 3 of the 43 patients
tent individuals, high titer B19V DNA is only detectable for enrolled [204]. All patients had produced neutralizing anti-
2–4 days and then drops to between 104 and 106 IU/mL as the bodies to parvovirus B19. Whether the cutaneous reactions
immune response develops. were due to the presence of insect cell proteins in the prepara-
Within 1 day of onset of the rash of EI, parvovirus B19 tion of the phospholipase activity of the VP1 was not resolved.
IgM and IgG are detectable by ELISA in serum samples, and Further efforts to develop a safe and effective parvovirus B19
diagnosis of EI is therefore based on IgM assays, ideally per- vaccine are currently on hold, but given the possible severe
formed by the capture technique [199, 200]. (Indirect assays consequences of parvovirus B19 infection, production of
to detect B19V IgM often give false positives due to cross- such a vaccine continues to be an important goal.
reacting antibodies or rheumatoid factor.) IgM antibody
remains detectable for 2–3 months following infection.
Parvovirus B19 IgG can also be detected by ELISA. With 2.10 Unresolved Problems
current IgG assays, IgG is also detectable within 1 day of
onset of rash and is probably present for life thereafter. As 2.10.1 Full Spectrum of Disease
more than 50 % of the population have IgG antibody to Although B19V is associated with a wide variety of diseases,
B19V infection, detection of B19V IgG is not helpful for the almost certainly the list is not complete. In addition, the discov-
diagnosis of acute infection. ery of P antigen as the receptor for B19V suggests a possible
Immunocompromised or immunodeficient patients with role for the virus in diseases previously unsuspected as related
chronic infection may not mount an immune response to to parvoviral infection. P antigen is found on thyroid tissue
B19V, and therefore, quantitative PCR is required for diag- [102], and B19V has been found in patients with Hashimoto’s
nosis. As with TAC, patients generally have high titer B19V disease and thyroid malignancy [205–207]. Whether it triggers
DNA (>109 IU/mL) detected. disease or is a bystander remains unclear [208].
The sensitivity level of detection of B19V has greatly Neurological disease has also been associated with parvo-
increased by the use of PCR, but at the risk of possible con- virus infection. Pruritus is not uncommon in fifth disease,
tamination and false-positive results confusing interpreta- and in one study, 50 % of patients with serologically con-
tion. Even in immunocompetent persons whose recovery firmed fifth disease experienced neurological symptoms,
from acute infection is uncomplicated, a sensitive assay can especially neurasthenia in fingers or toes [209]. One patient
probably detect B19V DNA for the rest of their lives [53, developed more significant disease with progressive weak-
129]. Thus, simple detection of B19V DNA in serum or tis- ness of one arm. Brachial plexus neuropathy has been
sues does not indicate active infection. described in other patients with B19V infection [210–212].
In those illnesses where B19V is known to be involved, the
full spectrum of disease is still uncertain. Parvovirus B19 is
2.9 Control and Prevention recognized as the cause of acute polyarthropathy, but its role
in chronic arthropathy and as a possible trigger for rheuma-
The humoral immune response plays a dominant role in the toid arthritis is still undetermined. Similarly, the role for
normal immune response to parvovirus, and antibodies are B19V as a cause of autoimmune disease, kidney disease, vas-
protective in both passive and active immunizations. Human culitis, and in utero B19V infection inducing constitutional
27 Parvoviruses 639

bone marrow failure such as Diamond–Blackfan anemia is fecal samples (HBoV2–4). However, the bocavirus DNA is
still under investigation. also commonly found in asymptomatic individuals, raising
concerns that they may simply be “passenger” viruses, acquired
2.10.2 B19V Vaccine Policy early in life and not necessarily pathogenic [219–223].
Apart from the problems in developing a vaccine without the
cutaneous side effects, the target populations for such a vac- 3.2.3 Serological Surveys
cine also remain to be determined. Should only patients at As with Parv4, several groups investigating human bocavirus
high risk of severe or life-threatening disease, such as sickle- have expressed viral capsids in insect cells [224–229] and
cell patients, be protected? Or, in view of the wide variety of developed serological assays to detect both IgM and IgG.
disease manifestations affecting all strata of the population, However, most assays probably measure cross-reacting anti-
should a universal vaccine policy be pursued? A universal bodies to any of the four different human bocaviruses, and
vaccination policy would have the added advantage of pos- few studies have tried to distinguish between the antibody
sible eradication of B19V from a community but would have responses [230, 231].
the disadvantages of high cost–benefit ratio.
3.2.4 Laboratory Methods
Although HBov1 can be grown in vitro in well-differentiated
3 Human Bocaviruses (HBoV) airway epithelial cells [232], replication is inefficient, and
for most cases, viral DNA is detected by PCR. HBoV1 DNA
3.1 Historical Background is commonly found in between 2 and 33 % of hospitalized
children with respiratory symptoms (see Jartti et al. [233] for
Human bocavirus was discovered in 2005 as part of a virus a review of published work), making it the fourth most com-
discovery program to identify the causes of lower respiratory mon infection identified. However, in many of the cases
tract infections in children [213]. Human bocavirus sequence (13–19 %) [233], the virus is found with other pathogens,
was first identified in two large pools of nasopharyngeal aspi- raising questions as to whether it is the main cause of symp-
rates submitted for diagnosis of respiratory tract infections in toms. Patients with active or symptomatic bocavirus infec-
young Swedish children. After sequence-independent spe- tions have higher concentrations of virus; therefore, in order
cific primed amplification (SISPA) two libraries of DNA to identify primary infection, it is important to perform quan-
were obtained, and 62 of the >800 clones analyzed contained titative PCR to determine the viral loads in respiratory secre-
bocavirus sequences. A specific human bocavirus PCR was tions and in serum or plasma samples, in combination with
developed, and testing confirmed the presence of the novel detection of bocavirus-specific IgM or IgG seroconversion.
bocavirus in 17 of 540 (3.3 %) of clinical samples. This respi-
ratory human bocavirus is now classified as human bocavirus
1 (HBoV1). Subsequently related viruses, human bocaviruses 3.3 Biological Characteristics of Human
2–4, have been detected in fecal samples [214–216]. Bocaviruses

HBoV1 has the typical structure of a member of the

3.2 Methods Involved in Epidemiological Parvoviridae, with nonenveloped icosahedral particles of
Analysis ~25 nm on diameter [234]. The full-length genome, includ-
ing the inverted terminal repeat sequences, has been cloned
3.2.1 Sources of Mortality Data into a plasmid and, when transfected into cells, leads to the
There have been no reported cases of fatal infection due to production of infectious virions with typical features [235].
any of the human bocaviruses to date, although there is a Similar culture or reverse genetics have not been described
single case of fatal type 7 adenovirus reported in a patient for the other human bocaviruses.
who also had bocavirus detected on a throat swab [217]. The full-length genome of HBoV1 is 5,543 nucleotides,
However, no viral titer or serology results were provided, with dissimilar hairpin sequences at the 5′ and 3′ ends. The
and the significance of the detection of the bocavirus is genome has three large open reading frames (as seen in other
unknown. There has also been an additional case of life- members of the bocavirus genus) encoding the nonstructural
threatening infection with bocavirus in a 4-year-old, with no protein (NS1), the capsid proteins (VP1 and VP2), and a sec-
other pathogen identified [218]. ond nonstructural protein, NP1 [236].
The capsid protein, VP2, has been expressed in insect
3.2.2 Sources of Morbidity Data cells and self-assembles to form virus-like particles that are
Most studies of human bocavirus infection are performed the basis of most serology assays. The three-dimensional
based on the detection of viral DNA in respiratory (HBoV1) or structure of these virus-like particles has been solved to
640 K.E. Brown

<0.79 nm. Although it shares the β-barrel structural core, a this nucleic acid represents infectious virus, and there is no
depression at the twofold axis, a protrusion at the threefold evidence of transfusion-transmitted infection.
axis, and a channel at the fivefold axis common to parvovi-
ruses, the surface morphology is different and appears much
“flatter” than other members of the group [237]. 3.6 Pathogenesis and Immunity
The coding sequences for the other human bocaviruses
(HBoV2 [214], HBoV3 [238], and HBoV4 [216]) is known, HBoV1 can be grown in human airway epithelia [235], and
but apart from HBoV3, the terminal sequences have not been cells in the respiratory tract are presumed to be the main site
elucidated. The different human bocavirus species show of replication during infection. Virus can also be detected in
between 10 and 30 % divergence, with increased genetic serum during primary infection [226, 247], and this finding
variation and evidence for recombination between HBoV2 almost certainly reflects high-level virus replication in the
and HBoV4 [216]. target tissues.
Following primary infection and local replication, there is a
serological response with IgG seroconversion and production
3.4 Descriptive Epidemiology of a short-lived bocavirus-specific IgM [226, 242, 248, 249].
Little is known about the pathogenesis of the fecal human
Human bocaviruses have a worldwide distribution and have bocaviruses.
been identified in every country where a search has been under-
taken. HBoV1 is predominantly found in respiratory secretions
and has been observed in 2–20 % of samples from children 3.7 Patterns of Host Response
with upper or lower respiratory tract disease [233]. Although
HBoV1 DNA can be detected throughout the year, primary Many of the published associations of bocavirus and disease
infection occurs predominantly in the winter and spring months associations are difficult to interpret because it is now clear
[213, 221, 239], as is true of many other respiratory infections. that there can be prolonged persistence of bocavirus DNA in
Based on serological studies using HBoV1 as antigen, most, if respiratory tract (or fecal) samples. Thus, early studies often
not all, individuals are infected in early childhood before the did not find a clear association with clinical presentation.
age of six [224, 225, 240]. More recent studies have indicated However, now that the criteria for diagnosing HBoV1 are
that some of the antibody detected will be cross-reacting to the defined (high viral load in respiratory secretions, DNA in
other human bocaviruses [226, 230], but even so, HBoV1 serum, and a serological response), many groups have shown
infections appear to be almost universal in childhood. that acute primary infection is associated with both upper
HBov2–4 are predominantly identified in fecal samples, and lower respiratory tract infection, and specifically with
both in patients (children and adults) with gastroenteritis and wheezing [247, 250, 251].
in healthy control subjects [216, 233]. HBoV2 appears to be In a study of 109 Finnish children followed up at 3–6
the most commonly identified species, followed by HBoV3 monthly intervals, primary HBoV1 infection diagnosed on
and then HBoV4 [226, 233]. This is also reflected in the sero- virological and serological grounds was associated with
epidemiology, with seroprevalence showing species-specific symptoms of respiratory tract infection in 61 % of children
frequencies as follows: HBoV1 > HboV2 > HBoV3 > HBoV4. [249]. Upper respiratory tract infection was the most com-
Adult sera from individuals in China, Pakistan, and Finland mon (60 %), with lower respiratory tract infection only being
showed similar seroprevalences: HboV2, 30–50 %; HBoV3, seen in 5 % of children. Acute otitis media was reported in
8–38 % (8 % in Finland); and HBoV4, 1–4 %. 46 % of children, and further studies are needed to see if this
is a true clinical association.
Similar criteria for the diagnosis of infection due to the
3.5 Mechanism and Routes of Transmission fecal bocaviruses have not been identified, and although
HBoV2–4 can be found in patients with acute gastroenteritis,
HBoV1 appears to be transmitted predominantly by the in controlled studies, they are found in healthy controls at
respiratory route, although detection of HBoV1 DNA in similar rates [222, 252].
urine [241, 242] and fecal samples [243, 244] suggests that it
may also be spread by fecal–oral route.
HBoV2–4 are found mainly in fecal samples and appear 3.8 Diagnosis
to be spread by the fecal route. Studies of sewage confirm
that HBoV2 at least is commonly found there [245]. It is now recognized that diagnosis of active human bocavi-
Although low levels of human bocavirus DNA have been rus infection should not be based on the detection of bocavi-
detected in serum from blood donors [246], it is not known if rus DNA in respiratory or fecal samples alone, because of
27 Parvoviruses 641

the persistence of DNA at these sites. For HBoV1, a firm 4.2.3 Serological Surveys
diagnosis should be based on the detection of viral DNA in Several groups have expressed the Parv4 capsid protein in
the serum, along with a serological antibody profile of recent insect [254, 255] or bacterial cells [256] and used these to
infection. This diagnostic profile generally includes evidence develop assays to detect antibodies for Parv4. However,
of IgG seroconversion, or detection of IgM, or low avidity these assays have generally only been used to test for anti-
IgG antibody. If serum is not available for serology and PCR, body levels in high-risk populations and not to look at sero-
then high titer (>104 genome copies/mL) HBoV1 DNA in prevalence in the general population.
respiratory secretions should be used [233].
Similar criteria have not been developed for HBoV2–4 4.2.4 Laboratory Methods
infections. Parv4 has not been grown in culture, and thus, diagnosis of
Parv4 infection is by the detection of Parv4 DNA by molecu-
lar techniques, usually PCR. As with parvovirus B19, low
3.9 Unresolved Problems levels of Parv4 DNA in serum or tissues likely represent pre-
vious infection and are part of the “bioportfolio” as described
There is still much to be learned about human bocavirus for erythroviruses [53].
infections, not least their pathogenesis and role in human dis- Acute infection can also be established by detection of
ease. With the recently developed criteria for HBoV1 diagno- Parv4-specific IgM by ELISA [254, 257].
sis, the true role of HboV1 as a human respiratory pathogen
can be determined. The significance of finding human boca-
viruses in the elderly or immunosuppressed is still unknown. 4.3 Biological Characteristics of Parv4

Although the virus has not been grown in culture, and the
4 Parv 4 full-length sequence is not known, it appears to have the
typical features of a member of the Parvoviridae. On EM the
4.1 Historical Background particles are nonenveloped 20–22 nm icosahedral particles
[258].
Human parvovirus 4 (Parv4) was also discovered in 2005 as So far 5,268 nucleotides of the sequence have been
part of a virus discovery program looking for new viruses in identified, and although this sequence represents the full-
plasma samples using sequence-independent single primer length genome, the inverted terminal repeat sequences are
amplification. The positive sample was from a daily injection incomplete [259]. The genome has two large open reading
drug user (IDU) with signs of acute viral infection who was frames, similar to B19V, but a very different transcription
HIV-RNA negative. profile, with a spliced RNA encoding the nonstructural
Testing of pooled plasma products from Europe and protein, and in the middle of the genome a second pro-
North America readily detected this virus in 4–5 % of pools, moter from which the RNA for the two capsid proteins
with viral loads varying from <100 copies/mL to 4 × 106 cop- (VP1 and VP2) are transcribed [260]. The ability to trans-
ies/mL. The prevalence may be significantly higher in other mit Parv4 infection with virally inactivated blood products
parts of the world. [257] suggests that Parv4 will have a similar inactivation
profile to parvovirus B19.
As with B19V, Parv4 does show sequence variation, and
4.2 Methodology Involved virus sequences can be divided into three main groups or
in Epidemiologic Analysis genotypes. Genotypes 1 and 2 (previously known as Parv5)
have been found in many countries [261–268]; genotype 3
4.2.1 Sources of Mortality Data appears to be predominantly found in sub-Saharan Africa
Parv4 infection is not known to be a fatal disease, although [269, 270].
the virus has been found in two patients with encephalitis of
unknown etiology [253]. However, Parv4 infection is rarely
looked for. 4.4 Descriptive Epidemiology

4.2.2 Sources of Morbidity Data In Europe and North America, Parv4 appears to be a rare
Very little information is available on the clinical features of infection outside certain high-risk groups—those who
acute infection with Parv4 because cohorts of recipients of share needles or receive blood products. However, only
blood products and IDUs, who are at high risk of acquiring been limited seroprevalence studies have been conducted
infection have received only limited attention. in the general population, and the true prevalence is not
642 K.E. Brown

known [254, 255]. Within high-risk groups, seropreva- rapid drop in Parv4 DNA titers, but with low levels (<104
lence varies widely with up to 95 % of HCV-HIV copies/mL) being detectable for many months.
coinfected intravenous drug users having detectable anti-
body in one study [271]. Parv4 DNA has also been
detected in a variety of different tissues from patients who 4.7 Patterns of Host Response
fall into these high-risk groups, confirming previous Parv4
infection [255, 272, 273]. Together these results suggest In the study of acutely infected hemophiliacs, the only repeat-
that the main route of acquisition of the virus in these edly observed clinical presentation was a rash in three patients
countries is through parenteral transmission. and unexplained hepatitis in two of them [257]. However, no
However, the epidemiology appears to be very differ- further information on the rash was provided, and no signifi-
ent in other parts of the world. In one study, Parv4 DNA cant increase in liver enzymes was reported. The original
was frequently detected in the sera of young children, index case had symptoms of an acute viral syndrome.
with approximately 12 % of 2-year-olds having detectable
Parv4 DNA [270]. Following this study, further serologi-
cal assays for Parv4 IgG have been performed, and sero- 4.8 Diagnosis
positive rates of 20–37 % have been found in adults in
sub-Saharan Africa [274]. Similar high levels of Parv4 Diagnosis is generally by the detection of Parv4 viral DNA
antibody have been described in elderly patients from the by PCR. Patients with acute infection appear to have tran-
Cameroon [275]. Parv4 DNA has also been detected in sient high levels of Parv4 DNA in serum. However, the dura-
nasal secretions and fecal samples from young children in tion of the high-level viremia before the development of an
Ghana [276]. IgM and IgG response is not known.

4.5 Mechanism and Routes of Transmission 4.9 Unresolved Problems

In Europe and North America, the contrast between high There are still many unsolved questions about Parv4, includ-
seroprevalence and detection of Parv4 DNA in tissues of ing its route of transmission and the main clinical presenta-
individuals at high risk of blood-borne infection com- tion of infection. Although this virus appears to be transmitted
pared to a virtual absence of seropositivity in the general by the parenteral route, there are very few data on the sero-
population is striking. It suggests that the main route of positivity rate among blood donors in most countries.
transmission in these countries is parenteral. However,
that seems less likely in other parts of the world, such as
in Africa. Although the high seroprevalence and detection References
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 Rhabdovirus: Rabies
28
Kira A. Christian and Charles E. Rupprecht

1 Introduction transsynaptic spread through several neurons to the central

nervous system (CNS), before infecting acinar cells of the sali-
Rabies is an acute, progressive encephalitis of mammals and vary glands and excretion by saliva into the oral cavity [7].
has the highest case fatality proportion of any conventional Onset of disease is initially nonspecific, consisting in humans
infectious disease. This disease is an ancient, reemerging of signs and symptoms compatible with a “flu-like illness,”
global zoonosis, caused by highly neurotropic viruses in the such as fever, headache, and general malaise. Following the
family Rhabdoviridae, genus Lyssavirus [1]. prodrome, an acute neurologic phase may include intermittent
Illness develops following a productive infection with bul- insomnia, anxiety, confusion, paresis, percussion myoedema,
let-shaped, enveloped virions that contain single-stranded, excitation, agitation, hallucinations, cranial nerve deficits,
non-segmented, negative-sense RNA [2]. The usual route of chorea, dysphagia, hypersalivation, piloerection, priapism,
virus transmission is via the bite of a rabid animal. Non-bite paralysis, and sometimes maniacal behavior [8]. Clinical pre-
exposures, such as direct mucosal contact, inhalation of virus, sentation may also include paresthesia at the site of the bite
inoculation with improperly inactivated vaccines, or transplan- exposure, and classically hydrophobia or aerophobia, mani-
tation of infected corneas, tissues, and organs, have occurred festing as phobic pharyngeal spasms following provocative
[3]. Historically, in the United States, the primary source of stimuli [8]. The clinical course is progressive, with death ensu-
human exposure to rabies virus was the domestic dog, which ing usually within days. One form of the disease, termed para-
still predominates as the major reservoir in developing coun- lytic, or “dumb rabies,” may also present as part of the clinical
tries. In developed countries, multiple wildlife species are spectrum, with the general sparing of consciousness, together
affected. For example, current rabies reservoirs in the United with ascending paralysis, progressive unresponsiveness, coma,
States include raccoons, skunks, coyotes, foxes, and bats [4]. and death. Once clinical signs are present, there is no proven
The incubation period usually ranges from 1 to 3 months after cure. Intensive medical support may prolong life, but typically
exposure, but can range from days to years [5]. Once virus is death ensues due to cardiac or respiratory failure. Exceptions
deposited in peripheral wounds from a bite, centripetal pas- to this general scheme are exceedingly rare, with few well-
sage occurs toward the central nervous system [6]. The virions documented cases of human survival from clinical rabies, usu-
may replicate locally or enter unmyelinated nerve terminals ally (but not always) with a history of either pre- or postexposure
and migrate by retrograde axonal transport to the neuronal cell prophylaxis (PEP). Survivors may have significant residual
body. After replication in the cell body of a primary neuron, neurologic impairment, but this is not found in all patients.
infection proceeds via retrograde axonal transport and Serological surveys and documented occurrences under labo-
ratory conditions in the animal reservoir species have sup-
K.A. Christian, DVM, MPH (*) ported the existence of acquired immunity, which presumably
Division of Global Disease Detection and Emergency Response, follows subclinical exposure, abortive infection, or survival of
Center for Global Health, Centers for Disease Control and Prevention, overt clinical rabies [9, 10].
Atlanta, GA 30333, USA
e-mail: [email protected]
C.E. Rupprecht, VMD, MS, PhD
Ross University School of Veterinary Medicine, St. Kitts, West Indes
2 Historical Background
LYSSA, LLC, Lawrenceville, GA 30044, USA
Rabies is an ancient disease of uncertain origin and one of
Global Alliance for Rabies Control, Manhattan, KS 66502, USA the oldest recognized infectious diseases [11]. As such, it is
e-mail: [email protected] difficult to provide more than highlights, given the fascination

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 651

DOI 10.1007/978-1-4899-7448-8_28, © Springer Science+Business Media New York 2014
652 K.A. Christian and C.E. Rupprecht

and dread it evoked in historians of the past. For a more thor- isolation of distinct viruses in Africa, serologically and
ough treatise, the interested reader is referred to Steele and genetically related to rabies virus [19], supported the hypoth-
Fernandez [12], Baer [13], and Wilkinson [14]. Most global esis of an African genesis [20], with adaptive radiation of
reference languages singularly denote the terms damage, several lyssaviruses throughout the Old World.
violence, fury, madness, or rage as literally synonymous Beyond mere geographic documentation, the history of
with this affliction. Many great civilizations refer to a disease rabies was also marked by a series of observations and spec-
akin to rabies, weaving a tapestry of overstated fact, legend, ulations regarding its cause, prevention, treatment, and diag-
and nightmare, exceedingly out of proportion to its actual nosis. For example, about 100 ad, Celsus treated animal bite
pathos. In the not-too-distant past, extreme ostracism, tor- wounds by cauterization. In 200 ad, Galen recommended
ture, or execution were inflicted on those even suspected of amputation of the bitten limb. Both had limited success (later
having hydrophobia. Democritus in 500 bc recognized rabies supported by laboratory animal research) [12]. During 1804,
in dogs and other domestic animals, as did many other Greek Zinke demonstrated transmission of rabies virus to a normal
and Roman scholars. Thus, it can be assumed that this viral dog by inoculation of infected saliva, considering it a toxin
disease, or a similar contagion, was well recognized and [12]. These observations helped lead to institution of muzzle
occurred throughout Asia, Europe, and, perhaps, Africa, dur- laws and stray dog control, which resulted in rabies elimina-
ing centuries of recorded history. tion from several countries such as Denmark, Norway, and
Early historical accounts focused primarily on individual Sweden by 1826, before the invention of rabies vaccination.
human or animal case reports. For example, wildlife rabies Animals have been central to progress in applied rabies
was recognized throughout the Middle East since biblical research since the late nineteenth century. For example, in
times [11]. However, few epizootics were well documented 1879, Galtier used the domestic rabbit as a suitable laboratory
until the Middle Ages. Dramatically, there were multiple host for rabies [12]. His observations enabled the later classic
accounts of rabid wolf attacks in Franconia during 1271 [12]. 1881 experiments in which Pasteur (with important primary
Perhaps such incidents were one of the origins of the “big contributions by collaborators such as Roux, Chamberland,
bad wolf” of the fairy tale. Throughout Europe, outbreaks and Thuillier) reported the characterization of a “microbe of
were recorded in wolves, foxes, and dogs, especially during infinite smallness” attenuated for the dog but having a uniform
the eighteenth and nineteenth centuries. These episodes con- (fixed) incubation period in the rabbit [21]. Pasteur distin-
tinued sporadically until World War II, when another major guished this adapted laboratory form of disease from that of
epizootic in foxes swept Europe, from east to west, at a rate the agent in nature, or “street virus.” He used this laboratory-
of approximately 30–50 km/year, the epidemiological reper- adapted “fixed” virus (based upon derived characteristics, not
cussions of which are still felt. to be confused with inactivation), grown in rabbit spinal cords,
Quite likely, lyssaviruses evolved in the Old World. In and dried for varying periods, to give graded doses of nonin-
most of the New World, the disease was largely unknown, at fectious to fully infectious virus for the immunization of ani-
least among dogs, until the early eighteenth century; one of mals. The first successful human rabies vaccination was
the first descriptions dates to 1703, from Mexico. Rabies was administered in 1885 to a boy aged nine years, Joseph Meister,
subsequently identified in the Caribbean by the mid- and late severely bitten by a presumably rabid dog, based on the ani-
1700s. By 1753, dogs in Virginia had been affected, and mal’s clinical derangement and pica [21]. This single historic
most of the Mid-Atlantic American and New England colo- event ushered in the era of rabies vaccinology, which was rea-
nies shortly afterward. Pioneer expansion in the early 1800s sonably successful by accepted standards of the day. Occasional
was replete with tales of “madstones” and “phobey cats.” In failures were attributed to either prolonged delays before vac-
South America, one of the first reports of rabies was in cine initiation or to the particular severity of an exposure.
Peruvian dogs during 1803. Besides typical incidents involv- Since Pasteur’s time, numerous improvements in safety
ing carnivores, Baer cites Spanish reports from the 1500s and efficacy of the early nerve tissue origin (NTO) biologics
onward that associated bat bites and human mortality in have been attempted. Such gradual attempts were partially
Latin America [13]. Thus, rabies viruses were likely imported frustrated, until the 1940s, by the lack of standardized vaccine
repeatedly into the New World, perhaps from the Old World potency evaluation [22]. For example, in 1908, Fermi devel-
circumpolar invasions predating recorded history, and more oped the first chemically treated rabies vaccine, unfortunately
extensively during European colonization in the sixteenth still with residual live virus. By 1919, Semple showed that
through eighteenth centuries, contemporaneous with major phenol might more fully inactivate rabies virus without
domestic animal outbreaks in Europe. Later documentation destroying its antigenicity. Semple’s vaccine was used exten-
of antigenic and genetic similarities between both Old and sively for at least 65 years but was replaced gradually in Latin
New World rabies viruses [15, 16] supports trans-Atlantic America and elsewhere by a suckling mouse NTO vaccine,
introduction by infected animals, in whom incubation peri- by the efforts of Fuenzalida and Palacios in 1955. By circum-
ods could exceed the length of the voyage [17, 18]. Continued venting the sensitization to myelin basic proteins found in
28 Rhabdovirus: Rabies 653

adult animal brains, the suckling mouse NTO vaccine had a potency of early inactivated vaccines plagued these first tri-
lower rate of neuroparalytic reactions. A number of studies in als. By 1948, a live virus vaccine using an attenuated Flury
the 1940s suggested that, besides vaccine, rabies immune strain of egg-adapted virus was successfully applied to dogs.
serum was also effective in preventing disease. The impor- A variety of both inactivated and live virus vaccines was
tance of combined therapy consisting of serum plus vaccine, shown subsequently to be effective in preventing rabies in
in combination with local wound treatment, had been demon- most of the major domestic animals. During the 1950s, ani-
strated most convincingly in those few cases where human mal management and mandatory dog vaccination programs
rabies occurred, despite intervention, when one of these fac- resulted in the gradual elimination of canine rabies in the
ets of an accepted protocol was altered or delayed [23]. Other United States and other developed nations [27]. This, cou-
major vaccine initiatives during the 1950s and 1960s moved pled with still-ongoing improvements in epidemiologic sur-
away from NTO vaccines, with the development of avian veillance, diagnostics, and preexposure immunization and
embryo vaccines, initially of dubious potency. Such alterna- PEP, has markedly decreased the number of human rabies
tive strategies eventually culminated with the first true cell cases in developed countries. Clearly, an extensive public
culture vaccine in the 1970s, the human diploid cell vaccine health infrastructure and proper allocation of resources are
(HDCV), with improved safety and efficacy [22, 24, 25]. required to minimize domestic animal rabies and maintain
Although the original Pasteurian use of an attenuated virus the vigilance necessary to recognize potential human rabies
vaccine in rabbit CNS tissue was discontinued by the 1950s, exposure for the prompt initiation of prophylaxis.
inactivated NTO vaccines, from small mammals or livestock,
were still used in parts of the developing world during the
twenty-first century, despite the availability of safer, potent, 3 Methodology Involved
albeit more costly alternatives. The further development of in Epidemiologic Analysis
purified avian embryo vaccines and Vero cell vaccines today
should compete economically and displace historical NTO 3.1 Sources of Mortality Data
vaccines altogether over the next few years.
Before the development of routine laboratory tests for There is no single definitive depository for human rabies data
rabies diagnosis, the clinical presentation of the biting animal at a global level. Data, if collected, may be available in
provided a primary motivation for human PEP. At the begin- national summaries, regional reports, or the peer-reviewed
ning of the twentieth century, scientists such as Babes and literature. Human rabies is a notifiable disease in the United
Van Gehuchten described lesions in the CNS, presumptively States, as in most other developed countries. Statistics for
related to rabies [12], but which were actually nonspecific, humans and animals are compiled from local and state health
and applicable for a set of other etiologies as well. However, departments by the Centers for Disease Control and
in 1903, Negri described intracytoplasmic acidophilic inclu- Prevention (CDC) and published in the Morbidity and
sion bodies (which he believed to be protozoa) in neurons of Mortality Weekly Report, as well as in an annual summary. In
rabid humans and other mammals, facilitating the early diag- Europe, numbers and distribution of human rabies cases and
nosis of rabies when brain tissue was collected, fixed, and cases in animals are published in the Rabies Bulletin of
stained appropriately. Histopathologic identification was a Europe. The World Health Organization (WHO) in Geneva
key procedure despite the relatively high rate of false-nega- and the associated Pan American Health Organization
tive observations until Goldwasser and Kissling introduced (PAHO) in Washington, DC, together with the World
the immunofluorescence diagnostic technique in 1958 [26]. Organization for Animal Health (OIE), collect data from
This technique is still in routine use today, as the global gold countries or political units on deaths attributable to rabies.
standard for rapid, sensitive, and specific rabies diagnosis. These data are distributed from national governments as a
These historical accomplishments reveal that rabies source of information on global rabies occurrence and the
research efforts focused primarily on the human as victim, general relative risk of infection, which, provided that proper
rather than on primary prevention in the animal reservoir. epidemiologic surveillance is ongoing, should be nil in coun-
While animals did serve as critical experimental subjects, tries with no reported cases during any one particular year
focused human vaccination preceded the serious consider- (Table 28.1). Notably, the amount of human PEP adminis-
ation of dog immunization, which was originally deemed tered in some countries, such as the United States, may be
impractical. It was not until 1919 that an attempt at mass vac- lacking in these global summaries, because reporting may
cination of dogs occurred with the use of Fermi vaccine in not be required by most localities or on a national level.
Japan, with the focus shifting to the source of exposure, com- Obviously, the temporal and spatial occurrence of rabies in
pared with the preceding three decades, during which many animals will affect exposure patterns in people. For example,
thousands of persons were treated with Pasteurian postexpo- the emergence of a raccoon rabies epizootic in the mid-
sure prophylaxis (PEP). However, problems associated with Atlantic and northeastern United States during the 1970s
654 K.A. Christian and C.E. Rupprecht

Table 28.1 Global disease surveillance: selected countries/political units reporting no cases of rabies during 2012
Region Places
Africa Cape Verde, Libya, Mauritius, Réunion, São Tomé and Principe, Seychelles
Americas
North Bermuda, St. Pierre and Miquelon
Caribbean Antigua and Barbuda, Aruba, The Bahamas, Barbados, Cayman Islands, Dominica, Guadeloupe, Jamaica,
Martinique, Montserrat, Netherlands Antilles, St. Kitts (St. Christopher) and Nevis, St. Lucia, St. Martin, St.
Vincent and Grenadines, Turks and Caicos Islands, Virgin Islands (the United Kingdom and United States)a
Asia and Middle East Hong Kong, Japan, Kuwait, Lebanon, Malaysia (Sabaha), Maldivesa, Qatar, Singapore, Taiwanb, United Arab
Emirates
Europe Austria, Belgium, Cyprus, Czech Republica, Denmarka, Finland, Gibraltar, Greece, Iceland, Ireland, Isle of Man,
Luxembourg, Netherlandsa, Norway, Portugal, Spaina (except Ceuta/Melilla), Sweden, Switzerland, United
Kingdoma
Oceaniac Australiaa, Cook Islands, Fiji, French Polynesia, Guam, Hawaii, Kiribati, New Caledonia, Northern Mariana
Islands, Palau, Papua New Guinea, Samoa, Vanatu
Sources: CDC [154], as well as PAHO/WHO reports
Bat rabies may exist in some areas that are reportedly free of rabies in other mammals
a
Bat lyssaviruses are known to exist in areas that are reportedly free of rabies in carnivores
b
Taiwan remained rabies-free during 2012, however, in July 2013 reported its first case of rabies in over 50 years to OIE with the identification of
a rabid ferret-badger, Melogale moschata (http://www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapFullEventReport
&reportid=13775, cited 24 July 2013)
c
Most of Pacific Oceania is reportedly “rabies-free”

significantly affected the epidemiology of human PEP, on the intensity and occurrence of animals with suspicious
diverting critical health resources as animal rabies cases clinical presentations and encountering humans or domestic
increased. Moreover, if predicted effects of climate change animals, would tend to be more reliable in detecting cases
models occur, an expansion of vampire bat distribution in the during an outbreak but less so during expected enzootic peri-
Americas could subsequently result in an increase in the ods. This bias may be greatest in localities in which the
number of human exposures requiring PEP administration responsibility for suspect animals, such as predominantly
[28]. To properly assess global and national needs and imple- sick or nuisance wildlife rather than domestic dogs, does not
ment appropriate use of PEP, efforts toward proposing a rest with any one particular agency. Historically, as vestiges
nationally notifiable reporting system for PEP should be from the time of enzootic canine rabies, an animal control
encouraged. officer may respond to domestic animal problems in a com-
When rabies surveillance is “adequate,” human death munity. However, without a mandated and funded local
rates may begin to approximate incidence rates of human response to suspect wildlife, a “shoot-and-bury” practice
infection. Generally, for statistical purposes the disease can may evolve to cope with rabies among wildlife species, with
be regarded as 100 % fatal, because the acute clinical course the result that fewer cases are recorded. Disease-specific
incidence rates are also virtually equivalent to prevalence incidence (or death) rates for animals are generally unavail-
rates. However, in most developing countries, human rabies able, since free-ranging wildlife populations are not well
is grossly underreported. When it is reported, human cases enumerated, and one cannot approximate the true popula-
may outnumber documented cases in animal species. Such tions at risk. Thus, in the United States, representation of the
limitations are not limited to developing countries, as human public health magnitude or burden of the disease, solely by
rabies is underreported even in the United States, consider- absolute case numbers of animal rabies reported, is inher-
ing that human cases have been diagnosed retrospectively ently biased and should be qualified in view of its obvious
from autopsy material. historical, scientific, and logistical limitations.
Where canine rabies occurs, human exposure and rabies Predictably, predominant animal reservoirs change over
incidence rates appear related to the local epizootiology of time and in geographical distribution. For example, before
the disease. However, the relationships between human World War II, dogs were the major rabid animal in the United
rabies mortality, virus exposure, and wildlife rabies cases are States. After dog rabies management began, the advantages of
less well established. For example, since most animal reser- surveillance in other species became more apparent, first dur-
voirs in the United States are free-ranging wildlife species, ing the 1960s with foxes and skunks, until the latter 1970s.
the number of animal rabies cases is variable and biased by Thereafter, since the incursion of raccoon rabies into the east-
surveillance efforts first developed for canine rabies detec- ern United States, the annual totals of rabid wild animals have
tion, prevention, and control. Surveillance, typically based surpassed historical records, demonstrating the magnitude of
28 Rhabdovirus: Rabies 655

the wildlife rabies problem in such particular geographic in relation to a known standard, as international units (IU)
regions [29]. Thereafter, the total areas affected, and the pro- per milliliter of sera. The RFFIT requires trained personnel
portion diagnosed rabid among raccoons tested, appear stable. and appropriate laboratory equipment for cell culture and
Based upon such observations, when animal outbreaks occur, manipulating live rabies virus, in addition to being somewhat
administrations of human PEP will increase dramatically. complex and time-consuming [32]. Alternatively, commer-
cial enzyme-linked immunosorbent assays (ELISA) have
been applied for simple, rapid screening of large numbers of
3.2 Sources of Morbidity Data sera. However, specificity may be less reliable, especially at
low antibody levels, and utility is determined by the source
Human rabies case morbidity is largely reflected in mortality and quality of crude antigen. The fundamental difference
data. In contrast, only a few documented cases of humans between these tests is that the ELISA is based on primary
surviving clinical rabies exist, some with serious neurologi- recognition of antigens, whereas the RFFIT and other neu-
cal sequelae. Morbidity that has otherwise been averted could tralization assays (such as the fluorescent antibody virus
be assessed through numbers of human PEP administered. neutralization test) are functional tests.
However, because reporting PEP in countries such as the The absolute interpretation of such rabies serological sur-
United States may not be required, information on potential vey data is not straightforward. Titers do not directly corre-
morbidity prevented by PEP is not available readily. Such late with protection against disease, because other factors,
occurrence may be approximated, as in developing countries, more difficult to measure and interpret, play a role in preven-
by the number of vaccine doses and human rabies immune tion of disease [4]. Further, minimal differences in the
globulin (HRIG) sold or used. Based on these imperfect data reported values of rabies antibody results might occur
(because of the vaccine’s utilization in both pre- or PEP pro- between laboratories that provide antibody determination
tocols and administration of HRIG on a per-weight basis), using variations in tests such as the RFFIT [5]. Obviously,
current national estimates range between 10,645 and 35,845 there is no known absolute “protective antibody” level for all
(average = 23,415) human administrations of PEP per year in humans or other animals [31]. Minimum standards for
the United States [30]. In some localities, PEP data are report- humans are based empirically on presumed protective activ-
able and partially quantifiable, whereas in many other places, ity of rabies-specific antibodies, e.g., virus-neutralizing anti-
vaccine and HRIG are available only through the state or local body (VNA), for a given exposure scenario and on repeatable
government, or private providers, from which the number of values for paired sera, as could be readily detected by refer-
PEP administrations may be approximated. Representative ence laboratories.
reporting of human PEP would be advantageous for proper With regard to rabies in animal reservoir species, a subset
assessment of the epidemiology of rabies, for national and of animals may be exposed to virus, may develop measur-
regional PEP needs, and to assess risk factors for exposure, as able viral antibodies, and are then immune to subsequent
well as for further definition of the overall economic impact of challenges. Such data do not support the existence of a “car-
rabies. These data tend to be more rabies-specific, as opposed rier state” in animals. Thus, serological surveys of wildlife in
to all of the other requisite morbidity factors associated with areas with enzootic rabies may reveal a low prevalence of
the trauma of the bite occurring after a rabid animal exposure, antibodies [33]. These incidents may occur among animals
and the secondary effects that may result thereafter (such as that are infected and will ultimately die of rabies or more
death, or serious morbidity, associated with the severity of likely among individuals that have developed immunity to
the attack itself, rather than from viral induced mortality or rabies, presumably following natural sublethal exposures.
morbidity, per se). These naturally occurring antibodies are distinct from those
due to purposeful immunization of free-ranging animals via
limited trap–vaccinate–release programs, regional oral rabies
3.3 Serological Surveys vaccination programs, or occasional parenteral vaccination
of wildlife with domestic animal vaccines by well-intentioned
In general, serology may be used to assess natural exposure wildlife rehabilitators [34].
to viral antigens and evaluate an induced response to immu- Clearly, multiple ecological factors will affect rabies epi-
nization. Historically, rabies serological evaluation [31] had zootiology, as related to host, agent, and environment. Among
been performed by antigen-function assays, such as a mouse carnivores, the small Asian mongoose provides one clear
neutralization test, by antigen-binding assays, such as an example of outbreaks influenced by host biology and distri-
indirect immunofluorescence test, or by antibody-function bution [35]. Mongooses were introduced for rodent control
assays, such as the hemagglutination test. Currently, one of onto most of the larger Caribbean Islands, including Puerto
the most widely used serological tests is the rapid fluorescent Rico, Dominican Republic, Haiti, Cuba, and Grenada.
focus inhibition test (RFFIT). Results are typically reported Though mongooses are wild, their population density is
656 K.A. Christian and C.E. Rupprecht

usually directly proportional to human activities [35]. As for Negri bodies, as are the Purkinje cells of the cerebellum
such, mongooses proliferated and are now deemed undesir- and pyramidal cells of the cerebral cortex. Limitations from
able due in part to predation on native fauna and the risk from this technique include a concern that Negri bodies may be
rabies in this introduced reservoir species. For example, 56 % absent in up to one quarter to one third of rabies cases, while
of the 73 rabies cases diagnosed in Puerto Rico in animals artifact and inclusions produced by other agents could be
during 2012 were attributed to mongoose [29]. Although poi- confusing and differentiated only after considerable labora-
soning has been practiced, such control was undesirable and tory experience.
ineffective over time. Many mongooses (as many as 55 % at Classically, animal inoculation with suspect diagnostic
the end of an epizootic cycle) have evidence of rabies anti- material was once used widely to support rabies diagnosis,
bodies, possibly as a result of sublethal virus exposure, and particularly because of the potential dramatic ramifications
such animals may be immune for life [48]. Removal through of false-negative results in human rabies exposure situations.
poisoning or other lethal means appears detrimental, which Laboratory rodents were used routinely for viral isolation, as
may simply enhance reproductive turnover, dispersal, and suckling and weanling mice are highly susceptible to infec-
increase the number of naive, susceptible animals. In view of tion by intracerebral inoculation. Typically, the time from
such levels of naturally occurring disease and probable conse- inoculation to illness varied from 7 days to 4 weeks, but viral
quent herd immunity, the nearly closed populations of these antigens in brain may be confirmed as early as 4 days follow-
nonindigenous mammals should be ideal candidates for oral ing inoculation. Confirmation in the inoculated animals of
vaccination. This would represent one means of disease man- rabies, as the source of illness and mortality, should always
agement to limit human and domestic animal exposures, be performed, because laboratory species may die of comor-
especially in a circumscribed island environment [36]. bid conditions or as a result of infections by other agents in
the original clinical specimen.
Besides the use of mice, murine neuroblastoma cell cul-
3.4 Laboratory Methods tures are as sensitive as laboratory animals for isolation of
field strains from saliva and brain of rabid animals and are
Several etiologies which cause encephalitis may be confused the method of choice in laboratories where cell culture facili-
with rabies. Epidemiologic studies should be based on labo- ties are available [38]. The presence of antigens in infected
ratory confirmation of suspected disease or death from cells is demonstrated by the FA test.
rabies. As summarized in the above historical description, In brief, the direct FA test uses a fluorescein dye conju-
laboratory techniques have evolved gradually over the last gated to rabies immune serum, which in turn is reacted with
century. Although variations are practiced in many laborato- acetone-fixed impressions from CNS tissue of the presumed
ries, the standard techniques of the WHO Expert Consultation rabid subject, including the brainstem and cerebellum or hip-
on Rabies (2005), and the current edition of the WHO pocampus [26]. The antigen–antibody reaction is detected by
Laboratory Techniques in Rabies, are recommended and microscopic observation of fluorescence under light of the
widely used [37]. Included in these texts are methods for the appropriate wavelength. The direct FA test is very rapid and
collection, preparation, and shipping of specimens; tissue reliable when used by an experienced laboratorian. The
and organ removal; laboratory animal inoculation; immuno- direct FA method is the test of choice for fresh or frozen tis-
fluorescent antibody (FA) procedures for detection of virus sue, because of test sensitivity, specificity, and economy.
antigens; serological determinations; cell culture propaga- Immunohistochemical testing, or a modified FA technique,
tion of virus; electron microscopy; vaccine production; can be applied on formalin-fixed tissues; however, the
potency and safety determinations; and specific virus identi- method is more cumbersome and cannot distinguish between
fication by antigenic and genetic techniques. For regulations different lyssavirus species. Also, RT-PCR methodologies
governing specific international or national shipment of can be used for diagnostic support but may be unreliable due
potentially infectious specimens, such as rabies, the local or to RNA degradation in these fixed tissues [39]. Brain tissues
national public health laboratory should be consulted for from suspect rabid animals continue to be submitted in for-
current updates. malin, and research continues to develop an ideal molecular
Historically, brain tissue from the rabid subject was exam- methodology to confirm rabies and to determine variant
ined for Negri bodies, which are specific intracytoplasmic characterization [39]. Significantly, in the Old World, other
inclusion bodies observed under the light microscope. These lyssaviruses related to rabies virus will cross-react in the
inclusions contain viral nucleoproteins, which accumulate in direct FA test, depending on the quality of the particular
quantities large enough to be detected after staining by the diagnostic conjugate. Specific monoclonal antibodies
Seller’s, Giemsa, Mann, or other methods, and can be visual- (MAbs) can be used to distinguish rabies virus from these
ized microscopically. Within the hippocampus, Ammon’s related lyssaviruses, but the public health consequences of
horn is one particular portion of the CNS usually examined identification are the same, regardless of exposure to any one
28 Rhabdovirus: Rabies 657

of these etiologic agents, in that appropriate medical care monocytosis which is compatible with a viral encephalitis,
and PEP considerations are necessary. may also occur [43]. Repeated samples should be taken for
Rather than reliance upon a history of relevant viral expo- antibody and antigen detection, because negative results in
sure and compatible clinical signs, proper laboratory-based early initial samples do not rule out rabies. Further, because
diagnosis is needed for case confirmation. Besides the direct of the risk of false-negative results with a potential human
FA test from the 1950s, during the early 2000s, a rapid exposure, intravitam testing of the rabies-suspect animal is
immunohistochemical test (RIT) to detect lyssavirus anti- inappropriate.
gens was developed. Modifications of a former indirect test In addition to classical laboratory procedures for detec-
led to a direct test, the dRIT, which uses a cocktail of highly tion of viral antigens and antibodies, recent progress has
concentrated and purified biotinylated anti-nucleocapsid been made in the application of molecular techniques, par-
MAbs produced in vitro, in a direct colorimetric staining ticularly nucleic acid detection and amplification of cDNA
approach, and allows a diagnosis to be made in <1 h. by RT-PCR [44–46], with subsequent generation of viral
Currently, the dRIT is an experimental procedure designed nucleotide sequences, leading to a greater understanding of
for consideration as a potential confirmatory test of the direct lyssavirus epizootiology and phylogeny [19, 43, 47].
FA test. Similar to the direct FA test, the dRIT can be per- However, the extreme sensitivity of molecular methods, such
formed on brain touch impressions, and based upon the as the RT-PCR technique, also greatly increases the probabil-
reagents used, viral antigens may appear as magenta inclu- ity of a false-positive diagnosis from laboratory contamina-
sions against a blue neuronal background. The test recog- tion. Moreover, because of lyssavirus heterogeneity,
nizes all variants of rabies virus and all representative false-negative results can occur if primer selection is inade-
lyssaviruses [40]. One advantage to the dRIT, compared with quate to compensate for heterogeneity. To date, universal
the direct FA test, is that the dRIT can be conducted by light primers for all known lyssavirus variants have not been
microscopy, compared with the FA test, which must employ clearly defined or standardized. Considering these factors,
the use of a fluorescence microscope [41]. The dRIT may be related costs, and the considerable expertise required for
used to enhance field surveillance among suspect animals, proper analysis and interpretation, such molecular tech-
particularly in support of national, regional, state, or local niques are not recommended for routine primary rabies diag-
oral vaccination programs. However, until the concept is nosis alone when CNS tissue is available, as opposed to
validated as sensitive and specific as the direst FA test, the laboratory confirmation in concert with other techniques.
dRIT should not to be used for public health decisions, in The routine sequence characterization of lyssaviruses from
those situations in which human or animal exposure has formalin-fixed tissues is possible but may be difficult, partially
occurred or is suspected, and local public health authority or as a function of the fixation time and resultant short fragments
other officials should be contacted for immediate and appro- of nucleic acid [16]. Optimization of these methods, beyond
priate diagnostic testing [41]. mere qualitative diagnostic methods for viral antigen analysis
While animals are diagnosed postmortem, rabies in preserved in such tissue, may be feasible by the adaptation of
humans can be diagnosed while the patient is still alive. techniques that would employ MAbs for retrospective virus
Human antemortem diagnosis can be made by virus isolation identification in archival material [48]. Besides determination
from saliva, antigen detection in biopsy tissue, or demonstra- of optimal specific reagents, corroborative data would have to
tion of virus-specific antibody (without prior vaccination). be generated on the sensitivity and specificity of such tech-
Selected FA tests are performed on human brain or skin biop- nique in comparison to fresh tissue analysis. Because of
sies, the latter usually taken from highly innervated areas, increased utilization for other viral diseases, such as HIV, influ-
such as the nape of the neck, where piloerector nerve plexi are enza, polio, etc., additional development of appropriate mod-
prominent [42]. Also, in some human rabies cases, corneal ern molecular methods is anticipated to improve the application
cells obtained from touch impressions may be antigen- to basic rabies diagnosis in developing countries.
positive during life, care being taken not to abrade the deli-
cate corneal tissue (a rationale to caution against this method
vs. other stated procedures). Antemortem diagnosis may also 4 Biological Characteristics
be made by showing a fourfold or greater rise in serum anti- of the Agents That Affect
body titer during the illness in the absence of vaccination or Epidemiologic Patterns
administration of rabies immune serum. Demonstration of
virus antibody in the cerebrospinal fluid (CSF) is also a reli- Rabies virus is the type species of the genus Lyssavirus;
able indicator of infection, even after vaccination. Vaccination additional and proposed members of the genus Lyssavirus
alone does not elicit CSF antibody. During viral infection, include those listed in Table 28.2.
other pathophysiologic changes in the CSF, such as an Rabies is essentially a “dead-end” disease in humans and
increase in specific proteins, and pleocytosis, particularly a several other species. While possible, human-to-human
658 K.A. Christian and C.E. Rupprecht

