Textbook of Veterinary Virology PDF

TEXTBOOK
OF
VETERINARY
VIROLOGY
Prof. S. N. Sharma
Dr. S. C. Adlakha
International Book Distributing Co.
TEXTBOOK
OF
VETERINARY VIROLOGY
Textbook of
Veterinary Virology
Prof S N Sharma
Ex Professor of Virology
Department of Veterinary Microbiology
Punjab Agricultural University
Ludhiana
Dr S C Adlakha
Ex President
National Academy of Veterinary Sciences
New Delhi
International Book Distributing Co.

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Preface
This book is intended to fulfil the need of veterinary students in
general and Post-Graduates in Microbiology in particular. besides the
veterinary disease Investigators and Practitioners of veterinary
medicine. Virology is one of those branches of science which has
experienced a tremendous growth during the last few years especially
in the area of Molecular Virology. The resultant information is spread
over a number of publications. An attempt has been made to present all
the relevant information in a concise manner including the latest
advances.
This book is divided into two parts: General Virology and
Systematic Virology. There is plethora of literature on general virology,
yet the authors have tried to present the basic principles of animal
virology in a concise manner with the hope that the reader appreciates
the nature of viruses, their pathogenicity. replication etc. In.the second
part information on infections of vertebrates has been given with
emphasis on the diagnostic and preventive aspects of virus infections of
domestic animals and poultry. The organization of chapters is
hierarchial and follows the taxonomy of animal viruses. A short family
description precedes each chapter. To present the material in a limited
number of pages, the authors have given only selected references at the
end of each chapter. There are more viruses in domestic animals and
birds than those discussed in this book; the viruses of little or no
pathogenic importance or viruses encountered as contaminants in
animal cell culture have been omitted. Greater importance has been
given to viruses of economic importance in India and other developing
countries of Asia and Africa.
The authors will feel rewarded if this book will meet the
requirements of the veterinary profession in the developing countries.
The suggestions for improvement of this book in a future edition are
welcome.
Authors
Contents
Abbreviations
xi
PART I
General Virology
1. Structure and Composition
Classification of Viruses
Viral Replication
Cultivation of Viruses
Viral Genetics
Viral Pathogenesis
7. Persistent Infections
8. Viral Immunity
9. Epidemiology of Viral Diseases
10.. Viral Tumorogenesis
11. Viral Vaccines and Antiviral Agents
12. Diagnosis of Viral Diseases
2.
3.
4.
5.
6.
3
13
34
46
55
65
76
81
87
94
102
115
PARTll
Systematic Virology
D.N.A. Viruses
13. Poxviridae
Vaccinia Virus; Cow Pox Virus; Buffalo Pox
Virus; Camel Pox Virus; Sheep Pox Virus; Goat
Pox Virus; Lumpy Skin Disease; Ecthyma (Ort)
Virus; Bovine Papular Stomatitis Virus; Milkers
Node Virus; Swine Pox Virus; Myxoma Virus;
Fibroma Virus; Fowl Pox Virus,
14. Parvoviridae
Bovine Parvovirus; Porcine Parvovirus; Feline
Parvovirus; Canine Parvoviruses.
129
150
\/iii
TeXlbook of Veterinary Virology

15. Papovaviridae
Bovine Papillomavirus type 1 and 2; Bovine
Papilloma virus type-3; Bovine Papillomavirus
type-4; Bovine Papillomavirus type-5; Bovine
Papillomavirus type-6; Canine Papillomavirus;
Rabbit Papillomavirus; Equine Papillomavirus.
16. Adenoviridae
Bovine Adenoviruses; Ovine Adenovirses;
Canine Adenoviruses; Infectious Canine
Laryngotracheitis Virus; Equine Adenoviruses;
Porcine Adeno-viruses; Avian Adenoviruses.
17. Hcrpesviridae
Bovine Herpes Virus-I; Bovine Herpes Virus-2;
Malignant Catarrhal Fever Virus; Bovine Herpes
Virus-3; Hcrpes Virus of Sheep; Herpes Virus of
Goats; Equine Herpes Viruses; Pseudorabies
Virus; Simian Herpes Virus-I; Canine Herpes
Virus; Fowl Hcrpes Virus-I; Duck Herpes
Virus-I; Pigcon Hcrpes Virus; Marek's Disease
Virus.
18. Unclassified DNA Virus
African Swinc Fever Virus.
159
164
176
206
RNA Viruses
19. Picomaviridae
Apthovirus; Enterovirus; Swine Enteroviruses;
Porcine Enterovirus-I; Porcine Enterovirus-9;
Bovjne Enteroviruses; Avian Encephalomyelitis
Virus; Duck Hepatitis Virus; Bovine Rhinovirus-I; Equine Rhinovirus 1 & 2.
20. Calciviridae
Vesicular Exanthema Virus; Feline Calcivirus.
21. Togaviridae
Alphavirus; Equine Encephalomyelitis Virus;
Pestivirus; Bovine Viral Diarrhoea Virus; Border
Disease Virus; Swine Fever Virus; Arterivirus;
Equine Viral Arteritis.
22. Flaviviridae
Japanese B Encephalitis Virus; Wesselsbom
Virus; Louping III Virus.
211
231
234
247
COnlellls
23. Reoviridae
Reovirus; Bovine Reovirus (type 1 & 3); Avian
Reovirus type 1 to 5; Rotavirus; Bovine Rotavirus; Orbivirus; Blue Tongue Virus; AfricIDl
Horse Sickness Virus.
24. Bimaviridae
Infectious Bursal Disease Virus.
25. Coronaviridae
Bovine Corona Virus; Canine Corona Virus;
Feline Infectious Peritonitis Virus; Porcine
Corona Viruses; Avian Infcctious Bronchitis
Virus.
26. Orthomyxoviridae
Equine Influenza Virus 1 and 2; Swine Influenza
Virus; Avian Influenza Virus; Fowl Plague
Virus.
27. Paramyxoviridae
Paramyxoviruses; Avian Parnmyxoviruses; New
Castle Disease Virus; Mammalian Parninfluenza
Viruses; Parainfluenza-l; Parainfluenza-3 Virus
in cattle; Parainfluenza-3 Virus in sheep;
Parninfluenza-5;
Morbillivirus;
Canine
Distemper Virus; Rinderpest Virus; Peste-DesPetits Virus; Pneumovirus; Bovine Respiratory
Syncytial Vims.
28. Rhabdoviridae
Vesicular Stomatitis Virus; Rabies Virus; Bovine
Ephemeral Fever Virus; Marburg Vims.
29. Rettoviridae
Oncoviruses; Bovine Leukaemia Virus; Feline
Leukaemia Virus; Murine Leukaemia Viruses;
Avian Leukosis Viruses; Avian Reticuloendotheliosis Viruses; Murine Mammary Tumour
Virus; Bovine Syncytial Virus; Lentiviruses;
Equine Infectious Anaemia Virus; Visna/Maedi
Virus; Caprine Arthritis-Encephalitis Virus:
Jaagsiekte (Ovine Pulmonary Adellomatosis
Virus).
251
264
267
278
284
309
322
Textbook o/Velerinary Virology

30. Bunyaviridae
Rift Valley Fever Virus; Akabane Virus; Nairobi
Sheep Disease.
31. Toroviridae
Breda Virus; Berne Virus.
32. Unclassified RNA Virus
Borna Disease Virus.
33. Unclassified Agents
Scrapie.
Index
347
356
360
362
364
Abbreviations
Ads
AEV
AGID
AHS
AIBV
ALV
ASFV
BAV
BDV
BEV
BHV
BLV
BPV
BPoV
BRV
BTV
BVD
CAEV
CAM
CCV
CDV
CE
CF
CHV
CIE
CK
CM!
CPE
CPV
ere
adenoviruses
avian encephalomyelitis virus
agar gel immunodiffusion
African horse sickness
avian infectious bronchitis virus
avian leukosis virus
African swine fever virus
bovine adenovirus
border disease virus
bovine ephemeral fever
bovine herpesvirus
bovine leukosis virus
bovine papilloma virus
bovine parvovirus
bovine rhinovirus
bluetongue virus
bovine viral diarrhoea
caprine arthritis-encephalitis virus
chorio-allantoic membrane
canine corona virus
canine distemper virus
contagious ecthyma
complement fixation
caprine herpesvirus
counter immuno electrophoresis
chicken kidney
cell mediated immunity
cytopathic effect
canine parvovirus
cytotoxic T cells
xii
CIL
EAV
EBV
REV
EHV
EIAV
ELISA
EM
F
FAV
FCV
FIPV
FMD
FPLV
H
HA
HI
IBD
IBH
IBR
ICH
IF
IFN
ILT
IP
IPV
mv
JSRV
Kb
LD50
LCM
MCF
MDV
MHV
MVV
N
NI
l'i1>V
OAV
PEV
Textbook of Veterinary Virology
cytotoxic T lymphocytes
equine arteritis virus
Epstein-Barr virus
equine encephalomyelitis virus
equine herpes virus
equine infectious anaemia virus
enzyme-linked immunosorbent assay
electron microscope/microscopy
fusion protein
fowl adenovirus
feline calcivirus
feline infcctious peritonitis virus
foot-and-mouth disease
feline panleucopenia virus
haemagglutinin
haemaggl utination
haemagglutination inhibition
infectious bursal disease
inclusion body hapatitis
infectious bovine rhinotracheitis
infectious canine hepatitis
immunofluorescence
interferon
infectious laryngotracheitis
immunoperoxidase
infectious pustular vulvovaginitis
Japanese encephalitis virus
jaagsiekte retrovirus
kilobases
50 percent lethal dose
lymphocytic choriomeningitis
malignant catarrhal fever
Marek's disease virus
major histocompatibility complex
maedi-visna virus
neuraminidase
neutralization index
Newcastle disease
ovine adenovirus
porcine enterovirus
Abbreviations
PI
PPR
PPV
REV
RIA
RSV
RVF
SN
SPY
SV
SVE
TGE
UV
VE
VN
VSV
parainfluenza
peste-des-petits-ruminants
porcine parvovirus
reticuloendotheliosis virus
radioimmunoassay
respiratory syncytial virus
Rift valley fever
serum neutralization
sheep pox virus
simian virus
swine vesicular exanthema
transmissible gastroenteritis
ultraviolet
vesicular exanthema
virus neutralization
vesicular stomatitis virus
xiii
PART!
GENERAL VIROLOGY
Chapter 1
Structure and
COlD position
The viral diseases of man and animals have been known for many
centuries. The science of virology emerged during the last decade of
last century. Ivanovski in 1892 reported that tobacco mosaic virus agent
could pass through filters which retained bacteria. In 1898 Beijerinck
showed that the tobacco mosaic disease agent differed fundamentally
from toxin and it diffused through agar and he used the term
'contagium vivum fluidum' - that it was liquid or soluble. He also
reported that only those plants which were growing and whose cells
were dividing could be infected. The disease causing agent must be
incorporated into the living protoplasm in order to propagate and it
cannot multiply outside cells. Loeftler and Frosch in 1898
independently reported that foot and mouth disease of cattle could also
be produced by a material passed through the filter which retained
bacteria. Twort (1915) and d' Herelle (1917) recognised that bacteria
also could be infected by filter passing agents.
Virology is now recognised as a basic biological science and
veterinary virology has grown immensely during the past few decades.
The subject of virology is divided into four main divisions i) Animal viruses - the viruses of man and animals.
ii) Insect viruses - the viruses of insects and worms.
iii) Bacterial viruses (Bacteriophages).
iv) Plant viruses - viruses of plants.
The real nature of viruses has been elucidated since 1930. Stanley
(1935) crystallized tobacco mosaic virus. Hershey and Chase (1952)
discovered that only DNA of bacteriophage entered its bacterial host
and only DNA was necessary for infection. Fraenkel-Conrat (1956)

proved that RNA of tobacco mosaic virus carried all the information for
growth. Since then an enormous upsurge in our knowledge regarding
the nature of viruses and its molecular biology has taken place.
Viruses have a very simple structure. The mature virus particle
(Virion) consists of a central core of nucleic acid surrounded by protein
coat. They vary in size ranging from 300 x 200nm of pox viruses to
20-25 nm of picornaviruses. Viruses can be distinguished from other
unicellular microorganisms (Table 1.1). Lwoff and Toumier (1966)
described the viruses having following five characters i) Possession of only onc type of nucleic acid, either DNA or
RNA.
ii) Reproduction solely from nucleic acid, whereas other agents
grow from the sum of their constituents and reproduce by
division.
iii) Do not undergo binary fission.
iv) Lack of genetic information for the synthesis of essential
cellular systems.
v) Use of ribosomes of their host cells.
Table 1.1
IMPORTANf PROPERTIES OF UNICELLULAR ORGANISMS AND VIRUSES
Property
1. Nucleic
acid (NA)
Bacteria Mycoplasma Chlamydia

DNA
and
RNA
DNA and
RNA
DNA and
RNA
2. Nucleic
acid(NA)
infectious
3. Ribol!omes +
4. Action of No
interferon
5. Metabolic
activity
6. Binary
fission
Rickettsia Viruses
DNA and Either DNA
or RNA, not
RNA
both
Some DNA
and RNA
viruses have
infectious
NA
action
No
action
Inhibifs
growth
No
action
Inhibits
replication
Structure and Composition
The criteria given above clearly distinguish viruses from other

microorganisms; the most important criterion is that viruses contain
only one type of nucleic acid. DNA or RNA and are completely
dependent on the host cell for their reproduction. Some viruses may
persist in their host cells by integration of their genome (DNA) or DNA
CQPy of their RNA into the genome of host cell. The viruses are not
'<lsceptible to antibiotics that act against specific steps in the metabolic
pathways of bacteria.
Physical structure
Morphology: The size of virus particles range from about the size
of smallest bacteria (300 nm) to about the size of largest protein
molecules (20 nm). The unit of length is nanometre (nm) which is equal
to 10-6 millimetres. For recording the size of very small structures
Angstrom unit (A 0 or AV) is used. One nanometre is equal to 10
Angstrom units. The viruses occur in many shapes and sizes. The
viruses were also known as 'ultrafiIterable viruses' or
'ultramicroscopic' since the viruses could pass the filters which
retained bacteria and could not be seen under the light microscope. The
viruses were measured by their capacity to pass through earthenware
filters. The use of earthenware fillers was replaced by collodion or
cellulose acetate membrane filters of gruded pore sizes. The membrane
filters are non toxic to cells in culture and do not alter the pH of the
medium and they do not adsorb large quantities of virus particles
during filtration.
Another procedure for determining the size of viruses is high speed
centrifugation. The rate of sedimentation 0f virus particles depend upon
its size and the density and' viscosity of the suspending fluid. The
relationship of sedimentation and size of virus particles IS governed by
Stoke's law. During later half of 1930's and 1940's electron
microscope made it possible to study the morphology and size of virus
particles. In 1959 negative staining to electron microscopy of viruses
transformed the knowledge of viral ultrastructure.
The simplest viruses consist of a single molecule of nucleic acid
(DNA or RNA) enclosed within or built into protein coat. the capsid.
The capsid and its enclosed nucleic acid constitute the nucleocapsirl
(Fig. 1.1). The capsid is composed of morphological uniL~ called
capsomeres. which are held together by nonconvalent bonds. The
Textbook ojVeterinary Virology
capsomeres consist of one or more molecules of polypeptidcs and are

seen in the electron microscope. In some of the viruses there is an
envelope of lipoprotein surrounding the nucleocapsid. The envelope is
acquired as the virus passes through or buds from host cellular
membrane~ and contains components of the host cell.
Fig. 1.1 Schematic Diagram of the Structure of a Virus
The assembly of capsomeres in a virion is defined by the nature of

bonds formed between individual capsomeres, which imparts symmetry
to the capsid. Two types of viral symmetry have been recognised, the
cubical symmetry and helical symmetry; and these constitute an
icosahedral capsid and a helical or tubular capsid respectively (Fig.
1.2). Some of the viruses have a combination of symmetries aand
various structures. These are called complex viruses.
Fig. 1.2 Schematic Representation of the Structure of a Virus

with Cubical (a) and Helical (b) Symmetry.
1. lcosahedral Symmetry: An icosahedron means a figure of 12

verticles (corners) and 20 faces each of an equilateral triangle. This
indicates that the capsid be build of 60 (or multiples of 60) equivalent
parts. Electron microscopic studies of icosahedral viruses show that
these viruses have a regular surface array of morphologic units and the
number is often greater than 60 but not generally a multiple of 60. The
icosahedron has axes of 2. 3 and 5 fold rotational symmetry passing
through its edges. faces and vertices. respectively (Fig. 1.3). The
icosahedron is a strong structure which encloses a maximum volume.
The total number of capsomeres can be calculated by the formula 10
(n-l)2+2, in which n is the number of morphologic' subunits between
and including those on any five fold axes. The number of capsomeres
(M) can also be calculated from the formula M = 10 T +2, in which T is
the triangulation number. the number of small triangles formed on the
single face of an icosahedron when each adjacent capsomere is
connected by a line. In adenovirus Tt =6 or T =25. giving a total of 252
capsomeres. In adenovirus particles, capsomeres on the faces and edges
bond to six neighbouring capsomeres and are called hexamers; those at
the vertices bond to five neighbouring capsomeres are called
pentomers. There are 240 hexamers and 12 pen tamers. Each penton has
1 fibre of filament in mammalian adenoviruses while there are 2
filaments in avian adenoviruses.
Capsid
~.J.....-;..--r-
"ucl .
..
..le
acid
Nuel~oeapsid
Co r~prot~in
Fig. 1.3
The lrosahedron. It h~ a Rotational Axes of

Twofold, Threefold and F'ivefold Symmetry.
2. Helical or tubular symmetry: The nIJcleocapsids of ~ever?l

RNA viruses have a helical symmetry, the capsomeres ~nd nuc1~lc aCid
molecules self-assemble. as helix. The morphologiC subumts are
arranged in their hmg axes In the nucleocapsid in a screwlike man.ner
with several units per turn ()f the helix. The flcxuous hel.lcal
nucleocapsid coil is always inside a glycolipoprotein envelope. poSSibly
to give the very tong nuc!eocapsids st;lbility.
Textbook o/Veterinary Virology
Viral envelope: The envelope is derived at host cell membranes-plasma membrane, nuclear membrane, endoplasmic reticulum and
Golgi complex during maturation by budding. The lipids of viral
envelope are acquired from the cell while the proteins are virus coded.
One kind of protein is glycoprotein peplomer (peplos means envelope)
or spike whi~e the other kind of protein is nonglycosylated and is found
on the inside of envelope of virions of several families and is knOW1l as
matrix protein. The matrix protein gives rigidity to the viron
morphology, e.g. the envelope of rhabdoviruses is closely attached to
the bullet shaped matrix protein that encloses nucleocapsid. In certain
other viruses like arena viruses, bunyaviruses and corona viruses there is
no matrix protein and are therefore more pleomorphic. The envelope is
also possessed by certain icosahedral viruses like African swine fever
virus, herpes viruses, toga viruses, flaviviruses and retroviruses. The
envelope is immunogenic and is required for infectivity but in some
poxviruses which have an envelope the infectivity is not dependent on
the envelope.
3. Complex symmetry: Some viruses like poxviruses have a
dumbell shaped core surrounded by complex membranes and other
icosahedral or helical symmetry. The retroviruses have a tubular
nucleocapsid surrounded by an icosahedral capsid enclosed within an
envelope.
Chemical structure
The essential components of infectious virus particles are nucleic
acid and protein. The enveloped viruses contain lipids and
carbohydrates in their glycoprotein peplomeres. The complex viruses
like pox viruses also contain lipids. The chemical composition of
viruses can only be determined when the viruses are obtained in a pure
form as the viruses contain host cell constituents. Most of the virus
particles are attached to cell debris having almost similar chemical
properties. Therefore, until 1950, without the introduction of more
refined methods of purification, the animal viruses were not purified
sufficiently for their chemical analysis. The other reasons of delay in
purification of animal viruses was the small quantity of virus material
available. With the introduction of tissue culture, it is now possible to
obtain sutlicient vIrUS for chemical analysis.
In the purificaiton of viruses differential ultracentrifugation leads
to their considerable purification. The important technique introduced

in 1950's is density gradient centrifugation where sucrose gradients
ensure finer separation of particles with different sedimentation
properties. Anotll.!r method which has proved of great value in
purification of viruses is equilibrium sedimentation in caesium chloride
and potassium tartrate which separate the particles according to their
buoyant density. Density gradients of these salts are prepared and the
mixture of virus and host cell debris is centrifuged in a high speed
centrifuge. The different particles take positions in the gradient
according to their buoyant density.
The viruses can also be separated from contaminating material by
using fluorocarbons or other organic solvents; mild detergents to
remove host cell material selectively - especially for removing lipid
material and denatured host protein. The enveloped viruses cannot be
purified by detergents or lipid solvents because they are disrupted due
to the action of these agents. The non enveloped viruses or naked
viruses are stable in lipid solvents or even in strong detergents like
sodium dodecyl sulphate.
1. Nucleic acid: Any particular virus contains either DNA or RNA
which may be either single stranded or double stranded and the genome
consists of either one molecule or several molecules. In most of DNA
viruses the genome consists of a single molecule while several RNA
viruses contain the genome of several molecules. The genome may be
of linear or circular configuration. The nucleic acid of certain DNA or
RNA viruses is infectious i.e. it can start multiplication cycle if
introduced into susceptible cell. In such cases messanger RNA
(mRNA) is transcribed from viral DNA in the nucleus by a cellular
transcriptase, while in the case of RNA viruses the viral RNA itself acts
as mRNA. In other virus families the extracted nucleic acid is not
infectious. Among DNA viruses transcription requires viral rather than
cellular transcriptase. Among RNA viruses when the viral RNA is of
minus (-) sense or is double stranded its transcription to produce
positive (+) sense in RNA requires a virion associated transcripitase
which is separated from nucleic acid by extraction procedures. In the
positive (-t) sense RNA viruses the viral RNA itself acts as its own
mRNA. The positive (+) sense RNA of retroviruses is not infectious
because replication of RNA occurs only after production of DNA
provirus by a virion associated reverse transcriptase.
10
The genome of all DNA viruses consists of a single molecule,

which is double stranded except in parvovilUses. The genome may be
linear or circular. The papovavirus DNA is a supercoilcd circle known
as superhelix, when an enzyme nicks one of the strand the DNA
molecule becomes a relaxed circle. The hepadnavirus genome is
partially double stranded as one of the strand of circular DNA is shorter
than the other. In case of linear DNA viruses during replication a
temporary circular configuration is adopted. The molecular weight of
DNA of different viruses varies from 1 to over 200 x 1()6.
The genome of RNA viruses may also be single or double stranded
and in some viruses it is as single molecule while in others it is
segmented. In arena virus and bimavirus, RNA consists of 2 segments,
bunyavirus RNA is of 3 segements, orthomyxovirus RNA consists of 7
or 8 segments, and reovirus RNA is of 10, 11 or 12 segments. All viral
RNA's are linear. The RNA of some of the viruses is said to have a
positive (+) sense (also known as polarity), that is the RNA has same
sense as mRNA. Picomaviruses, calciviruses, togaviruses, flaviviruses,
coronaviruses and retroviruses have positive (+) sense genome. If the
nucleotide sequence of the genome is complementary to mRNA it is
said to have a negative (-) sense. The (-) sense genome is with
paramyxoviruses, rhabdoviruses, arena viruses and bunyaviruses. All
these viruses have an RNA dependent RNA polymerase (transcriptase)
in the virion. In arena viruses and in one genus of bunyaviruses one of
the RNA segment is ambisense, i.e. part (+) sense and part (-) sense.
The molecular weight varies from 2 to 15 x 106 which is much less than
seen in most DNA viruses.
Some viruses contain host cell nucleic acid, e.g. some papoviruses
contain host cell DNA and arenaviruses contain cellular ribosomes.
Sometimes several copies of viral genome may be enclosed in a single
virus particle or the virus particles contain no nucleic acid, known as
empty particles or contain incomplete genome known as defective
interfering particles.
2. Proteins: The proteins make up 50-70% constituent of the
virion. The virus coded proteins are structural, i.e. they form part of the
virion, and non structural, i.e. proteins required during the replication
cycle of virion. The structural proteins provide a protective coat to the
viral genome. The vertebrate viruses contain sC'/eral proteins ranging
from 4 distinct species of proteins in foot arid mouth disease VIruses to
11
over 100 in ease of poxviruses. There is normally one copy of viral

nucleic acid in a virus particle but there arc many copies of each viral
protein. Apart from providing protective shell to the viral genome the
proteins have other properties as well.
Table 2.1
NATURE OF GENETIC MATERIAL OF VIRUSES
Family
Structure ofNucleic acid
Poxviridae
Parvoviridae
Linear ds DNA
Linear as DNA (-) sense, a hairpin structure at onc
end
Circular supcrhelical ds DNA
Linear ds DNA
Linear ds DNA
Linear ds DNA
Circular ds DNA with ss region
Linear ss RNA (+) sense
Linear ss RNA (-) sense
Linear ss RNA (-) sense
Linear ss RNA (-) sense. Genome segmental, 7 or 8
molecules
Linear ss RNA (+) sense, diploid genome
ss RNA (-) sense, segmented genome, 3 molecules
Linear ds RNA, segmented genome 10, 11, or 12
molecules.
Linear ds RNA, segmented genome, 2 molecules
Papovaviridae
Adenoviridae
Herpcsviridae
Iridoviridae
Hepadnaviridae
Picomaviridae
Ca1civiridae
Togaviridae
Flaviviridae
Coronaviridae
Rhabdoviridae
Paramyxoviridae
Orthomyxoviridae
Relroviridae
Bunyaviridae
Reoviridae
B irnav iridae
The surface proteins have an affinity for the specific receptors on

the surface of susceptible cells and contain anti genic determinants
which produce protec~ive antihody in the infected animal. Some of the
virus proteins have an enzymic activity, e.g. a protein in (-) stranded
RNA viruses acts as transcriptase. Glycoproteins make up the
peplomers projecting from thee~velope. There is a second type of
envelope protein which is nonglycosylated matrix protein that occurs as
a layer at the inner surface of lipId envelope in orthomyxoviruses,
paramyxoviruses and rhabdoviruses.
12
3. Lipid and carbohydrate: These constituents are found only in

the envelope except complex viruses like poxviruses. Lipids and
carbohydrates are derived from the host cells. Carbohydrate is the
major part of glycoproteins of peplomers. Glycoproteins act as
important antigenic determinants to which host immunity is directed.
References
FBNNER, FRANK, 1987. Veterinary Virology. Academic Press, New York.
BROWN, 1984. The nature of viruses. In Topley and Wilsons Principles of
Bacteriology, Virology arul Immunity, Vol. 4. Williams and Wilkins,
Baltomore.
FRED,
LAUFfER, M.A.; BANG, F. B.; MARAMOROSCH, K., AND SMITH, K.M., 1982.
Advances in virus research. Academic Press, New York.
Chapter 2
The object of virus classification is to make a systematic ordered
arrangement of viruses that have similarities and differences. Earlier
efforts to classify viruses arranged them according to host symptoms or
type of diseases and tissue affinities. This system had deficiencies e.g.
the same virus produces different disease syndrome in different hosts,
different strains of same virus can produce different syndromes in the
same host and different viruses can produce the same clinical picture.
A classification based on epidemiological data was also tried.
Enteric viruses: These viruses are acquired by ingestion and
replicate primarily in the digestive tract. The important enteric viruses
include rotaviruses, coronaviruses, enteroviruses and adenoviruses.
Respiratory viruses: These viruses enter the host by inhalation
and replicate in the respiratory tract. These viru~s include
orthomyxoviruses. rhinoviruses, paramyxoviruses, adenoviruses and
coronaviruses.
Arboviruses: Arthropod bovine viruses infect arthropods and
ingest vertebrate blood. These viruses replicate in arthropod host and
are transmitted to a vertebrate host by bite. These viruses replicate also
in verteblate host These include orbiviruses. bunyaviruses,
flaviviruses, togaviruses. rhabdoviruses and African swine fever virus.
The viruses classified on epidemiological data comprise viruses
belonging to different families with different physical and chemical
properties. Therefore, the most important criteria for classification are
the physical and chemical characteristics of the virion and its mode of
replication. The criteria for classification into different families are-
14
1. the kind of nucleic acid e.g. single or double stranded DNA or RNA
and mode of replication; 2. morphology of the virion including its size,
shape, nucleocapsid symmetry and presence or absence of
nucleocapsid, number of capsomeres and pH sensitivity.
The criterion of subdivision of families is controversial. Most
virologists agree that viruses should differ substantially in nucleic acid
sequence to be designated as different species but there is yet no
agreement on how such differences should be quantitatcd. Monoclonal
antibodies are of great value in the differentiation of viruses at species
level and below. There are other techniques being used for studying the
composition of viral nucelic acid to identify species and to understand
minor differences in viral s..rains. The techniques being employed are
molecular
hybridization,
oligonucleotide
finger
printing,
clectrophoresi~ in gels and nucleotide sequence analysis.
The International Committee on Taxonomy of viruses (ICTV) has
recommended that the highest taxonomic group is the family, and is
named with a suffix-viridae. Subfamilies have a suffix-virinae and
genera with a suffix-virus. Latinized names for families, subfamilies
and generic names are written in italics and vernacular names derived
from them are written in roman letters. It is still customary to use
vernacular terms rather than latinized binomials for viral species e.g.
Newcastle disease virus.
There are still viruses affet::ting man and animals which are still
unclassified but majority of these viruses known to man have been
assigned to one or another of the 20 families (Table 2.1). A brief
description of each family or vertebrate viruses is given below:
DNA viruses
Poxviridae: Pock means a pustule or ulcer. These are complex

large, brick shaped or ovoid virus particles measuring 300-450 x 170260 nm in diameter. The virions have an envelope containing lipid and
tubular and globular protein structures. All have an inner core which
contains a single linear molecule of double stranded (ds) DNA; 130280 Kbp. There are more than 30 structural proteins and several
enzymes aSSOCIated with the virus particle apart from 4 percent lipid
and 3 percent carbohydrate. The pox viruses replicate in the cytoplasm,
mRNA is transcnbed by a virion-associated transcriptase. Mature
particles are released from microvilli or by cellular disruption.
Classification o/Viruses
15
The family is divided into two subfamilies1. Chordopoxvirinae. comprises of vertebrate viruses and is
divided into 6 genera, which include animal pathogens.
2. Entompoxvirinae. comprises of insect viruses.
Parvoviridae: Parvoviruses are small about 20 nm in diameter,
have icosahcdral symmetry with 32 capsomeres. The genome is a
single stranded (ss) DNA with molecular weight 1.5-2.2 x Ht. The
virions are heat stable. The family comprises of 3 genera and members
of2 genera affect the vertebrate hosts. Genus parvovirus include animal
pathogens, while genus dependovirus includes defective viruses which
depend on adenovirus for replication. They occur in birds, animals and
human beings but arc not pathogenic. Replication takes place in
nucleus.
Papol'Qviridae: Pa stands for papilloma; po for polyoma and va
for vacuolating agent. These are noneveloped icosahedral with a
diameter of 45-55 nm. The genome is a single cyclic molecule of
double stranded DNA with molecular weight 3-5 x 106 The
replication takes place in the nucleus. There are two genera
papillomavirus and polyomavirus. Most species arc oncogenic.
At/ellm'iridae: Adeno means glands. The virions arc noneveloped
with icosahedral symmetry, 70-90 nm in diameter with 252
capsomeres. Vertex capsomeres arc distinct from others and carry 1-2
filamentous projections. The genome is a single linear molecule of
double stranded DNA with molecular weight 20-30 x 106
Adenoviruses replicate in the nucleus. The viruses arc usually
associated with respiratory and intestinal infections and sometimes with
eye infection. Many viruses arc conditionally oncogenic. There are two
genera in this family 1. Mastadenovirus, these arc mammalian
pathogens and 2. A viadenovirus, pathogenic for birds. A common
antigen is shared by all mammalian strains which differ from
corresponding antigen of avian strains.
Herpesviridae: Herpes means creeping. The herpes viruses are
enveloped 120-150 nm in diameter with icosahedral symmetry and 162
capfoomeres. The virion consists of a core in which genome is wrapped,
the icosah~dral nucleocapsid, a tegument surrounding the capsid and an
envelope. The genome is (ds) DNA with a moleuclar weight 80-150 x
106. The multiplication takes place in the nucleus and virion mature by
the addition of glycoprotein lipid membrane as the virus passes through
16
the inner lamellae of nuclear membrane into endoplasmic

reticulum. The family has been divided into 3 subfamilies 1.
Alphaherpesvirinae- which include infectious bovine rhinotracheitis
virus, bovine mammallitis virus, B virus, pseudorabies virus,
equine rhi~opneumonitis and equine coital exanthema virus, viruses of
dogs,
cats
and
chickens.
2.
Betaherpesvirinae-includes
cytorr.egaloviruses of man and animals. 3. Gammaherpesvirinaeincludes viruses associated with tumors like Marek's disease virus of
chickens.
The herpes viruses produce lifelong persistant infections usually in
the latent form. The excretion of virus from the host may be continuous
or intermittent without disease or episodes of recurrent clinical disease.
Iridoviridae: Irido means shining, iridescent. The viruses in this
family are icosahedral enveloped viruses measuring from 130-300 nm
in diameter. The genome is a single linear (ds) DNA with a molecular
weight of 130-160 x 1()6. The multiplication occurs in the cytoplasm
using virion associated transcriptase but nuclear involvement is needed
for viral DNA synthesis.
Hepadnaviridae: Hepa means liver and dna. It includes human
hepatitis B virus. The viruses are spherical particles with 42 nm in
diameter, consist of an icosahedral core which is 27 nm. The genome is
circular partially double stranded DNA molecule, which consists of a
long and short strand. The multiplication takes place in nucleus of
heptocytes.
Families of RNA viruses
Picornaviridae: The name picorna is originally derived from

poliovirus, insensitivity to ether, coxsackievirus, orphan virus,
rhinovirus and ribonucleic acid omitting one ('r'), but also consistent
with pico which means small and rna is ribonucleic acid. The viruses
are small noneveloped, icosahedral, 25-30 nm in diameter.The genome
is 1 piece linear single stranded (+) sense RNA with a molecular weight
about 2.5 x 106 The viruses replicate in the cytoplasm and functional
proteins are mainly produced by post-translational cleavage. The family
comprises of 4 genera 1. Enterovirus, these are stable at pH 3, include
poliovirus and large number of species that affect domestic animals; 2.
Rhinovirus, virus is unstable at pH 3; 3. Cardiovirus, comprises the
viruses of encephalomyelocarditis of swine and rodents; 4. Aphthovirus
comprises of foot and and mouth disease virus.
Calciviridae: Calix means cup. The calciviruses are noneveloped,
17
IcosahedIal with 32 capsomeres and measure about 35-40 nm if.

diameter. The genome is ss RNA in 1 piece. The family comprises of
one genus- Cl)lcivirus which includes vesicular exanthema of swine.
Togaviridae: Toga means cloak. These are small spherical
enveloped viruses, measuring about 60-70 nm in diameter. The single
molecular genome is single stranded RNA with a molecular weight 4 x
1()6 a!ld is (+) sense. The viruses multiply in the cytoplasm and mawre
by budding from cell membranes. The arthropod borne viruses multiply
in arthropods as well as in vertebrates. The family comprises of 4
genera-Alphavirus, Rubivirus, Pestivirus and Arterivirus.
FlavivirUtae: F1avi means yellow. The viruses included in this
family were formerly classified in togaviridae but now placed in new
family-Flaviviridae, mainly because of different strategy of
iephcation but in other respects the flaviviruses resemble the alpha virus
genus. These viruses are smaller measuring about 40-50 nm in
diameter. The family comprises of only one genus-Flavivirus.
Reoviridae: The family name is a single, respiratory enteric
orphan virus. The viruses are nonenveloped, icosahedral wilh a
diameter of 60-80 nm with two protein coats. The genome is ds RNA
in 10 to 12 pieces with a molecular weight of 10-16 x 1()6. The viruses
multiply in the cytoplasm. There are 3 genera in the family causing
infections of veterinary importance.
BirnavirUtae: Sigla bi-two and rna. The family comprises of
ic.osahedral symmetry and measure about 60 nm in diameter. The
genome is ds RNA which is linear and is in two segments. There is
only one genus in the family-Birnavirus, which includes the important
poultry virus of chickens, infectious bursal disease virus.
4renaviridae: Areana means sand. Arenaviruses acqui:'ed their
within
names because of presence of ribosomes incorporated
pleomorphic enveloped virions measuring about 50-300 nm in
diameter. The genome consists of two pieces of (-) sense ss RNA.
Coronaviridae: Corona means crown. The virions are enveloped,
pleomorphic with 75-160 nm in diameter. There are widely spaced,
pear shaped peplomeres in the lipoprotein envelope. The envelope
lacks a matrix protein and encloses a core of undetermined symmetry.
The genome is a single molecule of (+) sense ss RNA with a molecular
weight 5.5-6.1 x 1()6. The family comprises of only one genus which
includes important poUltry and other viruses of domesticated animals.
18
Orthomyxoviridae:
Myxo means mucus. The viruses are
enveloped, pleomorphic particles with 80--120 nm in diameter. The
nucleocapsid has a helical symmetry. The envelope has surface
projections of two types, a haemagglutinin and a neuraminidase. The
genome consists of eight segments of (-) sense ss RNA with a
molecular weight of 5 x 106 and is associated with viral transcriptase.
The family consists of one genus-influenza virus. Two important
species of viruses influenza A and influenza B are included in this
family.
Paramyxoviridae: The viruses are pleomorphic, enveloped,
usually spherical measuring about 150 nm in diameter with a helical
nucleocapsid symmetry. The genome consists of a single molecule of
(-) sense ss RNA with a molecular weight of 5-7 x 106. The virion
contains a transcriptase. The envelope contains two glycoproteins,
haemalglutinin and in some species with neuraminidase activity, and
fusion protein. The family is subdivided into 3 gencra. Paramyxovirus,
Morbillivirus and Pneumovirus. The family includ~s the viruses of
important pathogens of veterinary importancc.
Rhabdoviridae: Rhabdo means rod. The virus particles are
enveloped, bullet shaped or bacilliform measuring about 180 x 75 nm.
The capsid has a helical symmetry which is closely attached to
lipoprotein envelope, with surface projections. The genome is a single
molecule of H sense ss RNA with a molecular weight of 3.5-4.5 x 106
with transcriptase. The virus family comprises of two generaVesiculovirus and Lyssavirus besides ungrouped rhabdoviruses causing
disease in animals like bovine ephemeral fever virus.
Retrm'iridae:
Re stands for reverse and tr
stands for
tmnscriptase. This is a large family of enveloped viruses with
icosahedral core containing a helical nucleoprotein. There is reverse
transcriptase within the virion. The genome is diploid consisting of
inverted dimer of (+) sense ss RNA with a molecular weight of 3 x 106
(for one monomer). The ds DNA of copy of genome of the virus is
transcribed by the viral reverse transcriptase and is integrated into the
cellular DNA as an essential part of replicate cycle. Proviral DNA is
found in the DNA of all normal cells of many species of animals and
may produce virus under certain circumstances. These are known as
endogenous retroviruses. The exogenous retroviruses are transmitted
19
horizontally. The family is subdivided into 3 subfamilies, two of which

contain pathogens of veterinary importance.
Bunyaviridae: Buyamwera is a locality in Africa. These are oval
or spherical particles measuring about 90-120 om in diameter with a
nucleocapsid of helical symmetry. The genome is of 3 segments of
circular (-) sense ss RNA with a molecular weight of 3.2 and 0.5 x 1()6.
They replicate In-the cytoplasm and bud from Golgi body membranes.
Due to segmented genome the viruses readily undergo genetic
resortment and may produce antigenic shift. The family comprises of 5
genera.
Other viruses: A family Filoviridae has been proposed. There are
other viruses which are yet to be classified e.g. toro viruses ,
astroviruses, the unclassified virus of Borna disease and mysterious
agents, which cause the subacute spongiform encephalopathies which
include scrapie.
Filoviridae: The viruses included in this family are Marburg virus
and Ebola virus which resemble rhabdoviruses but the viruses are
pleomorphic and sometimcs very long. The genome is a single
molecule (-) sense ss RAN.
Toroviruses: Torus stands for an object like donut. The viruses are
aS30ciated with diarrhoea in horsces and calves. The viruses are
enveloped, disk shaped measuring 35 x 170 nm and contain a
nucleocapsid, probably of helical symmetry. The genome is a single
molecule of (+) sense ss RNA.
Astroviruses: Astro means star. The viruses are star shaped and
found in the faeces of calves, lambs and humans. The genome consists
of one molecule of ss RNA.
References
BROWN,
F. 1986. The classification and nomenclature of viruses. Summary of

results of meetings of the International Committee on Taxonomy of
viruses in Sendai, Sept. 1984, Intervirology 25, 141.
R.E.F. 1983. Classification and nomenclature of viruses. Fourth
report of the International Committee on Taxonomy of viruses.
Intervirology 12,1.
MATHEWS,
Table 2.2
CUSSIflCATION OF ANIMAL VIRUSES
Family
Genus
Species
Symml!lry
Presence
o/capsid
o/DlVelope
Nucleic
acid
No. 0/
Particle
capsomues diameter
(nm)
1
DNA VIRUSES:
Poxvlrldae
Sub-family
i.
Chordopoxvirinae
(Vertebrates)
Orthopoxvirus
Capripoxvirus
Variola virus
Vaccinia virus
Cowpox virus
Camel pox virus
Ectromelia virus
Buffalo pox virus
Horse pox virus
Monkey pox virus
Rabbitpox virus
Sheep pox virus
Goat pox virus
Lumpy skin disease
Leporipoxvirus Myxoma virus

Rabbit fibroma virus
Squirrel fibroma virus
Complex
dsDNA
associated
with virion
transcriptase
300-450
x
170-260
ii.
Parapoxvirus
Contagious ecthyma virus

(Orfvirus)
Pseudocowpox virus
Bovine papular stomatitis
virus
Suipoxvirus
Swine pOx virus

Yaba monkey tumorpox
virus
Avipoxvirus
Fowl pox virus

Canary pox virus
Pigeon pox virus
Sparrow pox virus
Starling pox virus
Junco pox virus
Quail pox virus
Turkey pox virus
Entomopox
virinae
Parvovlridae
32
18-26
Insect viruses
Parvovirus
Kilham rat virus

Bovine parvo virus
Porcine parvo virus
Canine parvo virus
Icosahcdral
ss +OfDNA
Contd.
72
45-55
Feline panleukopenia
Aleutian mink disease virus
Mink enteritis virus
Goose parvovirus
Papovarlridae
Dependovirus
Adeno associated virus

(AAV) type 1 human
BovineAAV
EquineAAV
CanineAAV
OvineAAV
Sirnia., AAV
AvianAAV
Densovirus
Insect parvoviruses
Papillomavirus Cotton tail rabbit

Papilloma virus
Bovine papilloma virus
type-1-6
Equine papilloma virus
Canine oral papilloma virus
Sheep papilloma virus
Goat papilloma virus
Deer papilloma virus
Icosahedral
dsDNA
(Circular)
Comd.
252
7~90
Rabbit oral papilloma virus

Porcine genital papilloma
virus
Human papilloma virus
Polyomavirus
Adenovirldae
Polyoma virus
SV40
K virus
Rabbit vacuolating virus
Mastadenovirus Adenovirus type 2

(Human)
Bovine adenovirus
types 1-8
Equine adenovirus
Porcine adenovirus
types 15
Canine adenovirus
types 12
Infectious canine
hepatitis virus
Ovine adenovirus
Simian adenovirus
Murine adenovirus
Icosahedral
dsDNA
linear
Contd.
2
Aviadenovirus
162
12~200
Fowl adenovirus
types 1-9
Inclusion body
hepatitis virus
Turkey adenovirus
.type 1-2
Quail bronchitis virus
(;oose adenovirus
types 1-3
Herpesvlridae
Sub-family
Alphaherpes
1.
virinae
Icosehedral
Simplex
virus
Herpes simplex virus

type I &2
Bovine mammilitis
virus
Poikilovirus
Pseudorabies virus
Equine rhinopneumonitis
virus 1
Varicelle virus
Varicella zoster virus
Other herpes
viruses
Bovine herpes virus 1,4

Equine herpes virus 2, 3, 4
Canine herpes virus 1
dsDNA
linear
Contd.
Porcine herpes viruses

Feline herpes virus 1
Ovine herpes virus
Caprine herpes virus
Simian herpes virus
Fowl herpes virus
Duck herpes virus
Pigeon herpes virus
ii.
Ul.
Betaherpes
virinae
Cytomegalovirus
Cytomegalovirus of multiple
animal species
Muromeglovirus
Cytomegalovirus of mice
Gammaherpes Lymphocrypto
virinae
virus
Theta
lymphcrypto
virus
Iridoviridae
Epstein-barr virus
Marek's disease virus
Turkey herpes virus
Ateline herpes virus-3
Saiminine herpes virus 2
Iridovirus
Insects viruses
Chloriridov irus
Icosahedral
dsDNA
1892-2172
200-220
ConlrJ.
Rana virus
Lymphocystis
virus
Amphibious viruses
Fish viruses
Unnamed
Note: African swine fever virus has been removed from the family iridoviridae and has not been placed in any other family.
Hepadnavlrklae
(Proposed family)
Hepadnovirus
Human hepatitis
B virus
Duck hepatitis B virus
Woodchuks and squirrels
hepatitis B viruses
Icosahedral
Human polio virus 1-3

