BSMT201 – Mycology and Virology (Laboratory)

Virology Introduction Third Semester A.Y. 2020-2021

Christine Torio, RMT Final Exam
June 9, 2020 3-1

OUTLINE  Submicroscopic, obligate intracellular parasite capable of

infecting any animal, plant, or bacterial cell.
I. EVIDENCE OF VRIAL DISEASE  Viruses are found in every ecosystem.
II. VIRUS  They are strict obligate intracellular parasites, incapable of
A. Spanish Flu Pandemic 1918-1919 replication without a living host cell.
III. PROTECTION FROM VIRAL INFECTION  Virus types are very specific, and each has a limited
IV. VIRAL STRUCTURE number of hosts it can infect; this is referred to as viral
A. Virions tropism.
B. Enveloped Viruses  Transmission of viruses from animals to humans still
C. Naked Viruses occurs, as demonstrated in the more recent viral
V. ILLUSTRATION OF A VIRAL PARTICLE outbreaks associated with the severe acute respiratory
A. Nucleic Acid Genome syndrome (SARS), West Nile, and influenza A H5 viruses, as
B. Viral Capsid well as the 2009 H1N1 virus, formerly known as the
C. Nucleocapsid Enclosed in a Lipid Envelope pandemic “swine flu.”
VI. VIRAL CLASSIFICATION BASED ON THREE BASIC  The influenza virus has proven to be one of the deadliest
PROPERTIES viruses to affect humans; its history dates back to the
VII. VIRAL REPLICATION 1700s in Italy. The virus was named to indicate disease
VIII. VIRAL PATHOGENESIS resulting from the “influence” of miasma (bad air).
IX. CLINICAL VIROLOGY LABORATORY
 The more recent influenza pandemic of the twentieth
A. Standard precautions and Biosafety Level 2
century was associated with a human influenza virus in
B. Specimen Selection and Collection
which genes reassorted in combination with an avian
C. Specimen for Diagnosis of Viral Diseases
influenza virus.
i. Throat, Nasopharyngeal Swab or
 The emergence of a new viral disease across a very large
Aspirate
geographical region (worldwide) with prolonged human-
ii. Bronchial and Bronchoalveolar Washes
to-human transmission is called a pandemic.
iii. Rectal Swabs and Stool Specimens
 To date, most of the pandemics recorded have been
iv. Urine
caused by an influenza virus. Pandemics result when an
v. Skin and Mucous Membrane Lesions
influenza virus undergoes a genetic shift and the
vi. Sterile Body Fluids Other Than Blood
reassortment of genes combines with those of another
vii. Blood
organism, usually an animal.
viii. Bone Marrow
 The resulting virus emerges as a completely new or
ix. Tissue
“novel” virus. The genetic changes in viral genomes may
x. Genital Specimens
result from antigenic shift (major changes that result in
xi. Serum for Antibody Testing
novel viral antigens) and/or antigenic drift (minor changes
D. Specimen Transport and Storage
that occur infrequently)

REFERENCES
A. Spanish Flu Pandemic 1918-1919
1. Christine Torio’s ppt
 This pandemic was associated with infection with a novel
2. Tille, Patricia M. 2014. Bailey and Scott’s Diagnostic
influenza virus of avian origin. After a period of adaptation
Microbiology. 13th ed. Elsevier Mosby, Missouri.
in humans, the virus emerged in pandemic form and was
responsible for more than 50 million deaths worldwide,
I. EVIDENCE OF VIRAL DISEASES including 500,000 in the United States.
 23 BC. The Eschunna Code of ancient Mesopotamia noted  What was so different about this pandemic was that it
“the bite of mad dogs to affect disease on humans.” affected young and healthy individuals, not just the very
 Homer, author of the Iliad, characterizes Hector as “rabid.” young or very old.
 Aristotle’s work, The Natural History of Man (4th century  The more recent influenza pandemic of the twentieth
BC) describes a “madness” in dogs that “causes them to century was associated with a human influenza virus in
become very irritable and all mammals they bite become which genes reassorted in combination with an avian
diseased.” influenza virus.
 All writers realized the communicable nature of something
unseen. These writings clearly refer to the rabies virus, III.PROTECTION FROM VIRAL INFECTION
transmitted through the saliva of an infected animal.
 Successful for some viral pathogens
 Vaccination (immunization) has proven to be a valuable
tool in the control of viral diseases such as yellow fever
and rabies and has been instrumental in the eradication of
II. VIRUS one of the most lethal viruses, smallpox.