Table 28.2 Viruses currently included in the genus Lyssavirus

Recognized and proposed species Predominant natural hosts Geographical range
Rabies virus (type species) Chiroptera, Carnivora Worldwide
Australian bat lyssavirus Pteropodid and insectivorous bats Australia
European bat lyssavirus, type 1 Insectivorous bats Europe
European bat lyssavirus, type 2 Insectivorous bats Northwestern Europe
Khujand virus Insectivorous bat, Myotis mystacinus Central Asia
Aravan virus Insectivorous bat, Myotis blythii Central Asia
Bokeloh bat lyssavirus Insectivorous bat, Myotis nattereri Germany
Irkut virus Insectivorous bat, Murina leucogaster Eastern Asia
Duvenhage virus Insectivorous bats Sub-Saharan Africa
Lagos bat virus Pteropodid bats Sub-Saharan Africa
Mokola virus Unknown (shrews, rodents?) Sub-Saharan Africa
Shimoni bat virus Insectivorous bat, H. commersoni Kenya
West Caucasian bat virus Insectivorous bat, Miniopterus sp. Southeastern Europe
Ikoma lyssavirus Unknown (isolated from civet, C. civetta) Tanzania
Recognized viral species are listed in italics, as adapted from Kuzmin and Tordo [155]

transmission via bite has not been reliably established [49]. area, regardless of affected host, be it the reservoir species
However, likely cases of human-to human transmission con- (e.g., raccoon) or mere “spillover” into an essentially dead
tinue to be reported [50]. A marked exception to the apparent end, but susceptible host (e.g., domestic cat). In short, all
rarity of direct human contagion from exposure to patient animal reservoirs are also vectors, but not all potential vec-
secretions, such as saliva, consists of at least eight human tors are reservoirs for rabies.
rabies cases resulting from the surgical implantation of Not all mammals are equally involved in rabies mainte-
infected corneas from donors that had succumbed to undiag- nance from an epizootiological perspective, but for poorly
nosed rabies [51, 52]. In addition, other cases have occurred understood reasons. Among the Carnivora, meso-carnivores,
in Europe and the United States from other organ and tissue ranging in size from the mongoose to the jackal or coyote,
transplants. For example, in 2004, four recipients of kidneys, are most significant. As one specific example of virus vagil-
a liver, and an arterial segment from a common organ donor ity, coyotes possess many host qualities ideal for the initia-
died an average of 13 days after the onset of neurologic tion of a rabies epizootic. Historically, during 1915–1917, an
symptoms following the development of encephalitis, and extensive outbreak of coyote rabies extended over large por-
antibodies against rabies virus were later found to be present tions of southeastern Oregon, northeastern California, west-
in three of the four recipients and the donor [53]. ern Utah, and Nevada [58]. Thereafter, rabies among coyotes
Biological characteristics of lyssaviruses have a measur- was reported only sporadically in the western United States,
able, although incompletely understood, effect on epizootio- despite the coyotes’ widespread distribution and abundance.
logic patterns among their animal host species [54]. Initially, Another major focus was detected in Texas during 1988 [59],
through classic serology, and later by MAb and genetic and the virus spread throughout southern Texas. Antigenic
analysis, translational research was possible to differentiate and genetic analysis of isolates obtained from this outbreak
serotypes, variants, and genotypes of rabies virus and related suggested the involvement of a rabies virus variant associated
lyssaviruses [2, 15, 16]. All mammals are believed suscep- with canine rabies along the United States–Mexico border
tible to lyssaviruses; however, prominent reservoirs among but apparently capable of sustained coyote-to-coyote perpet-
bats and carnivores perpetuate largely independent cycles of uation. Intensive oral vaccination progress initiated during
infection. Epizootiologically, such variant viruses are main- 1995 was able to eliminate this virus in the United States
tained by different host species. Complex virus–host inter- [60], which contributed to the United States being declared
actions lead to the emergence of viral characteristics that free of canine rabies virus transmission in 2007 [61].
are beneficial to self-perpetuation among these particular Another unique characteristic of virus infection is a pro-
species that manifest as regional epizootiological “compart- longed incubation period during which the infection is virtu-
ments” [55–57]. Thus, in countries with adequate labora- ally undetectable. In humans, although the incubation period
tory-based surveillance, such as the United States, there are is generally several weeks to months, unusually long incuba-
discrete geographic zones of raccoon rabies, skunk rabies, tion periods of 6 or more years have been described [17]. This
and so forth. Such viruses are fully transmissible to other potential for long incubation periods influences directly the
mammals within a region but genetically and antigenically management of exposed domestic animals. An exposed,
distinguishable as a single variant in a particular geographic unvaccinated domestic animal may be euthanized or held in
28 Rhabdovirus: Rabies 659

quarantine for 6 months [4], which extends beyond the major- western United States [29]); skunks (primarily in the central
ity of documented incubation periods for dogs and cats. United States and Canada [29]); procyonids, such as the rac-
Mechanisms for successful in vivo productive infections coon (in the southeastern, mid-Atlantic, and northeastern
are not fully appreciated. Neurotropism appears to be a criti- United States [29]); and mongoose species (in Asia, Africa,
cal facet of a successful strategy to partially evade immune and several Caribbean islands [35]). Other conspicuous carni-
detection [62, 63] with the ultimate end to ensure future viral vores, such as bears, wolves, and all of the felids, can serve as
progeny. Under the constraints of mammalian anatomy, there effective short-term viral transmitters intra- and interspecifi-
are fairly limited routes available to a virus for transit from cally, but insufficient documentation is available to suggest
an initial portal of entry (i.e., a bite in a peripheral muscle) to that these groups can continue to propagate virus or serve as
a primary portal of exit (e.g., saliva). One of the most com- reservoir hosts for unique viral variants, as opposed to pri-
mon mechanisms for other viruses to transit to such distant mary infection by the primary host. “Bat” rabies from infec-
sites is by viremia or spread via lymphatics. The passage tion by rabies viruses per se is a New World phenomenon
along both of these latter pathways may also initiate a vigor- only, described primarily among the insectivorous species of
ous immune response. An effective blood–brain barrier North America and the hematophagous vampires and frugiv-
would limit the utility of viremia in enhancing the spread and orous species from Latin America [69, 70]. Ultimate evolu-
perpetuation of a neurotropic virus. In contrast, all known tionary and ecological perpetuation of lyssaviruses appear to
lyssaviruses utilize neurotropism. Direct evasion, and sup- be driven by bats [71]. Distinct from the type species rabies
pression of host immune surveillance, appears to be a critical virus, related lyssaviruses have been diagnosed in Australian,
evolutionary strategy [64]. Long incubation periods may be African, and European bat species [72–75]. While the disease
one consequence of reliance on flow within neural circuitry is naturally maintained by relatively few taxa, rabies may
[65]. Adaptation to the CNS may also be a mechanism to affect any mammal, such as ungulates, and result in a largely
lower the risk of extinction in a host that may pursue a more “dead-end” infection. Contrary to popular belief, small mam-
solitary existence for short periods of its life history. Since mals, such as rodents (mice, rats, etc.) and lagomorphs (rab-
the reservoir host must eventually seek a breeding partner, an bits, hares, etc.), are not important in rabies, and cases are
opportunity for transmission is readily available, considering uniformly rare [76]. Further, rodents have not arisen as “miss-
the common use of teeth and oral mucosal contact prepara- ing link” reservoirs in any region in which domestic dog or
tory to typical mammalian copulation, especially among car- wildlife rabies has been controlled.
nivores and bats. Following bite inoculation, covert access to
the CNS via neuron-to-neuron passage would be one unique
mechanism for minimizing immune detection while reach- 5.1 Incidence
ing distant sites important for replication and transmission
[62, 66]. Lyssaviruses are rather fragile, maintained by direct To properly understand the occurrence of human rabies, the
transmission, and do not survive readily in the external envi- context of animal rabies is fundamental. Rather than a global
ronment, where inactivation is operative. Dependence on rabies pandemic, single or multispecies animal host groupings
fluid-sucking arthropod vectors is unnecessary, as are addi- are apparent when appropriate disease surveillance is practiced
tional structural proteins to ensure extra-mammalian envi- systematically. Combined with historical temporal and geo-
ronmental survival, because in vivo transmission is largely a graphical data, both antigenic characterization and nucleotide
direct host-to-host event. For example, virus may perpetuate sequence analysis can be used to “compartmentalize” viral iso-
fox-to-fox, bat-to-bat, and so on, irrespective of firm con- lates with different animal reservoirs responsible for their per-
straints imposed by either geography or season. petuation and to estimate relative risks of human disease from
differential animal exposures (Table 28.3). For example, during
2012, only one human fatality occurred; however, 6,162 animal
5 Descriptive Epidemiology rabies cases were reported in the United States (including the
District of Columbia and Puerto Rico) [29] (Fig. 28.1). In
Rabies is a zoonosis with diverse natural mammalian reser- contrast to fulminant canine rabies pre-World War II, >90 % of
voirs [54]. Predominant hosts are bats and mammalian carni- current animal rabies cases in the United States are from wild-
vores. These mammals include canids, such as the domestic life (Fig. 28.2, top). Most of this increase resulted from contin-
dog (as the principal global reservoir, particularly in equato- ued spread of a predominant raccoon rabies virus variant,
rial regions of Asia, Africa, and Latin America [27]), foxes following unrestricted progression of an outbreak initiated by
(in the circumpolar Arctic, Central and Eastern Europe, the animal translocation during the late 1970s to the Virginias from
Middle East, and scattered foci throughout the western United a nidus in the southeastern United States [77, 78]. Cases rise as
States [29, 67]), raccoon dogs (in eastern Europe [68]), jack- infected individuals within raccoon populations encounter
als (in Africa and Asia), and coyotes (historically in the naive conspecifics with dramatic results.
660 K.A. Christian and C.E. Rupprecht

Table 28.3 Rabies immunization: preexposure and postexposure

Criteria for preexposure immunization
Exposure category Nature of risk Typical populations Preexposure regimen
Continuous Virus present continuously, often Rabies research lab workers.a Rabies Primary course. Serologic testing
in high concentrations. Specific biologics production workers every 6 months. Booster
exposures may go unrecognized. immunization if antibody titer
Bite, nonbite, or aerosol exposures falls below acceptable levelab
Frequent Exposure usually episodic with Rabies diagnostic lab workers,a cavers, Primary course. Serologic testing
source recognized but exposure veterinarians and staff, and animal every 2 years. Booster
also might be unrecognized. Bite, control and wildlife workers in areas vaccination if antibody titer is
nonbite, or aerosol exposures where rabies is enzootic. All persons below acceptable level
who frequently handle bats
Infrequent (greater than Exposure nearly always episodic Veterinarians and animal control staff Primary course. No serologic
population at large) with source recognized. Bite or working with terrestrial animals in testing or booster vaccination
nonbite exposures areas where rabies is uncommon to
rare. Veterinary students. Travelers
visiting areas where rabies is enzootic
and immediate access to appropriate
medical care including biologics is
limited
Rare (population at large) Exposure always episodic. Bite US population at large, including No vaccination necessary
or nonbite exposure individuals in rabies-epizootic areas
Adapted from CDC [5]
Preexposure immunization. Preexposure immunization consists of three doses of HDCV or PCEC vaccine, 1.0 ml, IM (i.e., deltoid area), one each
on days 0, 7, and 21 or 28. Administration of routine booster doses of vaccine depends on exposure risk category as noted
Postexposure immunization. All PEP should begin with immediate thorough cleansing of all wounds with soap and water. Persons not previously
immunized: HRIG, 20 IU/kg body weight, as much as possible infiltrated at the bite site (if feasible), with the remainder administered IM; 4 doses
of HDCV or PCEC, 1.0 ml IM (i.e., deltoid area), one each on days 0, 3, 7, and 14. Persons previously immunized: Two doses of HDCV or PCEC,
1.0 ml, IM (i.e., deltoid area), one each on days 0 and 3. HRIG should not be administered. Preexposure immunization with HDCV or PCEC; prior
PEP with HDCV or PCEC; or persons previously immunized with any other type of rabies vaccine and a documented history of an acceptable
rabies virus-neutralizing antibody response to the prior vaccination
a
Assessment of relative risk and extra monitoring of immunization status of laboratory workers are the responsibilities of the laboratory supervisor
(see United States Department of Health and Human Services’ Biosafety in Microbiological and Biomedical Laboratories [137])
b
Routine Preexposure booster immunization consists of one dose of HDCV or PCEC, 1.0 ml/dose, IM (deltoid area), or HDCV, 0.1 ml ID (deltoid).
The acceptable antibody level is a 1:5 titer (complete inhibition in the RFFIT at a 1:5 dilution, approximately equivalent to 0.1 IU/ml). Boost if
titer falls below this level, as long as the person remains at risk of viral exposure

30

25
n=195

20
# of Deaths

15

10

0
1952
1954
1956
1958

1960
1962
1964
1966
1968
1970
1972
1974
1976
1978
1980
1982
1984
1986

1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012

Year

Fig. 28.1 Reported human rabies cases in the United States, 1952–2011 (Sources: Petersen and Rupprecht [81] and Dyer et al. [28])
28 Rhabdovirus: Rabies 661

Reported rabies cases in wild animals in the also lower likelihood of routine vaccination. Difficulties in
United States–2012 clinical recognition also increase the public health signifi-
cance of rabies in cats, as early nonspecific signs may easily
RACCOONS
be compatible with unrelated differential diagnoses, and
SKUNKS may be compounded by an abscess compatible with a bite
BATS wound in an outdoor cat [80]. By temporal comparison, 84
cases of rabies in dogs were reported in the United States
FOXES
during 2012, compared with 303 cases in cats reported dur-
RODENTS AND ing 2011 [79].
LAGOMORPHS
In contrast to areas enzootic for dog rabies with a conse-
OTHER
quent high case load (e.g., estimated at >20,000 annual
human fatalities in India alone), human rabies is rare in
n=5,643 developed countries. For example, between 1980 and 2011,
only 71 human deaths were diagnosed in the United States,
20 of which were imported. Of the 45 cases for which viral
variant data were available among those suspected of infec-
Reported rabies cases in domestic animals in the tion while in the United States during 1980–2011, 40 were
United States–2012 attributable to bat rabies virus variants. By contrast during
3 the same time period, of the 20 imported cases for which
84 variant data were available, 18 (90 %) were attributable to
dog virus variants [81].
CATS
LIVESTOCK
DOGS 5.2 Epidemic Behavior
OTHER
(2 BISON,1 LLAMA) Application of MAb and molecular technology to the study
257
of the epizootiology of the Lyssavirus genus (previously felt
to consist of a relatively homogeneous group of viruses) first
175 provided substantive evidence for considerable variation
n=519 within the group, based on the nucleoprotein (N) and glyco-
protein (G) protein reactivities among both fixed and street
Fig. 28.2 (Top) Reported rabies cases in wild animals in the United rabies viruses and between rabies viruses and related lyssavi-
States, 2012. (Bottom) Reported rabies cases in domestic animals in the
United States, 2012 (Source: Dyer et al. [28]) ruses [15, 82]. The use of MAbs was particularly useful in
determination of the extent of natural antigenic variation
among lyssaviruses as well as epizootics—such as the
During 2012, rabies was also reported in other important raccoon epizootics in the eastern United States described
wildlife, primarily skunks (1,539 cases) and foxes (340 below—either isolated from a variety of wildlife reservoirs
cases), in the Midwest and Alaska/northeastern United within a fairly restricted geographical area or between conti-
States, respectively, and rabies in bats (1,680 cases) was nents. In particular, through the use of genetic sequence
widely distributed [29]. In the eastern United States, rabies comparisons, distinctions became much more obvious
was reported in 1,953 raccoons during 2012 [29]. Historically, between those viruses isolated from bats and carnivores [82].
Hawaii is unique in the United States as never having
reported a case of indigenously acquired rabies, attributable
in part to its remote insular geographical isolation in Oceania 5.3 Geographic Distribution
and 120-day quarantine policy.
Domestic animals most at risk include those with a lower As illustrated in Table 28.2, rabies is distributed on all conti-
likelihood of parenteral vaccination but higher potential for nents, with the exception of Antarctica, but varies greatly
virus exposure (especially if poorly supervised and free- dependent upon the dynamics of certain viruses among par-
ranging), such as cats. During 2012, 257 cases of rabies ticular mammalian hosts and the degree of operational sur-
were reported in cats [29], compared with 303 cases during veillance, prevention, and control methods used in each
2011 (Fig. 28.2, bottom) [79]. Cats are significant in part geographical unit. Currently in the United States, the most
because they have begun to outnumber their canine counter- prominent wildlife rabies hosts include raccoons, skunks,
parts, owing to the increased popularity of cats as pets, but foxes, and bats [29].
662 K.A. Christian and C.E. Rupprecht

Through MAb and molecular laboratory techniques incubation period of approximately 1–3 months [84]. Clearly,
applied to public health and agricultural surveillance, fox the overt limitations of a passive surveillance system for
rabies outbreaks in Canada and Alaska appear related, as do domestic animal rabies may obscure temporal trends in wild-
the raccoon epizootics in the eastern United States [57, 78]. life reservoir species and consequent risks for humans, as
Skunk isolates group as distinct variants defining separate evidenced by the encroachment westward of the raccoon epi-
outbreaks in the north central/south central states and zootic from the eastern seaboard, described above.
California. Such investigations, when extended over large
geographic areas, lead to a better understanding of lyssavirus
dynamics and improved animal rabies management pro- 5.5 Age, Sex, Race, and Occupation:
grams, to distinguish recent viral introductions, potential Socioeconomic, Nutritional, and
vaccine failures, and so on. Genetic Factors
Antigenic or molecular characterization can also be
employed to investigate unusual or unexpected lyssavirus There do not appear to be reliable trends associated with
mortality, especially in domestic animals or humans that lack many risk patterns leading to viral exposures, with few
an obvious exposure history. The antigenic patterns or nucle- exceptions. Racial, genetic, nutritional, or socioeconomic
otide sequences obtained can be compared with variants differences in risk of exposure have not been fully investi-
from known animal reservoirs. For example, analysis of gated, and there does not appear to be a trend in occurrence
recent human rabies cases from the United States implicates or susceptibility with regard to these factors, as well. If an
variants of virus associated with insectivorous bats (e.g., active surveillance system for the reporting of PEP were
Eptesicus, Tadarida, Myotis, Lasionycteris, or Perimyotis established, these epidemiologic characteristics would be
spp.), indirectly or directly, in the etiology of infection. analyzed to further delineate sources of exposure and target
Unfortunately, bat rabies cases are not routinely identified to preventive measures. For a variety of reasons, presumably
species. Thus, case surveillance data alone do not provide related to exposure, there is a bias in gender (toward males)
adequate clues about unique variants affecting different bat and in age (toward children) among human rabies cases. For
species or species groups. More rigorous reporting standards example, in many developing countries children are the pri-
coupled with modern virological analysis will be necessary mary caretakers of animals and, accordingly, are more at risk
to provide further detail into epidemiologic facets of bat for exposure to.
lyssaviruses.
In contrast to most countries in North America and west-
ern Europe, where wildlife rabies predominates, Southeast 6 Mechanisms and Route
Asia, Africa, Latin America, and most developing countries, of Transmission
canids continue to be the principal causative vectors of trans-
mission to humans [83]. Additionally, transmission by hema- Lyssaviruses are transmitted by saliva from the bite of a rabid
tophagous bats, unique to the Americas, is an emerging animal, which may be infectious for days before obvious ill-
public health problem, in addition to being a historically ness, but not prior to brain involvement [5, 17, 45, 65]. If the
important disease of livestock, with widespread economic converse existed, thousands of human mortalities would be
implications [69, 70]. obvious following exposure to animals proven nonrabid by
routine postmortem diagnostic examination of brain mate-
rial. Moreover, if a true “carrier” state readily existed, in
5.4 Temporal Distribution which most infected animals remained healthy while excret-
ing large quantities of infectious material, human rabies
Human rabies occurrence is predicated in part by the sea- would not be rare and the current practice of confinement
sonal patterns of the infected animal species. In the absence and observation of the rabies-suspect biting dog, cat, or fer-
of an active or sensitive surveillance system for human PEP, ret would fail as an effective public health measure. Beyond
epidemiologically it would be reasonable to investigate a the domestic dog, cat, and ferret, reliable observation periods
potential association between increased animal movements, have not been established routinely because the shedding
seasonality of human outdoor activities (adjusted for age), period for virus is undetermined for most species [4].
and exposures resulting in PEP. Due to the exceedingly low Virus does not penetrate intact skin. Hence, touching a
occurrence of human rabies mortality in the United States, rabid animal does not constitute exposure. Other nonbite
there is no obvious meaningful historical trend in the tempo- routes of natural viral transmission have been documented
ral distribution of cases, with the exception of cases associ- very rarely and largely remain unimportant epidemiologically
ated with bats. Most bat rabies cases appear in late summer. [85]. For example, contamination of mucous membranes with
Associated human cases occur typically in autumn, after an infectious material is considered the major nonbite route of
28 Rhabdovirus: Rabies 663

exposure, and as previously discussed, transplant recipients of period during which the virus is virtually undetectable repre-
tissues or solid organs have succumbed, in which the donors sents a phase of extraneuronal infection [62] or coincides
had died of undiagnosed rabies [51–53]. In February 2013, a with direct infection of the nervous system [66]. During
patient in the United States died of rabies 18 months after these early phases of virus infection, the neuronal synaptic
receiving a deceased-donor kidney transplant. The donor, in transfer may occur in the form of ribonucleoprotein–tran-
retrospect, had displayed signs and symptoms consistent with scriptase complexes [64], rather than the assembly and pas-
rabies upon death: vomiting, upper extremity paresthesias, sage of complete virions per se, as regularly occurs in later
fever, seizures, dysphagia, autonomic dysfunction, and subse- phases of replication. After a relatively quiescent early
quent brain death. At the time of his death, his symptoms were phase, viral antigens can be found distributed in most parts
thought to have been caused by ciguatera, a foodborne toxin of the CNS [92]. A temporal relationship between viral RNA
[86]. The rabies viruses infecting the donor and deceased synthesis and immediate-early gene mRNA expression sug-
recipient of the kidney were found to be consistent with the gests an activation and upregulation mechanism may be
raccoon rabies virus variant and were more than 99.9 % identi- responsible for the burst of viral activity after a variable incu-
cal across the entire N gene (1,349/1,350 nucleotides), thus bation period [93].
confirming organ transplantation as the route of transmission Rabies is often considered as a disease due to prototype
[86]. Three additional persons had received organs from the neurotropic viruses. Multiple nonneural sites have also been
deceased donor: the other kidney, heart, and liver. All three associated with viral antigens following centrifugal transport
persons remained asymptomatic for the 18-month period from the CNS [62]. Studies suggest that the relative propor-
between organ receipt and administration of PEP, recom- tions of infectious virions will vary depending on the end
mended upon retrospective diagnosis of rabies in the donor, organ examined [94]. Typical exit portal tissues important in
and to date have not shown signs or symptoms of rabies. A transmission, such as salivary glands, support high viral
comprehensive public health investigation identified a further titers and contain only a minimal accumulation of matrix and
38 contacts of the donor and 17 contacts of the recipient who deviant viral production products, while multiplication in
were recommended to receive PEP; the investigation further other nonneural sites, such as adrenal glands and mucous
revealed that the kidney donor was an avid hunter and trapper glands of the nasal mucosa, produce only moderate viral
with frequent wildlife exposures and at least two raccoon concentrations but large amounts of viral nucleocapsid and
bites, for which he did not seek medical care, in February 2010 anomalous structures [94]. Further, antigens may be found in
and in January 2011 [87]. Four cases of apparent aerosol trans- free sensory nerve endings of tactile hair in a skin biopsy,
mission have been implicated: two individuals working with which is one of the best diagnostic methods of confirming an
extremely dense populations of bats in the confines of caves antemortem diagnosis of rabies in humans [95].
(vs. millions of cavers who may do the same) [88] and in two One relatively unique characteristic of lyssavirus infec-
laboratory workers who were exposed to high concentrations tion is consequent behavioral changes that may favor trans-
of aerosolized rabies virus [89, 90], and in 2002 a bat conser- mission, most notably irritability and the predilection to bite.
vationist in Scotland died of infection with European bat lys- Other more subtle behavioral changes may occur as well. For
savirus 2. It is posited that the individual acquired the virus via example, in the case of raccoon rabies, induction of juvenile
inhalation [91]. These considerations aside, there is a theoreti- vocalizations in an infected adult host has been observed,
cal risk of human-to-human transmission resulting from con- which may enhance investigation by conspecifics, bringing
tact with rabid patients [49, 50]. Although suspected on into close proximity another susceptible host.
occasion in both wild and domestic animals, neither the oral Mechanistically, behavioral manifestations of “dumb”
route nor other unusual means, such as transplacental trans- or “furious” rabies in a given host may be dependent on
mission, have been implicated definitively as important in the specific accumulation and differential effect of virus in
human rabies cases to date, partially because of the difficulty selective brain areas, and these two extreme clinical pre-
in ruling out the traditional bite route as the primary means of sentations may likely reflect differences in viral infection
exposure, especially in regions with enzootic dog rabies. within specific areas of the CNS. Further, an individual may
alternatively manifest both of these generalized forms [96].
Host incapacitation during clinical disease may be obvious
7 Pathogenesis and Immunity and dramatic. As such, lyssaviruses have been character-
ized as poorly adapted or imperfect parasites because infec-
The final outcome after viral exposure, leading to either tion typically results in host death. However, in an ecologic
death, or apparent immunity, is complex. Following local sense, the type of behavioral changes associated with classic
introduction at the site of a bite, the virus may remain local- rabies may ensure viral perpetuation regardless of ultimate
ized and undergo limited replication at extraneuronal sites, host outcome, provided transmission of progeny virus has
such as in muscle tissue. Currently, it remains unclear if this occurred before host demise.
664 K.A. Christian and C.E. Rupprecht

While the phenomenon of differential susceptibility of 8 Patterns of Host Response

mammalian hosts to various viral variants is clear, the mecha-
nisms underlying such observations are not. For example, free- 8.1 Clinical Features
ranging raccoons may have serum VNA detected even in areas
without raccoon rabies [33]. Moreover, while skunks inocu- The bite of a rabid animal may often be too superficial to
lated with a Midwestern skunk isolate succumbed within 4 or produce a productive infection. Even if exposure is effec-
5 weeks, raccoons infected with several greater concentrations tively transdermal, the bite must be concomitant with the
of virus did not succumb or shed detectable amounts of rabies presence of virus from the infected host, which may be
virus; 2 of 12 raccoons developed VNA and were the only two excreted only intermittently. A host is only considered poten-
that survived subsequent rabies virus challenge [97, 98]. Such tially infected once the virus has breached the skin or mucous
observations may help to explain the differential occurrence of membrane barriers. This is not synonymous with disease but
rabies in Midwestern skunks, without apparent perpetuation is partially dependent on viral load and variant characteristics
in raccoons. In the eastern United States, raccoons are compe- within a host ranging from fully susceptible to immune. Host
tent hosts for a different variant, which does not appear to be immune response following viral exposure may or may not
independently supported by skunks. Additionally, the relative result in the production of detectable amounts of VNA. While
resistance of the only North American marsupial, the opos- in theory only a single virion is necessary, a fully productive
sum (Didelphis virginianus), to rabies virus and its paucity of infection usually requires numbers of fully infectious parti-
acetylcholine receptors provide experimental data suggesting cles several orders of magnitude greater to produce disease.
that host susceptibility may be influenced in part by receptor “Inapparent” infection of long duration may occur rarely,
occurrence and relative abundance, to which virus may bind but has not been demonstrated to be of epidemiologic impor-
and be internalized [99]. tance, regardless of widespread speculation to the contrary.
Rabies VNA, induced solely by the viral glycoprotein, is Distinct from inapparent or so-called latent infection (which is
believed to play a major role in the pre- and PEP protection a misnomer compared to the latency of other truly persistent
against rabies [22]. However, comparison of absolute VNA herpesvirus or retroviral infections), the incubation period
titers and mortality in laboratory rodents immunized with associated with overt disease can be highly variable in rabies
whole virus vaccine or viral glycoprotein did not delineate a and prolonged, during which the host is not infectious [85].
clear role for VNA in vaccine-induced resistance to disease Productive infection, during which input virus replicates, may
[100]. Specific discrepancies between VNA titer and rabies result in viral encephalitis that manifests with neurologic signs
protection were also shown in wild carnivores where no firm and symptoms. Perhaps the only consistent feature of the
correlation existed between any known titer level of VNA resulting clinical presentation is that it is rarely typical from
resulting from oral vaccination and protection per se [101]. one host to another. The prodromal stage is uniformly acute,
Data from immunization experiments suggest that protective lasting from 2 to 4 days, including moderate fever, malaise,
activity may ultimately correlate with a vaccine’s ability to anorexia, headache, and nausea. Presenting symptoms in
induce immunologic memory. Lack of firm correlation between humans are often nonspecific. However, paresthesia at the site
absolute VNA titers and survivorship in immunized animals of the bite and aerophagia or hydrophobia (spasms of pharyn-
suggests that, in addition to VNA, other immune effector geal or inspirational musculature induced by air currents or
mechanisms may be involved in the protection against lethal anticipation of swallowing) are highly suggestive of rabies.
rabies virus infection in either pre- or PEP situations [22, 31]. Physical and mental status deteriorates rapidly. Not discount-
Studies have demonstrated that in addition to glycoprotein ing classical Pasteurian observations of spontaneous recovery
[2], the internal viral ribonucleoprotein (RNP) may also be in animals or the remarkable contemporary accounts of the
important to the induction of protective immunity [102, 103]. few human survivors [81], the case fatality proportion in prac-
In related studies following RNP immunizations, primates tice still approaches unity, once clinical signs are present.
developed a strong anti-RNP antibody response, were pro- Death is usually attributable to respiratory or cardiac failure.
tected against lethal rabies, and were primed for the induction
of a VNA response following exposure to antigens [104].
These experiments and others suggest that the RNP plays an 8.2 Diagnosis
important role in inducing both primary protective immunity
and cross-protective immunity against infection to heterolo- The incidence of more common infectious agents that result
gous strains. Since RNP is a strong inducer of nonneutraliz- in human encephalitis should be compared to the relative rar-
ing antibody, one of the functions of RNP-specific antibodies ity of rabies in the establishment of a diagnosis. Pairing of a
may be the promotion of viral RNP attachment via Fc recep- known animal exposure followed in several weeks’ time
tors to phagocytotic cells, stimulated by virus to produce with the appearance of classic signs and symptoms should
cytokines such as interferon, which inhibits viral replication. alert the astute infectious disease professional. However,
28 Rhabdovirus: Rabies 665

rabies should also be considered in any suspected viral century in the United States, canine rabies was enzootic
encephalitis of unknown origin, regardless of a definitive throughout the world [110]. In the early 1880s using virus
history of animal bite, especially once a compatible clinical obtained from a rabid dog and serially passed in rabbits by
syndrome is manifest. An exposure may have been unrecog- intracerebral inoculation, Pasteur vaccinated a series of
nized, forgotten, or discounted. In humans, differential diag- dogs and subsequently challenged them with rabies virus
noses include herpesvirus and arboviral encephalitis and resulting in acceptable results; additional challenge studies
poliomyelitis, among others. Although lyssavirus infection using monkey intracerebral inoculation allowed Pasteur to
and pathogenesis can be described experimentally, there demonstrate that dogs became resistant to additional chal-
remains no practical or reliable method for detecting expo- lenge with virulent wild rabies virus [111]. Post-Pasteur,
sure or infection in humans until the CNS has been exten- refinement and evolution of vaccine designed to protect
sively infected and there has been centrifugal distribution of dogs against rabies resulted in extensive vaccination cam-
virus via peripheral nerves. At this point, rabies may be diag- paigns implemented during the 1940s and 1950s and subse-
nosed in humans through positive FA results on brain biopsy, quent decline of rabies reported in dogs in the United States
full-thickness skin biopsy from the nape of the neck or on a [110]. Today in the United States, rabies vaccine is adminis-
corneal impression, virus isolation from saliva, detectable tered paranterally to dogs every 1 or 3 years depending on
antibody in the CSF or serum (the latter from an unvacci- local or state requirements. However, despite the successes
nated individual), or demonstration of viral amplicons in in the United States, canine rabies remains responsible for
samples of CNS, saliva, skin, or other tissues. Negative the most human rabies deaths globally. The domestic dog in
results do not rule out rabies. Optimal diagnostic material is developing countries is often unowned, “community”
fresh brain tissue for detection of viral inclusions in neurons. owned, or otherwise neglected by agricultural veterinary
Use of appropriate laboratory-based techniques is the only services [112]. Canine population mismanagement is one
method to confirm a suspected rabies case based upon clini- contributor: although vaccination campaigns in the develop-
cal appearance alone and/or a history of likely exposure to ing world have been found to achieve the WHO recom-
infectious material. mended vaccination coverage of ≥70 %, high dog turnover
rate in the population (i.e., introduction of new susceptible
individuals) contributes to enzootic canine rabies [83].
9 Prevention and Control Considering the success of the elimination of canine rabies
virus transmission in the United States, the burden of rabies
9.1 Basic Epidemiologic Methods deaths attributable to dogs throughout the developing world,
and availability of vaccine, surveillance, and laboratory
A modern preemptive public health strategy against rabies diagnostics, the need for a global effort to achieve the elimi-
encompasses prompt and proper PEP of humans bitten by nation of canine rabies transmission is increasing. A tool
infected animals, prevention of disease among companion developed in the context of promoting attention to the inter-
animal species by preexposure vaccination, and the ecologi- dependency of human and wildlife health, i.e., One Health,
cally sound management and control of rabies among free- aims to assist the global public health community in achiev-
ranging reservoirs. While a focus upon the exposed person is ing this goal and eliminate the approximate 55,000 deaths
an obvious and primary biomedical responsibility, the ulti- per year attributable to rabies through The Blueprint for
mate management of the host is the only way to selectively Rabies Prevention and Control [113].
eliminate rabies in animals. Management of animal rabies With a concentration upon wildlife, a vaccinia-rabies gly-
extends beyond domestic animals. Since the conception of coprotein (V-RG) recombinant virus vaccine [114–116] was
oral rabies vaccination (ORV) of wildlife during the 1960s at developed that has proved to be an effective oral immunogen
the CDC and the initial field experiment during 1978 in in raccoons [101, 117] and a variety of other important spe-
Switzerland with modified live rabies virus, the distribution cies [118–122] and provides long-term protection against
of millions of vaccine doses in Europe and North America rabies [120]. Advantages of this recombinant orthopoxvirus
attests to the utility of this technique, with local disease elim- vaccine included greater thermostability than attenuated
ination as one intended result [67, 105–109]; as evidenced by rabies viruses and the inability to cause rabies, because only
the elimination of fox rabies in many sites in Europe and the cDNA of the surface glycoprotein of the rabies virus was
southern Canada, and in the United States of a coyote rabies inserted within the recombinant virus [114]. The period of
virus variant [61]; the suppression toward elimination of the 1980s was marked by intensive, collaborative, safety
gray fox rabies, as exemplified in west-central Texas [60]; evaluations of the V-RG virus under laboratory conditions in
and the containment of raccoon rabies to the eastern states. North America and Europe [120, 123] that culminated in the
With regard to domestic animals, for millennia prior to initial limited field experiments by the end of that decade
the modern age and through the first half of the twentieth [124, 125].
666 K.A. Christian and C.E. Rupprecht

Despite the successes of V-RG in conferring immunity Addressing bat rabies control, the importance of bat
against rabies among several wildlife species including exclusion from human habitations and better public educa-
raccoons, V-RG is not efficacious for control of rabies tion to facilitate recognition of potential exposures cannot be
in several other important reservoirs, such as Mephitis overemphasized. Overall, bat conservation is very compati-
mephitis, the striped skunk [121]. Further, less-attenuated, ble with basic public health tenets.
first-generation rabies virus candidates, such as the rabies
virus strains SAD/ERA, may be associated with vaccine-
induced rabies in these species [126]. For safe, effec- 9.2 Basic Immunization Concepts
tive, and more comprehensive wildlife rabies control, a and Practice
human adenovirus type 5 (HAdV-5) vectored rabies virus
glycoprotein recombinant vaccine (AdRG1.3; ONRAB®) For practical purposes, rabies may be viewed as universally
has been tested for safety and efficacy in the laboratory fatal once clinical symptoms manifest. In the same context,
and used in the field in Canada and is currently being extremely effective human PEP consists of vaccine and RIG
considered for broader use throughout North America. if administered promptly and properly, after thorough wound
Adenoviruses belonging to the family Adenoviridae, sub- cleansing, if a bite is evident. Besides application in humans,
family Mastadenoviridae, are nonenveloped DNA viruses the extension of PEP to companion animals and livestock has
found ubiquitously among many mammalian species, also been investigated, such as in dogs [132, 133] and sheep
including humans [127]. The ONRAB® vaccine employs [134]. Moreover, further epidemiologic investigations to
a replication-competent HAdV-5 vector into which a include the use of PEP for animals should be conducted,
DNA copy of the ERA virus glycoprotein gene has been from a One Health perspective within the veterinary field,
inserted [128, 129] and, similarly to V-RG, is packaged considering the critical role of domestic animals in human
into sachets for oral administration to wildlife. Results society for food, fiber, security, companionship, transporta-
from field trials in Canada have indicated that ONRAB is tion, etc., among others.
stable both in vitro and in vivo, with a limited ability to As described earlier, minimum standards as opposed to an
replicate in an established model for human adenovirus absolute “protective antibody” for humans are based empiri-
infection and is suitable for environmental use as an oral cally on presumed protective activity of rabies-specific anti-
rabies vaccine for wildlife [130]. bodies and can be detected by reference laboratories. A
Some opponents to wildlife ORV espouse depopulation, RFFIT titer of ≥1:5 (approximately 0.10 international units
rather than vaccination, as a control method, citing the effec- [IU]/mL) or higher is evidence of adequate immunization in
tiveness of dog rabies control via compulsory muzzling, persons at either constant or frequent risk of exposure, at
movement restrictions, and stray animal removal in Europe 6-month or 2-year intervals, respectively, as a measure of
and North America during the early 1900s, before the utili- baseline immunity, as recommended by the United States
zation of efficacious vaccines en masse. Two components of Advisory Committee on Immunization Practices [85]. A
this technique—animal movement restrictions and muz- single booster vaccine dose is administered if the level is
zling—are inapplicable to wildlife, leaving only depopula- lower, based on a determination of apparent risk (Table 28.3).
tion. As a sole mechanism of control, depopulation may Preexposure immunization simplifies human PEP because of
decrease the local intensity of rabies or postpone the invasion priming of the immune response and is thought to provide
of a new geographic area, but it is unlikely to completely some degree of protection against unrecognized exposures,
eliminate rabies or permanently protect a region from an which by definition should be negligible if proper protocols
impending epizootic. are followed. Nevertheless, when pre-immunized persons
Several professional groups may view a number of wild- are knowingly exposed to rabies virus, two booster doses of
life species, including some rabies vector species, as nui- vaccine, administered on days 0 and 3, are required to induce
sance or pest species, and hence oppose ORV, citing the a sufficient anamnestic response to prevent development of
undesirable potential for increased numbers following elimi- disease. Also, rabies immune globulin (RIG) is contraindi-
nation of rabies, inappropriately viewing rabies as a form of cated under such circumstances, because it may interfere
wildlife population control. However, proportionate mortal- with development of an adequate anamnestic response [135].
ity due to rabies among the most prominent wildlife vectors Pre-immunized individuals should remain vigilant in recog-
in the United States, such as raccoons, skunks, and so on, has nizing potential viral exposures and seek appropriate PEP. If
not been investigated. In Europe, collective data suggest that actual viral exposures occur but are unrecognized, the pre-
rabies may not be a significant regulator of population size; immunized individual may still die of rabies, albeit very
there has been no substantial increase in red fox numbers rarely, as did one Peace Corps volunteer, who was bitten by
following the elimination of rabies over large geographic her own puppy, that died of a disease compatible with rabies
areas [131]. [136] (Table 28.4).
28 Rhabdovirus: Rabies 667

Table 28.4 Rabies postexposure prophylaxis (PEP) guide, United States

Animal type Evaluation and disposition of animal Postexposure prophylaxis recommendations
Dogs and cats Healthy and available for 10 days Should not begin PEP unless animal develops signs of
observation rabiesa
Rabid or suspected rabid Initiate PEP immediatelyb
Unknown (escaped) Consult public health officials
Raccoons, skunks, bats, foxes, other Regard as rabid unless geographic area is Initiate PEPc. Consider factors such as provocation,
carnivores, woodchucks known to be free of rabies or until animal is suggestive clinical signs, severity of wounds, type of
proven negative by laboratory tests exposure, and timeliness of test results (24–48 h) for
decisions regarding immediate initiation or to delay
pending test results
Livestock, rodents, and lagomorphs Consider individually Consult public health officials; bites of squirrels,
(rabbits and hares) hamsters, guinea pigs, gerbils, chipmunks, rats, mice,
other rodents, and lagomorphs almost never require PEP
Adapted from the ACIP
a
If clinical signs compatible with rabies develop during the 10-day confinement and observation period, the animal should be euthanized and tested.
Depending on circumstances, initiation of PEP may be delayed pending a positive report, if results may be obtained in 24–48 h
b
If the bite was unprovoked or resulted in severe wounds, treatment of the bitten person should begin immediately with human rabies immune globu-
lin (HRIG) and human diploid cell vaccine (HDCV) or purified chick embryo cell vaccine (PCEC). PEP may be discontinued if the test is negative
c
If available, the animal should be euthanized and tested as soon as possible. Holding for an observation period is not recommended since the
potential viral shedding period prior to clinical signs has only been determined for dogs, cats, and ferrets

Newer approaches to immunization from convention- Besides epidemiologic utility (for which they were origi-
ally prepared products may also include subunits such as nally generated), antirabies MAbs are also useful in the
DNA vaccination. For example, mice immunized intra- refinement of host immunization and PEP concepts. Rabies
muscularly with a plasmid vector expressing the rabies VNA are induced solely by the viral glycoprotein and play a
virus glycoprotein under a SV40 early promoter devel- major role in the immune protection. Hybridomas that
oped specific cytotoxic lymphocytes, thymic lymphocytes secrete rabies virus antigen-specific MAb have been gener-
(subset TH1), and rabies VNA and were protected against ated and can effectively neutralize fixed and street lyssavirus
rabies virus challenge [138]. Although many questions isolates. These MAbs were selected on the basis of isotype,
remain regarding the consequences of persistent “infec- antigen and epitope specificity, virus strain specificity, affin-
tion” with a plasmid, the effects of long-term stimulation ity, and neutralizing activity. Administration of such MAbs
of the immune system, the genetic stability and safety of protected animals when challenged with a lethal dose of
foreign promoters and nucleic acids, and the potential rabies virus in experimental PEP models [139]. These data
benefits are clear, which include simplicity and economy. strongly suggest that MAbs, alone or in combination with
Obviating the need for booster immunizations would be vaccine, may be an effective method of protection against
particularly useful for certain applications, such as live- clinically relevant lyssaviruses [140]. Such an approach has
stock in extensive range situations, for example, where several theoretical advantages over presently used hyperim-
protection against bovine paralytic rabies via vampire mune sera: first, in contrast to current products, compara-
bats in Latin America with current vaccines is prohibitive tively small volumes of MAbs would have to be inoculated
economically and logistically and could have similar util- for equivalent active protein content, because specific neu-
ity for humans in remote locations. tralizing activity per mass of protein is higher, so MAbs may
Viral sources as substrates for vaccine development could be optimal for lessening the trauma and pain of local wound
also be expanded. Compared to the conserved fixed virus infiltration with a source of passive antibodies, and second,
strains that form the basis for most rabies virus vaccine pro- safety issues arising from the possibility of adventitious
duction historically, considerable diversity is obvious among agents associated with human or animal blood products
lyssavirus antigens by MAb analysis. Nevertheless, even would be alleviated by bulk production under modern GMP
recombinant rabies virus vaccines that only express one viral conditions in cell culture. Despite the experimental progress
glycoprotein are able to fully protect animals from severe shown, the rationale and acceptability of heterogenous
experimental and field exposure against all of the major murine MAbs for future human rabies PEP have been ques-
rabies variants. Moreover, after considerable investigation, tioned on the grounds that a human anti-mouse response to
there is no firm evidence to suggest that current human rabies the MAbs would present a significant drawback by the con-
deaths are attributable to antigenic variation of street rabies sequent effects on kinetics and antigenic targeting. If murine
viruses, but rather they appear due to serious omissions in or other heterologous MAbs are used, human anti-species
basic PEP protocols [23]. responses might be expected, but these are not necessarily
668 K.A. Christian and C.E. Rupprecht

deleterious, because MAbs would only be used once in Self-replicating attenuated or recombinant vaccines offer
rabies PEP. As is the case for HRIG, MAbs would not be the greatest versatility for oral rabies vaccination of animals
readministered should the person be reexposed in the future. such as dogs. While a few candidate oral rabies vaccine prep-
In the previously vaccinated subject, rabies PEP in these sit- arations have demonstrated sound protective immunity in
uations consists of vaccine only on days 0 and 3. Moreover, captive dogs, no self-replicating viral system, conventional
if such MAbs are recognized as foreign antigens, with an or otherwise, may be considered completely apathogenic,
expected shortened serum half-life, this potentiated clear- especially in the context of the immunocompromised host
ance may be advantageous by minimizing the opportunity [142]. As an alternative, inactivated oral preparations should
for interference with an active immune response on behalf of maximize safety considerations, yet the significant quantities
the vaccinated host, while effectively neutralizing virus, required for minimal efficacy may be cost-prohibitive unless
prior to induction of host VNA. This is one of the primary novel adjuvants, delivery techniques, or production methods
reasons cited for homologous or human MAbs, as opposed are found [143].
to heterologous. As such, several homologous MAbs are Applied research on the oral or enteric administration of
under clinical evaluation [141]. affordable inactivated rabies biologics may diminish the
Given the above considerations, what is an appropriate public health concerns related to the current products under
gauge for immunoprotection for extension to humans? Mere consideration for application to free-ranging dogs, particu-
in vitro neutralizing capacity alone may not be fully indicative larly as they relate to human rabies deaths in developing
of protective efficacy in vivo. Some MAbs that do not neutral- countries. However, regardless of the approach, limited labo-
ize viruses in cell culture are still effective when administered ratory trials of oral canine vaccination may not directly
via PEP to animals [141]. Besides extracellular neutralization, equate to rabies control under field conditions, where a com-
other useful capabilities of MAbs may also be operative, such bination of ORV and traditional parenteral vaccination may
as the inhibition of intracellular viral spread and interference be necessary. This may be especially applicable when con-
in transcription of rabies viral RNA. Such questions, as well as sidering that the majority of successful canine rabies elimi-
the ability to consider the conduct of phase III clinical applica- nation programs have only involved traditional parental
tions, should require introspection as MAbs progress toward application, and not canine ORV.
regulatory approval for use in human PEP.

10.2 Alternatives to Current Biologics for

10 Unresolved Issues Human/Animal Disease Prevention?

10.1 Rabies Management in the While current biologics to prevent rabies in humans, domes-
Free-Ranging Dog: Oral Vaccination tic animals, and wildlife are extremely effective, progressive
(ORV) as a Solution? development toward new paradigms is necessary. The vigi-
lance necessary to minimize human rabies mortality neces-
Rabies management of animals is linked directly to human sitates an extensive public health infrastructure and requires
rabies prevention. One of the most cost-effective methods to the annual prophylaxis of tens of millions in the developing
prevent rabies in humans is the mass vaccination of dogs by world and tens of thousands of potential exposure cases
the parenteral route. Whether oral delivery of vaccination is throughout a developed country, such as the United States.
needed for application to dogs remains uncertain. During the Following a bite from an infected mammal, PEP of rabies in
past century, considerable progress has been made in the labo- humans includes proper wound care and the simultaneous
ratory development and field testing of ORV for free-ranging administration of multiple doses of an efficacious rabies vac-
wildlife disease prevention and control in Europe and North cine, together with a preformed antibody source, typically
America. Major species have included the red and gray fox, HRIG. However, this regimen presents certain obstacles.
raccoon, raccoon dog, and coyote. Oral vaccines tested in the Modern inactivated cell culture vaccines and HRIG are
field to date have included attenuated rabies (e.g., SAD/ERA/ vastly improved over historical NTO vaccines, but such
SAG) and recombinant glycoprotein (e.g., orthopox-, adeno-) products are relatively expensive (especially in the develop-
viruses. Especially with regard to any application for domestic ing world where they are most needed), are often in scarce
canine vaccination, intended biologics should at minimum be supply, and may carry a perceived theoretical risk of adventi-
pure, potent, efficacious (against a virulent, epidemiologically tious agent acquisition to the public. Major concerns in
relevant rabies virus challenge), and safe to target and nontar- rabies prevention have concentrated on the need for potent,
get species, especially humans, at risk for exposure to distrib- inexpensive PEP for humans, especially replacement for
uted, edible baits, or through direct, intimate contact with a more costly HRIG, while retaining activity against a wide
vaccinated host. variety of diverse lyssaviruses.
28 Rhabdovirus: Rabies 669

In contrast to the relatively impotent equine antirabies case histories of recovery after clinical signs in animals sug-
serum used in the past that resulted in high adverse reactions, gest that host defenses may be exploited to alter the end stage
such as serum sickness in up to 40 % of human recipients, of this otherwise fatal malady, if the pathophysiologic mech-
modern purified equine rabies immune globulin (ERIG) anisms of disease can be understood.
products are safer, more potent, and more affordable than As one now classical example, during 2004, a girl aged 15
older cruder products and may be at a fraction of the cost of years in Wisconsin rescued and released a bat, which had bitten
HRIG [144–146]. The ERIG products have been used effec- her on her left hand. The wound was cleaned with peroxide, but
tively in conjunction with vaccine in human rabies PEP, par- PEP was not administered. Approximately 1 month later, she
ticularly in developing countries under the continued threat developed a clinical syndrome consistent with infection with
of enzootic dog rabies. In surveys, less than 1 % of humans rabies virus, including generalized fatigue and paresthesia of
have reported adverse events, coupled with adequate efficacy her left hand, which later developed into diplopia, ataxia, nau-
[147]. By comparison, no ERIG has been available in the sea, and vomiting. On the fourth day of illness, blurred vision,
United States market for several years, although it was still left leg weakness, and ataxia were present which developed
licensed into the late 1980s. Due to its potency and lack of into fever, slurred speech, nystagmus, and tremors of her left
apparent significant local or systemic effects, purified ERIG arm [149]. She was admitted into a pediatric referral facility in
products were the only immediate alternative, should the Milwaukee. On the second day of care, CDC confirmed the
supply of available HRIG be threatened by shortages, con- presence of rabies virus-specific antibody in the patient’s CSF
tamination, or other limitations. Such use might be consid- and serum. However, attempts to isolate virus, detect viral anti-
ered a temporary antecedent until the availability of more gens, and amplify viral nucleic acids from two skin biopsies
novel replacements, such as MAbs. and nine saliva samples were unsuccessful [149]. An experi-
To be most effective, conventional PEP should be applied mental treatment protocol, later termed the “Milwaukee
prior to viral invasion of the nervous system. However, PEP Protocol” that combined anti-excitatory and antiviral drugs,
may still be effective even after virions access the nervous including ketamine, ribavirin, and amantadine, was adminis-
system. For example, in one experimental animal infection tered in conjunction with supportive intensive care [149].
study, analysis by RT-PCR has revealed the presence of Neither rabies vaccine nor HRIG was administered because of
rabies virus-specific RNA in the olfactory bulb and cerebral the patient’s demonstrated immune response and the potential
cortex of animals within 6 h of direct intranasal inoculation. for potential harm from a potentiated or altered immune
Yet, when animals were administered a neutralizing MAb up response [150]. After more than 70 days of hospitalization, the
to 24 h after inoculation, 80 % survived a challenge in which patient recovered, with only minor neurologic sequelae at the
all controls succumbed [139, 140]. Neither virus nor virus- time. While promising, this experimental protocol has been
specific RNA was detected when these survivors were eutha- attempted in several additional human rabies cases without
nized a month later. These data clearly demonstrate that success. Clearly, prevention of exposure, or prompt appropriate
virus “neutralization” and clearance from the CNS are com- prophylaxis after exposure, but before development of clinical
plex processes, less than fully understood [148]. Thus, if pro- signs, remains a primary focus in public health. While there is
duced in a cost-effective manner, antirabies MAbs may be no established therapy that is effective for patients who develop
useful in the future PEP of humans, as well as in economi- rabies, efforts should continue on basic viral pathogenesis
cally or otherwise important, unimmunized domestic ani- research, development of relevant surrogate animal models and
mals. Such biologics that abrogate infection even after virus protocols that mimic supportive intensive care, and experimen-
enters the CNS present exciting new possibilities for inter- tal applications in human cases where ethical/legal approvals
vention with significant advantages over historical poly- and modern facilities exist [151].
clonal rabies immune globulin.

10.4 Animal Translocation and the Threat of

10.3 Treatment of Clinical Human Rabies? Disease Introduction/Reintroduction?

While prevention of disease among reservoir hosts and vic- People who reside in true rabies-free areas enjoy a luxury
tims alike is still the optimal approach, when this fails, fur- of not having to be concerned with PEP following an ani-
ther alternatives regarding treatment of clinical rabies may mal bite. However, given the availability and rapidity of
be warranted. Historically, the individual experimental appli- modern transportation, alarming trends are evident in ani-
cation of RIG and Ig fragments, cytosine or adenine arabino- mal translocation on local, regional, and intercontinental
side, interferon, acyclovir, antithymocyte globulin, steroids, levels. Such deliberate translocation, but often with unin-
vidarabine, tribavirin, inosine pranobex, or ribavirin has not tended results, carries a significant risk of the emergence
demonstrated any substantive utility to date [8]. However, of infectious agents into new niches previously unavailable
670 K.A. Christian and C.E. Rupprecht

because of zoogeographic barriers. There are limited legiti- questionable efficaciousness, and ethical considerations.
mate reasons for purposeful animal translocation, such as Besides the carnivore issue, there has been a significant
for species reintroduction, research, education, or zoologi- renewed impetus to enact population management for other
cal captive breeding. Unrestricted importation of nonnative species as well, most notably humans. One of the major mod-
species, or translocation of native mammalian species dur- ern strategies under consideration has been fertility control by
ing the incubation stage of the disease, poses the threat of immuno-contraception [152, 153]. Contraceptive approaches
rabies introduction into rabies-free regions. Within rabies have pursued several avenues, including (1) eliciting an
enzootic countries, there is also a danger that other vari- immune response to ova, sperm, or zona pellucida antigens;
ants may be introduced into new areas, such as occurred in (2) disrupting spermatogenesis; or (3) interfering with repro-
the late 1970s with translocation of infected raccoons from ductive hormones. The ideal technique would entail a targeted
the southeastern United States, resulting in the initiation of approach, involving either a species-specific antigen or vector,
the mid-Atlantic raccoon rabies epizootic, which perpetu- although none has yet been identified. Application to dogs and
ates along the Atlantic seaboard states. The same potential wild carnivores may be an important adjunct to current man-
looms over the transportation of infected carnivores to unaf- agement techniques. Although the need appears obvious and
fected states for hunting, trapping or other recreational use. preliminary efforts are promising, considerations that must be
Translocation of infected wildlife (i.e., bats and canids), addressed include (1) the levels of herd immunity needed to
from enzootic rabies areas in one country to another, would preclude the persistence of either a subpopulation that evades
hamper public health efforts at wildlife rabies control, if exposure to these agents (e.g., bait avoidance) and prevent
they escape. Little is known about the clinical signs, incuba- “nonresponders” from gaining a significant reproductive
tion periods, or potentials for transmission of related agents, advantage; (2) the nontarget species issue, because of bait
such as other Old World lyssaviruses to New World hosts via competition; and (3) the escape of a transmissible agent and
importation of reservoir species; such establishment would propagated spread beyond an intended “pest” target popula-
be problematic, owing to the questionable vaccine efficacy tion (i.e., introduced red foxes in Australia) to other distant
against certain nonrabies lyssaviruses. Enlightened and tar- geographic regions (i.e., red foxes in North America) by delib-
geted education, enforced legislation, improved regional erate or unintentional animal or agent translocation. If such
surveillance, and a rapid, appropriate public health response concerns could be minimized, a recombinant vaccine could, in
will minimize the overall dangers associated with animal theory, not only immunize against an infectious agent of
translocation and will hopefully prevent an infectious nidus choice, but also simultaneously decrease host abundance as
from becoming an epizootic, as seen increasingly in island well. For these reasons, more limited, targeted parenteral
nations, such as Indonesia. application to animals may be more relevant than generalized
bait distribution in the environment.

10.5 Reservoir Population Management: Acknowledgments The authors would like to thank Jesse D. Blanton
Immuno-Contraception of Animals? and Jessie L. Dyer for their assistance in the preparation of this
manuscript.