Coxsackie virus AI-22,
Icosahedral
dsDNA
circular
42
RNA VIRUSES:
Picomavlrldae
Enterovirus
ss+ RNA
60
22-30
24BI-6
Echovirus 1-9, 11-21 & 29-34
Porcine enteroviruses 1-9
Simian enteroviruscs 1-18
Human enteroviruscs 68-11
Human hepatitis virus
(Human enteroviruses -12)
Avian encephalomyelitis virus
Contd.
ss + RNA
32
35-40
ss+RNA
linear
32
40-70
Duck hepatitis virus

Ovine enterovirus
Bovine enteroviruses
Rhinovirus
Human rhinoviruses lA, B2

Bovine rhinoviruses 1-2
Equine rhinoviruses 1-2
Cardiovirus
Encephalomyocarditis
virus of swine and rodents
Aphthovirus
AphthovirusO, A,C, SAT

1-3. Asia 1 and subtypes
Calclvlrldae
Calcivirus
Vesicular exanthema ~irus

Feline caJ-civirus
Norwalk virus of human
Icosahedral
Togav-irldae
Alphavirus
Sindbis virus
Western equine
encephalomyelitis virus
Eastern equine
Venezuelan equine
Chikunguniya virus
Icosahcdral
Conk!.
Rubivirus
Rubella virus
Pestivirus
Bovine virus diarrhoea

Border disease virus
Hog cholera virus
Lactic dehydrogenase virus
Arterivirus
Equine arteritis virus
Flaviviridae
Flavivirus
Icosahedral
Yellow fever virus
Japanese B encephalitis
virus
Russian summer spring
encephalitis virus
St. Louis encephalitis virus
Louping ill virus
Kyasanur forest disease virus
Dengue virus 1-4
Reovlridae
Reovirus
Reovirus type 1
Bovine reovirus types 1-3
Ovine reovirus
Feline reovirus
Simian reovirus
Avian reovirus types 1-5
Icosahedral
92
60-80
ss+RNA
linear
dsRNA
linear
10-12
pieces
Contd.
Orbivirus
Blue tongue virus types 1-24

African horse sickness virus 1-9
Ibraki virus
Epizootic haemorrhagic
disease virus of deer
Rotavirus
Nebraska calf diarrhoeal virus

Foal gastroenteritis virus
Piglet gastroenteritis virus
Simian rotavirus
Ovine rotavirus
Caprine rotavirus
Feline rotavirus
Avian rotavirus
Birnavlridae
Bimavirus
Infectious bursal
disease virus
Infectious pancreatic
necrosis virus of fish
Icosahedral
Arenavlrldae
Arenavirus
Lymphocytic choriomeningitis virus

Lassa virus
Helical
dsRNA
(segmented 2)
32
60
ss RNA Ambiscnse
(segmented 2)
linear or circular
50-300
Contd.
Coronavlrldae
Coronavirus
Orthomyxoviridae Influenza
virus
Avian infectious
bronchitis virus
Bovine coronavirus
diarrhoea
Canine coronavirus
diarrhoea
Feline infectious
peritonitis virus
Equine coronavirus
diarrhoea
Porcine haemagglutinating
encephlomyelitis virus
Turkey blue comb virus
Helical
ss+RNA
linear
75-160
Influenza virus
Equine influenza virus
1 and 2
Swine influenza virus
Influenza virus B
Influenza virus C
Avian influenza virus
Turkey influenza virus
Duck influenza virus
Unnamed
Helical
ss -RNA
(segmented
80-120
8)
Contd.
1
Paramyxoviridae
Rhabdoviridae
Paramyxovirus New castle disease virus

Mumps virus
Bovine parainfluenza 3
virus
Equine parainfluenza 3
virus
Canine parainfluenza 2
virus
Avian parainfluenza 2
virus
Morbillivirus
Measles virus
Rinderpest viru~
Canine distemper virus
Pneumovirus
Respiratory syncytial virus

Bovine and ovine respiratory
syncytial virus
Pneumonia virus of mice.
Vesiculovirus
Lyssavirus
Vesicular stomatitis virus

Rabies virus
Mokola virus
Bovine ephemeral
fever virus
Fish rhabdoviruses
Two unnamed
sub groups
Helical
6
ss-RNA
8
about
150-300
(Pleomorphic)
Helical
ss -RNA
180x75
Contd.
Retrovlrldae
Sub-family
Oncovirinae
i.
ii.
TypeC
oncovirus
Mammalian type C
oncoviruses
Avian type C oncoviruses
Reptilian type C
ollcoviruses
TypeB
oncovirus
Mouse mammary
tumorvirus
TypeD
oncovirus
Squirrcl monkey virus
Spumavirinae Spumavirus
ss+RNA
80-100
Helical
ss-RNA
90-100
Foamy agents
iii. Lentivirinae
Lentivirus
AIDS virus
Equine infectious anaemia
virus
Maedi & Visna viruses
Caprine arthritis virus
of sheep.
Bunyavlrldae
Bunyavirus
Bunyamwera virus
Akabane virus
Rift valley fever virus
Phlcbovins
Icosahedral
(segmented 3)
Contd.
Phlebotomus fever virus

of man
Nairobi Sheep disease virus
Uukuvirus
Rodents & Ticks
Hamavirus
Haemorrhagic fever with

renal syndrome virus
Unclassified viruses
Fllovlrldae
(Tentative)
Filovirus
Marburg virus
Ebola virus
Other unclassified RNA viruses

Torovirus
Breda and Berne
Unusual agents
Astrovirus
Calf. lamb & human

faecal viruses
Borna disease
virus
Horse & Sheep
Subacute
spongiform
Encephalopathies
Kuru. Scrapie.
Creutzfeld Jacob syndrome
Transmissible mink
encephalopathy
ss-RNA
Helical
ss +
79Q,-97Ox80
36x170
35
Viral Replication
virus. In certain virus families the majority of virus (90-99%) remain

cell associated and released only when the cell is disintegrated. The
eclipse period varies from 5-15 hours for DNA viruses and from 3-10
hours for RNA viruses.
--
Release
1000
QI
c
III
100
...
>
....
10
//'1
Ce II associated
virus
c;
:I
III
:I
QI
QI
:;:
III
:::I
0.1
/
~Cell
free virus
15
20
Eclipse
10
Hours after addition
of
vi r us
Fig.3.1 One Step Growth of Nonenveloped Virus.
The Fig. 3.2 represents the complete replication cycle of an

icosahcdral DNA virus. Following attachment the infecting virus
particle penetrates the host cell and is partially uncoated and viral
genome is exposed. The messenger RNA is transcribed and is
translated into early proteins that regulate the expression of viral and
cellular genome, and enzymes required for replication of viral nucleIc
acid. The viral nucleic acid replication takes place, late viral genes are
transcribed. The late proteins are predominantly viral structural
proteins, some of which are subject to post translational modifications
like glycosylation and cleavage. Assembly of virus particle take place
in the nucleus or cytoplasm. Enveloped viruses are completed by
budding through cellular membranes. Each cell produces several
thousand new virus particles.
36
Cell membrane
Cytoplasm
Virus
I
~
N,,,.ooaP'id@ +~
~
/.
Neuraminidase
Haemagglutinin
Ribosome
Fig. 3.2 Schematic

Representation
of Influenza
Virus
Reproduction 1 and 2. Penteration or Virion to Cell; 3.
Release or Ribonucleoprotein (RNP); 4. Entry or RNP
into Cell Nucleus; S. Replication or Viral RNA; 6. Viral
RNP; 7. Synthesis or Viral Proteins in the Ribosomes; 8.
Newly Formed Viral Protein; 9. Formation or Virion;
10. Virion Leaves the Cell by Budding.
Adsorption
The initial interactions between viruses and cells occur randomly,

although both are negatively charged and repel each other, this
attachment is reversible which is facilitated by cations and initial
concentration of infectious virus particles and cells. For firm binding
the virus must have surface capsid proteins or envelope spikes which
bind to specific receptor sites on cell membrane. ThIS specificity often
explains the host range of viruses. In caSe of myxoviruses the binding is
via haemagglutinin, an envelope glycoprotein, to glycoprotein or
glycolipid cellular receptors with oligosaccharide chains terminating in
N-acetylneuraminic acid. In picoma viruses, poliovirus binds only f.(I
human or primate cells, because only the homologous cells carry
Viral Replication
31
receptoIS which the relevant viral capsid protein attachment site can
bind. There is some specificity about the binding of virions to particular
cellular receptors; several different viruses may utilise the same
receptor.
Penetration and uncoating
The recent studies have shown that virions can enter the cells by
endocytosis, fusion and translocation. The majority of virions entering
the cell fail to set up infection because they are degraded by lysosomal
enzymes.
Endocytosis: Adsorbed virus is incoporated into endosomes which
are cytoplasmic vacuoles. Following attachment to receptors the virus
particles move down into coated pits, coated with clathrin, fold inwards
to produce coated vesicles that enter the cytoplasm and fuse with
lysosome and form a phagolysosome. In case of enveloped viruses the
envelope of endocytosed virion fuses with lysosomal membrane,
releasing the viral nucleocapsid into the cytoplasm.
Fusion: The fusion glycoprotein of paramyxoviruses enables the
envelope of these viruses to fuse directly with the plasma membrane.
This way the nucleocapsid is released directly into the cytoplasm.
Translocation: Certain noneveloped viruses are capable of
passing directly through the plasma membrane into the cytoplasm.
The uncoating of those viruses which enter by fusion, their
nucleocapsid is discharged directly into the cytoplasm. In case of
viruses with helical nucleocapsids, the transcription, begins from viral
RNA while it is still associated with nucleoprotien. In icosahedral
reoviruses the nucleic acid viral genome expresses while it is still
covered with protein membranes, only certain capsid proteins are
removed and it is not fully exposed from the core. The pox viruses are
uncoated in 2 stages, fIrstly upto core from which half genome is
transcribed, then completely following the synthesis of virus coded
uncoating protein. In case of picoma viruses, cQnformational changes
take place during the process of attachment. This results in the loss of
capsid proteins and the virion becomes susceptible to proteases. For
viruses which replicate in the nucleus, there is evidence that later stages
of uncoating take place in nucleus.
Viral synthesis
The naked viral genome codes for messenger RNA to produce
38
virus proteins on cellular ribosomes. The viral nucleic acid also codes
for new viral nucleic acid. The new viral nucleic acid associates with
capsid proteins to make nucleocapsid. In enveloped viruses additional
viral envelope glycoproteins become associated with host cell
membranes.
Messenger RNA production (Transcription): In Case of DNA
viruses which replicate in the nucleus, the cellular dependent RNA
polymerase II performs the function of transcription. In other viruses, a
virus coded and integrated component of the virus particle performs
this function, cytoplasmic ds DNA viruses carry a DNA dependent
RNA polymerase while ds RNA viruses have a ds RNA dependent
RNA polymerase. The (-) sense ss RNA viruses carry a ss RNA
dependent RNA polymerase. The viral RNA of (+) sense RNA viruses
binds directly to ribosomes and is translated.
a. DNA viruses: Particular part of genome is transcribed in
sequence, early genes ftrst and late genes later. The strategy in different
DNA viruses is as under:
i) ds DNA, cellular transcriptase: In papovaviruses, adenoviruses
and herpesviruses, the viral DNA is transcribed within the nucleus by a
cellular-dependent RNA polymerase. In adeno and herpes viruses there
are at least2 cycles and in each cycle the structural proteins of the virus
particles are made from mRNAs produced in last cycle of transcription.
Polycistronic RNA transcripts under cleavage and splicing to produce
monocistronic mRNAs.
ii) ds DNA, virion transcriptase: In case of pox viruses and
African swine fever virus which replicate in the cytoplasm, carry their
own transcriptase. The monocistronic mRNAs are directly transcribed
from viral DNA. The transcripts are translated directly into proteins in
3 cycles of transcription.
iiy ss DNA, cellular transcriptase: The (:..) sense ss DNA
(parvoviruses) requires the synthesis of a complementary strand to form
ds DNA. This transcription takes place in the nucleus and the
transcripts are processed to produce mRNA's before being exported to
the cytoplasm for translation.
iv) dslss DNA, cellular transcriptase, virion DNA po1ymerases:
The ss DNA of the genome of hepadnaviruses is ftrst repaired by virion
associated DNA polymerase and then DNA is converted into
Viral Replication
39
supercoiled ds DNA. Transcription of mRNA then takes place by

cellular RNA polymerase 11.
b. RNA viruses: The transcription of RNA viruses is more
complicated:
i) ss (+) sense RNA: The (+) sense ss RNA is itself infectious. In
picoma' and flaviviruses the genome acts as a single polycistronic
mRNA which is translated into a single polyprotein which is
subsequently cleaved to give individual viral polypeptides. In
alphavirus genus of Togaviridae which contains (+) sense ss RNA
molecule, only about two thirds of viral RNA at 5' end is translated. In
Coronaviruses part of virion RNA acts as mRNA and is translated to
produce a RNA polymerase which then synthesizes genome length (-)
sense strand. From this overlapping subgenomic RNA is transcribed, of
which only the nonoverlapping sequence is translated.
ii) ss RNA (-) sense, virion transcriptase: In paramyxo and
rhabdoviruses the (-) sense virion RNA is copied in two distinct ways,
the replication mode and transcription mode. Copying in the replication
mode produces a full length (+) sense strand which acts as a template
for the synthesis of new virion RNA. In transcription mode, 5
subgenomic (+) sense RNA's are produced and each serves as a
moncistronic mRNA. In orthomyxoviruses, btlnyaviruses and
arenaviruses the genome is segmented. Each segment is transcribed to
yield a mRNA which is translated into one or more proteins. In
orthomyxoviruses transcription of 8 RNA's occur in the nucleus called
as 'cap snatching'. A virion associated endonuclease enters the nucleus
and removes a short segment from the capped 5 terminus of cell
mRNA, this is transported to the cytoplasm where it binds to the virion
RNA and serves as a primer to initiate transcription.
iii) ds RNA, virion transcriptase: In reoviridae and birnaviridae
the ds RNA genome is segemented. Each segment is separately
transcribed into cytoplasm by virion assoiated RNA dependent RNA
polymerase. The ds RNA segments correspond to single gene.
Monocistronic mRNA are transcribed from each segment These
RNA's complex with a protein before each is copied to produce ds
RNA, which serves as a template for further mRNA's transcription.
iv) ss RNA (+)'sense, virion reverse transcriptase: In retroviruses
ss RNA (+) sense is transcribed into DNA by a viral RNA-dependent
40
DNA polymerase. the resulting RNA-DNA hybrid is converted into ds

DNA and gets integrated in cellular DNA. Transcription of RNA
occurs from the integrated DNA via cellular transcriptase. followed by
splicing of RNA transcript as well as cleavage of resulting proteins.
Virus. protein synthesis (Translation):
Viral proteins are
translated from viral mRNAs at ribosomes in the same fashion as cell
mRNAs produced their own proteins. In reoviruses. which have been
studied in detail. each monocistronic mRNA binds via capped 5'
terminus to 40s ribosomal subunit. which moves along mRNA
molecule until stopped at the initiation codon. The 60s ribosomal
subunit then binds together with methionyl tRNA and various initiation
factors and then the translation proceeds.
The proteins translated from early transcripts of DNA viruses
include enzymes and other proteins required for replication of viral
.RNA as well as proteins which suppress host cell RNA and protein
synthesis. The function of many early proteins of large DNA viruses is
still not known. The late proteins are translated from late mRNA. most
of which is transcribed from progeny viral nucleic acid molecules.
Most'of the late viral proteins are viral structural proteins and are often
produced in considerable excess. The regulation of synthesis of protein
is mainly at transcription level. With RNA viruses also early and late
proteins are made but the control is not generally as rigorous as in DNA
viruses and occurs at the level of translation.
The newly synthesized viral proteins migrate to various sites in the
cell where they are needed. The mechanism controlling such migration
are not known but probably resemble those employed for cellular
proteins.
Viral genome replication: Different mechanisms of DNA
replication are employed by each family.
The replication of single stranded DNA of parvoviruses takes place
by cellular polymerases. They are initially converted into double
replicative form due to a reaction primed by 3' terminus of infecting
DNA. Further DNA synthesis requires the binding of a virus coded
protein to the 5' terminus. The viral ss DNA appears to be produced
after nicks at 5' end and repeated rounds of synthesis.
The replication of linear double stranded adenovirus DNA also
require specialized terminal structure. The replication is initiated at
either end of the double strand. It is primed by an early adenovirus
protein. part of which remains covalently attached to the 5' terminus of
new DNA strand. Synthesis from 5' to 3' end proceeds continuously
Viral Replication
41
with displacement of parental strand of the same polarity. The

displaced strand serves as a template in the formation of double
stranded molecules by synthesis of complementary DNA strands.
In case of papovaviruses, especially SV40' the replication begins at
a unique site at which an early virus protein, T antigen, appears to
promote unwinding of the DNA helix (Fig. 3.3 (a) & (b)). DNA
Fig.3.3(a) Double Helix or DNA
GAT
I
I
I
I
I
I
-0- F-O-F-O-F-O-F-O-F-O
('/ ""
';'.
'<:>
~
C' . . . . . . ~
'/
.~
0,
'" '.'
,('0
<:>,
~"
'0
.~
. . .0,
"'...
'A',
('
~/'
0,
Flg.3.3(b) Schematic Representation of DNA Replication; Separation of

the Double Strand Is followed by the Addition of Free
Nucleotldes with the Formation -of Two Identical Chains; D.
Deoxyribose, F. Phosphoric Acid, A, G, C, T-Purine and
PyrimIdine Bases.
42
synthesis by host polymerases proceeds in both directions. The

simultaneous synthesis of both strands takes place in the direction
opposite to the movement of other. This is accomplished by repeated
initiation and synthesis of short DNA segments complementary to one
strand of template, followed by ligation of the segments to produce a
complete strand. The replication of DNA is semi-discontinuous, in each
direction one strand is produced as a continuous polymer, the other in a
discontinuous fashion in segments.
The herpes viruses specify a large number of enzymes involved in
DNA synthesis. The replicating DNA initially consists of circles and
linear forked forms, which are later replaced by large bodies of tangled
DNA.
The replication of poxivirus DNA takes place in the cytoplasm and
depends upon virus coded proteins. The replication begins at each end
of genome and involves a strand displacement mechanism, with the
formation of small DNA fragments covalently linked to RNA primers.
The replication of virion RNA requires first the synthesis of
complementary RNA, which then serves as a template for making more
virion RNA. In RNA viruses, where RNA is of (-) sense the
complementary RNA is of (+) sense and RNA polymerase is the virion
associated transcriptase used for transcription of subgenomic RNA. The
primary transcripts from (-) sense virion RNA are cleaved to produce
mRNA's, some remain uncleaved to serve full length template of virion
RNA synthesis.
In case of (+) sense virion RNA, complementary RNA is (-) sense.
Several RNA molecules can be transcribed simultaneously from a
single complementary RNA template. The resulting structure is known
as replicative intermediate, which is partially double stranded with
single stranded tails. The replication of picorna and calcivirus RNA is
initiated by a protein similar to that of adenovirus DNA. This small
protein VPg is covalentIy bound to S' terminus of (+) and (-) RNA
strands as well as virion RNA but not to mRNA. In retroviruses
replication needs the synthesis of double stranded DNA through reverse
transcriptase of virus RNA and this is integrated into cellular
chromosome to function in replication. A virion associated reverse
transcriptase using the RNA molecule as a primer produces a ss DNA
copy. The reverse transcriptase functioning as ribonuclease removes the
parental RNA molecule from DNA-RNA hybrid. The (-) sense ss RNA
can then occur from integrated (proviral) DNA.
Viral Replication
43
Reoviruses replicate their double stranded RNA by encapsidating

mRNA and copying each one of them to form double stranded
molecules within the subviral particle. All other viruses replicate their
genomes by forming complete transcripts of virion RNA which are
then used as templates for RNA replication.
Virion Assembly
a. Icosahedral viruses: The structural proteins of non enveloped
icosahedral viruses associaLe spontaneously to form capsomeres, which
self assemble to form empty procapsids into which the viral nucleic
acid is packed. The packaging of viral nucleic acid in adenovirus
mechanism is elucidated. One terminus of viral DNA is characterised
by a nucleotide sequence, which enables the DNA to enter the
procapsid bound to basic core proteins, after which some of the (;Ore
proteins are cleaved to make mature virion.
b. Enveloped viruses: The viruses with helical symmetry of
nucleocapsids and few with icosahedral nucleocapsids acquire envelope
Lhrough budding cellular membrane. The envelope contains from
glycoproteins. The mechanism of glycosylation of envelope proteins is
described in brief. In the budding of viruses from cell membranes, the
nucleic acid containing subviral particles interact with cytoplasmic
domains of glycoproteins to induce the modified membrane to envelope
them. The interaction between the helical nucleocapsids of myxo,
paramyxo and rhabdoviruses is mediated by additional viral proteins,
the M proteins. The icosahedral nucleocapsids interact with their
membrane associated glycproteins to yield virus particles containing
equal number of virus capsid proteins and each membrane
glycoprotein. The cellular glycoproteins are excluded from virus
assembly process. In final maturation of viruses containing number of
membranes, proteolytic cleavage of specific virus glycoproteins is also
required to form the infectious virus particles ego the fusion
glycoprotein of parainfluenza viruses and heamagglutinin of influenza
viruses.
Replication groups: The following replication groups can be
assigned' to the viruses.
i) The viruses with double stranded DNA. They mostly divide iDto
the nucleus except pox viruses. All these viruses produce mRNA by
DNA transcription, the enzymes used for transcription are packed in
44
virus particles like pox viruses or the nuclear polymerases of the cell
may be used with or without modification.
ii) This group contains single stranded DNA viruses. The
replication involves the formation of double stranded DNA replicative
forms. m,RNA is transcribed from one strand of this template. Cell
polymerases are required in transcription, which takes place in nucleus.
iii) The retroviruses produce mRNA by transcription of double
stranded DNA replicative intermediate. The DNA is produced by
reverse trariscriptase of genome RNA and is integrated with cell
chromosome. The integrated DNA is transcribed by cell enzymes like
other cellular genes.
iv) The double stranded RNA viruses produce mRNA by
conservative transcription of double stranded virus RNA using virus
specific enzymes. The replication of these viruses is cyptoplasmic and
host enzymes are not involved in RNA synthesis.
v) The negative stranded RNA viruses replicate exclusively in the
cytoplasm or may involve nucleus as in influenza viruses. The genome
may be single RNA molecule or segmented. Messenger RNA is
produced by transcription of genome RNA by virus specified enzymes
in case of viruses which replicate in the cytoplasm. In influenza virus
which involves nucleus for replication, the primers formed to initiate
transcription in nucleus are formed by cellular polymerases and both
virus and cell polymerases are concerned with influenza mRNA
synthesis.
vi) In the (+) stranded RNA viruses the initial expression of
genome requires direct translation of the infecting nucleic acid to
produce proteins concerned in RNA replication. Certain members in
this group like picorna viruses use only complete virus RNA as mRNA
throughout replication while others produce a subgenomic mRNA's to
amplify the synthesis of particular gene products.
Selected References
ALBERTS, B.; BRAY, D.; lawIs, I.; RAFP, M.; ROBERTS, K., and WATSON, lD.,
1983. Molecular biology of the cell. Garland, New York and London.
BALTIMORE, D., 1971. Expression of animal viral genomes. BacterioI. Rev. 35,
235.
BISHOP, D.H.L. and COMPANS, R.W., eds. 1984. Nonsegmented negative strand
45
Viral Replication
viruses; Paramyxoviruses and Rhabdoviruses. Academic Press, New

York.
R.W. and
COMPANS,
viruses;
BISHOP,
D.H.L. eds. 1984. Segmented negative strand
Arenaviruses,
Bunyaviruses
and
Orthomyxoviruses.
Academic Press, New York.

JOKLIK,
W.K., 1983. Structure and function of reovirus genome. Microbiol.

Rev. 45, 483.
D.l, 1981. Structural analysis of animal virus genome J. Gen.
Virol. 55,1.
McGEOGH,
SIMONS,
GAROFF, H. and HELENUIs, A., 1982. How an animal virus gets into
and out of its host cell. Sci. Amer. 246,46.
K.;
STRAUSS,
E.G. and STRAUSS, J.H., 1983. Replication strategies of single

stranded RNA viruses of eukmyotes. Curr. Top Microbiol. Immunol.
105,1.
Chapter 4
Cultivation of viruses
The viruses replicate in living cells only. Some of the viruses have
a restricted host range. Most of the viruses can be grown in the cell
culture system, embryonated hen's eggs or in the laboratory animals.
Experimental animals
The experimental animals in the case of veterinary viruses may be
homologous hosts or heterologous hosts. In human medicine the
homologous host cannot be used. Loeffler and Frosch used cattle for
earliest studies in viral assay of foot and mouth disease virus. The
natural host is still being used for studies ofpathogenesis, immunology,
vaccine trials, diagnosis and chemotherapy. The experimental animals
should be specific pathogen free. The animals should have no prior
immunity to particular virus. The experimental animals are used for
following purposes.
i) Virus isolation - For diagnostic purposes the experimental
animals are still used e.g. mice in rabies and Louping ill disease
diagnosis.
ii) To study pathogenicity and host immune reactions - This is
studied in homologous host e.g. pig in swine fever. The cost of using
the homologous host is very high and therefore inbred experimental
animals are used instead of homologous host, e.g. inbred mice used in
African swine fever. The laboratory animals used as models are:
a. Rabbits - The rabbits were used by Pasteur to adapt street virus
of rabies. In malignant catarrhal fever virus these animals react in the
similar manner as the cattle.
47
b. Guinea pigs - Guinea pigs react to foot and mouth disease virus
when inoculated intradermally in the foot pad. Primary vesicle is
formed on the foot pad and secondary vesicles appear in the mouth
following viraemia.
c. Ferrets - Ferrets are used in the study of pathogenesis of
distemper virus.
Other laboratory animals are also used in virus study or in the
preparation of antisera against different viruses.
iii) To test and develop viral vaecines-Mice, guinea pigs, rabbits
are used for attenuation of virus strains as well as for testing vaccines.
Foot and mouth disease virus vaccine is initially tested in guinea pigs
and finally in cattle and pigs.
iv) To raise manoelonal or polyclonal antibodies-Various routes
are employed to inoculate experimental animals with virus infected
material. The usual routes are intracerebral, intranasal, intradermal,
intramuscular, intravenous and subcutaneous. The route of inoculation
largely depends upon the nature of virus, its possible affinity for the
tissue, age and species of experimental animal. The experimental
animals used in virus work has been replaced to great extent by the use
of embryonating chicken and cell culture but still it is a useful method
for studying clinical manifestations, pathogenecity, pathogenesis and
epidemiology of animal virus diseases.
Embryonated ben's eggs
The embryonating hen's eggs are being used since 1931 when
Woodruff and Goodpasture cultivated fowl pox virus on the
chorioallantoic membrane. Bumet used chicken embryo for cultivation
of viruses very extensively. Nearly all the viruses known at that time
could be grown in the chicken embryos by various routes of
inoculation. The chicken embroys are still used for isolation and
cultivation of many avian and few mammalian viruses. This method is
more economical and convenient than animal inoculation. The use of
embryonated eggs have number of advantages.
a. It is readily available, cheap and easy to maintain.
b. Free from bacteria and many latent viruses.
c. Free from specific and non specific factors of defence.
d. Se\1sitive to viruses which do not produce infection in adult
birds.
The presence of virus of fertile eggs can be detected by changes in
embryos like mortality, deformities, haemorrhages of the embryos,
48
pocks and oedema of chorioallantoic membrane. specific antigens in

the fluids like haemagglutinis. complement fixation antigen etc.
In Fig. 4.1. a schematic diagram of developing chicken embryo is
shown.
_ - - - - - - - - - - - - - - S.rosa
~-------------Embryo
- - - - - - - - - - - - Allantoic cavity
~~~~~~~s~==== Amnion
Allantois
I'r~f;f~~~~'~i.z:~;:==
A mhioti c cavity
Somatopleure
;....,...~- Yolk sac
(Splon chnopleure)
It
I,..,.-.. . . .
Vitell ine membrane

~~~.--'~c.r:,.:...:...,4.~
.l...:...'--r-__- _...........,."':;"O:~_ _
Albumen
Seroamniotic cavity
Extraembryonic cavity
Five days embryo

_ - - - - - - - - - A lIan tois
:::i~~~~~~==== Shell
Serosa
;:;~~~;;:--- Allantoic cavity