BSMT 2021 – ORDONEZ VL| Page 1 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

 Many viral diseases such as influenza, acquired  encode the proteins required for viral penetration,
immunodeficiency syndrome (AIDS), and hepatitis transmission, and replication. The viral genome structure
continue to pose challenges in treatment, prevention, and determines the mechanism for viral replication.
control. Types of viral genome:
 “The science of virology will continue to evolve and  (+) sense strand RNA
clinicians will continue to rely on the laboratory scientist  (–) sense strand RNA
for the development and implementation of testing to  DNA genomes
diagnose, treat, and prevent viral disease”  Viral genomes may be single- or double-stranded
molecules.
IV. VIRAL STRUCTURE
B. Viral Capsid
A. Virions (virus particles) Parts
 protects the viral genome and is responsible for the
 An inner nucleic acid core, consisting of either ribonucleic tropism to specific cell types in naked viruses. Viral capsids
acid (RNA) or deoxyribonucleic acid (DNA). typically are composed of repeating structural subunits
 A protein coat that surrounds and protects the nucleic acid referred to as capsomeres.
(the capsid).  Capsomers associate to form the capsid and a
 In some of the larger viruses, a lipid-containing envelope characteristic symmetric structure.
that surrounds the virus. Most Common Capsid Structures:
a. Icosahedral capsids- cubical and have 20 flat sides
B. Enveloped Viruses b. Helical form- irregularly shaped capsids and are spiral
shaped.
 are very susceptible to drying out and destruction in the
environment, they typically are transmitted by direct
contact, such as respiratory, sexual, or parenteral contact. C. Nucleocapsid Enclosed in a Lipid Envelope
 This prevents exposure to the environment and successful  The envelope is responsible for viral entry into the host
propagation of the viral agent to another susceptible host. cell. During the infectious process, enveloped virions bud
from a host cell’s cytoplasmic, nuclear, or endoplasmic
C. Naked Viruses (No Enevelope) reticular membrane, and a portion of the membrane
remains attached to the virion as the viral envelope.
 are very resistant to environmental factors. Inserted into this viral envelope are viral proteins, such as
 Because of their stability, they typically are transmitted by hemagglutinin (HA), neuraminidase, or glycoprotein
the fecal-oral route. spikes.
 Many viruses have glycoprotein spikes extending from  The glycoprotein spikes assist in stabilization of
their surface. The term nucleocapsid often used to attachment for the lipid envelope and for attachment to
describe the nucleic acid genome surrounded by a the host cell to facilitate viral entry. Some enveloped
symmetric protein coat. viruses also contain a matrix protein that lies between the
envelope and the nucleocapsid.
V. ILLUSTRATION OF A VIRAL PARTICLE  The matrix protein may have enzymatic activities and/or
 Enveloped and nonenveloped virions have icosahedral or biologic functions related to infection, such as inhibition of
irregular (usually helical) shape. host cell transcription.