As most humans are infected with rabies virus after a bite, new
Disclaimer
focused animal management strategies appear obvious to min-
The findings and conclusions in this chapter are those of the
imize the burden of disease in people. Rabies is but one of a
authors and do not necessarily represent the view of the
multitude of factors that arguably may influence the density of
Centers for Disease Control and Prevention.
free-ranging carnivores over time [131]. One criticism of
wildlife rabies vaccination is a concern that by decreasing or
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 Rhinoviruses
29
Ian M. Mackay and Katherine E. Arden

1 Introduction (BECs) [12, 13], and smooth muscle cells [14] but not in
monocytes [15] or dendritic cells (DCs) [16]. The inflam-
Picornaviruses, which include the human rhinoviruses matory immune response they trigger very soon after infec-
(HRVs) and enteroviruses (EVs), are the most frequent tion has its greatest impact in the young, the elderly, those
cause of acute human illness worldwide [1]. HRVs are the with asthma or chronic obstructive pulmonary disease
most prevalent cause of acute respiratory tract illnesses (COPD), and in the immunocompromised. First infections
(ARIs) which usually commence in the upper respiratory usually elicit a stronger response. Antiviral interventions
tract (URT). ARIs are the leading cause of morbidity in chil- have been under development for decades; to date most have
dren under 5 years and occur in all seasons [2, 3]. ARIs met with varying degrees of failure or unacceptability.
linked to HRV infections are associated with excessive and Vaccines have been considered unachievable because of the
perhaps inappropriate antibiotic prescribing [4] and with large number of diverse and distinct viral types.
significant direct and indirect healthcare expenditure [5, 6]. There are 100 classically defined and recognized HRV
ARI incidence is highest in the first 2 years of life, with up serotypes grouped into two species, HRV-A and HRV-B,
to 13 episodes per year including up to six positive for an and a recently defined third species, HRV–C, containing
HRV, and it is not uncommon to average one infection per more than 60 genotypes identified and characterized entirely
child-month [3, 7–9]. In preschool-aged children, nearly by molecular means. Their cousins, the four enterovirus
50 % of general practitioner visits are for ARI [10], many of species (EV-A, EV-B, EV-C, and EV-D), are also found in
which are self-limiting. ARIs can often be managed in the the airways at times. Most systematic and mechanistic stud-
community with supportive care from parents, but compli- ies of HRV etiology and pathogenesis have been informed
cations can arise that require a medical visit for manage- by studies in adults, mostly prior to the discovery of HRV-Cs.
ment of asthma, otitis media, or sinusitis [11]. HRVs Adults exhibit reduced symptoms from HRV infections
replicate in nasal cells, sinus cells, bronchial epithelial cells because of prior exposure and the resultant protective
immune memory which that imparts (see Sect. 7.3).
I.M. Mackay, PhD (*) Furthermore, many modern studies (1) draw conclusions
Queensland Paediatric Infectious Diseases Laboratory, about lower respiratory tract (LRT) disease using URT spec-
Queensland Children’s Medical Research Institute, imens and (2) infrequently sample, doing so across small
Sir Albert Sakzewski Virus Research Centre,
cross sections of time. These limitations have hampered
Children’s Health Queensland Hospital and Health Service,
The University of Queensland, Herston, QLD 4029, Australia attempts to associate virus detection and disease. Current
thinking is that HRV-Cs may be key players in asthma exac-
Australian Infectious Diseases Research Centre,
School of Chemistry and Molecular Biosciences, erbations although our inability to culture them routinely
The University of Queensland, St Lucia, QLD 4072, Australia has hindered our progress in understanding their role. The
e-mail: [email protected] impact of the HRVs has been underestimated for decades,
K.E. Arden, PhD and the concept of the HRVs as a very large assemblage of
Queensland Paediatric Infectious Diseases Laboratory, genetically, immunogenically, antigenically, and temporally
Queensland Children’s Medical Research Institute,
distinct and stable viral entities remains rare; they are more
Sir Albert Sakzewski Virus Research Centre,
Children’s Health Queensland Hospital and Health Service, commonly considered a single variable virus, a view that
The University of Queensland, Herston, QLD 4029, Australia science does not support.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 675

DOI 10.1007/978-1-4899-7448-8_29, © Springer Science+Business Media New York 2014
676 I.M. Mackay and K.E. Arden

2 Historical Background Rhinovirus had been abolished and the HRV-A and HRV-B
species assigned to the genus Enterovirus within the family
The disease most commonly associated with the airways and Picornaviridae [35]. The HRV-Cs have been assigned a new
resulting from HRV infection is the common cold, a self- naming scheme based on genetic sequence in the absence of
limiting coryzal illness [17–19]. The term dates back to antigenic or serological data. While the sequencing of all
ancient Greece, but evidence that the syndrome and asthma, serotyped HRV genomes was completed in 2009, few of the
another disease most frequently due to HRV infection, has HRV-Cs or apparently novel HRV-As or HRV-Bs have been
been with us since ancient times can be viewed in writings similarly characterized, so the full spectrum of HRV
on the Ebers papyrus, a medical document written in the six- genomes, the rhinovirome, remains incomplete. In this chap-
teenth century BC [20, 21]. In 1930 the common cold was ter we have described individual serotyped HRVs as the
considered either to be due to exposure to the elements or to “classical” types, a type being the description for a single,
infection by bacteria [22]. It was later understood to be genetically stable, stand-alone HRV.
largely due to something in bacteria-free filtrates, and so the
search for viral causes began [23, 24].
The Common Cold Unit (CCU) was established in 3 Methods for Epidemiologic Analysis
Salisbury, UK, to seek solutions to the mysteries of the com-
mon cold, mostly through adult volunteer infection studies 3.1 The Pre-molecular Era
and careful systematic science [23]. The CCU functioned for
44 years (1946–1990), and it was here in 1953 that the first The original clinical definition of an HRV infection was writ-
in vitro culture of an HRV was achieved using lung tissue ten using data from cell and tissue culture and adult human
from a particular embryo (Fig. 29.1) [25, 26]. Propagation infection studies. After 1953 in vitro isolation methods
failed once this tissue was expended [22, 33]. employed a virus interference test to more easily determine
Once HRV isolation was possible, viral serotyping devel- successful isolation; cultures suspected of infection with an
oped and culture techniques were further refined. This leads uncharacterized HRV prevented infection by another, readily
to an international effort to characterize and name the HRVs titratable virus [36]. Later, Price (1956; the JH strain) and
[27–30]. then Pelon and co-workers (1957; 2,060 strain) developed
In 2006 renewed interest in HRV research was triggered culture systems that permitted HRV replication to be more
by the description of a distinct clade of HRV types [31] easily identified [37, 38]. The early HRVs were initially clas-
found using molecular typing. The resultant flurry of HRV sified as echoviruses (ECHO 28; later HRV-1) [39]. At the
research raised questions about many earlier paradigms of same time, propagation of the HGP (HRV-2) strain resulted
rhinovirology and of the role of established respiratory from using increased acidity, lowered cultivation tempera-
viruses in ARIs. The novel clade was proposed as a new spe- tures, and constant motion (rotation) [40, 41]. Despite the
cies, HRV-C, which was taxonomically confirmed in 2009 challenges [42], virus isolation was a more sensitive indicator
[32–34]. Prior to the discovery of the HRV-Cs, the genus of infection than an antibody rise in paired sera [43].

HRV-814
HRV-A1A
HRV-A16 Crystal structures
HRV-A3
HRV-A2

HRV-814
HRV-A2 Genomes
HRV-A1B
1930

1940

1950

1960

1970

1980

1990

2000

2010

2020

Fig. 29.1 A timeline of virus Influenza virus 1933 Avian influenza virus H5N1 1997
discovery from the human Coxsackive virus 1948 Human metapneumovirus 2001
respiratory tract. The date of each Echovirus 1951 SARS-CoV 2003
virus’s published description is Adenovirus 1953 HCoV-NL63 2004
shown, as are the dates the HRV HRV 1953 HCoV-HKU1 2005
crystal structures were defined HRSV 1956 Human bocavirus 2005
and the first HRV genomes HPIV 1956 HRV-C 2006
sequenced HCoV 1965 Swine influenzavirus H1N1 2009
29 Rhinoviruses 677

It was found that several cell lines and methods were HRV infections. The virological and immunobiological cost
required to encompass virus concentrations ranging from of this improvement is a paucity of low passage “wild” HRV
101 to 105 TCID50/mL [44–47] and growth differences isolates to work with; thus, many research findings from
among the different virus types. Additionally, cell age after recent years have employed easy to grow highly passaged
plating (<72 h), inoculum volume (relevant to the culture and adapted HRV isolates. The impact of virus adaptation on
vessel), medium pH (6.8–7.3), and cell density were impor- the reliability of data from use of such viruses is unknown.
tant factors for the reproducible appearance of HRV-induced PCR-based assays have dramatically increased the fre-
plaques and for higher virus yields [48–51]. The HRVs can quency of HRV detection [65–70]. The improved sensitivity
grow at temperatures above 35 °C (some prefer that under and reduced turnaround time have shown that HRVs, as a
certain conditions) [52], but rolling at 33 °C, preceded by a group, are usually the predominant viruses in ARI cases [71–
2–4-h stationary incubation period [41], has historically 73]. With reliable detection levels that extend from as few as
provided the highest yield and fastest in vitro HRV growth 102 TCID50/sample to well above clinically relevant loads,
[36, 50, 53, 54]. PCR can detect virus levels which are commonly shed dur-
Serodiagnosis grew increasingly impractical as the num- ing all stages of experimental infection studies [74, 75].
ber of serotypes increased [49, 55]. However, antibody-based The common understanding of the systemic [76–78] or
methods were essential for type-specific neutralization of symptomatic [79, 80] context of HRV detections was estab-
infection [56] from which early epidemiology data were lished during the era of culture detection, and PCR has chal-
derived and around which the HRV nomenclature system lenged these paradigms by detecting virus more often than
evolved in 1967 [28]. The first classical strains were offi- culture. HRVs are sometimes found in “healthy controls”;
cially named in 1967 [57], the last in 1987 [30]. however, it is likely that with more thoughtful definitions of
Today we know that cell culture-based methods are unre- “healthy,” these detections would reduce. It is not uncom-
liable for accurately representing respiratory virus epidemi- mon to experience a feeling that one is “coming down” with
ology; although enhanced by immunofluorescence, they are something that never develops further. This is likely due to a
still used [58]. The HRV-Cs have not been successfully cul- transient infection or reinfection by an HRV or other respira-
tured in any cell lines or primary cell culture, although many tory virus that is eliminated quickly by the host response. It
attempts have been described [32, 59–62]. In 2011 HRV-C15 is possible to correlate viral nucleic acid load at the sampling
and W23 (another HRV-C) were shown to grow using organ site with disease severity; however, this is made difficult by
culture [63]. Sinus tissue hosted increasing levels of viral the highly variable sampling efficiency of respiratory tract
RNA, as did adenoid, tonsil, and nasal polyp tissue, but much specimens which only permit the generation of reliable
less effectively, as measured by in situ hybridization [63]. quantitative PCR (qPCR) data if serial specimens are
The sinus organ culture system also allowed testing of the available [81].
first reverse engineered HRV-C (pC15) [63]. Isolation identi- The 5′ untranslated region (UTR; Figs. 29.2 and 29.3) is
fied HRVs in ~23 % of adults with ARIs, associated with 0.5 the most common target for diagnostic oligonucleotides
illnesses per year [64]. since the first HRV RT-PCR in 1988 [82], and the region has
retained relevance for virus detection by its adaptation to
reverse transcriptase real-time methods (RT-rtPCR) [53, 65,
3.2 The Molecular Era 66, 69, 74–76, 79, 83–103]. The 5′UTR is comprised of a
number of conserved sequence “islands” (Fig. 29.2) that per-
Because culture is inefficient and subjective and requires mit the robust detection of the majority of HRVs and those
expertise, even for the culturable HRV types, it is becoming “respiratory EVs” which can be regularly detected in the
an art lost to clinical laboratories the world over. It is unsur- respiratory tract [104, 105]. The detection of respiratory EVs
prising that PCR-based methods now prevail, providing a in no way detracts from the importance of supporting clinical
much improved understanding of the nature and scope of decision making using these assays. However, repositioning

A B C D E F
VP4
1 63-83 167-187 307-329 358-373 444-461 541-563 623
Andeweg et al., 1999
UTR
LU, 2008
212nt
GAMA, 1988 JOHNSTON/1993
390nt

Fig. 29.2 A schematic of conserved sequence regions in a generalized HRV 5′ UTR, based on a map described by Andeweg et al. [68]. The PCR
primers of broadly reactive conventional RT-PCR [82, 113] and RT-rtPCR [75, 114] assays are shown
678 I.M. Mackay and K.E. Arden

POLYPROTEIN
STRUCTURAL
NON-STRUCTURAL
P1 P2 P3
5’UTR 3’ UTR
VP4 VP2 VP3 VP1 2A 2B 2C 3A 3C 3D

HRV-C HRV-B HRV-A HEV

+
PRO
PRO ATpase POL
VPg

Fig. 29.3 A schematic of the ~7,200 nucleotide ssRNA genome and essential for genus- and species-level identification are underlined
key regions of a typical HRV member of the genus Enterovirus. The (dashed line) as are those which are more commonly used in the clinical
polyprotein and precursory (P1–P3) and 11 matured peptides are research setting (wavy line). The distinctively located HRV and EV cis-
named in genome boxes and functionally identified underneath. The acting replication elements are shown as stem loop structures and pro-
RNA is polyadenylated at the 3′ end and covalently bound to the virion tease (PRO) and polymerase (POL) functional regions are labeled
protein, genome (VPg encoded by 3B) at the 5′ terminus. Regions (Adapted with permission from McErlean et al. [33])

these primers or changing the method of employing them genetic diversity to confirm the identity of a clinical HRV
[106–109] may undermine assay performance, as evidenced type while also providing broad enough sensitivity to amplify
by predicted hybridization mismatches, uncommonly low the ~160 HRVs from a challenging biological substrate, clin-
detection frequencies [110], and by comparison of multiple ical specimens [128]. Screening of airway specimens for
primer sets using the same specimens [111]. The addition of HRVs is not routine [111] due to factors including cost and
an oligoprobe rtPCR method increases amplicon detection the perceived low clinical relevance of detection. Genotyping
sensitivity and specificity, identifying 100-fold fewer is mostly relegated to research facilities. Because of this,
TCID50/mL or 10 fold fewer genome copies than agarose gel HRV molecular epidemiology studies tend to be smaller and
detection of amplicon [75, 79, 112]. focused on a specific disease or research question.
Other molecular tools, capable of detecting multiple tar-
gets, have evolved in recent years [58, 70, 115–121], and
some have gone on to be approved for clinical laboratory use 4 Biological Characteristics
[122]. Microarrays can detect thousands of viral targets, but
are expensive for routine use (USD30–300 per sample) and Most in-depth molecular studies of HRV replication have
not sensitive enough to avoid a pre-hybridization PCR ampli- focused on a single HRV type. Generally, it is presumed that
fication when using clinical specimens. At their most robust, results can be extrapolated to the other HRV types and to the
microarrays, like PCR, rely on the existence of conserved in vivo situation. HRVs replicate in the cytoplasm (Fig. 29.4)
regions of sequence to detect unknown viruses allowing [129] with membrane-associated replication structures con-
them to detect previously unknown HRV types [123]. High- taining double-stranded RNA (dsRNA) replicative interme-
throughput or “deep” sequencing platforms have become diates (RI) which are formed in cells 4 h after infection [52,
less expensive and more readily available, and they have suc- 130]. Single-stranded infectious RNA forms after RIs start to
ceeded in finding new diversity within the HRV species accumulate [130]. Genomic RNA (plus strand) is the tem-
[124]. The experiments remain costly so have not yet found plate for complementary minus strand synthesis which in
a place for regular screening tasks and remain coupled to a turn is the template for new genomic plus strands that become
need for pre-PCR steps. Rapid protein- or virion-based incorporated into virions [131]. Virions are synthesized from
assays are not (yet) adequately sensitive [125, 126]. 4 to 7 h after infection and reach maximum release levels at
Because of the high number of HRVs and the high fre- 10–18 h [131].
quency of infections, genotyping methods have become an HRV replication in epithelial cells may shut off host cell
essential accompaniment for understanding HRV epidemiol- transcriptional activity via direct cleavage of transcription
ogy. Nucleotide sequencing of the VP1, 5′UTR+VP4+VP2 factors and nuclear pore complex components. Protease 2A
(called hereafter VP4/VP2), or 5′UTR region has replaced (2APRO) of HRV-B2 may directly cleave eukaryotic initiation
traditional serological methods, because of its speed and factor 4G (eIF4G) when bound to eIF4E [132, 133]. The
need for fewer specialized reagents compared to serotyping. eIFs have key roles in initiation and rate control of host cell
VP1 yields the most comprehensive subgenomic genotyping translation [132]. Host cellular protein production is virtu-
information and is essential for the minimal definition of a ally replaced by HRV-B14 proteins after only 6 h of infection
new HRV type [127]. The VP4/VP2 region (Fig. 29.3) is [134]. HRV-B14-infected cells also display reduced nuclear
considered easier to use because it encompasses sufficient importing and degraded nuclear pore complex (NPC)
29 Rhinoviruses 679

CYTOPLASM

NUCLEUS
A
AA

(+) RNA

A
AA

(+) RNA

(-) RNA
ATTACHMENT TRANSLATION
REPLICATION

HRV POLYPROTEIN (+) RNA

CLEAVAGE

MEMBRANE
P1 P2 P3 ASSOCIATED
REPLICATION
COMPLEX
VPO VP3 VP1 VIRAL
PACKAGING

CAPSID
ASSEMBLY

Fig. 29.4 Schematic of a general HRV attachment and entry process. protomers, pentamers, and finally capsids. Nonstructural proteins are
Genome replication in association with membranes produces the viral also released in these cleavages as well as through autoproteolytic
polyprotein which is co- and posttranslationally processed by 2APRO cleavage. Mature HRV virions packaged with an ssRNA genome escape
and 3CPRO into the proteins (P1–P3) and structural peptides (VP1–VP4; by cell lysis (Adapted with permission from Arden et al. [138])
VP2 and VP4 derive from the VP0 precursor protein) that assemble into

components [135]. This may represent another HRV strategy apparent from a wealth of immunobiological data that HRVs
for limiting the host response by preventing or reducing key still efficiently trigger a proinflammatory immune response
signaling pathway molecules (e.g., IRF-3, STAT1, NF-κB) that has considerable clinical impact among at-risk groups,
and shutting down host cell protein synthesis. Protease 3C and that their putative interruption of host cell machinery
(3CPRO) from HRV-A16 targets the nucleus and can disrupt does little to hinder this.
active and passive nucleocytoplasmic transport [129, 136].
Recombinant 2APRO protein from HRV-A16,HRV-A89,
HRV-B4, HRV-B14, HRV-C2, and HRV-C6 exhibited differ- 4.1 The Rhinovirus Genome
ing specificities and kinetics against eIF4G as well as NPC
components demonstrating functional diversity between The virion encapsulates an approximately 7 kb positive sense
HRV types [137]. This finding underscores the functional RNA genome (Fig. 29.3), which tends to be more adenine
diversity within the HRV species and the risk of extrapolat- and uracil (A+U) rich than the EV genome [139]. In particu-
ing too greatly from the study of single HRV types. It is lar, A+U more frequently occupies the third or “wobble”
680 I.M. Mackay and K.E. Arden

Fig. 29.5 The current spectrum of 168 complete HRV complete poly- ducted using MEGA version 5 (Poisson model, 500 bootstraps with
protein amino acid sequences available on the GenBank database. The consensus support shown at the nodes where space permitted [149])
alignment was conducted using MAFFT within Geneious Pro v5.6 (Reprinted with permission from Miller and Mackay [150])
[148]. The phylogenetic and molecular evolutionary analyses were con-

codon position. The single RNA “gene” acts as messenger There are 158 complete HRV polyproteins on the
RNA to encode the single multi-domain, proteolytically pro- GenBank database (Fig. 29.5). The first complete HRV
cessed “polyprotein.” The coding region is bracketed by genome sequence (HRV-B14) was described in 1984 [141]
UTRs which perform regulatory functions necessary for followed by HRV-A2 in 1985 [142] and HRV-A1b in
genome duplication [140]. These are very similar genomic, 1988[143](Fig. 29.3). In 2007 Kistler et al. added 28 genomes
transcriptional, and translational features to those of their [144] and Tapparel et al. 12, including one common to both
close cousins, the EVs. Most of the information currently studies [145]. Sequencing of the VP4/VP2 region was com-
required for virus identification by the International pleted for all classical strains in 2002 [146], and the com-
Committee on Taxonomy of Viruses (ICTV) can be found plete set of 1D regions were available in 2004 [147].
through analysis of the genetic features of HRVs (Fig. 29.3). Currently there are at least 50 named HRV-C VP1 regions
29 Rhinoviruses 681

available and 20 complete HRV-C genomes. Many more (Fig. 29.6) [33]. The HRV capsid shell is composed of 60
genomes are appearing as part of the Rhinovirus Consortium’s protomers, each comprising one copy of the viral proteins
efforts to complete and study the rhinovirome using high- VP4, VP3, VP2, and VP1. VP1, VP2, and VP3 (each
throughput sequencing technologies to genetically charac- ~30 kDa) are to some extent exposed on the capsid surface,
terize HRVs from their combined clinical specimen stores whereas VP4 (~7 kDa) is internalized and associated with
( http://www.international-rhinovirus-consortium.org/ ). viral RNA. Five protomers come together at a point around a
Many 5′UTR and VP4/VP2 sequences reside on the GenBank fivefold axis, and this cluster is called the pentamer. The five-
database, most of which are labeled using in-house labora- fold axis is circumscribed by a cleft referred to as the “can-
tory schemes rather than an approved nomenclature. Analysis yon.” VP1, VP2, and VP3 are each formed by a convoluted
of the full-length genomes supports the use of 5′UTR, VP1, set of protein sheets and loops [159]. The loops protrude
and VP4/VP2 subgenomic regions for useful representation beyond the external capsid surface and contain discontinu-
of HRV species and types [144, 147]. ous antigenic sites. Of the HRV types studied, four neutral-
Recombination, the process of genetic exchange which izing antibody immunogenic (NIm) regions have been
results in a chimeric genome [151], can only be detected in identified on HRV-B14 and HRV-A16: NIm-1A (located in
mature viruses after the fact, and it must therefore be VP1), NIm-1B (VP1), NIm-II (VP2 and VP1), and NIm-III
inferred indirectly through genomic analysis and compari- (VP3 and VP1) [159]. Antigenic sites identified on HRV-A2
son. Predictions of infrequent recombination among the are called A, B, and C [164]. The scope and location of anti-
HRVs [83] have been made based on examination of the genic and immunogenic moieties among the HRV-Cs is
available set of HRV coding and noncoding regions [152]. unknown. Using known receptor binding sequence as a guide
Intensive analyses reported that recombination is not a for computer modeling (Fig. 29.6), it has been predicted that
driving force for the evolution of HRV types [144, 153, when discovered, the receptor for the HRV-Cs will differ
154]. Some discrepancies are likely because of the different from the major and minor receptors defined for the HRV-As
number of sequences used, the different origins of the and HRV-Bs [33].
viruses used for sequencing, and the analysis methods
employed. HRV-C evolution seems to have been more
affected by prior recombination, than is apparent for mem- 4.3 Classification of the HRVs
bers of HRV-A or HRV-B. This is similar to the EV species
but with far fewer predicted recombination events than for The three HRV species within the genus Enterovirus are a
EV evolution [114, 151, 155, 156]. Most of the recombina- genetically, immunogenically, and antigenically diverse
tion proposed to have affected the HRVs occurred between assemblage of >160 viral types (Table 29.1). This accounts
HRV-C and HRV-A and is often found within the 5′UTR or for the combination of HRV-A1a and -A1b, exclusion of
at the 5′UTR/VP4 junction [83, 157, 158] but rarely in cod- HRV-87, which is actually EV-D68 despite confusion over
ing sequence (2A [158] or 3C [83]). The high sequence acid liability [169–171] and combination of HRV-Hanks
diversity among the individual HRV polyprotein coding which is actually HRV-A21 [147]. Serological studies indi-
sequences may keep recombination events to a minimum in cate that some HRV-A and HRV-B types may not be distinct
order to retain viral fitness [158]. The ability of HRVs to enough to deserve a unique identity [147]. Species within the
recombine in practice awaits empirical evidence; the extent genus share >70 % amino acid (aa) identity in the polyprotein
of recombination among all HRV or EV types and the fre- and in 2C+3CD and >60 % aa identity in P1 (Fig. 29.3) as
quency with which viable recombinants arise are entirely well as their host cell receptors, a limited natural host range,
unquantified. a genome base composition (G+C) that varies by no more
than 2.5 %, and a similar compatibility of proteolytic pro-
cessing, replication, encapsidation, and genetic recombina-
4.2 The Rhinovirus Capsid tion [172]. A variant of the same HRV type shares 87–88 %
aa identity or more in VP1 [129]. Much of the nongenetic
The 28–30 nm HRV virion has been visualized for only a criteria remain undefined for the HRV-Cs. In 2008 the genera
handful of HRV-A and HRV-B types (including HRV-A1a, Enterovirus and Rhinovirus were officially combined, retain-
HRV-A2, HRV-B3, HRV-B14, and HRV-A16), but no HRV-C ing the former genus name Enterovirus with the Human
structures have been empirically determined to date. The enterovirus C as the prototype species. A genus in the order
first, HRV-B14, was described in 1985 [159] followed by Picornavirales, family Picornaviridae, is at least 58 % dif-
HRV-A1a in 1989 [160], HRV-A16 in 1993 [161], HRV-A3 ferent in its amino acid identity from any other genus. In
in 1996 [162], and HRV-A2 in 2000 [163]. HRV-C structure 2009 a proposal establishing the species Human rhinovirus
has only been predicted using computer modeling, but their C was ratified by the ICTV. Formal HRV-C numbering com-
basic structure seems to be that expected of an HRV menced in 2010, and type numbers were initially assigned
682 I.M. Mackay and K.E. Arden

VP3
a b NImIII

MAJOR GROUP
VP1
NImIA & IB

VP3
ICAM-1 footprint

VP2
ICAM-1 footprint
+ NlmII

VP1
ICAM-1 footprint NYT

ISL
HRV-C3
MAJOR domains
(ICAM-1)
HRV-B14

c 69 48 26 d
Amino acid homology (%)
VP1
VLDL footprint,
Site A

MINOR GROUP
VP2
Site B
VP1
Site B

NFPK

VP1 A/P
Site C
VP2
Site B

HRV-C3
MINOR domains
(VLDL-R)
HRV-A2

Fig. 29.6 Predicted HRV-C3 pentamers compared to major (HRV- lar adhesion molecule (ICAM)-1 receptor footprints (red) [165, 166].
B14) and minor (HRV-A2) group HRV pentamers which have been Magnified areas of interest (boxed) highlight computer-based compari-
obtained from X-Ray crystallography. (a) HRV-C3 versus HRV-B14 sons to the equivalent HRV-C3 (orange) predicted structures of interest.
SimPlot data projected onto a space filling depiction of the predicted (c) HRV-C3 versus HRV-A2 SimPlot data projected onto the HRV-C3
HRV-C3 pentamer. Shading represents the amino acid identity (26– pentamer. The domains of interest are mostly shown within a single
69 %). The yellow-dashed triangle represents a single icosahedral asymmetric unit. (d) A minor group pentamer (HRV-A2, gray) includ-
asymmetric unit (T = p3 conformation) composed of VP1 and VP2 from ing antigenic sites (sites a–c, green) and very-low-density-lipoprotein
the same protomer and VP3 from an adjacent protomer. The major receptor (VLDLR) footprint (red) [167]. Attachment of the VLDL-R
group domains of interest are divided between two asymmetric units for involves adjacent VP1 molecules. Magnified VP1 area represents one
ease of viewing. Receptor (white) and antigenic (red) sites are shown in half of a VLDL-R footprint [168]. Amino acid substitutions (arrowed)
outline. (b) Bird’s eye view of a major group HRV pentamer in ribbon contributed to the differences between minor group sites b and c
form (HRV-B14, gray) with labeled antigenic neutralization sites (Adapted with permission from McErlean et al. [33])
(NImIA-III, green) and combined HRV-A16 and HRV-B14 intercellu-

based on the date of submission of relevant sequences to intermingling of some HRV-A and HRV-C types [114].
GenBank (HRV-C1, formerly NAT001; HRV-C2, f. NAT045; Nonetheless, careful application of sequence identity thresh-
HRV-C3, f. QPM; HRV-C4, f. C024, etc.; Table 29.1) [127]. olds when comparing clinical sequences to the GenBank
A clinical detection of an HRV-C can be considered a novel database (≥96 % identity required before assigning a clinical
type principally based on its VP1 sequence or provisionally detection to a particular type) succeeds in characterizing
(“C_pat,” Table 29.1) based on VP4/VP2 [146] and could be HRV species and types [9]. There are currently 50 types
confirmed as a variant of a previously characterized HRV-C within HRV-C (which includes the types once grouped
by identity thresholds to either region. The 5′UTR can be together under HRV-“A2,” HRV-X, and HRV-NY clades), 78
and still is used [173, 174] for HRV genotyping, but it is a HRV-A types, and 25 HRV-Bs. The most up-to-date informa-
more problematic region than VP1 or VP4/VP2 because of tion on current taxonomic trends can be found at the ICTV
the recombination activity that affects this region, especially Picornaviridae study group website (http://www.picor-
among the HRV-Cs [175]. This is presented as phylogenetic nastudygroup.com/).
29 Rhinoviruses 683

Table 29.1 ICTV-approved nomenclature for the members of the HRV species
Human rhinovirus
A B C
1M,B 34B 64B 3H,A C3 (f. QPM) C26 C_pat14 (f. SA365412)
2M,B 36B 65B 4A C10 (f. QCE) C27 C_pat15 (f. HRV-CO-1368)
7H,B 38B 66B 5A C1 (f. NAT001) C28 C_pat16 (f. RV1250)
8H,A 39B 67B 6H,A C2 (f. NAT045) C29 C_pat17 (f. RV1039)
9H,B 40B 68B 14H,A C4 (f. C024) C30 C_pat18 (f. RV546)
10H,B 41B 71B 17H,A C5 (f. C025) C31 C_pat19 (f. China/GDYY100/2008)
11H,B 43A 73B 26H,A C6 (f. C026) C32 C_pat20 (f. 202511)
12H,B 44B 74B 27H,B C7 (f. NY074) C33 C_pat21 (f. 202092)
13H,A 45A 75B 35A C8 (f. N4) C34 C_pat22 (f. 20264)
15H,A 46B 76B 37A C9 (f. N10) C35 C_pat24 (KR1868)
16H,B 47B 77B 42A C11 (f. CL-170085) C36 (f. NAT069) C_pat27 (f. PV68)
18H,A 49B 78B 48A C12 C37 (f. NAT059) C_pat28 (f. Cd08-1009-U)
19H,B 50B 80B 52A C13 C38 (f. tu34)
20H,B 51B 81B 69A C14 C39 (f. g2-11)
21H,B 53B 82B 70A C15 C40 (f. g2-25)
22H,B 54A 85B 72A C16 C41 (f. g2-23)
23H,B 55B 88B 79A C17 C42 (f. g2-28)
24H,B 56B 89B 83A C18 C43 (f. 06-230)
25H,B 57B 90B 84A C19 C44 (f. PNC40168)
28H,B 58B 94B 86A C20 C45 (f. PNC40449)
29M,B 59B 95A 91A C21 C46 (f. PNC40449)
30M,B 60B 96B 92A C22 C47 (f. K1091_301104
31M,B 61B 98B 93A C23 C48 (f. PNG7293-3193)
32A 62B 100B 97A C24 C49 (f. IN-36)
33B 63B N13 99A C25 C50 (f. SG1,SO5986)
M and H indicate early cell tropism-based classification (monkey, human) abandoned in favor of a sequential numbering system [177]. HRV types
were later divided into the major and minor groups defined by receptor tropism [184, 185]. Receptor-designated minor group HRV types are
underlined, and major group types are shown in bold. Antiviral groups (A and B) are labeled [165, 194]. HRV-A8 and HRV-A95 are also likely the
same serotype [147]. A full list of genetically close serotype pairings was presented by Ledford et al. [147] HRV-C nomenclature was defined in
2010 and currently includes a number of provisionally assigned types (pat) which are confirmed once preliminary VP4/VP2 data can be confirmed
with VP1 sequence and the provisional number removed (e.g., C_pat1 to C_pat13 have already been reassigned)

Historically a key feature distinguishing the HRVs from the and initiates uncoating [164, 182, 190]. ICAM-1 interacts
EVs was the instability of the HRV capsid in the presence of with its receptor, leukocyte function antigen-1 (LFA-1), and
acid and their lower preferred laboratory propagation tempera- plays a role in recruitment and migration of immune effector
ture (33–34 °C versus 37 °C for EVs). Over time HRVs have cells [191]. The minor group [184] of classical viruses employ
been subclassified in different ways. The first was based on members of the low-density lipoprotein receptor (LDLR) fam-
tissue tropism and host range. HRVs that preferred growth ily to attach to cells [167]. Binding of VLDL-R occurs outside
using monkey cells were called “M” strains and those (the of the canyon employing a different destabilizing and uncoat-
majority) that grew only in human cell cultures, “H” strains ing mechanism. Heparan sulfate may act as a receptor under
[56, 176–180]. These two groups correlate with receptor usage specific conditions [183, 192, 193].
[131] (Table 29.1) and possibly with the titer of the inoculum In 1990 Andries et al. defined, and Laine et al. refined,
employed [181]. In 1962 it was proposed to abandon this ter- two “antiviral groups” (A and B) based on their susceptibil-
minology in favor of a sequential numbering system [177]. ity to a panel of antiviral molecules [165, 194]. These group-
Picornaviruses recognize a variety of cellular receptors ings reflected the nature of the amino acid (and hence
[169, 182, 183]. HRV types are also subdivided into major nucleotide) sequence of the region interacting with the anti-
and minor groups defined by use of one of the two main recep- viral molecules. These antiviral groups can also be visual-
tor molecules [184, 185]. The capsid of the majority of clas- ized using phylogeny [194]. When sequences from other
sical HRVs (n = 89) [184] interacts with the amino-terminal subgenomic regions, including P1, 2C, and 3CD, were
domain of the 90 kDa intercellular adhesion molecule examined by phylogeny, the species were found, in most
(ICAM-1; CD54) [186–189]. Receptor binding destabilizes cases, to inversely correlate with antiviral grouping labels
the HRV capsid, probably by dislodging the “pocket factor,” (Table 29.1).
684 I.M. Mackay and K.E. Arden

r
ea
ar

ea
re

er
CLINICAL ENTITY

dl
ne

ut
id

O
In

M
URT

Nasal cavity
Otitis media
Nasopharynx Rhinitis
Coryza
Oropharynx
URT

Pharyngitis
Larynx Laryngitis
(voicebox)

LRT
Trachea (windpipe) Croup
[laryngo-tracheo-bronchitis]

Bronchus

Bronchioles Tracheobronchitis
Bronchitis

LRT
Bronchiolitis

Pneumonitis

Alveoli

Fig. 29.7 A schematic representation of the human respiratory tract. URT and LRT diseases associated with respiratory virus infection
The upper (shaded pink) and lower respiratory tract (URT/LRT) and the (Adapted from Mackay et al. [200] with permission from Caster
components of the ear are indicated as are the approximate locations of Academic Press)

Today, sequencing and phylogeny play a central role in (Fig. 29.7). The circulation of HRVs varies with population
species classification within the genus, and together, they are age, underlying disease, immunocompromise, over time, and
surrogates for the important biological classification criteria across distance. Circulation is influenced by the nature,
[146, 147, 165, 195–197]. For the HRV-Cs, first described as strength, distinctiveness, and memory of the immune
the “HRV-A2” clade (not to be confused with the single virus, response HRVs trigger and by the nature and prevalence of
HRV-A2, this naming scheme appeared after the HRV-C other concurrently circulating respiratory, and perhaps non-
clade’s name was proposed) of viruses in 2006 [31], sequenc- respiratory, viruses. With the recent discovery of the uncul-
ing of 5′UTR and VP4/VP2 has provided the bulk of HRV turable HRV-Cs came the realization that previous HRV
information from clinical studies. While culture in primary epidemiology was only reliable if conducted by one or more
sinus tissue has been reported [63], no receptor is yet defined. suitably broad-spectrum HRV PCR assays [111]; hence,
prior to 1988, detection of the full spectrum of ≥160 HRVs
did not occur. After 1988, the ability to detect all types very
5 Descriptive Epidemiology much depended on the nature of the PCR primers and detec-
tion methods used. The great number of distinct HRV types
HRVs are the most numerous and frequently detected of all has burdened the search for answers to epidemiology-related
the “respiratory viruses,” so-called because of their predomi- questions. However, as for other important respiratory
nant detection in and tropism for the human URT or LRT viruses including human respiratory syncytial virus (HRSV)
29 Rhinoviruses 685

and the influenza viruses (IFVs), the virus types within a spe- required, only viral nucleic acid which relaxes some limita-
cies show evidence of being both distinct and discrete viruses tions imposed by the need for rapid, careful, temperature-
that are independently recognized by their host and conse- controlled, and expensive transport requirements [64, 203,
quently independently infect their hosts. Each HRV type is 204]. Bronchoalveolar lavage samples are best for seeking
also genetically stable [144]. LRT etiologies, especially in adults where nasal wash viral
The HRV species circulate variably from year to year loads can be low compared to those in children, but this is an
with evidence of epidemics of distinct types. A prospective invasive method with some risk attached [205].
longitudinal cohort study over 6 months examined HRV fre-
quency and diversity in 272 specimens from 18 healthy chil-
dren (0–7 years of age) [198]. A median of three HRVs and 5.2 Host Population Distribution
a maximum of six were detected per child. A similar out-
come resulted from an Australian cohort study [9]. HRVs infect all people, all around the globe. Spread of
Genotyping reveals more of the HRV diversity at a single HRVs is most obvious and frequent from child to child and
site than culture ever could with molecular studies finding from child to parent [206]. In populations of mixed age, the
between 34 and 70 distinct HRVs at a single location [9, majority of HRV detections occur in children [128]. Among
128, 199]. The number of additional HRV cases that occur in 272 specimens from 18 healthy children, over a third
children outside of specifically defined symptomatic periods (37 %) were HRV positive. Children less than 5 years of
remain to be defined, with current studies indicating that a age (44 % of whom were HRV positive) were shown to
much higher number of HRV infections may occur. More have more HRV infections and a wider diversity of HRV
comprehensive investigation of HRV type and illness will types than children more than 5 years old (28 % HRV posi-
be undertaken during analysis of data from the Australian- tive) [198]. Healthy adults in the military [54, 207], at uni-
based Observational Research in Childhood Infectious versity [208], at home [209–212], and in the workplace
Diseases (ORChID) study (http://clinicaltrials.gov/show/ [209] have also featured prominently in historical, culture-
NCT01304914). based, and volunteer infection studies and heavily influ-
Interestingly, the HRV-Bs are often underrepresented, enced our view of HRV infection outcomes [64, 206].
even when accounting for the smaller number of known Although studies of children in hospital-based populations
HRV-B types [128]. A number of studies have not found any usually report more significant clinical outcomes (relating
robust patterns between the circulating HRV types or species to the LRT) [213] than community-based studies, these
and clinical outcome, but the majority of studies seeking this data are still broadly applicable. Hospital populations origi-
information are short and sample infrequently, limiting their nate from the community and reflect the more serious and
ability to find the patterns they seek [128]. perhaps first exposures to the virus. Hospital-based popula-
tions define the potential of a virus to cause severe clinical
outcomes. Disease at this end of the spectrum has the stron-
5.1 Specimen Collection gest influence on future prioritization of therapeutic
research and developments [214].
Studies into the relative sensitivities of nasopharyngeal aspi- Modern air travel contributes to the rapid spread of respi-
rates (NPA) and swab sampling methods produce differing ratory viruses as seen in their often frequent detection among
results, but generally, if seeking the best diagnostic yield for travelers [215] including those with febrile illnesses [216].
as many respiratory viruses as possible (i.e., seeking a labo- Apart from children, HRVs are found with the great clinical
ratory diagnosis to support clinical decision making), NPAs impact in the elderly (described as 60–90 years of age) with
are the sample of optimal choice. One study reported similar 50 % of ARIs positive for an HRV, sometimes with a greater
clinical sensitivities between swabs and NPAs for human burden of disease than IFVs [217]. Those with asthma or
coronaviruses (HCoVs), IFVs, and HRSV, but reduced sen- COPD are also affected by the ARI triggering exacerbations
sitivities using swabs for HRVs, human adenoviruses of wheezing illness (see Sect. 8.2). It is thought that this is
(HAdVs), human metapneumovirus (HMPV), or parainflu- not a different type of infection but rather a different response
enza viruses (HPIVs) [201]. A second study reported no dif- to infection by the host. Wheezing can also result from infec-
ference in sensitivities for HRVs, HAdVs, and HPIVs but a tion in atopic people who do not have underlying asthma or
reduced sensitivity for HRSV and IFVs when using swabs COPD. HRVs cause significant impact in the immunocom-
[202]. Nasopharyngeal washes also yield more viral culture promised, and this group is the only population to date that
success than either nasal or pharyngeal swabs. Nonetheless, has been found to host truly persistent HRV infections (see
many studies use nasal swabs as the sample of choice because Sect. 5.7). Because the HRVs are the largest group of viruses
they allow self-collection and involve much less discomfort to infect humans, it is not surprising that they confuse dif-
than NPAs, and PCR has meant that infectious virus is not ferential diagnoses during pandemics and have key roles in
686 I.M. Mackay and K.E. Arden

co-detections and asymptomatic disease. The study of HRVs 5.5 Coinfection

is the study of all respiratory viruses; while each can be con-
sidered in isolation, this will likely be detrimental to a greater The use of cell and tissue culture underestimated the fre-
understanding of respiratory virus pathogenesis. quency of multiple infections in patients, most likely because
the dominant virus out-replicated any others, or due to viral
load differences, specimen quality issues, differing cell tro-
5.3 Seasonality pisms, or the triggering of an antiviral state by the first virus.
When the majority of respiratory viruses are sought using
HRVs circulate throughout the year but usually with a PCR techniques, multiple virus-positive specimens can com-
bimodal peak in temperate locations in both hemispheres. prise a third of those tested [230], dropping to around a fifth
The highest peaks, mostly defined using adult populations, of ARI episodes when fewer viruses are sought [217]. There
are in the autumn (fall) and spring [64, 66, 211] (and, pecu- is sometimes an emphasis on the high number of HRV cases
liarly, on a Monday [218]). The major winter dip in HRV that are identified in the presence of another virus, and
prevalence closely coincides with the peaks of other respira- including HRV testing does raise the frequency of pathogen
tory viruses, particularly IFVs [219] and HRSV [66]. One detection above one per sample [231]. Coinfections, or, more
hypothesis states that a miasma exists in the school class- correctly for PCR-based studies, co-detections (since PCR
room, of particular relevance to those who suffer asthma cannot determine infectivity), have been found to either
exacerbations, and this miasma maintains immune stimula- increase [71, 232–236] or have no impact on the clinical out-
tion, which subsequently wanes among school children dur- come in their host [237–241], and so the issue of clinical
ing holidays, to be challenged anew upon return to school relevance of co-detections is still uncertain. In extreme cases,
[220]. It is clear that an interplay or interference takes place half of all HRV detections can be found concurrently with
between viruses at the population level, particularly evident another virus. On the surface, this is a significant fraction,
among RNA viruses. and yet 80 % or more of HRSV, HMPV, EV, and IFV detec-
There is a correlation between spiking spring and autum- tions and 71 % of HCoV-NL63 detections can be found in
nal HRV case numbers and an asthma exacerbation “season” the company of another virus [242]. Other studies find differ-
10–24 days after return to school from holidays, in a range of ent, but still higher proportions of co-detections involving
climates [220–223]. This was particularly obvious among non-HRVs [217]. Whether co-detections represent a particu-
asthma hospitalizations of children (5–15 years of age) in lar synergism between the involved viruses, a differential
Ontario, Canada, which peaked at weeks 37–39 across a capability to manipulate the host immune response, a sign of
decade [223]. Upon investigation, HRVs were the most prev- innocuousness for the most frequently involved virus [243],
alent of the viruses found in a 1-year analysis of emergency or a chance due to overlapping seasons remains unclear. It is
room presentations in Ontario [223]. HRVs also predominate clear, however, that co-detections are not an anomaly or an
during “hay fever season” [172]. Although a defined season- error due to “overly sensitive” PCR tests; they are evidence
ality is not always found in the tropics [224], this may some- of further biological complexity that, until recently, remained
times be due to testing that does not include HRVs [222, 225] hidden from us. Recent studies have shown that the initial
or only some HRVs [226]. impression of HRVs being overrepresented in these cases
was incorrect. Closer analysis of viral co-detections has
revealed patterns [231, 244]. These became clear when co-
5.4 Recurrence detections were examined bidirectionally, not just how many
HRVs were positive for virus X but also how many of virus
All the HRV types continue to circulate today, including X cases were positive for an HRV. Whether in a hospital or a
those named in the earliest of the nomenclature assignments. community setting, HRVs more often occur as the sole virus
At a single site during 12–24 months, 70 or more types can detected in ARIs [9, 244]. Considering their ubiquity, it is
co-circulate [8, 9] [174], dropping [198, 227] if the study interesting that relatively low numbers of concurrent detec-
time frame at the site is shortened. A recurring HRV type, tions occur [245, 246], supporting the concept that HRVs
defined using molecular tools, accounted for 1.6 % of any have a direct role in the clinical outcome of their infection
virus detected in a birth cohort followed for 12 months [8] [247]. The HRV partnership with host immunity may be a
and, in another cohort, occurred twice in two children, within mutualistic one, inadvertently imparting an advantage to the
a 6-month period [198]. host by protecting against more cytopathic respiratory viral
Within a given year and across different years, it is appar- pathogens, while the host provides a vessel for HRV replica-
ent that HRV species exchange predominance [9, 36, 60, tion and transmission. Studies of single respiratory viruses
227–229]. No evidence exists to satisfactorily explain this; without being in the context of the respiratory virome are of
however, herd immunity may be a factor. limited value in drawing conclusions about clinical impact.
29 Rhinoviruses 687

5.6 Virus Interference and the HRVs involved an HRV than would have been expected by chance
alone (p ≤ 0.05). For some period, RNA virus infection,
Much of the longitudinal epidemiology data previously especially the HRV group, may render the host less likely to
relied upon to form assessments of HRV significance was be infected by other viruses and, by extrapolating to the com-
acquired using culture-based techniques. With improved and munity level, help constrict the epidemic periods of other
more comprehensive testing, patterns can be seen among the viruses by reducing the number of fully susceptible hosts.
interactions of HRVs and other respiratory viruses. Virus interference as a feature of respiratory virus epide-
Virus interference is a type of virus-virus interaction miology can also be seen in results of other studies [256].
(VVI) that has been known for decades. VVI has recently During an 8-week period that spanned peak 2009 H1N1 pan-
been categorized into types [248]. At the population level, it demic influenza season in Wisconsin, it was influenza A
has been noted that during trials of live attenuated IFV virus (IFAV) that seemed to dominate HRV in children with
(LAIV) vaccines, an interferon (IFN) response was triggered asthma who were sampled weekly [236]. Whether this
that protected vaccinees against off-target viruses for 7 days reflects all IFV-HRV interactions or just those involving a
postvaccination [249]. This 1970 study went so far as to sug- novel IFV such as 2009 H1N is unclear. It was found that
gest such effects could be maintained for a prolonged period PBMCs from these children exhibited normal immune
using a regime of consecutive schedule vaccinations, each responses [236].
separated by 7 days or more, during times of a prolonged
epidemic [249]. A similar effect was produced using live EV
vaccines (LEV) to replace pathogenic EV types and interrupt 5.7 HRV Shedding and Persistence
outbreaks [250]. Orally administered LEVs succeeded in
their principal task but also reduced the incidence of ARIs Reports of subjects with continuous and extended (greater
during epidemics by 50 % overall [250]. This shows that than 2–3 weeks) periods of HRV positivity [3, 257] increased
immune activation in the gastrointestinal system generates as PCR methods replaced cell culture for HRV detection.
an anatomically distinct protective effect and there may be a This had only rarely been recorded using culture [54]. HRV
similar effect on the gut’s inflammatory status after respira- RNA has been detected days prior to symptoms commencing
tory virus infection. In contrast to the LAIV results, the off- and for as long as 5 or more weeks after they cease [3, 258–
target protective effect was reversed in a study using a 261]. Studies that only define the period between ARIs in
trivalent inactivated IFV vaccine [251]. The mechanism children as that time when specimens are RT-PCR negative
underneath these opposing outcomes is unclear. [3] will not detect overlapping serial infections (Fig. 29.9).
During the heyday (1960s) of tissue culture for virus stud- Epidemiology that incorporates HRV typing generally does
ies, a common biological assay for infection with HRV not find chronic shedding [204]. HRV shedding normally
involved attempted infection of the culture with an enterovi- ceases within 11–21 days, after signs and symptoms have
rus (EV) or HPIV-1 [252, 253]. Failure of the superinfecting stopped [3, 9, 44, 75, 85, 260]. Thus, the perception of per-
virus to grow heralded the likely presence of a non- sistence is probably due to serial or overlapping infections
cytopathogenic HRV. Virus interference has been used to by multiple untyped strains [8, 54, 210, 262]. Few studies
measure IFN in specimens through its inhibition of HRV [263] have suitably addressed persistence in HRV infections
growth [254]. More recently HRV-HAdV dual PCR-positive involving healthy subjects since pre- and post-sampling clin-
cases were found less often than expected and harbored ical data are rarely described [80, 264].
lower viral loads of HRV than did specimens from cases of To date, true persistence—an ongoing detection of a sin-
sole HRV infections [255]. Significantly, the majority of gle confirmed HRV type—has been limited to individuals
these instances of VVI involve RNA viruses [244]. It has with underlying immunosuppression or immune dysfunction
been shown that dual infections of peripheral blood mono- [260]. HRV-Cs were detected more than three times longer in
nuclear cells (PBMCs) with viruses other than HRSV immunocompromised young patients than in immunocom-
(including HRVs) induced immune responses similar to petent children, with a mean of 16 versus 53 days [265].
those of single infections, but coinfections including an Multiple detection of the same HRV type (100 % identical
HRSV resulted in reduced IFN-γ responses [71]. VVIs are HRV-1a sequence in each patient over time) extended to
affected by the ability of each to moderate the host response 4 months in hematopoietic stem cell transplant recipients.
against them.
Virus interference has also been identified in virus posi-
tives as a series of patterns among respiratory specimens 5.8 Asymptomatic Infections
tested for up to 17 respiratory viruses (Fig. 29.8) [9, 244].
Statistical analyses supported that many of the co-detections The proof of causality is as difficult to achieve as the proof of
occurred in patterns, in particular that fewer co-detections innocuousness when it comes to respiratory viruses and
688 I.M. Mackay and K.E. Arden

Fig. 29.8 A simplified

representation of the impact of a b
a
first respiratory virus infection on
subsequent respiratory virus
superinfections. Very shortly after
the host is infected, (a) the local
early innate immune response
creates an antiviral state in
neighboring cells (see Sect. 7.1),
perhaps also in distant epithelia,
mediated by circulating immune
cells. The resultant inflammatory
response (b) creates a shield of
sorts, reducing the likelihood of
infection by a superinfecting virus
mediated by viral stress-inducible
gene (VSIG) products

VSIG ACTIVATED
“SHIELDS UP”

ARIs. The definition of “well” subjects prior to or at the time obvious and measurable signs. Strict definitions help improve
of sampling or inoculation is sometimes not clear, especially patient management and the commencement or better direc-
for young children who cannot reliably report symptoms [3, tion of treatment or cohorting. However, in research studies
96, 204]. Often parents notice a symptomatic illness before the arbitrary degree of severity required for reporting a
an infection is detected in the laboratory [3], supporting the symptomatic event often overlooks very simple changes in
importance of diaries in longitudinal home-based commu- host biology due to a virus’s replication. These changes to
nity studies. Nonetheless, even with the support of telephone the norm are mild but nonetheless represent disease (a disor-
interviews and home visits, milder cold symptoms may be der of structure or function that produces specific symptoms
missed. It is not uncommon for an asymptomatic control to or that affects a specific location and is not simply a direct
subsequently become symptomatic or have been symptom- result of physical injury) in the literal sense. Such minor or
atic before sampling [8, 266]. Some studies employ sensitive short-lived, often unrecorded [3], indications of infection
symptom scoring systems [267], but the criteria for being include sinus pain, headache, sore throat, earache, watery
symptomatic are usually designed to describe and clearly eyes, fatigue, muscle aches and pains, and mood changes.
discriminate overt or more “severe” illnesses, those with Within families, HRVs are frequently transmitted from
29 Rhinoviruses 689

HRV-’X’ HRV-’X’
HRV-’Y’ HRV-’Y’
HRV-’Z’ HRV-’Z’

0 (First sample) 1 2 3 4 5 6(Last sample)