-......;~~- Yolk sac
-;-/-+,..~~~.....- Yo I k
~~~d~~:t:~t Allantoic
Amnion
~~~,~~
stalk
Albumen
Vitf'lIine membrane
Belly stalk
~~~~~~~~~~=====::: ASeroamniotic
11 an t oi c ca vi ty
cavity
Thirteen days embryo
Fig. 4.1 Schematic Diagram or Developing Chicken Embryo.
There are various routes of inoculation like yolk sac.

chorioallantoic membrane. allantoic sac. amniotic cavity and
intravenous.
49
The yolk sac method of inoculation is performed on 6-8 days old

embryos. Avian encephalomyelitis virus aand avian infectious
bronchitis virus can be grown by this route. There is dwarfing of
embryos inoculated with avian infectious bronchitis virus.
The allantoic cavity route of inoculation is done in 10-12 days old
embryos. Influenza virus and Newcastle disease virus are usually
cultivated. The virus titre is high and large quantities of virus can be
harvested. The amnio-allantoic fluid shows viml haemagglutination
when the virus grows.
The chorio-allantoic membrane method is widely used in the
veterinary virology. The herpes and pox viruses are usually grown. The
virus produces visible foci on the membrane usually known as 'pocks',
oedema or other abnormalities. The age of embryo is 10-12 days.
The amniotic sac method of cultivation is done at 10-14 days old
embryos. This method is employed in the primary isolation of influenza
virus and mumps virus. The growth of virus can be detected by
haemagglutination.
The intravenous route of inoculation is carried out in 13 days old
embryos. The bluetongue virus is grown by this route. The growth of
virus can be detected in the death of the embryo and other changes.
Cell culture
The cell cultures became commonly used after the introduction of
antibiotics and fungistatic substances. The use of laminar flow cabinets
is also very helpful in preventing the contamination of cultures as well
as the leakage of virus. However, the aseptic precautions are still
essential. The finger contamination is possible and this necessitates
careful handling of cell culture material. There are antibiotic resistant
mycoplasma which pose problem. The cultured cells are used for virus
isolation, virus titration, vaccine production and biochemical studies.
Since 1949 when Enders, Weller and Robbins reported that poliovirus
could be grown in non neural cells with the production of cytopathic
changes, large number of unknown viruses were isolated in cell
cultures. The advantage of cell culture over chicken embryos or
experimental animals are:
i) cell cultures can be produced in large quantities and stored at
-70C,
ii) cell cultures give a clear cytopathic effect,
50
Textbook of Veterinary Viro[o gy
iii) cell cultures can be grown in chemically defined medium

which is free from antibodies or other infections,
iv) cell cultures can be radiolabelled to study the details of virus
multiplication.
Production of cells cultures

The cells may be grown as explants of tissues or usually in
monolayer cultures and occasionally as suspension cultures.
a. Monolayer cultures: For almost all diagnostic and research
work monolayer cell c.ultures are used. Primary cultures are grown
from cells produced by partial proteolytic digestion of tissues like
kidney, testis, thyroid etc. Trypsin is used as proteolytic enzyme for
cell dispersion. The embryonic cells are often used because they grow
well and are easy to be disaggregated. The trypsin dispersed cells are
suspended in growth medium and seeded in glass or plastic containers.
The cells adhere to the bottom of these containers, flatten and
repeatedly divide until a confluent shcct of cells is formed. Secondary
cultures are produced by detachment and dispersal of primary
monolayer cells which are seeded in fresh growth medium. This cell
dispersion is done by trypsin and EDTA mixture. After several
passages the cells normally stop to grow but some cell lines have been
developed which can be passaged indefinitely. These continuous cell
lines become heteroploid and are not diploid in character. Some
continuous cell lines have been derived from neoplastic tissue e.g. Hela
cell line.
The cells in monolayer cultures in vitro differentiate into three
basic morphological types i) Epithelial polyhedral cells derived from ectodermal and
endodermal tissues like renal cortex, thyroid, amnion and epidermis,
ii) Fibroblast: Spindle shaped cells derived from mesoderm e.g.
connective tissue, embryonic muscle,
iii) Macrophages derived from macrophages and monocytes. They
can be grown as secondary cultures with difficulty.
The continuous cell cultures or cell line are mostly used in virus
work but primary cultures are still used to isolate some viruses because
the primary cultures are thought to be more susceptible to infection as
they carry the receptors which the cell lines lack.
b. Suspension cultures: This process .is used where large scale
production of the virus is required as in the case of vaccine production
Cultivation o/Viruses
51
or for biomedical studies. Some cell lines can he adapted to grow in

suspension where medium is continuously stirred and aerated, or is
stationary. Suspension cell cultures have also been grown from viral
and non-viral tumours like lymphoid tumours of Marek's disease,
bovine leukosis and canine mammary tumours. Hybridomas are also a
special type of suspension cultures.
c. Organ cultures: Explant cultures are occasionally used for
research purposes for cultivation of certain viruses. Cells may be grown
in vitro as explants of tissues such as respiratory or intestinal
epithelium. Tracheal ring cultures are most sensitive assay for avian
infectious bronchitis virus.
The cells are grown in tissue culture medium inside closed
containers. These containers are made of clear smooth non toxic glass
or plastic and sealed with screw caps or rubber bungs. Tissue culture
medium is an isotonic sterile solution of essential inorganic ions,
glucose, antibiotics and serum. The pH of the medium is maintained at
6.8 to 7.4. In recent years several growth factors have been identified
and certain cell lines can be grown in media that are chemically
defined. Defined media have advantages for the isolation of viruses that
are likely to be neutralized by antibody present in the normal serum but
this can be overcome by using foetal calf serum. The serum free media
is especially useful for cultivation of hybridoma cells used for
production of monoclonal antibodies.
Recongition of virus growth in cell cultures: The following
methods are commonly used for diagnostic work:
i) Cytopathic effect: The majority of viruses produce degenerative
changes in cell cultures. These changes can be recognised in unstained
preparations. The changes produced are rounding, retraction, syncytium
formation, detachment from surfaces. Different viruses produce
different cytopathic effects (CPE). Fixation and staining of cell
monolayers reveals further diagnostic details like inclusion bodies.
ii) Immunofluorescence or immunoperoxidase: Specific antisera
labelled with fluorescent dye or enzyme peroxidase can be used in
direct or indirect tests to detect the presence of cytopathic or non
cytopathic viruses in cell caltures.
Hi) Haemadsorption: Cultured cells infected with certain viruses
like orthomyxoviruses, paramyxo.... iruses and togaviruses which bind
from cytoplasmic membrane. acquire the ability to adsorb erythrocytes.
52
This ability is due to incorporation into plasma membrane of newly

synthesized viral protein that binds the red blood cells. The
haemadsorption can be prevented by prior addition of specific
antiserum to virus envelope. The haemadsorption can be demonstrated
both by cytocidal and noncytocidal viruses. This property
is
demonstrated very early (24 hours) when only a small number of cells
are infected.
iv) Haemagglutination: The medium of cell cultures infected
with some viruses like myxo, paramyxoviruses etc. acquire the property
to clump the erythrocytes of certain species of birds or animal. This
property is inhibited by specific antiserum (Haemaggl uti nationinhibition).
v) Interference: The replication of one virus in cells usnally
inhibits the replication of second virus. Interference operates between
leukaemia viruses and sarcoma viruses.
The assay of viral infectivity
The content of infectious virus can be assayed or titrated by
infecting cell cultures, chicken embryos of laboratory animals
or
natural host in serial dilution of virus suspension and observing
the
evidence of virus replication. The virus assays can be quantitative
or
quantal.
i) Quantitative assay: The quantitative assay is similar to bacterial
counts on agar plates. The viable bacteria multiples and forms a colony,
the colony count therefore, represents a direct estimate of the number
of bacteria. The plaque assay in monolayers of cultured cells is similar
to the colony count in bacteria.
a) Plaque assay: Dulbecco in 1952 introduced this technique for
quantitation of animal viruses. A suitable range of dilutions (2 fold or
5
fold) is inoculated on the monolayer cultures for an hour or so to allow
the virions to adsorb on the cells. The infected cells are then overlaid
with semisolid agar or methylcellulose gel, which localises the viral
spread. Then each infective virus particle gives rise to a localized focus
of infected cells that becomes visible with the naked eye after few days
of infection. This area of cytopathology is known as plaque. ~~ make
plaque more distinct, the cell monolayers are usually stained with
neutral red or crystal violei dye. The living cells are stained while
the
plaques appear as clean areas against the coloured background.
53
Infection with a single virus particle is responsible for a plaque. By

counting the number of plaques by a known volume of appropriate
dilution, the calculation can be made of the number of infectious virus
particles per milliliter of the original suspension which is expressed as
plaque forming units (PFU) per millilitre.
b) Transformation assay: Some oncogenic viruses which do not
kill cells but transform them i.e. the cells grow in an unrestrained
fashion to produce a heaped up mass of cells in a monolayer. These
transformed cells can also grow in a semisolid agar or methylcellulose.
The number of microtumours are counted and number of infectious
virions of oncogenic virus is calculated.
c) Pock assay: The pox group of viruses are grown on the
chorioallantoic membrane of chicken emrbyos. Each infectious unit
produces a 'pock' which is visible with the naked eye. The number of
pocks are counted in a known volume of particular dilution, a
calculation can be made to calculate the number of infectious units per
millilitre of original suspension.
ii) Quantal assay: This assay does not measure the exact number
of infectious virus particles in the inoculum, but only indicates the
presence or absence of infectious virus particles. Serial dilution of virus
are inoculated into number of replicate cultures, embryonated eggs or
animals. The virus, if present, replicates in appropriate time and
destroys the cell culture or kills the embryos or animals. Therefore each
host gives a single piece of information, whether the infectious virus
particles were present or absent in that particular dilution. The end
point of quantal titration is taken to that dilution of virus suspension
which infects 50% of inoculated hosts and the titre is expressed as 50%
infectious doses like infectious dose 50 (ID,J or tissue culture infective
dose 50 (TCIDsJ.
Assay based on other properties of virious

Haemagglutination: Many viruses contain virus coded proteins in
their outer coat which are capable of binding to the erythrocytes and
producing haemagglutination. Hirst in 1941 reported haemagglutination
in influenza virus for the first time. Haemagglutinin in influenza virus
is a glycoprotein. About 101 influenza virus particles are required to
produce haemagglutination, which can be read with the naked eye.
Thus haemagglutination is an indicator of the presence of large number
54
of virus particles but it is not sensitive to know the presence of small

number of virus particles. Large number of virus families are known to
possess the property of haemagglutination like, Adenoviridae,
poxviruses,. Togaviridae,
Flaviviridae,
Orthomyxoviridae,
Paramyxoviridae, Corona-viridae, Bunyaviridae, Rhabdoviridae and
Reoviridae.
Electron microscopy: Negative staining with potassium phosphotungstate makes it possible to count the number of virus particles by
election microscopy. In election microscope even the non infectious
virus particles are counted, therefore, the count of a given virus
suspension is always higher than in quantal or quantitative assay in
cultured cells or eggs.
Selected References
COOPER,
P.D., 1967. The plaque assay in animal viruses. In 'Methods in

Virology' (K. Maramorosch and H. Koprowski, editors) Vol. 3, 244311. Academic Press, New York.
FRESHNEY,
R.I., 1983. Culture of animal cells. A manual of basic technique.

AIan R. LissIWiley, New York.
STOKER, M.O.P. and MACl'HERSON, I.A., (1967). Transformation assay. In

'Methods in Virology' (K. Maramorosch and H. Koprowski, editors).
3,313-336. Academic Press, New York.
Chapter 5
Viral Genetics
Introduction
Viral genetics is concerned with elucidation of the precise structure
of the virus genomes to the extent to which determines the biological
properties and disease producing capacity of viruses. Viral genetics
also involves delineation of the pattern and origin of virus variation.
both in terms of virus evoluation and the temporal changes of
antigenicity and pathogenecity of viruses.
Effective genetic studies of animal viruses date from Dulbecco's
introduction of plaque assay in vertebrate cells and they are now
expanding under the impacts of improved cell culture technique. more
versatile and precise biophysical and biochemical techniques for
studying viruses and their components. The methods of studying virus
genetics were based primarily on the isolation of induced or
spontaneous mutants followed by their functional classification and by
genetic analysis using the methods of genetic engineering and
restriction endonucleases. Genetic engineering helps in molecular
cloning of fragments of viral nucleic acid and provide methods of
sequencing nucleic acid. It is now possible to determine the precise
structure of viral genes and even the entire genome of a virus. The
possibility of isolating more than one gene and the possibility of
mapping genes through shared genomic sequences has been increased
with cosmid technology. The development of cosmids have allowed
upto 50 kb of genomic DNA to be cloned in a single cosmid vector.
Cosmids are plasmids which contain (lambda) cohesive ends. a
56
replication ongm, an antibiotic resistance marker and a unique

recognition site into which genomic DNA can be ligated.
Mutation
Good phenotypic characters which are a prerequisite for genetic

studies are' provided by mutations that have phenotypic expression. All
the early work involved selection for phenotypic changes that reflected
a mutation in the particular gene responsible for that function.
Mutation can be defined as alteration or changes in gene, the
functional unit of genome. This changes in the gene is the source of
variation, it alters the properties of the virus and also causes phenotypic
changes of the virus. However, not all phenotypic changes that occur
with viruses are due to mutation. Many instances are known in which
the virus takes on certain phenotypic characters following the growth in
different'types of cells. Such host controlled variation has been found
in many groups of viruses such as orthomyxo and paramyxoviruses.
Mutations of animal viruses can occur spontaneously or can be
induced by various chemicals such as nitrous acid, 5-Bromodeoxyuridine (BUDR), hydroxylamine and nitrosoguanidine etc. The
frequency of mutation either spontaneous or induced is higher with
RNA than with DNA viruses because RNA polymerase and reverse
transcriptase (in case of retroviruses) are less accurate. The high
frequency of mutation usually causes rapid drift of the genome. As a
result an RNA virus strain is always a heterogenous collection of
different genotypes, the composition of which depends on the selective
conditions under which it is grown.
Induced mutation particularly chemical mutagenesis are important
for laboratory studies in viral genetics.
Types of mutation
Mutations can be classified according to the change in the nucleic
acid and the resultant mutants are classified by their phenotypic
expression. Types of mutations according to the change in the nucleic
acid are Silent mutation, Nonsense mutation, Frame shift and Deletion
mutations. In the first two types of mutations, the effect on polypeptide.
function is not adverse or may be variable but in the other two
mutations the polypeptide function is usually lost.
The mutants which are classified by their phenotypic expression
Viral Genetics
57
are conditional lethal, temperature sensitive, host dependance (host

range), plaque size, on cytopathic and pock types.
The conditioriallethal mutants are those which cannot grow under
certain non permissive (i.e. lack of some enzymes or other requirement
essential for the replication of a particular virus) conditions, but can
replicate under normal or permissive conditions.
The vast majority of the conditional lethal mutants of animal and
human viruses are temperature sensitive (ts) mutants. These ts mutants
are unable to multiply in susceptible cells at higher temperature. The ts
mutants are extremely valuable because they can be recovered in many
genes and they form the basis for most of our present knowledge of
animal virus genetics.
The Genome of Animal viruses
The nucleic acid or the genetic material of animal viruses is either
DNA or RNA. The coding of genetic information in RNA is unique and
is the predominant molecular form of the genome in animal and plant
viruses.
Viral nucleic acid may be linear or circular, single stranded (ss) or
double stranded (ds), covalently bonded to protein or present as unique
or diploid subunits according to the type of virus. On the basis of the
functional properties, the genome of RNA viruses may be represented
as plus or negative strand or both. The genome of plus strand non
segmented RNA viruses is infectious since it can function directly as
mRNA for synthesis of entire viral proteins and the polymerase
necessary for replication of the viral genome. The genome of negative
strand RNA viruses is not infectious because it cannot function as
mRNA. The nucleocapsid of negative strand viruses includes an RNA
dependent RNA polymerase as integral part of its structure so that
mRNA synthesis can be initiated. The genome of retroviruses although
possess plus strand and function as mRNA, but is not infectious
because it is dependent on preformed virus associated reverse
transcriptase for its replication. The genome of cytoplasmic viruses
(e.g. pox viruses) is not infectious and several enzyme activities
including DNA dependent RNA polymerase are associated with the
virion.
The Molecular weights (M.W.) of the genomes of RNA viruses
range from 1.5 x 1()6 of the parvovirus to the 169 x 106 of the poxvirus.
58
On the other hand the RNA viruses are more uniform and the M.W. of
the genome vary within a three fold range only.
The relative, coding capacity of a viral genome'provides a measure
of the minimum amount of genetic information present in the viral
genome. The ratio of the average nucleotide M.W. to the average
amino acid M.W. is taken to be 321:110 or 2.9:1 approximately. As a
result of thumb calculation the relation between genome M.W. and
gene product M.W. is 10:1 for single strand viruses and 20:1 for double
straDd viruses. Although the relative coding capacity assumes that
genes are unique and non overlapping, it remains useful ind~x for
comparison of the minimum genetic information content of the various
virus groups.
Number of gene in viruses
All DNA viruses and several groups or RNA viruses have genomes
consisting of a single molecule of nucleic acid but the genomes of some
RNA viruses consist of several pieces of RNA. If the gene is defined as
the nucleotide sequence that specifies a polypeptide it is possible to
estimate the number of genes in different viruses from our knowledge
of the amount of viral nucleic acid in the virus particle. In viruses with
fragmented genomes each molecule of RNA represents a single gene.
Mapping of the genome of animal viruses
The basic aim of virologists is to characterise the various functions
involved in virus multiplication and to identify the portion of the viral
genome that encode these functions. Great strides have been made in
the mapping of animal virus genomes, that is in identifying the portions
of their genome that encode specific proteins.
Viruses with a positive strand continuous genome that is translated
into a single polypeptide chain (e.g. polio) gene maps can be obtained
biochemically. A wide range of inhibitors of protein synthesis with
accurately defind modes of action have been available and are used for
precise gene sequence with RNA molecule by measuring the order in
which proteins are synthesized. Pactamycin for example specifically
inhibits inhibition of protein synthesis but permits completion and
release of polypeptides. A radio active amino acid is added to the
infected cell culture together with the antibiotic pactamycin, which
inhibits initiation of protein synthesis. The label will be incorporated
Viral Genetics
59
more by proteins corresponding to the distal 3' end part of the RNA
which has the highest chance of being still untranslated when the label
is added. The relative labelling of various proteins gives their location
in the uncleaved polypeptide chain.
The gene order for several negative strand virus has been
determined by measuring the effect of UV irradiation of virions on the
synthesis of the polypeptides specified by the various genes. The
method is based on the blocking of the progress of transcription by the
UV induced pyrimidine dimers, therefore, a gene at the 5' end on the
template strand has the highest sensitivity to the radiation because its
transcription is blocked by any dimer alorigwith the whole genome and
the synthesis of peptide undergoes the greatest reduction.
The location of genes encoding proteins of different viral genomes
can be determined by variety of techniques, among these, the genetic
recombination analysis and restriction endonuclease cleavage maps are
commonly used.
Genetic recombination between viruses

The relative order and location of genes can be specified by
measuring the frequencies with which pairs of mutants in the various
genes recombine with one another to yield wild type virus.
Several kinds of genetic recombination takes place when two
different virus simultaneously infect a same type of cell. This
recombination occurs between the newly synthesized nucleic acid.
Different types of genetic recombinations are intramolecular
recombination, reassortment, reactivation and marker rescue.
Intramolecular recombination: It involves the exchange. of
genetic material between different but closely related viruses.
Intramolecular recombination observed with all double stranded (ds)
DNA viruses and among RNA viruses particul&rly with foot and mouth
disease virus and polio virus. Increased frequency among DNA viruses
is presumably due to strand switching by the viral polymerase. In rare
cases intramolecular recombination occurs between unrelated viruses
e.g. SV40 and adenovirus. By the process of intramolecular
recombination the retrovirus also pick up cellular oncogenes and such
oncogenes are incorporated into the proviral DNA and subsequent
transcription of such oncogene into viral RNA converted into viral
oncogenes (Fig. S.la). The mechanism of recombination between
60
nonsegmented RNA genome remains 10 be elucidated. High frequency

of recombination is also observed with RNA tumour virus and
recombination is a normal consequence of replication of the genome.
Reassortment: Viruses with segmented genome undergo recombination by exchange of complete genome subunits. This form of
recombination is known as reassortment. This occurs when a particular
cell type is infected with two unrealated viruses. No DNA virus has
segmented genome, therefore, the phenomenon of reassortment is
confined to RNA viruses. Reassortment is an important source of
genetic variability. The major antigenic changes of influenza virus are
primarily caused by reassortment between different species (Fig. 5.1b).
Reactivation
This is applied to the condition during which there is production of
infectious virus particle when a cell is infected with two or more
viruses of the same strain and each of which has suffered a lethal
mutation. This type of multiplicity recombination leading to the
production of viable recombinants is observed among the members of
the poxviridae, orthomyxoviridae and reoviridae families.
Marker rescue
This refers to the genetic recombination between an infectious
virus and an inactivated virus of a related but distinguishable genotype.
A special kind of marker rescue called fragment rescue has been
extensively used for correlating the functional and physical maps of
viral DNA. It involves the introduction of a fragment of DNA
containing a specific mutation produced by recombinant DNA
technique along with the parental virus or its complete genome into a
susceptible cell. By this process the desired mutation can be introduced
into a portion of the progeny virions (Fig. 5.2).
Restriction endonuclease cleavage maps
In one method the genes are mapped by crossing parts of mutants
in different genes of virus strains that are related and characterized by
the DNA of wild type recombinant by restriction endonuclease
analysis.
In other method, mRNA from infected cells are hybridized 10
separate restriction endonuclease fragments. They are then dissociated
from the DNA, translated in cell free protein synthesizing systems an.1
Fig. 5.1(a) Genetic Recombination-Intramolecular Recombination.
Fig.5.1(b) Genetic Recomblnatlon-Reassortment.
0'1
62
Parental virus
oI
D~A
Fragments
;9 \+
.+~
+
O
+
o
Mutant
Fig. 5.2
().
Mutant
\..~mm
/ +/
~
and
fragments
""omb;"aHo"
Parental typt>
Fragment Rescue. Cells are Infected with Specific Restriction

Endonuclease Generated Fragments of Parental Virus DNA
and Its Mutants only the Fragment Carrying the Parental
Allele of the Mutated Gene (m) can give Parental Type
Recomblnants.
the proteins that are formed are identified. Since the position of the
viral genome of each restriction endonuclease cleavage fragment is
known the location of the gene coding for the protein that is translated
can be identified.
63
Viral Genetics
Genetic Engineering
Restriction endonucleases: These are the enzymes of bacterial origin

which have the property to cleave viral DNA at specific sites. The word
restriction was given in 1960 when it was observed that certain
bacteriophage failed tq grow in particular species of bacteria.
The failure to replicate inside the bacteria is due to the degradation
of the phage DNA by specific endonuclease of the bacteria. Several
hundreds of restriction endonucleases have been identified and purified
from various bacteria. Each of these restriction endonuclease
recognises a unique short (usually 4 to 6 nucleotide base pairs)
sequence of nucleotides and cleaves DNA into precise number of
fragments.
The DNA fragments produced by a panel of restriction
endonuclease can be analysed by gel electrophoresis and analysis of
these patterns can be used to identify viral sUbtypes.
Recombinant DNA:
The development of recombinant DNA
methodology has been facilitated by great improvement in the
technique of sequencing DNA. With the help of DNA sequencing
technique it is possible to get the exact picture of a DNA molecule and
large quantities of selected fragments of viral nucleic acid can be
obtained by the use of restriction endonucleases.
In recombinant DNA technique the desired DNA or copy DNA
from a virus is selectively cut into fragments by the use of specified
restriction endonuclease. Such viral DNA fragments are then inserted
into the DNA molecules usually of a bacterial plasmid DNA, and are
joined together with the help of enzyme ligase. The plasmid DNA
containing the foreign DNA is then placed into the vector bacterial
species. Inside the bacterium there is replication of the plasmid DNA
and there is production of many copies of the plasmid. Bacterium
containing the desired plasmid (i.e. the plasmid containing the foreign
DNA) is identitied, cloned and allowed to grow.
Recombinant DNA technique is applicable not only for DNA
viruses but also for RNA viruses. With the help of reverse transcriptase
it is possible to make DNA copy from either viral RNA or mRNA.
References
FENNER, F.; McAusLAN, B.R.; MlMs, e.A.; SA..\-1BROOK, J. and WHITE O.D., 1974.
The biology of animal viruses 2nd Ed. Academic Press, New York.
64
FENNER, F.; BACHMANN, P.A.; OmBs, E.PJ.; MURPHY, F.A.; STUDDERT, M.l. and
WHl1B, O.D., 1987. Veterinary Virology Academic Press, Inc.
(London) Ltd.
BROWN, 'F. and Wn.soN, S.O., 1984. Topley and Wilson's Principles of
Bacteriology, Virology and Immunity Vol. 4, 7th Ed. Edward Amold
Ltd. London.
HUNTER, E., 1978. Current Topics in Microbiology and Immunology. 79:295.

CONRAT, F.H., 'KIMBALL, PAUL, C. and LEVY, JAY, A., 1988. Virology, 2nd Ed.
Prentice Hall, Englewood Cliffs, New Jersey.
JOICLIK, W.K., 1980. Principles of Animal Virology. Appleton Century Crofts,
New York.
Chapter 6
Viral Pathogenesis
The understanding of pathogenesis of viral infections in host can
be best understood by studying virus induced changes in cultured cells.
The changes observed io cultured cells can be used to interpret the
changes in whole animal.
Viruses may be cytocidal or noncytocidal. The cytocidal or lytic
viruses produce morphological changes in cells known as cytopathic
effects (CPE). The noncytocidal or nonlytic viruses are non cytopathic
and produce little metabolic disturbance. The cytocidal and
monocytocidal viruses do not always lead to production of new virus
particles. In certian viral infections the viral genome either persists as
an episome or is integrated with the host cell genome. In such cases the
transformation of host cells takes place. These transformed cells may
produce tumours in experimental animals. The morphology of host
cells is altered and these transformed cells can be passaged indefinitely.
Certain noncytocidal viruses produce persistant infection. The infected
cells produce and release virions but cellular metabolism is not
affected, the infected cells continue to grow and divide. The various
types of interactions between virus and cell are summarised as under:
1)
Cytocidal infection: Changes in morphology of infected cells

(ePE).
2) Persistent non cytocidal mfection: No CPE, the virus and ceUs

continue to grow and divide.
3) Persistent noncytocidal non productive infection: No CPE.
Viral genome persists a3 episome or integrated. Virus is not
produced normally but by cocultivation with permissive cells,
irradiation or with chemical mutagens the virus can be
eJCpressed.
66
4)
Transformation: The cell morphology altered. It is produced

by RNA tumour viruses and rarely by DNA tumour viruses.
Cytopathic effect
The' lytic infection produces cytopathic effect (CPE) in cultured
cells which is an important diagnostic criteria. Several viruses produce
characteristic cytopathic effect. In the infected cells there is shut down
of cel1ular proteins, large number of viral macromolecules accumulate,
sometimes vlfal proteins are found in crystalline aggregates or
inclusions and these distort the cell morphology. The cell damage due
to virus infection can be due to the reasons given below:
a. Shut down of cellular protein synthesis: The cytocidal viruses
produce proteins early in infection which are responsible for stopping
the synthesis of cellular proteins and these proteins in turn affect the
synthesis of cellular RNA and DNA. This is incompatible with the
survival of cells. Some viruses like picornaviruses, poxviruses and
herpesviruses shut down the synthesis of cellular proteins rapidly and
these viruses are rapidly cytopathogeic. The adenoviruses shut down is
more gradual and with noncytocidal viruses like in retroviruses there is
no shut down and no cell death. There are certain viruses (flaviviruses)
which are cytocidal-and at the same time do not shut down the cellular
protein synthesis indicating that this is not the only mechanism
responsible for cytopathic effect.
b. Cytopathic effect of viral proteins: The capsid proteins of
certain viruses in high conct'ltrations are toxic to the infected cells. The
penton and fibre proteins of adenoviruses are toxic and may be the
cause of cytopathic effect. The cytopathic effect is also produced when
a large inocula is used to infect the cells.
c. Inclusion bodies: Certain viruses produce inclusion bodies in
the infected cells. These inclusions may be intranuclear or
intracytoplasmic and may be acidophilic or basophilic. The pox viruses,
paramyxoviruses, reoviruses, rabies virus produce intracytoplasmic
inclusion bodies while adenoviruses, herpes viruses and parvoviruses
produce intranuclear inclusion bodies. Canine distemper and rinderpest
viruses may produce both intracytoplasmic and intranuclear inclusion
bodIes in the same cell. The inclusion bodies are accumulation of viral
structural components. The basophilic inclusion bodies in pox viruses
are si.es of viral synthesis. In fowl pox and cowpox viruses the
inclusion bodies arc acidophilic and represent accumulations of viral
Viral Pathogenesis
67
proteins. In adeno and reoviruses the inclusion bodies represent

crystalline aggregates of virions which distort the cell. While in herpes
viruses the inclusion bodies are the result of late degenerative changes
and these produce margination of chromatin.
d. Cell fusion: The paramyxoviruses, herpesviruses, some coronaviruses and pox viruses produce syncytia due to the changes produced
in the cell membranes which result in fusion of infected cells with
uninfected cells. The syncytia are also produced by these viruses in the
tissues of infected animals.
In addition to the changes produced in cells due to spccific effects
of viral replication the virus infected cells also show non specific
changes like cloudy swelling. The cloudy swelling changes the
permeability of plasma membrane. The cell destruction is also the
result of leakage of lysosom:ll enzymes into the cytoplasm.
Persistent infections
The noncytocidal viruses replicate in the cells but do not kill them.
These viruses often produce persistent infections in the cells in which
they replicate. The cell metabolism is little affected. In several RNA
viruses like arenaviruses, retroviruses and some paramyxoviruses the
virions are released by budding from plasma membrane and the
persistent infection is produced. The infected cells yield the virus and
grow and divide for long periods but slow and progressive changes are
produced leading to cell death except retroviruses. In the animals the
cell replacement is rapid and slow death of cells due to persistent
infection has no effect. The persistently infected cells, however, lose
this capacity to carry out specialised functions and antigenic changes
are produced in the cell membrane of infected cells. These viruses may
also interfere with the secretions of immunoglobulins by lymphocytes
and hormones by somatotrophic cells e.g. cells of islets of Langerhans,
w~thout kilhng the cells concerned. Rhinovirus infection results in cilial
stasis and destruction of cilia subsequently but the cells are often not
killed. This lowers the resistance of respiratory tract to secondary
bacterial infection.
New antigens in infected cells
New virus specified antigens appear into the plasma membrane of
infected cells. The plasma membrane of cells infected with enveloped
RNA viruses incorporates viral heamagglutinin which is exhibited by
the property of haemadsorptioll (influenza virus, paramyxoviruses and
68
toga viruses). The virus specified proteins appear in the plasma

membrane in the early stage of infection with many viruses. The virus
coded antigens in the plasma membrane constitute a target for the virus
specified immune mechanisms which may destroy the cells before
significant number of virus particles are produced and thereby slow
down the progress of infections. In some cases, host immune response
may precipitate immunopathological reaction. In retroviruses infection,
transplantation antigens appear on the plasma membrane of the
transformed cells.
Cell transformation
The viruses of many DNA and RNA viruses change the growth
characteristic of cells. This alteration in cell morphology is called cell
transformation. The viruses producing cell transformation are related
with oncogenic ability of these viruses in animals. In the DNA virus
transformation the cells do not produce infectious viruses and the
infection is non productive while the infection with RNA viruses like
retroviruses, the transformed cells produce infectious virus and it is
productive infection. The virus DNA in transformed cells remains
integrated into the host cell DNA or may be episomal as in the case of
papilloma and herpcsviruscs. The transformed cells survive and can be
passaged indefinitely. These transformed cells have the capacity to
induce tumours in the nude mice, which have defective cellular
immunity. The transformation by DNA viruses is non productive and
certain virus specific antigens are regularly demonstrable. Tumour
associated transplantation antigens (TSTA) are located in the plasma
membrane while tumour (T) antigens are found in the nucleus.
Infection and spread of viruses in the body
The viruses enter the animal body through one of its surfaces, then
spread, either locally or through blood or lymphatic system to produce
systemic infection. For the maintenance of virus infection in nature the
infectious virus must be shed in the environment or taken up by a
vector or passed congenitally.
Routes of entry of virus in the animal body
Most viruses enter the host either through skin or mucous
membranes of respiratory and alimentary tracts, while a few viruses
also enter through urogenital tract and conjunctiva. The arthropod
borne viruses enter their host by direct inoculation by the bite of insect.
69
Viral Pathogenesis
Certain viruses produce the disease at the site of their entry without any
systemic spread, such as influenza viruses and rota viruses. The viruses
which produce disease at distant sites from their entry point, penetrate
the mucosal barrier and then spread within the host at the site where
viral replication and disease production takes place.
Table 6.1
PORTALS OF ENTRY OF VIRUSES IN THE ANIMALS
Route
Skin (abrasions)
Viruses
Pox viruses (fowl pox, cowpox, swine pox, contagious

pustular dermatitis, bovine papular stomatitis viruses),
herpesviruses, picornaviruses (swine vesicular disease
virus), Papillomaviruses.
Skin Bite by arthropod Fowlpox virus, swinepox virus, myxoma viruses,
Marek's disease, equine infectious anaemia virus,
Mechanical
rabies virus.
Rift valley fever virus, Nairobi sheep disease virus,
-do- Biological
Equine encephalitis virus, Japanese encephalitis virus,
Louping ill virus, Turkey meningoencephalitis virus,
Wesselsbron disease virus.
Herpes viruses, Equine arteritis virus, Bovine
Genital tract
papilloma virus.
Conjunctiva
Equine herpes virus-I, Infectious bovine rhinotracheitis virus.
Respiratory tract
Herpes viruses, Adenoviruses, Feline panleukopenia
virus, Canine parvovirus, Rhinoviruses, Apthoviruses,
Feline calcivirus, Parainfluenza viruses, Respiratory
syncytial viruses, Influenza viruses, Newcastle disease
virus, Canine distemper virus, Rinderpest virus,
Lymphocytic choriomeningitis virus, Hog cholera
virus, Pseudorabies virus. Marek's disease virus,
Bovine malignant catarrhal fever virus.
Coronaviruses, Rotaviruses, Astroviruses, ToroIntestinal tract
viruses, Adenoviruses, Enteroviruses.
Avian leukosis viruses, Avian encephalomyelitis
Congenital infection
virus, Infections bovine rhino tracheitis virus, Bovine
virus diarrhoea virus, Bovine leukemia virus,
Bluetongue viruses, Equine herpes virus-I, Equine
arteritis virus, Pseudorabies virus, Swine parvovirus,
Japanese encephaliti~ virus in swine, Hog Cholera
virus, Border disease virus, Rift Valley fever virus,
Canine herpes virus 1, Feline panleukopenia vim:.
Feline leukemia virus.
70
a. Entry through the skin: The skin is a strong barrier to

infection. The virus is destroyed by desiccation and by acids and other
inhibitors formed by skin microorganisms. The cornified epidermis is
impermeable unless broken by cuts, abrasions or punctures. Insect bite
is the main method of penetration.The mosquitoes, mites, ticks; sand
flies, fleas directly inoculate the viruses into the blood strcnm. Some of
the viruses are transmitted mechanically while others are transmitted
biologically. The bites of large animals, like dogs, jackals, wolves, bats
etc. introduce saliva containing rabies viruses into the tissues. The entry
through the skin is purely mechanical and virus does not play any part.
b. Respiratory tract: The respiratory tract is protected by defense
mechanism, the scavenging role of alveolar macrophages and
mucociliary blanket which removes particles. The initial lodgement of
air borne virus depends upon the anatomy of respiratory tra~t as well as
on the size of the droplet, which is very important. Large particles are
trapped by nasal hairs. Particles of about lO/lm in diameter arc
deposited on the nasal epithelium over the turbinate bones and those of
5/lm in diameter reach lung alveoli. Wherever the virus particle lands,
it has to make a contact with the surface of epithelium to initiate
infection. The contact occurs only by chance ?ecause the virus particles
entrapped in mucous and passed upwards to the pharynx or backwards
from nose by mucociliary escalator. The factors which affect the
chance of contact are thickness, flow rate and viscosity of mucus and
gaps in mucociliary blanket. Mucus secretion and ciliary movements
may be inhibited by changes in temperature, ion concentration and
humidity of the air. Many viruses remain localized to the respiratory
tract but some viruses enter via the respiratory tract following systemic
spread.
c. Alimentary tract: Several viruses enter the alimentary tract by
ingestion. The moving contents of intestine remove the .viruses which
do not adhere to intestinal epithelium. The mucus surface is acidic in
stomach and alkaline in the intestine and also contains phagocytes and
virus inhibitors such as proteolytic enzymes, bile and antibodies. The
heavy load of bacteria also may have a protective effect. Those viruses
which survive, infect the ahmentary tract, enteroviruses, reoviruses,
coronaviruses and rotaviruses are more resistant to bile and acid than
rhinoviruses, intluenza viruses and enveloped viruses which do not
produce intestinal intection. Some of the enteroviruses like avian and
Viral Pathogenesis
71
porcine encephalomyelitis viruses cause generalized infection rather

than the disease of the intestinal tract Parvoviruses produce diarrhoea
after reaching the cells of intestinal tract via blood.
d. Urogenital tract: Several important pathogens of animals like
infectious bovine rhinotracheitis, equine rhinopneumonitis and porcine
papilloma viruses infect this tract. Many viruses do not establish
infection presumably due to lack of interation with host cell receptors
and frequent flushing with the sterile urine.
e. Conjunctiva: The conjunctiva is protected by the secretions
from ~he lachrymal and other glands. Occasionally viruses infect the
eye (adenoviruses, herpesviruses and vaccinia virus). Experimentally
infection through this route is produced by large number of viruses.
The exact mechanism and determinants that enable viruses to
survive and penetrate mucous surfaces is little known as yet.
Virus spread within the host

Viruses may spread through a number of pathways depending on
the specific entry point and target organs involved. The viruses may
remain localized to the body surfaces through which they enter or they
may produce generalized infection due to viracmia and subsequent
localization in target organs.
Localized spread of infection: In tissues cell to cell infection
takes place with or without extracellular phase. The virus can spread
over mucous surfaces by moving mucus and lumen contents. Such
spread is not possible over the dry skin but some spread may take place
by scratching, rubbing and by fingers etc.
Spread through the lymph and blood system: The viruses which
survive the local inflammatory reaction reach the subepithelial tissues.
The virions enter the local lymphatics with the association of
phagocytes. Viruses enter the blood stream through damaged capillaries
at the initial lodgement site or later with lymph. The spread of virus to
other tissues takes place if the virus survives humoral and cellular
nefence mechanism in lymph and blood systems. The virus should have
a capacity to counteract the mononuclear phagocytes and lymphocytes
if< the lymph and blood, the fixed macrophages in lymphnodes, spleen
and liver. The viruses which lack the property for counteracting this
defence mechanism remain confine.d to the lodgement site. The size of
virus parii(' It: influences the efficacy of ingestion by reticuloendothelial
72
macrophages. The half life of circulating large viruses is less than small
viruses. The speed of blood and lymph flew also has a role in the
efficacy of ingestion by the reticuloendothelial macrophages. The
slower the. flow rate the greater is the chance of uptake. Certain viruses
like small pox rinderpest and polio replicate in lymph nodes and when
virus particles are discharged in the efferent lymph, they enter the
blood to produce generalized infection. The blood borne viruses are
protected from humoral defence mechanism by associating with
mononuclear lymphocytes, phagocytes, erythrocytes and other cells.
When the concentration of virus particles increase in lymph and blood,
the chances of breaching of biood-tissue junctions increase e.g. escape
in large amounts from the 'primary lodgement site and replication
within circulating and flxed phagocytes and lymphocytes. Some viruses
such as ectromelia and distemper of dogs may maintain their
circulating concentration by replicating in endothelial lining of lymph
and blood vessels. The original escape of virus from primary lodgement
site into the blood results in low viraemia and this primary viraemia is
followed by higher, secondary viraemia due to replication of virus in
primary target organ. This secondary viraemia also leads to infection of
secondary target organs like brain, placenta and skin. Infection of
central nervous system might follow from blood during viraemia
through the cerebrospinal fluid by either passing or growing through
the choroid plexus or virus might enter the cerebrospinal directly from
the blood. Viruses seem to cross the blood brain barrier easily in
immature host because of the thinner basement membrane. The blood
skin barrier is breached due to local inflammation. The blood borne
viruses localize in small vessels at the site of inflammation and pass
across capillary endothelium. The strength of maternal blood-foetal
junction varies with the type of placenta and stage of pregnancy. Few
viruses breach this barrier. The placenta may be infected but foetus
may still be protected e.g. blue tongue virus, while some viruses cross
this junction and infect the foetus.
Virus transport along nerves: The spread of virus from peripheral
sites to the central nervous system occurs along nerve flbres. The exact
pathway of virus transport along the nerve is not exactly known The
possibility in the transport of virus is sequential i.}fection of Schwann
cells, transit along with the tissue spaces betweea nerve flbres and
carriage up the axons.
Viral Palhogenesis
73
Virus shedding: The virus shedding occurs via one of the body
openings or surfaces involved in the entry of viruses. To maintain the
infection in populations the virus shedding is essential. The shedding of
virus from animal takes place via skin, respiratory tract, digestive tract,
urino-genital tract and milk etc.
a) Skill: Not many viruses are shed from skin lesions to cause
virus transmission. In case of foot and mouth disease, vesicular
stomatitis; pox virus and certain herpes virus infections, the virus is
shed from the vesicular or pustular lesions. The virus shed via saliva
and aerosal is more important in their transmission rather than those
shed via skin lesion. The skin is an important source of virus where
transmission is by direct contact. The localization of virus in feather
follicles is important in virus shedding of Marek's disease virus from
infected chicks.
b) Respiratory tract: The viruses causing diseases of respiratory
tract are shed in fluid expelled from the respiratory tract. Large droplets
fall rapidly and contaminate fomites while small droplets remain
airborne. Respiratory viruses are excreted both in nasal and oral
secretions. In certain virus infections the shedding of virus continues
even during convalescence.
c) Digestil'e tract: The enteric virus are excreted in the faeces.
These viruses persist for sometime outside body and are more resistant
to environmental conditions. The rotaviruses and enteroviruses are
relatively heat st.1ble.
d) Urogenital tract: A number of viruses are excreted in the urine,
ego rinderpest, canine hepatitis, foot and mouth disease virus. These
viruses replicate in tubular epithelial cells of kidney and excreted in the
urine. The arenaviruses are excreted throughout the life of infected
rodents. During coitus certain viruses are transmitted from male to
female and many viruses are excreted in the semen.
e) Milk: There are several viruses which are excreted in milk like
foot and mouth disease virus, mouse mammary tumour virus, some
tickborne flaviviruses etc.
f) Blood and organs: During viraemic stage the insect acquire
infection and spread a(bcviruses. Equine infectious anaemia and bovine
leukaemia virus are transmitted by contaminated needles and other
equipment. The pig and dog may be infected by consuming virus
containing meat. Hog cholera, African swine fever and vesicular
74
exanthema viruses are often transmitted by feeding the contaminated

garbage to the pigs.
Damage to host
The virus damage to host results in mild, severe or fatal illness or
oncogenesis. There is some information avaiable as to how the damage
occurs.
It has been shown that virus replication can take place in vivo
without producing morphological damage but biochemical changes in
such cells take place with pathological effects. Hence, although
apparently damaged cells attract attention first to explain the pathology
of the disease but it would not be wise to ignore any cell type showing
evidence of virus replication.
The manifestations of primary effects of some of the viruses may
be cell damage e.g. damage of nerve cells by rabies virus leading to
neurlogical effects, diarrhoea caused by rotaviruses and corona viruses
due to infection of intestinal epithelium. While many manifestations are
secondary effect of the original cell damage, inflammation is one such
general reaction resulting from complement activation, or liberation of
endogenous permeability factors from damaged cells. The haemostatic
response lowers circulating blood volume. The enteroviruses bring
about fluid disturbances. Fever is another reaction resulting from
endogenous pyrogen by viruses.
Inflammatory and vascular disturbances occuring later in virus
infection may be due to immunopathological reaction. Irtlymphocytic
choriomeningitis virus (LCM) infection of mice and Aleutian disease of
mink, immunopathology plays a majol role in disease manifestations.
Damage to the foetus

The foetal damage results from direct infection or indirectly from
the effect of virus disease on the mother. The cell damage to foetus, is
due to direct action of virus and primary and secondary manifestation
are the same as in the adult animal. However, these are amplified by
rapid metabolic and developmental changes occuring in the foetus. The
mitotic inhibition and chromosomal damage assumes more importance
in foetus in comparison to adult animal. The timing of infection during
foetal development is very important determining the nature and extent
of damage. The rubella virus infection In the first trimester of
75
Viral Pathogenesis
pregnancy, the malformations of foetus is followed directly from virus

destruction of certain cells within the affected organs. When organs
develop later in pregnancy malformations may be induced later in
foetal life. The cerebdlar hypoplasia produced by Kilham's rat virus
and feline panleucopenia virus may be the result of cytolytic action of
viruses on external germinal layer of cerebellum in laler period of
pregnancy.
The virus infection of mother also affect foetal development. Fever
produces abortions, stillbirths and foetal malformations. Changes in
foetal circulation may have rapid and severe affect on foetus.
Damage to infant
Maternal antibody protects infants against many virus infections
and also reduces the severity if the disease occurs. There are c~rtain
viruses which adversely affect the young animals. Rotaviruscs prGduce
enlentis in young animals more than adults.
References
BLOOM,
and RAGA-ZISMAN, B., 1975. Viral

immunopalhology. Academic Press, London.
B.R.
immunology
and
BlTRUS, W.H. and ALLlSON, A.C., 1977. Virus infection and the cell surface
North Holland and Publishing Co. Amsterdam.
COOMBS, R.R.A and GELL, P.G.H., 1969. Clinical aspects of immunology.
Blackwell Scientific Publications, Oxford.
DARl\'ELL, M.B. and KOPROWSKI, H. 1974. Mechanisms of virus disease.
Benjamin, Mento Park,. California.
DOHERTY, P.C. 1980. The molecular basis of microbial palhogenicity. Verlag
Chemie. Weinheim.
MTh1s, C.A. and WHITE, 0.0. V;ral palhogenesis and immunology Blackwell
SCientific Publications Oxford.
NollCDls, AL. and OLDSTONE, M.B.A, 1984. Concepts in viral pathogenesis.
Springer-Verlog, New York.
Tyrell, D A.J. 1983. liow do viruses invade mucus Surfaces? Phil. Trans. R.
Sco. London, B. 3() 3 75.
Chapter 7
Persistent Infections
Viruses of some families (herpes viruses) have been known to
cause infections that persist throughout the life of the infected animal.
The episodes of clinical disease might occur at long intervals. Certain
viruses produce chronic disease and the virus persists for months or for