Figure 2. Diagram of different Human DNA Viruses and Human RNA

Viruses.
A. Nucleic Acid Genome
Pathogenic Viruses

BSMT 2021 – ORDONEZ VL | Page 2 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

 size approx. 20 to 300 nm. removed; this may be by degradation of viral enzymes
 electron microscope (1930) or host enzymes or by dissociation. Uncoating is
 visualized viruses necessary to release the viral genome before the viral
 Improved magnification (more than 100,000 DNA or RNA is delivered to its intracellular site of
times) replication in the nucleus or cytoplasm.
 paved the way for viral classification based on 4. Macromolecular synthesis involves the production of
structural components nucleic acid & protein polymers. Viral transcription
leads to the synthesis of messenger RNA (mRNA),
VI. VIRAL CLASSIFICATION BASED ON THREE BASIC which encodes early and late viral proteins. Early
PROPERTIES proteins are nonstructural elements like enzymes,
1. viral morphology; and late proteins are structural components. Rapid
2. method of replication, including genome organization identification of virus in a cell culture can be
(whether the genome is RNA or DNA and single or double- accomplished by detecting early viral proteins in
stranded) infected cells using immunofluorescent staining
3. presence or absence of a lipid envelope. techniques. Replication of viral nucleic acid is
 The term means of replication refers to the strategy the necessary to provide genomes for progeny virus
virus uses to duplicate the viral genome. particles or virions. Macromolecular synthesis varies,
virions. Macromolecular synthesis varies, depending
VII. VIRAL REPLICATION on the organization of the viral genome.
5. Viral assembly is the process by which structural
 Viruses are strict intracellular parasites, reproducing or proteins, genomes, and in some cases viral enzymes
replicating only inside a host cell. The six steps of virus are assembled into virus particles. Envelopes are
replication, called the infectious cycle are: acquired during viral “budding” from a host cell
1. Attachment, also referred to as adsorption. It membrane. Nuclear ER* & cytoplasmic membranes
involves recognition of a suitable host cell and specific are common areas for budding. Acquisition of an
binding between viral capsid proteins (often the envelope is the final step in viral assembly.
glycoprotein spikes) and the carbohydrate receptor of 6. Release of intact virus particles occurs after cell lysis
the host cell. Each type of virus specifically recognizes (lytic virus) or by budding from cytoplasmic
and attaches to a specific type of host cell, allowing membranes. Release by budding may not result in
infection of some tissues but not others (viral rapid host cell death, as does release by cell lysis.
tropism) Detection of virus in cell cultures is facilitated by
2. Penetration is the process by which viruses enter the recognition of areas of cell lysis. Detection of virus
host cell. released by budding is more difficult, because the cell
a. By fusion of the viral envelope with the host cell monolayer remains intact. Influenza viruses, which
membrane. - This method not only provides a are released by budding with minimal cell
mechanism for internalizing the virus, but also destruction, can be detected in cell culture by an
leads to fusion between the infected host cell alternative technique called hemadsorption. Influenza
and additional nearby host cells, forming virus–infected cells contain virally encoded
multinucleated cells called syncytia. Detection of glycoprotein hemagglutinins inserted into the host
syncytia can be used to determine the presence cell’s cytoplasmic membrane, preparing for inclusion
of virus in cell cultures or stained smears of in the viral envelope at the time of release by
clinical specimens. cytoplasmic budding. Red blood cells (RBCs) added to
b. By phagocytosis by host cells (endocytosis) or the culture medium adsorb to the outer membranes
injection of viral nucleic acid of infected cells but not to uninfected cells. Each
infected host cell results in as many as 100,000
virions; however, as few as 1% of these may be
infectious or “viable” in the practical sense.
Noninfectious viral particles may result from errors or
mutations that occur during the infectious cycle.

VIII. VIRAL PATHOGENESIS

 illustrated by the mechanisms through which the measles
virus spreads in the body
Clinical Presentations of Virus Infection
1. acute viral infection, displaying acute viral infection,
displaying
Figure 3. Illustration of the viral infectious cycle 2. latent infection, which has no visible signs and symptoms,
3. Uncoating occurs once the virus has been but the virus is still present in the host cell in a lysogenic
internalized. It is the process by which the capsid is state (inserted into the host genome in a resting state)

BSMT 2021 – ORDONEZ VL | Page 3 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

3. chronic or persistent infection, in which low levels of virus Figure 6. Inverted microscope used to examine cell monolayers
are detectable and the degree of visible signs or symptoms growing attached to the inside surface beneath the liquid medium.
varies. The objective is under the glass test tube, facilitating observation
of the cell monolayer.