Monitoring period
(a) * *
(b) * *
(c) * * * * * * *

Fig. 29.9 The impact of HRV typing and of sampling based only on by the monitoring period. In this case a clinical diagnosis may record
symptoms. The example provided here diagrammatically represents a only a single symptomatic episode. Genotyping may not be performed,
single, hypothetical monitoring period, starting at time = 0, for a single and sampling may be intermittent, and so association between viral
individual. The period of potentially detectable HRV is indicated by an type or species and disease is impossible. In the study examples indi-
open box. If sampling occurred at each time point (0–6) and HRV posi- cated by (a) start and finish sampling or (b) symptomatic sampling,
tives were genotyped, it would be apparent that three different strains (asterisks mark sampling times in filled bars), the laboratory data would
infected the individual, although discerning HRV-X from HRV-Z at have made only one or two identifications, respectively. In the third
time point 3 would require a molecular cloning approach. Illness, in example, (c) frequent sampling of this type has previously led to con-
different forms, may have continued over the entire period depending clusions of HRV persistence or chronic shedding; when combined with
on the symptoms required/recorded and the period of time represented genotyping, it becomes apparent that different HRV types are present

children who are usually symptomatic [204]. Infants fre- disease upon repeat exposure to that virus. It should come
quently exposed to other children have more asymptomatic as no surprise then that HRVs, which usually cause brief
viral infections [8]. Among infected adult family members, infection anyway, could well produce only minor signs and
asymptomatic infections are more likely [204]. Among older symptoms upon reinfection. The unique and extremely per-
parents, whether their children live at home or not, asymp- sonal infection history of each member of a control group
tomatic infections are more frequent following HRV chal- cannot be determined unless they are part of a longitudinal
lenge than among adults without children or in younger cohort. So, what do cohort studies, supported by compre-
parents [268]. In a study of viral species in age-stratified hensive PCR-based testing, tell us about asymptomatic
cases and controls, significantly lower viral loads were found virus infections?
in those without the required symptoms [269]. QPCR may Some cohort studies do not look in asymptomatic chil-
prove useful to determine viral load cutoffs to address this dren, seeking samples only at times of symptomatic illness
issue in the future, although the respiratory tract is a difficult [66, 246, 272]. A birth cohort of children enrolled and sam-
tissue for qPCR [200]. pled when ill and every 6 months for 24 months identified
The high sensitivity of PCR-based methods has raised HRVs 14–28 % of infants and toddlers who had no nasal
concerns over the clinical relevance of a virus-positive symptoms (defined solely by the presence of rhinorrhea)
result [269]. It is clear that a proportion, around five to [273]. The Childhood Origins of ASThma (COAST) birth
28 % of study-defined asymptomatic control populations cohort followed 285 infants at high risk for allergies and
[90, 91, 269], are virus positive using sensitive PCR-based asthma for 12 months and identified HRV infections as pre-
methods. This may vary up to nearly 50 % of cases when ceding (mean age of first detection, 4 months) those of
stratified by age, virus, and season or when including high- HRSV (mean age at least 6 months), and HRVs were found
risk populations [8, 269]. Every respiratory virus, even in 35 % of asymptomatic versus 61 % of moderately to
IFVs and HRSV, can be found in cases without symptoms severely ill patients; the most frequently symptomatic chil-
at the time of specimen collection even after specific inocu- dren also had the greatest proportion of asymptomatic infec-
lation of adults [137, 269, 270]. This is a complex and tions [8]. In a study of 58 children with asthma sampled
incomplete story in need of more research, and so it is frus- weekly for 5 weeks during each of two peak HRV seasons,
trating that positivity in asymptomatic people is often used nearly two-thirds who were virus positive but not sensitized
to rank viral importance. Better data are required from to at least one allergen showed no asthma symptoms, and
asymptomatic controls for any conclusion to be drawn nearly half showed no ARI symptoms; in the children who
about causality [266], but this requirement often disregards were sensitized, less than one-third showed no asthma
the memory of a normal functioning protective host immu- symptoms, and only a fifth had no ARI symptoms [227]. A
nity. It is the host response that defines the degree of clini- convenience population of 15 healthy children (1–9 years
cal severity for the inflammatory disease that is the hallmark old) without asthma were followed during at least three sea-
of an ARI [271]. It is well known that previous exposure to sons, and picornaviruses were detected in 5 % of 740 speci-
a virus affords protection from the full clinical spectrum of mens (21 % of infections) not associated with symptoms,
690 I.M. Mackay and K.E. Arden

although 9 of the 25 infections came from households with 6.2 Self-Inoculation and Virus Survival
an infected sibling [3]. In summary, there is clear evidence
for the presence of HRVs in asymptomatic controls. A pre- It is well known from experimental inoculation studies that
cise proportion cannot yet be defined. Some study controls HRV infection can result from inoculation of the conjuncti-
show signs of a “lead-in” period where RNA positivity pre- val sac after virus is moved through the nasolacrimal duct
cedes an ARI defined on follow-up, while others may have [280]. In these studies virus was commonly delivered by
been defined as symptomatic if more symptoms had been aerosol or intranasal instillation of 0.25 mL to 5 mL of sus-
accounted for. pension [43–46, 280, 285–287]. In the laboratory, HRVs can
retain infectivity for hours to days on suitable, nonporous
solid surfaces, especially if the inoculum remains damp [47,
6 Mechanisms and Routes 287], which supports direct self-inoculation especially in the
of Transmission family setting and indirect inoculation via fomites [288]. In a
trial to define the movement of virus from a contaminated
6.1 Source of Infectious Virus donor to a recipient via multiple surfaces or by hand-to-hand
contact, 13 % (donor to objects to recipient) and 6 % (donor
HRVs have been found at extra-respiratory sites. Viremia to recipient fingers) of the virus recoverable from the donor’s
was determined in the blood of children with LRT infection fingertips were recoverable from the recipients’ [289]. Even
or pericarditis [274, 275], and HRV-C was more commonly under observation, eye rubbing (0.37 h−1–2.5 h−1) and nose-
associated with viremia than was HRV-A, supporting pos- picking (0.33 h−1–5.3 h−1) occur frequently [47, 290], sug-
sible increased pathogenicity [274]. Blood was also posi- gesting self-inoculation could outpace personal hygiene,
tive for HRV RNA and infectious virus from infants at particularly in the young.
necropsy [276, 277], and HRV RNA was detected in the
plasma of children with asthma, bronchiolitis, or common
cold [76]. An HRV was once isolated from feces [203], and 6.3 Airborne and Intimate Contact
more recently higher than expected loads of HRVs were Transmission
detected in fecal specimens from children with suspected
meningitis and fever of unknown origin [77], with gastro- It was once thought strange that ARIs were so common, but
enteritis [278], and in a child with pericarditis [275]. isolation rates for the expected viruses were so low [36, 291].
Nonetheless, the nasopharynx is still considered the main With a better understanding of the importance of preexisting
site of focal virus production [279], regardless of inocula- antibody (something common among the predominantly
tion route [280], and most studies of transmission routes adult volunteers used by many studies), the discovery of a
have centered on the URT. In contrast to IFV and HRSV, third, unculturable species of HRV (still causing ARIs but
HRV infection involves less destruction of tissue. Ciliated impossible to isolate or detect using antibody-based systems
epithelial cells are sloughed off in proportion to the severity for which no reagents existed), and a vastly improved diag-
of an HRV ARI, but this damage is minimal and does not nostic sensitivity, this is much less confounding. In the past,
occur during the viral incubation period or with subclinical household cross infection, determined by ARI, was low,
infections [137, 281]. The incubation period between infec- about five exposures to infected members required for infec-
tion and onset of virus shedding into nasal secretions is tion [17] despite viral loads in nasal washings peaking at
1–4 days with shed viral titers peaking in adults between 1.6 × 105 TCID50/mL [44]. Experimental transmission was
days 2 and 10 [44, 282]. The time until successful HRV also reportedly inefficient [45]. In contrast, “naturally”
transmission among adults in a childless family setting is close-quartered military populations, interacting over
usually 5–8 days and requires the donor to be shedding at 1–4 weeks, experienced rapid spread of HRVs to >50 % of
least 103 TCID50 at some stage, to have recoverable virus on the group [54]. The use of PCR recently clarified this dis-
the hands and in the nares, enough shared time, and a mod- crepancy, confirming that frequent transmission in families
erate to severe ARI [283]. is more common than culture-based studies had identified,
The lungs have been shown to host replicating HRV often resulting in asymptomatic infection among older sib-
[260], and the reader of such reports may be left with the lings and parents [204]. PCR has helped define the scope of
perception that detection of HRV replication in the LRT viral RNA, if not actual infectious virus, survival, and spread.
explains all LRT symptoms. However, relatively few studies Transmission studies require infectious HRV, and so the
seek or identify true HRV replication in the LRT. While HRV-Cs do not contribute to the historical data. Under
the overwhelming majority of LRT cases detect HRV crowded or intimate conditions and with more severe colds,
from the URT, a correlation between URT positivity and transmission reaches 38–100 % [283, 292]. In some studies,
LRT disease does exist [284]. both large- and small-particle aerosols proved inefficient,
29 Rhinoviruses 691

supported by a low isolation rate from saliva (39 % com-

pared to 65 % of hand washes and 50 % of nasal swabs)[44,
HRV
47, 293] and from only 8.3 % of participants exposed to
large-particle aerosols [293]. In other human donor-recipient TLR2

model studies however, aerosol proved to be the main trans-

mission route among antibody-free adults [46, 282]. The dis-
crepancy may have been due to insufficiently long or intense
exposure in the earlier aerosol experiments [45, 267]. Apart
from particle size, spread of virus by aerosol is affected by
existing nasal obstruction which can divert secretions from
the nares to contaminate saliva, the presumptive source of
virus in coughs and sneezes [44]. When exposed to 10 liters
dsRNA
of a small-particle aerosol, 101 TCID50 of HRV-15 was asso- MDA5
ssRNA
ciated with fever and prominent tracheobronchitis in
TLR7/8
antibody-free (<1:2) adult volunteers but not when delivered MyD88
via nasal drops or a coarse aerosol [46]. It has also been
found that simple breathing releases HRV RNA (the same
type was also identified from nasal mucous) from at least a NF-κB
P
third of adults and children with symptomatic ARIs and P
IRF3P
IRF7P
IRF7
IRF3
infectious HRV could be isolated from a fifth [294, 295].
It is apparent that HRVs accumulate at sites with heavy
human traffic, potentially forming a secondary source of
infection. HRV RNA can be detected from 32 % of ~47-hour-
ISGs (TNF, IL-6)
old filters placed to sample air in office buildings [296]. In
aircraft, high efficiency particulate air (HEPA) filters have
been found to harbor HRV RNA more than 10 days after they IFNα,β,λ

were removed for servicing [297].

NUCLEUS

7 Immunity
Fig. 29.10 A simplified representation of molecules involved in or that
HRV infections trigger a vigorous proinflammatory immune respond to the recognition and response to HRV infection of airway
response that is thought to drive the symptoms experienced as epithelial cells [302, 312–317]. IFN interferon, IRF IFN regulatory fac-
illness [271, 298, 299], but they do not seem to actively prevent tor, ISG IFN-stimulated gene, TLR Toll-like receptor, MDA5 Melanoma
or interfere with the host’s immune response the way most Differentiation-Associated protein 6; NF-κB Nuclear factor kappa-
other viruses have evolved to do. There may be a role for light-chain-enhancer of activated B cells, MyD88 myeloid differentia-
tion primary response 8
repeated challenge by HRVs and other respiratory viruses lead-
ing to inflammation and tissue remodeling. The host response
to HRV infection can be broadly broken into the innate (very the innate immune system (Fig. 29.10). Epithelial cells rep-
fast, encoded in the germ line, nonadaptive) and adaptive resent the front line against HRV invasion although alveolar
(slower to develop, reliant on T cells, B cells, and the genera- macrophages and DCs are better equipped to respond [300]
tion of antibody) responses. While the innate system is “always and do so despite not hosting HRV replication directly [16].
watching,” it is significantly amplified by virus infection. The Virus detection is mediated by pattern recognition receptors
adaptive response is initiated by the host’s first infection with a (PRRs) that have evolved to recognize conserved molecular
particular virus and then functions to limit subsequent infec- structures shared among diverse pathogens. Internal- or
tions through the production of neutralizing antibodies and surface-mounted PRRs include sentinels that specifically
amplification of existing cell-mediated immunity. recognize picornavirus RNA and protein and, in doing so,
trigger an immune circuit that results in the production of
IFNs and subsequently hundreds of IFN-stimulated gene
7.1 Innate Immunity and Interferon products. The innate response to viral infection hinges on
inducing two type I IFNs (initially IFN-ß then IFN-α),
After virus-receptor binding and internalization, the earliest secreted cytokines that produce antiviral, antiproliferative,
host cell immune response to an HRV infection is elicited by and immunomodulatory outcomes [301]. The type III IFNs
692 I.M. Mackay and K.E. Arden

(IFN-λ1 or IL-29, IFN-λ2 or IL-28A, and IFN-λ3 or IL-28B) an HRV incursion may depend on the method of virus
are also produced in response to viral infection in a range of approach [319]. TLR7 activation can reduce 2′5′OAS and
cells, although their receptor is not as widespread [302]. The MxA mRNA expression and IP10 protein in adolescents
type II IFN, IFN-γ, is produced by activated T cells and natu- with asthma compared to healthy controls [320]. TLR3 acti-
ral killer cells rather than in direct response to virus [303]. vation did not result in a similar disparity [320].
Detection of viral components triggers protein signaling cas- It has been suggested that HRVs may have evolved with
cades that regulate IFN synthesis through the activation of humans to such an extent that their symbiotic relationship
viral stress-inducible genes (VSIGs) [301, 304]. These are serves to help train the human immune system [321].
sometimes expressed constitutively but upregulated after Intriguingly, within the HRV species, there are differences in
IFN induction following HRV infection [305]. Released the type and level of host response induced [322] which may
IFN-ß binds to the IFN-α/IFN-ß receptor in an autocrine (the reflect receptor usage, route of entry and cell type infected,
same cell) and paracrine (neighboring cells) manner, starting HRV species, or the degree of laboratory-adapted virus used
a positive feedback loop for type I IFN production, the “sec- during in vitro studies.
ond wave.” VSIGs include the antiviral proteins protein
kinase R (PKR), 2′5′OAS/RNaseL, and the Mx proteins
[306]. IFN-α upregulates expression of MxA, 2′4′-OAS, and 7.2 Cellular Immunity and Inflammation
PKR [307]. The Mx pathway is also induced after virus
infection but is not constitutively expressed [307]. Depending After initial HRV infection, the innate response results in
on the sentinel system stimulated, there are different path- production of proinflammatory cytokines, vasoactive pep-
ways to VSIG activation. Those VSIGs with antiviral prop- tides, and chemokines that attract leukocytes, granulocytes,
erties (e.g., MxA, PKR, 2′5′OAS/RNaseL) inhibit different DCs, and monocytes (Table 29.2) [321, 323, 324]. The
stages of virus replication and strengthen an antiviral state in T-lymphocyte response to viral intrusion can be broadly cat-
the host. While this state is well known, the nature of its egorized as TH-1-like and TH-2-like. Other T-cell subsets
induction by different respiratory viruses and the impact of exist, but most work in relation to HRV has been conducted
induction upon the replication of other respiratory viruses on the earliest defined subsets. The TH-1 cellular response is
are topics for considerable ongoing research. important in managing cellular immunity and producing
One pathway to IFN induction relies on the IFN-upregulated interleukin (IL)-2 and IFN-γ. The TH-2 cellular response
cytosolic sentinels retinoic acid inducible gene RIG-I-like manages humoral immunity and stimulates B cells via IL4
receptors (RLRs) RIG-I (specific for IFAV and others) and (initiating production of IgE), IL5 (influencing eosinophils),
melanoma differentiation-associated gene 5 (MDA5, specific and IL13 (crucial component of allergen-induced asthma).
for picornaviruses and others) [306, 308]. These RNA heli- These two T-cell responses act in concert with epithelial-
cases recognize either RNA with a 5′-triphosphate or distinct derived chemokines (e.g., eotaxin) to promote the recruit-
dsRNAs, which results in activation of NF-κB leading to ment and activation of eosinophils and mast cells, contributing
“classical” type I IFN induction [301, 306]. Studies into the to chronic airway inflammation and the hyperresponsiveness
innate response to HRV infection have been limited to the use of airways to a variety of nonspecific stimuli [325]. TH-2
of a very few easily cultured types. It is presumed that the lymphocytes, opposing TH-1 lymphocytes, contribute to an
result can be extrapolated to most if not all types. This is yet to allergic inflammatory cascade, akin to what occurs to rid
be tested. RIG-I is degraded by HRV-A16 [309], IFN regula- humans of parasites [326]. The TH-1 response can also be
tory factor (IRF)-3 homodimerization is interfered with HRV- repressed by binding of microRNA, which leads to an altered
B14 which limits IFN-β induction [310, 311], and MDA5 is balance favoring a TH-2 state in mice and probably in humans
degraded by HRV-A1a but not HRV-A16 [312]. [327]. Regulatory T cells (Treg) suppress allergic inflamma-
Another pathway for recognizing HRV infection involves tory pathways and are therefore fundamental in protecting
the Toll-like receptors (TLRs), transmembrane PRRs that the airway from allergen sensitization [326].
terminate in an intracellular signaling region. The endo- Considerable immunobiological research has focused on
somally localized TLR3, TLR7, TLR8, and TLR9 recognize asthma exacerbation, with which HRVs are intimately
nucleic acids and are also involved in innate antiviral involved. Although upregulated by HRV infection, the TH-1
responses. TLR7 and TLR8 identify G/U-rich ssRNA from response is comparatively deficient in people with asthma
endocytosed viruses, while TLR9 recognizes unmethylated [328, 329]. This is problematic as an increased TH-1-like cyto-
CpG DNA present in DNA viruses [301, 318]. TLR2 and kine response, deduced from higher sputum mRNA IFN-γ/IL5
TLR4 are found on the cell surface and recognize HRV or values, speeds clearance of HRV and symptom amelioration
HRSV proteins, respectively [318, 319], and TLR3 recog- [85]. One possible cause of the TH-1 deficiency in people with
nizes dsRNA. TLRs operate mainly, but not exclusively, in asthma is inadequate maturation of type I and III IFN responses
plasmacytoid DC [301]. The particular TLR that notifies of due to reduced exposure to infections early in life [330]. The
29 Rhinoviruses 693

Table 29.2 Some important molecules involved in the response to HRV infection
Molecule Role
IFN-α/IFN-β (type I IFN) Produced by leukocytes and BECs; numerous subtypes; immunomodulator
IFN-γ (type II IFN) Produced by many cell types after viral infection, especially BECs, PBMC, and DCs; a key TH1 cytokine in
intracellular defense through stimulation of antiviral molecules; macrophage and NK cell activation and
B-cell proliferation
IFN-λ (type III IFN) Participates in creation of an antiviral state; produced by and influences the maturation of DCs
IL-1β Proinflammatory properties; enhances adhesion molecule expression including ICAM-1; induces IL-2
receptor
GM-CSF A granulocyte and monocyte growth factor
IL-2 Stimulates growth and differentiation of T and B lymphocytes and cytotoxic activity of NK cells and
monocytes
IL-4 TH2 differentiation, promotes IgE synthesis
IL-6 Activation, differentiation, and proliferation effects on T and B lymphocytes; induces C-reactive protein
stimulating pyrexia
IL-8/CXCL-8 Neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and
degranulation activity
IL-10 Anti-inflammatory factor produced by monocytes that acts by inhibiting proinflammatory cytokines IL-1,
IL-6, and TNF-α
IRF7 A master hub, regulating antiviral immunity
IP10/CXCL10 Chemoattractant for activated TH1 and NK cells
TNF-α Proinflammatory activity similar to IL-1 β; activates neutrophils; induces vascular permeability
MPC-1 A monocyte attractant
Bradykinin Potent inflammatory mediator, increases vascular permeability
TSLP3 An IL-7-like cytokine that activates myeloid DCs to induce naive T cells into TH2 cells producing IL-4, IL-13,
and TNF-α; induced by HRV in the presence of IL-4
BEC bronchial epithelial cells, DC dendritic cell, IRF interferon regulatory factor, IFN-γ inducible cytokine protein, NK natural killer, PBMC
peripheral blood mononuclear cells, IL interleukin, TNF tissue necrosis factor, TSLP thymic stromal lymphopoietin

“hygiene hypothesis” [331, 332] posits a pathway for an cytokine) and to inhibit the stimulation of IL12 (drives TH-1
asthma etiology described [325] in terms of the young, unchal- development) [337]. However, neither IL10 nor IL12 was sig-
lenged immune system, dependent on infections to stimulate nificantly induced in asthmatic adult volunteers in response
the development of its TH-1-like functions. One theory sug- to HRV-A16 compared to healthy subjects [338]. While
gests that HRVs play a central role in developing that effica- IFN-α was detected after transfection of DCs with HRV-B14
cious antiviral immunity, particularly in infancy, via their ssRNA, low TNF-α and IL12 levels were also noted [16]. It
frequent, usually mild self-limiting infections [333]. was posited that the reduced IL12 could indicate negatively
Genome-wide expression analysis of BECs from healthy affected local immunity possibly predisposing to secondary
and asthmatic adult subjects after HRV-A1a infection revealed infections [337]. Infection of stromal lung cells by HRV-B14
some significant differences that were found between cell triggered exaggerated levels of the pleiotropic IL11 (an IL6-
types and response to infection [61]. These included immune type cytokine), akin to those triggered by HRSV, which were
response genes (IL1B, IL6, IL8, IL1F9, IL24) and airway also detected in nasal secretions from children with wheez-
remodeling genes (LOXL2, MMP10, FN1) and an overall ing [339].
proinflammatory response and metabolic slowdown consis- Other cytokine changes have been identified in atopic
tent with proteolytic cleavage of transcription factors by some adult volunteers challenged with HRV-A16. G-CSF and
HRVs [133, 334–336] in the infected cells. This study further IL8 (chemo-attractant for neutrophils) levels rose in the
noted some similarities to gene expression changes observed URT (as examined by protein detection in nasal lavage) and
in brushings from people with mild asthma after allergen LRT (mRNA detection in sputum) with concomitant rises
exposure and in BAL cells from subjects with corticosteroid- in blood and nasal neutrophil numbers [85, 203, 340]. The
resistant asthma [61]. Overall, HRV replication and the host nasal epithelial cells of atopic individuals, especially in sea-
transcriptional response to it were similar in normal or asth- son, express more ICAM-1 than those of nonatopic adults
matic BEC cells [61]. This indicated, at least in adults, that [341] as do normal subjects infected by the major group
something beyond the epithelial cell is an important contribu- HRV-B14 [341]. By contrast, IFN-γ and IL8, which appear
tor to more severe clinical outcomes in asthma. later postinfection, downregulate ICAM-1 expression in
The application of inactivated HRV-B14 was found to pro- infected cells [191] and encourage infiltration of neutrophils
mote release of IL10 from monocytes (an immunosuppressive [342], respectively. Changes in ICAM-1 levels may modify
694 I.M. Mackay and K.E. Arden

T-lymphocyte-mediated cytotoxic or TH interactions with cross-reactivity to induce broad heterotypic responses

HRV-infected cells, upregulating receptor expression and against many other HRV strains, then an effective vaccine
encouraging eosinophil and T-cell infiltration into the lower could still be possible. In fact, boosting host immunity to an
airways of asthmatic individuals [187, 343]. HRV type by repeat infection does heighten immunity to one
or more other types [44, 353]. The highest of these hetero-
typic antibody titers develop against those types with the
7.3 Antibodies highest preexisting antibody levels [352]. The first descrip-
tion of a unifying HRV numbering system recounted the
Before an HRV can enter a cell, it must pass through a defen- appearance of minor serological cross-reactions, which were
sive barrier of secreted anti-HRV antibody, mostly IgA. The removed by modification of the technique [57]. Subsequently,
ease with which this passage occurs is proportional to the cross-reactions were better defined during experimental
progression of clinical disease. Healthy adult volunteers inoculation when multiple HRV immunogens and antigens
were found to develop IgA by at least 3 days to 2 weeks after were used to deduce the extent of heterotypic responses [56,
inoculation—about the same time as serum antibody—and 210, 352, 354].
retain peak levels for at least 8 weeks [178, 344–346], falling Less promising for HRV vaccinology was the description
faster than serum levels [347, 348]. There is also some evi- of antigenic variation within HRV types which suggested
dence for a degree of nasal immune memory [347]. that immunity to one variant of the type might not protect
Volunteers with pre-study serum antibody could still be against infection by other variants [355, 356]. The “prime
infected in some studies [46, 210, 292], but not in others strain” is a specific antigenic variant of a prototype HRV
[349]. Infection is more clear in volunteers without preexist- type that is neutralized to a lesser extent by antisera from the
ing nasal antibody to experimental challenge virus; they prototype, while yielding antisera that effectively neutralize
become infected, exhibit more severe ARI, and shed more both itself and the prototype [357]. Another form of this
virus for longer [292, 347]. IgA does not seem to modify ill- cross-neutralization is ascribed to the “intertypes,” which are
ness severity or virus shedding, but high levels prevent rein- HRV isolates that share a lower-level serological relationship
fection by the initiating virus type. Low levels or absence of with a pair of HRV strains, which themselves share neutral-
IgA does not prevent reinfection by the same HRV type, izing reactivity, e.g., HRV-A36 and HRV-A58 [358]. The
which may manifest as symptomatic or asymptomatic dis- low-level reciprocal neutralizing activity was not equivalent
ease [349]. in both directions; anti-HRV-A36 sera had a higher titer for
Older children, adolescents, and adults have greater HRV-A58 than anti-HRV-A58 sera did for HRV-A36 [358].
amounts of HRV-neutralizing antibody than young children Over 40 strains were linked directly by such one- or two-way
[42], accompanying a trend toward decreasing numbers of cross-reactions or indirectly through two or more strains.
symptomatic ARIs with increasing age [218, 350]. This fea- HRV-A67 and HRV-A28 are linked via HRV-A11, HRV-
ture raises an issue: did the use of older subjects in many A13, and HRV-A32 (anti-A11 serum neutralizes HRV-A28,
common cold studies underplay the pathogenic potential of anti-A13 neutralizes HRV-A11, and anti-A32 neutralizes
the HRVs because protective or partially cross-protective both HRV-A13 and HRVa-A67[358, 359]). A surrogate
antibodies moderated the impact of infection? Consequently, molecular method which provided insight into these interre-
quantifying levels of type-specific serum antibody became lationships, perhaps expanding upon them to identify useful
routine practice prior to some studies. Adult volunteer patterns for vaccine immunology purposes, would be most
studies determined that no infections resulted if preexisting welcome.
neutralizing antibody titers ≥1:16 existed; as levels grew In summary, heterotypic immunity and HRV intertypes
from 0, so did levels of resistance to infection [43, 210]. might be exploitable features of HRV immunobiology that
Adults were protected by serum titers of 1:3–1:8 [209, 210]. could confer maximum protection upon the host from the
The trend was interrupted by adults in the 20–39 year age minimum number of HRV types [358].
group, presumably because they had begun families and their
young children acquired and amplified currently circulating
types from the community and transmitted them into a 8 Pathogenesis and Host Response
household that was either immune naïve or lacking sufficient
antibody or cell-mediated memory for protection [351]. HRVs circulate in great numbers, and any specific roles for
Traditional vaccine strategies were quickly ruled out as a distinct HRV types in initiating disease remain to be defined.
prophylactic intervention for HRV illness because of the The relatively inconsequential common cold is the most fre-
extensive antigenic variability that is a hallmark of the genus quent manifestation of viral infection in humans, with 30 to
Enterovirus [178, 325]. However, if it were possible to iden- >80 % of colds positive for an HRV [69, 360, 361].
tify “master” strains [352] that exhibit sufficient antigenic Furthermore, ARIs due to HRV infection can exacerbate or
29 Rhinoviruses 695

result in a much greater burden of disease in those with sneeze results from trigeminal nerve irritation; sore throat is
asthma, COPD, or cystic fibrosis. Other complications likely due to the action of prostaglandins and bradykinins;
include otitis media, pharyngitis, and wheeze in atopic peo- and fever, psychological effects, fatigue, and myalgia are
ple without asthma. The role of viruses in the origin of some mediated by cytokines [367]. Hypertrophic adenoids have
of these diseases or their exacerbation is still unresolved. The also been found to have a high proportion of viral, especially
LRT disease may mask the URT nature of the infection, HRV, occupation regardless of host symptomatic state [372].
favoring clinical diagnosis of an LRT illness. Interestingly, Observation of natural culture-confirmed HRV colds in adults
during the 2009 H1N1 pandemic, much of the parent- noted that cough usually started by day 1 and was more per-
initiated healthcare visits from a birth cohort in the United sistent up to 9 days later [264, 373]. Rhinorrhea, sneezing,
States were not due to pandemic virus but HRV and HRSV and sore throat were reported by half or more of patients and
[362]. There is no known natural murine rhinovirus on which headache by at least a quarter of cases [207, 373]. As neutro-
to base a small animal model of HRV infection, and mice are phils accumulate at the site of primary URT infection, the
not natural hosts for HRVs. A recently developed model of myeloperoxidase in their azurophilic granules creates the
airway disease using mengovirus (a Picornavirus infecting yellow-green coloration of nasal mucus that was once consid-
rodents) may yield valuable in vivo airway infection and ered a sign of bacterial superinfection [271, 367]. A common
inflammation data [363]. cold caused by an HRV cannot be clinically distinguished
HRVs are often detected in neonates and infants with LRT from one that caused by any of the other respiratory viruses
signs and symptoms because the very young have narrow, [207, 371]. As is likely for a single HRV type, once the host
immature airways and are more significantly affected by air- has been infected by an HMPV, HPIV, IFV, etc., a secondary
way swelling, excessive secretions, and smooth muscle con- exposure to that same virus type will produce less severe clin-
traction [364]. This may also be due to the relatively naive ical outcomes due to pre-primed host immunity.
immunity of very young children. Much of the more severe
disease in HRV-positive children occurs in the youngest of
them. Some key examples are addressed below. 8.2 Asthma and Atopy

Asthma is a clinical diagnosis made on the basis of patient

8.1 Common Cold history, physical examination, assessment of airway obstruc-
tion or reversibility, and response to bronchodilators [374]. It
For the common cold, as for any illness, accurate epidemiol- is a complex chronic respiratory disease involving airway
ogy and burden of disease data underpin the prioritization of inflammation, airflow obstruction, and airway hyperrespon-
preventing, treating, and further researching the etiological siveness, which manifests as recurrent reversible attacks
agent. To assign funds for researching the agent, health pol- with deteriorating asthma control that are generated by inter-
icy makers also need to understand how efficacious and cost- actions between infectious agents and other environmental
effective the development of an intervention will be [365]. and genetic factors that remain incompletely characterized
The host immune response to HRV replication is the main [375]. The mechanistic role for HRVs in asthma inception
cause of the signs (quantifiable fever, rhinorrhea) and symp- and exacerbation is not yet defined [364, 376] but is being
toms (feeling of fever, myalgia, headache, fatigue, and mood revealed as the extremely complex interplay between inflam-
change) of a cold that the host experiences [321, 366, 367]. mation due to virus versus that due to atopy is explored
A feature of common colds is increased vascular permeabil- [315]. Possible virus-host interactions include (i) severe
ity which, enhanced by kinins, results in increased plasma HRV infection of healthy infants which may result in subse-
protein (albumin and immunoglobulin [Ig] G) levels in quent development of asthma; (ii) HRVs may trigger asthma
mucus, approaching the levels in serum [368]. Histamine in children with a genetic predisposition toward atopy; (iii)
levels do not rise in nasal secretions of otherwise healthy repeated mild infections may protect against more asthmo-
cold sufferers [368]. During the resolving phase of the ARI, genic/cytopathic viruses or the overdevelopment of the TH2-
glandular proteins (lysozyme, sIgA) predominate [346]. type response; and (iv) HRVs may simply exacerbate that
The common cold syndrome is also described as rhinosi- which already exists [377]. It is unclear if the risk of atopic
nusitis (the agglomeration of rhinitis and sinusitis since they asthma during infancy is increased by ARIs which affect the
frequently clinically coexist) [369, 370]. This consists of development of the immune system, or whether ARIs lead to
nasal discharge or rhinorrhea, nasal obstruction, sore throat, asthma development in children with a genetic predisposi-
sinus pain, headache, sneezing, watery eyes, cough, fever, tion to more severe responses to infection [325, 343, 378], or
fatigue, muscle aches and pains, and mood changes [367, a mix of both. In children with asthma, viruses have been
371]. These are caused directly or indirectly by viral infec- detected in at least 77 % of exacerbations (65 % picornavi-
tion; cough is the result of vagus nerve irritation by mucus; ruses, probably HRVs [379]) and in 50 % of adults [380].
696 I.M. Mackay and K.E. Arden

Acute wheezing episodes (including bronchiolitis and is compromised by incomplete tight junctions that are more
acute asthma) are a frequent, epidemic, and seasonal LRT sensitive to airborne pollutants [401] and most likely to aller-
manifestation of URT respiratory virus infection of children gens and respiratory viral infections. This is further specifi-
from all ages, especially during the first year of life [214, cally disturbed by HRV infection which reduces expression
381–385]. Bacteria are not major factors in wheezing exac- levels of tight and adherens junction proteins [402]. In those
erbations [386]. Wheezing is blamed for high socioeconomic with asthma, the presence of an HRV can induce illness that,
and healthcare costs, overuse of antibiotics, being the pri- while often more severe than in non-asthmatics, has been
mary cause of hospitalization among children, and, rarely, associated with significantly different HRV load or duration
for death [109, 387, 388]. of HRV RNA detection in people with asthma compared to
Traditionally HRSV infection has most often been the those without [403]. HRV-C types are often detected in more
virus causally associated with expiratory wheezing, wheezy serious clinical outcomes than HRV-A or -B [265] although
bronchitis, or asthma exacerbations because of the virus’s hospitalizations may be fewer for HRV-Cs than the other
well-known ability to infect the LRT, its more frequent species [404].
detection in some studies [386], and the low perceived likeli-
hood of URT viruses such as HRVs replicating in the warmer
LRT. Nonetheless, periods of epidemic wheezing in the 8.3 Acute Otitis Media
absence of high rates of HRSV detection are common [383,
389]. HRVs even predominated in some culture-based stud- AOM is diagnosed by middle ear effusion (otorrhea) with
ies of wheeze [384, 390]. The COAST study used sampling simultaneous signs and symptoms of ARI including fever,
criteria that were intentionally designed to investigate the earache, rhinitis, cough, sore throat, chest wheeze, nocturnal
role of HRSV in illness, but instead indicated that HRVs restlessness, irritability, poor appetite, diarrhea, and vomit-
were the most important predictor of subsequent wheezing ing. Transient abnormal (negative) ear pressure upon tympa-
in early childhood, and this is supported worldwide [224, nometry occurs in two-thirds to three quarters of
391, 392]. uncomplicated colds among healthy children [405, 406].
The asthmatic airway is characterized by an infiltration of AOM is a frequent reason for outpatient antibiotic therapy
eosinophils and Th2-type T cells (Th2 cells) [393]. In those which can reduce the time to resolution of symptoms in
with an atopic background, eosinophilia was more common, infants and has been attributed to reducing the overall hospi-
and the virus isolation rate was higher than in the nonatopic tal burden of AOM [407–412]. Since a longitudinal day-care
group [394]. The cytokine and eosinophil activation profiles study in 1982, the association between AOM and viral URT
for HRSV-induced wheezing differ from those induced by infection has been coalescing, and it is now clear that AOM
HRV in which IL5 is significantly higher in serum and nasal often occurs with or shortly after a viral ARI, most frequently
aspirates than for HRSV [118]. IP10 was the only cytokine in the young and occurring more often during winter than
significantly elevated in all symptomatic wheezing groups summer [413, 414]. The use of influenza vaccines reduced
[118]. Significantly higher rates of HRV detection with more AOM occurrence by a third during an epidemic period [415],
obvious LRT symptoms are more common in children with but the use of pneumococcal vaccine did not reduce the
asthma than in non-asthmatic populations [380, 388, 395, occurrence of AOM overall, just that relatively small fraction
396]. Exacerbations of asthma are often preceded by a (6 %) due to the target bacteria [416]. The isolation by cul-
symptomatic rather than asymptomatic HRV infection [17, ture and PCR detection of viruses from middle ear fluids and
379, 388, 397–399] although, in some instances, an exacer- the refractory nature of some AOM cases to antibiotic thera-
bation is the only sign of infection [400]. Reduced peak expi- pies confirmed that viruses play an important role in this ill-
ratory volume in children is especially associated with ness [409, 414, 417]. Studies relying on underperforming
detection of respiratory picornaviruses [379]. Severe culture-based techniques underestimated the role for viral
“wheezy bronchitis,” a historical term describing an acute ARIs [414, 418], but other studies using PCR techniques and
illness with preceding ARI and characterized by cough, including HRVs found them to be the most frequently
wheezing, breathlessness, and mucous production, was more detected virus in middle ear fluids and nasopharyngeal secre-
often positive for a virus than mild disease [394]. Even the tions [409, 417].
use of culture found that HRVs predominated in both URT The use of PCR has identified respiratory viruses, most
and LRT (sputum containing BECs) or combined respiratory often HRVs, in nasal secretions of 50–70 % of children with
tract samples [394]. Bacteria were often present with IFV, AOM [66, 413]. Because virus is often detected in the naso-
but not with HRVs [394]. pharynx at the same time as the middle ear fluid, the question
The airway epithelial cells form a physical barrier in addi- of the relevance of a PCR positive is a valid one [419].
tion to their roles in immune surveillance and regulatory Picornaviruses have been detected in 30 % of nasopharyn-
control [393]. However, the asthmatic bronchial epithelium geal swabs taken during cold season from AOM-prone
29 Rhinoviruses 697

infants and young children, and large quantities of HRV Adding the measurement of an inflammatory marker in the
RNA have been detected by in situ hybridization of adenoid serum, like IL-6, further improves the speed of predicting an
tissues from 65 % of children with recurrent AOM and/or infectious etiology for exacerbations of COPD [425].
adenoid hyperplasia [259, 420]. In a cohort of children fol-
lowed from 2 to 24 months and using culture-RT-PCR, HRVs 8.4.2 Pneumonia
in the URT were the second most frequent pathogens associ- Pneumonia is a disease that often occurs early in life, is
ated with AOM, after HRSV [66]. Viruses, most often HRVs responsible for millions of deaths each year [426], and is
(30.8 % of AOM with ARI), were also detected concurrently caused by viral and/or bacterial infections. A diagnosis of
with non-ARI periods associated with AOM episodes (14 % pneumonia requires a radiologically confirmed inflamma-
of AOM without ARI)[418] suggesting that AOM may be tory infiltration of the lung tissue. Childhood community-
the only manifestation of some HRV ARIs, just as wheezing acquired pneumonia (CAP) is common in developing
sometimes is. In the United States, 180 subjects were enrolled countries [427]. CAP also complicates existing chronic med-
and followed in a birth cohort until the first AOM episode or ical conditions and takes advantage of immunosenesence
between 6 and 12 months of age 362]. HRVs accounted for [428].
55 % of viruses detected and 69 % of specimens with a sin- The role of HRVs in contributing to the development of
gle virus detected. This dominance was maintained even bacterial pneumonia is likely underestimated [426, 429].
through the 2009 H1N1 influenza pandemic [362]. Determining an etiology is confounded by the rarity of
In the day-care AOM study mentioned above, primary obtaining LRT specimens, by short-term studies, and by the
acquisition of Streptococcus pneumoniae or Haemophilus complex milieu of viruses and bacteria involved. Less inva-
influenzae had minimal importance as an initiation factor for sive sampling of the URT permits more routine sampling and
AOM with effusion, but nasopharyngeal colonization was screening, and so convenience and reduced risk have led to
important [413]. Animal studies have shown that virus- the detection of putative pathogens in the URT with the gen-
bacteria interactions have a role in nasopharyngeal coloniza- eral assumption that they account for LRT disease, especially
tion and AOM development [414]. Positive correlation has in children under the age of 5 years [430]. Pneumonia studies
been made between HRV detection in AOM-prone children are complicated by the lack of a suitable control group; spu-
and Moraxella catarrhalis infection as well as a tendency tum is not produced from the healthy lower airway and nee-
toward the copresence of Streptococcus pneumoniae [259]. dle aspiration, while a gold standard is also a hospital
The presence of HRV-B14 was shown to increase adherence procedure with some risk [431].
of S. pneumoniae in human tracheal epithelial cell cultures Studies that are comprehensive and use sensitive molecu-
[421]. It is believed that these three bacterial pathogens can lar testing are also rare for the study of CAP etiology. When
colonize without symptoms until a viral ARI shifts the bal- used for CAP investigations, PCR methods almost double
ance toward a cytokine-mediated inflammatory state [419]. the microbiological diagnoses over conventional culture and
serology techniques, especially improving the identification
of mixed infections and fastidious viruses [432]. Rapid diag-
8.4 Other Diseases in Which HRVs Are nosis aids management and helps make decisions about
Often Detected treatment, while prolonged searching for an etiological agent
leads to further invasive testing [256, 433]. At least a quarter
8.4.1 Chronic Obstructive Pulmonary Disease of clinical CAP cases remain unsupported by microbiologi-
This disorder of older patients encompasses emphysema cal findings [241, 432].
(alveolar destruction) and chronic bronchitis (large airway Infections causing pneumonia vary with age and vacci-
inflammation with chronic mucous production) and describes nation status [433]. Viruses can be detected in up to 90 %
a long-term obstruction to airflow in the lung (compared to of infants (1–12 months of age) with pneumonia, and these
asthma which is a reversible obstruction with normal flow cases follow a seasonal pattern [427, 433]. Bacteria can
between exacerbations). While bacteria are found in half of also be detected in over 90 % of infants and older children,
all exacerbations, antibiotic therapies have often yielded the elderly, and those with severe CAP [432, 434]. Studies
poor outcomes [422]. HRV infections result in more COPD that predated the use of PCR pronounced HRSV, followed
exacerbations (~66 % of cases [92]) than any other virus by HRVs, the major viral contributors to CAP, with viruses
identified to date [422, 423]. An experimental human model comprising 27–72 % of childhood pneumonia cases [429,
of HRV infection in COPD provided preliminary evidence 434]. In the PCR age, the role of HRVs has received increas-
that HRVs cause exacerbations [286]. Viral culture associ- ing attention, and they are increasingly the major viral
ated symptomatic HRV infections with exacerbations among group detected from both URT and LRT (sputum) speci-
chronic bronchitics, including cases of isolation from spu- mens of children with CAP. This holds true even when
tum (LRT sample) in the absence of HRV in the URT [424]. studies extend across 1 or more years, which presumably
698 I.M. Mackay and K.E. Arden

would account for seasonal variation in virus prevalence significantly higher risk of recurrent wheezing in the sub-
[241, 256, 426, 434]. It is suspected that viruses such as sequent year than those in whom another virus is detected
HRV prepare the way for subsequent bacterial infection in [446]. HRVs were reported in over fivefold more cases of
some direct or indirect fashion [65, 259, 420, 435]. There bronchiolitis than HRSV among patients in a 2-year pro-
are laboratory data which support this [436] as well as spective cohort of very low birth weight infants in Buenos
observational data showing a high proportion of HRV- Aires, Argentina [448].
bacterial co-detections [256, 437].
Mixed infections including viruses are a possible cause 8.4.5 Sinusitis
of antibacterial treatment failure and sometimes a puzzle After a viral ARI, some proportion of infections may be
for physicians. Mixed infections occur frequently in LRT complicated by sinusitis (inflammation of the sinus
diseases such as pneumonia, which is not surprising since mucosa), the extent of which may be underestimated in
new techniques make it clear that the lungs are not the ster- children if the ARI is mild and unattended by parents [11].
ile environments we once thought [427, 434, 438, 439]. Symptoms may include sinus pain, headache, facial pain,
Viral-bacterial coinfections can comprise 15 % of patients, discolored nasal discharge, postnasal drip, cough, sore
while viral-viral (2–30 %) and bacterial-bacterial (1–7 %) throat, malaise, and sometimes fever (more so in children)
are much less common [230, 241, 256, 434, 437]. HRSV [367, 449]. The precise role for viruses and bacteria in
or HRV is often co-detected with S. pneumoniae in URT sinusitis is still unclear [450]. Sinusitis is a common comor-
samples [256, 437, 440]. HRV detections dominate in bidity in those with asthma [451]. The in situ presence of
younger children with pneumonia during peak HRV sea- HRV-B14 RNA in maxillary sinus epithelium was reported
sons, although frequently in co-detections with other in seven of 14 adults with acute sinusitis [452]. HRVs were
viruses [230]. also detected by PCR in half of adults with acute maxillary
sinusitis; half of the HRV positives were negative for any
8.4.3 Acute Bronchitis bacteria [65]. The common cold is often associated with
Acute bronchitis (less than 4-week duration in children) is computed tomographically confirmed sinus cavity occlu-
defined as a sudden cough that often results from large air- sion or abnormality in adults with self-diagnosed ARIs
way infection and frequently involves viruses. Croup or [367, 453]. Magnetic resonance imaging identified revers-
laryngotracheobronchitis (viral or recurrent [441]) is a com- ible abnormalities of the paranasal sinuses in a third of
mon LRT illness in children that includes the trachea and healthy adult volunteers following challenge with HRV-
larynx as well as the larger airways, resulting in a barking A39 [454]. Further evidence of the tropism of HRVs for
cough. Patients with croup most often have a viral infection sinus tissue comes from it being, so far, the only successful
with some role for HRVs, although the extent of this is host for in vitro HRV-C replication [63].
unclear [441, 442]. Despite testing, a third of cases remain
without a viral etiology [441]. Tracheobronchitis resulted 8.4.6 Cystic Fibrosis
from some HRV-A15 infection of volunteers [46]. Chest pain Culture- and serology-based testing has shown that virus
and cough have been reported in half or more of adults with infections in cystic fibrosis (CF) patients occur with the
HRV infection [207] as well as in children and adults with same prevalence as the general community, but the con-
HRVs detected during exacerbations of bronchitis, with or sequences of infection are more obvious or severe. These
without an associated ARI [443, 444]. include deterioration of lung function, cough, increased
expectoration and weight loss, and a synergistic increase
8.4.4 Bronchiolitis in bacterial growth or acquisition of new bacterial infec-
Bronchiolitis occurs seasonally, especially in winter, in tions [107, 455–458]. The mechanism behind the acqui-
infants (1–12 months of age), affecting the small periph- sition of new bacteria is still unknown and not always
eral bronchioles. Winter is the peak season for HRSV observed [459], but may involve a reduction in the host’s
circulation, but not usually for HRV. Bronchiolitis is a immune response or viral damage to the respiratory epithe-
clinical diagnosis encompassing various disease entities lium. There is circumstantial evidence that HRV infections
and is most often reported in association with detection of have been associated with respiratory exacerbations in
HRSV, a winter virus [445, 446]. However, HRVs make up cystic fibrosis patients [459, 460], albeit in very low num-
the majority of HRSV-negative bronchiolitis cases [128], bers by nonmolecular studies [461] and without a signifi-
and HRVs are co-detected with HRSV for which hospital- cantly different clinical outcome from non-HRV ARIs in
ization is prolonged compared to cases positive for either these patients [107]. Molecular methods have not yet been
virus alone [447]. Those children positive for an HRV applied regularly, thoroughly, and systematically, but they
during a clinically diagnosed bout of bronchiolitis have a generally find HRVs to be prominent among CF children
29 Rhinoviruses 699

with ARI-associated respiratory exacerbation and involved capsid-binding agents (Fig. 29.11). Additionally, oral deliv-
in mixed viral-bacterial infections [459]. ery can complicate drug safety because this route increases
the risk of systemic side effects compared to a nasal or topi-
cal route, but these risks must be considered alongside the
9 Control and Prevention disease to be treated; drug side effects are disproportionately
severe compared to a common cold than to a severe asthma
Hand washing and disinfectant wipes have been shown to be exacerbation. A systemic route is beneficial if an effect is
effective methods of interrupting transfer from fomites to the sought on HRV replication sites that are otherwise inacces-
nose or to conjunctivae [87, 288, 293]. However, with eye sible, such as those not associated with respiratory tract ill-
rubbing, face touching, and nose-picking occurring fre- ness [470].
quently [47, 290], self-inoculation often outpaces personal
hygiene, particularly in the young.
Hand disinfection is frequently recommended for preven- 10 Unresolved Questions and Problems
tion of HRV infection but has not been supported by con-
trolled clinical trials in a natural setting [462] despite good The recent discovery of the new species, HRV-C, has shone
results in experimental tests [463]. Ethanol-containing disin- a bright light on how little was known about the HRVs.
fectants were more effective than simple hand washing with The HRV-Cs and also the newly discovered HRV-As and
soap and water for removal of HRV-A39 inoculum, as HRV-Bs are fastidious in culture, with a single report of
assessed by culture, and the inclusion of organic acids HRV-C growth in primary sinus tissue, and the identity of a
afforded a residual antiviral effect [463–465]. However, con- cellular receptor still unknown. Thus, it is difficult to pro-
tinual hand washing with extra ingredients resulted in skin ceed in many areas, including basic virology, seroepidemi-
irritation [462]. The experimental testing [463, 464] may ology, immunobiology, and antiviral testing. Determination
have been biased by short study periods, the absence of a of the receptors for these new HRVs would aid the search
mucus carrier to mimic natural surface deposition and overly for a more accessible culture system. There would be great
stringent control over virus application/hand disinfection interest in a vaccine for some or all of the HRVs, but with
compared with the natural study. Additionally, the natural increasing evidence of the interactions between HRVs, their
setting study used PCR [462] which detects HRVs more hosts, and other respiratory viruses, it may not be wise to
often than culture. The disparity between outcomes may also interfere before we fully understand what the impact of
reflect the contribution of airborne HRV transmission. losing a constantly circulating HRV challenge would be.
Because of the absence of a vaccine or specific antiviral, Antivirals specifically targeting the HRVs may be a better
the most popular method of intervention in uncomplicated bet, but routine HRV testing and genotyping will first need
HRV ARIs is treatment of the symptoms. This is achieved to be more widespread as surveillance for antiviral resis-
using analgesics, decongestants, antihistamines, and antitus- tance will be an important component of monitoring the
sives. Due to a lack of studies, data are limited on the effec- success of any intervention.
tiveness of over-the-counter common cold medications for Studies to determine whether there are differences in clin-
children [466]. Anticholinergic agents have proven useful to ical and immunobiological impact between the many differ-
reduce rhinorrhea [467]. For controlling symptoms in those ent types are lacking but would greatly improve our ability to
with exacerbated asthma, most of which do not require hos- plan future routine testing, understand all the clinical
pitalization, bronchodilators and oral corticosteroids are the responses to the diverse HRVs and to outbreaks of ARI, and
main treatments [468]. The interruption of proinflammatory improve HRV epidemiology. It is interesting to note that the
immune responses or specific signaling pathways using ste- HRV-Bs are significantly underrepresented in HRV detec-
roids, or other novel therapeutics, may prove to be a more tions. We do not yet know their niche or clinical impact. It
robust approach for treating HRV infections; they have not may be possible that HRV-Bs are the most well adapted of
been successful for HRSV [325]. the HRVs, causing little to no detectable clinical impact, or
When initiated early in the illness, a combination of anti- they may create a different impact than that which we expect,
viral (IFN-α2b) and anti-inflammatory (chlorpheniramine) or they may be a species in decline.
components showed promise for interrupting nasal viral rep- The jury remains out on whether HRVs cause or are
lication and symptoms [469]. involved in the development of asthma or merely trigger
Antiviral agents (Table 29.3) require early application to exacerbations once asthma is established. With a very high
effectively precede the pathogenic immune response to HRV healthcare impact from asthma around the world and atopic
infection [325], but they often fail to reproduce their in vitro conditions that may be exacerbated by HRVs on the rise, this
successes in vivo. Most antirhinoviral drugs are based on is an important area for further investigations.
700 I.M. Mackay and K.E. Arden

Table 29.3 Preventive and therapeutic compounds affecting HRV infections

Therapeutic agent Primary role Effect Evaluation Reference
IFN-α Elicit cellular antiviral effects Decreased shedding if administered Toxicity [471–475]
within 24 h
Pirodavir (R77975) Capsid binder Intranasal formulation useful against Variable efficacy, irritation, [471, 476, 477]
both HRV antiviral groups; three to and mucosal bleeding
six doses per day
WIN54954 Capsid binder Broadly active in mice Reduced efficacy in humans [471, 478]
WIN56291 Capsid binder Active against HRV-C15 Effect only in organ culture [161, 479] [63]
so far
Pleconaril (WIN63843) Capsid binder Resolved symptoms 1–2 days FDA issued “not [471, 477]
earlier than placebo. Some types are approvable” letter because
resistant of side effects
Vapendavir (BTA798) Capsid binder Potent binding in animal models Good bioavailability and [477, 480]
safety profile in animals.
Phase IIa trial complete
Rupintrivir (AG-7088) 3C protease inhibitor Insignificant impact Discontinued [470, 471, 481,
482]
Enviroxime Replication inhibitor Potent anti-replicative activity in Side effects in vivo [406, 472]
vitro
Tremacamra Soluble ICAM-1 molecule Could reduce experimental cold [483]
symptoms and the quantity of virus
shed if administration occurs before
or after inoculation but prior to the
development of symptoms
Tiotropium Anticholinergic agent Reduced HRV-B14 viral load and No obvious change to cell [484]
(bronchodilator) RNA levels, decreased susceptibility viability in culture
of cells, reduced ICAM-1 mRNA
levels and IL-1β, IL-6, and IL-8
protein levels in culture
Levofloxacin Quinolone antibiotic Reduced HRV-B14 and HRV-A15 No obvious cytotoxicity in [485]
viral load (major group HRVs; not culture
the minor group virus, HRV-A2)
and RNA levels, decreased
susceptibility of cells, reduced
ICAM-1 mRNA levels and IL1β,
IL6, and IL8 protein levels in
culture
Pellino-1 Regulates IRAK-1 Controlled primary epithelial cell Did not cause unwanted [486]
non-IFN response to HRV-A1; shutdown of an antiviral
knockdown by siRNA reduced response. Target unknown
CXCL8 in primary BECs
Azithromycin Macrolide antibiotic Significantly increased IFNs and Modest effect in cell culture [487]
ISG mRNA and proteins resulting at relatively high
from HRV-A1 and HRV-A16 concentration. Mechanism
infection in primary BECs. Reduced unknown
HRV replication and release
FDA US Food and Drug Administration, ICAM-1 intercellular adhesion molecule 1, IFN interferon, IRAK-1 interleukin-1 receptor-associated
kinase-1, BEC bronchial epithelial cells
29 Rhinoviruses 701

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methods heat up the cold viruses. Rev Med Virol. 2010;20:156–76.
 Rotaviruses
30
Monica Malone McNeal and David I. Bernstein

1 Introduction 2 History of Rotavirus

Before the use of rotavirus vaccines, rotaviruses were The first rotaviruses to be described, based on pathology
recognized as the single most important cause of severe and epidemiology, were murine strains which were classi-
infantile gastroenteritis worldwide and were estimated to fied under the general description as the agents responsible
be responsible for >600,000 deaths annually [1–3]. for “epizootic diarrhea of infant mice,” i.e., EDIM [10, 11].
Today, rotavirus disease is much less common where the Murine rotaviruses were also among the first to be visual-
vaccines are available, but rotaviruses continue to cause ized by electron microscopy [12], along with viruses
disease and death in the most impoverished nations where obtained from the rectal swabs of monkeys that contained
vaccine is not yet available to most [4]. Transmission of viruses with comparable morphologic features [13]. These
rotaviruses occurs primarily by the fecal-oral route, pro- agents were described as 70-nm particles that had a wheel-
viding a highly efficient mechanism for universal expo- like appearance and were later designated “rota” viruses
sure that circumvents differences in cultural practices from the Latin word for wheel [14, 15]. In 1969, Mebus and
and public health standards [5]. Nearly all children less colleagues demonstrated the presence of these particles in
than 5 years of age have experienced at least one rotavi- stools of calves with diarrhea, thus associating these viruses
rus infection [6–8]. with a diarrheal disease [16]. The correlation between these
The most common symptoms associated with rotavirus viruses and severe diarrhea in young children was reported
disease are diarrhea and vomiting accompanied by fever first in 1973 by Bishop and colleagues, who used electron
[8, 9]. Rotavirus illness can be mild and of short duration microscopy to examine biopsy specimens of duodenal
or produce severe dehydration leading to hospitalization mucosa from children with acute gastroenteritis [17, 18].
and mortality if not treated. Severe disease occurs primar- Within a short time, other investigators confirmed the asso-
ily in young children, most commonly between 4 and 24 ciation between the presence of rotavirus in feces and acute
months of age, and the treatment of rotavirus illness is gastroenteritis.
largely limited to supportive measures such as oral or In addition to their distinctive morphologic features,
intravenous rehydration. human rotaviruses, along with their animal counterparts,
share a group antigen [19, 20] and are classified as mem-
bers of the Rotavirus genus within the Reoviridae family
[21]. In 1980, particles that were morphologically indistin-
guishable from established rotavirus strains but lacked the
M.M. McNeal, MS common group antigen were discovered in pigs [22, 23].
Laboratory of Specialized Clinical Studies, Division of Infectious
This finding led to the identification of rotaviruses belong-
Diseases, Department of Pediatrics, Cincinnati Children’s
Hospital Medical Center, University of Cincinnati, ing to six additional groups (B to G) based on common
3333 Burnet Ave. ML 6014, Cincinnati, OH 45229, USA group antigens, with the original rotavirus strains classified
e-mail: [email protected] as group A [24]. Recent sequencing of the gene respon-
D.I. Bernstein, MD, MA (*) sible for the group antigen has led to additional groups (F
Division of Infectious Diseases, Department of Pediatrics, to H) [25]. Only groups A to C have been associated with
Cincinnati Children’s Hospital Medical Center,
human diseases, and most known cases of rotavirus gastro-
University of Cincinnati, 3333 Burnet Ave. ML 6014,
Cincinnati, OH 45229, USA enteritis are caused by group A strains. Although there have
e-mail: [email protected] been large outbreaks associated with non-group A strains

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 713

DOI 10.1007/978-1-4899-7448-8_30, © Springer Science+Business Media New York 2014
714 M.M. McNeal and D.I. Bernstein

reported in China and Japan, particularly among adults genome segments range in size from approximately 660–
[26–28], group A rotaviruses are the strains to which vac- 3,300 base pairs with a molecular weights of approximately
cine development has been directed. 12,000–125,000 [29]. The known functions of these 12 pro-
teins are briefly described in Table 30.1.
Computer-generated images of rotavirus particles
3 Properties of the Virus obtained by cryoelectron microscopy show it is ca. 100 nm
in diameter and is composed of three concentric protein lay-
Rotavirus is a double-stranded RNA virus. When the genome ers depicted in Fig. 30.2 [32–34].
of rotavirus is extracted from viral particles and separated by The outer layer contains 780 molecules or 260 trimers of
polyacrylamide gel electrophoresis, 11 bands are visualized the VP7 glycoprotein and 60 trimers of the VP4 protein [35–37]
by silver staining as shown in Fig. 30.1. which forms spikelike projections that extend through and
Each rotavirus strain has a characteristic RNA profile 11–12 nm beyond the VP7 layer [32, 33]. The VP4 protein is
or electropherotype. The characteristic RNA electropho- anchored to the intermediate layer of the particle composed of
retic pattern of group A rotaviruses consists of four size the VP6 protein, also arranged as trimers. There are 132 aque-
classes containing segments 1–4, 5 and 6, 7–9, and 10 and ous channels in the outer layer that are positioned over 132
11 [5]. A short pattern is seen when the segment 11 runs channels within the perforated VP6 intermediate shell. Besides
slower than segment 10. interactions between the two layers of VP7 and VP6, both pro-
The genome of rotavirus encodes six structural proteins— teins interact with VP4 in these aqueous channels [38]. The
VP1 to VP4, VP6, and VP7—and six nonstructural proteins, innermost layer contains 120 molecules of the VP2 protein
NSP1 to NSP6 [29, 30]. Each segment encodes one rotavirus that interact with 12 molecules each of the viral transcriptase
protein except segment 11 which encodes both NSP5 and (VP1) and the guanylyltransferase (VP3) along with the 11
NSP6 using alternative open reading frames [31]. The segments of the double-stranded RNA genome [29, 39].