life and produce pathological effects. These persistent viral infections
are important as they are often important from epidemiological point of
view. They may be reactivated and cause episodes of disease, or may
lead to immuno pathological disease or may lead to neoplasia. For
convenience, persistent infections may be divided into three categories.
a) Latent injections: The virus is generally not demonstrable
except when reactivated to replicate, such episodes being sometimes
associated with recurrence of disease e.g. bovine herpes virus 1 and
cytomegalus virus.
b) Chronic injections: The virus is continuously demonstrated and
often shed. The disease may be chronic or absent or may develop later
with immunopathological basis e.g. African swine fever virus, Aleutian
disease virus, Hog Cholera virus etc.
c) Slow injections: Virus slowly Increases in concentration during
a long preclinical phase leading to slow progressively lethal disease,
e.g. Visna-maedia virus, caprine arthritis-encephalitis virus etc.
Latent infections
In herpes virus infections latency IS followed by recovery. The
mechanism of latency has been described in great detail in human
infections with herpes simplex. varicella-Zoster and EB virus infections
77
but the mechanisms is comparable for animal infections as well. During

primary infection with infectious bovine rhinotracheitis virus the virion
move to cranial or spinal ganglia alongwith the axons of sensory
nerves.The virus persists in ganglian neuron as episomal viral DNA.
Some of viral DNA is transcribed to mRNA but none is translated
except reactivation, when production of infectious virus takes place. In
recurrent bovine rhinotracheitis infections the virus moves down the
sensory nerves again till it reaches the nasal mucus membrane or the
skin, where further replication occurs in the epithelial cells and virus
shedding takes place. This is the mechanism which maintains the virus
from generation to generation in the bovine population. The
cytomegaloviruses establish latent infections in the salivary gland,
baldder epithelium and in monocytes and/or lymphocytes. The virus is
shed into the oropharynx and urine from which it is transmitted
directly.
Chronic infections
In chromc virus infections there is continuous virus production.
There may be no disease, chronic disease or disease occuring as late
complication. The following chronic diseases are being discussed
below in brief.
Lymphocytic choriomenigitis: Lymphocytic choriomeningitis
(LCM) is caused by arenavirus, is an example of persistent infection in
mice. The virus is transmitted horizontally and in utero. The mice are
normal at birth and appear normal for rest of their lives. Infected mice
have persistent viraemia and viruria, every cell of the animal is infected
and remains so throughout life. The circulating antibody is not detected
but immunological tolerance is not complete. A very low antibody is
produced which forms virion-lgG-complement complexes. These
complexes are infectious. There is no cell mediated immune response
to the virus. The inbred miGe late in life exhibit 'late disease' due to
deposition of antigen-antibody complexes in the renal glomeruli of the
kidneys.
Foot alld Mouth disease: In cattle recovery from the disease is not
complete as the virus is not elimianted. The virus is now known to
cause persistent infection of the pharynx of cattle, sheep, goats, and
other ruminants. The persistence of virus is not in all infected animals,
only few animals become the carriers. The cattle vaccinated with
78
inactivated vaccines may also become carriers if infected subsequent to

vaccination. The recovery of the virus from cattle and buffaloes have
been made even after 2 years of infection. The mechanism of
persistence is not known and its epidemiological significance is
difficult to assess. The transfer of infection from carrier to susceptible
cattle is doubtful but transfer of infection from carrier infected African
buffalo to cattle is known to occur.
Canine distemper: Canine distemper produces a disease known as
'old dog encephalitis' whic is similar to a disease among human beings
known as sclerosing panencephalitis due to measles virus. It is now
known that a few dogs after recovery from canine distemper virus
continue to harbour the virus in brain cells, where it replicates slowly
and eventually produces the disease known as old dog encephalitis. The
virus can be readily cultivated from the brains of the affected dogs.
Slow infections
.The term 'slow infections' is used to .describe such infections
which have large incubation perioo and cause a slow progressive
disease which is invariably a fatal disease. The virus can be recovered
from infected animals during incubation period as well as during
disease. Lentiviruses as well as certain unclassified agents produce
subacute spongiform encephalopathies.
VisnalMaedi: Vlsna/Maedi virus belongs to family retroviridae
and subfamily lentivirinae. These viruses cause chronic demyelinating
disease of central nervous system, chronic pneumonia in sheep and
chronic encephalitis and arthritis in goats. These viruses cause
persistent infection, mainly in circulating leukocytes and virus may also
exist as DNA provirus. An interesting feature of Visna/Maedi and
equine infections anaemia viruses is the antigenic drift in surface
proteins during the progress of infection in a single animal.
Subacute spongijorm viral encephalopathies: Subacute
spongiform viral encephalopathies are produced by five diseases with
similar clinicopathological features and causative agents. The diseases
are scrapie of sheep and goats, mink encephalopathy, wasting disease
of deer and elk, and kuru and Creutzfeldt-Iakob disease in humans. The
basic lesions i;; fl progressive vacuolation in neurons, an extensive
astroglial hypertrophy and proliferation, and spongiform change in the
gray matter. The scrapie infection of sheep is transmitted from mother
79
to lamb. The incubation period is very long, upto 3 years. When the
clinical symptoms appear the disease progresses slowly leading to
paralysis and death. The mice and hamsters can be infected
experimentally. _The incubation period is about 1 year in these
experimental animals. The disease is like any infectious disease, the
causative agent appears to be the size of small virus but there is absence
of any immune response and no effect of interferon. The scrapie agent
has a higher degree of resistance than conventional viruses. These
biological and physiochemical properties are also shared by agents of
other four subacute spongiform encephalopathie~.
Pathogenesis of persistent infections
There are several mechanism which bypass host defenses that
eliminate virus in acute infection. These factors are primarily related to
the virus on one hand and to the host defenses on the other, althogh the
two kinds of factors interact in some instances. The possible
mechanisms are detailed below:
Nonimmunogellic agents: The agents producing subacute
spongiform encephalopathies are uncharacterized agents which appear
to be non immunogenic, which do not induce interferon and also are
not susceptible to interferon action. It shows that the host cannot
control the replication and pathological effects of these agents.
Integrated genomes: The proviral DNA of retroviruses is
integrated and maintained from one generation to the next as part of
host genome. Such proviral DNA may be implicated in tumorigenesis.
In lentiviral infections provirus is not involved but viral infection
causes systemic disease.
Growth in protected sites: During latent phase most of
alphaherpes-viruses avoid immune elimination by remaining within the
cells of the nervous system, as DNA in ganglion cells during the
intervals between disease episodes and within axons prior to acute
recurrent disease episode. The beta and gamma herpes viruses persist in
lymphocytes and avoid immune elimination.
Antigenic variation: Certain retroviruses avoid hosts immune
mecha-nism by antigenic drift. During persistent infection, Visnal
Maedi and equine infectious anaemia viruses develop succession of
antigenic vatiants. These successive antigenic variants produce clinical
signs in cycles. The persistence of influenza virus in populations occur
80
by similar mechanism operating over a long period in succession in

animal hosts.
Modification of host defence mechanisms
The modification of immune response is achieved by several ways.

These may be ineffective antibodies, disturbance of function of cells of
immune system, avoidance of immune lysis of infected cells and
antigenic variation of the virus.
Defective antibody response: The viruses which cause persistent
plasma associated viraemia replicate in lymphoid tissue and
mactophages and induce production of nonneutralizing antibodies:
These antibodies produce immune complexes and cause 'Immune
complex disease' and also block immune cytolysis of virus infected
target cell by T cells. Persistent infections in congenital infections also
cause immunological tolerance.
Defective cell mediated immunity: The persistent infections may
be caused by partial suppression of hosts cell mediated immune
response as a result of many factors.
Growth in macrophages: In many chronic infections virus appears
to grow in macrophages. This causes impairment of humoral and cell
mediated immune response and impairment of phagocytic and
cytotoxic activities of reticuloendothelial system.
References
E.PJ., 1981. Persistent viral infections offood animals: Their relevance
to international movement of livestock and germ plasm. Adv. vet. ScL
Comp. Med. 25, 7l.
Hn..Ls, T.J., 1985. Herpes simplex latency. In ''The viruses" Vol. 3 p. 175,
Plenum New York.
MARY, B.W.J., 1985. Strategies of virus persistence. Br. Med. Bull. 41,50
MIMs, C.A.; CUZNER, M.L. and KEU..y, R.E. eds. 1984. 'Viruses and
demyelinating disease'. Acadenuc Press, London.
NOTKlNs, A.L. and OLDSTONE, M.B.A., eds. 1984. 'Concepts in viral
pathogenesis' Springer-Verlag, New York.
PRUSINER, S.B., 1982. 'New proteinaceous infectious partides cause scrapie'
Science 216,136-144.
WITfMANN, G.; GASKELL, R.M. and RZIHA, H.J., eds. 1984. 'Latent herpes
virus infections in Veterinary Medicine' Curr Top. vet. Med. Anim.
Sci. 27,1
GIBBS,
Chapter 8
Viral Imnlunity
The viruses are a group of organisms which must entcr a host cell
to proliferate, since they lack the necessary biochemical machinery to
manufacture protein and metabolize sugars. Some viruses also lack the
enzymes required for nucleic acid replication and are dependent on host
cell for this function also.
The illness produced by virus infections are as varied as the viruses
themselves. Illness may be acute, recurrent, latent (i.e. dormant
infection where the virus is not readily detectable but may recur) or
subclinical i.e. acute or chronic symptomless infection where the virus
is demonstrable.
The immune response may range from the apparently non existant,
of Kuru, to lifelong immunity or chronic immunopathology (e.g.
Hepatitis B).
In this chapter discussion will concentrate on those acute virus
infections, which usually evoke obvious immunity, since these are the
only ones about which there is reliable immunological data. It must
therefore be remembered that apart from an assortment of clinical and
clinico immunological observations, we have litttle undcrstanding of
immunological mechanism underlying the recurrent or latent or lifelong
subclinical virus infections.
Viral infection: A typical viral infection starts with local invasion
of an epithelial surface and they after one or more viraemic phases
results in infection of target organ. III the early stage of viral infection
host response is largely non antigenic. spel.ific and involve!; the
production of interferons (IFN's) and activating natural kiiier cells.
82
Once virus starts replicating the following immune responses are

triggered in the second phase production of humoral antibodies;
activation of regulatory T cells. (that can amplify or suppress effcctor
functions) and T cell mediated immunity effected by cytotoxic T cells
that are able to lyse virus infected cells or T cells that are able to induce
delayed type hypersensitivity. All this depends upon the species of the
virus and phase of infection. The relevance to protection or
immunopathology of the various effector systems of immune response
therefore depend on the phase of infection and on the biology of the
virus. Viral antigens are largely proteins or giycoproteins. The
glycoproteins are often glycosylated by the host cell during the budding
process. The internal antigens of the virus are not usually relevant to
protective immunity. Antigens which are expressed on surface of the
virion may be the potential targets for the immune response and so
antigens expressed on the membranes of infected cells.
The response to a viral antigen is almost entirely T cell dependent.
Even the antibody response requires T cells help.
Humoral response
Antibody is only capable of directly binding to extracellular
viruses. IgG and IgM antibodies are limited in their action to plasma
and tissue fluids, whereas secretary IgA may protect epithelial surfaces
and, therefore, is particularly important in protccting against viruses
which lack the viraemic phase. Antibody in association with
complement can cause lysis of cells carrying viral antigens or directly
damage enveloped viruses. Antibody dependent cellular cytotoxicity is
also very effective mechanism for killing virus infected cells which are
recognised by the presence of viral antigens on their membrane.
Effects or antibody
Antibody may upset the virus cell interaction which lead to
adsorption, penetration, uncoating and replication, for instance
following into phagocytic vacuole some viruses have envelopes which
interact with vacuolar membrane and cause the dissolution. The viral
nucleic acid is liberated into the cytoplasm. However, the essential
interaction with vacuolar membmne can be blocked if the virion is
coated with antibody. On the other hand, mice artificially made
agammaglobulinaemic, which produce no detectable antibody to
Viral Immunity
83
haemagglutinin, recover and are subsequently immune. Thus T cell

cesponses appear to be important.
Antibody dependent cell mediated cytotoxicity
Cells with cytotoxic potential, which also possess Fc receptor for
IgG may bind to and lyse target cells coated with antibody to relevant
class. These cells act by binding specific antibody on virus infected
target cells via their Fc receptors. They can specifically kill the virus
infected cells.
Cell-mediated immunity
In general it must be seen that protective immunity to a pathogen is
mediated by both cellular and hU'lloral immunity. Also the predominant
role of CMI versus humoral immunity can be ascribed to a variety of
pathogens but not to the mutual exclusion of one to the other. Infact
most immune responses in recovery from infectious agents involve the
interplay of both humoral and CM! compartments. Major functions of
CMI are to defend against pathogens that live faculative/obligatory
within the host cells (viruses, some protozoan, bacteria, fungi). A role
for CMI is also suggested in tumour immunity and other
immunological phenomenon such as allograft rejection, graft versus
host reaction.
The components of CMI consisting immune lymphocytes that act
directly i.e. cytotoxic T lymphocyte (CfL or Tc) and immune T
lymphoytes primarily TDTH cells, possibly Tc accessary cells (mostly
monocytes also release lymphokines that recruit and activate
macrophages and Nk c~lls) to provide effective immune functions
independent of antibody. Both T cell effector functions (Tc and TDTII)
are regulated through TH and Tc circuits. These regulatory T cell
effectors (TH and TS) are also important in B cell antibody response.
Cytotoxic T cells
erc
is component of the cellular arm of the immune response

The
whose chief function appears to be the destruction of virus infected
cells an important means by which host recovers from infection. The
are particularly important where the load of infection is high.
Cytotoxic T cell are anti genic specific usually belong to OKT8
subSt~t and are restricted to class I MHC molecules, however virus
erc
84
TeXlbook o/Velerinary Virology
specific killer T cells of OKT4 helper inducers subset have also been
detected in long term cultures. Interferon gamma and other
]ymphokines are produced by CTC and T4 after stimulation by virus
infected cel1 of influenza virus which activate macrophages, stimulate
growth and maturation of proginator cells and antibody forming B
cells.
In Tc responses there is requirement for endogeneous antigen
synthesis pathway, where the antigens are normally presented in
association with MHC-l molecules, perhaps because body cells that are
natural infection targets having MHC-l molecule. An exception
however in measles virus system for example MHC-II restricted Tc are
important in immunity and are induced by endogenous antigen
synthesis pathway.
Perhaps the most surprising aspect of CTC immunobiology has
been the recent observation that with some viruses the principal target
antigens are not the transmembrane viral glycoproteins found
abundantly at the cell surface. While some but not all transmembrane
proteins can act as CTC target In influenza virus system, thus so far the
most compre-hensively studied, internal viral proteins and even non
structural proteins act as the major target antigens. The advantage of
this may be recognition of early proteins that provides the host with
means of destroying infected cells before the completion of a
replication cycle and spread of new virus to neighbouring cells.
Delayed type hypersensitivity: In contrast to elL class 11 restricted
T cel1 do recognise viral glycoproteins as principal targets. These
glycoproteins are usually late proteins and are expressed not only on
the infected surface but also on the surface of virion. Thus, they form a
logical target for antibodies that act primarily in extracellular defence
capacity. In addition Class II restricted cells subserve a protective role
by mediating a non specific inflammatory response which in turn effect
immunity. The essential feature of TDTH component of CMI is that
immunity is mediated primarily through various lymphokines which
are released on interaction between immune T cells and antigen. These
lymphokines recriut and activate accessary cells primarily macrophages
and NK cells. Activated accessary cells nonspecifically can destroy or
limit the intracellular pathogen (virus, bacteria, protozoa) and
neop]astic cells. Exogenous pathway of antigens presenting cells is
required for induction TDTH response. Different lymphokines may be
85
Viral Immunity
produced by different lymphocyte subsets. Some lymphokines such as

tumour necrotic factor (TMF), lymphotoxin (L1) act directly to destroy
the target cells. Others act an macrophages to recruit/assist (MCF),
immoblise (MIF), activate (MAF/gamma IFN) macrophages and
activated macrophages are very potent non specific effectors of CM!
response to eliminate viral infected cells.
Amongst various lymphokines released by immune TDTH is
transfer factor (TF) which is an interesting lymphokine that has
potential both for non infectious form and immunoprohylaxix and
immunotherapeutic.
Non-specific immune response
Interferons are family of related' cell regulatory glycoproteins
produced by many cell types in response to virus infection, double
stranded RNA, endotoxin and a variety of antigens and antigenic
stimuli. Interferons released from virus infected cells binds to receptors
on neighbouring cells and induce an antiviral state which helps to
isolate infective foci. The mechanism may involve inhibition of viral
protein or nucleic acid synthesis. IFN also particularly inhibits cell
growth (suggesting a possible anti tumour activity) and exerts selective
effects on protein synthesis and immune response. Thus interferon may
contribute to decrease in cell mediated responses seen early in virus
infections.
Immunopathology
The immune response to viruses can cause damage to host via the
formation of immune complexes or by direct damage to infectf'.d cells.
Complexes can form in fluid phase, or following capping and stripping
of virus expressed on cell surfaces. Chronic immune complex
glomerulone-phritis can occur in mice infected neonatally with lympho
choriomenigitis virus (LCM).
References
M.B.A., 1980. Antibody mediated destruction
virus infected cells. Adv. Immumol. i, 311-331.
SISSOMS J.O. and OOLDSTONE,
of
R.M. and DoHERTY, P.C., 1979. MlIC restricted cytotoxic T. cells.

Studies on biological role of polymorphic major transplantation
antigens determining T cell restriction specificity, function and
responsiveness. Ad". Immuno!. 27, 51177 .
ZINKER:'lAGEL,
86
DENAM, A.M., 1983. Viruses and immurwpathology. In Immunology in

Medicine Halbrow E.J. and Reeves W.G. (eds) Gurune & stratton.
HAWHOV, D.W., 1984. Cytotoxic T lymphocytes in herpes virus
infections. Vet. Immunol. Immunopalhol. 6: 35-36.
Rous B.T. and
NOTKINs, A.L., 1975. Viral immunology and immurwpathology. Academic

Press, New York.
Chapter 9
Epidemiology of Viral Diseases
The science of epidemiology is the study of factors affecting the

health and disease in an animal population or group of animals.
Epidemiology unravels the mechanism of disease distribution, transmission, survival of viruses in animal population and their control. The
viruses are intracellular organisms, different families of viruses use
different mechanisms for their transmission and survival. Many virus
families span both vertebrate and invertebrate animals. At cellular level
the viruses depend upon multiple cell structures including the
membranes and nucleic acid. The epidemiological studies enable us to:
1. Determine the magnitude of the disease including the
locations, time of outbreak, as well as the age, sex and breed
of ammals affected in relation to population at risk;
2. Indentify etiologic agents by isolation and serologic studies;
3. Plan control measures such as quarantine, sanitation of the
environment, slaughter of affected and incontact animals
besides immunization of susceptible population at risk and
4. Evaluation of effectives of control measures by statistical
analysis.
Epidemiologic tools
The tools used by epidemiologist to study viral diseases arc
virologic, serologic, molecular and ecologic. In the itlitial stages the
only way of studying the transmission of viruses wa<; !hI;: homologous
hosts and later on the experimental hosts like monkeys, mice, baby
mice, guinea pigs and chicken embryos were found to be .u$Cful. The
88
cell culture method proved to be very useful and opened up extensive

epidemiologic approaches. The approaches like antigen detection,
detection of nucleicacid and electron microscopy have paved the way
for epidemiologic study of those viruses which are not easily
propagated.
The serology is as important to epidemiologist as the isolation of
the virus. The serum antibodies appear within a week after infection
with most of viruses. The serologic tests to detect these antibodies are
neutralization, complement fixation, haemagglutination-inhibition, gel
diffusion and enzyme linked immunosorbent assay.
In the recent past molecular techniques have been developed and
are of importance in epidemiological studies. The monoclonal
antibodies are being used to distinguish rapidly and unambiguously
between closely related viruses. The detection of nucleic acid is another
sensitive tool in detection and identification of viruses. One of the
potential and sensitive method used for identifying fine differences
among virus strain is nucleic acid hybridization. By using these
molecular methods the virologists have uncovered the modes of spread
and true life cycles of many viruses.
The transmission of viruses depends on the interaction of the virus
with host and this interaction is modified by host behaviour and
environment. The ecology surrounding each virus is different. The
viruses use fascinating, complicated and ingenious mechanisms for
their survival, transmission and causation of disease.
Virus host environment interactions
The clinical manifestation of disease due to viral infection is the
result of a dynamic interaction among the causal agent, the host and the
environment. These factors cannot be singled out in explaining the
epidemiology of a virus disease.
Viral agent
The major factors in viral diseases are virulence, transmissibility of
persistency of infection.
The virulence i.e. the ability of viral agent to produce disease, is an
inherent characteristic of the viral agents. Some virus strains are more
pathogenic to a population than others. Transmissibility of the virus is
also related to virulence.
89
Host factors
The host factors which play a part in the epidemiology are age,
breed, sex and immunity status.
The age is most important factor which plays an important role in
viral diseases. In certain viral infections like neonatal diarrhoea due to
rotavirus and infectious bovine rhinotracheitis, young calves are more
susceptible. The age factor is also relevant to acquisition of immunity
through consumption of colostrum etc.
Certain breeds of animals show more susceptibility to some
infections. For example, hill cattle are more susceptible to rinderpest in
comparison to cattle from plains in India. The sex has little bearing on
viral epidemiology.
Immunity is an important factor which determines the outcome of
the disease. Protection is gained by active as well as passive immunity
besides non-immunologic resistance. In a herd, the immunity wanes at
different intervals with the result that the probability of disease
introduction also rises and falls. The population immunity is influenced
by the introduction of new animals or new borns
Environmental factors
The environment influences both the causal agent as well as the
host. The important environmental factors are discussed below.
Population density: Extreme crowding plays an important role in
determining the mortality rates due to viral infections especially among
young animals. The range cattle have a lower mortality rate than calves
in feed lots.
Nutritional level: The direct effect of status of level of nutrition in
viral diseases is not clear. The other environmental factors which
influence viral epidemiology among animals are managerial practices,
immigration etc.
Perpetuation of viruses in nature
The viruses can survive outside the host only for a limited period.
For the perpetuation of viruses, repeated infectious cycles in
susceptible hosts are essential. It is not always necessary that virus
infection will result in clinical disease. The immune status of the host
and viral pathogenesis are also important factors in the perpetuation of
viruses in nature in case of certain diseases. In certain disea~s like foot
and mouth disease, carrier animals also act as a source of spread of
infeclion to susceptible livestock.
90
Perpetuation by short cycle

Certain viruses are highly contagious but their transmission period
is usually short. A new susceptible population is required to maintain
the continuity of the virus cycle. Some of the important diseases in this
group include,rinderpest, foot-and-mouth diseases, Newcastle disease,
canine distemper etc. Due to short life span, the turn over in animals is
much higher than that in man. This change in popUlation results in
higher availability rate of susceptible animals.
There are certain viruses which are resistant and can survive for a
long time in the external environment like fowl pox virus, foot and
mouth disease virus.
Many viruses which are responsible for chronic infections,
perpetuate in the hosts and are transmitted to new hosts continuously
for a long period. Examples are herpes and adeno viruses, rabies among
bats etc.
The perpetuation of arthropod borne viruses by insects is the usual
mechanism of overwintering. Some of the viruses may perpetuate by
congenital transmission like, leukemia, hog cholera, bovine viral
diarrhoea etc.
The transmission of viruses
The spread of viruses from infected to healthy animals depends
upon the mode of shedding of viruses by the affected animals. The
major routes for transmission of viruses are:
1. Contaminated skin or mucous membrane
2. Droplet infections originating from respiratory tract
3. Alimentary tract-faecal or oral transmission
4. Genitourinary tract
5. Vertical
6. Horizontal
1. Contaminated skill or mucous membranes: The continuity of
the skin must be broken for viruses to establish infection via skin. The
break in skin, or mucous membrane may be accidental or may be
inflicted by insects or other animals. The accidental damage to the foot
may be due to hard ground (foot and mouth disease) while rough
fodder may break the mucous membra!le of the mouth (foot and mouth
disease virus, papilloma viruses). Damage to teats by milking or
sucking facilitates establishment of certain viruses like orf (contagious
Epidemiology o/Viral Diseases
91
ecthyma) virus in sheep and herpes mammalitis virus in cattle. Bites

from rabid dogs and bats transmit rabies virus. The mosquitoes, ticks,
culicoides transmit orb iviruses, togaviruses, bunyaviruses, where the
viruses by biting insects also take place like fowl pox virus and
myxomatosis virus in rabbits.
2. Respiratory tract: The particles of 0.5-0.7 m size are optimal for
droplet infection because smaller than this size dry out while larger
ones fall out. Primary replication usually takes place in the nasal
epithelium but high concentration of virus results in primary inhalation
into the bronchi and lungs. Viruses gaining access through this route
spread only within respiratory tract like influenza, parainfluenza, pox
and respiratory syncytial viruses in cattle and those that disseminates
via circulation to other sites like distemper virus in dogs and Marek's
disease in pOUltry. There are certain viruses which may be confined to
respiratory tract or disseminate according to virulence e.g. Newcastle
disease virus.
3. Alimelltary tract: The transmission of viruses by alimentary
tract is from fomites or faecal contamination. TIle primary site for
these viruses is usually oropharynx epithelium like foot and mouth
disease virus. Only those viruses which are resistant to acid pH of
stomach as well as the effect of bile proteolytic enzymes can establish a
primary infection in the intestine. These viruses are picorna, rota and
parvoviruses.
4. Gellitourinary tract: The genital tract is a common route of
infection for certain viruses like herpes viruses, sheep pox etc.
Viraemia leads to transplacental infection. Immunotolerant excretors
are important in perpetuating the virus infection like border disease of
sheep and mucosal disease in cattle.
Incubation period and dissemination of virus

The incubation period is defined as the time taken from the entry
of the virus to the appearance of pmhogenomic symptoms. During this
period the virus replicates and spreads from primary to secondary site
of infection. The incubation period is generally measured in days but in
slow viruses like VisnaIMaedi the incubation period may be months or
years.
The main route of dissemination is via the circulatory system as
free or cell-associated virus. The virus liberated by the lysed cells
enters via lymphatic or directly into blood circulation to give rise 10
viraemia e.g. fOOL and mouth disease virus, Newcastle disease virus etc.
92
The free virus or infected cells may be ingested by macrophages. The

macrophages with the infectious virus transport to the local lymph
nodes or other secondary sites, e.g. distemper virus, rinderpest virus,
herpes viruses etc. The viruses may replicate in lymphocytes leading to
leucopenia.
Table 9.1
TRANSMISSION OF ANIMAL VIRUSES
Mode
Viruses
Infective material
Intimate
contact
Pox viruses
Herpes viruses
Papova viruses
Influenza
Rhinoviruses
Aphtho viruses
Coma viruses
Adenoviruses
Entero viruses
Adenoviruses
Rota viruses
Astro viruses
Intestinal corona
Herpes viruses
Dried, scales, vesicle, fluid etc.
Airborne
droplets
Oral
transmission
Genito
Urinary
MTV
Vertical
LCM
HOrizontal)
Vertical
Hog cholera, BlueTranspla.
tongue
cental
By arthropod
vectors
like mosqllitoes
Culicoidis etc
3) Mechanical Fowl pox. rabbit
transmission myxoma
b) Biological
Alpha viruses
transmission Flavi viruses
BunyaviIidae
and Reoviruses
(Orbiviruses)
Nasopharyngeal
Discharges, Saliva
Faeces. Saliva
Wat~
Food
Flies
Contaminated
Semen and other excretions
Blood
Blood, secretions and excrctions

from affected animals
-do-
93
References
BROWN,
F. and
WILSON, G., 1984. Principles of Bacteriology, Virology and

Immunity. Vol. 4. Edward Arnold.
FENNER, F.; McAuSLAN, B.R.; MIMS, C.A.; SAMHROOK, J. and WHITE, D.D., 1974.
The Biology of animal Viruses. Academic Press, New York.
KAHRS, R.F., 1985. Viral diseases of cattle. Iowa University Press.
S.B. and DlITfA, S.K.. 1981. Veterinary Virology. Lee and Febiger.
Philadelphia.
MOHANTY,
Chapter 10
Viral Thmorogenesis
Tumor viruses are known to transform cells in vitro and induce

tumors experimentally in animals while some tumor viruses naturally
produce tumors in animals. The studies on tumor viruses have led to the
identification of tumour viruses and cellular homologous of several
retrovirus oncogenes that appear to play a role in human cancer. The
tumor viruses replicate their genomes and express their genes using
cellular enzymes. A large number of DNA viruses naturally infect
several animals and human beings and transform cells in culture or
induce tumors in experimentally infected animals. These viruses arc
DNA tumor viruses and belong to five families given as under:
i) Polyomaviruses: simian virus 40 (SV~, mouse polyomavirus
(PY) and human viruses.
ii) Papilloma viruses: animal papillomaviruses, human
papilloma -viruses.
iii) Adenoviruses: simian, bovine and human adenoviruses.
iv) Herpesviruscs: primate, equine, chicken, frog, rabbit and
human.
v) Hepatitis-B like viruses: human hepatitis B virus, Woodchuck
hepatitis virus; ground squirrel hepatitis virus and duck
hepatitis B virus.
The genomes of DNA tumor viruses have a wide range of size
Polyoma and Papilloma viruses have a small circular duplex,
superhelical genomes of about 5.3 and 7.9 kilobases (Kb).
The genomes of adeno and herpes viruses are larger and consist of
linear duplex molecules of 30-38 Kb and 120240 Kb, respectively.
The genome of human hepatitis B like viruses, consist of duplex
Viral Tumorogenesis
95
molecule of about 3.2 Kb that contains a single stranded gap. The

members of polyomavirus, adenovirus and herpesvirus exhibit two life
styles (a) productive infection of cells that are permissive for virus
replication and death of the cell and (b) transformation with a low
efficiency of small proportion of cells that are nonpermissive for virus
replication. The cells productively infected with polyoma, adeno and
herpes viruses express the entire viral genome while the cells
transformed by these viruses and papilloma viruses express only a
subset of viral genome that include the transforming genes. For
studying the productive infection of papilloma viruses there is no
suitable cell culture system. The viral genome that encodes viral
transforming genes is small. Cell transformation is the result of viral
DNA integration into cellular DNA and expression of specific viral
genes encoded in the transforming region. No specific sites of
integration on the host cell genome or viral genome have been
identified in DNA viruses.
The cell transformation by polyoma viruses as well as by
adenoviruses is a multistep process, which involves at least two viral
coded
functions (i) a cell immortalization or 'establishment' function and (ii) transformation function required for the expression of fully transformed
state.
Polyomaviruses: Polyomaviruses(SV40' PY, JCV) transform
cultured cells and induce tumors in experimental animals but they do
not induce tumors in their native host. The transformation region of
SV40 codes for large and small T antigen. The expression of large T
antigen is required for initiation of cell transformation and the
continued expression of large T antigen is required for maintenance of
transformed phenotype. The expression of small T antigen is not
essential for cell transformation but plays an enhancing role under
certain conditions. PY codes for large, middle and small T antigen. The
expression of middle T antigen is essential for the initiation and
maintenance of transformation of primary cultures, both middle T
antigen and the N-terminal half of large T antigen are required for
transformation.
Popillomaviruses: Papilloma viruses in contrast to polyoma
viruses and Ads produce cancer in their host species. Virus replication
is regulated by the differentiated state of host cell. The papilloma virus
genom~ is maintained in the transformed cells as free replicating
episomal viral DNA molecules and not as integrated viral DNA as in
96
Textbook a/Veterinary Virology
the case with cells transformed by polyoma viruses and Ads.

Adenoviruses: All human Ads have the capacity to transform
rodent cells in vitro but only members of groups A, B and D induce
tumors experimentally in animals. The Ad transforming genes are
located in the left 11 percent of viral DNA genome.
Herpes viruses:
Alpha, beta and gamma subfamilies of
herpesviruses transform cells in culture but only gamma herpersivruses
induce tumors experimentally in laboratory animals. Several
herpesviruses cause cancer in their original hosts, e.g. Marek's disease
of chickens, Lucke's carcinoma of frogs. In human beings there is
epidemiological evidence that Epstein-Barr virus(EBV) is responsible
in the development of Burkitt's lymphoma(BL) and nasopharyngeal
carcinoma(NPC). The genomes of EBV are found in BL and NPC
tumors. The cells of BL tumors exhibit a translocation of chromosome
8 to one of the three chromosomes. In each case the human Cmycogene is located at the cytogeI1etic breakpoint. It may be possible
that C-mycogene plays a role in multiple carcinogenesis that is initiated
by infection of B lymphocytes with EBV.
In comparision to polyoma and adenoviruses, very little is known
about the putative transforming genes and transforming proteins
encoded by members of herpes virus group. Recent studies identified
regions of herpesvirus genome that possess transforming activity. The
viral DNA sequence is not detected in the transformed cells. However,
the cells transformed by e.quine herpesivrus type 1 have been shown to
retain a specific region of the viral genome.
Related funtions among transforming genes of DNA tumor viruses

and oncogenes of retroviruses and cancer cells
The recent studies have provided an evidence that cell
transformation induced by DNA tumor virus transforming genes, by
oncogenes of retroviruses and cellular oncogenes cloned from human
cancer cells is a multistep process that involves 2 or 3 biological
functions. The first function is the immortalization of primary cells.
The immortalization function is expressed by N-terminal portion of the
PY and SV40 large T antigen genes by the AD EIA genes and the myc
oncogene. The myc oncogene has been found in several types of human
tumors. The second class of functIon is known as transformation
function. This function is expressed by PY middle T antigen gene, AD
Viral Tumorogenesis
97
EIB transformation region and ras oncogenes. Transforming proteins

are generally found on cellular membrane structures. The third function
in oncogenic transformation can be supplied by spontaneous cellular
change or by transforming gene function encoded in some DNA tumor
virus genomes.
Common pathways of oncogenesis and cell transformation
The cells transformed by tumor viruses possess new properties.
The origin of transforming genes of DNA tumor viruses is not known.
However, the transforming genes of retroviruses(oncogcnes) appear to
have evolved from cellular genes, since cellular homologous of
retrovirus oncogenes are found in numerous vertebrate species. In
recent observation it has beeen found that the portions of Ad El <\
transfomring genes are structurally related to two retrovirus oncogenes
myc and myb. It is interesting observation because myc and EIA gene
products are both nuclear proteins that immortalize primary cells.
DNA tumor virus transforming genes and retrovirus oncogenes
encode interchangeable and complementary functions. The oncogenic
activity of human cancer oncogenes can be complemented by DNA
tumor virus transforming genes. This suggests that DNA tumor virus
transforming gene encode functions that mimic those of cellular genes.
Tumor virus genes or oncogenes that encode the transformation
function have no effect on primary cells but they often transform
established cell lines by introduction of viral or cellular gene encoding
the immortalization function, spontaneous mutation or exposure to
chemical carcinogens. The PY middle T antigen cannot transform
primary cells but it can transform established cell lines or cells that
have been immortalized previously by PY large T antigen. The Ad EIA
function can substitute for the PY large T antigen. Thus the
immortalization gene of one DNA tumor virus can wmplement the
transformation gene of second DNA tumor viIUs. The immortalization
function of DNA tumor virus genes and retrovirus Myc oncogene can
complement the transformation function of viral and cellular
oncogenes.
Recent studie,s have indicated that same cellular genes may be
activated by DNA tumor viruses, by retroviruses and by chemical
carcinogens. This suggests that there are limited number of pathways to
oncogenesis and aberrant regulation of smali group of cellular genes
98
Texthooko/Veterinary Virology
may be the basis of malignancy. The analysis of the libraries of CDNA

clones prepared from S V40 transfomed cells indicated that some cell
mRNA species are differentially expressed in SV40 transfored cells.
Retroviruses
Relroviruses induce a variety of neoplastic disease in vertebrate
hosts. The retroviruses have single stranded RNA viral genome and
RNA dependent DNA polymerase (reverse transcriptase). This virus
family includes: Type C oncovirus-mammalian type C oncoviruses;
Avian type C oncoviruses; Type B oncovirus-mouse mammary tumor
virus; Type D oncovirus-squirrel monkey-virus.
Retroviruses have been extensiVely studied and have been
molecularly cloned and complete sequence of viral genome is known.
These viruses possess a number of unusual properties as compared to
most other viruses. Retroviruses typically establish a chronic infection
in susceptible host cells. To induce the chronic infection, the viral
genome has developed a unique strategy for replication. The virus
replicates in host celI through a double stranded viral DNA
intermediate which is integrated into host cell DNA. Another feature of
the replication of retroviruses is that certain viruses behave as cellular
genes and they are carried in the germ line as endogenous proviruses
(integrated viral genomes linked to host cell-DNA). Certain other
retroviruses are spread horizontally e.g. human T-cell leukemia virus
(HLTV) which is associated with an adult form of human lymphoma!
leukemia. Some of the retroviruses carry genes which are closely
related to cellular genes and these genes endows the virus with the
capacity to induce transformation of tissue culture cells and enhance
the tumorigenic potential of the virus. These genes are called as
transforming genes or oncogenes (V-onc) and their cellular
counterparts are known as C-onc. Each one gene has been given a 3
letter name such as Src, Ras etc. The comparison of V-onc gene and Cone gene indicate that virus has transduced the C-onc gene with minor
genetic changes and placed it under viral regulatory elements.
Virus properties
Retrovimses are 80-130 nm in diameter and consists of an inner
electron dense core (nucleoid) surrounded by an envelope composed of
lipid containing unit membrane. The core consists of nucleoprotein
ViralTur.norogenes~
99
surrounded by icosahedral protein capsid, an outer coat core protein is

located between capsid and the envelope. The envelope has
glycoporteion protections or spikes with knobs. The infectivity is
extremely sensitive to lipid solvents and detergents and at 56"C for 30
min. Virions contain the reverse transcriptase. The viral RNA genome
is composed of two positive sense single stranded RNA. The two ~A
subunits are bound noncovalently to each other near their 5' ends. A
specific cell coded tRNA is essential for virus replication. The presence
of two viral RNA copies indicates that retroviruses are diploid.
Retroviruses are divided into 3 subfamilies: (i) oncoviruses, (ii)
lentiviruses (lente means slow) and (iii) spumaviruses (spuma means
foam).
The oncoviruses have been isolated from almost all vertebrate
species. The members of the subfamily are intially grouped according
to the host species from which they have been isolated and then by
morphogenesis of their virions (Type A, B, C or D). Most of these
viruses belong to C-type viruses. The C-type viruses develop as they
bud from plasma membrane. The mature-C type particles are 80-100
nm with a centrally placed core inside the envelope. The B-type
particles develop their inner core in the cytoplasm and acquire their
envelope as they bud from plasma membrane. The mature particles
measure about 125 nm and contain an eccentrically located electron
dense core. The D-type particles develop in a similar manner as B-type.
However, the mature D type particles resemble C type particles and are
more closely genetically related to C-type viruses. A type particles are
found intracellularly and are devoid of infectivity. These particles lack
of a lipid containing outer envelope because they do not bud from the
membrane. There are two types of A particles intracisternal and
intracytoplasmic. The intracisternal forms are derived from endogenous
viral genomes and are apparently defective. Some intracytoplasmic A
type particles are precursor forms of B & D type particles.
The retroviruses have a simple structure and genetic organisation.
There are four identifiable genes-those that are essential for virus
replication and V-onc genes, which enhance the oncogenecity of virus
but are not essential. There are only three viral genes whIch are
required for viral replication: gag for group specific antigen and
encodes core protems, pol(polymerase) encodes for reverse
ti1lnscriptase and env which encodes for viral envelope proteins. Their
100
5' to 3' order is gag-pol-env. The retroviruses containing V-onc gene

are replication defective: since cells infected with a V-onc containing
virus will overgrow those cells not infected by this type of virus.
Transfonnation competent defective viruses are always isolated as one
component of mixed virus population that also contains a replication
competent retrovirus. Certain isolates of RSV are both replication
competent and transformation competent. The viruses that contain Vonc genes, most carry only one V-onc, although some possess two. The
V -onc gene may be located in different regions within the viral
genome.
Most retroviruses demonstrate a high degree of specificity in the
range of host cells that they can successfully infect. The transmission of
many retroviruses are acquired by horizontial spread. These viruses are
known as exogenous. Some retroviruses are transmitted vertically as
heritable pro-viruses whose genomes are localised to different chromosomes. These viral genomes are called as endogenous. Under natural
conditions, most endogenous viruses have low pathogenicity and are
widely distributed. The endogenous viral genomes represent an
opportunistic parasitism of the host genome. The endogenous
proviruses are usually less oncogenic for the animal in which they are
found than are exogenous viruses.
The retroviruses have a high frequency of genetic recombination
during coinfection with two closely related viruses. The retroviruses
have a capacity to integrate their genomes at multiple sites within the
host genome which suggests that these viruses may be capable of
inducing mutagenesis of host encoded genes via insertional
mutagenesis. In vitro insertional mutagenesis has also been
demonstrated by using exogenous infection by Moloney leukemia virus
in rat cells.
Palhogenesis: The V -onc viruses induce disease after a long latent
period. These viruses usually induce one or several fonns of malignacy
but infection with some retroviruses leads to some wasting and
neurological disorders. In ALV infected chickens the disease is induced
after a latent period of several months despite continued high levels of
virus. It suggests that either the target cells are rarely infected by ALV
or the turnors arise as a multistep genetic process. The recent studies
lent a support to latter possibility. The endogenous viral loci and host
genes also have a significant influence on the outcome {If virus
101
Viral Tunwrogenesis
infection. High levels of virus with env glycoprotein are required for
tumor formation.
The transformation competent (V-Onc +) retroviruses are found
frequently in tumor tissues of many species but are isolated rarely.
These viruses are isolated as a mixture with the helper independent
virus that has transduced the V -Onc gene. Under natural conditions VOnc + viruses are not usually transmitted to second animal. The V-Onc
+ retroviruses induce a characteristic range of tunors after a short latent
period. The V -onc gene is required for tumor induction but role of other
viral sequences is not yet known. The C-onc genes from which V-onc
genes are derived are conserved in eUkaryotic cells. Initially C-Onc
genes were discovered by finding the normal cells of many species
which contained sequences and proteins homologous to V -Onc genes
and their protein products. It is speculated that C-Onc genes are
normally involved in cell growth, division and differentiation. The
coding sequence of almost all C-onc genes are segemented. The
recombination mechanism by which retroviruses transduce C-onc gene
is not known. It is likely that integration of viral DNA near C-onc gene
is involved. Inspite of the fact that C-onc gene and V -onc gene are
similar at aminoacid level, C-onc genes carries out some important
normal cell functions while V-onc genes are highly oncogenic. There
are three possible explanations for the oncogenic property of V-onc
genes: (i) increased expression of a normal gene product (ii) mutation
within V -onc gene coding sequences and (iii) the contribution of viral
protein coding sequences. The importance of mutations within the Vonc protein coding sequences for its oncogenicity has been shown for
ras genes.
References
FENNER, FRANK, 1987. Veterinary Virology. Academic Press, London.
TEDDER, R.S.and ROBIN, A., 1990-ln Principlies of Bacteriology, Virology and
Immunity VolA, edited by LH collier and Morage. Timbury, Edward
Amold, London.
GREEN, MAURICB, 1985. In Fields Virology edited by Bemard N.Fields et al.
Raven Press, New York.

LowRY, D.R., 1985.1" Fields Virology edited by Bemard N. Fields et al. Raven
Prp-ss New York,
Chapter 11
Virus Vaccines and Antiviral Agents
The reduction of economic losses due 10 viral diseases among the

domestic animals in the recent past has been due 10 management
practices, hygiene and sanitation and immunization. The most
important of these measures is by vaccination and this has brought
down the losses to great extent in animals. The sanitary methods of
control of virus infections have often been applied with great success
e.g. control of rinderpest, swine fever, r~bies, foot and mouth disease in
United Kingdom accomplished by Slaughter of affected animals.
The history of the development of vaccines is about 200 years old.
The first vaccine was used by Edward Jenner in 1798 against small
pox; about a century before the is solation of viruses. The next viral
vaccine was developed by Louis Pasteur in 1884 against rabies. The
most notable development in this country was goat adapted rinderpest
virus vaccine by Edward in 1923. Several outstanding vaccines have
been produced in the last 30 years employing cell culture.
Vaccination has played a major role in the control of many virus
dilleases among men and animals. The viral vaccines have traditionally
been classified into two major categories-live attenuated and
inactivated. Immunity 10 viral infections-depends on the development
of an immune response to antigens present on the surface of virions or
virus lOfected cells. An immune response to the nonsurface antigens of
the .virus play a minor role in the immunity to viral infection. The
immune response to certain internal proteins of the virus may play a
\:ooperative role in the development of effective resistance.
Viral Vaccines and Antiviral Agents
103
Attenuated or live virus vaccine

Most of the attenuated vaccines have been developed by growing
the virus in a host other than the natural host until the virulence for
natural host has been reduced to a level which allows it to be used
safely in that host. Some naturally occuring virulent viruses have also
been used as vaccines. The original vaccine (Vacca means cow)
introduced by Jenner in 1798 for control of small pox among human
beings, used cow pox virus. In Veterinary practice herpes virus of
turkeys is used to protect chickens against Marek's disease, bovine
rotavirus by oral routc is used to protect piglets against porcine
rotavirus infection, naturally occuring lentogenic strains of newcastle
disease virus are used to protect chickens against virulent Newcastle
Disease virus. Virulent viruses by unatural route are also used as
vaccines. Infectious laryngotracheitis virus via cloaca is used to prot~t
chickens, contagious ecthyma virus by scarification of inguinal region
of sheep is used to vaccinate sheep against orf virus.
Viruses from one species may undergo replication without
producing disease in another species but elicit protection e.g. measles
virus when inoculated in young dogs protects them against canine
distemper virus.
Most of the live virus attenuated vaccines have been derived
empirically by serial passage of virulent virus through heterologous
nost, laboratory animal, embryonated eggs or through cell culture.
During the process there is progressive loss of virulence for the original
host but maintains the capacity to immunize the animals against
virulent virus. It is important to fix the degree of attenuation, so that the
vaccine virus retains the capacity to replicate sufficiently to produce the
desired level of immunity. An alternative method of developing
attenuated vaccine strains is to select temperature-sensitive mutants.
Temperature sensitive mutant vaccines have been developed for bovine