Figure 7. Class II biosafety cabinet used in a clinical virology

laboratory.
Figure 4. Clinical representation of virus infection
A. Standard Precautions and Biosafety Level 2
IX. CLINICAL VIROLOGY LABORATORY  conditions are needed for community and most
nonretroviral laboratories. Requirements include standard
 Laboratory scientists familiar with cell culture, enzyme
microbiologic practices, training in biosafety, protective
immunoassay, immunofluorescence methods, and
clothing and gloves, limited access, decontamination of all
molecular methods (e.g., PCR), in addition to other
infectious waste, and a class I or II BSC. Some viruses
common laboratory techniques.
should not be propagated in Biosafety Level 2 laboratories,
 Large equipment- laminar flow biologic safety cabinet including influenza H5N1, SARS coronavirus, hemorrhagic
(BSC), fluorescence microscope, inverted bright field fever viruses, and smallpox.
microscope, refrigerated centrifuge, incubator,
 The laboratory should always be notified if rare agents
refrigerator and freezer, roller drum for holding cell
representing danger to laboratory workers are suspected
culture tubes during incubation, and enzyme or molecular
(e.g., SARS coronavirus, H5N1 avian influenza virus,
testing instrumentation
hemorrhagic fever viruses).

B. Specimen Selection and Collection

 Specimen selection depends on the specific disease
syndrome, viral etiologies suspected, and time of year.
 Selection should include the proper specimen source and
the correct sample volume and timing of collection.
 Specimens for the detection of virus should be collected as
early as possible after the onset of symptomatic disease.
Virus no longer be present as early as 2 days after the
appearance of symptoms
Figure 5. Roller drum used to hold cell culture tubes during
 Swab specimens should not contain chemicals or other
incubation. Slow rotation continually bathes the cells in the
compounds that may be toxic to cultured cells and
medium.
therefore are unsuitable for viral specimen collection.
Calcium alginate swabs interfere with PCR, the recovery of
some enveloped viruses, and fluorescent-antibody tests
and therefore should not be used
 Selecting a specimen based on disease is confusing and
specimen selection based on the suspected viral etiology
complicated.

BSMT 2021 – ORDONEZ VL | Page 4 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

 Sterile body fluids, especially CSF and pericardial and

pleural fluids, may contain enteroviruses, HSV, VZV,
influenza viruses, or CMV. These specimens are collected
aseptically by the physician and sent to the laboratory for
processing.

VII. Blood
 Viral culture of blood is detect CMV; HSV, VZV,
enteroviruses & adenovirus occasionally may be
encountered. CMV viremia is associated with peripheral
blood leucocytes. Five to 10 mL of anti-coagulated blood
collected in a whole blood tube is needed.
Figure 8. Viruses Detected by Culture, PCR, or Assay for Antigen  Heparinized, citrated, or EDTA anticoagulated blood is
acceptable for CMV detection. Citrated blood should be
used when other viruses are being considered. EDTA
C. Specimens for Diagnosis of Viral Diseases
should be used for samples collected for nucleic acid
I. Throat, Nasopharyngeal Swab, or Aspirate testing, because other anticoagulants may interfere with
the enzyme functions required for PCR amplification.
 Nasopharyngeal aspirates are superior to throat or Serum may be used for serologic tests and nucleic acid
nasopharyngeal swabs for recovering viruses. assays.
 Swabs are considerably more convenient. Throat swabs
are acceptable for the recovery of enteroviruses,
VIII. Bone Marrow
adenoviruses, and HSV, whereas nasopharyngeal swab or
aspirate specimens are preferred for the detection of RSV  Bone marrow for virus detection should be added to a
and influenza and parainfluenza viruses. Rhinovirus sterile tube with anticoagulant. Heparin, citrate, or EDTA
detection requires a nasal specimen anticoagulants are acceptable. As previously described for
blood, EDTA should be used if the specimen is intended for
II. Bronchial and Bronchoalveolar Washes nucleic acid testing. Specimens are collected by aspiration.
Except for parvovirus B19, most viruses are detected more
 Washings and lavage fluid collected during bronchoscopy readily from sites other than bone marrow.
are excellent specimens for detecting viruses that infect
the lower respiratory tract, especially influenza viruses and
IX. Tissue
adenoviruses.
 Tissue specimens are especially useful for detecting viruses
III. Rectal Swabs and Stool Specimens that commonly infect the lungs (CMV, influenza virus,
adenovirus, sin nombre virus), brain (HSV), and
 Stool and rectal swabs of fecal specimens are used to gastrointestinal tract (CMV). Specimens are collected
detect rotavirus, enteric adenoviruses (serotypes 40 and during surgical procedures. Fresh tissue is preferred for
41), and enteroviruses. Many agents of viral nucleic acid assays, but formalin-fixed and paraffin
gastroenteritis do not grow in cell culture and require PCR embedded tissues may be used after removal of the
or electron microscopy for detection. paraffin (deparaffinization) and extraction.