4 Virus Entry and Replication

1
Rotaviruses must attach to and enter a cell prior to replication.
2,3 The VP4 protein on the outer shell is involved in cell attach-
4 ment. Cellular receptors have not completely been determined,
but studies have indicated that sialic acid, heat shock cognate
5 70 protein (hsc70), ganglioside compounds, and integrins,
including α2β1, are probably involved in virus attachment and
entry [40–45]. A recent study has shown that a rotavirus hav-
ing a specific VP4 protein (P[14], see classification below)
5 was able to bind to a glycan characteristic of A-type histo-
6
blood group antigen (HBGA) [46]. Additional studies have
shown that other rotavirus strains having different VP4 pro-
teins, including strains that commonly infect humans, were
able to bind to specific HBGAs found in saliva and milk [47,
48]. HBGAs have been shown to be important receptors for
7 noroviruses. This finding may have importance to determining
8
9 susceptibility to rotavirus infections in the human population.
Rotaviruses are activated by cleavage of the outer capsid
VP4 protein by trypsin-like proteases into proteins termed VP5*
and VP8* which remain associated with the virus. Studies to
determine how rotavirus enters a cell after attachment have sug-
10 gested endocytosis or a process similar to enveloped-virus fusion
as the mechanism for viral internalization, but again, the process
is not fully elucidated [43, 49, 50]. From a number of studies, it
11 has been shown that the outer capsid proteins, VP5* and VP8*,
Fig. 30.1 Polyacrylamide gel electrophoretic patterns of genomic undergo conformational changes that may also involve the VP7
RNAs obtained from group A human rotaviruses and visualized by sil- protein during attachment and entry into the cell [49–52].
ver staining. The patterns demonstrate the characteristic four size
For replication to begin, the outer capsid proteins must be
classes of RNA separated into groups of 4, 2, 3, and 2 segments each.
Human rotavirus strains included (from left to right) lane 1, Wa; lane 2, removed to yield a double-layered particle. The RNA-dependent
248 strain; lane 3, 456 strain; lane 4, DS 1; lane 5, Wa.
30 Rotaviruses 715

Table 30.1 Rotavirus gene segments and properties of the encoded proteins
RNA segment Encoded protein Properties of protein
1 VP1 Structural inner core protein
RNA transcriptase and RNA binding
2 VP2 Structural inner core protein
RNA binding
3 VP3 Structural inner core protein
Complexes with VP1, RNA binding, guanylyltransferase, and methyltransferase activity
4 VP4 Outer capsid protein
Receptor binding, hemagglutinin, and neutralization protein
5 NSP1 Nonstructural protein
RNA binding, possible suppression of host innate antiviral response
6 VP6 Intermediate capsid protein
Group and subgroup antigen, necessary for transcription
7 NSP3 Nonstructural protein
RNA binding and control of viral translation, inhibits host translation
8 NSP2 Nonstructural protein
Multifunctional enzyme activity, interacts with NSP5 in formation of viroplasm
9 VP7 Outer capsid protein
Neutralization protein
10 NSP4 Nonstructural protein
Multifunctional protein, essential for replication, transcription and morphogenesis, enterotoxin
11 NSP5 Nonstructural protein
Binding to NSP2 to form viroplasm
NSP6 Nonstructural protein
Not encoded by all rotavirus strains, RNA binding, role not clearly determined

RNA polymerase (i.e., the VP1 transcriptase) associated with

the inner shell is then stimulated to synthesize the 11 viral
VP4 mRNAs that are capped by VP3 [53–55]. The mRNAs are
extruded from the virus cores through channels in the VP2 and
VP6
VP6 protein layers at the 12 vertices of the viral particles and
subsequently translated into viral proteins [56, 57]. A number of
VP7
studies using the technique of adding “short interfering RNAs”
VP2 (siRNAs) have helped to elucidate the various steps of viral rep-
lication and how the different viral proteins are involved [58].
Two nonstructural proteins, NSP2 and NSP5, have been found
to be involved in the formation of large inclusions or viroplasms
[59]. Particle assembly may be initiated by the formation of
complexes within the viroplasm that contain plus-strand RNAs
from the 11 genome segments along with VP1 and VP3.
Although the mechanism is not fully known, the virus assem-
bles and packages one of each of the plus-strand RNAs within
individual precursor viral complexes. Upon interactions with
VP2, the minus-strand RNA is synthesized and the 11 ds RNA
genome segments are produced [60]. VP6 trimers interact with
Fig. 30.2 Computer generated image of the triple shelled rotavirus
particle obtained by cryoelectron microscopy. The cut away diagram the VP2 core and a double-layered particle is completed [61–
shows the outer capsid composed of VP4 spikes and VP7 shell, inter- 63]. The double-layered particles then bud into the rough endo-
mediate VP6 shell, and inner VP2 shell surrounding the core contain- plasmic reticulum (ER) after their association with the NSP4
ing the 11 double stranded RNA segments and VP1 and VP3 proteins. transmembrane glycoprotein [64]. VP4 is either added prior to
(Courtesy of Dr. B. V. V. Prasad, Baylor College of Medicine,
Houston, TX.) entry into the ER or sometime thereafter. The other rotavirus
716 M.M. McNeal and D.I. Bernstein

glycoprotein VP7, which becomes sequestered within the rough produced can contain genes from either parent. Although
ER, is added to complete the formation of mature viral particles numerous human isolates appear to be animal strains or
[65, 66]. These mature viruses accumulate within the lumen of animal-human rotavirus reassortants as determined by
the rough ER until cell lysis occurs. sequence analyses, the number of cross-species infections
appears to be low [88–91].

5 Serotypes
6 Epidemiology of Rotavirus
The two outer capsid proteins, VP7 and VP4, contain the only
neutralization epitopes for this virus [67, 68]. Early studies 6.1 Disease Burden
using sera from hyperimmunized animals in cross-neutralization
assays described a number of different serotypes from infected 6.1.1 United States
humans and animals based on the VP7 protein [68, 69]. Viruses Before the use of vaccines, nearly every child was infected
were given a G type referring to the glycosylated structure of with rotavirus by the age of 5, and rotavirus illness resulted
this protein. When animals were hyperimmunized, most of the in 20–70 deaths annually in the United States [92–94].
neutralizing antibody was directed against the VP7. However, Infection with rotavirus often resulted in more severe illness
after oral infection, VP4 appeared to be the dominant neutral- than other pathogens, and therefore, rotavirus accounted for
ization protein [70–73]. A dual serotyping scheme for both a higher percentage of gastroenteritis episodes requiring
VP7 and VP4 was therefore developed. medical intervention [95, 96]. During peak rotavirus sea-
VP7 serotypes are determined by cross-neutralization and sons, 70 % of all gastrointestinal hospitalizations were due to
by using panels of monoclonal antibodies [74–76]. VP4 rotavirus-associated gastroenteritis [97]. It is estimated that 1
serotype determination, however, is more difficult [77–79]. child in 7 required a doctor’s visit at a clinic or emergency
Two numeric systems were established to classify the VP4 room, and about 1 in 75 were hospitalized because of a rota-
protein, or P type referring to the protease sensitivity of this virus illness [93, 98]. Based on these estimates, rotaviruses
protein. The P serotype is based on neutralization assays were responsible for 5–10 % of all gastroenteritis episodes in
using antisera against recombinant-expressed VP4 proteins children under 5 years of age leading to over 50,000 hospi-
or viruses with specific VP4 genes [80, 81]. A second clas- talizations. Depending on how hospital surveillance studies
sification system, one that is now more widely used to char- were conducted, these estimates may be lower than the actual
acterize rotavirus isolates, is based on sequence analyses, numbers of rotavirus-related hospitalizations [92, 99, 100].
and rotaviruses are described as different genotypes [82, 83]. Similar estimates of the disease burden due to rotavirus have
The P serotype is indicated by an open number and the geno- been reported in studies conducted in European countries
type is noted with a bracketed number. For example, the [101–103].
most common P type worldwide belongs to serotype P1A
and genotype 8 and is therefore designated P1A [8]. 6.1.2 Worldwide
Currently, rotaviruses are most often only classified by geno- Globally, rotavirus disease has an even more dramatic and
typing for the P protein, i.e., P [8]. To date, 27 G types and significant impact on infant health. Although rates of rotavi-
35 P types have been identified on the basis of sequence rus illness among children are similar throughout the world,
analyses from animal and human isolates [84–86]. Recently, the resulting mortality differs substantially. It is estimated
all 11 gene segments of rotavirus isolates have been that rotavirus illness is responsible for over 600,000 deaths
sequenced. The data from this analysis have importance in annually, representing 5 % of all deaths in children younger
determining the evolution of different rotavirus strains [87]. than 5 years of age worldwide [2]. More than 90 % of these
However, as discussed below, only a few G- and P-type com- deaths occur in Africa and Asia (Fig. 30.3). More than
binations account for the vast majority of human infections. 100,000 deaths occur in India and sub-Saharan Africa and
Rotaviruses, similar to other viruses with segmented 35,000 occur in China [2, 104, 105].
genomes, have the ability to form reassortants. This ability to
reassort has contributed to the diversity of G and P types
found throughout the world. In addition, as will be discussed, 6.2 Distribution of Human Rotavirus
a number of reassortant viruses have been used as vaccine Serotypes
candidates including the currently licensed vaccine,
RotaTeq™. During replication, if a coinfection has occurred, Early studies using cross-neutralization assays and monoclo-
gene segments from each parent virus can be incorporated nal antibodies to determine serotypes found that four main G
into new progeny viruses. The gene segments from different types (G1, G2, G3, and G4) accounted for 90 % of the strains
viruses can segregate independently, and the new viruses isolated from humans. However, with the development of
30 Rotaviruses 717

Fig. 30.3 Each dot on this figure represents 1,000 deaths. It is estimated that there are over 600,000 deaths worldwide. Edited from Parashar
et. al. Emerg Infect Dis. 2003;9:565–72

surveillance studies using reverse transcription-polymerase from 52 countries on five continents. The four common G
chain reaction (RT-PCR) genotyping and automated nucleo- types, G1, G2, G3, and G4, with either P[8] or P[4], repre-
tide sequencing of VP7, for G type, and VP4, for P type, sented over 88 % of the strains identified worldwide during
more strains have been identified and genotyped. The global this period. Serotype G9, containing either P[8] or P[6], was
distribution of G and P serotypes can vary greatly by location found to be an emerging strain in a number of different loca-
and time or, as was found in some studies, vary little over tions. In the second review by Gentsch et al. [85], data from
sequential seasons [74, 106]. Over 42 different P-G combi- studies conducted in 35 countries, involving over 20,000
nations of human strains containing 10 different G types and strains, were analyzed. In this study the four common strains,
11 different P types have been described [85]. G1P[8], G2P[4], G3P[8], and G4P[8], represented 72 % of all
In North America, Europe, and Australia, the G1P [8] strains with G9 containing either P[6] or P[8] identified in
strain has been responsible for over 70 % of rotavirus infec- over 2 % of the isolates. In South America and Asia, the inci-
tions. In European countries, G1, G2, G3, G4, and G9 were dence of G1P[8], about 30 %, is lower than in the United
the predominant G serotypes found, although the incidence States and Europe, while in Africa, it was even lower (20 %).
varied by country [107]. Similar findings were reported from In Latin America [110], the most common G types were G1
Central and South-Eastern Europe [108]. In the United (34.2 %), G9 (14.6 %), and G2 (14.4 %), while the most com-
States, the most prevalent strain in the pre-vaccine era was mon P types were P[8] (56.2 %), P[4] (22.1 %), and P[1]
G1P[8] (78.5 %) followed by G2P[4] (9.2 %), G9P[8] (5.4 %). A recent review of rotavirus infections in Africa sug-
(3.6 %), G3P[8] (1.7 %), and G4P[8] (0.8 %) [109]. Rarer gested that strain diversity was increasing and identified a
genotypes included G2P[6], G6P[9], and G3P[9]. These total of 24 P/G combinations [111]. A review of studies from
studies also demonstrate that strain circulation can vary China showed that G1 was the overall predominate strain but
greatly in the same location over time. that G3 viruses were becoming more common [112, 113].
A more global perspective has been described in two In summary, although the most common G types still
reviews. A review by Santos and Hoshino [84] summarized remain, G1 through G4, common strains now also include
rotavirus serotypes from 124 studies performed between G5, G8, G9, G10, and G12 (especially G9) in various regions
1989 and 2004 and included data on 45,571 strains collected around the world [109–111, 114, 115]. The most common
718 M.M. McNeal and D.I. Bernstein

types for the P protein are P[8] and P[4] but also include the infected with rotavirus than other causes of gastroenteritis,
more recently described P[6] and P[1] as well as P[9] and while in another study, the severity of rotavirus diarrhea was
P[14]. The effect of rotavirus vaccine introduction in coun- almost twice that of other causes of diarrhea [136]. The dis-
tries around the world on the diversity of circulating rotavi- ease lasts about 4–8 days, with a range between 2 and 22
rus strains remains to be determined. Surveillance programs days [137], while virus shedding has ranged from 4 to 57
are important to monitor the changes in rotavirus strains. days with a peak at about day 3 [138, 139].
Virus replication was initially thought to be limited to the
intestine, but it is now known that both antigenemia and vire-
7 Effect of Age and Seasonality mia are common [140–145]. The dissemination of virus may
be responsible [142] for other clinical manifestations includ-
Severe human rotavirus disease occurs most commonly ing encephalitis and meningitis [146, 147], upper and lower
between 4 and 24 months of age [7, 116], but the age may be respiratory infections including otitis media and pneumonia
lower in less developed countries [117, 118]. Neonatal rota- [134, 148, 149], and mild hepatitis [150]. Recently, cases of
virus infections, which are often asymptomatic, occur and encephalitis have been confirmed by identification of rotavi-
appear to be endemic in some newborn nurseries [119, 120]. rus RNA in the cerebrospinal fluid using reverse transcription-
The asymptomatic nature of neonatal rotavirus infections is polymerase chain reaction (RT-PCR) analysis [147].
due, at least partially, to protection from transplacental anti- Rotavirus infections are also often more severe in immuno-
body that can persist for the first months of life. The onset of suppressed persons, including bone marrow transplant recip-
rotavirus disease in infants has been reported to coincide ients, patients infected with human immunodeficiency virus,
with the decline of maternal IgG antibody [121]. and those who are malnourished.
Older children and adults, including elderly patients, are The treatment of rotavirus gastroenteritis is mainly
susceptible to reinfections with rotavirus. Infections typi- supportive, aiming to restore fluid balance in dehydrated
cally cause mild disease but rarely can be severe and require patients. Emphasis has shifted away from intravenous rehy-
hospitalization [122–126]. The reduced severity of rotavirus dration to oral rehydration with glucose-electrolyte solu-
disease in older children and adults is due primarily to the tions. However, should oral rehydration efforts fail, or in
immunity induced by previous rotavirus infections. One of cases of severe dehydration and shock, fluids are admin-
the interesting and somewhat unanticipated results of the istered intravenously. Other experimental approaches to
introduction of rotavirus vaccines has been the impact on treatment include bismuth subsalicylate [151, 152] and
disease in older children and even adults [127]. probiotics (reviewed in [153, 154]). Racecadotril (acetor-
As with other respiratory and enteric viruses, distinct sea- phan), an enkephalinase inhibitor with antisecretory and
sonality is associated with rotavirus disease [29, 128–131]. antidiarrheal properties, has been shown to be effective in
This seasonality is particularly evident in temperate climates, the treatment of diarrhea in adults and children [155–157],
where rotaviruses are responsible for the large increase in while another drug, nitazoxanide, licensed to treat Giardia
hospitalizations and deaths due to diarrheal diseases found intestinalis and Cryptosporidium parvum infections [158,
during the winter season [97]. The seasonality of rotavirus 159], has shown some effectiveness in the treatment of
disease is less apparent in tropical climates, but disease is still rotavirus infection.
more prevalent in the drier, cooler months [129]. The cause
for the seasonality of rotavirus disease remains unknown.
9 Pathogenesis

8 Clinical Disease and Treatment The incubation period for rotavirus is approximately 1–3
days [132]. In children with symptoms, the onset is often
Following an incubation period of 1–3 days [132], the onset abrupt, with fever and vomiting followed by explosive,
of rotavirus disease is often abrupt with fever and vomiting watery diarrhea. Vomiting may precede the diarrhea in
followed by explosive, watery diarrhea. About two-thirds of approximately half the cases [160]. Fever occurs commonly
children with rotavirus disease have diarrhea, vomiting, and during rotavirus illness, with estimates of between 45 and
fever [133–135], but children can also present with any two 84 % of patients [134, 135]. The disease is usually self-
or only one of these symptoms [9]. Stools are non-bloody limited, lasting 4–8 days, although the duration of symptoms
and generally lack fecal leukocytes, but mucus may be found. ranged between 2 and 22 days in a Guatemalan study [137].
Rotavirus infections are typically more severe than those In a study conducted in the United States, 63 % of hospital-
caused by other viral gastrointestinal agents and therefore ized children with rotavirus infection had fever, vomiting,
more likely to result in hospitalization [134, 135]. In one and diarrhea at presentation, but children also presented with
study [134] vomiting and diarrhea lasted longer in patients combinations of only one or two of these symptoms [9].
30 Rotaviruses 719

After fecal-oral transmission of rotavirus, infection is initi- replication of rotavirus had been shown, the general assump-
ated in the upper intestine and typically leads to a series of tion was that rotavirus pathology is strictly intestinal.
histologic and physiologic changes. Studies in calves revealed However, instances of non-gastrointestinal rotavirus-associ-
that rotavirus infection caused the villus epithelium to change ated disease, including the association with abnormal liver
from columnar to cuboidal, which resulted in a shortening function, as well as respiratory and nervous system involve-
and stunting of the villi. The cells at the tips of the villi ment were reported [175–180]. This spread was explained
became denuded, while in the underlying lamina propria, the when it was reported that both antigenemia (presence of
number of reticulum-like cells increased and mononuclear rotavirus protein in the blood) and viremia (presence of live
cell infiltration was observed. The infection appears to start at rotavirus in the blood) were found during rotavirus infection
the proximal end of the small intestine and then advances dis- in several strains of animals and in humans [140]. Since that
tally [161, 162]. From the few studies examining the patho- time, this observation has been confirmed by a number of
logic changes in the intestines of humans, the changes studies in humans [144, 145, 181–184].
appeared to be similar to those found in animals [163, 164].
Although rotaviruses cause severe diarrhea in numerous
species, including humans, the mechanisms responsible for 10 Immunity
the illness have not been completely determined but are due
to multiple factors (reviewed in [165]). From various studies After more than 30 years of research involving studies of
in animals, malabsorption of carbohydrates, sodium and cal- natural rotavirus infection, vaccine studies, and animal stud-
cium fluxes, retarded differentiation of uninfected entero- ies, the mechanisms of immunity to rotavirus infection remain
cytes, activation of the enteric nervous system, and toxin incompletely understood. Humoral antibody responses
(NSP4) have been found to be involved in causing diarrhea. include an early IgM response followed by the production of
It is clear that infection of mature enterocytes of the small rotavirus IgG and IgA [185, 186]. Infection also induces local,
intestines leads to damage and functional abnormalities, but intestinal antibodies that are predominantly IgA [187–190].
the degree of damage does not appear to correlate with the T cell responses have been difficult to measure in humans, but
severity of diarrhea. Further, diarrhea can occur before evi- several rotavirus proteins have been identified that contain T
dence of intestinal pathology. Thus, although damage to the cell epitopes and have been used to measure T cell responses
mature enterocytes leads to loss of the enzymatic activities of after rotavirus infection [191–193].
the brush border leading to malabsorption of electrolytes and As stated before, virus neutralization epitopes are found
glucose/amino acids, other mechanisms are also involved. on the VP4 and VP7 proteins located on the surface of the
Diarrhea has been induced in infant mice and rats by virion, and neutralizing antibody to the infecting virus can be
intraperitoneal inoculation with the rotavirus NSP4 protein detected after infection or vaccination. However, VP6
as well as with a 22-amino acid peptide derived from this appears to be the most immunogenic protein, although anti-
protein [166, 167]. NSP4 possesses membrane destabiliza- body made against VP6 is not neutralizing. One of the
tion activity that may result from increased intracellular cal- important questions concerning the development of immu-
cium concentrations, resulting in cytoskeleton disorganization nity to rotavirus infection is whether or not neutralizing or
and cell death [168–170]. NSP4 is also an enterotoxin that serotype-specific antibody is necessary for protection.
binds to surface receptors and initiates signal transduction Studies evaluating natural rotavirus infections were used ini-
pathways that leads to mobilization of intracellular Ca and tially to understand rotavirus immunity and the role of
chloride secretion. NSP4 increases the levels of intracellular serotype-specific antibodies. Many investigators reported
calcium [171] by activating a calcium-dependent signal that natural rotavirus infections produce incomplete protec-
transduction pathway that mobilizes transport of this ion tion, but most showed that previous infections protected
from the endoplasmic reticulum [172, 173]. against severe disease [194–199]. In a large study conducted
Another factor that may have a role in rotavirus-induced in Mexico, protection from both rotavirus reinfection and
diarrhea is the enteric nervous system underlying the villus rotavirus diarrhea increased with each new infection [198].
epithelium. It has been reported that rotavirus infection can However, sequential infections even with the same serotype
activate this system in mice and drugs that block nerve activ- have been reported. In an early study reporting rotavirus dis-
ity, attenuate rotavirus-induced fluid secretion in vitro, and ease with reinfection by the same serotype, the investigators
attenuate diarrhea in vivo [174]. Whether this is a major noted that protection of young children in a Japanese orphan-
mechanism of diarrhea occurring after rotavirus infection in age lasted 6 months then declined after 1 year. This study
humans remains to be determined. noted a close correlation between titers of serotype-specific
The tissue tropism of rotavirus infection in humans was antibody and protection [200]. This study is often cited to
thought initially to be restricted to the villi of the small intes- support the idea that neutralizing antibody is necessary for
tine. Because no consistent evidence of extraintestinal protection. While it is generally accepted that if present at a
720 M.M. McNeal and D.I. Bernstein

sufficient titer, neutralizing antibody will protect, it is still strains circulating, overall health of the child, or duration of
unclear whether only neutralizing antibody is necessary for protection. Studies of the monovalent G1P[8] vaccine,
protection. Rotarix™, discussed later, provide strong support for cross-
Although reinfections with rotavirus are common, other protection between serotypes.
studies have shown that protection lasts at least 1 year. Due to the difficulties associated with human studies, ani-
Neonates infected within the first 2 weeks of life were pro- mal models using mice, rabbits, and gnotobiotic pigs have been
tected against severe disease but not against reinfection in one developed [203–205]. Early studies in mice showed that while
study [119]. In another study, infants who developed a symp- CD8 T cells are involved in the resolution of an infection, anti-
tomatic or an asymptomatic rotavirus infection during the first body is necessary for protection against reinfection [206–208].
year of the study were protected against contracting a subse- In one study, mice given an oral immunization of a fully hetero-
quent rotavirus illness or even an asymptomatic reinfection typic rotavirus were almost completely protected from shed-
during the following year [195]. Similarly, in another study, a ding when later challenged, and this protection was dependent
natural rotavirus infection in the first year was found to be on the ability of rotavirus-specific IgA to be transported through
93 % protective against a symptomatic reinfection in the sec- intestinal epithelial cells or be present at the intestinal mucosa
ond year. This protection occurred even though the G1 strains [209]. In a recent study using mice deficient in IgA, shedding
that circulated during the first year were responsible for only of murine rotavirus after primary infection was prolonged, and
66 % of rotavirus disease in the second year [197]. In a recent mice were not protected against subsequent infection with the
cohort study conducted in India, it was found that rotavirus same strain of virus [210]. The role of rotavirus-specific serum
infections generally occurred early in life with 56 % of chil- IgG is not clearly defined but has been shown in some animal
dren infected by 6 months of age. Reinfections were common, models, using passive transfer of IgG antibodies, to have some
and protection against moderate or severe disease increased protective effect [211]. If these animal studies mirror what is
with the order of infection. However, protection was only needed in humans, then local antibody may provide protection
79 % after three infections and there was no evidence of and may involve antibody that is not serotype specific.
homotypic protection [201]. Protection from the current vac- Serum levels of rotavirus-specific IgA appear to be a good
cines appear to last at least 2–3 years (discussed below). correlate of protection, because it appears to parallel anti-
Correlations of serotype-specific neutralizing antibody and body levels in the gut [190, 212]. However, studies in vacci-
protection have not been supported in other larger studies. In the nated infants showed only a low correlation between
largest study, conducted in Bangladesh during a 2-year period protection from disease and circulating rotavirus-specific
when the four major G serotypes circulated, the titers of both memory B cells expressing α4β7 intestinal homing pheno-
preexisting homologous and heterologous neutralizing antibody type [190, 212, 213].
were significantly lower in patients with acute rotavirus disease
than in matched control subjects. Further analysis, however,
could not find a correlation with serotype-specific neutralizing 11 Control and Prevention
antibody, and protection seemed to correlate better with the
magnitude of the response rather than with specific neutralizing 11.1 Rotavirus Vaccines (Table 30.2)
responses [202]. Differences in the studies evaluating the role of
serotype-specific antibody and the protection provided by natu- Public health institutions around the world made the devel-
ral infection may be due to many factors including the viral opment of an effective rotavirus vaccine a high priority [214].

Table 30.2 Selected rotavirus vaccines used in clinical trials

Candidate Origin G and P type Comments
RIT4237 Bovine G6P[1] In small trials, safe but variable results. No efficacy in developing countries.
Discontinued development
WC3 Bovine G6P[5] Safe but variable results. Virus used to make reassortants containing human genes
MU18006 (RRV) Simian G3P[3] Some incidence of fever in vaccinees. Inconsistent protection. Virus used to make
reassortants containing human genes
LLR Lamb G10P[12] Used in China, limited efficacy data
RRV-TV Simian G1,G2,G3,G4,P[3] Licensed as Rotashield™ (Wyeth) and then withdrawn because of association with
intussusception
RV5 Bovine G1, G2, G3, G4, P[8] Currently licensed as RotaTeq™ (Merck)
RV1 Human G1, P[8] Currently licensed as Rotarix™ (GlaxoSmithKline)
116 E Human G9, P[11]
30 Rotaviruses 721

Numerous cost-effectiveness analyses in many countries have 50 %, even in developing countries, to moderate (20–50 %)
documented the need and benefit of a rotavirus vaccine [105, to nonexistent [238]. This virus was later used to make the
215–217]. Because natural rotavirus infections can induce reassortants containing human rotavirus VP7 genes that
excellent protection, at least against severe rotavirus disease, became Rotashield™ vaccine, the first licensed rotavirus
vaccine efforts have been directed mostly at the development vaccine that was subsequently withdrawn from the market
of live attenuated rotavirus vaccines given orally. The intro- (see below).
duction of rotavirus vaccines has profoundly altered the bur- One additional animal strain that has been developed into
den of rotavirus disease in the United States [218–221] and a vaccine is a G10P[12] strain isolated from a lamb, desig-
wherever it has become available [222–224]. nated LLR. This vaccine produced by the Lanzhou Institute
Early efforts concentrated on the use of animal rotavirus of Biological Products was introduced in China [239] but
strains, labeled the Jennerian approach because it relies on the was only recently demonstrated to be effective [240].
natural attenuation of animal viruses in humans for safety and
largely heterotypic immune responses for protection. The ini- 11.1.2 Reassortant Vaccines
tial efforts with animal rotavirus vaccines yielded inconsis- Because of the belief that homotypic immunity, or the devel-
tent results. Therefore, in an attempt to make the vaccines opment of serotype-specific antibody responses, might
more closely related to human strains, human rotavirus genes increase the protection seen with rotavirus vaccines and
coding for the proteins that induce neutralizing antibody, VP4 because of a lack of consistent protection after vaccination
and VP7, were introduced into these animal strains by creat- with heterotypic animal strains, the next vaccines developed
ing reassortant viruses as described earlier. Another approach were reassortant vaccines. These vaccines contained the
was the use of attenuated human rotaviruses, either isolated genes encoding VP7 or the VP7 and VP4 proteins of human
from asymptomatic infections in human neonates or attenu- rotavirus strains, with the remainder of the genes from an
ated by cell-culture adaptation and passage. animal strain. As discussed earlier, these proteins were cho-
sen because they induce neutralizing antibody.
11.1.1 Animal Strains
The first vaccine trial was conducted 10 years after the iden- Rotashield™
tification of rotavirus as an agent of severe diarrhea. This The simian strain, RRV, was used as the background to make
vaccine candidate was based on a bovine rotavirus strain, three different reassortant viruses containing the VP7 gene
RIT4237, which is a G6P[1] virus [225]. The initial studies from human strains D (G1 serotype), DS-1 (G2 serotype),
of the RIT4237 vaccine produced variable results. The vac- and ST-3 (G4 serotype) with the G3 serotype represented by
cine was safe and effective in Finland, thus supporting the RRV itself. The initial trials with monovalent viruses yielded
hypothesis that heterotypic protection was possible since this varying results [241]; therefore, all four strains were incor-
virus shared neither G nor P serotypes with the predominant porated into a tetravalent vaccine, RRV-TV, later licensed as
human types and also suggested that protection may be Rotashield™. Extensive evaluations of the RRV-TV were
greater than immunogenicity would indicate [226, 227]. completed before licensure. In two large trials conducted at
However, later studies in developing countries and in a centers across the United States, the RRV-TV vaccine was
Navajo reservation were disappointing [228–231], and ulti- found to be safe, with the subjects developing a slight
mately, the development of this vaccine was discontinued. increase in temperature after the first dose. Protection against
The initial studies of another bovine vaccine, WC3, con- severe disease was 80 % [241, 242]. Similar results were
ducted in Philadelphia appeared promising, during a season reported from Finnish studies except that fever occurred
that had predominantly G1 strains [232]. Unfortunately, later somewhat more commonly and efficacy was somewhat
trials in Cincinnati [233] and in less developed Central enhanced [243]. Protection similar to that seen in the US
African countries [234] showed no significant protection. studies was also demonstrated when it was evaluated in a less
This virus was later used to develop reassortant vaccines and developed country, Venezuela [244].
is the backbone of the RotaTeq™ vaccine now used around As a result of these studies, RRV-TV or Rotashield™ was
the world. licensed in the United States in 1998 and recommended for
The next major vaccine candidate was a simian (rhesus general use in all infants [245]. However, less than 1 year
monkey) termed MMU18006 or more commonly known as after licensing, 15 cases of intussusception that occurred
RRV. This vaccine is a G3P[3] strain and thus shared the G shortly after vaccination were reported to the Vaccine
type with a human strain. RRV vaccination was associated Adverse Events Reporting System (VAERS). Intussusception
with mild side effects, including low-grade fever and mild is a form of intestinal blockage caused when a segment of the
diarrhea, especially when it was given to older children who bowel prolapses into a more distal segment of the intestine
had lost maternal antibodies [235–237]. Protection with this [246]. Initial publications reported an increased risk of the
vaccine was also inconsistent, ranging from greater than development of intussusception from 3 to 14 days after
722 M.M. McNeal and D.I. Bernstein

receipt of the first dose of Rotashield™ and a smaller risk true for all rotavirus vaccines, the vaccine appears to less
after administration of the second dose [247–249]. The ini- effective in less developed areas of the world [269, 270]. In
tial estimate that one case of intussusception attributable to the poorest settings such as Africa and Asia, efficacy has
vaccination with RRV would occur for every 4,670–9,474 only been 40–50 % [265, 271, 272]. In general this correlates
infants vaccinated was later revised to approximately 1 in with the lower immune response in these settings [273, 274].
10,000 [250]. The risk of intussusception appears to be age The reasons for this decrease are not well understood.
related and increased substantially in children greater than Possible contributing factors include higher levels of trans-
90 days of age [251, 252]. Based on these findings, the use of placental and breast milk antibody that could inhibit vaccine
Rotashield™ was discontinued. Because of these findings, virus replication, malnutrition, micronutrient deficiencies,
the FDA also recommended that future trials using live oral interfering gut flora, and differences in the epidemiology
rotavirus vaccines should be conducted with more than [275].
60,000 children to ensure that the risk of intussusception Post-licensure evaluations of the vaccine have shown sub-
from a new vaccine would be less than was attributed to stantial decreases in rotavirus diseases wherever evaluated
Rotashield™ [253]. [224, 276, 277]. Surveillance studies and laboratory moni-
The pathogenic mechanism underlying the association of toring conducted in the United States have shown remark-
the Rotashield™ vaccine and intussusception has not been able reductions in rotavirus disease every year since vaccine
defined [254]. Several studies have investigated the possible introduction [219, 278]. Interestingly this has included not
infectious etiology of intussusception, but these studies have only those immunized but older children and adults suggest-
had a small sample size, and several different pathogens, ing a high level of herd immunity [127, 219, 278]. Safety has
including adenoviruses, have been identified [255, 256]. It is also been monitored. Except for a small increase in the rates
unclear if wild-type rotavirus causes intussusception as there of intussusception identified in Australia (relative risk 5.3
is a lack of seasonal variation for intussusception that might and 3.5 for the 1–7 and 1–21 days after the first dose of
be expected to occur if rotavirus was a major cause of intus- RotaTeq™, respectively, in infants 1 to <3 months of age),
susception [257–259]. the vaccine has been shown to be safe [279]. The increased
risk detected in Australia has not been confirmed in studies
RotaTeq™ conducted in the United States [280].
Initially, a monovalent vaccine containing the VP7 gene of a
human G1 rotavirus (WI79-9) and the remainder of the genes Bovine UK Reassortant Vaccine
from WC3 was developed after the inconsistent results with The bovine UK strain, which is also a G6P [5], similar to
WC3. This vaccine was reported to be effective [70, 260], WC3, was used to make a reassortant with G1, G2, G3, or
but because of the idea that serotype-specific protection was G4 human rotaviruses on a 10-gene UK background. The
necessary, a quadrivalent vaccine containing three viruses tetravalent UK vaccine appears to be safe and immunogenic
with gene substitutions of the VP7 gene with human G1, G2, [281]. Clinical trials are ongoing to evaluate this UK reas-
or G3 and one virus containing a VP4 gene substitution with sortant vaccine. There has been additional work done to
a human P[8] was developed and shown to be safe and effec- develop UK-based reassortants containing additional VP7
tive [261]. Finally, by adding a reassortant virus containing a genes for some of the emerging viruses such as G8 or G9
human G4 VP7 to the above four viruses, a pentavalent vac- strains, creating a hexavalent reassortant vaccine [282].
cine, RotaTeq™, was developed by Merck Research
Company [262]. 11.1.3 Human Strains
Because of the association of Rotashield™ with intussus- The use of human strains for vaccine candidates is based on
ception, the large pivotal trial of RotaTeq™ included over the data that show that natural infection can provide protec-
70,000 infants from 11 countries. The vaccine was shown to tion against subsequent infection or disease. The human
be safe and, unlike Rotashield™, it did not induce fever strains considered as vaccine candidates are either naturally
[263]. Most importantly, there was no association with intus- attenuated, as thought to be the case with neonatal strains, or
susception. The vaccine was highly effective, reducing all attenuated by culture adaptation and multiple passage. The
G1–G4 rotavirus gastroenteritis by 74.0 %, severe rotavirus candidates discussed below are each composed of one strain
gastroenteritis by 98.0 %, and hospitalizations and emer- and, therefore, rely on homotypic and heterotypic mecha-
gency room visits by 94.5 % [263]. RotaTeq™ was licensed nisms of protection.
in 2006 by the US Food and Drug Administration (FDA) for
use in infants. Neonatal Strains
Since the pivotal trial was published, there have been The first natural history study showed that asymptomatically
multiple trials of RotaTeq™ conducted around the world infected neonates had a subsequent reduction in the fre-
[264–268]. Efficacy has been excellent, but as appears to be quency and severity of rotavirus diarrhea, thereby generating
30 Rotaviruses 723

interest in the use of neonatal strains as vaccine candidates and did not interfere with other concomitantly administered
[119]. One of the first human strains to be tested as a rotavi- childhood vaccines [295, 296]. In the initial efficacy trial
rus vaccine was a neonatal, G1P[6] strain identified as M37. conducted in Finland, the vaccine was 73 % protective
Several small studies showed that the vaccine was safe but against all rotavirus gastroenteritis and 90 % protective
only moderately immunogenic, inducing neutralizing anti- against severe illness even though a relatively low dose was
body responses to the M37 strain but not to other G1 strains used [297].
[283]. In an efficacy study of Finnish infants, there was no Similar to RotaTeq™, the pivotal vaccine trial with
protection against predominantly G1 strains, and this vac- RIX4414 involved over 63,000 infants but was performed in
cine candidate was not pursued [284]. Latin America and Finland [298]. The RIX4414 vaccine
RV3, another vaccine candidate, is a G3P[6] human rota- (106.5 median cell-culture infective dose) given in a two-dose
virus that was isolated in a nursery in Melbourne, Australia, schedule at approximately 2 and 4 months of age was safe,
where it caused endemic, asymptomatic infections in new- did not induce fever, and again, most importantly, was not
born infants in the 1970s. Neonates infected with this virus associated with intussusception. During the entire study,
were 100 % protected against severe rotavirus disease, there were 25 cases of intussusception, 16 in the placebo
caused primarily by heterotypic G2P[4] strains, for their first group and 9 in the vaccine group. Efficacy data from a subset
3 years of life [119]. The vaccine was moderately immuno- of 20,000 infants from this trial showed 85 % protection
genic and protective in later studies [285, 286]. This vaccine against severe rotavirus diarrhea and hospitalization.
is also being evaluated in clinical trials. Protection against more severe gastroenteritis was 100 %. It
Two other more recent vaccine candidates were derived was also demonstrated that protection was high (86 %) not
from two neonatal strains isolated from asymptomatically only against severe rotavirus diarrhea caused by G1P[8]
infected neonates in India in the mid-1980s: one from New strains but also against G3P[8], G4P[8], and G9P[8] strains
Delhi and the other from the Bangalore region of India. Both which all shared the VP4 P[8] genotype. Efficacy against the
of these strains are natural reassortants between bovine and few G2P[4] infections (a strain that is not matched for either
human strains. The strain from New Delhi, 116E, a G9P[11] VP4 or VP7) was 41 %. However, in an integrated analysis,
strain, contains a VP4 gene segment from a bovine rotavirus efficacy against G2[P4] was 71.4 % against severe disease
and the other ten genes are from a human strain [89]. The and 81.0 % against disease of any severity [299]. More
other strain, I321, is a G10P[11] strain and contains nine recently a study in Mexico showed efficacy of over 90 %
genes of bovine origin and only gene segments for two non- against a fully heterotypic G9P[4] rotavirus strain in Mexico
structural proteins from a human strain [287]. Both I321 and [300].
116E vaccine candidates were tested in a small, placebo- In a subsequent trial of RIX4414 conducted in six
controlled trial, which showed only the 116E strain elicited European countries, protection was 87 % against any rotavi-
significant immune responses [288]. Therefore, only the rus gastroenteritis, 96 % against severe disease, and 100 %
116E strain is being pursued as a vaccine candidate in India. against hospitalization due to rotavirus. In this study, effi-
The most recent trials showed good immunogenicity for this cacy against G3, G4, and G9 strains was similar to that
vaccine candidate [289], and efficacy trials are under way. against G1 strains and was over 95 %. Efficacy against the
unrelated G2 strains was 85 %, suggesting that heterotypic or
Rotarix® nonneutralizing antibody responses are also involved in pro-
This vaccine was first licensed in Mexico in 2005 and has tection [301, 302]. Similar to other rotavirus vaccines, effi-
since been approved in countries around the world including cacy in less developed countries is decreased. In a study of
developed, less developed, and developing nations [223, African infants, efficacy against severe disease was 61.2 %
290]. The product is based on the attenuated human strain, and was even lower in Malawi infants, 49.4 % compared to
89–12 obtained from an infant with rotavirus gastroenteritis 76.9 % in South Africa [303]. Despite this lower efficacy, it
in Cincinnati, Ohio, and is a G1P[8] strain, representing the is important to understand that, because of the higher mortal-
most common strain worldwide. The isolate was attenuated ity rates, more lives will be saved in counties like Malawi
by multiple passages in tissue culture. Results of a multi- compared to those with lower mortality despite the lower
center efficacy trial showed that two doses of this vaccine efficacy with either vaccine.
provided 89 % protection against any rotavirus disease and The post-licensure effectiveness of Rotarix® has been
100 % protection from severe disease [291]. The 89–12 verified in several studies (reviewed in [223, 264]). Perhaps,
strain was further purified by limiting dilution and passaged most importantly, vaccination has been associated with
in tissue culture by GlaxoSmithKline. The final product was reduced mortality from diarrheal-associated disease in
called RIX4414 and is now marketed as Rotarix® Mexico [304] and in Brazil [305].
(GlaxoSmithKline), a two-dose oral vaccine. Initial testing Similar to RotaTeq™, post-licensure safety of Rotarix® is
showed that the vaccine was safe, immunogenic [292–294], being monitored. Studies conducted in Mexico and Brazil
724 M.M. McNeal and D.I. Bernstein

[307] revealed an increased rate of intussusception after the vaccines prove to be ineffective or have safety issues, nonliv-
first dose in Mexico (1 per 51,000 infants) and a small increase ing vaccines may be further developed.
only after the second dose in Brazil (1 per 68,000 infants). In an
Australian study that evaluated both RotaTeq™ and Rotarix®,
there was an increased relative risk for intussusception of 3.5 References
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206. McNeal MM, Barone KS, Rae MN, Ward RL. Effector functions of Immunogenicity and safety of live oral attenuated bovine rotavi-
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237. Losonsky GA, Rennels MB, Kapikian AZ, et al. Safety, infectiv- 259. Chang EJ, Zangwill KM, Lee H, Ward JI. Lack of association
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 Rubella Virus
31
Walter Orenstein and Susan E. Reef

1 Introduction was followed by reports of Australian [6], Swedish [7],

American [8], and British [9] epidemiologists and teratolo-
Rubella (German measles) is usually a mild febrile viral rash gists confirming his observations and also noting infants
illness in children and adults. However, infection early in presented with heart disease and deafness. Thus, the charac-
pregnancy, particularly during the first 16 weeks, can result teristic congenital rubella triad was established.
in miscarriage, stillbirth, or an infant born with birth defects After Gregg’s discovery, it was 20 years before the isola-
known as congenital rubella syndrome. tion of the causative agent, rubella virus. During this time,
various estimates of the risk of fetal disease after maternal
rubella were made. However, the wide range of estimates
2 Historical Background stemmed from the absence of a definitive diagnostic test and
consequent misdiagnosis of rubella in the mother. In late
German physicians, in the mid-eighteenth century, were the 1962, the rubella virus was isolated by two different groups:
first researchers to distinguish the rubella disease from other Weller and Neva [10] in Boston and by Parkman, Beuscher,
exanthems. Even though they named it Rotheln, rubella is and Artenstein [11] in Washington, DC.
recognized today by its common English language eponym In 1962–1965 a worldwide rubella epidemic started in
German measles [1]. In 1841, a British physician reported an Europe and spread to the United States. In the United States
outbreak in a boys’ school in India and coined the term an estimated 12.5 million cases of rubella occurred in the
rubella, a Latin diminutive meaning “little red” [2]. Even United States, resulting in 2,000 cases of encephalitis, 11,250
though, in the late nineteenth century, rubella was considered fetal deaths, 2,100 neonatal deaths, and 20,000 infants born
different from measles or scarlet fever [3], not until 1941 the with CRS, a constellation of birth defects that often includes
significance of rubella was noted. In 1941, Norman McAlister blindness, deafness, and congenital heart defects. The eco-
Gregg, an Australian ophthalmologist, linked congenital cat- nomic impact of this epidemic in the United States was esti-
aracts to maternal rubella. In his practice, Gregg had noticed mated at $1.5 billion [12, 13]. The pandemic led to the
an unusual number of infants with cataracts [4]. It is noted recognition of an expanded congenital rubella syndrome
that a crucial clue was a conversation he overheard in his (CRS), which added hepatitis, splenomegaly, thrombocyto-
waiting room between 2 mothers who were discussing the penia, encephalitis, mental retardation, and numerous other
rubella they both had sustained in pregnancy during the anomalies to the already described deafness, cataracts, and
Australian outbreak of 1940 [5]. Gregg’s original observation heart disease [14, 15]. The pandemic also made it obvious
that a vaccine was needed, and many groups set to work.
The global epidemic spurred development of rubella vac-
cines and emphasized the need to develop and implement
W. Orenstein, MD (*)
Department of Medicine, Emory University School strategies for using these vaccines to prevent this devastating
of Medicine, 1930-001-1AA, Room 446, health burden [3]. Between 1965 and 1967, several attenu-
1462 Clifton Rd, Atlanta, GA 30322, USA ated rubella strains were developed and reached clinical tri-
e-mail: [email protected]
als [16–18]. In 1969 and 1970, rubella vaccine was introduced
S.E. Reef, MD in Europe and North America. Since the late 1970s, vaccina-
Vaccine Preventable Disease Eradication and Elimination Branch,
tion has had a major impact on the epidemiology of rubella
Global Immunization Division, Center for Global Health, Centers for
Disease Control and Prevention, Atlanta, GA 30329-4018, USA and CRS resulting in the interruption of endemic rubella
e-mail: [email protected], [email protected] virus transmission in 2001 [19].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 733

DOI 10.1007/978-1-4899-7448-8_31, © Springer Science+Business Media New York 2014
734 W. Orenstein and S.E. Reef

3 Methodology In the United States, the most recent use of post-

vaccination serosurveys was to support the documentation of
3.1 Mortality Data elimination of rubella and CRS [25]. Two nationwide serop-
revalence studies were conducted through the population-
Deaths associated with rubella are rare enough that they have based National Health and Nutrition Examination Survey.
no impact on the epidemiology of the rubella. Sera were tested for rubella immunoglobulin G antibodies
during 1988–1994 and 1999–2004. From the earlier to the
later period, the overall age-adjusted rubella seroprevalence
3.2 Morbidity Data in the US population 6–49 years of age rose from 88.1 to
91.3 % was statistically a significant increase. Additional
In the United States, rubella and CRS became nationally analyses showed that seroprevalence either remained at the
reportable to the National Notifiable Disease Surveillance same level or higher for the groups (i.e., children of both
System (NNDSS) in 1966. In 1969, Centers for Disease sexes, women of childbearing age) that were targeted for
Control and Prevention established the National Congenital vaccination.
Rubella Syndrome Registry (NCRSR). The reporting effi-
ciency for clinical cases is estimated to be only 10–20 %. As
part of the process for documenting the elimination of rubella 3.4 Laboratory Methods
and CRS in 2004, the adequacy of surveillance was evaluated
by reviewing five different sources. The conclusion was that 3.4.1 Virus Isolation/Detection
the surveillance system was adequate and was able to support In persons with acquired rubella, the rubella virus can be iso-
the elimination of endemic rubella transmission [20]. lated from the blood and nasopharynx during the prodromal
period and from the nasopharynx for as long as 2 weeks after
eruption. However, the likelihood of virus recovery is sharply
3.3 Serological Surveys reduced by 4 days after the rash. Rubella virus can be iso-
lated using several different cell lines: Vero cell line, primary
Serological surveys of healthy population groups have been African green monkey kidney cells, or the RK13 cell line.
of major importance for understanding the pre-vaccine Through the World Health Organization Global Laboratory
rubella epidemiology including age specific to identify the Network, laboratories are trained to use either Vero/SLAM
target age groups and strategy for vaccine introduction [21]. cells or Vero cells to isolate rubella virus [26, 27]. The
In addition, serosurveys are used to monitor the impact of the method for detection of the rubella E1 glycoprotein is by
vaccination program, provide evidence for modifying the using monoclonal antibodies in either an immunofluorescent
vaccination strategy, and support the documentation of elim- or an immunocolorimetric assay.
ination of rubella/CRS [22]. Another recently developed method for virus detection is
Prior to the licensure and introduction of the rubella vac- the polymerase chain reaction (PCR). The most important
cine, the World Health Organization sponsored collaborative roles of RT-PCR in rubella control are to characterize the
serosurveys in 1967–1968 assessing the rubella seropositiv- virus genetically and to detect inactivated virus. RT-PCR can
ity in five continents; however, most of the studies were con- be used to detect virus before the IgM is positive and used to
ducted in the Americas and Europe. In 12 of the 25 studies detect inactivated particles when the person is shedding only
conducted, the seropositivity rate was 80 % or greater among small amounts of virus. The polymerase chain reaction
women aged 17–22 years of age [23]. In the United States, (PCR) has been adapted to the detection of rubella RNA by
serological surveys conducted in the pre-vaccine era showed reverse transcription and amplification.
seropositivity ranging between 80 and 92 %.
In some regions and countries, post-vaccination serosur- 3.4.2 Antibody Tests
veys are used to monitor the vaccination program. However, Sera
interpretation of serological studies can be complicated due After the isolation of the rubella, laboratory confirmation of
to variations in the sensitivity of the assays. In the European rubella infection was available. Over the last 40 years, sev-
Region, European Sero-epidemiology Network, the aim is to eral different testing methodologies were developed
standardize the serological survey of eight vaccine prevent- (Fig. 31.1). These include the following: neutralization
able diseases in 22 countries [24]. By standardizing the assays (NT), hemagglutination-inhibition (HI) assays,
methodology, international comparisons can be made to enzyme immunoassay (EIA), single radial hemolysis (SRH),
allow to evaluate the effectiveness of different immunization and latex agglutination. NT assays were the first to be devel-
programs and to coordinate vaccine policy to ensure that oped [28], but they are seldom used today, as they are
adequate levels of immunity exist throughout Europe. demanding and require use of cell cultures. HI assays were
31 Rubella Virus 735

Fig. 31.1 Rubella and CRS, Rubella and CRS, United States, 1998-2012
United States, 1998–2012 400 12
(*By year of birth)
CRS Rubella
350
10