vi-rus diarrhoea, bovine infectious rhinotracheitis etc. but there are
prob~ems with reversion to virulence. The cold adapted mutants appear
to be more stable but are of low immunogenicity.
The attenuated vaccines multiply in the host and thus produce a
long lasting immunity. The attenuation of viruses is achieved in the
laboratory by adapting the viIuses to different animal hosts, chicken
embryos and cell cultures often after many serial passages. These
104
vaccines can be administered to animals parenterally, orally or

intranasally. Intranasal and oral vaccine3 also induce the synthesis of
IgA by the locallymphoiq ceJIs.
The live vaccines have. certain disadvantages as well. The best
attenuated vaccines even cause a small number of clinical cases. There
is a great concern regarding the viruses to revert to parent virulent form
when it multiplies in the natural host. Stringent tests are therefore,
necessary to ensure that the product is innocuous. There is also danger
of natural spread to incontacts. The attenuated vaccines can also be
contaminated by latent viruses from the cell cultures of chicken
embryos used to grow the vaccine virus: live polio vaccines prepared in
monkey cells contained SV40 virus. There is also the problem of storage
and transportation at low temperature to maintain potency.
Naturally avirulent viruses may also be used as vaccines. Turkey
herpes virus is successfully used for vaccination against Marek's
disease. The virus does not produce clinical signs in turkeys or
chickens.
Inactivated or killed vaccine
The inactivated vaccines work on different principle from

attenuated ones. A sufficient amount of viral antigen is administered in
a series of intramuscuI.ar or subcutaneous injections to produce
sufficient immunity to prevent disease.
Inactivated viral vaccines consist of tissue suspensions derived
from artificially infected animals, chicken embryos or cell cultures. The
virions in these vaccines are usually killed by destroying their
infectivity by physic,al or chemical agents. Formalin usually in the
concentration of 0.05 to 0.25% has been used to inactivate the viruses.
Formalin denatures the viral proteins to some extent. The other
inactivating agents which inactivate the nucleic acid but not the viral
proteins are considered better agents such as p-propiolactone,
ethylenimines and ethylene oxide. These agents are now being used in
the preparation of viral vaccines. However, formalin is still being
preferred because of its easy availability and being cheap. Physical
agents like ultraviolet light are also used for virus inactivation. It is
important to note that unless all nucleic acid is inactivated the mutants
may arise.
105
Table 11.1
COY".PARISON OF INACDVATED AND AITENUATED VIRAL VACONES
Sr. Characteristics
Inactivated vaccine.f
Attenuated vaccine
There is a possibility of
reversion to virulence in
the inoculated hosts. The
virus may spread to other
susceptible hosts.
The live vaccines may
contain certain adventitious viruses.
The protection is rapid
often high and long
lasting.
No.
1.
Safety
Safe as all virus activity

is completely destroyed.
2.
Freedom from
extraneous
agents
Protection
The vaccines may be

free from extraneous
agents.
The onset of protection
is slow, short lived and
the quality of protection
is not good.
Such vaccines must be
inoculated in usually
large volumes and
by parenteral route only.
3.
4.
Dose and route

of administration
5.
Stability
The vaccines are stable

at room temperature.
6.
Quantity of virus
The quantity of viral

antigen acquired is
more and su<.:h vaccines
are costly to produce.
Can be administered by
oral or respir atory routes.
The volume acquired is
often small and cost of
administration is low.
These are not stable at
room temperature
unless in freeze
dried form.
The antigen required is
less and therefore
cost is less.
The killed virus vaccines do not multiply in the animals and are
safe. The inactivating agents in the vaccines also eliminate the
contammating agents. The inactivated vaccines are useful even in
pregnant animals or in those situations where live vaccincs cannot be
used. There are certain difficulties encountered with these vaccines.
The vaccine must contain high concentration of viru~ antigen to
produce immunity, such immunity is of short dUffltion and must be
boosted. These vaccines do not induce satisfactory levels of IgA and
106
1'exJbook of Veterinary Virology
usually produce circulation antibodies against surface components of

virus particles. In certain cases the rabies inactivated vaccine (samples
vaccine) may produce severe allergic encephalitis in man. Adjuvants
are used to enhance immunologic response to killed vaccines. The
adjuvants allow a slower release of antigen and its degradation and also
stimulate phagocytosis and other activities of reticuloendothelial
system. It is likely that certain adjuvants enhance cell mediated or
humoral immune response. Mineral oil. gels like aluminium hydroxide.
quail A. aluminium phosphate or alum are widely used in veterinary
vaccines. Recently aviridine (a lipoidal amine) has been shown to have
good adjuvanticity in foot-and-mouth disease vaccine:Virulent virus by
abnormal route is sometimes used as vaccine. Orf virus is used by
scarification on the thigh of lambs or infectious laryngotracheitis virus
by applying on the cloacal mucosa. This practice is dangerous because
virulent virus is shed which can infect non immune animals.
Isolated immunogens
Nucleic acid of virion is infectious and does not play any part in
immune response. while proteins in the capsid and envelope are
responsible for producing immunity. Instead of using whole virion.
only the relevant protein against which the immunity is produced is
inoculated. Such vaccines are known as subunit varcines. Recent
advances in molecular biology and peptide synthesis provide a basis for
producing large quantities of pUrified viral proteins or synthetic
peptides for use in vaccination. Two separate approaches to the
production of viral antigens are being currently exploited. One involves
the production of synthetic peptide representing immunologically
important domains of surface antigen. The second approach involves
the use of clOned viral DNA inserted into a suitable vector to produce
viral protein in a bacteria or virus or a yeast cell.
Synthetic peptides~ Rapid progress in determinin~ RNA and DNA
sequence of various viral genes has simplified the selection of peptides
with potential antigenic activity. In the recent past. major antigcnic
sites on protective proteins of a number of viru~s have been identified.
The amino acid sequence of viral protein can be determined. Parts of
proteins. oligopeptides can be synthesized chemically. It is possible to
synthesize short peptides which correspond to the antigenic
determinants to which neutralizmg antibodies bind. There is possibility
that relatively invaria~ or even buried sequence which are not normally
immunogenic when presented in situ in the virion. may be capable in
107
isolation, of eliciting neutralizing antibodies. This may have the

advantage of cross protection against heterologus serotypes.
The feasbility of using synthetic ~ptides for inducing immunity in
"
animals was desmonstrated for foot-and-mouth
disease virus (FMDV).
An icosapeptide corresponding to the major antigenic site of VP1 of
FMDV was coupled to keyhole limpet haemocyanin which served as
carrier and was inoculated with Freund's adjuvant to guinea pigs. These
guinea pigs withstood challenge with virulent FMDV. These
immunogenic peptides of FMDV did not protect the cattle.
Although the synthetic peptide approach has a considerable
potential, there are obstacles which remain to be resolved. The
synthetic peptides are poor immunogenic. The adjuvants and liposomes
potentiate the immune response to most of peptides. A single peptide
may be insufficient to induce immunity because large surface antigens
usually contain several distinct immunologic domains that elicit
protective immune response. In certain cases it may not be possible to
identify small peptide that are protective.
Cloned viral DNA: The viral genes can be cloned in prokaryotic
cells using a plasmid or bacteriophage vector. The'-doned viral DNA
can be expressed as viral protein in prokaryotic or eukaryotic cells. VP1
protein of foot-and-mouth disease virus has been expressed in bacteria,
E.coli, accounting for 17% of protein produced in transferred bacteria.
When mixed with Freund's incomplete adjuvant, the fusion protein
induces resistance in cattle and pigs to challenge with virulent foot-andmouth disease virus. Plasmid vectors can also be engineered to express
unfused viral proteins such as rabies virus surface glycoprotein.
Allti-idiotype vaccille: The amino acid sequence of antibody
molecule which binds to an antigen is known as its idiotype. The
antibodies raised against the idiotype are known as antiidiotype
antibodies. This antiidiotype antibody binds to same idiotype.
Therefore, the antiidiotype could be used as vaccine. The antiidiotype
antibodies raised against hepatitis B virus surface antigen elicit an
antiviral antibody response in animals.
Antiviral Drugs
While most of the bacterial diseases of man and animals have been
brought under control by the use of antibacterial agents, the same
cannot be said about viral diseases since anuviral drugs in general do
not have the same degree of efficacy as antibacterial drugs. Antiviral
drugs have limited use in man in certain advanced count:-ies. Recent
108
laboratory investigations have shown that the growth cycle of many

RNA and DNA viruses can be interrupted in cell culture and sometimes
even in experimental animals. Despite these successes most of the
antiviral drugs have proved to be highly cytotoxic and cannot be safely
used againsJ viral diseases in man and animals. Another important
reason is the close association of viruses with host cells during
replication. Short lived nature of many viral diseases is another obstacle
so that by the time the disease is diagnosed the viral multiplication has
largely been completed. To be effective, antiviral drugs have to be
administered early in the viral infection.
For antiviral therapy, the compound must be capable either of
preventing entry of a virus in the host cell or specifically inhibiting
some essential steps in virus replication, maturation etc.
Inhibitors of viral entry
Amantadine and its derivates have successfully been used in
human influenza and have also been shown effective against swine
influenza. The drug apparently prevents the penetration of virus into the
host cell although the exact mechanism is not fully known. This
compound also may act against the viral transcriptase. Methyl
adamantanemethylamine hydrochloride, a compound closely related to
amantadine is also effective against influezna A viruses.
Purines and Pyramidine antagonists:
The halogenated
pyramidines are the group of substances known to inhibit DNA
synthesis. The halogenated deoxyuridines namely 5-iodo-2deoxyuridine (IUDR) and its bromine equivalent (BUDR) are
incorporated into the viral DNA in place of thymidine and produce
defective (fradulent) non functional DNA molecules. The !UDR acts in
the final stages of viral replication. It is possible that !UDR inhibits the
action of DNA dependant RNA polymerases and blocks in the
formation of mRNA. These drugs are administered locally in human
cases of herpetic ulceration of cornea, unfortunately these are too toxic
to be given parenterally in man. BUDR inhibits DNA synthesis by
blocking the synthesis otthymidilic acid.
Thiosemicarbazones: lsatin-B-thiosemicarbazone (IBT) and paminobenzylaldehyde are potent inhibitors of poxviruses and
adenoviruses. Some derivatives are also active against certain
enteroviruses. The drug has no action on DNA synthesis and probably
acts late in viral replication cycle at the level of translation and
interferes in the synthesis of viral mRNA.
109
ArabinojurRosyl nucleosides: Adenine arabinoside is effective

against some herpes viruses and to some extent against pox viruses.
The drug is effective as a topical agent against herpetic keratitis. Its
action is inhibition of DNA synthesis by interfering with the reduction
of cytidylic acid to deoxycytidylic acid.
A cyclo vir: Acyclognanosine, commonly known as acyclovir is a
guanine derivative with an acyclic side chain. This is one of the newest
drugs which is more promising antiherpes viral agents. The drug is
phosphorylated to monophosphate by a herpes virus specified
thymidine kinase in herpes virus infected cells but this does not occur
in uninfectedcells. The monophosphate is converted to triphosphate in
herpes virus infection cells to a greater extent than in healthy cells.
Benzimidazoles: Benzimidezole and guanidine interfere with the
synthesis of single stranded viral RNA without affecting the cellular
RNA synthesis. These drugs prevent the multiplication of many
enteroviruses in vitro. The drug has no protective effect in animal
models due to rapid emergence of drug resistant mutants.
2-deoxy-D-glucose and glucosamines: The drug inhibits the
multiplication of many viruses that mature at the cell membrane like
orthomyxo, paramyxo and herpes viruses. The drug acts perhaps by
interfering with the synthesis of virus specific glycoproteins.
Antibiotics: Certain antibiotics like rifamycin, tolypomycin and
streptovaricin display antiviral activity against some pox viruses and
oncoviruses by interfering with maturation and by inhibiting the
activity of reverse transcriptase in OACoviruses. Actinornycin-D inhibits
DNA transcription and mitomycin-C inhibits protein and DNA
synthesis but these antibiotics are very toxic and cannot be used
paren terall y.
Interferences
In 1957 Issacs and Lindenmann found that allantoic fluid of eggs
that had been exposed to irradiated influenza virus posses an interfering
property with the multiplication of infectious homologous virus distinct
from the virus itself. The substance responsible for this interference
was named as 'interferon'. The nature and mode of action of interferon
is complex. The interfervns are host coded proteins and there are three
distinct types of interferons, one formed primarily in leukocytes (IFNa), another in fibroblasts (IFN-P) and third formed in stimulated
lymphocytes (lFN-y). The norma! cells do not synthesize interferon, but
the cells can be induced to do so by a variety of inducing agents.
110
Interferon blocks the multiplication of many different RNA and DNA

containing viruses both in eggs and in cell culture. The DNA
recombinant technology has been effectively used for cloning of the
genes for human interferon (IFN) in bacteria, yeasts and mammalian
cells.
Characteristics and types of interferon: The interferon is a small
soluble protein (mol wt. 25-45,000 d) containing nearly all amino acids.
The IFN is relatively non toxic, thermostable and active through a wide
range of pH. It is a weak antigen and can be neutralized by antiviral
serum. The IFN is inactivated by proteolytic enzymes like trypsin but is
not affected by receptor destroying enzyme, RNase, DNase or
periodate.
There are three classes of human interferon. IFN-a. and IFN-/3
predominantly produced by leucocytes and fibroblasts in response of
viral infection or other inducing agents, while IFN-y is produced by
unsensitized lymphoid cells in response to mitogens and by sensitized
lymphocytes when stimulated with specific antigen. IFN-/3 and IFN-y
species are glycoproteins in nature while IFN-a. are not glycoproteins.
IFN-/3 and IFN-yare stable at low pH (pH 2 at 4C) and retain activity
in the presence of sodium dodecyl sulphate (SDS).
There appears to be two highly conserved domains in interferon
molecules. One in the aminoterminal half of the molecule and probably
contains the site that binds to cell surface receptor, while the other
biologic functions.
The interferons are produced by almost all vertebrate species. The
best studied interferons are those of humans, rodents and birds. The
three classes of human interferons are immunologically quite distinct.
The interferon appear to be larger than 17000 dallon molecules, when
isolated from the cells. The reasons may be that some interferons are
glycoproteins, some naturally occuring interferons may be dimers and
under certain conditions, interferons may associate with other proteins.
The interferons are host species-specific, the basis for spcdficity
probably resides in the specificity of the interferon cell surface receptor
interaction. The specificity sometimes is very narrow. All interferons
possess antiviral and anticellular activity. The interferons cause
interference with the multiplication of viruses and regulate a variety of
cellular functions. The induction of antiviral state by interferons differs
against various VIruses both with type of cell and type of interferon.
The activity of IFN-yis fur more potent than IFN-a. and IFN-/3.
111
The production of interferons: The nonnal cells do not synthesize

interferon due to the absence of transcription of interferon genes.
However, interferon is transcribed spontaneously in some cells. All
such cell lines are lymphoblastoid cell lines, some produce lFN-y but
most produce lFN-a.. The interferon produced is homogenous but may
be the product of single interferon gene. The amount of interferon
produced by interferon-relating cell lines is too small and cannot be
utilized for practical purpose, The important interferon inducer is virus
infection. Both RNA and DNA containing viruses induce interferon
production, but RNA containing viruses are good interferon producers
than DNA containing viruses' except pox viruses. The interferon
production in the infected cells starts approximately 4 hrs after
infection at 37'C and reaches a peak when the viral protein synthesis is
at maximum and then declines.
The second type of interferon inducer is ds RNA. All naturally
occuring ds RNAs of viruses, replicative forms of ss RNA of animal
and bacteriophages, synthetic ds polyribonucleotides like poly I: poly C
or poly lC are interferon inducers. The ss RNA or DNA or ds DNA or
RNA-DNA hybrid molecules do not induce interferon production. The
ds polyribonucleotides which induce interferon production have the
properties (i) have stable secondary structure (ii) relatively resistant to
ribonuclease (iii) have higher molecular weight and (iv) have minimum
effective chain length of 100 residues.
The thrid type of interferon inducers are viruses those are unable to
replicate such as viruses in non permissive cells and inactivated viruses.
Thele is enormous variation in response to these inducers depending on
the nature of cell and of the interferon inducer.
There is another important group of interferon inducers consisting
of metabolic activators and inhibitors namely mitogcns, metabolic
activators etc.
The cells treated with very little interferon produce more interferon
than untreated cells, called as priming. Priming is used extensively to
increase the large scale production of lFN-a and IFN-/3.
Biological effects of interferons: Interferons have varied biologic
effecis, namely antiviral and cell multiplication-inhibitory activities
including antitumor activities, immunomodulation, inhibition of certain
synthetic cellulaa' activities and enhancement of others, toxicity
enhancement etc. The effects of interferons are exerted at plasma cell
membranes Vla combination with specific receptors.
112
Texlbook o/Veterinary Virology
Interferon has an antiviral activity. The viral infections cause sever

disease in animals treated with antibodies against interferon than in
normal cells. The interferon protects cells from effects of virus
infection afjd also renders infecticn abortive. In vaccinia virus infected
cells, host protein synthesis is inhibited more quickly in interferon
treated cells than .in untreated cells, marked cytotoxic effects are
induced in interferon treated cells and cells die. In other virus systems it
is not the case. In many cell systems, interferon treated cells are
protected against virus infection. Protection by interferon depends on
the concentration of interferon and multiplicity of infection. Large
doses of interferon protect against low multiplicity of infection while
high multiplicity of infection overcome the protective role of
interferon. Under natural conditions in the body, interferon protects
many cells from cytopathic effect of virus infection but does not
completely eliminates the infection. The interferon may be a factor in
establishment and maintenance of persistent infections.
Interferons do not actually inhibit virus multiplication but induce
the cells to synthesize proteins that are effectors of antiviral state. The
interferons do not induce the antiviral state if messenger RNA or
protein synthesis are inhibited. The interferon-induced inhibition of
virus multiplication, is due to interference with the ability of parental or
early viral mRNA molecules to be translated with the result that the
infection is aborted. This has proved in case of vaccinia virus infected
with L-cells and reoviruses. In some system it ~ not easy to detennine
whether the tmnscription or translation of early viral mRNA is
inhibited to greater extent. In the cells infected w.il.h VSV and SV40 it
was observed that transcription rather than translation of early viral
mRNA is inhibited. However, under strict precautions it was found that
in VSV infected cells the .effect of interferon treatment was on
translation of mRNA transcribed from parental viral genome. In case of
SV40 infected cells with SV40 DNA instead of whole virus, translation
of early viral mRNA is inhibited much more than transcription. When
interferon was added to the cells late in the lytic cycle of SV40
multiplication, the rate of mRNA was not inhibited but mRNA
translation was inhibited. These results suggested that interferon
inhibits the uptake, penetration and uncoating of SV..,. These studies
were ,:;arried out in vilro and it was found that cause of inhibition was
the presence of an inhib~tory factor and the inhibitory factor is
associated with ribosomes. Kerr and his associates demonstrated that
113
inhibitor of translation in extracts of interferon treated cells is the

induction by interferon of at least three separate enzymes and a further
requirement for ds RNA in their enzymes and a further requirement for
ds RNA in their activation. These enzymes are 2-SA synthetase, RNase
and protein kinase. Two of these enzymes require ds RNA for their
activation. The ds RNA is persumed to be formed as an intermediate or
by product in replication of all RNA and DNA viruses. The ds RNA
also appears to be essential for induction of IFN.
The enzyme 2-SA synthetase or 2-SA polymerase, catalyses the
synthesis of 2-SA from ATP. It is active only when bound to ds RNA.
Its products are a family of oligoadenylates.
RNase L: Lengyel and his colleagues observed that in the presence
of ds RNA and ATP both reovirus mRNA and cellular mRNA were
degraded more rapidly in extracts of IFN treated cells. It was further
reported that addition of 2-SA to an in vitro protein synthesizing system
reduced the translation of mRNA which is attributable to an
endoribonuclease that is activated by 2-SA. The function of 2-SA is to
bind to latent endonuclease and activate the enzyme to destroy both
viral and cellular mRNA and rRNA.
Protein Kinase: A second pathway, independent to 2-SA
synthetase/RNaseL, is inhibited by interferon in the presence of ds
RNA. A ds RNA-dependent protein kinase was found to phosphorylate
at least one protein. The protein phosphorylated by the activated kinase
was shown to be the subnit of the eukaryotic peptide chain initiation
factor, eIF-2. Phosphorylation makes this factor inactive and the
peptide chain cannot be elongated.
The inhibition of protein synthesis by IFN may also occur by the
inhibition of methylation of viral mRNA. The S' terminal guanosine
'cap' is unmethylated apparently as a result of IFN induced changes in
the levels of S-adenosylmethionine and S-adenosylhomocysteine.
Effect of Interferon on Transformation by Tumor viruses: SVAO
DNA tumor virus has been extensively studied. It has been observed
that pretreatment of cells with interferon markedly reduced the efficacy
of cell transformation. It was found that IFN differentially affected the
expression of early viral genes whether viral DNA was integrated or
not, thus, SVI transformed cells can be passaged in presence of
infection for many generations with no effort on T antigen synthesis,
while interferon inhibits T-antigen formation in lyticaHy infected cells.
The lytic SVI multiplication cycle was remarkable in that viral DNA
114
replication and mRNA translation were sensitive to interferon added

hours after infection. In other viral multiplication cycles the antiviral
state must be established before infection for interferon to cause
inhibition. In retroviruses IFN acts before integration, it prevents either
the synthesis or the integration of proviral DNA.
Prospects for clinical use of interferon: There is little scope of
IFN finding an extensive scope in infections like influenza and herpes
virus infections but interferons may be useful in life threatening
diseases like rabies, herpes virus encephalitis and to eliminate
persistent infections caused by certain group of viruses. The remarkable
effect of IFN on immunoregulatory mechanism has focussed attention
in IFN as lymphokines. The immunoregulatory mechanism of IFN-y
and the cell inhibitory activity of lFN has raised hope that IFN may be
useful as anticancer agent.
For therapeutic use, large amount of IFN is required and such large
amounts of lFN cannot be prepared in vitro. Now with the latest
biotechnological techniques large amounts of cloned IFN will become
available. Unfortunately the IFNs cause some undersirable effects like
fever, hypertension, myalgia, tachycardia and impaired liver function
but such side effects have to be weighed against life threatening
diseases.
References
FENNER, FRANK, 1987. Veterinary Virology. Academic Press, New York.
STEWARD,
M.W. and HOWARD, C.R., 1987. Synthetic peptides a next generaJion

ofvaccines. Immunology today 8,51-58.
EsPOsrro, IosEPH I. and MURPHY, FREDRICK A., 1989. Infectious recombinant

vectored virus vaccines. In advances in Veterinary Science and
comparative Medicine. Vol. 33,196-249.
MURPHY,
BRIAN, R.and CHANOCK, ROBERT, M., 1985. Immunization agai/'lSt

viruses. In Virology edited by N.N. Fields et al. Raven Press, New
York p. 349-370.
Wn.soN, G.; MILES, A and PARKER. M.T., 1984. Principles of Bacteriology,

Virology and Immunity. Edward, Amold.
TIZARD,
I.R., 1977. An introduction to Veterinary Immunology. W.B.

Philadelphia.
Saunder~.
Chapter 12
Diagnosis of Viral Diseases
The clinical diagnostic virology involves visualisation of the virus

particles by electron microscopy. virus isolation. serological diagnosis
through qualitative and quantitative detection of specific antibodies and
direct assay for viral antigens. A recent criterion has been added which
involves the detection of viral specific nucleic acid sequences. The
choice of diagnostic method depends on nature of samples. pre-existing
reagents and the type of virus to be detected. The diagnosis is important
for undertaking suitable control measures.
Collection of material for diagnosis
They key to diagnostic success is the examination of suitable
samples. The extra expense involved in collecting the proper material
from several animals at the appropriate stage of disease and its despatch
to reference laboratory to confirm the diagnosis is negligible when
compared with unsuitable .samples collected for disease diagnosis.
Tissues should be collected under sterile conditions. Organs which arc
source of bacterial contamination should be sampled last e.g.
alimentary tract. The site of collection of specimen is related to the
clinical signs and knowledge of pathogenesis of the suspected disease.
In live animals the material from 'target organs' should be collected.
(i) Nasal or nasopharyngeal swabs-for the v:ruses infecting
respiratory tract e.g. ortho and paramyxoviruses. herpes virus and
adenoviruses. The swabs are broken off immediately after collection in
the transport medium which compnses of buffered saline or cell culture
116
medium containing bovine serum and antibiotics and fungistatic agent

to suppress the growth of contaminants.
(ii) Vesicle fluid, scab or epithelium-for foot-and-mouth disease,
pox viruses etc.
(iii) Faecal and cloacal swabs-for enteric viruses e.g.
enteroviruses. parvoviruses etc.
At postmortem virus may be isolated from target organs which
contain high concentration of the virus and are less contaminated.
(i) Lymph nodes in rinderpest or mucosal disease. The lymph
nodes are less likely to con~n contaminants.
(ii) Spleen in Newcastle disease. infectious bursal dise.'lse. swine
fever. rinderpest etc.
(iii) Liver in canine infectious hepatitis. adenovirus. enterovirus
infections etc.
(iv). Foetuses in transplacental infection e.g. equine herpes virus
(foetal-liver and lung). border disease.
(v) Central nervous system in rabies.louping ill and pseudorabies.
The specimens, after collection. are labelled and despatched to the
laboratory with its history for the provisional diagnosis. The specimens
are despatched over ice in thermosOasks or in thermocole containers. If
the transit time is longer the ice should be replenished during transit or
the material may be transported over dry ice (-70"C). For particularly
labile viruses like herpes viruses. coronaviruscs etc . the- cell culture
may be taken to the animal.
Electron Microscopy
The visulatisation of viruses, causing disease, by electron
microscope (EM) is very logical. The use of electron microscope has
been gradual. EM is used with success in detecting viruscs in faecal
contents and in detecting viruses involved in acute gastroenteritis.
There are intrinsic faults with EM:
(i) The instrument is expensive and is not available in all the
diagnostic laboratories.
(ii) A high concentration of virus particles must be present in lhe
samples. It is estimated that a lowest concentration of 10'
particles per: millilitre is essential.
(iii) The morphology may not be dislinct lO allow exact viral
diagnosis.
Diagnosis o/Viral Diseases
117
The EM has also a unique advantage. The viruses which grow with
difficulty and for those viruses in culture for which there are no
immunoassays like parvoviruses in the brain, astroviruses, calciviruses,
the detection is by EM. The inethod is fastest and is least expensive in
case the EM is available. The visualisation after negative staining, such
as phosphotungstic acid, of virus particles in field samples is a rapid
way of diagnosis. The time required is about 15 minutes. A further step
is the use of immunoelectron microscope to identify antigenically the
virus strain implicated in the disease. The time required is about 3
hours.
Most of the recently discovered viruses as rotavirus, coronavirus
were detected by electron microscopy. The samples which often give
positive results are vesicular fluid, cutaneous lesions, faeces, liver and
tissue culture supematants. The viruses which are easily observed are
parapoxvirus, herpesvirus, papiIlomavirus, rotavirus, coronavirus,
parainfluenza virus etc.
Virus isolation
Inspite of recent development of rapid methods for detection of
viral infcctions, most laboratory diagnosis of viral disease still depends
on isolation and identification of viral agents. It is probably one of the
most sensitive method in the diagnosis of viral infections if properly
collected material is used. The three major systems for isolation of
viruses from clinical samples arc tissue culture, embryonated eggs and
experimental animals. Out of these the most sensitive method is tissue
culture.
A single monolayer type is not sensitive to the growth of all the
viruses. Most diagnostic laboratories choose different tissue cultures
depending on the suspected viruses. The choice of cell lines and to use
embryonated eggs or laboratory animals like suckling mice depends on
the availability of cell lines, cost considerations and the laboratory set
up. The major types of tissue cultures used are primary cultures, diploid
cultures or continuous cell lines. The primary cuilures acquire new
properties which render the cells susceptible to infection by viruses,
when in lhe native state they might not have been. The secondary cell
cultures are similar in properties to primary cultures. The diploid cell
cultures are fibrobJastic in nalure and are sensitive to number of viruses
which arc difficult 10 culture from clinical specimens. Established cell
118
lines are derived from either normal tissues or malignant tumors, they
are heteroploid cells. These cell lines acquire unique characteristic of
growth or sensitivity to viruses. The inoculum of the suspected
specimen js inoculated in several tubes containing tissue culture and are
incubated either stationary at 36-37C or roller drum at 33C. The
medium is usually changed after an adsorption period of several hours
to one day, to prevent the non specific toxicity. The cultures are
periodically examined for signs of developing viral growth. The
frequently used sign of virus growth is cytopathic effect (CPE). Most
viruses produce characteristic morphologic changes like lysis (necoris),
Inclusion formation, cytomegally giant cell(or syncytium), particularly
myxo and paramyxoviruses are recongiscd by haemadsorption of
guinea pig or other erythrocytes on the monolayer. Rubella virus can be
detected by its capacity to interfere with the growth of second challenge
virus.
The final identification of virus isolates usually depends on the
serologic tests and sometimes on morphologic, biophysical or biologic
characterization like acid stability, plaque formation etc.
The embryonated eggs at onc time were widely used in viral
diagnosis but due to development of tissue culture techniques, which
offer superior sensitivity, the use of chicken embryo has become less.
The use of suckling mice and guinea pig is still being continued for
enteroviruses, apthoviruses, alphaviruses, flaviviruses, arenaviruses,
orbiviruses and rhabdoviruses.
Serological methods
The serologic tests are used to detect either antigen or antibody
depending upon whether a standard antiserum or standard antigen is
used. The serum samples are heat inactivated to remove
complement(C) and non specific inhibitors. The serologic tests
commonly used for viruses are as under:
1. Virus neutralization test (VN).
2. Immunofluorescence (IF) and immunoperoxidase(IP) tests.
3. Radioimmunoassay (RIA).
4. Immuno-electron microscopy.
5. Enzyme linked immunosorbent assay (ELISA).
6. Agar gel double immunodiffusion test (AGD).
7. Complement fixation test (CFI).
119
8. Haemagglutination inhibition (HI) test

9. Haemadsorption inhibition test (lID!).
1. Virus neutralization (VN) test: VN tests depend on'the ability

of antibodies to neutralise virus infectivity, as shown by inoculation
into a sensitive host system in which the virus produces recognisable
effects. In some cases addition of homologous or guinea pig
complement 10 virus and its antiserum mixture enhances neutralization.
The VN test is performed in 2 ways. In alpha procedure a constant
concentration of antibody is added to an equal volume of serial ten fold
dilution of virus. The alpha procedure is generally used to identify the
virus. In beta procedure, constant concentration of virus is added to an
equal volume of serial two fold dilutions of antiserum. The beta
procedure is generally used 10 measure antibody titre. In both
procedures after an incubation of 1-2 hours at 37C, the mixture is
inoculated in the suitable host system. The tests are scored and
antibody titre calculated by several methods. A 50% reduction in
plaque assay, a 50% protection or a neutralization index of 2 is
considered significant. VN tests are normally most specific and
sensitive: the antibodies involved arc directed against surface antigens
of the virus which arc usually type or subtype specific. The neutralizing
antibodies usually persist for long periods in the absence of reinfection.
In the serotyping, reciprocal weak neutralization may occur between
two serotypes but if monoclonal antibody is used instead of
conventional hyperimmune serum, the serotyping becomes very clear
and specific since there is no cross reaction.
2. Immunofluorescence (IF) and immunoperoxidase(IP) tests:
The IF is used to detect antigen in virus infected tissues, cell cultures or
impression smears and to detect viral antibodies. The viral antigens can
be visulaized in cells by specific antibody conjugated to a fluorescent
dye like fluorescin isothiocynate or rhodamine B. The indirect test is
more in use where the dye is conJugated to a species antiglobulin. Thus
one conjugate can be used with any antiviral sera of that species. In the
direct method the conjugated antibody is applied onto the specimen and
incubated at 37C for 30 min. The specimen gives fluorescence if a
specific reaction occurs. The indirect test is also used for antibody
screening.
A modification of the test is immunoperoxidase (JP) test when the
120
antibody is conjugated to an enzyme which will change a colourless

substrate into a coloured one as in ELISA test. This test has the
advantage that only light microscope is needed to detect antibody
binding and moreover, the preparations (slides) can be maintained for
long periods.
3. Radioimmuno assay (RIA): The detection of antibody to viral

antigen is performed by radioirnmunoprecipitation. The test involves
detecting virus being fixed to polypropylene microwells and binding
antibody by radio labelled antiglobulin. The test is rapid, extremely
sensitive and specific but due to short life of some of the chemicals
means that reagents have to be repeatedly prepared and tested. RIA also
detects antigen antibody binding sites by different test procedures e.g.
antibody by immunoprecipitation of radiolabclled antigen with
antiglobulin coated beads.
4. Immunoelectron microscopy: Some viruses which are difficult
to culture have been diagnosed by electron microscopy, e.g. ro~viruses
in faeces of animals. The irnmunoelectron microscopy involves
reacting the virus with its antiserum. The virus particles are thus
clumped and are readily identifiable by negative staining and can also
be concentrated by low speed centrifug~tion. Electron dense ferritin or
gold can also be linked to antibody so that virus antigens can be
localised in ultrathin sections of the cells.
5. Enzyme linked immuno-sorbent assay (ELlSA): The test was
developed from RIA with the additional advantage of the samples
being scored for colour either by eye or spectrophotometer. ELISA is
used either to detect antigen or antibody. It is a rapid sensitive, specific
test and is not hazardous. For detecting antibody, wells of microtiter
plate are coated with antigen and are than washed to remove excess
antigen. The antiserum is added and is allowed to react with antigen.
After an incubation period, the excess unattached antibody is removed
and wells are washed again. Then enzyme conjugated antiglobulin is
added 10 the well. After further incubation an enzyme substrate is
added. The attached anzyme causes a colour change in the substrate
which may be measured spectrophotometrically. For antigen detection
the wells of microtitre are precoated with antibody and the plate
blocked with bovine serum albumin as before. The specimen e.g. tissue
Diagnosis o/Viral Diseases
121
extract, plasma or tissue culture fluid is added and the viral antigen is
tied with the antibody. The unbound material is washed, off. An enzyme
labelled antiviral antibody is subsequently added and binds only if first
solid phase antibody has already captured antigen. The antigen can be
linked to the filling of antibody sandwich in a positive test.
6. Agar gel double immunodiffussion test (AGD): This test is

used to detect viral antigens and antibodies. The glass slides arc
covered with agarose (1 %). On hardening, wells of desired pattern are
cut. The antigen and antibody are added in the appropriate wells and
incubated. The antj.gen and antiserum diffuse toward each other in the
gel resulLing in the fonnation of precipitin lines at the zone of
equiValence. If the two antigens are identical both the precipitin lines
fuse at their continuous ends. When antigens are cross reacting they
fuse but produce a spur like projection and extend towards cross
reacting antigen. If both antigens arc unrelated the precipitin lines cross
each other. The quantitation of viral antigen or antibody can be done by
modification of AGDP known as radial immunodiffusion test. When
antigen is added to a well in the gel medium containing specific
antiserum, a ring of precipitation appears around the well. The diameter
of the ring is dependent on the concentration of antigen. By measuring
the diameter of the ring, the concentration of the antigen can be
detennined.
7. Complement fixation test (eFT): The fonnation of antigenantibody complexes results in binding and fixing of guinea pig
complement. The fixation of complement is tested by adding sheep rbc
sensitized with the homologus antiserum or haemolysin. The sensitized
rbe will lyse if unfixed complement is present. The CFf can be carried
out within a day and it detects antibodies to internal viral proteins as
well as to envelope proteins. The antigen in the test can be inactivated
if the virus is infectious for the worker in the laboratory. The CF
antibodies persist for a short period and their presence indicates that the
infection is recent. Some of the sera or antigen are anticomplementary
and the test is tedious to perfonn.
8. Haemagglutination inhibition (HI) test: The ability to cause
agglutination of erythrocytes of many species of animals and birds is
found in many virus families. This property of haemagglutination is
122
inhibited by the specific antiserum of the virus. The ill test is used
either to type antigen or measure antibody. The test is carried out in
plastic plates with multiple wells. The agglutinated rbe produce a
widespread covering with serrated edge after setting while non
agglutinated cells form a button. If the test is used to type an isolate
then standard hyperimmune or monoclonal antisera are used. ill
antibodies are rapid, specific and sensitive, the ill antibodies appear
during late clinical phase of infection and persist for long periods in
animals. In ill tests even inactivated virus can be used. The HI titre of
the seriserum is expressed as reciprocal of the highest dilution of
antiserum that completely inhibits HA.
9. Haemadsorption-inhibition test: Some virus infected cells

adsorb red blood cells of certain species of animals because of virus
envelope proteins. This is known as haemadsorption. This can be
inhibited specifically by viral antiserum e.g. para influenza viruses. This
test is performed in a manner similar to ill test.
Detection of viral nucleic acid
The detection of homologous nucleotide sequences offer a valuable
tool for recognition of certain viruses. Advances in molecular
biological techniques have been made and many of these techniques are
useful in clinical virology.
The use of PAGE (polyacrylamide gel electrophoresis) has proved
to be a sensitive method for detection of viruses belonging to the family
Reoviridae.
Molecular biological techniques
l. Restriction endonuclease digestion of DNA and analysis of

fragment patterns: The restriction e'lzyme like DNA endonculease is
capble of recognising specific DNA sequence of 4-6 nucleotide bases.
After digestion the DNA molecules are cleaved into different
subgenomic fragments of different sizes. The pattern of DNA
fragments becomes specific for each DNA species or virus, which are
isolated by using gel electrophoresis and ethidium chloride.
2. DNA cloning lechnology: The DNA sequences are copied in an

ill vivo system to produce identical clones. By using reversc
uaD3Criptase, CDNA can be produced from RNA molecules. The.
123
cloned DNA sequences of interest can be amplified through replication

of vector.
3. Nucleic acid hybridisation: The hybridisation can occur with

both DNA and RNA. The complementary base pairing of DNA
molecules is separated from each other by denaturation which is
accomplished by heating or raising the pH. Cooling allows
reassociation or renaturation. On introduction of foreign DNA molecule
with known sequence(probe), Ienaturation of the known sequence or
probe contains a shared sequence complementary with the denatured
DNA. This process of nucleic acid hybridization occurs both with DNA
and RNA. The DNA duplex can be detected if the probe is labelled
with radioisotopes or other markers such as biotin or enzymes. The
formation of a duplex of probe and target DNA may not be done
complementarily and certain amount of mismatching is allowed. The
nucleic acid hybridisation reaction can be carried out in solution since
detection of specific DNA in body fluids is the common application.
The DNA probes can also be used to detect target DNA immobilised on
nitrocellulose membranes or nylon membranes (dot blot, slit blot or
Southern blot hybridsation). The target DNA can also be detected in
formalin fixed tissues. This method is suitable for detection of
oncoviruses associated with cancer.
Recent advances in the automated synthesis of oligonucleotide
probes has made chemically synthesised short DNA sequences a
practical alternative to cloned gene. Traditionally radioactiv~ labelling
of probes (32P or 35S) is now been replaced with non radioactive
labels as some of the radioactive labels have short half lives and are
risky. The commonly used non radioactive label for DNA probes is
biotin which can be detected with conventional avidin-enzyme
conjugates or antibiotin antibodies. The DNA-RNA duplex after
hybridisation using monoclonal antibodies have also been developed.
4. Nucleic acid sequencing: The ultimate method by which the
structure, chemical and physical properties and functions of gene
products can be analysed is by determining the nucleotide sequence of
gene. It is a powerful method for the analysis of the organisation of
specific information in viruses. Two methods are involved in DNA
scquencing.
124

5. Expression of cloned DNA sequence: The strategy for cloning
and expression of DNA sequence involves the insertion of DNA

downstream from a host promoter sequence so that RNA polymerase of
the host cell can recognise the cloned gene and initiate transcription. In
certain cases the native protein encoded by the DNA insert may be
unstable in the host and expression of the gene product as part of fusion
protein may be desirable. Such fusion proteins usually consist of the
gene product of interest together with the gene product of the host
6. Polymerase chain reaction: Polymerase chain reaction (PCR)

involves in vitro enzymic amplification of specific targed DNA
sequences. After cycles of heat denaturation of target DNA, primer
annealing and primer extension, the number of target DNA molecules
increase exponentially. The amplified DNA is then analysed and
detected. The Characterisation and specificity of PCR product is
achieved by using DNA probes in Southern or dot blot hybridization.
Applications of molecular techniques in clinical virology

Detection and idetltijication of viruses: The molecular techniques
are most useful for those viruses which are difficult to be diagnosed by
conventional methods. The hybridisation techniques and probes
specific for number of these viruses have been developed. The PCR has
offered a new dimension in diagnostic virology. The use of synthetic
nucleotides as probes for detection of viruses in clinical specimens is
not required for cloning and purification of viral nucleic aeids for
probes.
Epidemiological studies of viral infections: The molecular
epidemiology has helped to trace the movement of viruses between
host populations. The molecular bioliogical techniques like
comparisons of oligonucleotide fmgerprints of viruses form different
regions or protein and nucleic acid sequences solves epidemiological
pattern resulting from antigenic shift or drift of influenza virus.
Prevention and treatment of viral infections: The cloning and
expression of viral specific proteins has led to lQe development of
vaccine production. These techniques provided tools for studies of
virulence factors associated with virus infections and offered new
approaches for engineering vaccines. Specific mutations can be
Diagnosis a/Viral Diseases
125
introduced into viruses leading to the development of attenuated

vaccine strains. In addition, isolated genes or portions of genes
responsible for neutralization of antigens of viruses can be used for the
development of subunit vaccines.
References
HITCHNER, S.B.;
DoMERMUTII,
C.H.; PURCHASE, H.C. and Wll.LIAMS, I.E., 1975.
Isolation and identification of avian pathogens. Arnold Printing

Coporation, Ithaca, N.Y. New York.
LENNETIE,
E.H. and SCHMIDT, N.I., 1979. Diagnostic procedures for viral,

rickettsial and chlamydia! infections, 5th Ed. American Publi-:: Health
Association, Washington, D.C.
A summary of serological tests used to detect common infectious diseases of

animals. Veterinary Medicine and small Animal Clinician. 766172561730, 1981.
BACIIMA.'l, P.A., 1983. New Development in Diagnostic Virology. Current
Topics Hicrobiol. Immunol. 104:
GARDNER, P.S. and MCQUllIN, 1., 1980. Rapid virus diagnosis. 2nd Edn.
Butterworth, London.
LE1'INETTE,
E.H., 1985. Laboratory Diagnosis of viral infections. Dekker, New

York.
McNULTY, M.S. and McFERRA."I, J.B., 1984. Recent Advances in virus

Diagnosis. Martinus NijhotI, The Hague.
Wll.ER, I. and DOUGAN, C., 1989. DNA probes: A new role in diagnostic
microbiology. 1. Applied Bactcrio. 67, 229-238.
WRlGIIT, P.A. and WYNFORD, T.D., 1990. The polymerase chain reaction,
limitations in diagnosis and research. J.. Pathol. 162, 99-117.
PART II
SYSTEMATIC
VIROLOGY
D.N.A. VIRUSES
Chapter 13
Poxviridae
The name of the family has been derived from 'POC' meaning a
vesicular skin disease. The family is divided into six genera as given
below and include more than 30 viruses among vertebrates.
Table 13.1
Pox
VIRUSES (FAMII.Y POXVIRIDAE)
Genus
Species
Orthopoxvirus
Vaccinia virus-type species

Cow pox virus
Camel pox virus
Buffalo pox virus
Monkey pox virus
Rabbit pox virus
Horse pox virus
Ec_lTomeJia virus
Sheep pox virus-type species
Goat pox virus
Lumpy skin disease virus
Myxoma virus-type species
Rabbit fibroma virus
Hare fibroma virus
Squirrel fibroma virus
Swine pox virus-type species
Yaba monkey tumor pox virus
Elephant pox virus
Ecthyma virus-type species
Capripox virus
Leporipox virus
Suipox virus
Parapox virus
130
Genus
Avipox virus
Species
Bovine papular stomatitis virus
Milker's node virus
Fowlpox virus-type species
CllI1ary pox virus
Pigeon pox virus
Turkey pox vims
Quailpox virus
The members of this family are largest of all viruses, brick shaped
or ovoid virions measuring 220-450 x ]40-266 nm and can be seen
under the light microscope. The surface structureo(}f some of the virions
is of diagnostic relevance. The virions have an external coat containing
lipid and enclosing one or two lateral bodies and a core. The central
core or nucleoid contains the genome. The virion contains about 30
structural proteins and several enzymes. The nucleic acid is a single
molecule of double stranded DNA of molecular weight in the range
between 150 and 240 x 106 daltons. The virus particles are built from
more than 30 structural proteins and several viral enzymes, to be used
with the nucleic acid synthesis and processing which includes a DNAdependent transcriptase. There are about ten major antigens in the virus
particle, one antigen cross reacts with most pox viruses of vertebrates.
There is genetic recombination within genus and non genetic
reactivation within and among other genera of this family. The
multiplication takes place in the cytoplasm in special viroplasm
factories and produce B type inclusion bodies. The cytoplasmic
accumulations produce A type inclusion bodies. Mature particles are
released from microvilIi or by cellular disruption.
The members of some genera are ether resistant while those of
other genera are ether sensitive. The pox viruses withstand drying for
months and even storage at room temperatures. They are destroyed by
moist heat at 60 0 e within 10 minutes. They are also resistant to many
common disinfectants. The members of orthopox viruses agglutinate
red blood cells from turkeys and about 50 percent of fowls. The
haemagglutinin is lipoprotein in nature and separable from the virus
particles. It does not elute from agglutinated red cells and specific
antibodies inhibit agglutination.
Poxviridae
131
Most of the pox viruses grow readily in embryonated eggs and

produce pocks on the chorioallantoic membrane. They can also be
cultivated in cell culture like chicken embryo fibroblasts, bovine
kidney, rabbit kidney and HeLa cells.
The viruses of this family are pathogenic for man and many
species of animals and birds resulting in the formation of focal lesions
which are often proliferative in character. The spread of infection is by
respiratory route or through the skin. Some members are also
mechanically transmitted by arthropods.
Vaccinia Virus
The name of vaccinia virus is derived from the Latin word 'Vacca'
means cow, after it was isolated from cowpox in 1796 by J enner. It has
been cultured in laboratories to produce small pox vacine. It is not
possible to establish if the virus was a. genuine cow pox virus or a
naturally occuring vaccinia virus. The detailed antigenic structure
seems to indicate that vaccinia is more closely related to variola than to
cow pox. The vaccinia virus is widespread in animals and cause local
affections and generalised diesase. The infection is also transmitted
from affected animals to human beings. The virulent strains are
particularly dangerous to immunosuppressed persons.
Properties of the }'irus: In the genus of orthopoxvirus, all members
are interrelated as found by cross protection, neutralization and other
serological tests. Ectromelia shows the lowest degree of relationship.
The vaccinia virus is labile to chloroforms and ether. The scabs remain
infected for several weeks in the tropical environment. The cell free
virus is inactivated at 50C in 1-2 hours.
The virus IS serologically uniform. The virus particle contains
about 17 antigens which fall in 3 groups. (i) Heat labile antigens (L) (ii)
Heat stable antigens(S) (iii) Nucleoprotein antigens (NP). The NP