IV. Urine X. Genital Specimens

 CMV; mumps, rubella, and measles viruses;  Genital specimens often are required for detection of HSV
polyomaviruses & adenoviruses can be detected and human papillomavirus (HPV). Genital swabs should be
used for ulcerations and placed in appropriate viral
V. Skin and Mucous Membrane Lesions transport media.
 Enteroviruses, HSV, VZV, and in rare cases CMV or pox
viruses can be detected in vesicular lesions of the skin and XI. Serum for Antibody Testing
mucous membranes. Once the vesicle has ulcerated or  Acute and convalescent serum specimens may be needed
crusted, detection of the virus is difficult. Collection of to detect antibody to specific viruses. Acute specimens
specimens from cutaneous vesicles for detection of HSV or should be collected as soon as possible after the
VZV may require a Tzanck smear if PCR testing is not appearance of symptoms. The convalescent specimen is
available collected a minimum of 2 to 3 weeks after the acute
specimen. In both cases, an appropriate specimen is 3 to 5
VI. Sterile Body Fluids Other Than Blood mL of serum collected by venipuncture.

BSMT 2021 – ORDONEZ VL | Page 5 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

D. Specimen Transport and Storage

 Processed immediately
 Specimens for viral isolation should not be allowed to sit at
room or higher temperature.
 Specimens should be kept cool (4°C) and immediately
transported to the laboratory.
 If a delay in transport is unavoidable, the specimen should
be refrigerated, not frozen, until processed.
 Every attempt should be made to process the specimen
within 12 to 24 hours of collection.
 For storage up to 5 days, specimens are held at 4°C.
 Storage for 6 days or longer should be at –20° or
preferably–70°C.
 Specimens for freezing should first be diluted or emulsified
in viral transport medium. Significant loss of viral
infectivity may occur during prolonged storage, regardless
of conditions, especially for the more labile enveloped
viruses.
 If a commercial kit is used for viral identification (e.g.,
nucleic acid testing), the specimens should be transported
and stored according to the manufacturer’s instructions.
Figure 9. Laboratory Processing of Viral Specimen. HDF, Human Figure 10. Serology Tests for Hepatitis Viruses

diploid fibroblast; HEp-2, human epidermoid; PMK, primary

monkey kidney. *All inocula into tissue culture tubes are 0.25 mL
volumes.

BSMT 2021 – ORDONEZ VL | Page 6 of 7

BSMT 2-01 3-1 VIROLOGY INTRODUCTION

Figure 11. Algorithm for the processing of viral specimens based on

specimen type and suspected virus.

Figure 12. Viral Syndromes and Common Viral Pathogens

BSMT 2021 – ORDONEZ VL | Page 7 of 7