Number of Rubella Cases

Number of CRS Cases*

300
8
250

200 6

150
4
2004
100
Elimination
2
50 Documented

0 0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Year

developed in 1967 [29] and results were shown to correlate Oral fluid sampling can also be used for detecting RNA. In a
well with NT. However, HI is also labor intensive. HI is no recent study, the use of oral fluids for detection of RNA was
longer recommended for routine testing; however, the HI is superior to testing of sera or oral fluid for rubella IgM within
used as a reference method to establish a calibration standard the first few days after rash onset [36]. Oral fluid samples are
for other rubella assays. Other more recent assays developed easy to collect, noninvasive, and more acceptable to the pop-
included enzyme immunoassay (EIA), single radial hemoly- ulation. Its use enables field workers to obtain more com-
sis (SRH), and latex agglutination, which have been used plete sampling of suspected cases.
extensively for rubella antibody screening [30].
Nowadays, EIA is the most frequently used test for rubella
antibody screening and diagnosis, as it is a sensitive and an 4 Biological Characteristics
adaptable technique and is readily automated. EIA can also of the Vaccine
be adapted to detect class-specific antibodies (e.g., IgM,
IgG1, IgG3, IgA) and is the method of choice for detection Rubella virus is a member of the Togaviridae family and the
of rubella-specific IgM [31–33]. Indirect and M-antibody genus Rubivirus. Rubella virus is a single-stranded envel-
capture EIAs are available commercially for detection of oped RNA with a single antigenic type. It measures 50–70 nm
rubella IgM. Another adaptation of EIAs is the IgG1 avidity in diameter and has two envelope proteins (E1,E2) and a core
assay. The avidity assays are useful for diagnostic purposes, protein (c). The core protein is surrounded by a single-layer
particularly to distinguish primary rubella from rubella rein- lipoprotein enveloped with spike-like projections that con-
fection and to identify persistent IgM responses and nonspe- tain the two glycoproteins, E1 and E2. Humans are the only
cific IgM [34]. The most common diagnostic method known reservoir.
employs a denaturating agent (6–8 M urea or DEA) to elute The rubella virus is relatively temperature labile but is
low avidity antibody from antigen-antibody complexes in an more heat stable than measles virus; it is inactivated after
EIA (reviewed by Thomas et al. [34]; Best and Enders [30]). 30 min at 56 °C, 4 min at 70 °C, and 2 min at 100 °C. It
Depending upon the strength of this binding, the complex degrades rapidly with conventional freezing at −20°, but the
formed may or may not be easily dissociated. Antibody avid- virus is stable at −60 °C and below and when freeze-dried
ity is low after primary antigenic challenge, becomes higher with stabilizers. When stabilized with protein it can be
with time, and usually involves IgG antibodies [51, 52]. repeatedly frozen and thawed without loss of titer. Lipid sol-
However, IgG avidity assays can be difficult to establish, vents, weak acids and alkalis, and UV light inactivate the
standardize, quality control, and interpret; they are therefore rubella virus. It is also susceptible to a wide range of disin-
recommended only for laboratories experienced in using fectants and is inactivated by 1 % sodium hypochlorite, 70 %
these assays [35]. ethanol, and formaldehyde [37].
Over the last 15–20 years, the study of molecular epide-
Oral Fluids miology has evolved. In 2005, a systematic nomenclature
Since the early 1990s, the use of oral fluid sampling has been was adopted by the WHO [38, 39]. The genetic characteriza-
used successfully for the detection of rubella antibodies as tion of rubella virus has identified two clades that differ by
part of the surveillance system in the United Kingdom [35] 8–10 % at the nucleotide level. Clade 1 is divided into 10
736 W. Orenstein and S.E. Reef

genotypes (1a, 1B, 1C, 1D, 1E, 1 F, 1G, 1 h, 1i, and 1j), of occurred among older adolescents and young adults, with
which 6 are recognized and 4 are provisional (designated by outbreaks occurring among students in high schools, col-
lowercase letters). Clade 2 contains 3 genotypes (2A, 2B, leges, and universities and among persons on military bases
and 2C) [40]. For rubella isolates collected before 2000, iso- and workers in hospitals. Rubella incidence was highest
lates from North America, Europe, and Japan are closely among young adults [48]. In addition, the number of reported
related to each other and form clade I, whereas clade II com- CRS cases increased, from 23 in 1976 to 57 in 1979; how-
prises some strains from China, Korea, and India [41]. ever, the annual number of CRS cases never reached the level
However, in China between 2001 and 2007, there was a shift reported during the 1960s in the pre-vaccine era. Serologic
of genotypes toward predominance of IE and 1F [42]. Of studies at that time suggested that 10–20 % of adults
rubella isolates collected between 2005 and 2010, three gen- remained susceptible to rubella [49].
otypes (1E, 1G, 2B) had wide geographic distribution The resurgence of rubella and its increased incidence
whereas others occurred sporadically or were geographically among young adults focused attention on the need for addi-
restricted [40]. tional strategies. In 1978, the changing epidemiology of
The genetic differences between clades do not appear to rubella prompted the Advisory Committee on Immunization
translate into antigenic differences, despite amino acid changes Practices (ACIP) to additionally recommend that rubella vac-
of 3–6 % in viral proteins. Isolates from CRS cases are not cine be targeted to susceptible postpubertal females, in addi-
genetically distinct from isolates from acquired rubella. tion to adolescents, persons in military service, college
students, and persons in certain work settings (e.g., hospitals)
[50]. Efforts to increase overall childhood vaccination cover-
5 Descriptive Epidemiology age to greater than 90 % for all vaccine-preventable diseases,
including rubella, had begun in 1977, with the first National
5.1 Incidence and Prevalence Childhood Immunization Initiative [51]. In 1978, a program
was undertaken to eliminate indigenous measles in the United
Our understanding of pre-vaccine era epidemiology of States; the use of combined vaccines, either measles-rubella
rubella in the United States is from surveillance conducted (MR) vaccine or measles-mumps-rubella (MMR) vaccine,
from 1928 to 1983 in 10 selected areas in the United States. was encouraged. These efforts to increase immunity among
During the 1962–1965 the United States experienced an epi- selected adults and children resulted in substantial decreases
demic with an estimated 12.5 million cases of rubella, result- in the numbers of both rubella and CRS cases. During 1977–
ing in 2,000 cases of encephalitis, 11,250 fetal deaths, 2,100 1981, reported rubella cases declined from 20,395 to 2,077.
neonatal deaths, and 20,000 infants born with CRS, a con- During 1979–1981, reported CRS cases decreased from 57 to
stellation of birth defects that often includes blindness, deaf- 10 [52]. For the 1981–1982 school year, rubella vaccination
ness, and congenital heart defects. coverage was 96 % for children entering school (i.e., into kin-
In 1969, live-attenuated rubella vaccines were first dergarten or first grade) in the 50 states and the District of
licensed in the United States [43], and a vaccination program Columbia [53]. Efforts to maintain high coverage through
was established with the goal of preventing congenital infec- enforcement of school immunization laws produced a con-
tions, including CRS. Before the introduction of vaccine, tinuing decrease in reported rubella cases.
rubella incidence was highest among children aged <9 years In 1979, a new formulation of live-attenuated rubella vac-
[44]. The new rubella vaccination program targeted a dose of cine (RA 27/3) replaced the previous rubella vaccines in the
vaccine to children aged 1 year to puberty [45]. To increase United States. RA 27/3 vaccine had been determined to
coverage among school-aged children rapidly, mass cam- induce higher antibody titers and produce an immune
paigns were conducted, particularly in schools. By 1977, response more closely paralleling natural infection than pre-
reported vaccination levels were approximately 60 % for vious vaccines [54].
children aged 1–4 years, 71 % for those aged 5–9 years, and By 1979, rubella vaccination had eliminated the characteris-
64 % for those aged 10–14 years [46]. The number of tic 6–9-year epidemic cycle of rubella in the United States [52].
reported rubella cases declined 78 %, from 57,686 cases in In 1980, national health objectives for 1990 were established
1969 to 12,491 cases in 1976. As anticipated, the greatest for rubella and CRS, calling for reductions in the annual num-
decreases in rubella occurred among persons aged <15 years; ber of rubella cases to fewer than 1,000 and CRS cases to fewer
however, incidence declined in all age groups, including than 10 [55]. During the 1980s, the number of reported rubella
adults. This decrease in rubella also resulted in a decline in cases continued to decline steadily, and overall incidence con-
the number of reported CRS cases, from 68 cases reported in tinued to decrease in all age groups. By 1983, the 1990 objec-
1970 to 23 reported in 1976 [47]. The total number of rubella tives already had been achieved, with 970 rubella cases and
cases continued to decline overall during the late 1970s; four CRS cases reported. During the early 1980s, outbreaks
however, in subsequent years a resurgence of rubella continued to be reported in health-care settings, universities,
31 Rubella Virus 737

workplaces, and prisons. In 1981, ACIP recommendations The incidence of CRS has been evaluated mainly in devel-
increased emphasis on targeting these settings to ensure vacci- oped countries over the last 60 years. Initially in the United
nation coverage among students and staff members [56]. States, the incidence of CRS roughly paralleled the incidence
In 1988, state health departments reported an all-time low of rubella in individuals over 15 years; however, with the
of 225 cases of rubella; however, in 1989, a total of 396 cases interruption of endemic rubella virus transmission, CRS
were reported, and in 1990, the number increased to 1,125 cases became very rare and occurred mainly among mothers
[57]. Most cases were associated with outbreaks that who are foreign born [61].
occurred in settings where unvaccinated adults congregated, In most developing countries, there is little documenta-
including colleges, workplaces, prisons, and in religious tion to illuminate the epidemiology of either rubella or CRS.
communities that did not accept vaccination. Outbreaks The epidemic pattern for developing countries is similar to
among these populations accounted for 56 % of CRS cases the developed countries with cycles 3–7 years. Globally, it is
in the 1990s. In 1989, a goal was established to eliminate estimated that approximately 103,000 infants with CRS were
indigenous rubella transmission and CRS in the United born in 2010 with the greatest burden in regions where
States by 2000 [58]. With establishment of the 1993 rubella vaccine uptake is limited. A review of worldwide
Childhood Immunization Initiative, the number of annual data concerning CRS revealed rates in developing countries
rubella cases continued to decline in the mid-1990s. varying between 0.6 and 2.2 per 1,000 live births, similar to
Outbreaks continued to be associated with settings where rates seen in developed countries before universal vaccina-
adults had close contact; however, the demographic charac- tion [62, 63]. It has been estimated that the incidence of CRS
teristics of rubella patients changed. Before 1995, most per- is 0.1–0.2 per 1,000 live births during endemic periods and
sons with rubella were non-Hispanic; beginning in 1995, 1–4 per 1,000 live births during epidemic periods [64].
most were Hispanic [59]. Beginning in 1998, data on country Where rubella virus is circulating and women of childbear-
of origin were collected for rubella patients. These data ing age are susceptible, CRS cases will continue to occur.
revealed that, during 1998 and 1999, approximately 79 and
65 % of patients whose country of origin was known were
foreign born. Of these, 91 % in 1998 and 98 % in 1999 were 5.2 Epidemic Behavior
born in the Western Hemisphere, and 43 % in 1998 and 81 %
in 1999 were born in Mexico. These persons were either Rubella usually occurs in a seasonal pattern, with epidemics
unvaccinated or their vaccination status was unknown. every 5–9 years. However, the extent and periodicity of
During 1998–2000, a total of 23 CRS cases were reported to rubella epidemics is highly variable in both industrialized
CDC. The infants in 22 (96 %) of these cases were born to and developing countries. From published literature, epi-
Hispanic women, and 22 of the mothers with known country demics have been reported every 6–7 years in Hong Kong
of birth were born outside the United States. The countries of [65] and São Paulo, Brazil [66]; every 4–5 years in Panama
origin of these mothers were Mexico (14 mothers), [67]; and every 4–7 years in Argentina [68] and Bangkok,
Dominican Republic (four), Honduras (two), Colombia Thailand [69–71].
(one), and Philippines (one). Since 2001, the annual numbers
of rubella cases have been the lowest ever recorded in the
United States: 23 in 2001, 18 in 2002, seven in 2003, and 5.3 Geographic Distribution
nine in 2004. Approximately half of these cases have
occurred among persons born outside the United States, of Prior to the establishment of the rubella and CRS elimination
whom most were born outside the Western Hemisphere. goal in the Region of Americas, rubella had a worldwide dis-
During 2001–2004, four CRS cases were reported to CDC; tribution. However, in 2009, the last endemic rubella case in
the mothers of three of the children were born outside the the Region of the Americas was documented in Argentina
United States. In 2004, the panel convened by CDC con- [72]. Rubella continues to circulate in the Eastern
cluded that sustained transmission of rubella has been inter- Hemisphere. In 2012–2013, rubella epidemics have been
rupted. Since 2004, the United States has maintained documented in several countries (i.e., Romania [73], Poland
elimination of rubella and CRS. From 2005 to 2011, a [74], Japan [75], Ethiopia) in three different continents.
median of 11 rubella cases was reported each year in the
United States (range: 4–18). In addition, two rubella out-
breaks involving three cases, as well as four total CRS cases, 5.4 Temporal Distribution
were reported [60]. In 2012, as part of the documentation
process for PAHO, the United States convened an indepen- Prior to the elimination of endemic rubella virus in the United
dent external panel to evaluate if elimination of measles, States, the largest number of rubella cases occurred in late
rubella, and CRS had been maintained. winter and spring, in both high and low incidence. Because
738 W. Orenstein and S.E. Reef

of the acceleration of measles control, the understanding of 7 Pathogenesis and Immunity

rubella seasonality can be documented in developing coun-
tries. Using the measles case-based surveillance in African Although the pathogenesis of postnatal (acquired) rubella
region, rubella seasonality could be detected with variation has been well documented, data on pathology are limited
of seasonality by subregion [76]. In the West subregion, dur- because of the mildness of the disease. Primary implantation
ing 2003–2009, marked seasonality of rubella occurred each and replication in the nasopharynx are followed by spread to
year with sharp increases in reporting during January with the lymph nodes. This is followed by viremia and shedding
peaks in March–April followed by sharp declines in May, of virus from the throat.
leading to troughs during October– December each year. For acquired rubella, the rubella virus induces both circu-
However, in the South subregion, a distinct annual seasonal- lating and cell-mediated immune (CMI) responses. HI and
ity was observed with consistently few cases reported during NT antibodies develop very rapidly and may be detectable,
January–June each year, followed by gradual increases in while the rash is still specific antibodies, rubella-specific
June–July and peaks in September–October. IgM appears first and is closely followed by IgG1, IgG3, and
IgA [31]. IgM is transient; it peaks on about day 7 and
persists for 4–12 weeks after illness and occasionally for
5.5 Age and Sex about a year [77].
The role of CMI in protection from rubella has not been
In the pre-vaccine era in the United States, rubella was pri- determined. Rubella infection induces a fall in total leuko-
marily a disease of school-aged children; however, rubella cytes, T cells, and neutrophils, and a transient depression in
occurred also in preschool children. In many countries, this lymphocyte responses to mitogens and antigens (e.g., puri-
is the pattern for rubella infection. However, in other coun- fied protein derivative, PPD), but the mechanism responsible
tries such as Caribbean islands and Southeast Asia, young for the mild immunosuppression has not been elucidated.
adult females show high susceptibility, which can result in Studies of cytokine secretion demonstrate the strongest
cases among pregnant women with subsequent CRS. responses in persons with a recent history of rubella [78, 79].
In the pre-vaccine era, there were no differences in attack Lymphoproliferative assays show that CMI responses
rates by sex for children. In the post-vaccination era, in coun- develop a few days after onset of rash and persist at low lev-
tries where adolescent girls were targeted for vaccination, els for many years.
outbreaks among adolescent and adult males have been doc- The pathology of CRS in the infected fetus is well defined,
umented [71]. However, in countries that have not targeted with almost all organs found to be infected; however, the
females only in vaccination, attack rates in males and females pathogenesis of CRS is only poorly delineated. In tissue,
are similar. infections with rubella virus have diverse effects, ranging
from no obvious impact to cell destruction. The hallmark of
fetal infection is chronicity, with persistence throughout fetal
6 Mechanisms and Routes development in utero and for up to 1 year after birth.
of Transmission The immune response to the intrauterine rubella infection
starts while in pregnancy. However, the development of the
Rubella virus is spread from person to person via respiratory fetal humoral immune system appears to be too late to limit
droplets. Individuals with acquired rubella may shed virus the effects of the virus. Cells with membrane-bound immu-
from 7 days before rash onset to ~5–7 days thereafter. Both noglobulins of all three major classes—IgM, IgG, and IgA—
clinical and subclinical infections are considered appear in the fetus as early as 9–11 weeks gestation [80].
contagious. However, circulating fetal antibody levels remain low until
After primary implantation and replication, subsequent mid-gestation, despite the presence of high titers of virus. At
viremia occurs, which in pregnant women often results in this time, levels of fetal antibody increase, with IgM anti-
infection of the placenta. Placental virus replication may body predominating [81]. As in the case with other chronic
lead to infection of fetal organs. Infants with CRS may shed intrauterine infections, congenital rubella infection may lead
large quantities of virus from bodily secretions, particularly to an increase in total IgM antibody levels [82]. At the time
from the throat and in the urine, up to 1 year of age. Outbreaks of delivery of infected infants, levels of IgG rubella antibod-
of rubella, including some in nosocomial settings, have orig- ies in cord sera are equal to or greater than those in maternal
inated with index cases of CRS. Thus only individuals sera, even if the infant is born prematurely. IgG is the domi-
immune to rubella should have contact with infants who nant antibody present at delivery in rubella-infected infants
have CRS or who are congenitally infected with rubella virus and is mainly maternal in origin. In contrast, the IgM levels
but are not showing signs of CRS. are lower but are totally fetus derived.
31 Rubella Virus 739

8 Patterns of Host Response (2003), and Tunisia [85], encephalitis was seen more com-
monly, with an estimated rate of 1 in 300 to 1 in 1,500 cases.
In acquired rubella, the ratio of inapparent to apparent infections Long-term sequelae with such progressive rubella panen-
has been estimated to be from 1:1 to as high as 6:1 [83, 84]. Age cephalitis (PRP) are rare. PRP has similarities to subacute
probably influences the clinical expression of infection. Children sclerosing panencephalitis (SSPE) caused by measles.
usually develop few or no constitutional symptoms.
8.1.2 Congenital Rubella Infection
The risk of congenital infection is related to the gestational
8.1 Clinical Manifestations age at the time of maternal infection. The outcome of a pri-
mary rubella infection during pregnancy includes the follow-
8.1.1 Acquired Infection ing: spontaneous abortion, stillbirth/fetal death, infant born
The average incubation period is 14 days with a range of with CRS, infant born with congenital rubella infection with-
12–23 days. During the first week after exposure, there are out congenital defects, and birth of a normal infant.
no symptoms. During the second week after exposure, there The most common defects of CRS are hearing impair-
may be a prodromal illness consisting of low-grade fever ment (unilateral or bilateral sensorineural), eye defects (e.g.,
(<39.0 °C), malaise, mild coryza, and mild conjunctivitis, cataracts, congenital glaucoma, or pigmentary retinopathy),
which is more common in adults. Postauricular, occipital, and cardiac defects (e.g., patent ductus arteriosus or periph-
and posterior cervical lymphadenopathy is characteristic and eral pulmonic stenosis). Other clinical manifestations may
typically precedes the rash by 5–10 days. Children usually include microcephaly, developmental delay, purpura, menin-
develop few or no constitutional symptoms. Rarely, rubella goencephalitis, hepatosplenomegaly, low birth weight, and
may mimic measles in its severity of fever and constitutional radiolucent bone disease (Table 31.1).
symptoms, but Koplik’s spots are absent. Children with CRS may develop late-onset manifestations
At the end of the incubation period, a maculopapular ery- including endocrine abnormalities (e.g., diabetes mellitus,
thematous rash appears on the face and neck. The rubella thyroid dysfunction), visual abnormalities (e.g., glaucoma,
rash occurs in 50–80 % of rubella-infected persons and is keratitic precipitates), and neurological abnormalities (e.g.,
sometimes misclassified as measles or scarlet fever. The progressive panencephalitis), in addition to developmental
maculopapular erythematous rash of rubella starts on the manifestations which include autism [86].
face and neck and progresses down the body. The rash, which When pregnant women are infected with rubella during
may be pruritic, usually lasts between 1 and 3 days. The rash the first 11 weeks of gestation, up to 90 % of live-born infants
is fainter than measles rash and does not coalesce, and it may will have CRS; thereafter the rate of CRS declines until 17–18
be difficult to detect, particularly on pigmented skin. weeks’ gestation when deafness is the rare and only conse-
Rubella disease is usually mild, resulting in very few quence. Reinfection with rubella may occur, but if this occurs
complications apart from the serious consequences of con- early in pregnancy, transmission to the fetus is rare, and the
genital rubella infection. Transient joint symptoms (e.g., risk of congenital rubella defects is probably less than 5 %.
arthritis, arthralgias) may occur in up to 70 % of adult women
with rubella. They usually begin within 1 week after rash
onset and typically last for 3–10 days, although occasionally 8.2 Serological Responses
they may last for up to 1 month. Other complications include
thrombocytopenic purpura (1 in 3,000 rubella cases) and Antibodies to rubella virus develop promptly and can some-
encephalitis (1 in 6,000 rubella cases). In outbreaks in the times be detected on the day of rash onset. The IgM and IgG
Kingdom of Tonga (2002), the Independent State of Samoa classes rise rapidly; IgG persists, but IgM begins to wane

Table 31.1 Common transient and permanent manifestations in infants with congenital rubella syndrome
Transient manifestations Permanent manifestations
Hepatosplenomegaly, hepatitis Hearing impairment (deafness)
Thrombocytopenia with purpura/petechiae Congenital heart defects (e.g., patent ductus arteriosus, pulmonary arterial stenosis)
Dermal erythropoiesis (blueberry muffin syndrome)
Long bone radiolucencies Eye defects (cataracts, pigmentary retinopathy, congenital glaucoma,
Intrauterine growth retardation microphthalmos)
Meningoencephalitis Central nervous system involvement (e.g., microcephaly, mental and motor delay, autism)
Interstitial pneumonitis
740 W. Orenstein and S.E. Reef

(see Sect. 7). However, in a small percentage of persons, IgM 9.2 Response
persists for long period of time [30]. This persistence can be
confused with acute infection. To help differentiate IgM 9.2.1 Clinical Reactions
associated with acute infection, testing for seroconversion of Vaccines can develop mild rubella, including rash, lymph-
IgG or avidity testing is recommended. adenopathy, fever, sore throat, and headache. However, the
incidence of each of these side effects varies directly with
age, being almost absent in infants and increasing with age.
9 Control Fortunately, the minor side effects are seldom severe enough
to cause days to be lost from school or work [92–94].
9.1 Vaccine Development In a double-blind study of vaccination with MMR in
twins, there was a 1 % incidence of arthropathy and little
As noted earlier, the development of vaccines was spurred by evidence of other reactions [95]. In 1991, the Institute of
the 1962–1965 epidemics in Europe and the United States. Medicine of the National Academy of Sciences published a
Shortly after the isolation of rubella virus, investigators committee report on four possible adverse effects of rubella
attempted to develop an inactivated virus vaccine, but their vaccine: acute arthritis, chronic arthritis, neuropathies, and
attempts were unsuccessful. Either the vaccines were not thrombocytopenia [96]. The committee concluded that
antigenic or if antibodies were produced, it was questionable RA27/3 causes acute arthritis. With regard to chronic arthri-
if the preparation was contaminated with live virus [87]. tis, the committee stated doubtfully, “The evidence is consis-
With the issues of the inactivated vaccine, several groups tent with a causal relation between the currently used rubella
were interested in developing a live-attenuated vaccine in the vaccine strain (RA27/3) and chronic arthritis in adult women,
1960s. Parkman and colleagues were the first to successfully although the evidence is limited in scope and confined to
attenuate RV with 77 passages in African green monkey kid- reports from one institution.” Since that time, large vaccina-
ney cell cultures and to give the attenuated strain HPV77 [88]. tion campaigns conducted in millions of Latin Americans,
Between 1969 and 1970, three vaccines were licensed in the including women of childbearing age, have not been accom-
United States, including HPV-77.DK12 (dog kidney), HPV- panied by additional reports of significant chronic arthropa-
77.DE5 (duck embryo), and Cendehill (rabbit kidney) [46]. thy [64].
The Cendehill vaccine was licensed in Britain in 1969, and In 2011, IOM was asked to review adverse events associ-
shortly thereafter, the RA 27/3 vaccine (human diploid cells) ated with several vaccines. In their report, the IOM con-
was licensed in Europe. In Japan, the initial vaccines licensed cluded that the evidence is inadequate to accept or reject the
were the Takahashi (rabbit kidney) and Matsuura (Japanese causal relationship between MMR vaccine and chronic
quail-embryo fibroblasts) vaccines. Three additional vaccines arthralgia or arthritis in women [97].
were licensed in Japan: Matsuba (rabbit kidney), DCRB 19
(rabbit kidney), and TO-336 (rabbit kidney) [89]. 9.2.2 Shedding of Virus
By 1979, all three of the vaccines licensed in the United Because of the risk of spreading of vaccine virus to suscep-
States were replaced by RA27/3. RA27/3 vaccine generally tible persons including pregnant women, considerable effort
induces higher antibody titers and produces an immune has been made to detect the spread of vaccine virus to sus-
response more closely paralleling natural infection than the ceptible contacts. Early contact studies documented no evi-
other vaccines. HPV-77.DK12 was withdrawn due to the higher dence of spread to susceptible contacts. However, there was
incidence of side effects as compared to other vaccines. a rare asymptomatic seroconversion that could not be
After the development and licensure of the initial rubella explained fully [98, 99].
vaccines globally, additional vaccines were licensed in vari- Virus has been recovered from breast milk of women vac-
ous geographic locations. In 1980, a rubella vaccine (BRD-2) cinated postpartum. Transmission of the virus to the infant
was developed in the People’s Republic of China using a local has been documented, but the infection is asymptomatic and
RV strain from a child, isolated in human diploid cells. In a transient.
trial comparing the BRD-2 vaccine and RA 27/3 vaccine, the
seroconversion rate and mild side effects were similar [90]. In 9.2.3 Serological Response
Japan, currently, five different rubella vaccines are in use, Rubella vaccine is usually administered ≥12 months of age,
including the TO-366 vaccine [91]. Even though additional since maternal antibodies have usually disappeared by that
vaccines have been licensed and developed, RA27/3 contin- age. The seroconversion rate for children ≥12 months is
ues to be the most widely used vaccine strain globally. >95 %. The age at first vaccination does not appear to be as
Rubella-containing vaccine is available as either a single critical for rubella as for measles vaccine. Passively trans-
antigen or combined with measles (MR), measles and mumps mitted maternal antibodies to rubella have been found in
(MMR), and measles, mumps, and varicella (MMRV). approximately 5 % of infants from 9 to 12 months and 2 %
31 Rubella Virus 741

from 12 to 15 months of age. Studies of rubella-containing have introduced rubella vaccine by the end of 2015, and all
vaccine administered at 9–12 months of age has demon- GAVI eligible countries will have introduced it by 2018.
strated a seroconversion rate of >90 %. Rubella vaccine is With GAVI support, the goal of rubella eradication may be
usually offered to children with measles vaccine (MR) or within reach.
measles and mumps vaccines (MMR).
9.3.2 Vaccination in Pregnancy
Although there is now abundant evidence for the safety of
9.3 Rubella Vaccination Strategies: Their RA 27/3 for the fetus, pregnancy remains a contraindication
Impact on Rubella and Congenital to rubella vaccination, and women are advised to take pre-
Rubella cautions against pregnancy for 1 month (28 days) after vac-
cination. Prior to the efforts to eliminate rubella from Latin
9.3.1 Epidemiological Approach America, there was limited data on vaccination of unknow-
The goal of rubella vaccination programs is the prevention of ingly pregnant women [102]. To eliminate rubella and CRS
the intrauterine infection that causes CRS. There initially in the region of the Americas, countries conducted cam-
were two basic approaches: the US (indirect protection) (see paigns in adult. As part of the campaigns in several countries,
Sect. 5.1) and the UK (direct protection). However, with over women who were vaccinated and subsequently learned that
30 years of experience with introducing rubella vaccine into they were pregnant at the time of vaccination were followed
countries, the strategies have evolved. up. On the basis of serological evaluation, 2,894 (10 %)
In 2000, the WHO convened a meeting to review the women were classified as susceptible at the time of vaccina-
worldwide status of CRS and its prevention [100, 101]. Since tion; of their pregnancies, 1980 (90 %) resulted in a live
the previous international meeting on CRS and rubella in birth. Sera from 70 (3.5 %) of these infants were rubella IgM
1984, some of the changes included availability of more data antibody positive, but none of the infants had features of
on the CRS disease burden in developing countries, an CRS as a result of rubella vaccination. The maximum theo-
increase in the number of countries with national rubella retical risk for CRS following rubella vaccination of suscep-
immunization programs, and advances in laboratory diagno- tible pregnant women was 0.2 %. In all the available literature
sis. In 1996, only 83 countries/territories used rubella vac- on vaccination of pregnant women, approximately 3,000
cine in their national immunization programs. Since the susceptible women with live births have been followed up
2000 meeting, additional countries have introduced rubella- and none of the infants had features of CRS.
containing vaccine, two WHO regions (Regions of the
Americas and Europe) have established rubella elimination 9.3.3 Persistence of Vaccine-Induced Immunity
goals by 2010 and 2015, respectively, and one WHO region Studies on the long- term persistence of antibodies after
(Western Pacific) has established an accelerated rubella con- rubella immunization of susceptible persons have docu-
trol and CRS prevention goal by 2015. As of 2010, this num- mented that immunity probably persists for life in the major-
ber had increased to 130 countries. ity of vaccines. Although antibody titers fall over time,
In 2011, the WHO rubella vaccine recommendations sometimes to very low levels, immunological memory per-
were updated [64]. The WHO recommends that countries sists, and a secondary immune response will occur on expo-
that have not introduced rubella vaccination take the oppor- sure to rubella.
tunity offered by accelerated measles control and elimination Follow-up studies have shown that 95–100 % RA27/3
to introduce rubella vaccine. The measles vaccine strategy vaccines are seropositive 10–21 years after immunization
platform provides the opportunity to use combined vaccine [103]. The high seroconversion rate, the persistence of anti-
and an integrated measles-rubella surveillance system. The bodies, and an amnestic response when revaccinated do not
preferred strategy for introduction of rubella vaccination is support the need for a second dose of rubella vaccine.
to begin with MR/MMR vaccine in a campaign targeting a However, based on the indications for a second dose of mea-
wide range of ages together with immediate introduction of sles- and mumps-containing vaccine, a second dose of MMR
MR/MMR vaccine into the routine program. In 2011, GAVI is now offered in most industrialized countries, and this helps
(formerly the Global Alliance for Vaccines and Immunization) to boost low rubella antibody concentrations.
opened a window for introduction of rubella-containing vac-
cine into GAVI eligible countries (for more on GAVI, see 9.3.4 Reinfection
Chap. 1. Of the remaining 63 countries that had not intro- Reinfection is usually subclinical and is more likely to occur
duced rubella vaccine in 2011, 51 (81 %) are GAVI eligible. in persons with vaccine-induced immunity than in those
GAVI funding will support MR vaccine for catchup cam- whose immunity is naturally acquired. It is not due to anti-
paigns targeting children 9 months to 14 years 11 months genic variants of rubella virus [104]. Reinfection is defined as
and introduction grant. It is estimated that 30 countries will a significant rise in antibody concentration in a person with
742 W. Orenstein and S.E. Reef

preexisting antibodies. In a clinical situation, preexisting anti- 15. Cooper LZ, Krugman S. Clinical manifestations of postnatal and
bodies can be confirmed by testing an earlier stored serum, congenital rubella. Arch Ophthalmol. 1967;77:434–9.
16. Meyer HM, Parkman PD, Hobbins TE, et al. Attenuated rubella
but if no such serum is available, evidence of preexisting anti- viruses: laboratory and clinical characteristics. Am J Dis Child.
body may be accepted if there are at least two previous labo- 1969;118:155–69.
ratory reports of antibodies ≥10 iu/ml obtained by reliable 17. Prinzie A, Huygelen C, Gold J, et al. Experimental live attenuated
techniques (not HI) or a single result of antibodies ≥10 iu/ml rubella virus vaccine: clinical evaluation of Cendehill strain. Am J
Dis Child. 1969;118:172–7.
obtained after documented rubella vaccination [105]. 18. Plotkin SA, Farquhar J, Katz M, Buser F. Attenuation of RA27/3
The concern for reinfection is a pregnant woman as it might rubella virus in WI-38 human diploid cells. Am J Dis Child.
lead to fetal infection. Several challenge tests have been con- 1969;118:178–85.
ducted with some attempting to document viremia. In one of 19. Reef SE, Redd SB, Abernathy E, Kutty P, Icenogle JP. Evidence
used to support the achievement and maintenance of elimination
these studies [106], the viremia was detected in persons with of rubella and congenital rubella syndrome in the United States.
low levels of antibody titers. The risk of congenital infection J Infect Dis. 2011;204 Suppl 2:S593–7.
following reinfection in the first 12 weeks of pregnancy has 20. Averhoff F, Zucker J, Vellozzi C, Redd S, et al. Adequacy of sur-
been estimated in the United Kingdom to be about 8 %, while veillance to detect endemic rubella transmission in the United
States. Clin Infect Dis. 2006;43 Suppl 3:S151–7.
the risk of congenital rubella defects is probably no more than 21. Upreti SR, Thapa K, Pradhan YV, Shakya G, et al. Developing
5 %, which is considerably less than the >80 % risk of primary rubella vaccination policy in Nepal–results from rubella surveil-
rubella during the same period of pregnancy [107]. Thus, it is lance and seroprevalence and congenital rubella syndrome stud-
important to be able to use laboratory tests to distinguish rein- ies. J Infect Dis. 2011;204 Suppl 1:S433–8.
22. Pebody RG, Edmunds WJ, Conyn-van Spaendonck M, Olin P,
fection from primary rubella in pregnancy. et al. The seroepidemiology of rubella in Western Europe.
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23. Cockburn WC. World aspects of the epidemiology of rubella. Am
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 Part III
Viruses Causing Acute and Chronic Syndromes
and/or Malignancy
 Hepatitis Viruses: Hepatitis B
and Hepatitis D 32
Alison A. Evans, Chari Cohen, and Timothy M. Block

1 Hepatitis B Virus with a long incubation period [6], identification of the agent
itself eluded researchers until the mid-1960s when Blumberg,
The hepatitis B virus (HBV) virus is a small, partly double- Alter, London, and colleagues discovered a serum protein,
stranded enveloped DNA virus and is the etiological agent of named the “Australia” antigen, present in the blood of some
human hepatitis B infection. HBV is the world’s most com- Australian aborigines, individuals with Down syndrome,
mon cause of chronic viral hepatitis and hepatocellular carci- and oncology patients [7, 8]. The initial evidence suggested
noma (HCC). The latter condition is covered extensively in that the presence of this protein might be a marker of risk
Chap. 34. It is estimated that there are up to 400 million for development of leukemia. Through an exhaustive series
people chronically infected with HBV around the globe, of epidemiologic studies, it became clear that the Australia
most of whom are unaware of their infection status [1–3]. antigen might instead be associated with an infectious agent.
The virus is >50 times more transmissible than HIV, but an This hypothesis took precedence when it was demonstrated
effective vaccine is available and is having an impact on the in a clinical study that a subject previously negative for the
global burden of disease caused by hepatitis B [1]. The viral antigen became positive, coincident with biochemical evi-
infection and its control has been the focus of intensive sci- dence of liver inflammation [4, 9].
entific research since its discovery. Further studies led to the visualization of the virus parti-
cle (called the Dane particle) by electron microscopy [10,
11] and demonstration of its transmissibility in animal stud-
1.1 Historical Background ies and via blood transfusion [4, 9]. Using banked blood
from transfusion recipients, Prince [12] showed that the cir-
Hepatitis as a syndrome has been recognized since ancient culating antigen was first detected during the incubation
times, but the distinction of two forms – one usually transmit- period for posttransfusion hepatitis, was likely a component
ted via the fecal-oral route and one more often parenterally of the virus particle, and was persistently detected for long
transmitted – began to emerge in the early- to mid-twentieth periods after apparent recovery in some patients, consistent
century [4, 5]. Although it could be shown that the parenter- with what was then referred to as serum hepatitis, i.e., a hep-
ally transmitted agent had properties consistent with a virus atitis with a long incubation period and an asymptomatic car-
rier state [12]. Studies by Krugman et al. in institutionalized
A.A. Evans, ScD (*) developmentally disabled patients had separately identified
Department of Epidemiology and Biostatistics, sera that were capable of transmitting serum hepatitis with
Drexel University School of Public Health,
clinical characteristics similar to those seen by Prince [5].
3215 Market Street, Philadelphia, PA 19104, USA
e-mail: [email protected] Since “infectious hepatitis” transmitted via the fecal-oral
route was known as hepatitis A, the serum hepatitis virus was
C. Cohen, MPH, DrPH(c)
Public Health Research, Hepatitis B Foundation, called the hepatitis B virus (HBV). The Australia antigen
3805 Old Easton Road, Doylestown, PA 18902, USA was shown to be the surface protein of the viral particle, now
e-mail: [email protected] called hepatitis B surface antigen or HBsAg. The test for
T.M. Block, PhD HBsAg quickly became an important tool for prevention of
Department of Microbiology and Immunology, posttransfusion hepatitis by its use in screening blood
Drexel College of Medicine and Baruch S Blumberg Institute
donations [9].
of The Hepatitis B Foundation,
3805 Old Easton Road, Doylestown, PA 18902, USA Two major discoveries quickly followed confirmation of
e-mail: [email protected] the association of HBsAg with serum hepatitis. The first of

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 747

DOI 10.1007/978-1-4899-7448-8_32, © Springer Science+Business Media New York 2014
748 A.A. Evans et al.

these was the observation that noninfectious HBsAg parti- reporting sites. The peak prevalence was found in the 25–54-
cles could be used to produce an effective vaccine against year age group and in Asian/Pacific Islanders [18].
HBV [13]. Shortly after this came the confirmation that
chronic HBV infection is a major cause of hepatocellular 1.2.3 Serologic Surveys
carcinoma worldwide [14, 15]. General population serosurveys have examined the preva-
lence of HBV markers in many populations worldwide
[21–23]. These data have been used to define the general
1.2 Methodology Involved epidemiologic patterns of chronic HBV infection seen
in Epidemiologic Analysis worldwide (see Sect. 1.4). In a 2012 systematic review, Ott
et al. [24] used 396 published articles with data from general
1.2.1 Source of Mortality Data populations to estimate global HBsAg prevalence at 3.7 %.
Mortality from acute HBV infection is uncommon although Surprisingly, age-specific prevalence was highest in children
as many as 1 % of the primary infections lead to decompen- in West Africa (~8.5 %). East Asian age-specific prevalences
sated liver disease and failure and death. However, most exceeded those of West Africa only in adults age 45 and
HBV-related deaths are from hepatocellular carcinoma older.
(HCC) or liver cirrhosis (LC). The World Health Carefully constructed large-scale national serosurveys in
Organization (WHO) estimates that 600,000 individuals China, conducted in 1992 and repeated in 2005, have been
worldwide die each year as a result of HBV infection [16]. used to estimate the impact of disease control measures, par-
In the United States, the number of deaths is approximately ticularly immunization, on age-specific prevalence of HBV
3,000 per year [17]. In vital statistics data, HBV-related markers. In these two studies, the prevalence of HBsAg in
deaths may be undercounted because HCC and LC deaths children under 5 years old declined from 9.7 % in 1992 to
are often not attributed to specific viral etiologies on death 1.0 % in 2005, but no change was seen in prevalence in adults
certificates [18, 19]. Among 1,788 deaths in the United age >20 years. All age groups showed a reduced prevalence
States where HBV was listed as a cause of death, the demo- of anti-HBc, however, suggesting that overall exposure to
graphic groups with the highest standardized mortality rates HBV has been reduced in the population through immuniza-
were found in males (approximately 3-fold higher than tion of infants and other public health measures [25]. Within
females) and non-White/non-Black racial groups (approxi- countries such as the United States, general surveys such as
mately 5-fold higher than Whites). Age- specific mortality the National Health and Nutrition Examination Surveys
rates peaked in the 55–64-year-old age group [18]. In areas (NHANES), good indicators of the health status of the
of the world where the prevalence of chronic HBV infection majority of the population, have underestimated prevalence
is high, its impact on mortality is reflected directly in HCC of HBV and other diseases that affect the minority or disad-
and LC death rates. vantaged groups not fully included in the sampling approach.
The CDC-adjusted estimate of chronic HBV seroprevalence
1.2.2 Sources of Morbidity Data in the United States was 1.2 million persons in 2012 [17].
Acute HBV infection is a reportable disease in many parts of The true prevalence may be as high as 2–2.2 million, the dif-
the world. In populations where the infection is endemic, ference being largely due to undercounting of foreign-born
however, the majority of infections occur at younger ages persons [26, 27].
and are likely to be asymptomatic and to result in chronic
infection, which is not generally included in reportable dis- 1.2.4 Laboratory Diagnosis
ease statistics. In recent years, the United States has seen a A number of laboratory tests are available for the detec-
dramatic decline in reported acute HBV cases, falling from tion of current and past HBV infection and immunity. The
8,036 cases nationwide in 2000 to 3,350 in 2010. In 2010, markers and their interpretation in combination are summa-
the age group with the highest incidence rate for reported rized in Table 32.1. Most markers are detected by standard
cases was the 30–39-year-olds. Among those with risk factor enzyme immunoassay (EIA) or radioimmunoassay (RIA)
reported, sexual behaviors (sexual partner with known or and reported as either positive or negative. Some markers
suspected HBV infection, multiple sexual partners, and/or may also be quantitated by these methods.
men who have sex with men) were found more frequently HBsAg is the viral surface (envelope) glycoprotein coat.
than injection drug use and medical or occupational expo- While HBsAg is not itself infectious, its detection in the cir-
sure [18]. Chronic HBV has been a reportable disease since culation is an indication of current infection and the potential
2007, but only a few states routinely report cases. Because of for infectiousness to others. It is present in large amounts in
the difficulties in determining chronicity, enhanced surveil- the circulation of most infected persons, both as full viral
lance programs provide the most reliable data [20]. In 2010, particles and as large numbers of empty particles, round or
the CDC received reports of 10,515 chronic cases from eight filamentous in shape, which contain no viral DNA [28, 29].
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 749

Table 32.1 Patterns of serologic markers of HBV infection and their usual significance [1, 31]
HBsAg Anti-HBs Anti-HBc IgM anti-HBc HBeAg Anti-HBe HBV DNA
e antigen,
Viral Total antibody IgM class antibody associated Antibody
glycoprotein coat Antibody to HBsAg to viral core to viral core with viral core to HBeAg Viral DNA
Susceptible − − +/− − − − −
Natural immunity − + + − NA NA −
(past infection)
Vaccine-induced − + (≥10 mIU/mol − − NA NA −
immunity considered adequate)
Early acute + − + + + − +
infection
Chronic infection + − − − +/− +/− +/−
Presence for ≥6
months indicates
chronic infection
Resolved or − + + − − − −
resolving infection
“Occult” infection − − + − +/−
NA not applicable

These subviral particles may be present at 100-fold or more a level of ≥10 mIU/ml is an indicator of protective immunity
excess in relation to viral particles [29, 30]. [31]. While rare, the co-occurrence of HBsAg and anti-HBs
HBsAg is detectable in serum beginning 1–2 months after positivity have been reported in some chronic infections [36].
infection, sometimes much earlier, and prior to the onset of Antibody to hepatitis B core protein (anti-HBc) is non-
symptoms. Persistence for 6 months or longer is an indica- neutralizing, is detectable in all HBV infections, and persists
tion that chronic infection has been established. In acute indefinitely. It is not detected in persons who are immune to
infection, HBsAg levels begin to subside after the onset of HBV through vaccination [31]. It arises first as IgM class
symptoms and disappear by 1–5 months after infection, as anti-HBc, after the appearance of HBsAg in both acute and
symptoms resolve [31, 32]. In chronic infection, HBsAg chronic infection [28, 32]. A high level of IgM anti-HBc is
remains detectable for extended periods, often for the life- an indication of recent infection, and a predominantly IgM
time of the infected individual. In general, circulating anti- response may be detectable for a short time even after resolu-
gen load decreases after the first few months of chronic tion of HBsAg in acute infection. Thus, high-titer IgM anti-
infection but remains easily detectable by standard assays HBc in the absence of HBsAg is accepted as the laboratory
[28, 32]. Quantitation of circulating antigen load is emerging component of the case definition for reportable acute HBV
as a clinical marker of disease stage or treatment response in infection [31, 37]. Nevertheless, IgM anti-HBc may also
chronically infected individuals [30]. occasionally be detected in chronic HBV infection as well,
The so-called occult HBV, i.e., apparently HBsAg- often in the course of seroconversion from HBeAg to anti-
negative HBV infection (defined by HBV DNA in the liver HBeAg positivity [38].
or blood), has been described in some populations. In some Hepatitis B e antigen (HBeAg) is a soluble protein
cases HBsAg is present but in a variant form not recognized secreted from HBV-infected hepatocytes and encoded in the
by standard commercial assays. In other cases, HBsAg is open reading frame (ORF) for the C protein, with an alter-
present but suppressed to very low levels by host immuno- nate initiation site. Its precise function is not fully under-
logic state, viral mutation, and/or coinfections with other stood. Its presence is not necessary for viral replication, but
viruses (e.g., HIV, HCV). In some cases, serologic markers clearance of circulating HBeAg usually coincides with a
such as anti-HBc and anti-HBs may be absent as well [33]. reduction in viral load [28]. HBeAg is present during acute
Clinically, occult infections are significant because blood infection but clears usually prior to the clearance of HBsAg
and body fluids from patients may still be infectious and [39]. In chronic infection, it persists for a variable period,
some occult HBV patients are at high risk of HCC and influenced by host characteristics and viral genotype. In
chronic liver disease [34, 35]. some chronically infected persons, HBeAg may reappear
Antibody to HBsAg (anti-HBs) becomes detectable in the and disappear repeatedly over the course of infection [40].
serum of acutely infected individuals after the decline of The appearance of antibody to HBeAg (anti-HBe) accom-
HBsAg [32]. It indicates the development of protective panies or follows clearance of HBeAg in both acute and
immunity in natural infections and in response to HBV vac- chronic infections. In chronic infection, this is usually an
cination. It can be quantitated by routine clinical assays, and indicator of transition to less active disease, evidenced by
750 A.A. Evans et al.

lower liver transaminases and low to undetectable viral load. packaged in the cytoplasm, after which the viral poly-
In 10–30 % of anti-HBe seroconversions, however, active merase reverse transcribes the template into the DNA in the
hepatitis will continue, with elevated liver transaminases and partially double-stranded form found in viral particles in
detectable viral load. In another 10–30 % who initially have the circulation. In addition to these particles, smaller subvi-
less active disease after the development of anti-HBe, more ral particles composed of envelope proteins but no genetic
active disease returns at a later time [41]. material are secreted from infected cells in great excess
HBV DNA can be detected in circulation and in liver tis- [28]. In a chronic infection, as many as 10 [11] viral parti-
sues. Commercial assays using hybridization or amplifica- cles per day may be released into circulation, with a half-
tion methods for quantitation of circulating viral load are life of approximately 1 day [46].
now widely available on several commercial platforms. Serotypic variants of envelope proteins, designated as adr,
Many assays are able to detect viral loads as low as 6 IU/ml adw, ayr, and ayw, have long been noted in HBV infection.
(30 viral copies/ml) [42]. HBV DNA is first detected in While these do not appear to strongly associated with differ-
blood very early in both acute and chronic infection, the ear- ences in clinical disease, they vary considerably by geogra-
liest of the virus-specific markers. In acute infection, it drops phy and have been used epidemiologically to study
to very low levels by the time of HBsAg clearance, but using transmission and potential vaccine escape mutants [47, 48].
very sensitive methods, HBV DNA can still be detected at Genotypic variants of HBV have been classified into ten
times in the circulation of subjects who otherwise appear to different types (A–J) with >8 % diversity at the nucleotide
have cleared infection [28, 43]. In chronic infection, viral level, each with a characteristic geographic distribution [44,
load is high initially but may drop to low or undetectable 49, 50]. Genotypes A–H are considered to be the major ones
levels later [28]. Very high viral loads (>200,000 IU/ml) worldwide and have been the most extensively studied.
occur when the host immune system does not recognize the Subgenotypes (diversity of 4–8 %) have been described for
virus as foreign. During active immune response, viral loads most of the major genotypes. HBV genotypes differ in the
diminish and may drop to at or below the limits of detection frequency with which clinically significant mutations in the
when disease moves into the inactive phase [41]. Viral isola- precore (PC), basal core promoter (BCP), and pre-S1 regions
tion or viral culture are not used in the routine detection or are found [41, 47, 49]. The clinical significance of different
monitoring of HBV infection. HBV genotype and mutation combinations is an area of
ongoing research. This is briefly reviewed below (Sect. 3.19).
HBV remains intact under many environmental condi-
1.3 Biological Characteristics tions, but under most circumstances it can be handled safely
of the Organism at Biosafety Level 2. It is resistant to freezing, ether, and acid.
When dried on surfaces at room temperature, it can remain
HBV is classified as a Hepadnavirus, a family of DNA intact for at least 1 month. Boiling for at least 1 min or heating
viruses that also includes mammalian (woodchuck, ground to 60 °C for at least 1 h will inactivate the virus as will stan-
squirrel) and avian (duck) viruses that are similar in structure dard autoclaving or dry heat methods. Hypochlorite, glutaral-
and in their hepatotropism. Viral replication occurs in hepa- dehyde, formaldehyde, alcohol, and some other commonly
tocytes through a reverse transcription step that is unique to used disinfectants can inactivate HBV with sufficient expo-
this family of viruses [44, 45]. Infections of cells other than sure time. HBsAg is resistant to UV irradiation [1, 45, 51].
hepatocytes, if they occur, do not appear to cause significant
disease [29]. Humans appear to be the only natural host for
HBV, but infections of nonhuman primates have been pro- 1.4 Descriptive Epidemiology
duced in the laboratory [31].
The circular, partially double-stranded viral genome is The World Health Organization (WHO) has described
small, ~3,200 nt, and encodes four distinct proteins in over- three epidemiologic patterns of HBV infection based
lapping reading frames. These are the polymerase (P), the on the prevalence of serologic markers (Fig. 32.1). In the
envelope (S), the nucleocapsid or core (C), and the X lowest-risk regions (North America, Australia, Northern/
protein. Within the S ORF, three different surface proteins Western/Central Europe), the prevalence of HBsAg is <1 %
are encoded by different initiation codons – the S, pre-S1, in the general population and 4–6 % for anti-HBs. The
and pre-S2. Similarly, the C region encodes the core intermediate-risk regions (Eastern Europe, Central/South
(HBcAg) and e antigens (HBeAg) [28, 44]. Upon entry into America, Mediterranean, Southwest Asia) have HBsAg in
the host cell, viral genomes contained in core particles are up to 7 % and anti-HBs inasmuch as 55 %. In the highest-
repaired to form covalently closed circular DNA (cccDNA), risk regions (Southeast Asia, China, sub-Saharan Africa), the
which in turn acts as the transcription template for viral HBsAg prevalence may be as high as 20 % and anti-HBs
mRNA and genomic viral RNA. Genomic copies are positivity is nearly universal, ~95 % [52].
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 751

High (HBsAg prevalence ≥8%)

Intermediate (HBsAg prevalence 2%–7%)

Low (HBsAg prevalence <2%)

Fig. 32.1 Worldwide areas of high, intermediate, and low prevalence of HBsAg (From WHO [53])

1.4.1 Prevalence and Incidence comprise a significant proportion of chronically infected

The prevalence of chronic HBV infection is the best indica- individuals in the population. The low prevalence areas rep-
tor of the geographic burden of disease (Fig. 32.1). In areas resent about 12 % of the world’s population [1, 31, 54].
of high prevalence, i.e., ≥8 % HBsAg positive, 70–90%of
the population may become infected with HBV prior to 40 1.4.2 Epidemic Behavior
years of age. In these areas, infection in the perinatal period Widespread epidemics of hepatitis B infection have not been
or early childhood is common and is responsible for the bulk reported frequently. Small outbreaks may go unrecognized
of chronic infections. Acute hepatitis B is rarely seen since if links between apparently sporadic cases are not identified.
most people are exposed early in life. High rates of LC and In recent years, detected outbreaks in the United States have
HCC in adults in these populations reflect the impact of these been most frequently associated with nosocomial exposures
early life infections. The high prevalence areas include about in nonhospital settings. Improper infection control proce-
45 % of the world’s population [1, 31, 53]. dures in the use of blood glucose monitoring equipment have
In areas of intermediate prevalence (2–7 % HBsAg posi- been involved in multiple outbreaks in long-term care facili-
tive), acute hepatitis B is more common since age at infec- ties. Hemodialysis and other outpatient medical settings have
tion is shifted to older groups. Lifetime risk of infection may been implicated as well [55, 56]. Similarly, in the United
be as high as 20–60 %, including both perinatal/early child- Kingdom, outbreaks are most frequently associated with
hood infections and parenteral and sexual exposures later in nosocomial exposures [57] but have also been documented
life. The intermediate areas comprise about 43 % of the in groups with shared risk behaviors such as injection drug
world’s population. use and heterosexual intercourse [58]. In the past, nosoco-
In the low prevalence regions (<2 % HBsAg positive), mial infections caused by infected health-care workers’ con-
most new infections occur in adults through parenteral, sex- tacts with patients have been reported but have decreased in
ual, and occupational routes. Lifetime infection risk is incidence with the widespread use of HBV vaccination and
<20 %. Immigrants from higher-risk areas of the world may universal precautions against blood-borne infections [54].
752 A.A. Evans et al.