antigen crossreacts with other poxviruses. The protective antigens are
localisd in the virus coat.
Cultivation: Vaccinia virus grows well in the developing chicken
embryos by chorioallantoic route of inoculation, producing pock
lesions. The pocks are discrete when inoculum is diluted. The virus can
also be grown in cell culture like chicken embryo fibroblast (CEF),
rabbit kidney, rabbit testes etc and cell lines like HeLa and L cells.
Cytopathic effect (CPE) appears after 48 hours of inoculation and
132
includes fonnation of giant cells, basophilic and eosinophilic inclusion

bodies in the cytoplasm.
Vaccinia virus was initially propagated in calves for vaccine
productio~. The virus was also propagated in sheep, goats, rabbits and
donkeys.
Epidemiology: During small pox vaccination period among human
beings, the infections among animals originated from children after
primary vaccination. The pustules after becoming dry, fall as scabs and
survive for a long time. The dried scabs get mixed with dust and spread
in the fonn of airborne particles.
Pathogenesis: Vaccinia virus infections in animals develop as
local, generalized or latent infection. The local infection is most
common, lesions develop on restricted areas of mucous membranes,
skin and on other organs. A generalised disease develops in
immunosuppressed animals, there is massive multiplication of virus in
the primary target organs which results in viraemia and lesions develop
on the skin, mucous membranes and internal organs. The latent
infections are rare and virus occasionally persists in lymphoreticular
tissues without being secreted.
The typical pustules which develop on the skin or mucous
membranes pass from efforscences into papules, blisters and
subsequently into pustules when necrosis of blisters, central region
occurs. The pustules dry up and fall as scabs after several days of
period. In internal organs a localized proliferation occurs at the site of
virus multiplication followed by ballooning degeneration and lysis of
affected cells. The intracytoplasmic A & B types of inclusion bodies
can be detected.
After an incubation period of 3-7 days the lesions develop in about
5 days. If there is no bacterial infection the pustules dry up and fall off
after one week. The lesions are mostly confined to mucous membranes,
skin of the muzzle udder and rarely in the genital tract.
Immune reaction: After recovery the animals acquire strong
immunity. Bot~ cellular and humoral immunity play a part in
protection. The humoral immunity is transferred from mother to
newborn through colostrum.
Diagnosis: The quick diagnosis can be done by electron
microscope detection of vaccinia virus Particles in samples of skin or
mucous membrane pustules. The diagnosis can also be arrived at by
Poxviridae
133
CAM inoculation of chicken embryos for appearance of pock lesions or

by inoculation of cell cultures to observe typical CPE or detection of
immunofluorescene. The vaccinia virus can be distinguished from
cowpox virus by inoculation of chickens by feather follicle methods.
The vaccinia virus produces typical localized pock lesions while there
is no reaction in case of cowpox virus. The virus can be distinguished
from parapox viruses by electron microscopy, pock morphology on the
CAM, and CPE in cell cultures and immunofluorescence and other
serological methods. The detection of antibodies in paired serum
samples by HI and neutralization also confirms the diagnosis.
Cow Pox Virus
The cowpox is a benign contagious disease of cattle which is
distinct form the viruses of vaccinia and milkers nodule. The disease
occurs mostly is milking cows and the lesions restricted to particular
areas of animal body like muzzle, udder, testes or scrotum on the one
hand and systemic disease with pock covering the whole body on the
other hand. The disease is transmitted from affected animals to human
beings. Like vaccinia virus, cow pox virus has a wide host range in
laboratory animals.
Properties of the virus: The morphology and physico-chemical
characters of the virus are similar to vaccinia(Table 1). The cowpox
DNA is double stranded with a MW of 80 x 106 daItons. The virus
particle is brick shaped with a diameter of 250 x 300 nm. Cow pox
virus is very closely related to vaccinia and cannot be differentiated by
complement fixation test There are reports indicating the presence of
distinct biological strains. The virus particle has nucleoprotein (NP)
and LS antigen like vaccinia. The haemagglutinin of cowpox virus
clumps turkey and fowl red blood cells. Immunologically, cowpox
virus is a single serotype. The serological test with monoclonal
antibodies are used to differentiate cowpox virus from other orthopox
viruses.
Cultivation: The virus grows well on the chorioallantoic
membrane of 7-13 day old embryonated eggs. The pocks are
characterized by an intense haemorrhagic reaction. The virus can alSO
be grown in cell culture of chicken, human and bovine cells and
produces A & B type inclusions. The virus re~dily infects guinea pig,
mouse, monkey and many other species including man.
134
Epidemiology: Cow pox is mostly transmitted by direct contact

The virus passes into the environment through dried pustules from skin
and mucous membranes, secretions from eyes, respiratory tract and
digestive tract The virus enters the body through broken skin or via the
mucous membranes of upper respiratory and digestive tract. The virus
is indirectly transmitted during milking. A large number of domestic
and zoo animals are susceptible to cow pox virus. The virus is also
transmitted to humans, where it causes local affections in the form of
pocks on hands, arms and face. Systemic disease with fever,
lymphangitis, lymphadenitis, conjuncti-vitis and disseminated pox
exanthema may also develop. In recent years the disease has been
reported in human with no contact with cattle. It is, therefore, suspected
that reservoirs of cowpox infections have been observed in various
carnivorous species (Felidae), cats, leopards, lions, -ocelots, cheetahs,
jaguars etc. The infection has also been reported from laboratory rats.
The infection with virus similar to cowpox also ocur in wild rats.
The virus is excreted in urine and faeces. It has been concluded by
Marennkikova et al (19841 that various species of wild rodents are a
hidden natural reservoir for cowpox virus, maintaining the chain of
infection and final hosts being ungulates, carnivores and humans.
Palhogenesis: The pathogenesis of cowpox is similar to vaccinia
virus infection. The incubation period is 3-7 days. The cattle may show
slight fever and anorexia followed by appearance of lesions on teats
and udder which pass through all stages of pox infection. True cow pox
lesions are 1-2 cm in diameter and are thick, tenacious and yellow
brown to red in colour. The lesions are mostly confined to teats and
lower parts of udder. In severe cases the lesions may spread to inside of
the thighs and rarely to perineum, vulva and mouth. In bulls, the lesions
u~ually appear on the scrotum. Recovery results in an active immunity
and calves born from immune dams acquire protection through
colostrum. In man the diseaSe is self limiting and benign but in
unvaccinated adults the disease may be severe. Cytoplasmic inclusion
bodies (acidophilic)' are present in the infected tissue. In comparision to
vaccinia inclusion bodies, they are large, compact, less, granular and
occur only in small numbers.
Immune reaction: The recovered animals have a long lasting
immuity, more than vaccinia virus infection. The cellular immunity
disappears faster than Immoral immunity. Newborn animals receive
maternal antibodies through colostrum.
Roxviridae
135
Diagnosis: The diagnosis is based on the appearance of finn dark

red circular lesions with raised edges and depressed centres on the teats
and udder. Confinnatory diagnosis can be arrived at by isolation of the
virus from lesiogs and carrying out serological tests for identification of
virus. The appearance of haemorrhagic pock lesions on the CAM of
embryonated eggs is of diagnositc value.
Control: There is no specific method of control of cow pox. The
use of live virus vaccines for healthy animals after an outbreak of
cowpox in a herd of zoo animals is preferred. Either attenuated strains
of cow pox or attenuated vaccinia virus strains should be used For
primary vaccination two parenteral injections at an interval of 3-4
weeks are recommended.
Buffalo Pox Virus
It is a mild viral disease of buffaloes with lesions on teats, udder,
thighs and in few cases in lips and nostrils. The disease affects
buffaloes only. The disease was reported for the first time in India by
Sharma (1934). Since then the disease has been reported from many
states of this country. The disease has also been reported from
Indonesia, Italy, Pakistan, Russia and Egypt.
Properties of the virus: The buffalo pox virus resembles vaccinia
in morphology. The size of the mature virus particle varies from 280 to
230 nm x 200 to 250 om. The virus particles are brick shaped with a
lipid coat and complex symmetry. The virus is heat labiled at 56C for
30 minutes the virus loses its infectiyity. The virus is sensitive to
chloroform and bile salts but resistant to ether. There is a close
antigenic relationship between buffalopox, cowpox and vaccinia
viruses. There is no serological relationship between buffalopox, sheep
pox, goat pox and camel pox viruses. A common group (NP) antigen
reacts with antisera of sheep pox, pig pox and fowl pox viruses. There
is complete cross protection between cowpox and buffalo pox. The
buffalopox virus contains at least 4 major components, protein soluble
antigen (LS), nucleoprotein antigen (NP), haemagglutination.,(HA) and
the factors responsible for infectivity. The HA can be inhibited both by
buffalo pox and vaccinia virus antiserum. It is immunologically
uniform and cross reacts both with vaccina and cowpox as well as with
other orthopoxivurses. It is more closely related to cowpox and
vaccinia then to other orthopox viruses. However, the biological
properties of buffalo pox virus differ from those of cow pox and
vaccinia viruses.
136
Cultivation: The virus grows well on the CAM of 12 day old

chicken embryos producing large raised or flat, white or grey pocks,
without haemorrhage. Suckling mice is susceptible, the virus can also
be cultivated in chicken embryo fibroblast and HeLa cells.
Epidemiology: The virus is species specific. Experimentally
buffalo calves, rabbits, guinea pigs and suckling mice can be infected.
The virus can infect man through contact with infected buffaloes. The
mode of transmission and other epidemiological features appear to be
the same as in cowpox.
Pathogenesis: The ~sease occurs in epidemic forms in generalised
and localised forms. The lesions are seen on the teats, udder, medial
aspect of thigh and sometimes on the quarters, lips and around the
nostrils. In generalised form, the lesions are seen all over the body.
Thickening of teats, stenosis of milk duck and mastitis are common
sequelae.
Diagnosis: The diagnosis is based on the clinical symptoms and
isolation and characterization of the virus.
COlltrol: No vaccine is available to control the disease.
Camel Pox Virus
Camel pox is a severe disease of camels and mainly affects the
young animals. The disease is characterised by generalised exanthema.
The disease resembles small pox in man rather than cow pox.
Camelpox has been known since Middle Ages. The disease is prevalent
in India, Pakistan, Egypt, Iran, Iraq, Kenya, Algeria, the Sahara, Sudan,
Somalia and Soviet Union. The disease results in heavy losses in these
countries amongst animals aged 2-3 years almost every year. The
disease causes high mortality loss in condition and fall in milk and
meat production.
Properties of the virus: Camel pox virus is a separate species in
orthopox virus genus. The morphological and biological properties are
similar to other orthopoxviruses. The virus of camel pox is brick shaped
and measures 280 x 100 nm. It is highly resistant to natural
environment; the virus is inactivated by dry heat of lOO-110C in 1
hours. Some strains are sensitive to chloroform but resistant to ether.
Different strains of virus isolated from different parlS of world are
similar antigenically. The strains isolated from Iran and USSR show
haemagglutinating activity while the Egyptian strain did not reveal this
Poxviridae
137
property. The cross neutralization tests showed an immunologic

relationship between camel pox, variola a'ld cowpox viruses.
Cultimlion: The camel pox virus grows on CAM of chicken
embryos and majority of strains produce small, opaque and white
pocks. Some strains also produce haemorrhagic pocks. The virus also
grows on a variety of cells like kidney and testis of calf, camel, lamb,
pig and chicken embryo fibroblast and BHK cells. The CPE includes
production of giant cells.
Epidemiology: Camelpox is transmitted both directly and
indirectly. The virus contaminates the environment when pustulates in
the skin and mucous membranes become dry and fall off. The
secretions of eye and upper respiratory and digestive tract is also
important in the spread of virus. Virus in dry scabs and crusts remains
infectious for 4-50 months or longer. A few cases of human infections
have been reported but these reports are not reliable.
Camel pox virus spreads rapidly in camel populations and affects
young animals in particular. There is high mortality rate besides loss in
conditon and milk
Palhogenesis: Camel pox virus is pathogenic for chicken embryos
and mice by intracerebral inoculation. The incubation period is to-IS
days. Benign as well as malignant forms of disease occur. The foals are
more susceptible than the adult animals. In young animals the disease
appears to be generalised. The lesions appear on the lips, nose, eyelids
and other parts of the body. The pustules may also appear in the
mUf:ous membrane of mouth and nose, Corneal opacity may also occur.
The mortality rate varies from 4 to 7% in young animals.
Diagnosis: The diagnosis depends upon the history, course of
disease and clinical signs. Confirmation is by isolation and
characterisation of virus based on pock morphology on CAM,
pathogenicity of adult mice by intracerebral routes and CPE in cell
cultures resulting in gi:mt cell formation etc.
Control: No prophylactic vaccine is available at present.
Attenuated vaccinia strains are recommended. For primary vaccination,
two doses at an interval of 3-4 weeks of MV A vaccinia strain are
recommended. Revaccination at an interval of 2-3 years is sufficient.
Some camel owning tribes still use 'Variolation' for protecting the
animals. The dried scabs from affected animals arc collected and dried
and kept for about one year. The dried scabs in milk are inoculated -in
the lips of animals.
138
Table 13.2
DIFFERENTIAL CHARACTERISTICS OF ORTIIOPOXVIRUSES
S. Characteristics
No.
Vaccinia
Cowpox
Buffalo
Came/pox
1.
Isolated from
Man
Buffalo,
man
Camel
2.
Pock morphology
on CAM
Opaque,
white flat
haemorrhagic ulcer
Cow,man
and large
felines
Haemorrhagic
Small
opaque
white
3.
Ceiling temperature
for CAM growth (0C)
Inclusion type
Antigenic specificity
Pathogenicity
Baby mice
Chick mortality
41.0
40.0
Large
opaque,
white
hearnorrhagic
ulcer
38.5
BandA
V.
B
Va,Vc
B
Va, Vc
+++
+++
+++
+++
+
v-
V. - Vaccinia
4.
5.
6.
Variola
Va'V.
t+
38.5
Sheep Pox Virus

Sheep pox (SP) is a highly contagious and often fatal disease of
sheep. The lesions in generalized cases are associated with
haemorrhagic inflammation of respiratory and gastrointestinal mucosa
and high mortality. The earliest record of disease dates back to the 2nd
century using serological tests. The animals recovered from infection
with one isolate were resistant to challenge with any other isolates,
whether derived from sheep or goat. The genome of sheep pox, goatpox
and lumpy skin disease of cattle are very closely related. The term
'capripox' is proposed to include the viruses of these pox diseases of
ruminants.
Cultivation: The virus can be cultivated in sheep. The reports of
cultivation in chick embryo are conflicting. SPY produces CPE in
various cell cultures of sheep origin like testes, embryonic kidney and
thyroid as well as in goat testes and kidney and bovine testes and
Poxviridae
139
kidney. The virus produces inclusion bodies in the cytoplasm of cells

which are basophilic and/or eosinophilic in nature.
Epidemiology: The disease spreads by contact with infected sheep,
their aerosals, nasal secretions, saliva or dried scabs. The air borne
infection spreads to 20-25 yards. The virus may remain viable in wool
for 2 months and on contaminated pens upto 6 months. Mechanical
transmission by biting insects may play a part.
Pathogenesis: The incubation period varies from 4-7 days. The
disease usually causes systemic reaction, with high temperature and
eruptions on the cheeks, nostrils, lips, ears, groins, udder and lips of
vulva. The lesions begin as macules with slight oedema of sUITouding
skin. The macules develop into papllles and then become pustules. The
pustules mayor may not become vesicles. In more severe cases, lesions
in buccal, digestive and respiratory mucosa also develop. The mom lity
rate may reach upto 80%. When the clinical symptoms subside, the
temperature falls and during resolution of pocks, there is itching and
shedding of wool and exfoliation of epidermis. The course of the
disease is 3-4 weeks. The postmortem examination reveals
haemorrhagic inflammation and ulcers in mucous membrane of
respiratory and gastro-intestinal tracts. Nodules may be present in lungs
and kidneys. There is gelatinous oedema of subcutaneous and
intramuscular tissues. The incubation period in natural infection is 4-8
days.
Diagnosis: The clinical diagnosis is based on the history and the
symptoms. The confirmation is based on the isolation of virus in
susceptible sheep or cell cultures. The detection of antibodies in the
serum of recovered animals can be done by neutralization, complement
fIxation, gel diffusion and fluorescent antibody tests.
Control: Animals which recover from natural infection develop
solid immunity. Ovination had been in use since 18th century. Infected
scabs powder in glycerine saline had been in uS'e in this country. Serum
virus mixture had an improvement over ovination but there is risk of
actual spread of disease. Inactivated vaccines even when incorporated
with aluminium hydroxide as adjuvallt do not produce an effIcient
immunity. A chinese strain of sheep pox adapted to CAM of chicken
embryos is reported to be avirulent and induces a high level of
protective immunity. In Russia an avirulent strain (K) grown on CAM
of chicken embryos is reported to produce immunity upto 5 months.
140
The latest vaccine developed from Kenya isolate having no clear

preference for sheep and goats. The isolate is attenuated in lambs testis
and BHK cells. The vaccine is equally protective in sheep and goat
pox. The trials carried out in Middle Ea~;t have proved the efficacy of
new vaccine
Goat Pox Virus
Goat pox is an epizootic disease resembling sheep pox. The
disease is characterised by fever, mucopurulent nasa! discharge and
generalized cutaneous eruptions. The disease is preva;~nt in North
Africa, the Middle East, Parts of Europe, India and the Far East. In
India the disease is of economic significance causing significant losses
in goat producing regions. Naturally the virus affects the goats but there
are reports that some strains may produce disease among sheep also.
Human beings may get a mild disease.
Properties of the virus: Not much is known about this virus but
many workers agree that the virus is similar to sheep pox. The virus
remains viabl~ for many months in the dried state. The virus is ether
sensitive. Goat pox and sheep pox viruses share a common antigen as
revealed by immunodiffusion and complement fixation tests. Some
cross reaction has also been reported between goat pox and contagious
pustular, dermatitis virus, cross protection between sheep and goat pox
has also been reported by some workers.
Cultivation: The goat pox virus grows readily in cell cultures of
lamb kidney, kid kidney, and testis and produce CPE similar to sheep
pox. The reports regarding cultivation of goat pox virus on CAM of
chicken embryos are conflicting. Few strains may be able to grow on
the CAM of the embryonated eggs.
Epidemiology: The virus is host specific but some strains are
believed to infect sheep as well. The disease spreads by contact with
the infected animals or infected fomites. The disease may also be
spread by insects mechanically.
Pathogensis: Following incubation period of 5-14 days small
papules appear mostly on the hairless parts of the body like nose,
mouth, around the eyes, udder, testes, scrotum, inner aspect of thighs.
The lesions develop into vesicles and crusts and subsequently heal in 34 weeks. The disease is more common in young animals or in lactating
animals. The mortality rate varies from 0 to 50%.
Poxviridae
141
Diagnosis: Diagnosis is based on the course of the disease and

clinical symptoms. Confmnatory diagnosis can be arrived at by
isolation of virus in cell culture and further characterization. Gel
diffusion test is helpful in detecting specific precipitnogens in affected
tissues.
Control: Chicken embryo adapted strains are being used in certain
countries. Goat pox strains attenuated in cell culture are being
developed.
Lumpy Skin Disease
It is an acute febrile disca"se of cattle which is characterised by
eruption of skin nodules of varying sizes. There is drop in milk yield
and abortions occur among pregnant animals. The morbidity rate varies
from 5-45% but mortality rate never exceeds 1 per cent. The disease is
known to affect cattle only in different countries of African continent.
The natural infection and transmission mode is not known. The virus
resembles vaccinia in morphology. The virus can be adapted on the
CAM of chicken embryo and in a wide variety of cell cultures. Strains
of sheep pox virus adapted in cell culture give good immunity and have
proved to be useful in controlling the disease.
Ecthyma (Ort) Virus
Orf or cC>ntagious pustular dermatitis or contagious ecthyma (CE)
is highly infectious disease of sheep and goats chamcterised by
d~velopment of pustular and scabby lesions on muzzle aRd lips. The
disease is prevalent all over the world. The incidence among kids and
lambs of 4-6 months is very high. In this country the disease occurs
wherever sheep and goats are raised. The disease is transmitted to man,
where it results in local infections.
Properties of the virus: The virus particles are elongated oval
measuring 250 x 160 nm. The virion consists of thick outer wall and
centrally placed core. The negatively stained particles look like a ball
of wool. The arrangement of filaments on the outer membrane is
different than orthopoxviruses. The tubular subunits are arranged
regularly like a single coiled up rope. Lateraly'body surface protein and
internal body are smaller. The parapox viruses replicate in their own
factories in the cytoplasm where 'virions are found in several
developmental stages. The structural elements of orf and vaccinia are
very much alike. The virus resists desication and survives for years in
142
dried scabs. The virus is inactivated by chlorofonn but is only slightly

sensitive to ether. Haemagglutination has not been reported. The virus
has antigenic cross reaction with other members of parapox genus like
pseudocowpox. Some antigenic relationship has been reported between
orf virus and sheep and goat pox viruses but orf virus appeared to be
nearer to sheep pox rather than goat pox. All orf strains isolated from
sheep, goats and man are immunologically uniform. Restriction
analysis revealed genetic heterogenicity among virus isolates.
The virus has been reported to grow in chicken embryos. It grows
well in bovine and ovine cell cultures. Testis cells are particularly
suitable, the CPE includes formation of granular cytoplasmic inclusions
consisting of acidophiloc paranuclear mass and irregular basophilic
peripheral area The virus also grows in rabbits but other laboratory
animals are not susceptible.
Epidemiology: Orf is highly cont<'lgious among sheep and goats.
The transmission is by contact, aerogenic, Or virus remaining infectious
in the ground in the dried scabs. The virus is indireclty transmitted by
way of carcasses or processed meat from latently infected animals or
slaughtered during viraemic phase. The virus can also be transmitted to
lambs by infected ews at birth or later. Latently infected sheep and
goats are virus reservoirs. Contagious ecthyma is of zoonotic
importance as human beings also get infected. The virus induces
proliferative local lesions on the skin and mucous membranes.
The mortality in sheep and goats is usually not more than 1% but
morbidity reaches 100%. Early weaning; crowding weaken the animals
defences and increase the virulence of virus as a result of rapid
passages. This gives rise to more severe form of disease with mortality
ranging 20-50%.
Pathogenesis: The disease mainly affects lambs and kids and runs
a cyclic course. The virus enters the body through skin or mucous
membranes. After primary multiplication of virus at portal of entry,
viraemia develops and primary organs of affinity become infected. The
virus gets generalised in second viraemic phase. Lesions mostly occur
on hairless parts of the body, lips, mouth and sometimes udder and
interdigital region of the feet. Lesions like papules, pustules and ulcers
develop in 34 days. The course of the disease is 3-4 weeks.
Cytoplasmic inclusion bodies occur in the affected cells. Papular
lesions also develop in human beings.
Poxviridae
143
Diagnosis: Diagnosis can be made on the clinical symptoms and

contagious nature of disease. The antigen in the lesions can be detected
by gel diffusion and complement fIxation test The rapid and reliable
diagnosis can be made by electron microscopy.
Control: The live virus vaccine is available in certain countries.
Lambs and kids are vaccinated at 1 month of age and again
revaccinated 2-3 months later.
Bovine Papular Stomatitis Virus
Bovine papular stomatitis is a mild generalised viral disease of
cattle. The disease is widespread and occurs world-wide. Latent
infections are frequent in cattle. The virus belongs to genus
parapoxvirus. It is similar to ovine orf virus antigenically and
morphologically. The clinical and physical properties resemble those of
orfvirus.
Bovine papular stomatitis is a cyclic generalised disease. When the
latent infection is activated the disease produces lesions on the mucous
membrane of the muzzle palate, oesophagus, rumen and on the udder.
The incubation period is 2-5 days. The disease is mild, initial
erythyematous changes on mucous membrane of the muzzle develop
into erosive and proliferative stomatitis. There is no formation of
blisters. The rapid and reliable diagnosis can be made by electron
microscope. The virus can be propaged in cell cultures of foetal lung,
testes and kidney of cattle or sheep, CPE with gaint cell formatio!1 and
granular lysiS is produced. The viral antigen is detected by
immuofluorescence. No specific preventive measures are available
against this virus.
Milker'S Node Virus
Milker's node is a zoonotic disease transmitted to man by close
contact with cows, sheep and goat. The lesions on the udders of cows
frequently transmit the disease to milkers. The virus can be propagated
in in cell cultures of foetal lungs, kidneys, skin from cows and sheep. A
characteristic CPE with giant cell formation and lysis is produced.
Swine Pox Virus
Swine pox is usually a benign disease of young pigs characterised
by transient fever and typical pox like lesion on the body. The disease
144
occurs naturally in pigs especially young and suckling piglets. The

disease is world wide in distribution.
Properties of the virus: The disease is caused by 1 antigenically
different viruses. One is probably vaccinia and the other is true swine
pox virus.' The swine pox virus is similar in morphology to other pox
viruses measuring 320 X 280 nm. The virus is relatively heat stable and
survives'lO-12 days at 37C. The virus of swine pox is antigenically
distinct from vaccinia although by agar gel diffusion test a minor
component is shared with vaccinia virus. There is no cross protection
between vaccinia and swine pox.
Cultivation: The virus does not grow on the CAM of chicken
embryos. It replicates only in cell culture of swine origin like pig
kidney, pig testes, embryonic porcine lungs and brains. The CPE
consists of nuclear vacuolation and intracytoplasmic inclusion bodies.
Epidemiology: The virus is transmitted by contact rarely as biting
insects play a role in mechanical transmission of the virus. The pig
louse is of major importance in transmission of the disease where the
virus survives for a year. Other insects arc also involved in the mode of
transmission.
Palhogenesis: The incubation period in swine pox is 3-7 days. The
disease is accompanied by slight fever for 1-7 days. Small papules
especially on the ventral abdomen appear. The lesions may also appear
on the inner aspect of limbs. The papules develop into vesciles and
subsequently scab formation takes place. The healing usually take 2-4
weeks. The mortality rate is very low and rarely rises upto 3 per cent.
The disease produced by vaccinia is similar to the disease
produced by swine pox virus.
Diagnosis: The disease is usually diagnosed on clinical grounds.
The infestation of pigs with lice is suggestive of swine pox. Virus
isolation may be attempted and differentiation of pig pox and vaccinia
be done on serological tests. Immunodiffusion test with swinepox
antigen gives 2 precipitating lines with swinepox convalesent serum.
Control: Recovered animals develop solid immunity. No
vaccination is carried out. The best control is by good hygiene and
eradication of lice from the swine.
Myxoma Virus
Myxomavirus causes an infectious disease of rabbits. The virus
Poxviridae
145
causes a fatal disease in domestic, Belgian and Angora rabbits,

however, cottontail and jackrabbits are resistant.
Properties of the virus: The virus is rectangular in shape which .is
similar to vaccinia and measure about 280 x 230 nm. The negatively
stained particles show a beaded appearance and consist of mass of
threads or tubules. The virus is sensitive to ether and to a pH less than
4.6. It is susceptible to heat and inactivated at 55C. It is resistant to
sodium deoxycholate and survives for many months in the skin of
infected animals. The virus cross reacts with rabbit fibroma, hare
fibroma and squirrel fibroma viruses in immunodiffusion test. The
virus grows and produces CPE in a vareity of cells like rabbit, guinea
pig, squirrel, chicken and human tissues. It produces microscopic
plaques in rabbit kidney monolayer cell cultures. The virus produces
pocks on the CAM of chicken embryos. The virus can be propagated in
the brain of suckling mice without showing clinical signs.
Palhogenecily: Following infection in rabbits there is high fever
and conjunctivitis followed by generalized tumor like swelling on the
skin. The death may take place in 48 hours with virulent strains. The
animals that survive show generalised oedema of head, nose and lips.
The mortality varies from 25 to 90%.
Epidemiology: The mosquitoes or the fleas act as mechanical
vectors and possibly by direct contact
Diagnosis: A diagnosis can be made from characteristic clinical
signs and classical lesions and high mortality. The virus isolation can
be done in cell cultures and chicken embryos.
Control: Live attenuated vaccines are used. Shope's fibroma virus,
used as a vaccine, causes a local fibroma and cross protects against
myxomatosis. The rabbits should be protected from mosquitoes and
fleas. The disease is of seasonal nature.
Fibroma Virus
Fibroma virus is immunologically closely related to myxoma virus.
The vaccination of rabbits with fibroma virus confers protection against
myxomatosis. The natural transmission probably takes place by
mosquitoes or other biting insects as in myxomavirus. In natural
infections in cotton tail and domestic rabbits, firm spherical loosely
attached turnors occur on the skin and are usually found on the foot. In
domestic rabbits the growth is rapid and regression takes place 10-12
days after infection and quickly disappears.
146
Fowl Pox Virus

Fowl pox also known as avian diphteria is a serious disease
causing heavy economic losses. The disease occurs in tow forms:cutaneous and diphtheritic. The disease mainly affects chicken but
other poul'try like turkeys, guinea fowl, peacocks, pigeons, water fowl,
pheasants, canaries and sparrows may be affected. The disease is
prevalent in all the countries of the world and is widespread in India
where outbreaks occur.
In recent past several workers have reported the breakdown of
immunity in fowl pox vaccinated flocks, heavy economic losses in
terms of mortality, drop in egg production and meat occur due to fowl
pox in this country.
Properties of the virus: The elementary bodies or Borrel bodies
resemble in morphology to other pox viruses and measure about 330 x
200 nm. The virus is resistant to drying and is inactivated at 50C in 30
minutes. The virus withstands 1 percent phenol and 1 in 100 formalin
for 9 days but is inactivated readily by 1-2 per cent NaOH. The virion
is inactivated by chloroform but is resitant to ether: however, some
strains have been reported to be moderately sensitive. The avian pox
viruses are not related to other mammalian pox viruses although NP
antigen is shared by all pox viruses. There is cross neutralization
among avian pox viruses. Fowl pox virus is closely related to pigeon
pox. Fowl pox virus haemagglutinates fowl rbc and also exhibits
haemadsorption. During the .work conducted at Punjab Agricultural
Universitx on six strains of fowl pox virus, the strains were compared
with SDS-acrylamide gel electrophoresis. The result revealed some
antigenic differences both in number of protein bands and total
molecular weight. Antigenic separation of the three strains of fowl pox
in DEAE-cellulose column also revealed slight differences in their
elution profile. Some difference in extracellular and intracellular virus
has also been reported.
Cultivation: The virus grows readily on CAM of developing
chicken embryos and produces pock lesions. The virus also grows on
chicken embryos fibroblasts and produces intracytoplasmic inclusions.
The virus has also been adapted in chicken embryo dermis cells.
Epidemiology: The disease affects all ages but is mostly seen in 512 months old chickens, although chickens few days or few weeks old
also get affected. The virus is very stable and survives in dried scabs
Poxviridae
147
and exudates for many months. The infection occurs through

mechanical transmission of the virus to injured skin. The virus does not
penetrate the intact skin. Blood sucking mthropods like mosquitoes of
the genera culex and aedes transmit the virus from bird to bird. The
mosquitoes remain infective for several weeks. Virus may also enter
through minor abrasions in the mouth caused by rough feed or through
feather follicles and also through injuries to comb, wattles or skin as a
result of fighting, pecking, scratching etc. Poultry ticks or flies may
also take part in transmission.
Pathogenesis: The virus mainl>: affects chickens of all ages but
chickens are more susceptible from 5-12 months of age. Other poultry
such as turkeys, guinea fowl etc. may be naturally or experimentally
infected. Although avian poxes have been isolated from number of
avian species but each avian pox is more pathogenic for its own host.
The incubation period is 4-10 days. The characteristic lesions of
cutaneous form arc mostly found on the comb, wallies, legs, feet, vent
and around the eyes. The lesions first appear as white foci which
develop rapidly into nodules. The lesions may coal~sce and become
rough and dark brown. The base of the lesions are inflammed and
become haemorrhagic after about -2 weeks. After another one week or
so scab formation takes place. In the diptheritic form, slightly elevated
white opaque nodules develop into mucous membranes of pharynx and
larynx resulting respiratory distress of the host.
Diagnosis: Typical lesions in comb, wattles, legs etc. are
suggestive of the disease diagnosis. The diptheritic form of disease has
to be identified from other disease causing respiratory distress. The
confirmatory diagnosis can be arrived at by isolating the virus in
chicken emrbyos or cell culture. The neutralization test is not practical
in convalescent serum samples because the concentration of
neutralising antibodies is low. The specific antibodies can be rev('aled
by complement fixation and immunodiffusion tests.
Control: The recovered birds develop a solid immunity. Two types
of live virus vaccines are in use, fowl pox and pigeon pox. These
vaccines arc prepared from infected CAM of chicken embryos. Fowl
pox vaccine prepared in tissue culture is also in use. Fowl pox vaccine
contains live attenuated virus of chicken embryos or tissue culture
origin and can be given to birds by feather follicle or wing web method.
The age at vaccination by cutaneous method is 4 to 8 wecks. The oral
148
route of va~cination gives successful results with certain strains like

HPl (German) and this can be effectively used even when the chicks
are 5 days old.
Pigeon pox vaccine of chicken embryo origin does not provide a
long lasting protection though it is comparatively safer. The
vaccination Can be done even in younger chicks less than 4 weeks old
as well as in laying flocks.
References
1984. Poxviruses. In Principles of bacteriology, Virology and
Immunity Vol.4. Edited by F.Brown and Sir Graham, WiIson, Edward
Amold, London.
BAXBY, DEVRlcK,
BLACK, D.N., 1986. The capripox virus genome. Revue Scientifique a

Technique OIE. 5, 495-501.
BUXTON, A. and FRASER, G., 1977. Animal Microbiology Vol.2. Oxford
Blackwell Scientific Publications.
FERNANDES, G., SHARMA, S.N. and TANWANI, S.K., 1981. Studies on tissue
culture adapted fowl pox disease vaccine with particular reference to
immuno-genicity of intracellular and extracellular virus. Indian
Veterinary Journal. 58: 599-604.
Firtal Report 1980-85. Development of suitable vaccine for sheeppox and
characterization of its immune response in sheep. Department of
Veterinary Microbiology and Public Heath. G.B.Pant University of
Agriculture and Technology, Pantnagar.
Firtal Report 1980-83. Immunologic characterization of buffalopox virus and
immunity in buffalopox. Department of Veterinary Microbiology and
Public Health. G.B.Pant University of Agriculture and Technology,
Pantnagar.
Firtal Report 1983-88. Biochemical and biological studies on fowl pox virus
with a view to evolve a suitable vaccine. Department of Veterinary
Bacterio-Iogy and Virology, Pubjab Agricultural University,
Ludhiana.
KrrcHING, R.P., 1986. The control of sheep and goat pox. Revue Scientifique et
Technique OIE. 5,503-511.
LAL, S.M. and SINGH, I.P., 1977. Buffalopox-A review. Tropical Animal Health
and Production 2: 107-12.
Poxviridae
149
Virus infections of ruminants, 1990. Edited by Z.Dinter and B.Morein. Elsevier

Science Publishers B.V.
Mohanty, S.B. and Dutta, S.K., 1981. Veterinary Virology. Lea and Febiger,
Philadelphia.
NAKANo, J.H., 1977. Comparative diagnosis of poxvirus diseases. In
comparative diagnosis of viral diseases VoU. Academic Press Inc.
New Yorl.
P ANDEY, R.; KAUSIIIK, A.K. and GROVER, Y.P., 1985. Biology of Ortlwpox virus
infections of domestic ruminants. In Progress in Veterinary
Microbiology and Immunology. Edited by R.Pandey, S.Karger,
Basel. Vol1, pp. 199-228.
SHARMA, S.N., Nn..AKANTAN, P.R. and DHANDA, M.R., 1966. A preliminary note
on pathogenicity and antigenicity of sheep and goat pox viruses.

Indian Veterinary Journal. 43,673-78.
SINGH, LP., PANDEY, B and SRIVASTAVA, R.N., 1979. Sheep pox: A review.
Veterinary Bulletin: 49: 145-53.
TANTAWJ, H.H., 1974. Comparative studies of camel pox, sheep pox and
vaccinia viruses. Acta Virologica, 15: 347-381.
TAYLOR, W.P., 1985. Clinical and antigenic relationship between isolates of
sheep and goat pox viruses. Tropical Animal Health Biology 17: 6479.
TRlPATHY, D.N. and HANSON, L.E., 1975. Immunity to fowlpox. American
Journal of Veterinary Research. 36: 541-44.
TRIPATHY, D.N.; HANSON, L.E.; CARANDFLL, B.A., 1981. Pox viruses of
Veterinary Importance diagnosis of infections. In Kurstak,
comparative diagnosis of viral disease. Vol. 3A, Academic Press
London.
Chapter 14
Parvoviridae
The members of this group are small sized non enveloped viruses
measuring 18-26 nm in diameter and consist of an icosahedral capsid
and probably 32 capsomeres. The genomes consists of a single
molecule of single stranded DNA with a moleeular weight of 1.5-2 x
1()6 daltons. Members of genus parvovirus has a (-) sense DNA. In
some genera the single strands of both polarities are encepsidated and
form double stranded molecules upon nucleic acid extraction. In
addition genus parvovirus the Dependovirus and Densovirus belong to
this family and found in the insects. Most of the infectious agent of
Veterinary importance belong to genus Parvovirus.
Members of the genus Parvovirus replicate in the nucleus of
cycling cells. The replication is dependent either on certain helper
functions from the host cell or from helper virus (Dependovirus). Two
main overlapping transcription units have been identified, from which 3
major mRNA species are transcribed. DNA replication occurs via a
double stranded replicative form initiated by self priming mechanism.
Following DNA replication, viral capsids assemble into which ss DNA
is packed. Accumulation of both empty and full progeny virions can be
found in the nuclei of infected cells. Intranuclear inclusion bodies can
occasionally be seen in infected cells.
Parvoviruses are remarkably stable to environmental conditions,
hence disinfection of contaminated premises is difficult to achieve.
They resist 60o~ for about 1 hour and are stable at pH 3-9. The
pathogenesis of parvoviruses is determined by the age of the infected
animal. Following the infection of foetus (Pig or cat) or newborn (dog
voviridae
151
or cat). the virus may be pantropic. In older animals a narrower range

of cells is affected. Following natural inf~on there is rapid immune
response.
Bovine Parvoviruses (BPOV)
In cattle two genera-Parvovirus and Dependovirus are found.
Bovine parvoviruses are associated with diarrhoea in calves and some
problems like abortions. Members of genus dependovirus are defective
but multiply in the presence of adenovirus or herpesvirus.
Dependovirus have not been found to be associated with pathogenic
conditions.
In 1959 Abinanti and Warfield isolated the virus from the intestine
of calves. The virus was later identified (1970) as parvovirus. The
strain was called HADEN (Haem ADsorbing ENteric) virus (type-I. A
Japanese strain different from HADEN strain is considered to represent
type-2.
Properties of the virus: The virus particles are small measuring
18-25 nm in diameter. They are extremely resistant to chemical and
physical inactivating agents. The most reliable disinfection is achieved
by. cholrax 0.5% or ethylene oxide (10% ethylene oxide and 90% CO2).
All the isolates agglutinate guinea pig and human type 0 erythrocytes.
The isolates from cattle are antigenically related or identical to
prototype BPOV-l. One Japanese strain appears to be different and is
separated as BPOV-2. Bovine parvoviruses differ antigenically from
parvoviruses of man and animals.
Cultivation: The virus can grow in actively dividing cells. Primary
cell cultures of bovine foetus spleen, kidney and lungs are used. Foetal
calf testicle, lymph nodes and adrenal are equally suitable. The CPE in
dividing foetal lungs is distinct and reproducible. After 24 hours of
inoculation, the infected cells were swollen, refractile and exhibited a
stellate appearance and ultimately showed rounding and detached from
glass surface. Intranuclear inclusions with unique morphology are
formed and can be seen after staining.
Epidemiology: The evidence of parvovirus infection of cattle by
detection of antibodies (neutralising and haemagglutination) has been
:reported from USA, England, Australia, Algiers, Japan and Brazil. The
cattle are the reservoir and remain as source of infection for calves.
Vertical transmission has been proved through detection of significant
152
antibody titres in foetal serum and isolation of parvovirus from the

tissues of naturally occuring abortion. This has been confirmed by
experimental inoculations of pregnant cows or direct foetal
inoculations.
Pathogenesis: The calves excrete the virus after 24-48 hours of
experimental infection by oral as well as intravenous route. The virus
can be isolated from mucosal scrappings of the duodenum and lower
levels of intestine for 6 days. Viraemia associated with leukocytes
develop after oral inoculation. Infections ofintestinal cells occur during
systemic phase. The infection of cells in the cortex of adrenal glands,
the thymus, lymph nodes and heart muscle become pronounced during
systemic phase. Calves aged 2-8 months become diarrhoeic with 4-7
days of oral or intranasal infection while calves infected by intravenous
route develop more severe diarrhoea.
The virus when inoculated intravenously in pregnant cows or by
direct foetal inoculation, can be isolated from blood leucocytes 6 days
post infection as well as from placenta and foetus. Foetuses of first
trimester of gestation are highly susceptible. The foetuses of 2nd and
3rd trimester can also be infected. If foetuses survived, they developed
antibodies and from the calves born alive, virus is isolated irregularly.
The BPOV seroreactor cows commonly experience higher titres of
embryonic mortality and require more services per conception than did
non reactor cattle.
Most calves with BPOV have catarrhal enteritis. Foetuses aborted
during first and early second trimester of gestation are oedematous and
have increased amounts of pleural and peritoneal fluid. Intranuclear
inclusions are formed in the cells of small intestine, liver, lymph nodes
and cerebellum.
Calves with naturally occuring infections show clinical signs in
age from 1 week to 12 months. Calves show diarrhoea, the calves
which survive diarrhoea develop antibodies but virus can be isolated
intermittently from the faeces. The parvoviruses have also been isolated
from faeces of clinically normal young cattle. The sick calves show a
rise of temperature upto 41.2C and are listless.
Diagnosis: The virus can be isolated in bovine foetal lung and
bovine foetal spleen from faeces or intestinal scrappings. Fluorescent
antibody test can be applied for locating viral antigen is tissues from
infected animals and in cell cultures inoculated with suspected viral
Parvoviridae
153
material. The ID test can be used for detecting rising titres in the serum
of animals.
Control: No vaccine is available at present.
Porcine Parvovirus (PPV)
The porcine parvoviruses are responsible for reproductive failures
like abortions, still births, teratological defects, return to service and
mummification. The PPV infection is now ubiquitous in pig herds
throughout the world. The first isolation of the report of the virus was
from swine fever virus vaccine in Germany in 1966.
Properties of the virus: The virion measures 20-28 nm in
diameter. It has an icosahedral symmetry and is non enveloped. The
virus withstands a temperature of 56C for 30 minutes and is stable at
pH 3-9 for 90 minutes. The virus is resistant to ether and chloroform.
The virus agglutinates guinea pig, rat, chicken, rhesus monkey and
human red blood cells. The haemaglutinating(HA) activity is dependent
on temperature. The highest titres are obtained at 4C. The different
strains have been shown to be antigenically similar by ID and SN
techniques.
Cultivation: The virus grows in young actively growing porcine,
primary kidney cell cultures. Primary testicular, thyroid, foetal kidney
and continuous cell lines have also been used. The virus produces CPE
and intranculear inclusions as early as 16 hour post inoculation.
Pathogenecity: The virus appears to be primarily responsible for
reproductive disorders in swine. The virus has been isolated from herds
with a history of stillbirths, neonatal losses, infertility, abortions,
myofibrillar hyperplasia, vaginal discharges from sows and semen with
respiratory disorders and loss of condition. The virus has also been
isolated from routine kidney cultures of normal piglets and foetuses.
The PPV crosses the placenta of pregnant sows and aft~r artificial
infection by oral, intramuscular and intravenous route. The sequel of
infection depends on the time of gestation. The Pathogenic effects of
the foetuses are restricted to gestational age of 45-50 days. Death
usually occurs before the onset of immune-competence which is
estimated at 70 days of ges~tion. Transplacental infection with virus
and viral antibodies also take place. The virus is also suspected to cause
infertility and congenital defects. The infection of postnatal animals
mostly results in mild clinical or subclinical signs. In experimental
cases, pneumonic lesions are also produced.
154
Epidemiology: Majority of pig herds in UK and Australia are

infected. PPV is resistant to adverse circumstances and persists in
vacated pig houses for about 10 weeks. The highest concentration
exists in gi!t houses where breeding continues. The virus spreads
through the transfer of animals at the time of active infection. Piglets
infected in utero are source of infection for about 9 weeks. S.pecific
antibody to PPV has been reported in serum of cattle, sheep, cats,
guinea pigs and rats. Serocon-version and faecal excretion of PPV
Occurs when the rats are experimentally infected. Infected rats may
possible be a source of transmission. The oral route is the usual route of
virus transfer. Since the virus has been isolated from semen and vaginal
mucous it is probable that veneral route may be important in transfer of
virus. Immune tolerance has also been reported and these animals may
be responsible for veneral transfer of virus.
Diagnosis: PPV induces high haemagglutination inhibition(HI)
and serum neutralizing(SN) antibody thres in infected pigs. The HI test
is conveniently applied in the diagnosis of infected herds. Active
immunity is characterised by persistent HI titres in excess of 256 that
continue for 4 years or even more. Litters infected in utero show HI
titre of more than 256 at 10-12 weeks of age while piglets with passive
immunity have a lower antibody titre. Dead foetuses of over 70 days
have an antibody response to PPV. The heart blood and tissue extracts
from mummified foetuses show HI titre. The foetuses less than 70 days
show antigen in most tissues by fluorescent antibody or by HA
technique. Isolation of virus can be made from tissues of aborted foetus
(mesentric lymphnodes, ileum, liver, placenta) in actively growing
porcine kidney cells. The observation time is upto 30 days. The
autoculture of tissue such as kidney readily reveal evidence of PPV
within 1-21 days.
Control: There is no vaccine available.
Feline Parvovirus: Feline Panleucopenia Virus (FPL V)

Feline leucopenia is an extremely contagious and fatal disease of
cats characterized by high fever, anorexia, vomition, depression anrl
leucopenia. The disease has a world wide distribution and is an
important disease of cats in Europe and North America. The disease
has also been reported in Panther cubs from this country. The virus was
isolated for the first time in 1964 from the spleen of a leopard.
Parvoviridae
155
Properties of the virus: The virions are morphologically similar to

other parvoviruses and measure 20-25 nm in diameter. The virus is
resistant at 56C for 30 minutes and to ether, chloroform and trypsin.
All the strains of the virus are antigenically homogenous. By virus
neutralization test it has been shown that this virus is closely related to
mink enteritis virus and canine parvovirus. The virus haemagglutinates
porc;ine red blood cells at 4C.
Cultivation: The virus can be cultivated in rapidly dividing celIs
of feline origin. The virus produces weak transitory CPE in feline
kidney cell culture and produces type ~A' intranuclear inclusion bodies.
The virus does not grow in fertile hen's egg or in laboratory animals.
The virus also replicates in cell cultures of lion, tiger and mink tissues.
Pathogenecity: Feline panleucopenia is a contagious disease of
young cats but cats of all ages are affected. The incubation period
varies from 4-10 days and mortality rate in affected animals exceeds
60-70 percent and may even reach 100 percent. The clinical symptoms
range widely from paracute to mild cases. In paracute cases death
occurs in 24 hours. In acute cases lhe course of the disease is 5-7 days.
The symptoms are high fever, anorexia, marked depression and
dehydration. Vomition is common and diarrhoea may occur in 2-4
days. Before the temperature there is an initalleucocytosis followed by
decrease of Jymphocytes and polymorphs. The leucopenia becomes
pronounced with the progress of disease. The virus can pass the
placenta and induce abortion. The small intestine is inflammed,
thickened and oedematous. There is necrosis of intestinal mucosa, and
mesenteric lymph nodes. The red marrow of long bones becomes
semifluid in consistency and cellular elements are considerably
decreased. The disease can be transmitted to minks. It is considered that
feline panleucopenia, mink enteritis and feline ataxia are caused by the
same virus.
Epidemiology: All secretions and excretions contain the virus.
Recovered cats act as carriers and shed virus in urine and faeces for
about 1 year. Under natural conditions the virus spreads by direct
contact with infected animals or contaminated material. The natural
portals of entry are respiratory and oral route. The fleas and other biting
insects may act as mechanical carriers because the virus remains in the
blood of affected animals.
156
Diagnosis: The provisional diagnosis can be arrived at the clinical

signs, presence of leucopenia and presence of intranuclear inclusions in
the intestinal epithelium. Virus can be isolated from spleen and other
tissues in feline kidney cells. A rise of antibody titre in paired serum
samples can be detected by HI test.
Control: Live attenuated and inactivated vaccines are available.
The attenuated vaccines are derived from FPLV or mink enteritis virus.
The immunity produced is superior to inactivated vaccine. Two doses
of vaccine are given, the flrst dose at the age of 9-10 weeks and second
dose 2-6 weeks later. The live vaccine is not recommended for kittens
less than 4 weeks and for pregnant animals.
Canine Parvoviruses
Canine parvovirus-l also called as minute canine virus was
identifled in the faeces of dogs in 1967 but is not associated' with any
major cause of disease in dogs, although it may cause mild diarrhoea.
Canine parvovirus-2 which is antigenicaIly distinct from canine
parvovirus-l was rccognised in 1978 as a cause of haemorrhagic
gastroenteritis simuItanasouly from USA, Australia and UK.
Subsequently the disease has become enzootic in dogs through out the
world. In India the disease was reported by Balu and Thangaraj in
198], in and around Madras city. The disease has also been reported
from other parts of the coun try.
Canine parvovirus (CPV) is a small, non-envelop"ed, icosahedral
virus measuring approximately 20 nm .in size. The icosahedral capsid
encloses single stranded DNA genome. The virus is relatively stable. It
is satable at pH 3-9 and at 56C for 60 minutes as well as in the
presence of lipid solvants. The virus agglutinates pig and rhesus
monkey red blood cells at 4C and 25C and not at 37C. The
haemagglutination is inhibited by CPV antisera. The virus can be
grown in actively dividing dog epithelial ceUs.
The epidemiology of disease is similar to feline panleukopenia.
The virus is excreted in high concentrations in faeces. Three age related
syndromes have been recognised. Generalised neonalal disease is rare.
The panleukopenia but cerebraUar hypoplasia found in cats is absent.
Myocarditis is recognised as acute disease in pups characterised by
sudden death without any clinical signs. Apart from diarrhoea which
can be haemorrhagic, vomition often occurs. In acute cases there is
157
Parvoviridae
sudden death while in less acute cases, respiratory problems occur and
pulmonary oedema develops. The morbidity rate is very high upto
100% while mortality rate is upto 10%. The demonstration of virus by
haemagglutina-tion and haemagglutination inhibition test of faeces and
serum samples confirms the diagnosis.
Vaccination with either attenuated live virus or inactivated
vaccines is effective. The problems are erlcoutered in devising effective
vaccination schedules because of variable levels of antibodies
transferred to pups from mothers.
References
ABINANTI, F.R. and W ARFIFLD, M.S., 1961. Recovery of haemadsorbing virus
(HADEN) from the gastrointestinal tract of calves. Virology. 14,
288-289.
ACHERMANN, 0., 1982. Canine parvovirus infection and its prophylaxis. The
Blue book Dec. 1982 pp. 1-9.
APPEL., M.J.G.; COOPER, B.J.; GREISEN, H.and CARMICHAEL, L.E., 1978. Status
report. Canine Viral enteritis. J.Amer. Vet. Med. Assoc. 173: 15161518.
BUXTON, A. and FRASER, G., 1971. Animal Microbiology. Vo!.2. Oxford,
HOGGAN, M.D., 1971. Small DNA viruses. In comparative Virology edited by
K.K. Marmorosch and Kurstak. Academic Press, Inc. London
JEE, H.S. and JOIINSON, R.H., 1976. Porcine parvovirus: A review Veterinary
Bulletin 46: 653-660.
KAHRS, R.F., 1985. Viral diseases of eaU le. Kalyani Publishers. Ludhiana.
KEu..Y, W.R., 1978. An enteric disease of dogs resembling feline pan

leulwpenia. Aust. Vet. J54: 593-595.
MENGEUNG, W.L., 1975. Porcine parvovirus. In diseases of swine. Edited by
H.W.Dunne and A.D.Leman. Ames, Iowa, Iowa State University,
Press. 1975
MOHANTY, S.B. and DUTl'A, S.K., 1981. Veterinary Virology, Lea and Febiger,
Philadelphia
M.J., 1978. Growth of an autonomously
(feline pauleulwpenia): Kinetics and
morphogenesis. Archieve Virology 57: 107.
O'SHBA, J.D. and
replicating
STUDDERT,
parvovirus
158
Textbook o/Veter inary Virology
STORZ, J., 1990. Bovine parvoviruses. In Virus infections of ruminan

Js, edited
by D. Zintcr and B.Morein. Elsevier Scinece Publishers B.V.
Amsterdam.
STORz, J.; Leary, 1.1.; CARSON, J.H. and BATES, R.C., 1978. Parvovi
ruses
asrociated with diarrhoea in calves. Journal American Veterinary
Medical Association. 173: 624-627.
WOODS,
G.T., 1975. Bovine parvovirus-l, Bovine syncytial virus and bovine

respiratory syncytial virus and their infections. Advances in
Veterinary Science and comparative Medicine.
Chapter 15
Papovaviridae
The family includes two Papilloma virus and Polymavirus. The

name Papova was derived by connecting ftrst two letters from the
names of three viruses-Papillomavirus. Polyomavirus and Vacuolating
agent. Papillomaviruses are tumorigenic causing papilloma in different
species of animals for centuries but the viral etiology was rccognised in
1907. A possible bovine polymavirus was demonstrated in cultured
kidney cells of newborn calf. Polymavirus produces tumors in mice.
The vacuolating agent is a simian virus also known as SV40.
Papillomaviruses are nonenveloped. icosahedral measuring 50-55 nm
in diameter. Polyoma-viruses are smaller measuring about 45 nm in
diameter. The capsid is formed of 72 well discernible capsomeres in a
skew arrangement The viral core contains a single molecule of
infectious circular double stranded DNA with a molecular weight 3-5 x
1()6 daltons. The G+C content is 41 to 49%. They are ether stable and
withstand heating for 30 minutes at 56 to 65C. The papilloma viruses
are species speciftc but different types may be associated with various
forms of papillomatosis in a single animal species. The subdivision of
papillomaviruses into six types is made by serology and by comparing
restriction endonuclease cleavage patterns of their DNAs and DNADNA homolgy following hybridization. The replication occurs in the
nucleus but viral DNA is not integrated into cellular DNA. For growth
of papillomaviruses permissive cell type is lacking but established cell
lines respond to infection or transfection with foci of transformation.
Papillomaviruses. which were once thought to be of minor
importance in animals and man are now being recognised significant
160
oncogenic factors in some neoplastic entities. Laryngeal papilloma and

cancer, as well as carcinomas of the cervix and skin in man, skin cancer
in goats, eye cancer in cattle and equine sarcoid have been associated
with papillomavirus infection. An internal capsid antigen is common to
all papillomaviruses. The bovine papillomavirus (BPY) 1 and 2 can
serve as eukaryotic vectors in genetic engineering to insert foreign
genetic material (DNA) into cells in culture.
The warts of skin are usually self limiting benign tumors, which
occur usually in young aminals. The bovine papillomavirus 1 and 2
(BPY-l and BPY-2), the ovine papillomavirus, the deer fibroma virus
and European elk papillomavirus produce fibroblastic tumor in
hamsters. The BPY -1 and BPY -2 can also cause fibroblastic tumor in
horses. The papillomavirus infects the basal cells of epithelium. Some
of cells degenerate while others are stimulated to excessive growth and
wart formation. The new virus particles are found in the degenerating
cells and therefore there is much infective virus at the surface of the
wart. The virus is resistant and contaminates the faeces and animal
houses. The wounds on skins, tattoo instruments and hypodermic
needles lead to infection in cattle.
Bovine Papillomavirus types 1 and 2
Bovine papillomavirus type 1 and 2 (BPY-l and 2) are world wide
in distribution and cause fibroblastic tumors of the dermis in addition to
epithelial hyperplasia in cattle less than 2 years of age that are housed
in close contact with each other. The tumors commonly known as
fibropapillomas are common on the skin of neck, legs, back and
abdomen and are of various sizes. The tumors have a cauliflower like
appearance and a fibroma base in the dermis. The fibropapiIlomas of
the penis and vulva-vaginal mucosa have a smooth surface with less
epithelial proliferation. The virus is concentrated in the outer
keratinized epithelium and when shed contaminates the fences and
animal houses. Experimentally growing fibroblastic tumors of brains in
cattle and hamsters and polyploid tumors of urinary bladder can be
produced in calves with BPY. In certain parts of world bovine
haematuria is assoicated with bracken fern and BPY infection.
Yirus is transmitted between animals by contaminated halters, nose
rings, grooming equipment, rubbing posts and other articles
contaminated with diseased animals. The virus gains entry through
Papovaviridae
161
abrasions in the skin by direct contact with infected animals etc. The
disease is more common in housed animals than in cattle on pastures.
The warts are characterisitic and laboratory diagnosis is seldom
necessary. The virus particles can be demonstrated by electron
microscopy.
The lesions usually disappear spontaneously. Formalinized
suspensi.on of bovine warts provide a vaccine for prophylactic immunization when warts are a problem in a herd The efficacy of the
vaccine is difficult to assess.
A situation has been reported in which fibropapillomas of the
oesophagus and fumen harbour the genome of BPV -2 but neither the
mature virus nor its antigens could be dctected. This suggests epithelial
transformation to neoplasia without production of infectious virus.
Uovine Papillomavirus type-3
A typical warts in cattle which lack the dermal fibroma component
and tend to persist rather than regress involve adult as well as young
animals. The atypical papillomas contain a virus similar in morphology
with BPV-I and 2 but immunologically distinct. The vaccine
containing formalinized atypical warts does not control the natural
incidence of the disease.
Bovine Papillomavirus type-4
On the west coast of Scotland with much growth of bracken fern,
papillomas and carcinomas are found on the tongue, pharynx,
oesophagus, rumen and intestine of cattle. The tumor extracts produce
typical papillomas of the oesophagus, palate and skin. The causative
agent has been characterized as BPV type 4. The BPV -4 is found in the
papillomas of alimentary tract but alimentary carcinomas had no
demonstrable virus or viral antigens. Similar carcinomas of the
alimentary tract have been observed in enzootic haematuria areas of
Brazil and Colombia.
BPV -5 is the cause of papilomas on the teats of cows.
BPV -6 has been characterized in teat epithelial papillomas.
Virus particles resembling BPV have been found associated with
ocular lesions in Australian cattle.
162
Fibropapillomas of muzzle and legs in sheep in England have been

reported due to papillomavirus.
Canine PapilIomavirus
This type of wart usually begins on the lips and spreads to buccal
mucosa, tongue, palate and pharynx of puppies. The warts resolve
within few weeks and do not affect the dogs over 1 year of age.
Formalinized wart suspensions may have a prophylactic but no curative
value.
Rabbit Papilloma viruses
There are two distinct viruses causing papillomas in rabbits. The
oral papilloma virus is similar to that of bovine or other
papillomaviruses. The other virus or shope papillomavirus is found in
cotton tail rabbits where virus particles are readily detected.
Experimental transmission of shope virus to domestic rabbits results in
formation of cutaneous tumors with maiked virus where virons are not
detected but infectious viral DNA is present.
Equine Papillomavirus
Warts may be seen on the skin around lips and muzzle of young
horses between 1-3 years of age. The lesions may perisist upto 18
months. The agent is readily tran~missible to horses. The sarcoids are
also produced by the same virus. All ages of horses may be affected
and lesions persist for about 8 months.
References
BUXTON, A. and FRASER, G., 1977. Animal Microbiology, Vol. 2. Oxford
HuCK, R.A., 1969. Bovine papillomatosis: Synonymous warts. Infections
verrucae, Veterinary Bulletin 35475-477.
JARRETI, W.F.H.; Mc NEIL, P.E.; LAIRD, H.M.; 0' NEIL, 1.; MURPHY, 1.; CAMPO,
M.S. and MOAR, M.H., 1980. Papilloma viruses in benign and

malignant tumors of cattle. Cold Spring Harbor Conference on cell
Proliferation. 7: 215-222.
LANCASTER, W.D. and OLsON, C., 1982. Animal papillomaviruses. Microbiol.
Rev.46, 191-207.
MELNICK, 1.L., 1962. Papovavirus groups. Science 135: 1128.
PFIsTER, H., LINZ, V.;
GISSMANN,
L.; HUCHllIAUSEN, B.; HOFFMAN, D. and
Papovaviridae
163
ZURHAUSEN, H., 1979. Partial characterisation of new type of bovine

papilloma virus. Virology .96, 1-8.
S.B. and DUTIA, S.K., 1981. Veterinary Virology. Lea and Febiger,
Philadelphia.
MOHANlY,
OLsON,
C., 1990. Papillomaviruses. In virus infections of ruminants, edited by

D2inter and BMorein, Elsevier Science Publishers B.V.Amsterdam.
RUSSELL,
P.H. and EOINGTON, N., 1985. Veterinary viruses. The Burlington

Press (Camb). Ltd. Foxton, Cambirdge.
Chapter 16
Adenoviridae
Adenoviruses for the nrst time were isolated in 1953 by Rowe and
his colleagues in uninoculated cultures of human adenoid tissue (adcn
means glands) of children. Adenovirus have been isolated from man,
monkeys, cattle, swine sheep, dogs, mice and birds.
The virion is nonenveloped with icosahedral symmetry, 60-90 nm
in diameter. The capsid comprises of well discernable protein subunits
(capsomcres) surrounding a core of DNA and internal arginine, rich
proteins. The 252 capsomeres of the capsid are arranged into an
icosahedron having 20 triangular facets and 12 vertex capsomercs. The
12 capsomeres at the vertices have 5 neighbours called pen tons. Each
vertex capsomere carries 1-2 ftlamentous projections. The 240 nonvertex capsomeres have six neighbouring capsomeres and are called
hexons. The adenoviral genome is a single linear molecule of double
stranded DNA of mol. wt between 20 - 25 x 1()6 daltons. The G+C
content is 48-61 %. The terminal nucleotide sequences of each strand
are inverted repetitions. There are at"least 10 polypeptides in the virion
with molecular weights ranging from 5K to 120K. After uncoating the
viral DNA is transcribed into early proteins and late proteins. DNA
replication occurs by strand displacement; transcription in the nucleus
is followed by splicing into mRNA's. The mRNAs migrate into the
cytoplasm, where the structural polypeptides are synthesized. The
assembly of virus particles take place in the nucleus and release of
matured virus particles take place by disintegration of the damaged
cells. The host cells DNA, RNA and protein synthesis is effIciently
Adenoviridae
165
stopped by the infection. Some members of the family haemagglutinate

rbe. The virion is resistant to ether and relatively stable below 50C.
The family has 2 genera:- 1. Mastadenovirus-includes all
mammalian' adenoviruses and share a common antigen which is not
possessed by other genus 2. Aviadenovirus-includes all adenoviruses
of birds and these aviadenovirus do not haemagglutinate rbe of rats and
other species of animals. The adenoviruses can be cultivated in cell
cultures of natural host or closely related species. The CPE is
characterized by rounding or clumping of affected cells resembling
'bunches of grapes'. The CPE is produced late, 7-10 days longer. Since
the virus multiplies in nucleus, the nuclear changes include fonnation
of basophilic or acidophilic inclusion. The inclusions represent
aggregation of proteinaceous material and crystal of mature and
immature particles.
Bovine infections with adenoviruses are inapparent but in calves
pneumonia, enteritis or pneumoenteritis, conjunctivitis or keratoconjunctivitis are produced either solitary or as membrers of miscellaneous
microbial flora. In sheep adenoviruses produce enteric and respiratory
disease. Among the dogs the diseases are infectious canine hepatitis
and canine laryngotracheitis. In poultry the virus is associated with
drop in egg production, inclusion body hepatitis and respiratory
problems. Many' adenoviruses are capable of inducing malignant
tum.ors in newborn hamsters. The oncogenic activity of adenoviruses is
closely 'associated with base composition of nucleic acid. Highly
oncogenic viruses have a low, 48-49 percent G+C content and mildly
oncogenic types have 50-51 percent of G+C content while nononcogenic types have the highest G+C content, 55-60 percent.
Bovine Adenoviruses (BA V)
Properties of virus: The bovine adenoviruses known so far have
been classified into nine serotypes. These serotypes have been divided
into two subgroups based on the replication of viruses either in calf
kidney or testicle cells. These two subgroups also differ in their
antigenic properties. The new bovine isolates are constantly being
reported and isolated from apparently healthy cattle as well as among
those with clinical illness. Seroconversion or rising titres among
healthy cattle have been observed.
Members of subgroup 1 readily replicate in bovine kidney cell
166
cutures. They ar~ heat sensitive and have a complement fixing antigen
common to human adenoviruses. The members of subgroup 2 are not
easy to isolate and require few blind passages in calf testicular cells and
do not hav.e a complement fixing antigen with human adenoviruses.
The member of this subgroup invariably require calf testicular cell
culture for their replication. Some bovine adenoviruses are highly
cytopathogenic and some are less cytopathogenic. Most of the viruses
produce intranuclear inclusions in cell culture. Typing of bovine
adenoviruses is based first of all on cross neutralization tests. Only few
serotypes show haemaggluti-nating activity. Rat erythrocytes are
agglutinated by types 1, 2, 4 and 7. Bovine erythrocytes are
agglutinated to low titre. The two subgroups are differentiate~ by
complement fixation and immunodiffusion tests. It has recently been
reported that certain ovine adenovirus isolates cross neutralised with
BAV type 2 and infected both sheep and cattle under experimental
conditions.
Bovine adenoviruses show remarkable stability outside their hosts.
They are resistant to pH 2, pH 11, trypsin (0.25%), heating for 30 min.
at 50C., NaOH at 0.5-2% concentration inactivate the virus and are
good disinfectants.
Cultivation: Subgroup 1 viruses replicate in wide range of cultured
mammalian cell types, while subgroup 2 viruses grow in calf testicle
cells. Single inclusion are seen in subgroup-l and mulLiple inclusions in
the nucleus in subgroup-2
Epidemiology: The BAV is ubiquitous among cattle popUlation
and it appears that cattle are potential reservoir. Serological evidence
suggests a high incidence of infection with certain serotypes being
replaced by others from time to time. The prevalence of antibodies is
more in cattle which have suffered from respiratory disease. The
transmission in calves is by direct contact Actually infected animals
may shed the virus in nasal and conjunctival secretions, faeces and
urine.
Palhogenesis: The BAV was originally considered to be non
pathogenic for cattle but now it has been proved that the viruses are
associated with several diseases of cattle. Observations on
experimentally and naturally infected cattle indicate tJtat BAV is
associated with diarrhoea, enteritis, pneumoenteritis, pneumonia,
keratoconjunctivitis and weak calf syndrome. After infection there is
Adenoviridae
167
viraemia. The incubation period by experimental inoculation is 2-3

days but in natural cases it may be longer and possibly about 2 weeks.
Infection occurs mostly in calves between 3 weeks to 4 months old.
There are no gross or histopathological changes pathogenomonic for
disease syndromes caused by BAV. Intranuclear inclusions are found in
the organs after infection in the early stages.
Immune reaction: The adenoviruses are potent antigens. About 14
polypeptides are associated with the virion. High titre of antibodies
appear after infection. Maternal antibodies provide passing protection
against the homologous virus type.
Diagnosis: Isolation of virus from nasal and eye swabs and rectal
swabs must be attempted. On post mortem, various organs, kidneys,
testicles are homogenised and used for virus isolation. Fourfold
increase in SN antibody titres between acute and convalescent serum
samples confirms the diagnosis. Viral antigens can be demonstrated by
CIEP and direct IF tests.
Control: The progress in immunoprophylaxis against BAV is
slow. Bivalent adenovirus vaccines have been developed. The viruses
are inactivated Wilh p-propiolactone or formaline and aluminium
hydroxide and saponin used as vaccines. The vaccination is carried out
in pregnant cow and also in the calves or in the calves alone. The cows
are vaccinated twice in the last trimester of pregnancy and their calves
are vaccinated at 6-8 weeks of age. Vaccination is repeated in calves
10-12 weeks old. Vaccination of calves alone is also practised in
certain parts of Europe.
Ovine Adenoviruses (OA V)
Adenoviruses have been isolated from normal as well as from
pneumoenteric diseases of sheep. The OA V are responsible as the
causative agent of enteritis and respiratory infections in lambs.
Properties of the virus: The virus particles are resistant to heat
The exposure at 56C for 30 min. does not inactivate the virus although
there is a fall in titre of the virus. Divalent cations significantly
decrease heat resistance. All straIns are resistant to pH 3, ether and
sodium deoxycholate. Sodium hypochlorite and formaline produce
complete inactivation.
Sheep adenoviruses have been classified into six serotypes so far.
All serotypes agglutinate rat erythrocytes. All the strains share a soluble
168
group specific antigen with human adenoviruses. The six serotypes

could be distinguished by cross neutralization tests.
Cultivation: Ovine adenoviruses readily replicate in various ovine
and other Jl?ammalian cell cultures such as lamb kidney, testis, lung
cells, calf kidney and testis cells, pig kidney cells and in MDBK cell
line. OAV-6 preferably multiplies in lamb testis cell cultures. The CPE
appears 14-24 hours of post infection and comprises of increased
refractibility rounding of cells followed by lysis.
Epidemiology: The ovine adenoviruses are largely confined to
their host species. However, BAV-2 was observed among lambs
causing pneumoenteritis. The presence of adenoviral antibodies in
sheep populations have been reported from several countries all over
the world including India. Adenoviral pneumoenteritis causes grtat
losses in lambs due to mortality, increased food consumption and
retarded growth. Virus is shed mainly with the nasal discharge, faeces
and urine. Direct contact is most important in the spread of infection.
Fattening lambs in units with continuous replacement are most likely to
develop the disease because the passage of virus is uninterrupted
resulting in permanent chain of infection and enhanced virulence.
Crowding, insufficient ventilation, heat stress aggravates the disease
outcome. The disease is frequently accompanied by other viruses and
bacteria. Simultaneous infection with reovirus and/or parainfluenza-3
virus is common. Pasteurella haemolytica. corynebacteria and
Mycoplasma ovipneumonia is also common.
Inapparent infection is common. Where clinical disease occurs
morbidity may reach 100%. The disease occurs in 2-12 week old lambs
and losses may be 30-40% in suckling lambs and 10-15% in fattening
lambs. About 10% of losses occur in acute phase of disease while 90%
in second phase when bacterial infections complicate the process.
Pathogenesis: The disease can be reproduced by intranasal and
intratracheal inoculation of lambs. The virus replicates in the
respiratory and intestinal tract, from 4 day post infection, pathological
changes appear in other organs. Lesions are found in nasal mucous
membranes. lungs, intestines, lymph nodes, spleen, kidneys and liver.
The virus is excreted in the nasal discharge and faeces from 2-3 days of
post infection onward. In naturally infected lambs the virus can be
isolated in the acute phase of disease. Later lambs become carriers and
virus can be isolated occasionally from animals with antibodies. The
virus is intcrmitently excreted in faeces.
Adenoviridae
169
In nature the disease starts with dia.rrhoea after an incubation

period of 7-8 days. The respiratory symptoms follow 2-3 days of
diarrhoea. Sneezing, nasal discharge, conjunctivitis and lacrymation are
common. Diarrhoea stops after about 7-8 days but respiratory
symptoms persist, turning chronic in most cases.
Lesions of focal interstitial pneumonia are common. The mucous
membrane of small intestine is generally congested and covered with
viscous exudate. The retropharyngeal, peribronchiolar and mesentirc
lymph nodes are swollen. Greyish white foci are seen in the cortex of
kidneys.
Diagnosis: Virus isolation is attempted from nasal secretions
faeces and urine. The samples for virus isolation should be collected
early in acute period of disease. Lamb kidney, thyroid and testicle cells
are suitable for virus isolation. The CPE appears after one or two
passages. Viral antigens can be detected in the organs and infected
tissue cultures by direct immunofluorescence test. Serologic screening
can be done by gel precipitation test, immunofluorescence test and
ELISA. Neutralisation can be carried out against different serotypes of
ovine as well as bovine adenoviruses.
Control: Inactivated, bivalent adenovirus vaccines are available in
Europe. Two doses are given 10 or 42 days following fIrst dose.
Regular vaccination has reduced the losses due to pneumoenteritis.
Pregnant ewes are vaccinated twice with a 6 week interval. The second
dose to be given not later than 3 weeks before lambing. The lambs born
out of these sheep are actively vaccinated around 5 weeks of age and
given a booster vaccination 10 days later.
Canine Adenoviruses (Infectious canine hepatitis virus (ICHV,
Fox encephalitis virus
Infectious canine hepatitis is a febrile disease which mainly affects
young dogs and foxes and cause centrilobular necrosis of liver.
Experimentally the disease can be produced in guinea pigs, wolves,
racoons etc. The ferrets are resistant. The disease is widespread in
Europe, North America and other parts of world. Rubarth in 1947, fIrst
recognised this virus as a <;linical entity in dogs. The disease was
reported from India by Nair (1955) and Mukerjee and Mehrotra (1983).
Properties of the Virus: The virus has a morphology similar to
other members of adenoviruses. The virus particle measures 55-80 nm.
170
The virus is resistant to 0.5% phenol for several days and survives for
10-14 weeks at room temperature and for 6-9 months at 4"C. The virus
is inactivated by 0.2 per cent formaline within 24 hrs. The virus
agglutinates human '0' guinea pig and rat rbe. The virus shares a
common complement fixing antigen with human adenoviruses.
Cultivation: The virus is readily propagated in primary dog kidney
and testicular cells. The virus can also be propagated in cell cuitures of
pig, ferret, racoon and guinea pig origin. The CPE is characterized by
rounding, swelling of infected cells and formation of intranuclear
inclusions.
Epidemiology: The virus is excreted in the secretions from the
respiratory tract as well as in urine and faeces. The excretion in the
urine continues for a long time, at least 200 days after the acute phase
of illness. The virus is spread by direct contact of healthy animal with
infected animal or indirectly from contaminated surroundings and
clothings.
PalllOgenesis: The dogs of all ages are susceptible although young
dogs are more susceptible. Experimentally, guinea pigs, wolves,
racoons and coyotes are susceptible. Depending upon the virulence the
incubation period varies from 2-10 days. In severe cases, the dogs
suddenly die with acute abdominal pain, diarrhoea and vomiting in 1214hrs. In less acute cases there is high fever, apathy; diarrhoea,
vomiting, transient corneal opacity, enlargement of tonsils and
submaxillary lymph nodes and leukopenia. The dog assumes tucked up
position. The acute disease is usually fatal within a week but mild
forms of the disease also occur.
In foxes, the disease is characterized by acute encephalitis with
convulsions followed by paralysis, coma and death.
On postmortem the animal shows subcutaneous oedema, hacmorrhagic exudate in peritoneal cav~ty and intestinal tract. The liver is pale
and enlarged. The gall bladder wall is oedematous and vcry thick. The
spleen is enlarged and haemorrhagic. Microscopically, the liver cells
show necrosis and dilation of sinusoids. The intranuclear inclusions are
seen in the liver cells.
Diagnosis: The clinical picture and post mortem findings are
helpful in reaching at the diagnosis. The presence of intranuclear
inclusions in smears or tissue sections is suggestive of ICHV. Viral
isolations can be made in primary kidney cultures from blood, tonsils,
Adenoviridae
171
conjunctival sac, urine and nasal secretion of affected animals. The

paired serum samples collected from affected animals are titrated for
the presence of rising antibodies by complement-fixing and
haemagglutination-inhibition antibodies. The fluorescent antibody test
is useful for detection of ICHV antigen in the tissues of affected dogs.
Control: Both live and inactivated vaccines are being used for
immunization. The virus was modified by serial passages in dog kidney
cultures and .subsequently in pig kidney ceh cultures. The modified
virus alone or in combination with attenuated distemper virus is used as
a vaccine. Canine immunoglobulins confer passive immunity in pups
for about 3 weeks and can be used in exposed animals.
Infectious Canine Laryngotracheitis Virus
A virus isolated by Ditchfield and his associates in 1962 was
reported from the cases of respiratory illness in dogs. The virus is
antigenically related to ICHV but is distinct antigenically on
haemagglutination inhibition and neutralisation tests. The virus is
considered to be an antigenice variant of ICHV.
Equine Adenoviruses
Adenovirus infection among foals is worldwide. The viruses casue
a disease of upper respiratory tract in foals below 3 months. The
affected foals develop cough, dysponea, fever and conjunctivitis. The
course of the disease is 10 to 56 days. The foals above 5 months of age
develop mild signs.
Porcine Adenoviruses
The role of porcine adenoviruses is not clear. The virus has been
isolated from pig intestinal tract, brain and its kidney cell cultures.
Experimentally, the virus produces a clinical respiratory illness in pigs.
Atleast 4 serotypes of porcine adenovirus have been reported.
Avian Adenoviruses
Avian adcnoviruses can be isolated with ease from both healthy
birds and those suffering from a variety of disease syndromes. The.
avian adenovirus infection in chickens was recognised in 1954 and
appears to be prevalent throughout the world. The virus is prevalent in
the poultry flocks in this country as well. The avian
adenoviruses(AAV) have also been reported in turkeys and pheasants,
quail, ducks and geese apart from the fowl.
172
Properties of the virus: Avian adenoviruses have a structure in

common with other members of the group. The virus measures 70-75
nm in diameter. The molecular weight of DNA is 30 x 1()6 daltons and
G+C content is 54% which is slightly greater than G+C content of the
highly oncogenic human viruses (48-49%) but less than the non
oncogenic types (55-69%).
AAV show remarkable resistance to heat. Most of the strains resist
56C for 3 hours. The stability to heat is greater when they are
suspended in monovalent cations compared to divalent cations. The
virus particles are also resistant to .ultraviolet irradiation. It is also
resistant to ether, chloroform, sodium deoxycholate and trypsin. The
virus is stable over a wide range of pH (pH 2-9). Some serotypes
agglutinate rats, sheep, and chicken erythrocyt~ but this property is not
consistent. The AAV do not share a common complement-fixing
antigen with human adenoviruses or other mammalian viruses. On the
basis of serum neutralization test the fowl adenoviruses have been
typed into 12 serogroups.
Cultivation: Most of the AAV are readily propagated in chicken
embryos and cause death and stunting of chicken embryos. Chicken
kidney, chicken embryo liver, chicken embryo fibroblasts and duck
kidney support the gowth and produce CPE. The virus causes the
nucleus to enlarge and become filled with basophilic granules. Later,
intranuclear inclusion bodies of compact basophilic granules are
formed.
Epidemiology: The AA V are excreted in the faeces and to a lesser
extent in naso-oral secretions. The excretion of the virus continues for
weeks in the faeces. The virus being resistant to various physical
chemical agents, it is likely that virus present in the litter reinfects the
birds. Under natural conditions the virus is not highly contagious
within a flock. However, if other respiratory viruses are present, the
spread of virus within a flock is very quick. The major source of spread
could be the egg transmission of the virus. The virus has been isolated
by many workers from uninoculated chicken embryos. The AA V are
also present in cell cultures produced from embryos or chicks. The wild
birds can be infected with FA V (Fowl adenovirus) but it is unlikely that
other avian species than domestic fowl plays a major role in the
epidemiology of FA V, however they could be important in introducing
virus into an uninfected flock kept for producing SPE eggs.
Adenoviridae
173
Pathogenesis: It is clearly known that avian adenoviruses have a

widespread distribution throughout the internal organs of the host and
mayor may not be associated with a mild disease. The pathogenecity of
different strains varies considerably. The association of other microbial
agents increase tne severity of the disease outcome. Following initial
multiplication at the site of virus entry there is viraemia resulting in
virus spread to virtually all organs. The blood brain barrier normally
prevents entry of virus into the central nervous system. The main sites
of virus replication appear to be in the trachea and caeca.
Fowl ade~oviruses (FAV) have been associated with inclusion
body hepatitis, aplastic anaemia, haemorrhages, mild respiratory
disease and decreased egg production. Some FAV are oncogenic to
newborn hamsters.
Inclusion body hepatitis: Inclusion body hepatitis (IBH) was first
recognised in USA in 1963. The disease mainly occurs in broilers and
mortality rate varies from 2-10%. At post mortem the liver is swollen,
light brown to yellow with haemorrhages, there is marked anaemia,
icterus of skin, subcutaneous fat deposits, haemorrhages of various
organs and pale inactive bone marrow. Eosinophilic inclusion bodies
are present in hcpatocytes. Adenoviruses of different serotypes have
been isolated from the cases of IBH. The chicks which suffer from
infectious bursal disease become immunosuppressive and lead to IBH
outbreaks.
Respiratory disease: There is an association between FAV and
respiratory disease of chickens. The infection with other
microorganisms might activate latent adenoviruses. Experimentally the
disease can be reproduced in very young chicks with mild respiratory
disease. The clinical signs are enhanced by simultaneous inoculation of
Mycoplasma galliseplicum.
Effect on egg production: Infection of hens with FAV cause a
10% fall in egg production for 3 weeks. In Netherlands
haemagglutinating adenovirus of duck origin was isolated in 1976 from
a flock experiencing fall in egg production ranging from 30 to 85% and
production of high percentage of soft shelled eggs. The dropped egg
production started at 29-31 weeks of age which did not return to normal
and eggs were smaller and of poor shell strength for a short period of
time. The haemagglutinating virus resulted in less production of shell,
soft shelled eggs and also drop in production after an incubation period
174
of 7 days. The haemagglutinating virus known as EDS-76 virus or BC14 virus has bren reported from other parts of world like USA,
Australia. The virus has recently been reported from this country from
Andhra Pradesh, Orissa and Punjab. The seroprevalence of EDS-76
virus infection in poultry flocks has been reported from different parts
of this country.
Oncogenicity 0/Jowl adenoviruses: The FAV produces tumors in
newly born hamsters. The virus also transforms human and hamster
cells in vitro. Other avian adenoviruses do not show oncogenicity.
The adenoviruses are also responsible to produce certain
pathological conditions in other species of birds like quail bronchitis,
haemorrhagic enteritis of turkeys, respiratory tract infections in
pheasants etc.
Diagnosis: The clinical signs are not helpful to reach at diagnosis.
Virus isolation is done in chicken embryo liver cells or chicken embryo
kidney cell cultures. The identification can be done by virus
neutralization or fluorescent antibody test. The serum antibodies can be
detected by gel diffusion or by virus neutralization test.
Control: Immunization is not possible against FAV' because of
several serotypes. Attempts to control EDS76 virus causing drop in
egg production is centred around the use of inactivated vaccine. The
killed vaccines are available for control of egg drop syndrome.
References
BELAK, S. and PALFI, V., 1974. An adenovirus isolated from sheep and its
relationship to bovine adenovirus. Arch. Ges. Virusforsch. 46: 336369.
BELAK, S., 1990. Ovine adenoviruses. In virus infections of ruminants, edited
by D.Zinter and B.Morein Elsevier Science Publishers
B.V.Amsterdam.
BURKI, F., 1990. Bovine adenoviruses.ln virus infection of ruminants edited by
D. Zinter and B. Morein Elsevier Science Publisher D. V. Amsterdam.
CABASSO,
CHATIY,
V.I, 1962. Infectious canine hepatitis virus. Annals N.Y. Academy

of Sciences.IOI, 498-514.
M.S., 1985. Studies on egg drop syndrome virus (EDS076) in chicks in

Andhra Pradesh. Ph.D. Thesis, Andhra Pradesh Agri. Univ.
Hyderabad.
Adenoviridae
175
DnmlR, Z., MORElN, B., 1990. Virus infections of rwninants Elsevier Science
Publishers, Amsterdam
DUBEY, S.C. and SHARMA, S.N., 1985. Ovine adenovirus pneumoenteritis in
lambs in India. Indian Journal of Animal Sciences. 55: 878-879.
GREWAL,G.S.; SHARMA, S.N. and DEKA, B.C., 1981. Inclusion body hepatitis in
broiler chickens. Indian 1. Poultry Science 16; 51-56.
McFERRAN, 1.B. and ADAIR, B.M., 1977. Avian adenoviruses-A review. Avian
Pathology6: 189-217.
MOHANTY. F.e.; VERMA. K.C.; PRADHAN. H.K. and KUMAR, RAM, 1984. Egg
drop syndrome (EDS-76) in India, Seroprevalence of EDS-76 virus
infection in poultry flocks. Indian J.Poultry Science19: 15-18.
MOHANTY, S.B. and DUTI'A, S.K., 1981. Veterinary Virology, Lea and Febiger,
Philadelphia.
MUKHERJEE, S.C. and MEHR01RA, M.L., 1983. Studies on infectuious canine
hepatitis. Indian J.Vet Pathology. 7: 57-59.
NAIR, K.P.C., 1955. Infectious canine hepatitis in Madras State. Indian
Veterinary Journa131: 243-244.
RUSSEL. P.H. and EDINGTON, N., 1985. Vaterinary viruses 1st Edition. The
Burlington Pr~ss (Cambridge) Ltd.
SHARMA, KRJSHAJ'IA, SHARMA, S.N.; SAJ\filYAL, D.S. and BAXI, K.K., 1984.
lsolation and characterization of some avian viruses from ovaries of
domestic fowl. Indian 1. Animal Science 54: 977-979.
STAUBER, E.; BENS HAW, H.W.; BORO, C.; MATTSON, D. and FRAl"K, F.W.,1976.
Isolation of a serogroup two adenovirus from calf with weak calf
syndrome. Canadian I.Comparative Medicine40, 98-103.
Chapter 17
Herpesviridae
The family derives its name from Greek word herpein-to creep.
The family is subdivided into 3 families. Alphaherpesvirinae,
Betaherpesvirinae and Gammaherpesvirinae. There are about 20
herpesviruses which produce disease among vertebrates.
The mature virus particles are large, 180-200 nm in diameter when
enveloped. The capsid is about 100 nm in diameter and consists of 162
capsomeres, 12 pentameric and 150 hexameric arranged in icosahedron
form. The nucelocapsid is closely surrounded by another layer
consisting of protein termed as integument which carries short
projections. In the core a fibrillar protein spool is present onto which
the DNA is wrapped. The genome is a linear double stranded DNA
with a molecular weight between 80 and 150 x 1()6 daltons. The DNA
has random nicks and gaps along the length. The guanine and cytosine
(G+C) content varies from 45 to 74 percent in different viruses. The
DNA of each herpes virus can be distinguished from ,other viruses of
the family by characteristic pattern of cleavage with restriction
enzymes. More than 25 structural polypeptides with molecular weights
between 4K and 20K have been identified, some of which are
glycosylated or phosphorylated. The envelope contains lipids. It is
suggested that lipids present in the cell before infection are
incorporated into the virus envelope. Polyamines have been detected in
herpesviruses. Thymidine kinase in increased amounts is found in cells
infected with the virus.
Virus DNA is transcribed in the nucleus, mRNA is translated in the
cytoplasm. Virus DNA is also replicated in the nucleus and is spooled
Herpesviridae
177
into preformed immature nucleocapsids. The assembled virus particle

leaves the nucleus by budding from the nuclear membrane. The virus
particle is modified by inserted viral glycoproteins that forms the
envelope. Eosinophilic intranuclear inclusion bodies are the remanants
of virus factories. In case of cytomegalus virus infected cells,
basophilic cytoplasmic inclusions are also found.
The viruses are heat labile and are inactivated at 50C within 30
minutes. Most viruses do not show property of haemagglutination.
Serologoical cross reactions have been reported in different members of
this family on the basis of neutralization, complement fixation,
precipitation and cross protection tests. The viruses grow in cell
cultures derived from a wide range of animal species. Some ;)f the
viruses grow on developing chicken embryo and produce pocks on the
chorioallantoic membrane. Generally the lesions produced are
proliferative type which later become necrotic. Many members of the
family grow in the central nervous system and produce latent
infections.
Bovine Herpes Virus-I (BHV-I)
Bovine herpesvirus-l (BHV-I) refers to all isolates which are

serologically related to infectious boyine rhinotracheitis virus and
infectious pustular vulvovaginitis virus (IBR/IPV). IBR is an acute
contagious disease of cattle characterised by fever dysponea rhinitis
and other inflammatory changes of upper respiratory tract inclUding
abortion and still birth. The virus in found worldwide. IBR was
described as a new respiratory tract disease and was isolated by Greig
et al. (1958) and Kendrick et al. (1958). The virus is prevalent in USA,
Canada, Britain, Germany, Australia, Newzealand and African
continent. The existance of infection from India was reported by
Mehrotra et al. (1976). The antibodies against this virus are present
among the cattle population of many organised farms of this country.
Properties of the virus: The outline core in negatively stained
preparations appears to be polygonal measuring about 130-180 nm in
diameter. The capsid is made up of 162 capsomeres. The envelope
consists of a double membrane which is similar to host cell membrane
and is about 200 nm. The genome is double stranded DNA with a
molecular weight of 54 x 1()6 daltons and G+C ratio ranging from 7172%. The virus contain lipid in their envelope which renders it
178
susceptible to many disinfectants, especially solvents like ether,

acetone, alcohol and chloroform. The virus is extremely susceptible to
0.5% NaOH, 0.01 % HgC~, 1% chlorinated lime, ] %phenol derivates
and 1% quaternary ammonium compounds. These chemicals inactivate
the virus within seconds. Formaline (5%) inactivates the virus within
one minute. The virus is relatively thermolabile and is inactivated
within 21 minutes at 56C. In the environment the virus survives for 30
days in winter, while in the buildings it survives for 6-13 days in winter
and 5-9 days in spring. The virus is stable at temperatures below -65C
but less stable at -20C if storage exceeds one year. The virus is slowly
inactivated at 4C and at 37C it survives for about 10 days. It is
important to note that virus survives in the semen when stored and can
contaminate virus free semen when infected semen is stored in the
same container. For virus survival in the environment, humidity is
important. The virus is stable between pH 6-9.
A one way serological relationship has been reported between
BHV -1 and pseudorabies virus. When the genomes of these viruses
were compared, the homology was found to be approximately 8%. One
way relationship also exists between BHV-l and goat herpes virus
(BHV -6). By complement fixation and gel diffusion tests the virus is
reported to show antigenic cross-reactivity with equine herpes virus-I.
The BHV-l, Marek's disease virus and Burkitts lymphoma virus share
a common antigen. Minor antigenic difference is possibly the reflection
of immunological drift. All IBR and IPV isolates are basically similar,
however, diverse clinical manifestation suggested strain differences.
Recently differences in IBR and IPV virus strains have been shown by
restriction endonuclease digestion of their DNA.
Cultivation: The virus multiplies in a wide variety of cell cultures
but for isolation, the bovine tissues like bovine kidneys and testes cell
cultures are most useful. The infected cells become rounded and
refractile and form syncytia. Large intranuclear inclusion bodies are
formed in the infected cells. The virus does not grow in embryonated
eggs.
Epidemiology: The BHV-1 is' widely distributed among cattle
population all over the world but the host range IS limited. Many wild
species of animals have been found seropositive but clinical signs have
only been obseved in cattle. Ferrets have been found susceptible in
USA but not in Europe. The rabbits are being used as experimental
llerpesviridae
179
models. Experimentally neonatal hamsters and skunks are susceptible.

Serologically goats and sheep have also been reported positive. The
wild ruminants in Africa and in zoos appear to be virus reservoir.
Various virus strains are harboured by most animals following infection
and after a period of persistence reach a state of latency from which
they can emerge from stress. The BHV -1 has recently been isolated
from soft shelled ticks (Ornithodorus coriaceus) collected from deer
bedding areas in Western USA. Cattle and deer graze together in this
region. Both these species were seropositive. The ticks may harbour the
virus for a long period of time or the virus may replicate in the ticks or
mechanical transmission may take place.
Cattle of all ages are susceptible especially newborn calves without
maternal antibodies. The virus is excreted through nasal, ocular
secretions and placenta of aborted animals. The virus spreads by
contact. The outbreaks usually take place whe;i the new animals excrete
the virus but it is not certain whether these animals are responsible for
perpetuation of virus. The BHV -1 is capable of establishing latent
infection in recovered animals and these animals continue to excrete
the virus for a ling time. The tissues in which the virus establishes
latency and how the reactivation of virus takes place is not yet known.
The virus spread also takes place through contaminated semen, natural
service, the teaser bull or herdsman. The semen becomes infected with
preputial washings. Antigenic drift is another factor which may
contribute the virus to survive in nature but this aspect needs detailed
study.
Pathogenes;s: The virus enters the body via the mucous
membranes of the upper respiratory tract via mucous membranes of the
genital tract, by way of conjunctival epithelium and may be through
soft shelled ticks. The virus is transported to other tissues by infected
leucocytes. The excretion of virus in an infected animal is usually 1016 days but persistence for longer periods have been observed,
especially in calves when passive immunity is on the verge of
detection.
The virus may cause different types of clinical manifestation.
Respiratory form: The natural infection varies in severity.
Following an incubation period of 4-6 days, the disease manifests with
a sudden rise of temperature. The affected animals show anorexia,
depression, nasal discharge, coughing, open mouth breathing and
180
Textbook of~'eterinary Virology
dysponea. The nasal mucosa is severely inflammed. New born calves

may die due to necrosis of liver. The morbidity is high and varies from
30-39% while mortality rate does not exceed 3%. The course of illness
varies from 7-14 days.
Genital form: The genital form results following introduction of
virus to the mucosa of the genital tract by mating or through other
external agents. After an incubation period of 48 hours the first pustule
appears, after 2-7 days and then, further pustules develop. Pustules may
from a yellowish membrane which detach leaving white necrotic
material on the mucosa of vulva and vagina. This form of the disease
predisposes to further bacterial infection and may involve the uterus.
There is no abortion but there is infertility in the affected animals.
Abortion: Abortion is a sequel to respiratory form of disease. The
virus presumably reaches the foetus through the infected leucocytes and
the lymphatic channels of lymph nodes. Abortions usually occur
between 4th and 7th month of gestation. Foetuses are invariably dead
when expelled and the placenta may be temporarily retained. Abortions
may also take place due to administration of live virus vaccines.
Conjunctivitis and keratoconjunctivitis: Conjunctivitis is usually
bilateral and is a common clinical manifestation of typical IBR. There
is excessive ocular discharge, the conjunctiva is hypeeramic. In
secondary bacterial infection, keratitis and corneal ulceration occur.
The BHV is associated with ocular carcinoma but definite experimental
proof is lacking that it is the etiological agent.
Mastitis: The virus has also been isolated from cattle suffering
from mastitis. Experimentally the virus may produce mastitis.
Encephalitic form: The virus has been isolated from encephalitis
in calves. The animals show ataxia, depression followed by periods of
excitement characterised by running and terminating into stumbling
and falling. The animal develops clonic spasm of legs, neck and lumbar
muscles, become blind, develop coma and die within 3-4 days of onset
of symptoms. A few animals recover but become blind.
Disease of alimentary system: Diarrhoea may be a clinical sign
and often fatal in young calves. The virus has been isolated from adult
cattle with enteritis.
The concurrent outbreak of IBR/lPV have not been recorded
frequently, however, an outbreak of IBR and IPV has been reported.
fJ erpesviridae
181
The Virus has also been isolated from naturally occuring outbreak
of respiratory disease.
Immune reaction: The antibodies appear following infection and
persist for years. Higher titres appear after respiratory tract infection
while lower titres appear after genital tract infection. No correlation
exists between the levels of immune globulins, virus excretion and
severity of clinical sings. When the antibody titres decrease the virus
latency is terminated and infectious virus is shed. Immunoglobulin of
all the three classes are present in the respiratory and genital tract
discharges with higher IgG titres during oesterus.
The cell mediated immunity plays a major role in body defense
against IBR/IPV infections.
The maternal antibodies persist upto 3-4 months. The antibodies in
nasal secretions of calves are in lower concentrations and rarely persist
beyond the third week. The calves are 'recommended to be vaccinated
at 3 week of age.
Diagnosis: The onset of clinical signs and course of disease are of
diagnostic value. The confirmatory diagnosis can be arrived at by
isolation of virus. The choice of material for virus isolation depends on
the form of infection. In live animals with respiratory from of IBR,
nasal swabs made of gauze should be collected at an early stage of
disease. In case of IPV, gauze swabs fixed to long forceps are placed
into the vagina, left for at least one minute and rotated before removal.
The material kept at 4C should be transported to the diagnostic
laboratory. Liver spleen, lymphnode and brain can also be collected for
isolation of virus. The serum sample alongwith other material should
also be collected for detection of antibodies, second sample of serum is
collected after 2-3 weeks of the collection of first sample. For virus
isolation primary bovine tissues are used. In bovine kidney cell culture
the CPE is apparent after 24-36 hours and is characterised by rounding,
increased granularity and characteristic bunch of grapes. Within 79-96
hours there is complete destruction of cell monolayer. Cowdry type A
inclusion bodies are seen. The serum neutralization test can be used to
characterize the virus. FAT is useful in detecting virus antigen in
tissues.
Control: Numerous attenuated and inactivated vaccines are
available all over the world. If the disease is confirmed to small number
182
of herds, strict control programme offers a best chance of elimination.

In case higher percentage of herds are infected a prophylactic
programme is the best solution.
The fi,rst attenuated live vaccine was employed by intramuscular
route in late fIfties. The vaccine proved to be dangerous in pregnant
animals because the vaccine strains were not properly attenuated. The
emergency vaccination is recommended when the heard is stricken with
the disease and diagnosis is made before all animals have contracted
the field virus. The vaccines against BHV-l contain other components
as well like parainfluenza-3 virus, bovine viral diarrhoea and
adenovirus and sometimes Pasteurella or Haemophilus are also
included. The vaccination schedule recommended is that first
vaccination be given to calves between 3-4 months of age in case the
calves had received antibodies in colostrum. Otherwise, the vaccination
in calves may be given at any age. Second vaccination is recommended
4-6 weeks later followed by yearly vaccination. The vaccination
prevents severe clinical signs but not infection with the possibility of
excretion of virus following stress.
Inactivated vaccines are also used to control the disease. If the
inactivated vaccine contain low antigen content, the immunity
produced is of low grade, and if these animals pick up infection the
virulent virus multiplies and is excreted in large amounts without
showing clearcut signs of the disease. These animals become latent
carriers and spread the disease.
Bovine Herpes virus-2 (Bovine Mammillitis virus)

It is an ulcerative infection of teats and udder of dairy cows. The
virus was first isolated in 1957 from South Africa. The disease has been
reported from different parts of Africa, Europe, USA and Australia.
Properties of the Virus: The virus particle is similar in
morphology to other herpes viruses. It is sensitive to ether and
chloroform. The virus is antigenically indistinguishable from the
Allerton virus, the causative agent of lumpy skin disease in Africa but
can be distinguished from infectious bovine rhinotracheitis and
malignant catarrhal fever viruses. The virus has a serological