1.4.3 Age products; body secretions that contain blood, semen, vaginal
Age-specific risks of infection with HBV differ by popu- secretions; and cerebrospinal, peritoneal, pericardial, syno-
lations, as described above in Prevalence and Incidence. vial, and amniotic fluids [67]. Saliva of infected persons con-
Age at infection is a strong determinant of chronicity in tains small amounts of virus, and transmission through bites
HBV infection. Infants infected perinatally have a risk of is possible, while other contacts with saliva (e.g., through
developing chronic infection of about 90 %. When infec- kissing) have not been shown to transmit infection. Breast
tion occurs after the perinatal period through about 5 years milk from infected mothers has not been shown to be a
of age, the risk of chronic infection is reduced to 25–30 %. source of HBV infection but caution is recommended if nip-
In older children and adults, about 5–10 % of infections ples are cracked or bleeding [68, 69]. Natural transmission of
become chronic [59, 60]. Most chronic infections are HBV by urine, feces, tears, sweat, or respiratory droplets has
asymptomatic for long periods following infection. In not been reported [31, 70].
acute infections, the development of symptoms is also
age dependent, with young children showing symptoms in 1.5.1 Direct Parenteral Exposure
only 5–15 % of infections but older children and adults in Without prophylaxis, mother-to-child transmission occurs
33–50 % [60, 61]. most often when mothers are HBeAg positive and/or have
high circulating viral load during pregnancy. Babies born to
1.4.4 Sex these mothers have rates of infection as high as 70 % [71,
HBV infection rates are higher in males in many populations 72]. In mothers who are HBsAg positive without HBeAg,
due to two factors. The male predominance in acute infection perinatal transmission risk is lower, about 10 % [31]. Most
may be due to higher numbers of males in risk groups for vertical transmission occurs in the perinatal period, but a
parenteral exposure such as injection drug users. The preva- small proportion of cases show evidence of intrauterine
lence of chronic infection is also higher in males in most transmission [73, 74].
populations worldwide, even where prevalence of markers of Other parenteral exposures contribute to HBV infection
past HBV infection does not differ by gender. This may be as well. In medical settings, transmission from patient to
due to a predisposition in males to develop chronic infections patient has been frequently documented where infection
or to a higher prevalence of other predisposing factors among control practices or blood donation screening methods are
men, e.g., immunosuppression [52, 61, 62]. inadequate [59, 75]. Unsafe injections in medical settings
were estimated to be responsible for 21 million new HBV
1.4.5 Race infections worldwide in 2000, nearly one third of all new
Race may be considered a risk factor for HBV infection infections [76]. Injection-associated risk can be attributed to
insofar as it reflects origins in populations of high ende- the reuse of needles and other devices used for immuniza-
micity, i.e., Asians, Pacific Islanders, Alaska Natives, and tions, finger-stick devices, and acupuncture needles [61].
Africans. Hemodialysis settings are particularly problematic because
of the high potential for environmental contamination [77,
1.4.6 Occupation 78]. Transmission from infected health-care providers has
Persons employed in any setting where there is risk of con- been reported, particularly in the past when universal precau-
tact with blood or body fluids are at increased risk of HBV tions and immunization were less frequently used. In recent
infection. Health-care and public safety workers [63, 64], years in the developed world, such transmission has been
embalmers [65], staff in residential facilities for the develop- reported only rarely, and risk appears to be found largely in
mentally disabled [63], and body piercers and tattoo artists individuals with high viral loads who performed invasive
[66] are among those for whom measures to prevent blood- procedures [79].
borne infections, including HBV immunization, have been Injection drug users are at risk for HBV infection through
recommended. sharing of injection equipment, particularly in communities
where there is prevalent infection and low rates of immuni-
zation [31]. Receipt of tattoos in prison or other settings
1.5 Mechanisms and Routes where sterile equipment is not available has also been shown
of Transmission to be associated with HBV transmission, while tattoos per-
formed with sterile equipment are not associated with trans-
Humans are the only natural reservoir of HBV infection. mission [80–82].
Transmission occurs through parenteral routes, percutane-
ously or permucosally. Body fluids and tissues of infected 1.5.2 Inapparent Parenteral Exposure
persons that have been shown to contain HBV and are there- While HBV infections occur in settings without obvious
fore potential sources of infection include blood and blood direct parenteral exposures, it is thought that inoculation
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 753

occurs through openings due to skin conditions, injuries, or Shortly after exposure, there is high-titer viremia, fol-
insect bites rather than through intact skin or mucosal sur- lowed by the detection of HBsAg, usually at 4–10 weeks
faces [59, 83]. The ability of HBV to remain infectious for after infection, and then HBeAg. Humoral immune response
long periods in the environment contributes to infections is first detected as anti-HBc, several weeks after the appear-
that occur through inapparent parenteral means. Horizontal ance of viral antigens, by which time most of the hepato-
transmission may occur within families, particularly among cytes have been infected [28, 29, 92]. Infected individuals
children due to their high viral loads [60, 70, 84] and from with immature or suppressed immune response are more
infected mothers to their children even after the perinatal likely to develop immune tolerance to infection and become
period [54]. Prolonged sharing of living quarters and com- chronically infected without evidence of significant liver
munal use of personal items such as razors and toothbrushes damage [28, 92].
is associated with infection in households where at least In acute transient infections, specific T lymphocyte
one person is already infected [67, 85]. Risk of infection responses to many HBV antigens are detectable only after
continues through early childhood in children of infected several weeks of infection during which viral particles and
mothers [54]. antigens are present circulating at very high levels [28, 90].
Unprotected sexual contact is a common means of trans- Once this response is initiated, circulating viral load begins
mission of HBV infection, in both heterosexuals and men to drop, and infection is rapidly cleared from nearly all hepa-
who have sex with men (MSM). In age-adjusted HBV marker tocytes [28]. The exact means by which clearance is accom-
prevalence data among US adults, increasing numbers of plished is not fully understood but appears to require the
lifetime sexual partners, earlier age at first intercourse, and rapid replacement of infected hepatocytes killed by CTLs
MSM were associated with higher prevalence of HBV infec- along with releases of cytokines that reduce viral replication
tion [86]. Nonimmune sexual partners of HBV-infected per- in the remaining infected cells without killing them [28, 29,
sons have a high rate of incident infection [87]. History of 93]. Circulating HBsAg and HBeAg diminish as well and
other sexually transmitted infections and anal intercourse generally clear by 4–6 months after infection [32]. While
have also been identified as risk factors. transaminase levels may rise to very high levels during this
period leading up to clearance, it appears to occur without
massive destruction of infected hepatocytes [28, 29]. Even
1.6 Pathogenesis and Immunity after apparent recovery, patients who have had acute HBV
infections may have detectable HBV DNA in circulation for
Pathogenesis in HBV infection is primarily but not exclu- long periods of time [28, 92].
sively confined to the liver. Extrahepatic manifestations such In infections that become chronic, the specific cellular
as polyarteritis nodosa, glomerulonephritis, joint inflamma- immune responses to viral antigens, especially cytotoxic T
tion, and skin rashes can occur in both acute and chronic lymphocyte responses, are much weaker and narrower than
infections [88, 89] as a result of deposition of immune com- in acute infections [90]. Humoral response, however, is simi-
plexes [90]. While there is some evidence that viral replica- lar to that seen in acute infection except in that anti-HBs is
tion is possible in cells other than hepatocytes, there is no rarely detected [28]. An immunosuppressive role for HBeAg
consistent evidence that this contributes to pathogenesis has been postulated as partial explanation for the failure to
[29]. Moreover, HBV replication is usually not directly cyto- clear infection [29]. Because chronic infection is a more
toxic to hepatocytes. During incubation, there is a long likely outcome in infections of very young or immunosup-
period during which viral replication is ongoing, but there pressed individuals, the functional capacity of host immune
are no symptoms, no biochemical evidence of liver inflam- response clearly has a role as well. Over time, however,
mation, and no detectable immune response [28, 90]. It is chronically infected individuals may exhibit many changes
when immunologic response to infection is detected that in circulating markers of viral infection, liver damage, and
there is evidence of liver damage. This is consistent with the immune response. These are dependent on both host and
idea that it is the immunologic response rather than the virus viral characteristics.
itself that is largely responsible for hepatocellular damage
[28, 29]. Possible exceptions to this may be in fulminant
hepatitis B where some viral variants associated with this 1.7 Patterns of Host Response
outcome have in vitro characteristics that may be cytotoxic
in vivo [91] and in fibrosing cholestatic hepatitis, a rare out- HBV infection produces a variety of host responses ranging
come after liver transplant in HBV-infected patients [92]. in severity from self-limited asymptomatic infection to life-
Otherwise, the observed sequence of appearance of serologic time chronic infection. In acute symptomatic infection, signs
markers indirectly supports the lack of direct cytopathic and symptoms are nonspecific and similar to other forms of
effect of HBV infection. acute hepatitis, including malaise, nausea, vomiting, rash,
754 A.A. Evans et al.

joint pains, and abdominal pain. Acute hepatitis may occur markers and the patterns associated with different clinical
with our without jaundice [67]. manifestations of infection are summarized in Table 32.1.
Chronic infections often do not begin with obvious symp- Table 32.2 summarizes the serologic patterns seen in the dif-
toms, and their manifestations differ between individu- ferent phases of chronic infection [2, 41].
als and over the same individual’s lifespan. Three defined
phases of chronic infection – immune tolerant, immune 1.7.2 Clinical Outcomes of Infection
active, and inactive hepatitis – are defined by the combina- In acute, self-limited infections, about 70 % are asymptom-
tion of HBeAg/anti-HBe, viral load, and evidence of liver atic. Among the remaining 30 %, symptoms range from mild
inflammation. to severe and can persist for some months [32, 39]. Recovery
Chronic infections occurring later in life usually begin in from infection is accompanied by the appearance of anti-
the immune-active phase with HBeAg present [41, 94]. HBs, an indicator of immunity to reinfection [63]. Some 1 %
Immune tolerant is the initial phase for individuals infected of acute infections develop into fulminant hepatitis with
perinatally, and it may persist for decades with positive massive hepatic necrosis [67]. Risk of fulminant hepatitis is
HBeAg and high viral load but little or no evidence of liver increased by coinfection with other hepatotropic viruses,
damage [95]. Many of these individuals eventually progress e.g., HCV or HDV [2].
to the immune-active phase in adulthood though the trigger- Chronic infections often begin with no obvious symp-
ing events for this transition are poorly understood [95]. toms, especially in infants and young children. Some decades
These later acquired infections usually begin in or quickly may pass before signs and symptoms of liver inflammation
transition to the immune-active phase with some evidence of occur as the host transitions into the immune-active phase.
immune response to infection. In this phase, viral loads are Chronic infections do sometimes resolve spontaneously, at a
lower than in the immune-tolerant phase but still readily rate of clearance of HBsAg 0.5–1 % per year, usually during
detectable, and HBeAg is present. Immune-mediated liver the immune-inactive phase [41]. It is estimated that 10–30 %
damage results in elevations of ALT and histologic evidence of chronically infected individuals will eventually develop
of liver inflammation as well as fibrosis in some. The risk of LC or HCC [15, 39, 99]. In addition to disease activity, age,
the most serious morbidity and liver-related mortality gender, family history, and lifestyle habits (e.g., alcohol con-
increases with the duration of the phase. For some, however, sumption) can affect risk [100–102]. Conversion and rever-
there is successful transition to less active disease, indicated sion between HBeAg positive/negative or high-titer HBV
by HBeAg to anti-HBe conversion, reduction of viral load, DNA positive/negative are also associated with higher risk of
and normalization of liver enzymes. Prior to this transition, HCC [40, 103].
there may be a transient hepatitis flare. After HBeAg clear- There is considerable evidence for variation in the natural
ance, most individuals enter the inactive hepatitis phase, history of disease for different HBV genotypes and subgeno-
characterized by lower viral load, normalization of ALT, and types, but the full range of comparisons is not possible
reduction of histologic evidence of liver damage [2, 41]. because of the geographic specificity and rarity of some vari-
After seroconversion to the anti-HBe-positive state, a sub- ants. Moreover, some observed differences in natural history
set of individuals will continue to have moderate to high between genotypes/subgenotypes may not be direct effects
viral loads and evidence of liver inflammation. This is of genetic variation. For example, a variant that appears to be
referred to as anti-HBe-positive active hepatitis and is asso- more likely to cause chronic infection may do so indirectly
ciated with ongoing hepatocellular damage [96, 97]. Others through prolonging the HBeAg+ period in chronic infection
who convert to anti-HBe positive and enter the inactive phase which would in turn lead to higher rates of perinatal trans-
may experience one or more reversions to HBeAg-positive mission and, consequently, younger age of infection, which
immune-active phase, during which viral load rises and liver itself is associated with increased risk of chronic infection.
inflammation increases [96, 98], and the risk of cirrhosis is There are also known differences between genotypes and
high compared to those who sustain transition to inactive subgenotypes in the prevalence of viral mutations that are
hepatitis [97]. independently associated with disease progression [49, 104,
105], and these cannot always be taken into account in natu-
1.7.1 Serologic Patterns of Infection ral history studies due to limitations of sample size or labora-
The incubation period of acute HBV infection is usually tory resources.
between 60 and 90 days from the time of infection to the Despite this caveat, within populations some clear geno-
onset of jaundice or ALT elevation [32, 63]. The first detect- type/subgenotype differences in natural history have become
able serologic marker is HBV DNA, followed by HBsAg, apparent. The best studied is the comparison outcomes in
both of which appear before the onset of symptoms. The genotype B vs. genotype C in Asian populations where both
length of the incubation period is related to both the amount are found. In Taiwan, adults with genotype B chronic infec-
and route of inoculation as well as host factors [67]. Serologic tion had higher age-specific prevalence of HBeAg and higher
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 755

Table 32.2 Characteristics of the phases of chronic HBV infection

Immune tolerant Immune active Inactive hepatitis
HBeAg Present May be present or absent Absent, conversion to anti-HBe+
Viral load High (>200,000 IU) >20,000 IU/ml (HBeAg+ form) Low or undetectable (<2,000 IU/ml)
>2,000 IU/ml (HBeAg− form)
Usual ALT level Normal Elevated Normal
Liver biopsy findings Normal or minimal Inflammation, often with fibrosis Minimal inflammation, resolving fibrosis
inflammation/fibrosis
HBsAg Present throughout Present throughout Present but may resolve
Duration Often many years Variable Variable
Risk of cirrhosis/HCC Very low Increases with duration Low but increases with reversion
while in phase to immune-active phase
Adapted from [2, 41]

incidence of spontaneous clearance of HBeAg compared to explored. Equally important is further study of the approxi-
genotype C patients [49, 106]. Infection with genotype C in mately 60 % of chronically infected individuals who never
Taiwan is also associated with higher risk of HCC in develop serious liver disease [32, 39, 41]. Protective factors
HBeAg− individuals [102]. are not well understood.
Studies in Alaska have been particularly revealing because
several HBV genotypes are represented in Native Alaskan
populations, facilitating within-population comparisons. In a 1.8 Control and Prevention
large longitudinal study with a median follow-up time of 20
years, Livingston et al. identified subjects with genotypes A, 1.8.1 Immunization
B, C, D, and F at baseline, and age-adjusted HBeAg+ status The development of the first HBV vaccine was undertaken
was associated more strongly with genotypes C, D, and F by Blumberg and Millman in 1969, based upon the observa-
than with A and B. For those HBeAg+ at baseline, genotype tion that some infected persons appeared to develop protec-
C subjects were much less likely than those with other geno- tive antibodies upon recovery and that antibodies to HBsAg
types to have seroconverted to HBeAg− by the end of fol- were usually absent in those with apparent chronic infections
low-up. Genotype C patients also had a significantly older [111]. Empty HBsAg particles, i.e., particles that do not con-
age at HBeAg− seroconversion than all other genotypes tain viral DNA, are abundant in the circulation of chronically
(47.8 years vs. 16.1–19.4 years) [107]. infected individuals. The first vaccine was produced by the
Within genotype A, there are three defined subgeno- separation and purification of these particles from donor
types, A1–A3. Of these, A1 is the most common genotype sera. The purified particles were demonstrated to produce a
in sub-Saharan Africa. In a case–control study in South protective immune response in HBV-naïve individuals [13,
Africa, Kew et al. [108] found that genotype A was asso- 111]. A vaccine was formulated and in randomized, placebo-
ciated with a 4.5-fold risk of HCC compared to all other controlled trials was shown to be highly effective in prevent-
genotypes. Moreover, all of the genotype A cases who were ing clinical hepatitis or asymptomatic antigenemia [4, 112].
subgenotyped were subgenotype A1. Genotype A cases also Safe and effective vaccines against HBV infection have
occurred in significantly younger individuals than the cases been available since 1981. Their use as a routine vaccination
of other genotypes; this observation is consistent with the has been shown to reduce incidence of acute and chronic
known pattern of HCC in this part of Africa, where the risk infection. In Taiwan, the impact of universal infant HBV
in younger, HBeAg− men appears to be higher than in other immunization, begun in 1984, has been reflected in reduc-
parts of the world [105]. tions in HCC incidence in the vaccinated age cohorts [113].
The significance of HBV genetic variation is an evolving Worldwide, however, many populations at risk for HBV do
area of research. Of particular importance are studies that not have good access to routine immunization. In 2011, 180
include both genotyping and viral mutant detection and rep- countries had included HBV vaccine in routine childhood
resent the full range of variability seen in HBV worldwide. immunization programs. The average three dose series com-
The functional significance of many genotype and subgeno- pletion rate in these countries is 75% [114]. Using estimates
type differences has yet to be characterized. There are indi- of prevalence of HBsAg and HBeAg among women of
cations in some clinical studies that viral genetic variants childbearing age, Goldstein et al. [115] modeled the poten-
may play a role in treatment response to antiviral therapies, tial impact of different HBV immunization strategies on
especially interferons [109, 110]. The potential for HBV HBV-related mortality globally and by region in the year
variants to inform future drug discovery remains to be fully 2000 birth cohort. Potential global reduction in HBV-related
756 A.A. Evans et al.

mortality was estimated for 90 % vaccine coverage at pregnant women [122] and doses of both HBV vaccine and
between 68 and 84 %, depending on whether the vaccine HBIG in birthing centers. Routine delivery of a birth dose
was given at birth. of HBV vaccine is recommended in all infants in the United
Early HBV vaccines were derived from the empty HBsAg States, regardless of known maternal HBV infection status, to
particles in the blood of infected donors. Current vaccines provide early protection in case of erroneous or unavailable
are made from yeast-derived recombinant subunits of HBsAg maternal HBsAg status at birth [121]. This is the approach
and contain no viral DNA. The vaccine does not contain thi- recommended by the WHO as well in countries where peri-
merosal. It may be delivered as a single-antigen vaccine or in natal HBV infection risk is high [53]. In regions where many
combination with other antigens. It is given intramuscularly, births occur outside of medical facilities, HBV vaccine doses
and three doses are usually the recommendation though can be made available to community health workers and
these may vary with different vaccine manufacturers and birth attendants. Vaccine administration as soon as possible
age/risk groups. after birth is recommended for maximum protection. In these
When given as directed in healthy persons, the vaccine settings it is usually not possible to provide HBIG as well,
is both highly immunogenic (stimulates the production of due to cost of purchase and maintaining appropriate storage
antibodies) and has a high protective efficacy (reduces infec- conditions [53].
tion rates in vaccinees). In infants, 80–95 % develop pro-
tective antibody titers after two doses, and 98–100 % after 1.8.3 Treatment
three doses. Protective antibody titers are seen in up to 95 % There are seven widely approved antiviral therapies for
healthy vaccinated children after three doses. In adults, chronic HBV infection, two interferons and five nucleos(t)
immunogenicity of standard vaccine regimens decline with ide analogs, designed to inhibit activity of the viral DNA
age, so that by age 60 only 75 % of those receiving three polymerase [123]. These treatments are effective in reduc-
doses have protective titers. For this reason, older adults tion of viral load and/or HBeAg seroconversion in some
and individuals of any age with chronic conditions causing patients, and expert groups worldwide have produced
immunosuppression (e.g., hemodialysis) may require higher detailed guidelines for the selection of appropriate patients
and/or increased numbers of doses [31]. In those who do pro- for their use [124–131]. The relatively short-term endpoints
duce protective antibody, the vaccine is 80–100 % effective achievable with antiviral therapies, however, do not neces-
in preventing HBV infection or clinical hepatitis. Antibody sarily translate into long-term reductions of morbidity and
titers decline with age, but in most cases an anamnestic mortality from HCC and liver cirrhosis [2]. Antiviral treat-
response is elicited when individuals are exposed to HBsAg, ment may reduce but does not eliminate the risk of HCC
demonstrating that immune memory remains intact [116, among chronically infected individuals who achieve long-
117]. Booster doses are not recommended for most popula- term control of viral replication and improvement of liver
tions [61, 118]. histology [132, 133, 134]. Not all treated patients reach these
endpoints, however, and those who do often require costly
1.8.2 Prevention of Perinatal Transmission long-term therapy to achieve it, bringing with it risk of anti-
Transmission of HBV infection from mother to child during viral resistance mutations and medication side effects [133].
the perinatal period is a major cause of chronic HBV infec- Many chronically infected individuals who might benefit
tions worldwide, particularly in populations with high serop- from antiviral treatment lack access to it because they are
revalence of HBsAg with HBeAg and/or high viral loads. unaware of their infection status, are not receiving appropri-
Several approaches to prevention of perinatal transmission ate medical monitoring, or cannot afford the medications [3,
have been proposed in different settings, their use being 135]. Moreover, the majority of chronically infected indi-
dependent on a population’s resources and rate of perinatal viduals do not fall within the current antiviral treatment
vs. other forms of HBV transmission [53]. When the infec- guidelines since available medications have not been shown
tion status of the mother is known, the most effective inter- to be effective in some classes of patients, notably those in
vention to prevent perinatal infections is the administration the immune-tolerant or immune-active phases [136].
of the first dose of hepatitis B vaccine (“birth dose”) and one
dose of hepatitis B immune globulin (HBIG) within 24 h of
birth (preferably within 12 h), followed by completion of the 1.9 Unresolved Problems
standard 3-dose regimen for Hep B vaccine. This strategy
has been shown to be 85–95 % effective in preventing peri- Since the discovery of HBV, great strides have been made
natal HBV transmission in infants of infected HBeAg- in reducing its impact on human health. Nevertheless,
positive mothers [119–121]. hundreds of millions of people worldwide are chronically
Implementation of this strategy, however, depends on infected with HBV. Millions more will become infected
the availability of accurate prenatal HBsAg screening for every year. In addition to immunization, public health mea-
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 757

sures for prevention of perinatal HBV transmission and pre- 2.2 Methodology Involved
vention of exposure to blood-borne agents have the potential in Epidemiologic Analysis
to reduce or eliminate new HBV infections if implemented
appropriately and consistently. For those already chronically 2.2.1 Morbidity and Mortality Data
infected, current antiviral treatments have the potential to It is estimated that about 15 million people worldwide are
substantially reduce the burden of disease and mortality, but infected with HDV, mostly in areas of high HBV prevalence
they are not widely available in endemic populations due to or in immigrants from those areas [149]. Population-level
cost and medical care access limitations [137]. Even in the data on HDV epidemiology is not widely available since it is
developed world, many chronically infected individuals do not usually part of reportable disease surveillance systems.
not know their infection status or do not receive appropriate Exceptions to this are found in areas of higher prevalence of
medical management for it [3]. Cost reduction for antiviral HDV infection, including Italy, Greece, and Australia [150–
agents and better education of medical professionals on their 153]. Even in areas where the disease is reportable, HDV
appropriate use will require new resources and strategies prevalence and disease burden may be underestimated since
within the already burdened health-care systems. screening and diagnosis are undertaken only in those known
Continued basic research in HBV virology, immunology, to be infected with HBV. In the United States, some states
and therapeutics are needed. Despite intensive research, include HDV in reportable disease statistics but consider
key elements in the natural history of infection such as how these to be underestimates of prevalence or incidence [154].
the virus gains entry into hepatocytes and how infections In the United Kingdom, Cross et al. [155] noted that many
are cleared are still unknown [29, 90, 138]. Currently avail- laboratories do not routinely test HBV-infected patients for
able antivirals are not effective in many groups of chroni- HDV markers. This is likely the case in many populations,
cally infected individuals. Novel approaches and new and it would lead to underestimation of disease burden [156].
targets for therapeutics are needed [136, 138, 139]. The The impact of HDV is also hard to ascertain from vital statis-
staging of liver disease, assessment of treatment response, tics mortality data since liver disease and HCC deaths are
and early detection of cirrhosis and HCC in the clinical set- often not attributed to specific viral etiologies in death cer-
ting would be more accessible and safer for patients with tificates. Furthermore, in vital statistics data, HBV-related
the use of noninvasive methods in the place of liver biopsy, deaths may be undercounted because HCC and LC deaths
and further work in this area is needed to identify better are often not attributed to specific viral etiologies on death
approaches [139, 140]. certificates [19].
Worldwide eradication or elimination of HBV may be an
achievable goal [141]. To achieve it, however, will require 2.2.2 Surveys
not only basic science and clinical advances but public health Because of the unique relationship with HBV infections,
innovation and actions to bring the means of HBV control to HDV prevalence surveys worldwide have focused in popula-
all populations. tions at risk for HBV infection. Geographic areas of HDV
endemicity have been identified in the Mediterranean,
Middle East, and parts of Asia and Africa [156–158]. Both
2 Hepatitis Delta Virus (HDV) within and outside of those geographic areas, high preva-
lence of HDV infection has been reported among injection
2.1 Historical Background drug users, people with many sex partners, and other groups
at high risk for blood-borne infections in some populations
First reported by Rizzetto et al. [142] in 1977, the hepatitis [159–162]. Prevalence of HDV infection worldwide had
delta virus (HDV) was initially identified as a novel been declining in some areas where HBV immunization cov-
antibody-antigen system found in the blood and liver of erage is good, e.g., many parts of Southern and Western
persons infected with HBV and associated with more Europe [159]. However, the decline of prevalence in Europe
severe liver damage. The delta antigen was detected in the observed through the 1990s appears to have halted more
nuclei of hepatocytes. Transmission studies in chimpan- recently [156], possibly due to immigration from areas of
zees demonstrated that the so-called “delta agent” was dis- higher prevalence [158, 163]. Prevalence may be increasing
tinct from HBV but dependent on the presence of HBV in some parts of the world not originally considered endemic
infection [143–145]. Further studies characterized the areas for HDV because of immigration from known areas of
agent as a single-stranded circular RNA virus, defective in endemicity [159]. There is some evidence that more effective
that it cannot establish or maintain infection without the treatment for HIV infection has indirectly led to either stable
copresence of HBV. HDV does not share sequence homol- or increasing prevalence of HDV infection among these
ogy with HBV but is similar to some known plant viroids high-risk groups in Europe because of increased survival
[146–148]. [160].
758 A.A. Evans et al.

2.2.3 Laboratory Diagnosis HDV cannot survive or replicate without the presence of
Laboratory criteria for diagnosis of HDV infection require HBV. With a genome of 1.7 kb, HDV is the smallest known
the detection of antibody to HDV (anti-HDV), HDV RNA, animal virus and the only known animal virus to have a
and/or HDV antigen (HDAg) in blood or liver tissue. circular RNA genome. Because of these unique qualities,
Commercial availability of these tests is variable. For epide- HDV is classified as its own separate genus, Deltavirus, of
miologic studies of the prevalence of HDV exposure, total which it is the only known member [165]. Initially, HDV
anti-HDV is the best single marker; however, this approach has classified into three major genotypes worldwide, each
may underestimate prevalence of recent infections (<30 with both geographic and demographic differences in prev-
days) [164] and past infections [157]. Anti-HDV is not an alence [169]. Recent work has further broken down the
indicator of protective immunity against HDV infection. genotypes into on into eight clades: HDV-1 is found world-
Clinically, the distinction between HDV/HBV coinfection wide; HDV-2 (HDV-IIa) is localized to Japan, Taiwan, and
(i.e., infections occurring simultaneously) and superinfection Yakutia, Russia; HDV-3 to the Amazon basin; and HDV-4
(HDV infection occurring in established chronic HBV infec- (HDV-IIb) also to Taiwan and Japan; and HDV-5, HDV-6,
tion) is considered important. Upon initial infection in either HDV-7, and HDV-8 are found in Africa [170]. Coinfections
of these, HDV RNA and HDAg are at least transiently present with more than one HDV genotype have been reported
in the liver and blood. Detection of HDAg in blood is not [171]. The clinical significance of HDV genotypes and
always feasible, however, because of the presence of neutral- genetic variants is not fully understood. Humans are the
izing antibodies [165–167]. Unless there is documentation of only known natural host for HDV infection though experi-
existing HBV infection, it can be difficult to distinguish co- mental infections of chimpanzees and woodchucks infected
and superinfections at presentation. Coinfections usually with HBV or WHV (woodchuck hepatitis virus) are possi-
have a longer incubation period – 3–7 weeks from exposure ble [67, 171].
to ALT elevation and onset of symptoms – depending on the
size of the inoculum. Superinfection incubation periods are
shorter – as little as one week [159, 168]. 2.4 Descriptive Epidemiology
Coinfection: When HBV/HDV infections occur simultane-
ously, HBV markers (HBsAg, HBeAg, HBV DNA) are detect- 2.4.1 Prevalence and Incidence
able in the blood before any HDV markers appear. HDV RNA It is estimated that 5 % of those infected with HBV are
and HDAg appear transiently, and HDAg is seen in blood only also infected with HDV, leading to a worldwide preva-
in about 25 % of patients [165]. A more useful marker in coin- lence of approximately 15 million individuals. Good epi-
fection is IgM anti-HDV (IgM class antibody to HDV), which demiologic data on HDV prevalence are not available for
appears during the acute phase in >90 % of coinfections [167]. much of the world, and highly affected subpopulations
Resolution of acute coinfection is accompanied by a rise in may not be reflected in national-level data. In 2001, the
IgG anti-HDV (IgG class antibody to HDV). Persistence of WHO designated the areas of highest prevalence as the
IgM anti-HDV beyond the acute phase indicates the develop- Mediterranean Basin, Middle East, Central Asia, West
ment of chronic infection. In resolved coinfection, all HDV Africa, Amazon Basin, and specific islands of the South
markers including IgG anti-HDV may eventually clear. As a Pacific. In contrast, most areas of East Asia have lower
result, there is no consistent marker of past infection [157]. prevalences. In North America, Australia, and Western
Superinfection: When an individual already infected with Europe outside of the Mediterranean Basin, prevalence is
HBV is superinfected with HDV, HDV RNA, and/or HDAg, very low [168]. Even in areas of the world where it is sus-
detection in serum coincides with a decline in HBsAg, some- pected that HDV is endemic, such as sub-Saharan Africa,
times to undetectable level [166]. This may lead to difficul- few or no data on its prevalence or geographic distributions
ties in diagnosis in the early phase of disease since HDV are available [172].
testing is often not considered unless the presence of HBV The global picture of HDV endemicity does not necessar-
infection is evident. Appearance of HDV DNA is followed ily coincide with that of HBV infection [149]. This may be
by detection of anti-HDV. The persistence of IgM anti-HDV due to assortative contact patterns within populations of
is more frequently found in superinfection rather than in HBV-infected persons. For example, HDV is rare among
coinfection and is an indicator of chronicity. HBV-infected chronic liver disease patients in Hong Kong
except in those who are injection drug users, where HDV
prevalences of >90 % have been reported [173]. Among
2.3 Biological Characteristics HBV-infected persons across the Asia Pacific region, the
highest HDV infection rates have been reported among those
HDV is a defective single-stranded RNA virus that requires with behavioral risk factors (e.g., sexual activity, injection
HBV to establish infection. Specifically, HDV uses the pro- drug use) and in some medical settings (e.g., hemodialysis)
tein coat of HBV (HBsAg) to encapsulate its RNA genome. [174]. Since the discovery of HDV in the late 1970s, some
32 Hepatitis Viruses: Hepatitis B and Hepatitis D 759

populations (e.g., in the Amazon basin and Southern Italy) Nevertheless, some infected individuals are asymptomatic
have seen a clear reduction in HDV prevalence because of and have little to no histologic evidence of liver disease [95,
HBV immunization and measures to prevent transmission of 149, 157, 181]. The pathogenesis of HDV infection is not
other blood-borne diseases [156]. In other populations where well understood. Damage to infected hepatocytes is increased
HDV had previously been rare, there have been reports of with the level of HDV replication, but whether this is a direct
increasing prevalence and HDV-associated acute hepatitis cytopathic or indirect immune-mediated effect is not clear
outbreaks due to migration or increases in injection drug use [169]. HDV replication requires the presence of HDAg, but
[149, 161, 175, 176]. HDAg appears not to be cytopathic [182, 183]. As with HBV
infection, adaptive immune response in HDV infection is
2.4.2 Specific Risk Groups thought to play a role in pathogenesis, but this is not fully
Individuals infected with hepatitis B are at risk for acquiring understood [159].
an HDV superinfection. Those who are susceptible for HBV
infection, especially those who are in high-risk groups for
hepatitis B infection, are at risk for developing HBV/HDV 2.7 Patterns of Host Response
coinfection.
Initially, HDV infection presents with signs and symptoms
Ethnicity/Race of acute hepatitis, whether in co- or superinfection with
Those individuals born in parts of the world where HDV is HBV, with variable severity but generally more severe than
prevalent are at higher risk for HDV infection. There is no what is seen in HBV monoinfection [159]. This initial phase
evidence for a biological difference in risk due to race or may be more severe than in other viral hepatitides, with
ethnicity. 2–20 % leading to fulminant hepatitis [169]. In most (80–
95 %) of HBV/HDV coinfections, the course is self-limited
Behavioral and results in clearance of both viruses [159, 169]. Only
Behavioral risk factors for HDV infection are similar to 2–5 % become chronic. In HBV/HDV superinfections, as
those for other blood-borne infections, i.e., injection drug many as 20 % may resolve spontaneously [169], but 70–80 %
use, high-risk sexual activities, close contact with persons at become chronic infections with more rapid progression of
high risk, and unsafe tattooing and piercing [161, 173, 175, liver disease than is seen in HBV monoinfection [149, 159].
177, 178]. Chronic HDV infection leads to cirrhosis in up to 80 % of
cases, and it is associated with greater risk of developing
Other chronic liver disease and dying from liver disease than HBV
HBV-infected individuals who receive unscreened blood or infection alone [168]. The association of HDV infection with
blood products are at increased risk of HDV infection, par- the development of HCC is not clear. A review by the
ticularly in higher prevalence areas. Other exposures in med- International Agency for Research on Cancer (IARC) in
ical settings may carry risk [178]. HBV-infected hemodialysis 1994 found the evidence for HDV’s carcinogenicity inade-
patients in developing countries, for example, may be at high quate [184]. Nevertheless, several cohort studies since that
risk for superinfection [179]. time have shown a risk of HCC in HBV/HDV infections of
2–6-fold over HBV infection alone [185, 186]. Whether this
is a direct carcinogenic effect of HDV or an effect mediated
2.5 Mechanisms and Routes through the higher risk of cirrhosis in HDV infection remains
of Transmission to be seen.

HDV is transmitted similarly to HBV, through direct contact

with blood and infected body fluids. There is a risk of HDV 2.8 Control and Prevention
transmission through sexual contact, though it is lower than
HBV [159, 171]. HDV is rarely transmitted vertically from There are no specific measures for prevention of HDV. HBV
mother to infant [168]. Horizontal intrafamilial transmission immunization and general measures to prevent blood-borne
may occur through inapparent percutaneous or permucosal infections are the most commonly used and effective means
means but not through casual contact [180]. of prevention. Individuals who are already chronically
infected with HBV may reduce the risk of HDV superinfec-
tion by undergoing antiviral treatment for HBV as well as
2.6 Pathogenesis and Immunity avoiding behaviors that increase the risk of parenteral infec-
tions. In those where HDV/HBV infection is already estab-
In the clinical setting, HDV infection is associated with more lished, treatment with interferon-alpha has shown some
severe liver disease compared to infection with HBV alone. benefit [149].
760 A.A. Evans et al.

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 Hepatitis Viruses: Hepatitis C
33
Ponni V. Perumalswami and Robert S. Klein

1 Introduction hepatitis [8, 11–14]. Leading up to the discovery of HCV, both

cohort and case-control studies of persons with NANB hepati-
Hepatitis C virus (HCV) is a bloodborne human pathogen tis demonstrated specific risk factors associated with acquiring
with a current global prevalence rate of approximately the disease, including transfusion of blood and blood products,
2–3 %, representing approximately 130–180 million infected occupational exposure to blood, sex with known infected part-
individuals [1–5]. Infection with HCV may cause acute hep- ners, injection drug use, and sex with multiple partners [15–21].
atitis, but the majority of persons with acute infection are Together, these studies led to the conclusions that the primary
asymptomatic. Persons chronically infected with HCV are at etiological agent of NANB was predominantly both transfu-
risk of chronic hepatitis and of developing serious hepatic sion related and community acquired.
complications such as cirrhosis, hepatocellular carcinoma
(HCC), or liver failure. In the developed world, HCV is the
leading cause of cirrhosis and primary liver cancers [6, 7] 3 Methods for Epidemiologic Analysis
and the major cause of end-stage liver disease leading to liver
transplantation [8, 9]. A recent report commissioned by the 3.1 Sources of Morbidity and Mortality
Institute of Medicine (IOM) of the National Academies esti- Data
mates that up to 75 % of HCV-infected persons have not
been diagnosed [10]. Morbidity and mortality data on HCV infection are acquired
from a variety of sources including death certificates, hospi-
tal admissions, surveys, research studies, and required
2 Historical Background reporting of cases. In the USA, HCV is a separate reportable
disease, and health-care providers, hospitals, and laborato-
The recognition of what was eventually identified as HCV ries are required to send reports of cases of HCV infection to
began in the 1970s with the first descriptions of posttransfu- state and local health departments that include them within
sion hepatitis not attributable to hepatitis A virus (HAV) or their jurisdiction.
hepatitis B virus (HBV). This disease entity was termed at that Among HCV-infected persons, there are a rapidly increas-
time “non-A, non-B” (NANB) hepatitis [8, 11, 12]. Hepatitis ing number of deaths, which in the USA now surpass deaths
C virus was discovered in 1988 and was shown to be the among HIV-infected persons. US multiple-cause mortality
primary etiologic agent of parenterally transmitted NANB data from 1999 to 2007 of approximately 21.8 million death
certificates demonstrated an increase in the mortality rate
from HCV infection over that time period [22]. A statisti-
cally significant average annual age-adjusted mortality rate
P.V. Perumalswami, MD, MCR (*)
Division of Liver Diseases, Mount Sinai Medical Center, increase of 0.18 death per 100,000 persons per year related
1468 Madison Avenue, Annenberg Bldg, Rm 21-42, to HCV was observed (P = 0.002). In addition, the relatively
1123 New York, NY 10029, USA young ages at death (45–64 years) of most HCV-infected
e-mail: [email protected]
persons portend a large and ever-increasing health-care bur-
R.S. Klein, MD den in the years to come. In the USA alone, estimates fore-
Division of Infectious Diseases, Department of Medicine,
cast a peak in the number of incident cases of end-stage liver
St. Luke’s and Roosevelt Hospitals,
425 West 59th Street, Suite 8B, New York, NY, USA disease due to HCV of approximately 38,600 in the year
e-mail: [email protected] 2030, 3.9 times the predicted annual incidence in 2010 [23].

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 765

DOI 10.1007/978-1-4899-7448-8_33, © Springer Science+Business Media New York 2014
766 P.V. Perumalswami and R.S. Klein

The annual number of liver transplants and deaths are fore- made: Central and East Asia and North Africa/Middle East
cast to peak about 2–3 years later at approximately 3,200 are estimated to have a high prevalence of HCV infec-
transplants and 36,100 deaths. Of the approximately 2.9 mil- tion(>3.5 %), and South and Southeast Asia; sub-Saharan
lion pre-cirrhotic patients infected with chronic hepatitis C Africa; Andean, Central, and Southern Latin America;
in 2005, it is estimated that without treatment, 24,900 or Caribbean; Oceania; Australasia; and Central, Eastern, and
0.9 % will have died from hepatitis C by 2010; 379,600 or Western Europe have moderate prevalence (1.5–3.5 %),
13.1 % by 2030; and 1,071,229 or 36.8 % by 2060. Globally, whereas Asia Pacific, Tropical Latin America, and North
estimates indicate that up to four million persons are newly America have low prevalence (<1.5 %).
infected each year, 170 million people are chronically
infected and at risk of developing cirrhosis and liver cancer,
and 350,000 deaths occur annually due to all HCV-related 3.3 Laboratory Diagnostics
causes [24, 25].
There are two different types of assays that are used in the
diagnosis and evaluation of HCV infection— serologic and
3.2 Surveys molecular assays. Serologic assays detect antibody to HCV
(anti-HCV) and can be used to screen for and diagnose prior
The National Health and Nutrition Examination Survey or current HCV infection. Serological assays include widely
(NHANES) refers to a series of assessments that have peri- used enzyme immunoassays (EIAs) with specificities greater
odically collected data on the health and nutritional status of than 99 % and therefore remain the best screening tests for
a large sample of adults and children in the USA. The infor- the diagnosis of hepatitis C. False-negative anti-HCV testing
mation includes HCV prevalence, based on interview, physi- may occur, albeit rarely, in the setting of severe immunosup-
cal examination, and laboratory testing, allowing clinicians pression such as HIV infection, solid organ transplantation,
to target at-risk groups with educational services and thera- agammaglobulinemia, or in patients on hemodialysis [29–32].
peutic options. The latest NHANES survey from 1999 to False-positive anti-HCV with EIA tests may also occur, par-
2002 included 15,079 total participants and is the largest epi- ticularly in populations with a low prevalence of HCV infec-
demiological study ever conducted on HCV prevalence in tion. Signal to cut-off ratios (>4.0 associated with true HCV
the USA. According to NHANES, 1.6 % or 4.1 million per- exposure or infection) with EIAs can be used to help guide
sons in the USA, most of whom were born between 1945 and whether further confirmatory testing with recombinant immu-
1964 were anti-HCV seropositive [3]. The survey, however, noblot assay (RIBA) can be helpful. The RIBA test assesses
sampled from a noninstitutionalized civilian population and the serological reaction of a patient to multiple HCV antigens
did not include certain high-risk persons, namely, the incar- and is a confirmatory test to the anti-HCV EIA test. A posi-
cerated, homeless, nursing home residents, hospitalized tive RIBA in conjunction with a positive anti-HCV antibody
patients, those in active military service, and immigrants. test indicates a true past or present infection with HCV.
The survey also missed some groups with an expected high A negative RIBA in conjunction with a positive anti-HCV
prevalence of HCV infection (e.g., health-care workers and antibody test indicates a false-positive anti-HCV antibody
persons on long-term hemodialysis) because of their under- test, and the patient can be reassured without further testing
representation in the sample studied. As a result, data from [33]. In persons with positive HCV screening tests, molecu-
NHANES may have underestimated the true prevalence of lar assays to detect viral nucleic acid, and therefore active
HCV in the USA by at least one million infected persons viral infection, are recommended and can be either qualita-
[26, 27]. tive or quantitative. Real-time polymerase chain reaction
Globally, the prevalence of HCV varies geographically. (PCR)-based assays and transcription-mediated amplification
Recent developments in modeling allow the seroprevalence (TMA) assays have largely replaced qualitative molecular
of anti-HCV to be used to estimate the global burden of dis- assays and are widely used for monitoring during response-
ease for HCV infections. Specifically, an international col- guided therapy. These assays have sensitivities of 10–50 IU/
laborative, The Global Burden of Diseases, Injuries, and mL and have excellent specificity (98–99 %). Serologic and
Risk Factors 2010 (GBD2010) Study, is an effort to estimate molecular assays do not assess disease severity or progno-
the global burden of HCV infection. The GBD Study defined sis. Additionally, the most current consensus proposal distin-
21 regions such that detailed data in one country can plausi- guishes six genotypes based on phylogenetic cluster analysis
bly be extrapolated to other countries in the region in order to of complete genomes. Genotype testing is performed in the
create burden estimates [25, 28]. Based on a systematic evaluation of HCV infection in order to maximize the chance
review and meta-analysis of primary national data sources of successful treatment outcome for each individual patient
and articles published for peer review between 1980 and as treatment type, duration, dose, and effectiveness are
2007, the following global estimates of HCV have been influenced by genotype. While phylogenetic analysis with
33 Hepatitis Viruses: Hepatitis C 767

direct sequencing of an HCV genomic region is considered USA, genotype 1 is the most common (75 %). In Europe, geno-
the gold standard for identifying different HCV genotypes, types 1 and 3 are most common, while in Egypt genotype 4
this method is expensive and time consuming. For this rea- predominates. Additional variants, known as quasispecies, are
son, commercial genotyping kits were developed for routine present in infected individuals and are a result of the high error
determination of HCV genotypes. rate of the viral RNA polymerase during replication.

4 Biological Characteristics 5 Descriptive Epidemiology

A member of the family Flaviviridae, HCV is classified within 5.1 Morbidity and Mortality
a separate genus, Hepacivirus, due to differences from other
members of the family in the organization of the structural Global morbidity and mortality from HCV infection are a tre-
proteins that make up the amino terminal third of its polyprot- mendous public health burden. Available estimates indicate
ein. The virus has an enveloped, positive-sense, single-strand that worldwide, there were 54,000 deaths and 955,000 dis-
RNA genome of approximately 9.6 kb in length with extraor- ability adjusted life years associated with HCV infection [25].
dinary genetic diversity [34]. The RNA contains a single large The major burden from HCV infection comes from sequelae
open reading frame that encodes for a 327 kD polyprotein of from chronic infection. Estimates indicate that three to four
approximately 3,000 amino acids that is flanked by nontrans- million persons are newly infected each year, 170 million
lated segments [35]. The polyprotein is processed by host cell people are chronically infected and at risk of developing liver
peptidases and viral proteases that cleave it into structural and disease including cirrhosis and liver cancer, and 350,000
nonstructural proteins needed for viral replication [36]. The deaths occur each year due to all HCV-related causes [24, 25].
5′-end includes structural proteins (core, E1, and E2) that
encode nucleocapsid and envelope proteins, and the 3′-end
includes nonstructural proteins (NS2, NS3, NS4A, NS4B, 5.2 Prevalence and Incidence
NS5A, and NS5B) required for replication (see Fig. 33.1).
The nonstructural proteins have distinct enzymatic functions The available data suggest that the global prevalence of HCV
including production of proteases (NS3, NS4A), helicases infection has increased from 2.3 % (95 % uncertainty inter-
(NS3), and polymerases (NS5B). The HCV genome contains val [UI]: 2.1–2.5 %) to 2.8 % (95 % UI: 2.6–3.1 %) and >122
both highly conserved and highly variable regions. Six major million to >185 million between 1990 and 2005 [4, 24].
genotypes of HCV have been described, and there are subtypes These estimates are based on systematic reviews of pub-
within each genotype. The genotypes are differentiated by lished prevalence data, including volunteer blood donor
sequences in the relatively conserved core, E1 and NS5B studies, and therefore they may underestimate the true popu-
regions [36]. Pairwise differences in the nucleotide sequences of lation prevalence [37]. Although HCV infection is endemic
the six HCV genotypes are on the order of 31–33 % and the worldwide, there is a large degree of geographic variability
geographic distribution of specific genotypes varies [34]. In the in its distribution (see Fig. 33.2).

HCV RNA

positive-strand RNA virus

9600 nucleotides - 3011 amino acids

structural non-structural

C E1 E2 NS2 NS3 4a NS4b NS5a NS5b

5’UTR p7 3’UTR
IRES
core envelope protease serine membranous ? RNA-dependent
proteins protease, web RNA polymerase
helicase
cofactor for NS3

Fig. 33.1 HCV RNA (Data from Heim [262])

768 P.V. Perumalswami and R.S. Klein

Prevalence of
Hepatitis C
Virus infection
> 2.9%
> 2%–2.9%
1.0%–1.9%
< 1.0%
No Data

Fig. 33.2 Geographic prevalence of hepatitis C virus infection (Data from the CDC accessed online September 19, 2012 from http://www.cdc.
gov/immigrantrefugeehealth/guidelines/domestic/viral-hepatitis-figure5.html)

The prevalence of anti-HCV antibody is quite variable reported in the setting of hemodialysis and blood or plasma
throughout the general population, with the highest rates donation [48–52]. However, many outbreaks have occurred
among persons with repeated percutaneous exposures through in other health-care settings as a result of poor infection con-
injection drug use. Additional risk groups include hemophili- trol practices; failure to use aseptic technique during prepa-
acs, patients on hemodialysis, patients transfused with ration or delivery of therapeutic injections has commonly led
unscreened blood and blood products, inmates of long-term to cross-contamination from reused needles and syringes,
correctional facilities, and persons with occupational exposure multidose saline vials, infusion bags, heparin solutions, and
[38–45]. Data regarding the incidence of HCV infection are pain treatments [53–64].
difficult to obtain, since most acute infections (60–70 %) are Additionally, outbreaks of acute HCV infection have been
asymptomatic and there is no widely available test to distin- reported in IDUs and HIV-infected MSM [65, 66].
guish acute from chronic infection [46, 47]. Surveillance data
from the CDC demonstrate a decrease in the annual number of
incident cases (see Fig. 33.3). These data are derived by adjust- 5.4 Geographic Distribution
ing rates from the Sentinel Counties Study of Viral Hepatitis
(1982–2006) and the Emerging Infection Program (2007) for Data from the WHO and CDC concur in their indication that
underreporting and asymptomatic infection. the prevalence of HCV infection varies in different geo-
graphic regions [67]. Data from GBD2010 estimates higher
prevalences of HCV in East and Central Asia (3.7–3.8 %),
5.3 Epidemic Behavior Eastern Europe (2.9 %), North African (3.6 %), and central
and west sub-Saharan Africa (2.3–2.8 %), while prevalence
Outbreaks of hepatitis C are limited in scope because is lower in North America (1.3 %) and Latin America (1.2–2.0 %)
of the predominantly bloodborne route of transmission. [5, 68]. Additionally, there are variances with respect to age
Historically, most early outbreaks of HCV infection were and peak prevalence [24]. The highest prevalence of HCV is
33 Hepatitis Viruses: Hepatitis C 769

Fig. 33.3 Decrease in the number 60,000 Incidence of acute Hepatitis C, by year
of acute HCV cases, by year in the United States, 1982–2010
USA (Data from the CDC.
Accessed online September 19, 50,000
2012 from http://www.cdc.gov/

Estimated number of cases

hepatitis/Statistics/index.htm)
40,000

30,000

20,000

10,000

0
19 19 19 19 19 19 19 19 19 20 20 20 20 20 20
82 84 86 88 90 92 94 96 98 00 02 04 06 08 10

Year

15 % in Egypt, while the lowest reported prevalences are HCV-positive cases in this survey were born between
<1.0 % in the UK and Scandinavia [24, 67]. 1945 and 1964. Among young injection drug users, the
annual incidence of HCV infection ranges from 10 to 36 %
[77–81]. The overall age distribution of disease is likely
5.5 Temporal related to patterns of exposure (injection drug use in young
and middle-aged adults; transfusion in older adults) and
Countries with similar overall prevalence of HCV infection possibly to age-specific variation in clinical expression of
have different age-specific prevalence patterns; three main disease.
patterns have been described [67]. The first pattern, found in This is in contrast to the age-specific prevalences of HCV
the USA and Australia, is characterized by a low age-specific infection which increase steadily with age in many countries
prevalence among persons less than 20 years and greater than as outlined above [72–74, 82–84]. This suggests a cohort
50 years with the majority of HCV infection occurring in per- effect in which the risk for HCV infection was higher in the
sons 30–49 years old. This pattern suggests that most HCV past. Where the emergence of HCV infection occurred in the
transmission occurred in the last 20–40 years and primarily in distant past, the burden of HCV-related chronic disease
young adults [3, 69, 70]. The second pattern, found in a num- might already have reached its highest magnitude. However,
ber of countries including Turkey, Spain, Italy, Japan, and changes in disease transmission patterns that result in
China, is characterized by low age-specific prevalence in chil- younger persons acquiring infection could lead to future
dren and young adults with the majority of infections occur- increases in chronic disease as this cohort ages. In Egypt,
ring in persons over 50 years old [71–74]. This pattern where there has been an ongoing high risk for decades, the
suggests a cohort effect where most HCV transmission high magnitude of the current burden of HCV-related chronic
occurred 30–50 years ago. The third pattern, found in Egypt, disease is predicted to continue [85].
is characterized by high prevalence of infection among per-
sons in all age groups with HCV infection increasing steadily
with age [75, 76]. This pattern suggests increased risk in the 5.7 Sex
distant past followed by ongoing high risk of transmission.
Data from the USA demonstrates that HCV infection is
more common in men (2.1 %) than women (1.1 %). It is
5.6 Age not well understood why this difference exists, although
differences by gender in risk factors such as injection drug
Acute hepatitis C infection may occur in all age groups but use likely contributes [3]. Males with HCV infection are
appears to occur mostly among young adults. According to more likely (2.5 times) to develop cirrhosis and hepatocel-
US data from NHANES, prevalence of anti-HCV increased lular carcinoma when compared with females, suggesting
with age from 1.0 % in those 20–29 years of age to a peak that estrogen may have protective effects against fibrosis
of 4.3 % in those 40–49 years of age [3]. Two-thirds of progression [86–88].
770 P.V. Perumalswami and R.S. Klein

5.8 Race replicons, each of which contains an adapted HCV genome

encoding a selectable marker [96]. However, replicons do not
Hepatitis C occurs worldwide in all racial/ethnic groups reproduce other aspects of the life cycle such as virion produc-
studied. In the USA, data from the most recent NHANES tion and infection [96, 97]. Two additional advances came in
demonstrated a higher overall prevalence among non- 2003 and 2005 with the development of retroviral pseudopar-
Hispanic black persons (3.0 %) compared with non-Hispanic ticles bearing unmodified HCV glycoproteins (HCV gp) and
white persons (1.5 %) and was almost entirely attributable to the discovery that a genotype 2a isolate (JFH1) from a patient
differences among older participants [3]. According to with acute fulminant hepatitis could undergo the complete
Armstrong and colleagues, this finding suggests that younger virus life cycle in cell culture, respectively [98].
non-Hispanic black persons may not be subject to the dispro- Direct percutaneous exposure to blood is the most efficient
portionately high burden of disease that was seen in the pre- mode of HCV transmission. Higher rates of transmission
vious generation. However, the authors do note that the small occur with large and repeated percutaneous exposures such as
number of younger anti-HCV-positive participants limits injection drug use and with transfusions or transplantation
definitive conclusions [3]. Although sparse data make com- from infectious donors (see Fig. 33.4) [99]. Lower rates of
parisons difficult, observed racial differences in prevalence transmission occur with single, small dose percutaneous expo-
or mortality rates are likely due to the differences in expo- sures such as accidental needlesticks [91, 99]or by mucosal
sures and risk factors in different locations. exposures to blood- or serum-derived fluids (e.g., birth to an
infected mother, sex with an infected partner) [99–101].

5.9 Occupation
6.1 Iatrogenic Exposure
The route of acquisition of HCV is similar to that of hepati-
tis B virus (HBV) in the developed world, with exposure to Transfusion-associated HCV infection was a major worldwide
blood playing the principal but not exclusive role in transmis- risk before HCV testing became available in 1989. Prior to
sion. HCV is not transmitted as efficiently as HBV through screening of the blood supply, transmission of HCV occurred in
occupational exposures to blood and is largely confined 5–18 % of people receiving a transfusion [102–104]. In some
to health-care workers who have sustained contaminated groups who were transfused large amounts of blood or pooled
needlestick injuries. The average incidence of anti-HCV blood-derived products prior to screening, such as hemophili-
seroconversion after accidental percutaneous exposure from acs, prevalence of HCV infection ranged from 59 to 99 % [105,
an HCV-positive source is 1.8 % (range: 0–7 %) [89–91]. 106]. The risk of acquiring HCV via blood transfusion has been
Transmission has been associated with hollow-bore needles, virtually eliminated in countries that have implemented routine
deep injuries, HIV coinfection, and a high titer of HCV in the HCV testing of donors (less than one in a million per unit trans-
source patient’s blood [89–93]. Study of dentists has dem- fused) [107]. However, some countries have not prioritized
onstrated an increased risk of HCV infection compared to blood transfusion safety and/or lack the resources to implement
controls (1.75 % vs. 0.14 % OR 12.9, 95 % CI 1.7–573), donor screening, and in these countries blood transfusion
particularly among oral surgeons, as has also been shown for remains an important source of infection [108].
HBV [94]. Worldwide, there is a substantial preventable burden of
HCV due to iatrogenic transmission. There is a high preva-
lence of transfusions, reuse of needles and syringes, needle-
6 Mechanisms and Routes stick injuries among health-care workers, and unnecessary
of Transmission medication injections [109, 110]. It has been estimated that
contaminated health-care injections cause approximately two
HCV entry into hepatocytes is assumed to be a multistep pro- million of the HCV infections acquired annually and may
cess that requires sequential interactions between cellular fac- account for up to 40 % of all HCV infections worldwide
tors and viral proteins. Replication depends on viral and host [111]. In addition to unsafe injection practices, in some coun-
proteins and occurs in association with intracellular mem- tries, poor or nonexistent infection control in health and den-
branes [95]. Historically, HCV research has been hampered by tal care facilities may be a source of HCV transmission [112].
a lack of adequate in vitro and in vivo model systems. In vivo In Egypt, where the prevalence of HCV is the highest in the
study of HCV infection is restricted to humans and chimpan- world, the reuse of glass syringes during the parenteral ther-
zees due to a lack of small animal models. Another major limi- apy campaigns to control endemic schistosomiasis is a widely
tation in the study of the HCV life cycle is the inability to suspected to be the source of iatrogenic transmission [113].
culture wild-type strains of HCV efficiently in cell culture. However, there may have been considerable other concurrent
Much has been learned regarding RNA replication from iatrogenic exposure at the time [114]. More recent evidence
33 Hepatitis Viruses: Hepatitis C 771

Fig. 33.4 HCV infection by route of HCV Infection By Route of Transmission

transmission (Data from the CDC. Accessed
online September 19, 2012 from: http://
commons.wikimedia.org/wiki/ Unknown
File:Hepatitis_C_infection_by_source_ 10 % Injection drug use
(CDC)_-_en.svg) 60 %
Other (hemodialysis;
health care work;
perinatal)
5%

Transfusion
(before screening)
10 %

Sexual
15 %

in Egypt supports a continuation of iatrogenic exposures 2011). Indirect drug sharing and preparation practices includ-
including unsafe medical and dental practices that is contrib- ing back loading and using cotton (filters), cookers (drug solu-
uting to ongoing HCV transmission [112, 115]. tion containers, such as spoons), and rinse water have all been
Iatrogenic transmission of HCV is not limited to resource associated with HCV transmission [81, 118–120].
poor countries. Outbreaks of HCV transmission have occurred
in resource-rich countries as well [55, 56, 59, 61–63]. Most of
these outbreaks were reported in the setting of hemodialysis 6.3 Sexual Transmission
and poor infection control leading to cross-contamination
from reused needles and syringes, multidose saline vials, Sexual practices and exposures predictive of anti-HCV posi-
infusion bags, heparin solutions, and pain treatments. tivity include the number of recent and lifetime partners,
high-risk sexual practices, other STDs (HSV-2 and
Trichomonas), and HIV infection particularly in MSM [71,
6.2 Injection Drug Use 121–128]. The magnitude of risk for transmission of HCV
infection by sexual activity has been controversial. Sexual
Injection drug users (IDUs) have the highest overall preva- transmission of virus occurs when infected body secretions
lence of HCV infection compared with any other risk group. or infected blood are exchanged across mucosal surfaces
Worldwide, it is estimated that 16 million people injected [99]. The presence of virus in body secretions or blood is
drugs in 2007 [116]. Data from one systematic review sug- necessary but may not be sufficient for transmission to occur
gests that ten million IDUs globally are HCV seropositive [101]. Other factors that may influence transmission include
[117]. Anti-HCV prevalence is noted to be as high as the titer of virus in body fluids, the integrity of the mucosal
60–80 % in IDUs. Injection drug use has been the leading surfaces, and the presence of other genital infections. Studies
mode of transmission during the past four decades in the to detect HCV RNA in semen, vaginal secretions, cervical
USA and Australia and now accounts for most newly smears, and saliva have yielded mixed results [121, 122, 125,
acquired infections in many countries in Europe. Eastern 129, 130]. Studies have demonstrated that when HCV RNA
Europe, East Asia, and Southeast Asia have the largest pop- is detected in these secretions, it is present in lower concen-
ulations of IDUs infected with both HBV and HCV. trations than in blood. Low levels of virus in genital secretions
The WHO recommends that IDUs be a key target group for may be one reason that HCV is sexually transmitted less effi-
prevention and treatment of HCV (WHO Resolution A63/15 ciently than HBV or HIV. Also, the absence of appropriate
772 P.V. Perumalswami and R.S. Klein

target cells in the intact genital tract may require the presence increased risk of vertical transmission of HCV with the mode
of abnormal mucosa for transmission. A review of the litera- of delivery except in women who are also infected with human
ture demonstrated that the risk for sexual acquisition of HCV immunodeficiency virus (HIV). The increased risk of perinatal
appeared to be related in large part to HIV coinfection [131]. HCV transmission in mothers coinfected with HIV may be
There have been at least two cross-sectional studies demon- related to higher HCV viral loads in coinfected persons as a
strating increased risk of acquiring HCV infection among result of HIV-mediated immunosuppression [145]. Other fac-
heterosexual HIV-infected persons [132, 133]. tors may increase perinatal transmission in HIV-coinfected
The clearest and least equivocal sexual risk behavior that mothers. One such factor is facilitation by HIV infection of
leads to HCV transmission is unprotected sex among HIV- HCV entry into blood cells with subsequent replication;
positive men who have sex with men (MSM) [131]. The risk another is concomitant use of injection drugs by coinfected
of HCV transmission by sexual activity differs by the type of mothers [151–153]. The reason for increased risk of vertical
sexual relationship. Persons in long-term monogamous part- transmission in coinfected mothers who are IDUs is not known.
nerships are at lower risk of HCV acquisition (up to 0.6 % It has been postulated that greater maternal peripheral blood
per year) than persons with multiple partners or those at risk mononuclear cell (PBMC) infection confers an increased risk
for sexually transmitted infections (up to 1.8 % per year) for vertical transmission. Internal fetal monitoring and pro-
[101]. Data from the CDC Acute Hepatitis Sentinel County longed rupture of membranes should be avoided if possible in
Surveillance program demonstrated that 18 % of individuals all HCV-infected pregnant women. Patients should be advised
with acute HCV infection reported no other risk factor except that breastfeeding is permissible since there is no evidence that
sexual contact with an anti-HCV-positive person in the pre- breastfeeding increases risk of HCV transmission.
ceding 6-month period or multiple sexual partners.
HIV coinfection appears to increase the rate of HCV
transmission by sexual contact although the precise mecha- 6.5 Hepatitis C Virus Transmission
nism is unknown. Cross-sectional studies from the USA to Health-Care Workers
have demonstrated a two- to fourfold higher risk of hetero-
sexual HCV infection in HIV-infected than in HIV-uninfected HCV is not transmitted efficiently through occupational
subjects [132, 134]. Epidemics of sexually transmitted HCV exposures to blood. Nosocomial transmission is largely con-
infection among HIV-infected MSM have emerged in fined to health-care workers who have sustained contami-
Northern Europe, the USA, and Australia in the last decade nated needlestick injuries. The average incidence of anti-HCV
[65, 128, 135–138]. Risk factors for transmission in HIV- seroconversion after accidental percutaneous exposure from
infected MSM include multiple partners, fisting, use of sex an HCV-positive source is 1.8 % (range: 0–7 %) [89–91].
toys, and presence of genital ulcerative disease [139]. This risk is intermediate between that resulting from similar
exposures to HIV (~0.3 %) and in a susceptible person from
exposure to HBV (6–30 %) [154]. Transmission has been
6.4 Mother-to-Child Transmission associated with hollow-bore needles, deep injuries, HIV coin-
fection, and a high level of HCV viremia in the source patient
Prior to the testing of blood for HCV in 1992, blood transfusion [92, 93]. Transmission rarely occurs from mucous membrane
was the predominant mode of acquisition for HCV infection in or non-intact skin exposures to blood, and no transmission to
children. Since then, mother-to-child transmission has become health-care workers has been documented from intact skin
the leading source of HCV infection in children where blood exposures to blood [155]. The prevalence of HCV infection
screening is routine. The rate of perinatal transmission of HCV among health-care workers is no greater than in the general
is 4–7 % per pregnancy and requires detectable HCV RNA in population, averaging 1–2 % [67]. Even more rarely, HCV-
maternal serum at delivery [67]. The exact timing of transmis- infected health-care workers have transmitted HCV to
sion of HCV from the mother to the child is unknown. Both in patients through needlestick injuries or other percutaneous
utero and intrapartum infections are possible. The outcome of exposure, with an extremely low risk—averaging about
perinatal transmission of HCV in twin pregnancies is often dis- 0.5 %, even for those episodes involving surgeons.
cordant, with transmission of HCV more likely to affect the
second twin and with neonatal HCV RNA undetectable at
delivery; these observations suggest that intrapartum transmis- 6.6 Hepatitis C Virus Transmission
sion may be more frequent than in utero transmission [140]. in Other Settings
Risk factors for vertical transmission include higher maternal
serum HCV RNA levels (above ten [6] copies per mL), pro- Percutaneous exposure to blood can occur in a variety of other
longed labor after membrane rupture, internal fetal monitoring, practices, during which transmission of HCV may occur.
and coinfection with HIV [100, 141–150]. There is no apparent These exposures include but are not limited to tattooing, body
33 Hepatitis Viruses: Hepatitis C 773

piercing, intranasal drug use, ritual scarification, circumcision, 7.2 Chronic

acupuncture, and cupping. There are currently insufficient
data to determine the extent to which these risks factors may Individuals with chronic hepatitis C (CHC) are at increased
contribute to overall HCV transmission. risk of liver-related morbidity and mortality. The majority of
persons with CHC are asymptomatic. The most common
symptom of CHC infection is fatigue [172, 173]. Other
7 Pathogenesis and Immunity symptoms may include myalgias, nausea, anorexia, arthral-
gias, and difficulty concentrating. Progressive hepatic fibro-
The majority of people with acute HCV infection progress to sis leading to cirrhosis is the major complication of chronic
chronic infection (85 %) [156–159]. The key features of HCV infection and accounts for almost all HCV-related mor-
acute and chronic hepatitis C virus infection are reviewed in bidity and mortality [9]. In patients who develop cirrhosis,
this section. symptoms include worsening fatigue and anorexia, fluid
overload, difficulty concentrating or confusion due to hepatic
encephalopathy, and gastrointestinal bleeding. Physical
7.1 Acute Infection examination findings can include signs of chronic or end-
stage liver disease including palmar erythema, spider angio-
The natural history of HCV infection generally begins with mas, ascites, and asterixis. Laboratory changes include
a subclinical and unrecognized acute phase following infec- abnormal liver tests including transaminase elevations.
tion. Symptomatic acute hepatitis occurs within 2 weeks to
6 months of infection in only a minority of cases (10–15 %)
and is clinically indistinguishable from acute hepatitis of 8 Patterns of Host Response
other causes—with a combination of fever, fatigue, loss of
appetite, nausea and vomiting, dark urine, jaundice, and Persons with CHC are at risk for progression to cirrhosis,
liver tenderness, accompanied by elevated levels of serum liver failure, and/or hepatocellular carcinoma (HCC).
bilirubin and alanine and aspartate aminotransferases (ALT However, the disease course in persons with CHC is gener-
and AST, respectively) [160]. The majority of cases of HCV ally protracted and variable (see Fig. 33.5) [174]. The risk of
infection progress to chronic infection, but in up to 15 % of developing cirrhosis is 5–25 % over 25–30 years [175, 176].
people, infection may resolve spontaneously. Evidence sug- There are a number of host, viral, and environmental factors
gests that patients with symptoms in the setting of acute HCV that are associated with disease progression.
infection tend to have a higher rate of spontaneous clearance Host factors associated with more rapid fibrosis progres-
[161–163]. Other factors that might contribute to spontane- sion include older age (above 40 years old), longer duration
ous clearance include infection in infants and young women, of infection, male gender, obesity, hepatic steatosis, insulin
selected host polymorphisms near the gene encoding IL28b, resistance, and hepatic iron overload [67, 86, 176–186]. Age
infection with HCV genotype 3, having a low peak viral load, appears to be the predominant risk factor for more accelerated
Caucasian race, and rapid decline in viral load within the first fibrosis progression [86, 177]. Data from Poynard and col-
four weeks of diagnosis [161–166]. Persons with alcohol leagues indicate that median time to cirrhosis was 44 years in
consumption and HIV coinfection are less likely to spontane- patients infected at under 20 years of age, 30 years in patients
ously clear acute HCV infection [159, 163, 167]. infected between 31 and 40 years of age, and 12 years in
Early detection of progression to chronic HCV infection patients infected over the age of 50 years. The concept that
can be difficult, as it almost always occurs in the absence the aging liver is more susceptible to fibrosis is also supported
of symptoms. The differentiation of acute from chronic by experience with liver transplantation, where older donor
HCV infection depends largely on the clinical presentation age is a strong risk factor for rapid fibrosis in the graft
including presence of symptoms, recognition of a time- [187, 188]. A number of mechanisms have been proposed to
limited exposure, and documentation of ALT elevation and explain this: increased vulnerability to oxidative stress, a
its duration. After exposure, HCV RNA is usually detect- reduction in hepatic blood flow, and mitochondrial capacity
able before antibody appears in serum. HCV RNA can be [189–191]. Additionally, there are genetic factors that influ-
identified as early as 7–21 days following exposure, whereas ence the natural history of HCV and treatment response. Both
anti-HCV is generally not detectable before 8–12 weeks the innate and adaptive immune responses are important for
[30, 156, 168, 169]. When symptoms of acute HCV infection clearance of an acute HCV infection. After an HCV infection,
occur, they may include malaise, fatigue, myalgias, nausea, the innate immune response is initially important for control-
and right upper quadrant pain. Fulminant hepatic failure is a ling viral replication with the adaptive immune response
rare presentation of acute HCV infection unless other under- peaking at 8 weeks after infection [192]. Ultimately, a coordi-
lying chronic liver disease is present [170, 171]. nated effort between the innate and adaptive immune
774 P.V. Perumalswami and R.S. Klein

Fig. 33.5 Natural history of HCV

infection (Reprinted with HCV Infection
permission from International
Journal of Medical Sciences.
Chen and Morgan [174]. Available Acute Infection,
20–30 % with symptoms
from http://www.medsci.org/
v03p0047.htm)
Clearance of HCV RNA, Fulminant Hepatitis,
15 %–25 % Rare

Chronic Infection,
75 %–85 %

Extrahepatic
Manifestations

Chronic Active
Hepatitis

Cirrhosis,
10 %–20 % over 20 years

Decompensated Cirrhosis, HCC, 1 %–4 % per year

5-year survival rate of 50 %

responses is necessary to eliminate HCV from the liver [192]. HCC, is expected to continue to increase through the next
Genetic variation of the host immune system may affect decade [216]. Patients with HCV-related cirrhosis are at
fibrosis progression rates by way genetic factors that may increased risk of hepatic decompensation with signs/
influence activators of hepatic stellate cells, the family of pro- symptoms of liver failure and/or portal hypertension
teolytic enzymes known as matrix metalloproteases (MMPs), including ascites, portosystemic encephalopathy, and portal
and the tissue inhibitors (TIMPs) of MMPs. hypertension-related gastrointestinal bleeding [217, 218].
Coinfection with HIV and/or HBV is also associated Patients with HCV-related cirrhosis are at an approxi-
with progression of CHC [193–198]. While viral interac- mately 3 % per year risk for developing hepatocellular car-
tions with interference between HBV and HCV in patients cinoma [219–221].
with HBV-HCV dual infection with inhibition of the repli-
cation of one of the viruses have been described in several
studies [199–205], this phenomenon is incompletely 8.1 Extrahepatic Manifestations
understood, and the mechanism by which this occurs is yet
to be established [206, 207]. Dual infection with HBV and Chronic HCV infection can have serious consequences for
HCV is characterized by one virus dominating over the organ systems other than the liver. HCV infection may affect
other and can result in more accelerated progression to cir- the central nervous system, endocrine system, lymphatic
rhosis [193, 194]. Approximately 15–30 % of HIV-infected system, eyes, kidneys, blood vessels, skin, joints, and
persons are coinfected with HCV [208]. With HIV coin- peripheral nerves [222, 223]. Approximately 38 % of
fection there is an increased rate of end-stage liver disease, patients with chronic HCV infection will manifest at least
hepatocellular carcinoma, and death, in addition to the one symptomatic extrahepatic manifestation during the ill-
increased progression to cirrhosis [195–198, 209–211]. ness [224]. Most extrahepatic manifestations of chronic
Accelerated fibrosis progression is also seen with high HCV infection are immunologically mediated, and chronic
alcohol intake (daily consumption >50 g of alcohol) and infection seems to be necessary for their development. The
marijuana use [212–215]. most prominent manifestations include mixed cryoglobuli-
While the incidence of HCV infections is decreasing in nemia (MC) vasculitis, lymphoproliferative disorders, renal
the USA and other developed nations, due to the prolonged disease, insulin resistance, type 2 diabetes, sicca syndrome,
course of CHC, the number of infected people with com- and rheumatoid arthritis-like polyarthritis. Presence of these
plications related to end-stage liver disease is still increas- conditions can have a substantial impact on patient manage-
ing. The prevalence of hepatitis C-related cirrhosis and its ment as they can affect morbidity and mortality (for a
complications, including hepatic decompensation and detailed review, see [223]).
33 Hepatitis Viruses: Hepatitis C 775

9 Control and Prevention prominently mediated by cellular immunity [229]. Current

HCV vaccine approaches include peptide, plasmid DNA,
9.1 Public Health Approaches recombinant proteins, and vector-based vaccines. Virally
vectored vaccines are the most promising in terms of T-cell
Prevention of HCV-related liver disease should remain a key induction—in particular adenoviral and modified vaccinia
focus of clinical and public health interventions [10]. Major Ankara vectors. An understanding of the combination of
steps to prevent new HCV infections have been taken in sev- functions possessed by the T-cell population is needed to
eral countries including routine screening of blood donors, effectively assess HCV vaccine efficacy. Parameters of inter-
implementation and enforcement of standard precautions, est when assessing a vaccine-induced HCV-specific T-cell
eliminating reuse of injection equipment, and facilitating population include breadth, cytokine production, cytolytic
safer drug injecting behaviors with adoption of harm reduc- capacity, magnitude, phenotype, and proliferative capacity.
tion programs. Interventions using combined strategies of A major challenge in HCV vaccinology will be the assess-
education, behavioral interventions, substance-use treatment, ment of vaccine efficacy in well-characterized at-risk popu-
syringe access, and syringe disinfection can reduce the risk of lations. Early phase vaccine trials are currently underway.
HCV seroconversion by 75 % [225]. Therefore, efforts to pre-
vent infection, reduce harms, and treat HCV-infected IDUs
are essential. These steps should be extended worldwide. As 9.2 Treatment
part of secondary prevention, HCV-infected individuals
should be counseled to minimize their risk of transmitting The first successful therapies for HCV infection became
HCV and referred for medical evaluation and consideration available with the use of interferon to treat NANB hepatitis
of antiviral therapy. Of course, the latter approaches rely on in 1986 [230]. Sustained virological response (SVR) is anal-
the identification of persons with chronic HCV infection. In ogous to a cure and the goal of treatment. An SVR is defined
the USA, the CDC estimates that although 27 % of the popu- by the absence of detectable HCV RNA in a patient’s blood
lation was born during the interval 1945–1965, they account 6 months after the conclusion of antiviral therapy. Viral gen-
for approximately three-fourths of all HCV infections and otype testing is performed in the evaluation of HCV infec-
73 % of HCV-associated mortality, and they are at greatest tion in order to maximize the chance of successful treatment
risk for hepatocellular carcinoma and other HCV-related liver outcome for each individual patient as treatment type, dura-
disease. As a result, CDC has recently revised prior recom- tion, dose, and effectiveness are influenced by genotype.
mendations to include onetime testing for persons born dur- Historically, persons infected with HCV genotypes 1 and 4
ing 1945–1965 without prior ascertainment of HCV risk have required longer durations of treatment and have had
[226]. This recommendation for onetime screening in persons lower SVR rates. Since the early treatments, significant and
born during the 1945–1965 has now been endorsed by the US rapid progress has been made in establishing effective ther-
Preventive Services Task Force [227]. apy for HCV infection. There are now an increasing number
To address occupational risk of HCV infection, individual of potent options for treatment.
institutions should establish policies and procedures for The standard of care for HCV treatment for the last decade
HCV testing of persons after percutaneous or mucosal expo- until 2011 has been a combination of pegylated interferon and
sures to blood, and they should ensure that all personnel are ribavirin [30]. Pegylated interferon alfa is a potent inhibitor of
familiar with these policies and procedures. Health-care pro- HCV replication that acts by inducing interferon-stimulated
fessionals who provide care to persons occupationally host genes that have antiviral functions, and given its diverse
exposed to HCV should be knowledgeable regarding the risk actions, it is not associated with viral resistance [95]. Ribavirin
for HCV infection and appropriate counseling, testing, and acting synergistically with pegylated interferon alfa is an inte-
medical follow-up. gral component of the treatment for HCV, although the mech-
Finally, while there is currently no approved vaccine for anism of action is not well known. Treatment for HCV
HCV, attempts toward developing a vaccine are currently infection varies according on genotype.
underway. Despite difficulties associated with extreme vari- The first major advancement in HCV treatment in over a
ability and mutability of hepatitis C virus (HCV), several decade, occurred in 2011 with introduction of the first
vaccines that prevent initial infection or viral persistence, or generation direct-acting antiviral agents (protease inhibitors)
that clear viremia in individuals with chronic HCV infections, in combination with pegylated interferon and ribavirin
are currently in development [228]. The likely goal for a pro- [231–235]. Challenges with these new regimens included
phylactic vaccine against HCV is to present persistence of additional side effects, increased pill burden, increased
the virus, rather than to prevent primary infection. The is drug-drug interactions, and antiviral resistance [95].
based on the fact that a small proportion of people infected Additionally, use of these agents in real world practice had
with acute HCV spontaneously clear it and this appears to be lower SVR results than reported in registration trials [263].
776 P.V. Perumalswami and R.S. Klein

At the end of 2013, another major advancement of HCV injuries [249]. Although no immediately prophylactic regi-
therapy was realized with approval of second generation direct- men exists for HCV, effective treatment is available. When a
acting antiviral agents, Sofosbuvir and Simeprevir [236]. needlestick, percutaneous injury from other sharp instru-
Sofosubuvir, a nucleotide analogue NS5B polymerase inhib- ments, or mucosal exposure to blood occurs, the human
itor, is a component of the first all-oral, interferon-free regi- source of the exposure should be tested for antibody to HCV
men approved for treating chronic hepatitis C. Interferon-free (anti-HCV), and all repeatedly reactive results by enzyme
therapy for treatment of hepatitis C reduces the side effects immunoassay should be confirmed by RIBA for anti-HCV. If
associated with use of interferon [237, 238, 239, 240, 241]. the source is anti-HCV positive, the exposed person should
Many additional agents with different mechanisms of be tested for anti-HCV and alanine aminotransferase level at
actions, improved safety profiles and cure rates greater than baseline and follow-up (e.g., at 4–6 months) [250, 251]. For
95 % in registration trials are currently in various stages of earlier diagnosis of HCV infection, testing for HCV RNA
clinical development [242, 243]. The short-term prospects may be performed at 4–6 weeks. There are no recommenda-
for continued improvements in treatments with other direct tions for restriction of activities during the postexposure
acting antivirals in conjunction with pegylated interferon and follow-up period. There is consistent evidence that treatment
ribavirin are in the pipeline and are quite promising, and reduces the risk that acute hepatitis C will evolve to chronic
these advances will likely lead to increased rates of cure in infection [252, 253]. Currently, the American Association
the coming years [244–246]. Additionally, all oral interferon- for the Study of Liver Diseases (AASLD) practice guidelines
free regimens are now on the horizon, which will lessen recommend treatment initiation in patients with acute HCV
treatment side effects and likely increase patient adherence if serum HCV RNA is not eliminated spontaneously within
and tolerability [240, 241]. However, it is important that both 12 weeks of HCV transmission [30]. When HCV infection is
patients and providers be aware that there is no protective identified early, the individual should be referred for medical
immunity and reinfection is possible with ongoing risk management to a specialist knowledgeable in this area.
behavior and exposure.
Successful treatment of HCV infection eliminates trans-
missibility by eradication of viral infection (i.e., cure). 10 Unresolved Problems
It is reasonable to assume that transmissibility is likely
reduced by reduction in HCV viral load even if cure is not Although effective diagnostic tools and treatments are avail-
accomplished. able, HCV remains a major public health problem. In many
countries, including the USA, HCV-associated disease is the
9.2.1 Perinatal HCV Infection leading indication for liver transplantation, and it is a lead-
Immune globulin and antiviral agents are not recommended ing cause of HCC in the USA [37, 89, 254]. HCC and cir-
for postexposure prophylaxis of infants born to HCV-positive rhosis have been increasing among persons infected with
women. Children born to HCV-positive women should be HCV [255, 256], and these outcomes are projected to
tested for HCV infection. Early diagnosis of HCV infection increase substantially in the coming decade [23, 257]. HCC
can be done by testing for HCV RNA at age 1–2 months and is the fastest growing cause of cancer-related mortality, and
should be repeated after 3 months within the first year [100, infection with HCV accounts for approximately 50 % of
153, 247, 248]. Umbilical cord blood should not be used for incident HCC [7].
the diagnosis of perinatal HCV infection because cord blood Transmission of HCV infection continues despite current
can be contaminated by maternal blood. Testing of infants prevention strategies. Early diagnosis of persons with HCV
for anti-HCV should be performed no sooner than infection has been difficult. Because 80 % of acute HCV
15–18 months of age, when passively transferred maternal infections are asymptomatic, and more than 60 % of persons
anti-HCV declines below detectable levels. If positive for with chronic HCV infection are asymptomatic [156, 163],
either anti-HCV or HCV RNA, children should be evaluated early diagnosis has relied on screening for asymptomatic
for the presence or development of liver disease, and those infection and disease. Most persons with HCV do not know
children with persistently abnormal ALT levels should be they are infected, are not evaluated for treatment, and do not
referred to a specialist for medical management. receive needed care (e.g., education, counseling, and medi-
cal monitoring) [258–260]. In the USA alone, 75 % of
infected persons remain unaware of their infection [261].
9.3 Postexposure Management Additional strategies for and resources to support prevention
and treatment of HCV are still needed. Education of persons
It has been estimated that in the year 2000, 16,000 HCV at risk, implementation of standard precautions and infection
infections may have occurred worldwide among health-care control, and screening of blood donors for HCV are partially
workers due to their occupational exposure to percutaneous or entirely lacking in many parts of the world.
33 Hepatitis Viruses: Hepatitis C 777

Finally, not all persons with HCV infection progress to 18. Cossart YE, Kirsch S, Ismay SL. Post-transfusion hepatitis in
end-stage liver disease or cirrhosis. However, we currently Australia. Report of the Australian Red Cross study. Lancet.
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Acknowledgments We would like to acknowledge Dr. Scott L. The increasing burden of mortality from viral hepatitis in the United
Friedman, Fishberg Professor of Medicine, Dean for Therapeutic States between 1999 and 2007. Ann Intern Med. 2012;156:271–8.
Discovery and Chief, Division of Liver Diseases at Mount Sinai School 23. Rein DB, Wittenborn JS, Weinbaum CM, Sabin M, Smith BD,
of Medicine, for his review and input on this chapter. Lesesne SB. Forecasting the morbidity and mortality associated
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 Hepatitis Viruses: Hepatocellular
Carcinoma 34
Ju Dong Yang, Roongruedee Chaiteerakij,
and Lewis R. Roberts

1 Introduction tion of telomerase reverse transcriptase. In HBV- and HCV-

induced hepatocarcinogenesis, the interplay between HBV
Liver cancer is the fifth most common cancer in men and and HCV, the host immune response to infection, and chronic
the Eighth in women. The estimated number of new liver inflammation in the liver contribute to malignant trans-
cancer cases worldwide has increased by 70 % between formation. HBV and HCV are involved in carcinogenesis
1990 and 2008. It has been projected that the number will directly through induction of genetic mutations and through
increase by 30 % to one million new cases in 2020. Viral oncogenic effects of viral proteins. Indirectly, both viruses
hepatitis is the most common cause of hepatocellular car- share some common carcinogenesis pathways, in particular,
cinoma (HCC) in the world. Hepatitis B virus (HBV) is the chronic inflammation, oxidative stress, and tumor suppressor
most common etiology of HCC in most Asian and African gene p53 inactivation. The goal of HCC surveillance is to
countries and accounts for more than half of all HCCs in the decrease HCC mortality through the early detection of HCC
world. Hepatitis C virus (HCV) infection is the leading cause in asymptomatic patients. In real-world practice, the major-
of chronic liver disease and HCC in most Western countries ity of at risk individuals are not under HCC surveillance and
including the USA. The prevalence of HCV infection has HCC is usually detected at a late stage when patients become
increased and the proportion of patients with liver cirrhosis symptomatic from advanced HCC. There is strong evidence
or HCC from HCV has also increased in the USA over the that HCC surveillance increases the early detection of tumors
past several decades. Carcinogenesis is a complex and multi- and improves survival in patients with HCC. However, the
step process involving a sequence of tumor initiation, promo- efficacy of HCC surveillance is still controversial mainly
tion, and progression over several years. Common molecular because of the lack of robust level 1 evidence supporting the
mechanisms of HCC occurring in the cirrhotic liver include use of HCC surveillance. Noninvasive diagnosis of HCC in
cell-cycle dysregulation, sustained angiogenesis, apoptosis individuals with cirrhosis can be made by contrast-enhanced
resistance, and cellular immortalization through reactiva- four phase CT (non-contrast, arterial, portal, and delayed
phases) or dynamic contrast MRI. Arterial enhancement and
J.D. Yang, MD, MSc portal venous or delayed phase washout are highly specific
Division of Gastroenterology and Hepatology, characteristic features of HCCs. Percutaneous liver biopsy is
Mayo Clinic College of Medicine, indicated when lesions (>1 cm) develop without background
200 First Street SW, Rochester, MN 55905, USA
liver cirrhosis or dynamic imaging modalities show incon-
e-mail: [email protected]
clusive results. The treatment of HCC depends on multiple
R. Chaiteerakij, MD, MSc
factors, including the tumor extent, severity of liver dys-
Division of Gastroenterology and Hepatology,
Mayo Clinic College of Medicine, function, performance status of the patient, socioeconomic
200 First Street SW, Rochester, MN 55905, USA support, and the availability of different types of treatment.
Department of Medicine, Faculty of Medicine, Surgical resection, liver transplantation, and local ablative
King Chulalongkorn Memorial Hospital, treatment are potentially curative treatment modalities for
Thai Red Cross Society, Bangkok, Thailand patients with early-stage HCC. Transarterial chemoembo-
e-mail: [email protected]
lization and radioembolization are locoregional treatment
L.R. Roberts, MBChB, PhD (*) modalities applicable for patients with intermediate-stage
Division of Gastroenterology and Hepatology,
Mayo Clinic College of Medicine,
200 First Street SW, Rochester, MN 55905, USA The authors Ju Dong Yang and Roongruedee Chaiteerakij have equally
e-mail: [email protected] contributed to this work.

R.A. Kaslow et al. (eds.), Viral Infections of Humans, 785

DOI 10.1007/978-1-4899-7448-8_34, © Springer Science+Business Media New York 2014
786 J.D. Yang et al.

HCC. Sorafenib is a targeted systemic treatment that is meta-analysis found that HBV and HCV infections increase
offered to patients with advanced stage HCC. HCC risk by 13.5-fold and 12.2-fold, respectively, while
HBV and HCV co-infections additively increase the HCC
risk by 51.1-fold [18].
2 Historical Background of the
Association Between Viral Hepatitis
and Hepatocellular Carcinoma 3 Incidence of HCC

A possible association between hepatitis B virus (HBV) Worldwide, liver cancer is the fifth most common can-
infection and hepatocellular carcinoma (HCC) development cer in men and the eighth in women [19]. The global inci-
was first reported in the 1950s [1]. Subsequently, the associa- dence of HCC has been rising, from 437,408 cases in 1990
tion between HBV infection and HCC was supported by sev- to 782,000 in 2012. HCC cases are projected to increase
eral epidemiologic studies in the 1970s, mostly from Africa further to 997,955 new cases by 2020 and 1,251,520 new
and Asia [2–6]. A causal relationship between HBV infection cases by 2030 [19]. The trends in HCC incidence reflect the
and HCC was confirmed by a Taiwanese prospective study temporal and geographical changes in prevalence of risk
showing that HBV-infected patients had a 98.4-fold increased factors for HCC. The rising incidence is primarily occur-
risk for developing HCC compared to individuals without ring in countries with low and intermediate HCC incidence.
HBV infection [7]. There is also a lower but nevertheless In the USA, the annual HCC incidence has been shown to
increased risk of HCC associated with chronic HBV carrier have increased by threefold between 1975 and 2005 (1.5–
status [8]. The relationship between hepatitis C virus (HCV) 4.9 cases per 100,000 persons) [20]. Indeed, a study from
infection and HCC was first noted in the early 1980s [9, 10]. Olmsted County, Minnesota, has confirmed the increasing
As HCV (then described as non-A, non-B chronic hepatitis) trend of HCC incidence, showing that the incidence of HCC
became endemic in Japan in the late 1940s and early 1950s, increased from 3.5 to 6.9 per 100,000 person years between
the majority of the early evidence of a causal link between 1976 and 2008 [21]. In England and Wales, the HCC inci-
HCV and HCC was from studies performed in Japan. Due dence increased by 46 % between 1971 and 2001 in males
to the lag time of 20–40 years between the establishment of (1.8 to 2.7 cases per 100,000 persons) and increased by 8 %
chronic HCV infection and the development of HCC, a dra- in females (0.8 to 0.9 cases per 100,000 persons) [22]. The
matic increase in HCC-related mortality in Japan occurred rising trend in HCC in these countries is mainly attributable
beginning in the 1970s and most of these HCCs developed in to HCV infection. It has been projected that the HCV-related
patients with non-A, non-B hepatitis [11]. Subsequent to the HCC incidence in the USA will peak in 2015–2020 and then
identification of the hepatitis C virus in 1990 and the devel- gradually decline [23].
opment of specific assays for the infection, among those with In contrast to the rising trends of HCC incidence in low
chronic non-A, non-B hepatitis, antibody to HCV (anti-HCV) and intermediate prevalence areas, the opposite trends have
was detected in 94.4 % [10]. Epidemiologic studies from been observed in high-prevalence countries. For example, the
Spain and Italy also reported that 61–75 % of HCC patients age-standardized HCC incidence in Japan declined steadily
were positive for anti-HCV [12–14]. The initial causative between 1995 and 2005 from 41.5 to 24.0 cases per 100,000
role of HCV infection in HCC development was supported persons among men and from 10.8 to 7.3 cases per 100,000
by a Japanese study showing that HCV RNA was detected persons among women [24]. These declines are attributed
in HCC tissue and the surrounding noncancerous liver tis- to the marked reduction in the incidence of HCV infection
sues of patients with HCC [15]. A meta-analysis of 32 case- after implementation of a nationwide primary prevention
control studies published between 1993 and 1997 concluded program [24]. Similarly, a gradual decline in HCC incidence
that chronic HBV-infected patients had 13.7-fold increased has also been observed in Taiwan where HBV is the major
risk for HCC compared to individuals without chronic HBV cause of HCC. Within 10 years after initiation of a nation-
infection, and HCV-infected patients had 11.5-fold increased wide vaccination program, the HCC incidence in children
risk for HCC compared to individuals without HCV infec- aged 6–14 years had decreased dramatically from 0.76 per
tion [16]. This meta-analysis also reported that the risk for 100,000 in the period from 1982 to 1986 to 0.36 per 100,000
HCC increased to 165-fold for those infected with both HBV in the period from 1990 to 1994 [25].
and HCV, indicating a synergistic interaction of HBV and The overall incidence of HCC rises sharply after age of 40
HCV coinfections on the risk for HCC development [16]. and increases with age in both genders [19]. The incidence
Given the evidence for the association between HBV and peaks at age 70 and then declines [20]. The age distribution
HCV infections and HCC risk, both HBV and HCV were of HCC varies worldwide. Several factors, including gender,
classified as human carcinogens by the International Agency age at HBV or HCV infection, and coexistence of other risk
for Research on Cancer (IARC) in 1994 [17]. A different factors, account for the global variations in the peak age of
34 Hepatitis Viruses: Hepatocellular Carcinoma 787

onset. The peak age of onset in Africa, another region where prevalences of HBV and HCV are about 6 and 3 %, respec-
HBV is a major cause of HCC, is generally younger than in tively [27]. Most countries in Asia, except for Japan, Pakistan,
other parts of the world, suggesting that viral, genetic, envi- and Mongolia, have a higher proportion of HBV-related HCC
ronmental, or other risk factors may modify the risk for HCC than HCV-related HCC. HBsAg seropositivity among HCC
development. For example, the peak age of onset in Mali is patients was 27–64 % in India, 53 % in Taiwan, 52–66 % in
40 years [26]. Turkey, 58–60 % in Thailand, 59–67 % in China, 62–82 %
in Korea, and 61–93 % in Vietnam, whereas anti-HCV posi-
tivity was approximately 2 % in Vietnam, 5–15 % in Korea,
4 Geographical Variation of HBV 3–10 % in China, 4–53 % in India, 19–29 % in Turkey, 28 %
and HCV-Induced HCC in Taiwan, and 5–10 % in Thailand among HCC patients in
these countries [28]. By contrast, only 18 % of HCC cases in
Although HCC develops in all regions around the world, Japan and approximately 30 % of HCC cases in Pakistan and
substantial global variation of HCC incidence is observed Mongolia were positive for HBsAg. Japan had the highest
(Fig. 34.1). Over 85 % of HCC cases occur in East and proportion of HCC cases positive for anti-HCV (68 %) and
Southeast Asia and in Middle and Western Africa, whereas anti-HCV positivity of HCC patients was 45 % in Pakistan
the incidence is much lower in Europe (with the exception of and 40 % in Mongolia. Of the Asian countries, HBV and
South Europe), South America, North America, Australia, HCV coinfection was most commonly found in Mongolia
and New Zealand [19]. The variation in geographical dis- (25 %) [28]. Similar to Asian countries, most countries in
tribution of HCC is explained by the prevalence of specific Africa have a much higher prevalence of HBV-related HCC
risk factors, in particular, chronic HBV and HCV infec- cases than HCV-related cases. HBsAg positivity in HCC
tions. A strong correlation between HCC incidence and cases was approximately 65 % in Mozambique, 60 % in the
prevalence of chronic HBV and HCV infections has been Gambia, 45 % in South Africa, and 42 % in other African
reported (Fig. 34.1). It was estimated that 75–80 % of HCC countries. The only exception is Egypt where the propor-
cases worldwide are associated with either chronic HBV tion of HCC cases with HBsAg+ was only 10 %. Egypt
(50–55 %) or HCV (10–25 %) infection, while the global had the highest proportion of HCC cases with anti-HCV+

<2.4 <4.5 <6.0 <8.7 <73.8

Fig. 34.1 (a–c) Worldwide incidence of HCC and prevalence of HBV and HCV
788 J.D. Yang et al.

<2 % 2 %–7 % ≥8 %

Not included in WHO region <1.0 % 1.0–1.9 % 2.0–2.9 % >2.9 %

Fig. 34.1 (continued)

34 Hepatitis Viruses: Hepatocellular Carcinoma 789

(69 %), whereas all other African countries reported a pro- [19]. The mortality to incidence ratio of HCC was 0.98 in
portion of 5–25 % of HCC cases with anti-HCV+ [28]. The 1990 and remained high at 0.95 in 2012 (Fig. 34.2) [31]. It
initial spread of HCV infection in Japan was thought to be has been predicted that the number of liver cancer deaths will
due to mass treatment campaigns for endemic Schistosoma increase to 929,368 in the year 2020 and to 1,175,804 in 2030
japonicum beginning in the 1920s [29]. Similarly, the high [19]. The 5-year survival rate for liver cancer worldwide is
prevalence of HCV-associated HCC in Egypt is thought estimated at 15 % [32]. There are substantial geographical,
to be related to the iatrogenic transmission of HCV in that regional, and local variations in HCC survival, in large part
population during mass treatment campaigns for endemic related to access to surveillance and therapy. Although the
Schistosoma mansoni [30]. number of liver cancer deaths has been increasing due to the
Contrary to Asian and African countries, in most European increasing incidence of HCC, the overall survival in patients
countries except for Greece, the proportion of HCC patients with HCC appears to have improved over time. A study from
with positive anti-HCV is greater than those with positive Olmsted County, Minnesota, showed that overall survival in
HBsAg. Anti-HCV positivity in HCC patients was 48 % in patients with HCC has improved over the past three decades,
Spain, 43 % in Italy, 36 % in Austria, and 25 % in Belgium with an increasing proportion of patients receiving cura-
and the UK, while the proportion with positive HBsAg+ was tive treatment over time and improvement in survival from
approximately 10–20 % in these countries. In Greece, the a median survival time of 3 month in 1976 to 9 months in
proportion of HCC patients positive for HBsAg and anti- 2008 (P = 0.01) [21].
HCV was 56 and 16 %, respectively [28]. In Germany, the
proportion of anti-HCV-positive cases (25 %) was close to
that of HBsAg-positive cases (22 %). It is important to note 6 Etiology of HCC
that the proportions of HCC cases negative for both HBsAg
and anti-HCV among European countries were relatively 6.1 Hepatitis B and Hepatitis C Viruses
higher compared to the proportions in Asian countries (35 %
in India and 5–25 % in all other Asian countries) and in HBV and HCV infections are the most common causes of
Africa (less than 30 %). Strikingly, in Sweden 82 % of HCC HCC in the world. Alcohol is the next most common eti-
cases were seronegative for HBsAg and anti-HCV, in con- ology of HCC. Due to the rising epidemic of obesity and
trast to the proportion that was seronegative for both viruses metabolic syndrome, NAFLD (nonalcoholic fatty liver dis-
in other European countries (32–52 %) [28]. The high pro- ease) is now recognized as one of the four major etiologies
portion of HCC cases seronegative for both viruses reflects of HCC [33].
the fact that risk factors other than chronic HBV or HCV Variations in incidence of HCC in different racial and
infection, particularly alcohol use and increasingly fatty liver ethnic populations are generally related to the underlying
disease, are of substantial importance in contributing to HCC prevalence of the major risk factors in these populations.
development in Europe. The proportion of HCC cases with In the USA, Blacks and Hispanics have a 2.5 and 2.0 times
positive HBsAg or anti-HCV varies greatly among countries higher incidence compared to Whites, respectively [34]. In
in North, Middle, and South America. In the USA, HCV addition to the different risks of HCC, the clinical features
accounts for 30–50 % and HBV accounts for 5–15 % of and presentation also differ among different races. Caucasian
HCCs. In Brazil, 40 % of HCC cases were seronegative for carriers of HBsAg tend to develop HCC at an older age in a
both HBV and HCV viruses and 37 and 18 % of HCC cases background of underlying liver cirrhosis, while Asians and
were positive for HBsAg and anti-HCV, respectively. In Peru Africans tend to develop HCC at a relatively early age with
and Mexico, 44 % of cases had positive HBsAg, 20 % of less cirrhotic change of the liver [23]. This difference may be
cases had positive anti-HCV, and 30 % of cases were sero- explained by the difference in age at which HBV infection is
negative for both HBV and HCV viruses [28]. acquired in the different populations as well as genetic varia-
tions of host and HBV genotype between the different races.
It is well known that persistent HBV replication is associ-
5 Mortality ated with increased risk of HCC [35, 36]. HBV genotype C
is the most prevalent genotype in Asians and known to be
In 2012, an estimated 746,000 worldwide deaths were due to associated with the presence of hepatitis B virus e antigen
liver cancer [19]. Liver cancer was the second leading cause (HBeAg), a decreased rate of spontaneous HBeAg clearance,
of cancer-related death in males, the sixth cause of cancer higher rates of HBeAg reversion after HBeAg clearance, and
death in females, and the second most common cause of an increased risk of HCC independent of HBV DNA lev-
death from cancer overall [19]. Liver cancer is considered els [37–40]. Several publications from Asian countries have
to be the second most lethal cancer, after pancreatic cancer, identified male sex, older age, elevated serum alanine ami-
which had the highest mortality to incidence ratio of 0.96 notransferase (ALT), positive HBeAg status, elevated serum
790 J.D. Yang et al.

Fig. 34.2 Worldwide

incidence and mortality of
HCC in males and
females

HBV DNA level, and HBV genotype C as independent pre- USA have also increased over the past several decades [45].
dictors of HCC development [41, 42]. The risk for HCC A population-based study in Olmsted County, Minnesota,
can also be affected by long-term changes in serum levels showed that HCV is the main driver of the increasing inci-
of HBV DNA or ALT. A study from Taiwan showed that dence of HCC, accounting for 45 % of HCCs between 2001
persistently high HBV DNA levels (1,000,000–10,000,000 and 2008 [21]. Individuals with positive anti-HCV have a
copies/mL) are associated with a substantially increased 28-fold increased risk of HCC compared to individuals
risk of HCC, with a hazard ratio (HR) of 16.8 (CI, 7.3–38.4) with negative anti-HCV [46]. In Taiwan, a population-
compared to controls. The ALT level was also significantly based study showed cumulative lifetime (age 30–75 years)
associated with HCC risk [43]. incidences of HCC for men and women with positive anti-
Hepatitis C is the leading cause of chronic liver disease HCV were 23.73 and 16.71 %, compared to lifetime inci-
and HCC in most Western countries including the USA dences for HBsAg-positive individuals of 27.38 and 7.99 %
[21, 44]. The prevalence of HCV and the proportion of for men and women, respectively. HCV and HBV coinfec-
patients with liver cirrhosis or HCC induced by HCV in the tion increases the risk of HCC; the cumulative lifetime
34 Hepatitis Viruses: Hepatocellular Carcinoma 791

incidences of HCC in dually infected men and women were an important new risk factor for HCC in many developed
38.35 and 27.40 %, respectively [47]. countries [61, 62]. Obesity has been associated with a signif-
HCV transmission by blood transfusion was common in icantly increased risk of mortality from HCC [63]. A study
the USA and other highly developed countries prior to the using the US Scientific Registry of Transplant Recipients
introduction of screening; the risk of HCV transmission from for primary adult liver transplant recipients between 2001
a blood transfusion has been negligible in the USA since the and 2009 showed that NAFLD was the third most common
implementation of donor blood screening for HCV in 1990 indication for liver transplantation in the USA and is on a
[48, 49]. More recently, the most common route of transmis- trajectory to become the most common indication. A US
sion has been by percutaneous exposure to blood via illegal referral center study showed that NAFLD is the third most
intravenous or intranasal drug use and via high-risk sexual common cause of HCC [33]. Importantly, there is substan-
behaviors [50, 51]. Transmission of HCV among injection drug tial and increasing evidence suggesting that NAFLD can
users (IDUs) remains problematic, as suggested by a report that lead to the development of HCC in the absence of estab-
the prevalence among them in England and Wales rose from lished cirrhosis [64, 65]. The annual incidence rate of HCC
47 % in 1998 to 53 % in 2006 [51]. Iatrogenic transmission is in patients with NASH cirrhosis was reported to be 2.6 %
not uncommon as a route of HCV transmission, even in more [66]. Diabetes has been shown to be associated with a two-
developed parts of the world. In a study from Spain, hospi- to threefold increased risk of HCC in multiple cohort and
tal admission was the most common risk factor preceding the case-control studies [67]. A more recent large cohort study
diagnosis of acute HCV infection, which usually progresses to showed that well-controlled diabetes (HgbA1C <7 %) in
chronic HCV infection [52]. Although mother to infant vertical patients is associated with a 44 % reduction in the risk of
transmission is rare, a high HCV titer in the mother is known to developing HCC compared to poorly controlled diabetes
increase the frequency of HCV transmission [53, 54]. (HgbA1C >7 %) [68]. It is not clear however whether dia-
betes has a direct carcinogenic effect in the liver or whether
it indirectly increases the risk of HCC by promoting hepatic
6.2 Alcoholic Liver Disease inflammation and fibrosis. Metformin and statin drugs have
been shown to be associated with a decreased risk of HCC,
Alcoholic liver disease is the second most common risk and metformin treatment was independently associated with
factor for HCC in the USA [55]. Although alcohol intake an 80 % decrease in HCC occurrence in a cohort of patients
is known to increase the risk for HCC, the threshold dose with HCV-induced cirrhosis [69, 70]. A more recent meta-
and the minimal duration of use associated with risk of HCC analysis which included approximately 105,495 patients
are not well established [56]. The risk of developing HCC with type 2 diabetes showed that metformin was associated
appears to be lower in patients with alcoholic cirrhosis com- with an estimated 62 % reduction in the risk of liver cancer
pared to patients with cirrhosis from other etiologies; in a among patients with type 2 diabetes (P < 0.001) [71]. Fish
recent Danish nationwide cohort study, the 5-year cumula- oil also appears to be protective against the development
tive HCC risk for the alcoholic group was only 1.0 % (95 % of HCC. A Japanese population-based prospective cohort
CI, 0.8–1.3 %) [57]. It is known that females are more sus- study of 90,296 subjects showed that consumption of n-3
ceptible than males to liver injury from alcohol intake due to polyunsaturated fatty acid (PUFA)-rich fish and individual
differences in alcohol metabolism [58]. One quarter of alco- n-3 PUFAs was associated with a 30–40 % risk reduction of
holic patients in the USA have HCV infection [59]. Not sur- HCC, in a dose-dependent manner. These inverse associa-
prisingly then, coexisting viral hepatitis and alcohol intake tions were similar irrespective of HCV or HBV status [72].
appears to increase the risk of HCC [56, 60]. A population- Statin use was associated with a 26 % decrease in risk of
based study from Olmsted County, Minnesota, has shown HCC in a matched case-control study nested within a cohort
that HCV patients with significant alcohol intake develop of VA patients with diabetes [73]. On the other hand, insulin
HCC at an earlier age compared to those without significant treatment for diabetes has been shown to be associated with
alcohol consumption [21]. Secular trends in per capita alco- an increased risk of HCC [70, 74]. Based on the available
hol consumption in different countries or regions may there- evidence, no definitive statement can be made about the con-
fore have significant impact on trends in HCC incidence. tribution lipid derangement (fatty liver) on the risk of HCC
due to viral hepatitis.

6.3 Obesity-Related Nonalcoholic

Steatohepatitis 6.4 Aflatoxin

Obesity-related nonalcoholic steatohepatitis (NASH), or Aflatoxin B is a mycotoxin which is a frequent food con-
nonalcoholic fatty liver disease (NAFLD), is emerging as taminant in sub-Saharan Africa and Eastern Asia and is a
792 J.D. Yang et al.

well-known risk factor for HCC [75]. Aflatoxin B has a syn- exhausted liver regeneration, some hepatocytes escape from
ergistic effect with HBV in HCC development by inducing senescence through activation of the telomerase enzyme or
p53 mutations (most commonly a G-to-T transversion in alternative mechanisms, leading to cellular immortalization
codon 249) and activating oncogenic pathways [76]. [86]. It appears that generation of a genotoxic inflamma-
tory tumor microenvironment with a high concentration of
reactive oxygen species leads to the accumulation of genetic
6.5 Other Cofactors alterations in immortalized hepatocytes, as well as to epi-
genetic alterations in DNA methylation, histone modifica-
A positive family history of liver cancer increases the risk tion, and noncoding RNA expression that contribute to the
of HCC by two- to threefold independent of viral hepatitis development of the tumor phenotype [87]. The combina-
[77]. Males are at two to four times greater risk for HCC tion of oncogenic alterations, including oncogene-activating
than females. The male predilection varies around the world gene mutations, gene amplification, and increases in onco-
and is usually higher in high-incidence areas [19]. The sex gene expression, as well as tumor suppressor inactivation by
discrepancy in HCC incidence is explained by two factors, mutation, genetic loss, or epigenetic mechanisms, results in
namely, the difference in rates of exposure to known risk fac- dysregulation of the