relationship with herpes simplex virus-l and 2 of man.
Cultivation: The virus does not grow in the emrbyonated chicken
eggs. It readily grows in bovine, sheep, pig and cat cells derived from
Ilerpesviridae
183
kidney, testes, thyroid and lymph nodes. The CPE includes large
syncytia with basophilic or acidophilic Cowdry type A inclusions in the
nucleus. The virus does not produce characteristic CPE on established
cell cultures.
Epidemiology: The exact nature of transmission is not known but
it has been hypothesized that heifers may harbour a latent infection.
The virus within the herd is transmitted during milking by
contaminated hands or milking machines. Transmission by flies and
other insect vectors has also been suggested.
Pathogenecity: The incubation period is 5-10 days. The virus
causes a vesicular skin disease which mainly affects the udders and
teats of milking cows and heifers before calving. Variable size of
vesicles develop on the teats, udder and perineum. A certain percentage
of cows develop mastitis. Suckling calves may develop lesions on the
muzzle. The disease is self limiting and occurs in autumn or early
winter. The lesions develop as raised circumscribed Plaques and there
is thic~ening of teat wall. There is severe local cutanous oedema and
erythema of the affected skin. The vesicles sometimes rupture and form
necrotic painful ulcers.
Diagnosis: The clinical picture of the disease is suggestive of the
diagnosis. Th~ virus can be isolated in cell culture and further
characterised. The exudate from the lesions can be used for virus
isolation.
Malignant Catarrhal Fever Virus (MCFV)
It is an acute generalized disease of cattle and buffaloes,
characterized by high fever, profuse nasal discharge, severe hyperaemia
of respiratory and alimentary epithelia, extensive lymphoid hyperplasia,
keratocon-junctivitis, encephalitis and rapid loss of condition. The
disease is prevalent in most parts of the world. The incidence is
generally low and sporadic although the incidence is higher in Africa
and several explosive outbreak have been reported from USA and
Canada. The disease was reported fIrst time by Parihar et al. (1975)
from Punjab. Subsequently several outbreaks have been reported from
the same area in Faridkot district of Punjab. Singh et al. (1979) have
reported the epidemiological features of this disease in Punjab.
Properties of the virus: The virus is very fragile and rapidly
destroyed by putrefaction and heat. The cell free virus is inactivated by
184
lipid solvents and detergents. It is relatively stable in culture media

containing serum. The infectivity is lost within 10-15 minutes at 56C.
At -70C the virus maintains the infectivity titre for several months.
The virus is highly cell associated. The highest titres of virus are found
in buffy coat of blood, lymphnodes and other tissues of
reticuloendothelial tissues. There is no evidence of significant
immunological variation between the strains of the virus derived from
cattle or wild animals although there are reports that virus strains are
not antigenically homogenous.
Cultivation: In wildebeest cattle and buffaloes, the infectivity in
tissues is cell associated. The cell free infectious virus is found in
ocular and nasal secretions of wildebeest. The rich source of virus are
Iymphnodes, thymus, spleen, bone marrow and buffy coat from
peripheral blood. The most sensitive cell culture for virus isolation was
considered to be primary or secondary monolayers prepared from calf
thyroid where it produces syncytia, vacuolization, clumping of nuclei
and formation of A type inclusion body in nucleus. Recently it has been
found that calf ~estis cells are more satisfactory. The CPE is more easily
detected and high yields of cell free virus is obtained. The virus does
not grow in chicken embryos. The virus can be transmitted serially in
young susceptible calves.
Epidemiology: The disease primarily affects cattle and buffaloes
but is being increasingly encountered in farmed deer from which it is a
major disease hazard. The most important experimental host is the
domestic rabbit.
The majority of cases of MCF occur when there is a close contact
between sheep and indicator host. When infected sheep are introduced
in cattle sheds, the incubation period estimated from time of
introduction of sheep is about 96 to 185 days. Cases of MCF continued
to appear upto 3 or 4 months after removal of sheep from close contact
with cattle. The reports of multiple cases of MCF have supported the
thesis that sheep are probably reservoir hosts of the virus but there are
also reports where investigators failed to incriminate sheep. There are
possibilities that other species act as inapparent carriers. The wildebeest
and wild ruminants also act as reservoirs. The virus crosses the placenta
in some reserrvoirs through excretions, particularly nasal discharge
ocular secretion and placental excretions. The disease generally is not
transmitted by contact among cattle. Vectors like flies, ticks, and lice
Herpesviridae
185
are unlikely to play any role in transmission of MCF. The details of

virus transmission are not well understood.
The majority of cases of MCF occur in young adults but animals of
all ages, sex and breed are susceptible.
Pathogenesis: The portal of entry of MCF virus is probably upper
respiratory tract, nasal mucosa and/or tonsil. Aerosal or intratracheal
route of infection in cattle and rabbits is successful. The congenital
transmission takes place frequently in reservoir host but has not been
reported in cattle. The congenital transmission has been demonstrated
only once in cattle.
A long incubation period is characteristic of this disease. It is about
19.5 + 3.7 days experimentally. In practice it is essential to wait for
about 2 months before concluding that animal has not been infected by
a parenteral inoculation. In cattle viraemia is detectable from 9-17 days
after inoculation of virulent blood. The viraemia is assoicated with
cellular fractions of the blood and not with the plasma, erythrocytes or
platelets. The disease MCF is being considered as immunopathological
condition. The basis for this are-{a) The lesions are not attributable to
the virus as shown by absence of virus or antigens; (b) The long
incubation period and prepatent viraemia suggest a requirement for
sensitization to virus or virus associated antigens; (c) The lesions are
similar to virus induced autoimmune or lymphoproliferative conditions
and (d) Association of lymphocytic infiltration with vascular and
epithelial lesions. The MCF pathogenesis appears to be immune
deregulation attributable to virus infection causing dysfunction of
natural killer cells and uncontrolled proliferation of lymphoblastoid
elements in many tissues. Some workers have suggested that lesions of
MCF may be due to a type III or Arthus like hypersensitivity reaction
or a combination of type III and type IV reactions.
The disease is characterized by high fever, severe inflammatory
and degenerative lesions of upper respiratory tract, entire digestive tract
and eye. There is severe inflammation of oral and nasal mucosa with
diffuse necrosis. The lesions are found on the lips, hard and soft palate
and cheeks. There is inflammation of eye, lacrimation, eyelids become
swollen and corneal opacity develops. There is enlargement of lymph
nodes and they become haemorrhagic. The meningites of central
nervous system are oedematous and encephalomyelitis may be present.
The disease often terminates in fatal encephalitis in 5-12 days after
186
onset of pyrexia. The brain, myocardium, lungs show severe

congestion. In some cases there is abomastitis and enteritis. The
morbidity rate is very low.
The .leS~ons in the alimentary tract are epithelial necrosis and
erosions followed by hlceration in the pharynx, on the soft palate,
oesophagus, on the folds of reticulum and pillars of rumen. In
abomasum congestion and haemorrhagic erosions of fundic folds and
pyloric region are common. The intestines (Calcum, and upper colon)
exihibit congestion. There is congestion, diptheritic membranes,
erosions, haemorrhages on the turbinates and septum and sometimes in
the trachea. The lymphnodes show enlargement. The spleen is
enlarged. The mucosa of urinary bladder and vagina show congestion,
necrosis and erosions while the kidneys are swollen. There is.
destruction of small lymphocytes in the lymphopoietic tissues,
proliferation and infiltration in many organs with large lymphoblastoid
cells and angiitis affecting components of the walls of arteries and
veins.
Immune reaction: The few caule that survive infection arc
resistant to challenge for several years, develop neutralizing antibodies
in their serum. The neutralizing antibodies appear in the late stage of
disease. There is evidence that the neutralizing antibodies do not avert
the fatal outcome of the disease. The role of CMI in MCF is not known
properly but the failure of neutralizing qntibodies to protect indicates
that CMI plays an important role in immue response. Delayed
hypersensitivity has been reported in MCP.
Diagnosis: The diagnosis is based on clinical signs and pathogenic
changes. Virological diagnosis is well established and based on the
recovery of virus from sick or killed animals and on the demonstration
of antibodies in the scrum of animals collected at late stage of disease
or during convalescence in the very few surviving animals. The virus is
isolated from buffy coat of infected blood in cell culture. The CPE is
neutralized by specific antiserum.
COlltrol: All attempts to immunize cattle with attenuated virus
have failed. The inactivated virus preparations combined with
adjuvants produce high level of neutralizing antibodies in cattle but
there is resistance to parenteral challenge by cell free or cell associated
virus. In rabbits, the inactivated vaccines provide protection against cell
free virus given intravenously but not against virulent spleen
f/crpesviridae
187
suspensions. Few workers have claimed to have isolated strains safe

and attenuated for cattle, which confer immunity to challenge but have
to be confirmed. In the absence of vaccines, the prevention of MCF
depends on the prevention of close contacts between susceptible and
reservoir hosts. This means avoidance of housing or herding together of
sheep and cattle or deer. In the zoological gardens, wildebeest should
be separated from many susceptible species.
The Movar type of Bovine Herpesviruses (DHV-3)

A new serotype of bovine herpesvirus nonpathogenic to calves was
isolated in 1967. Subsequently agents similar to this virus were isolated
from USA, Africa and Europe. The pathogenic role of these viruses is
minor or insignificant. These viruses have been named as BHV-3.
Cattle of all ages and breeds are susceptible. The morbidity rate is 2030% while mortality is very low. Transmission is by direct contact. The
virus is assoicated with bovine respiratory disease complex. The
disease may be inapparent, mild or acute. The virus induces mild
clinical signs of tracheitis in young calves. In assoication with
Pasteurella multocida the disease is quite severe. The acute disease is
characterised by fever. dyspnea, COUgh, hyperpnea and copious nasal
discharge.
Herpesvirus of Sheep (CHV -1)

The virus is associated with plllmoRary adenomatosis of sheep.
The disease has a long incubation period of 6-9 months and the disease
is characterized by chronic progressive pneumonia with the
development of adenomatous in growths of the alveolar walls. The role
of CHV -1 virus in pulmonary adenomatosis is not clear as the agent of
this disease Uaagsiekte) has now been found to be a retrovirus.
Herpesvirus of Goats (CHV-2)

Herpes viruses have been isolated from kids with severe
generalized infection of genital lesions in goats, acute and chronic
pneumonia, wartlike lesions of eyelids and proliferative lesions around
mouth and hard palate and dermal lesions. Experimentally the virus can
cause abortions. The restriction endonuclease DNA fragment pattern is
quite different from that of BHV-l virus but the DNA of CHV-2 shared
a high degree of base s('.quence homology. The common feature of the
188
herpesvirus about latency and reactivation has been shown by using

corticosteriod treatment in goats.
Equine Herpesviruses
Three antigenically different herpes viruses are known to infect
horses. Equine herpes virus-l (EHV-l) is the most important and causal
agent of an important disease of horses known as equine
rhinopneumonitis. The equine herpes virus-2 (EHV -2) mayor may not
be associated with any disease while equine herpes virus-3 (EHC-3) is
the cause of coital exanthema. Morphologically these viruses are
similar to one another and exhibit some genetic homology but they
differ in immunological properties.
Equine Herpes Virus-l (Equine Rhinopneumonitis Virus)
The virus produces highly infectious disease of horses
characterized by fever, respiratory catarrh and abortion in mares. In
nature, only equines are affected. The disease has been reported from
USA, Germany and many other countries of Europe, Japan and India.
In 1965 Sharma and his associates reported the disease on the basis of
histopathological examination. The isolation was reported in 1976 by
Jain and others from Hissar. The EHV-l infection is reported to be
endemic in various equine farms in this country.
Properties of the Virus: Morphologically the virus resembles the
other members of the family and possess DNA containing core
enclosed in icosahedral capsid and is further enclosed in an envelope.
The virus particles are about 150-198 nm. The virus does not survive
for long period outside the host but can be stored at -18C for a year.
The virus is inactivated by heating at 56C for 10 minutes. The virus
haemagglutinates equine red blood cells. Earlier EHV-l strains were
regarded antigenically similar but strains isolated from abortion cases
are said to be more infective, grow better in cell culture and are
released to greater extent in comparison to the strains isolated from
respiratory infections. Recent findings revealed by restriction
endonuclease analysis of the genomes of the isolates suggest that
strains from abortion cases are different from those isolated from
respiratory cases.
Cultivation: The virus does not grow directly in the chicken
embryos. The virus can be adapted to grow on the CAM of chicken
Herpesviridae
189
embryos by alternate serial passages in hamster kidney cell cultures and

CAM of chicken embryo. The virus multiplies in human amnion, HeLa
and horse, sheep, pig, cattle, cat and chicken embryonic tissues. The
CPE produced is rounding and ballooning of infected cells,
multinucleated syncytia and acidophilic 'A' type intranculear
inclusions. Plaque formation occurs on cells of several species.
Unweaned hamsters and unweaned mice can also be infected. The virus
produces a fatal hepatitis. In pregnant hamsters the virus may produce
upper respiratory tract infection and abortion.
Epidemiology: The introduction of an infected horse may cause
abortion. Under natural conditions virus may be maintained- in the
upper respiratory tract of carrier animals. There is a possibility that
virus may give rise to latent type of infection and spread by veneral
contact as in the case of infectious bovine rhinotracheitis virus. Recent
evidence has shown that antigenic difference between EHV -1 strains
exist. This may be another important factor in the epidemiology of the
disease.
Pathogenicity: The incubation period in natural infection varies
from 9-60 days. Acute respiratory disease occurs mainly in foals,
weanlings and yearlings. The disease is rarely fatal unless characterised
by profuse serous nasal ~ischarge which later becomes mucopurulent.
Distinct herpetic lesions develop in the respiratory tract characterised
by necrosis of respiratory epithelium and intranuclear inclusions.
Pregnant mares may abort mostly at 6-11 months of gestation after 14120 days of exposure to virus. The natural route of infection is
presumed to be via the upper respiratory tract followed by cell
associated viraemia. The lesions in early abortions are severe autolysis
of foetus while in late abortions there is jaundice, petechiation of
mucous membranes, excessive pleural fluid, pulmonary oedema and
spleenic enlargement The lesions observed in mares are distention of
regionallymphatics.
The neurologic from of disease involves the central nervous
system and is assoicated with enzootics of respiratory disease or
abortion. The clinical sings are ataxia. weakness and paralysis.
The Tatent infection in EHV -1 has not been demonstrated so far but
there is indirect evidence that some horses must be carriers and that
latent infection can be reactivated.
190
Diagnosis: The presumptive diagnosis is based on the clinical

symptom~
and history of abortion. The presence of intranculear

inclusions in the exfoliated epithelial cells of the respiratory tract and in
the histopathological sections of lungs, liver, spleen thymus,
myocardium, kidney of aborted foetus is of value in arriving at
diagnosis. Antibodies in paired sera samples can be detected by virus
neutralisation, counter immunoelectrophoresis, indirect fluorescent test
on the cryosections of frozen lung and live tissues of aborted foetuses.
Control: The immunity resulting from natural infection in foals
suffering from respiratory infection is of short duration, while the
immunity against abortion in mares is more durable. Live and
inactivated vaccines are available in foreign countries. Live, virulent,
hamster adapted vaccine given intransally in horses is a good
immunogen but may induce abortion in pregnant animals. Attenuated
tissue culture vaccines are available. Two vaccinations are administered
for primary immunization and then annual administration of vaccine at
2 months pregnacy and again at 6 months of pregnacy. These vaccines
produce satisfactory immunity and are safe for pregnant mares.
Inactivated vaccine mixed with oil adjuvant have been developed.
These vaccines produce satisfactory immunity and are safe for pregnant
animals ..
Equine Herpesvirus-3 (Equine Coital Exanthema)
Equine coital exanthema is an acute, usually rare disease
characterised by formation of pustular and ulcerative lesions on the
vaginal and vestibular mucosa, on the skin of penis, prepuce and
perineal region and occasionally on teats, lips and respiratory mucosa.
The causative agent is equine herpesvirus-3. The virus shows no
serological cross-reactivity with other equine herpesvirues but shares
antigens with equine herpesvirus-I. The virus grows in cells of equine
origin and produces large plaques, the virus remains cell associated.
In coital exanthema the genital lesions are extensive but there are
no systemic signs. The incubation period is short about 2 days and in
uncomplicated cases healing is usually complete in 14 _days. Abortion
and infertility is not associated with EHV-3. Affected stallions show
decreased libido. The virus can also cause subclinical respiratory
infection in yearling horses. The presence of disease at the stud farm
disrupts the breeding schedule. The control measures consist of
removal of stallions from service and symptomatic treatment.
lIerpesviridae
191
Pseudorabies Virus (Porcine herpes virus-I)

Pseudorabies or Aujeszky's disease or mad itch or infectious
bulbar paralysis is primarily a disease of swine but occurs naturally in a
wide range of animals including swine, cattle, sheep, dogs, cats, minks,
ferrets, foxes and rats. In all animals excepting adult swine the disease
is characterised by pururitis, paralysis and fatal termination. Mild
illness also occurs in man. The disease is enzootic in many countries of
Europe like Bulgaria, Czechoslovakia, Denmark, France, Hungary,
Italy, the Netherlands, Poland. The disease is also prevalent in U.K and
U.S.A. There are reports of disease outbreaks from China, Algeria,
Tunisia and Angola.
Properties of the virus: Morphologically the virus is
indistinguishable from that of other viruses of herpes group. It is more
thermostable and resistant to pH changes than other herpes viruses. It
may survive for 2-7 weeks in infected premises and upto 5 weeks in
meat. It surives only for few hours on material contaminated with
faeces and urine. It is sensitive to ether and chloroform and is rapidly
inactivated by 0.5-1 % NaOH. The recommended disinfectants are 5%
phenol, sodium and calcium hypochlorites, 2% formalin, 2% sodium
hydroxide, trisodium phosphate and quartemary ammonia. There is no
report regarding its property of haemagglutination. All the strains of the
virus are reported to be antigenically similar by restriction
endonuclease technology, the DNA of different strains was found to be
slightly different. The virus is antigenically related to B virus.
Cultivation: The virus grows readily on CAM of embryonated
eggs and produce pocks after 3-4 days of infection. Many strains are
lethal to embryo. The virus grows in a" wide variety of cells derived
from swine, caule, rabbits, dogs, monkeys and chicken embryos.
Rabbit kidney is highly susceptible. Cowdry type A intranuclear
inclusions and polykaryocytes are seen in infected cells.
Epidemiology: Naturally fatal encephalomyelitis is produced in
cattle, sheep, goats, dogs, cats, deer, rabbits, rats and mice. The
morbidity rate is high in pigs but pigs in first few weeks of life
succumb to pseudorabies but in older pigs the mortality is negligjble.
The pig is the principal reservoir and almost all cases of disease in
cattle are due to cohabitation with swine. Latent infection occurs in
swine and inapparently infected pigs are source of infectious virus.
192
Cattle are considered dead-end-hosts of the virus and there is no

evidence of interbovine transmission. The cattle and other ruminants
rarely transmit the disease to other animals unless their tissues are eaten
by susceptible carnivores. The rats and mice are poor shedders of the
virus and do not play a role in the spread of the virus. The carnivores
may serve as a source of infection to other species after they have
become infected by ingesting infected meat.
Pathogenesis: The virus naturally enters into domestic ruminants
via break in the skin caused by bite or other wounds and occasionally
by aerosal infection. Experimenta~ly cattle can be infected by any route.
In prodromal phase the swine shed the virus in saliva and transmit to
cattle by muzzling or biting the flank, vulva or anal region while
wandering with them. Peroral infection in cattle may occur via
contaminated feed or water. Respiratory transmission ,occurs in poorly
ventilated areas from swine to cattle. The virus enters the central
nervous system via peripheral nerves causing neuronal damage
resulting in local pururitis and encephalomyelitis. The virus has been
isolated from nasopharynx, lungs, and vagina of affected call1e.
Persistant latent infections capable of reactivation are common in adult
swine but have not been reported from cattle.
The clinical signs in affected cattle are encephalomyelitis
characterized by intense pruritis leading to self mutilation. The
incubation period in natural cases is 4-7 days. The clinical signs are
short which last about 10-24 hours. There is a general lack of typical
and constant gross lesions. The site of pruritis is generally lacerated.
There is accumulation of cerebrospinal fluid and meningeal congestion
but lesions in the viscera are not significant The lungs may show
congestion, oedema and haemorrhages.
Diagnosis: Typical cases of disease in callle and sheep are easily
detected, however, diagnosis in pigs is difficult. Subcutaneous
inoculation of specimens from clinical cases (brains, lungs, tonsils and
kidney) into rabbits usually produce intense itching and death in 3-5
days. Isolation of virus in cell culture is a fruitiful method. Fluorescent
antibody technique can also be adopted to brain tissue of the affected
animals. In pigs serologic tests like virus neutralisation are useful to
detect the presence of antibodies.
Control: Swine recovered from the disease develop neutralizing
antibodies which are transferred to new born piglets through colostrum.
JJerpesviridae
193
Maternal antibodies protect the pigs for about 2 months. Attenuated and
inactivated vaccines have been used in pigs with promising results.
Experimentally attenuated and inactivated vaccines made for pigs have
been used in cattle. The cattle vaccinated with inactivated vaccine
develop marginal serologic response but remain resistant to challenge.
The disease in cattle and sheep can be prevented by keeping pigs out of
contact from these animals. Eradication by slaugher and quarantine of
infected pigs after the incidence of disease in swine is reduced to
manageable level.
Simian Herpes Virus-! (B virus)

The virus is naturally found in normal Asiatic monkeys especially
rhesus and cynomologus species. The virus causes a fatal ascending
myelitis of man.
Properties: The virus measures 100 nm in diameter and is
morphologically similar to herpes simplex virus. The B virus antiserum
neutralises human herpes simplex virus but not vice versa.
Cultivation: The virus grows in the chicken embryos and produce
pocks on the choroallantoic membrane. The virus also replicate in cell
cultures of rabbit, monkey and human cells.
Epidemiology: The virus spreads by direct contact in the monkeys
especially when the animals are crowded together in captivity. The
virus sets up a latent infection as is the case with herpes simplex virus
in man. It has been shown that about 10 percent of newly caught rhesus
monkeys have the antibodies against B virus and this percentage goes
upto 60 percent when the animals are crowded in captivity. The saliva
and central nervous tissue of clinically healthy animal contain the virus.
The virus may also be recovered from the primary monkey kidney cell
cultures.
Pathogenesis: The naturally infected monkeys may show herpes
like lesions on the dorsal surface of tongue and the lips. The lesions in
the monkeys develop commonly when the animals are crowded. Man
usually gets infected through the bite of a monkey but one laboratory
worker became infected after handling glassware used for tissue culture
of monkey kidney. The incubation period in man is 10-20 days. At the
site of bite there is local inflammation and the virus reaches the central
nervous system by way of peripheral nerves and cause an acute
encephalomyelitis and death. There is focal necrosis of lymphnode,
adrenals, spleen and liver.
194
Diagnosis: The diagnosis can be arrived at by isolating the virus

from saliva and brain of monkeys or brain and spinal cord of infected
human beings.
Control: There is no specific treatment for human beings. The
vaccinatio" of man with formalised vaccines has been unsuccessful.
Hyperimmune B virus antiserum be given after the bite and should also
be inoculated at the site of the bite.
Canine Herpes Virus (Canine tracheobronchitis virus)
This is a newly recognised virus of puppies causing an acute fatal
generalised viral infection 'of neonatal and infant puppies. The virus
was reported in 1865 from United states but now it has been reported
from Canada and Europe.
Properties of the virus: The virus has a similar morphology like
other herpes viruses. The virus particles are ether sensitive and acid
labile. The virus is not antigenically related with other members of the
group but a low degree of cross reaction has been reported with herpes
simplex virus.
Cultivation: The virus readily grows in dog cells, like dog kidney,
lung and uterine cells. The CPE arc produced as early as 16 hours after
infection.
Epidemiology: The transmission of virus is not airborne. Infected
dogs excrete virus in saliva, nasal secretions and urine. Infection can
occur transplacentally or by contact.
Pathogenecity: The disease causes anorexia, laboured breathing
and abdominal pain. The mortality may reach upto 80% in puppies upto
1 week of age. In dogs of few weeks age the disease is mild or
inapparent. Focal areas of necrosis and haemorrhages are found in the
liver, kidney and lungs. The incubation period is 3-8 days.
Diagnosis: The virus can be isolated in dog kidney cells and
identified by neutralization test.
Control: No vaccines arc available.
Fowl Herpesvirus-1 (Avian Infectious Laryngotracheitis virus)

Infectious laryngotracheitis (ILT) is highly contagious respiratory
disease of poultry characterised by respiratory disease, gasping,
expectoration of blood stained exudate and high mortality. All breeds
and ages of birds are affected. The disease was reported for the first
Herpesviridae
195
time from USA in 1925. It has now been reported from several
countries of the world. The disease was reported by Singh et al. (1964)
from this country. The infection is widely prevalent in this country
where chickens are raised. The disease mainly occurs in a mild or
subclinical form, however, there are isolated reports of acute form as
well.
Properties of the virus: The physical, chemical and biological
properties of the virus are similar to other members of this group. The
double stranded genome has G+C content of 45 to 50%. The viruS is
one of the resistant poUltry viruses. It is readily destroyed by 3% cresol
and i % NaOH. It is ether sensitive and is readily destroyed by exposure
to 55C. The virus does not have the property of haemagglutination.
The virus appears to be antigenically homogenous although some
variation in serum neutralization has been reported with certain strains.
Variation in pathogenicity of different strains has been reported. Some
strains are highly pathogenic while others are non pathogenic or mildly
pathogenic.
Cultivation: The virus grows in embryonating chicken eggs and
produces opaque pocks with central areas of necrosis. The embryos are
stunted and death occurs in 2-12 days after inoculation. The virus also
grows in chicken embryo cell cultures, chicken embryo kidney, lung
and respiratory epithelium and produces CPE characterized by
syncytial formation and cowdry type A inclusion bodies.
Epidemiology: Infectious laryngotracheitis virus is usually
introduced into a flock by carrier bird and is transmitted by aerosal and
inhalation, less commonly by ingestion. The virus is not transmitted
through egg and insect vectors do not play any role. The carrier state in
the recovered birds exist and the recovered birds may harbour the virus
for more than 2 years. Outbreaks following contact between vaccinated
and nonvaccinated brids have been reported.
Pathogenesis: The chickens of all ages are susceptible but the
disease is most common in birds of 4-18 months of age. After an
incubatin period of 2-8 days there is mild coughing and sneezing which
is followed by nasal and ocular" discharge, dyspnea, gasping and
coughing and depression. There is haemorrhagic tracheitis and the bird
extends its head and takes a prolonged inspiration. The coughing may
be associated with expectronation of bloody mucus and frank blood.
Morbidity is about 100%. The mortality with virulent strains may be
196
50-70% and with strains of low virulence about 20%. The strains of
low virulence are associated with conjunctivitis, ocular discharge,
swollen infraorbital and nasal sinuses and lowered egg production.
The principal lesions are found in the larynx and trachea and which
include haemarrhagic inflammation and oedema. In advanced cases,
caseous exudate and diptheritic membrane may be seen in these organs.
The inflammation in the bronchi, lungs and air sacs may also take
place. Microscopically there may be loss of cilia, cellular infllteration
and necrotizing tracheitis. Type A intranuclear inclusions may be
present in early stages of disease.
Diagnosis: In acute form of disease, characterisitc repiratory
symptoms and high mortality are often diagnosed. The mild cases are
difficult to diagnose. Demonstration of type A intranuclear inclusions
in tracheal or conjunctival tissue is si.gnificantly diagnostic. The most
common method of diagnosis is by virus isolation from clinical
material (tracheal exudate, lung suspension) in chicken embryos.
Typical pocks, are produced on the CAM after 4-5 days of inoculation.
The virus can be characterized by serum neutralization and agar gel
diffusion test The fluorescent antibody test can be applied for
demonstration of antigen in the tissues.
Control: Recovered birds are reportedly immune for 1 year. The
vaccination is recommended in enzootic areas since vaccination results
in a carrier state. Both virulent and avirulent vaccines have been used.
The rust approach of vaccination was adopted in 1930, the young
susceptible birds used to be mixed up with known recovered birds
before laying stage. This was followed by inoculation of wild virus in
the cloaca. It produced immunity without serious respiratory signs. In
1950s it was known that strains of low virulence occured and these
viruses were then introduced for cloacal vaccination. When more
attenuated strains grown in cell culture became available, other routes
of vaccination like inoculation by infraorbital sinus or by intranasal and
eye drop procedures were followed. Mass vaccination by aerosal and
drinking water administration have also been developed. Immunization
with attenuated vaccines protect birds against clinical disease but does
not protect against infection with virulent virus or development of
latent carrier status for either the virulent or vaccine viruses.
The eradication of the qisease is achieved by complete
depopulation and disinfection of infected premises.
l/erpesviridae
197
Duck Herpesvirus-l (DHV-l) (Anatid Herpesvirus)

(Duck Plaguevirus, Duck virus enteritis)
It is an acute fatal contagious disease of ducks, geese and swans,
and is of economic importance of duck producing areas. The disease
was reported by Baudet in 1923 from Netherlands. Now it is recognised
as a major disease .of ducks throughout Europe, North America, China
and India. The disease from this country was reported in 1964 by
Mukerji et al fr:om West Bengal. The disease outbreaks have been
reported from other areas of this country as well. In addition to
domestic ducks, wild ducks, geese, swans are other water birds that are
equally susceptible.
Properties of the virus: The virus is similar in morphology and
other physical and chemical characters with herpes viruses. The virus is
ether and chloroform sensitive and does not haemagglutinate duck,
chicken, sheep red blood cells. All the strains of the virus are
antigenically homogenous.
Cultivation: The virus grows on the chorioaIlantoic membrane of
duck embryos and kills the embryos within 4 days with extensive
haemorrhages. The virus can also be adopted to grow in chicken
embryos and chicken embryo fibroblast cells. The adapted virus
becomes attenuated for ducks.
Epidemiology: Natural infection occurs in domestic and wild
ducks, geese, swans and other water fowl. Migratory water fowl may
contribute to spread within and between continents. The virus is
transmitted by direct or indirect contact through infected water.
Transmission does not occur through eggs but arthropods have been
suspected to transmit the virus. The virus has been isolated from wild
ducks upto one year after infection.
Pathogenesis: The incubation period is 3-7 days. A sudden, high
and persistent mortality may be the first sign. Morbidity and mortality
vary from 5-100%. Most ducks develop clinical signs and die. There is
drop of egg production of about 25 to 40%. In affected birds there is
viraemia, photophobia, thirst, diarrhoea, nasal and ocular discharge.
Ingested virus causes enteritis and viraemic spread leads to vasuculitis
and widespread focal necrosis. Blood is present in the body cavities
including gizzard and intestinal lumens and petechial haemorrhages in
many tissues. Intranuclear, inclusions are readily demonstrated in
hapatocytes, intestinal epithelium and lymphoid tissues.
198
Diagnosis: Clinical and gross postmortem findings can be

confmned by fmding herpesvirus inclusion bodies or positive
immunofluorescnece. The virus can be isolated in duck embryos and
identified }>y virus neutralization test
Control: The virus produces adequate immunity after natural
infection. The inactivated vaccines do not give satisfactory immunity.
A chicken embryo attenuated strains when administered
subcutaneously, produce good immunity.
Pigeon Herpesvirus
It causes conjunctivitis: respiratory distress, diarrhoea, dehydration
and emaciation with lesions in liver and other organs of pigeons. The
virus grows in chicken embryo by CAM route and also in chicken
embryo kidney and liver celJs. Pigeons 1-6 months of age are most
often affected. Focal necrosis and A type intranuclear inclusions are
found in the liver and kidneys.
Marek's Disease
Viru~
(MDV)
It is a transmissible virus disease which mainly affects domestic

fowl and is characterised by mononuclear infiltration around peripheral
nerves and to lesser extent in skin, muscle, iris and internal organs. The
disease affects mainly domestic poultry and is common in young birds
of 2-5 months old. The first detailed account of the disease was
published in Holland in 1914. The specific herpesvirus etiology was
established in 1967. The disease occurs worldwide. In this country the
disease was reported in 1970 and is prevalent in all parts of this
country. Mohanty et al.(1973) reported annual losses to the tune of 40
million rupees to poultry industry of this country. Marek's disease is an
important model for the study of tumorigenic potential of
herpesviruses.
Properties of the virus: Two types of virus particles, onc is naked
virion measuring 35-100 nm and second type of virion particles are
enveloped and measure 150-170 nm in size, have been described. The
enveloped virion of MDV are of two types, one of which is found in the
nucleus of infected cells and is 150-180 nm in diameter and presumed
to be noninfectious. The second type found predominantly in cytoplasm
and in feather follicle epithelial cells is infectious and measure 250-280.
nm. The genome is double stranded DNA with a moleular weight of
Herpesviridae
199
lxlO' daltons. The G+C content is 46 percent. In cultured cells the

virus is not readily demonstrated but preparations from lysed feather
follicle epithelium reveal the presence of particles measuring between
275-400 nm in diameter. The virus survives for a long time at 60C in
growth medium containing 10% calf serum and 7.5% dimethyl
sulphoxide. All the strains isolated have been found to be antigenically
homogenous but some differences have been reported. The virus cross
reacts with herpes simplex, Aujeszky's disease virus, IBR and EpsteinBarr virus. The highly oncogenic MDV strains and their attenuated
variants have been placed in one group and low o~cogenic or non
oncogenic strains in second group based on immunodiffusion tests. The
herpes virus of turkey (HVT) forms a third antigenically related group.
There is certain DNA homology between HVT and MDV viruses.
Cultivation: The virus can be cultivated in susceptible chickens,
chicken embryos and cell cultures. When day old chicks are inoculated
with suspected material the lesions can be detected after 2-4 weeks in
ganglia, nerves and certain visceral organs. By fluorescent antibody
technique many tissues of chicks show the viral antigens. The virus can
also be cultivated on the CAM of susceptible chicken embryos or by
yolk sac route. The virus produces discrete white pocks on the CAM.
The virus grows in chicken kidney (CK) and duck embryo fibroblasts
(DEF) and produces characteristic CPE in 6-14 days. The CPE consists
of rounded and fusiform refractile cells and polykaryocytes which have
inclusions in the nucleus. When cells from infected chicken kidney are
grown, characteristic CPE is produced. The tumour, spleen, kidney,
buffy coat or whole blood from affected birds is suitable material for
virus isolation.
Epidemiology: Most of the birds at the time of maturity have
antibodies in MDY although the infection persists and virus is shed in
the dander of feather follicles. Congenital infcction does not occur. The
disease occurs most commonly in young birds between2-5 months of
age. If the chickens are exposed on the day of hatching the disease
develops at 3 weeks of age. Natural transmission is air borne through
inhalation. The infectious virus is present in oral, nasal and tracheal
secretions and in feather follicle epithelium. Genetic resistance, age and
viral strains influence the outcome of infection. A small percentage of
infected birds develop clinical MD. During rust few days of life, chicks
are very susceptible to the virus. The virus matures in the feather
200
collicle and is shed through desquamated cells into the environment.

The infected feathers remains infected for 6 weeks or more. Blood or
tumor material containing live, intact cells can transmit the disease.
Infected whole cell cultures may transmit the disease but not cell free
fluid as the virus is cell associated. Occasionally MD has also been
reported from free flying birds like crow and myna.
Pathogenesis: The outcome of infection of chickens by MDV is
influenced by virus strain, route of infection, dose, age, sex, immune
status and genetic susceptibility of the chickens. Subclinical infection
with virus shedding is common. The infection is acquired by inhalation.
The epithelial cells of respiratory tract are productively infected and
contribute to cell associated viraemia involving macrophages. By sixth
day there is productive infection of lymphoid cells in a variety of
organs. During the second week after infection there is persistent cell
associated viraemia followed by proliferation of T lymphoblastoid cells
and after few days cells begin to die. The lesions result from infiltration
and proliferation of T lymphocytes which may result in leukemia and
inflammatory cell response to the lysis of nonlymphoid cells by the
virus. Enlargement of one or more peripheral nerve trunks is one of the
most constant gross pathological finding. The nerves are 3 times their
normal diameter and show loss of striations, oedematous, grey or
yellowish and somewhat translucent in appearance. The celiac, cranial,
intercostal, mesentiric, brachial, sciatic and greater splanchnic nerves
are mostly involved. The enlargement of the nerve is usually unilateral.
Lymphoma-tous lesions affects the gonads, lungs, liver, spleen, kidney
and thymus.
The behaviour of MDV with lymphoid and non lymphoid cells
differs considerably. In non lymphoid cells infectious virus particles are
produced in feather follicle epithelium or non infectious virus particles
or viral antigen is produced in other epithelial cells or in cell culture. In
both cases the death of cell takes place. In lymphoid cells the
transformation of cells take place with extensive proliferation without
cell death. In these cells the presence of virus particles or the
expression of viral antigen is not detected but in some Iymphoblastoid
cell lines a low percentage of cells may produce viru~ particles or viral
antigen.
~arek's disease is a progressive disease with variable signs. In
neurolymphomatosis or classical disease there is paralysis of one or
Herpesviridae
201
both legs or wings. The early sign shown by the birds is incoordination.
one leg held forward and other backwards. There is drooping of wings
and lowering of head or neck. If vagus nerve is involved there may be
dilation of crop and gasping. The acute marek's disease occurs in
explosive outbreaks in large number of birds. The clinical signs are
depression followed by ataxia and paralysis of some birds. In ocular
lymphomatosis the iris of one or both eyes is gray in colour and the
pupil is irregular ecentric. The cutaneous form of disease is recognised
by nodular lesions upto 1 cm in diameter seen particularly at feather
follicles.
Immune reaction: The infected nonlymphoid cells contain viral
antigens while infected lymphoid cells contain Marek's associated
tumor specific antigen (MATSA). These antigens can be detected by
immunofluorescence and immunodiffusion test. MATSA can be
detected by using immune sera prepared from chickens or rabbits
immunized with transplantable tumor cells or cultured lymphoblastoid
cells but not by antiserum derived from chickens that have recovered
from MD. MATSA has been associated with cells transformation. The
birds develop both humoral and cell mediated immunity (CM!) after
infection with MD. The virus specific antibodies appear 1-3 weeks after
infection and persist throughout the life of bird. The presence of
antibody does not have a significant protective effect but may decrease
the severity of the disease. The resistance to MD is primarily due to
CMI response. T cells probably play a double role in MD by providing
target cells for transformation by MDV and also by participating in
immune mechanism against the development of lymphoid tumors.
MDV has an immunosuppressive effect. The progeny of infected birds
acquire passive immunity which disappears within 3-4 weeks.
Diagnosis: A tentative diagnosis is arrived at on the basis of
symptoms and lesions. In classical form there is unilateral or bilateral
paralysis of legs or wings, curling of toes, torticollis, dilatation of iris.
In acute forms symptoms are rapid depression, emaciation and death.
Most cases show oedematous swelling of vagus and sciatic nerves and
of brachial, coeliac and lumber plexus.
Detection of viral antigen by immunofluorescence is the reliable
diagnostic procedure.
MDV can be isolated by inoculation of susceptible chickens, cell
cultures and embryonated chicken eggs. The specimens of choice are
202
intact viable cens from buffy coat, spleen, tumour tissue or other
lymphoid cens. Virus can also be isolated from skin and feather tips
which contain cell free virus. The day old susceptible chicks ate
inoculated intraperitoneally. After 18-21 days the birds show gross or
microscopic lesions. The virus isolation and detection of serum
antibody ftom the inoculated chickens should be undertaken. MDV
produces characteristic CPE in duck embryo fibroblast (DEF) and
chicken kidney (CK) cen culture. Characteristic plaques are produced
in 6-14 days and consist of rounded refractile cells and polykaryocytes
with cowdry type A inclusions. The virus produces pocks on the CAM
of chicken embryos. Identification of virus isolated can be confirmed
by immunofluorescent technique.
The antibodies can be demonstrated in the sera of infected or
recovered birds by agar gel precipitation test, indirect fluorescent
antibody test or passive haemagglutination test.
Control: MD is the first virus induced malignacy to be controlled
by vaccination. There are three types of live vaccines available. These
are herpes virus of turkeys (HVT). apathogenic strain of MDV and cell
culture attenuated MDV. All these vaccines are equally effective. The
vaccine virus persists in the vaccinated birds and does not prevent
superinfection, replication or shedding of virulent MDV but prevents
clinical disease. The resistance is life long in vaccinated birds. The
mechanism of protection is due to cell mediated immunity response.
The vaccinated chickens probably develop lymphopro1iferative lesions
containing Marek's associated tumour specific surface antigen
(MATS A) and a CMI response directed against MATSA protects the
chickens against subsequent lymphoma formation by the ,:,irulent virus.
The most extensively used vaccine is HVT. Chickens are generally
vaccinated at hatching and should be reared in isolation. The hygienic
procedures help to delay the exposure of the chickens and thus allow
immunity to be established.
References
ALLAN,
G.P.; YEARVAN, M.R.; TURllUEN, L.W.; BRYANS, J.J. and McCOLLUM,

W.H., 1983. Molecular epizootologic studies of equifU! herpes virus-l
infections by restriction endonuclease fingerprinting of viral DNA.
American Journal of Veterinary Research. 44 (2): 263-27l.
BARAHONA,
H.; MELl!NDEZ, L.V. and MELNICK, J.L., 1974. A compendium of
Ilerpesviridae
203
herpesviruses isolaJed from non human primates. Intervirology. 3:

175.
BAUDET, A.E.R.F., 1923. Mortality in ducks in the Netherlands caused by a

fiIJrable virus fowl plaque. Tijdschi Diergeneesk. 50: 455-459.
BUXTON, A. and FRASER, G., 1977. Animal Microbiology. Vo1.2. Blackwell
Scientific Publications. Oxford.
CAMPBELL, T.M. and STUDDERT, MJ., 1983. Equine herpesvirus type-1 (EHV1).
Veterinary Bulletin53: 135-145.
CHURCHll.L, A.E. and BlOGS, P.M., 1967. Agent on Marek's disease in tissue
culture. Nature215: 528-30.
DEWNETT~D.P.;
BARASA, 1.0. and JOOIINSON, R.H., 1976. Infectious bovine

rhinotracheitis virus: studies on the veneral carrier status in range
cattle. Research in Veterinary Scicnce20: 77-83.
DEv PRAKASII and RAJYA, B.S., 1970. Avian leucosis complex. I. Demographic
studies. Il. Pathoanatomy and serum lactic dehydrogenase level of
Marek's disease in natural infection. Indian Journal of Animal
SciencesAO: 282-296.
DEw, C. and McFERRAN, lB., 1966. Experimental studies on Aujesky's disease

in cattle. Journal of comparative Pathology and Thcraeutics. 76: 379385.
GmBS, E.P.J. and RWEYEMA.\iU, M.M., 1977. Bovine herpes viruses. Part. I.
Bovine erpesvirus 1. Part 11. Bovine herpesviruses 2 and 3.
Veterinary Bulletin, 47: 317-43 and 411-425.
GREIG, A.S.; BAUMISTER, G.L.; MITCIIFLL, O. and BARKAR, C.A.V., 1985.
Cultivation in tissue culture of an infectious agent from coital
exantheme of cattle. A preliminary report. Canadian Journal of
Comparative Medicine. 22: 119-122.
GREWAL, GURDEV, SINOIl and SINGII, BALWANT, 1976. Incidence of Marek's
disease virus infection in domestic fowl5 of Punjab (India). Avian
Diseases. 20: 191-194.
HANSEN, L.E., 1978. Laryngotracheitis. In M.S.Hofstad, B.W.Calnek, C.F.
Helmboldt. V.M. Reid and H.W. Yoder, JR (Editors), Diseases of
POUltry. Oxford a:ld IBH Publishing Co.New Delhi pp. 607-618.
Infectiuous bovine rhinotracheitislinfectious pustular vulvovaginistis. 1984.
Publication of Division of Pathology, Indian Veterinary Research
Institute, Izatnagar, U.P.
204
JAIN, N.C.; MANCHANDA, V.P.; GARO, D.N. and SHARMA, V.K., 1976. Isolation
and characterisation of Equine herpes virus type-I. Veterinary
Record.99: 57.
JAIN, N.C.; SB1lJ, P. and PRASAD, G., 1986. Herpes virus infections. In National
symposium on 'Current status of herpes virus infections in man and
animals in India' held at at Department of Veterinary Microbiology,
Haryana Agricultural University, Hisar.
KAHRS, R.F., 1985. Viral disease of cattle. Kalyani Publishers, Ludiana.
HAY, H.G. and TITI'SLER, R.P., 1925. Tracheolaryngitis in Poultry. Journal of of
American Veterinry Medical Association. 67: 229-23l.
MEHROTRA, M.L.; RAJYA, B.S. and KUMAR, S., 1976. Infectous Bovine
rhinotracheitis (IBR). Keratoconjuctivitis in calves. Indian fournal of
Veterinary Pathology. 1: 70-73.
MEHROTRA, M.L.; KUMAR, S. and RAJYA, B.S., 1981. Note on passive
of
infectious
haemagglutination
test
for
detection
bovinerhinotracheitis infectious pustular vulvovaginitis (lBRIPN)
virus antibody. Indian Journal of Animal Scineces. 51: 559-569.
Mll.LER, NJ., 1955. Infectious necrotic rhinotracheitiS of cattle. Journal of
American Veterinary Medical Association. 126: 463-467.
MOHANTY,. G.C.; AcHARJYo, L.N. and RAJYA, B.S., 1973. Epidemiology of
Marek's disease (MD). Stuides on the incidence of M.D. precipitins
in some zoo birds. Poultry Scinece 52: 963-966
MOHANTY, S.B., 1990. Pseudorabies virus. In virus infections of ruminants
edited by Z. Dinter and B. Morein. EIsevier Science Publishers B.V.
Amsterdam.
MOHANTY, S.B. and DUTIA, S.K., 1981. Veterinary Virology. Lea and Febiger,
Philitdelophia.
MUKERJI, A.; DAS, M.S.; GHOSH, B.B. and GANGULY, lL., 1963. Duck plague in
West Bengal I and /I. Indian Veterinary Journal. 40: 457-462.
NAZARIAN. K.; LINDEHL, T.; KLElN, G. and LEE, L.F., 1973. Deoxyribonucleic
acid of MareKs disease virus in virus induced tumors. Journal of
Virology. 12: 341-346.
PARIHAR, N;S.; RAJYA, B.S. and Gll.L, B.S., 1975. Occurrence of malignant
catarrhalfever in India. Indian Veterinary Journal. 52: 857-859.
PLOWRIGHT, W., 1990. Malignant catarrhal fever virus. In virus Infections of
Herpesviridae
205
ruminants, edited by D. Zinter and B. Morein. Elsevier science

Publishers, B.V. Amsterdam.
H.G., 1972. Recent advances in thl: knowledge of Marek's disease.
in veterinary Science and comparative Medicine. 16: 22953.
PuRCHASE,
Advan~es
SAMBY AL, D.S. and BAXI, K.K., 1979. Isolation of Marek's disease virus from
tissues of birds suspected for Marek's disease. Poultry Advisor, 12:
17-19.
SHARMA, G.L.; LALL, IM and BHALLA, N.P., 1965. Histopathological evidence
of equine viral abortion in India. Indian Journal of Veterinary
Science and Animal Husbandry 35: 18-23.
Singh, G.; Singh, B.; Guta; P.P. and Hothi, D.S., 1979. Epizootiological
observations on malignant catarrhal fever and transmission of the
disease in buffalo calves. (BubaIus bubalis). Acta Veterinaria Bruno.
48: 95-103.
SINGH, S.B.; SINGH, G.R. and SINGH, C.M., 1964. A preliminary report on the
occurrence of infectious laryngotracheitis in poultry in Punjab.
Poultry Science. 43: 492-494.
SlRAUB, P.C., 1990. Infectious bovine rhinotracheitis virus. In virus infections
of ruminants, edited by D. Zinter and B. Morein. Elsevier Science
Publishers B.V. AmsterdaIJ)..
STUDENT, M.I; SIMPSON, T. and Rozilllian, B., 1981. Differentiation of
respiratory and abortigenic isolates of equine herpes virus I by
restriction endonucleases. Science. 214: 562.
WrITHMAN, G.; GASKELL, R.M. and RZIHA, H.J., 1984. Latent herpes virus
infections in Veterinary Medicine. In a seminar in the commission of
European Communities held at Tubingen. FRG. Martinus, Nijhoff
Publishers, Boston.
Chapter 18
Unclassified DNA virus

African swine fever Virus (ASFV)
The African swine fever virus which was previously included in
family Iridoviridae has now been taken out of this family but not yet
been placed under any other family. African swine fever virus produces
highly contagious fatal disease in pigs with some similarities to swine
fever. The disease causes serious economic losses and is indigenous to
African continent. The disease first appeared in 1910 in East Africa and
Africa and then it spread to other territories of the continent. ASF was
reported in 1957 from Portugal. A number of isolated outbreaks have
been reported from Spain, France and Italy. In 1971 the disease was
reported from Cuba. The disease has not been reported to exist in this
country.
Property of virus: The virus particle measures 200-220 nm in
diameter and consists of electron dense centrally placed nucleoid
surrounded by a hexagonal outer shell. The virion acquires an
additional outer envelope from plasma membrane. The genome consists
of single stranded DNA, which codes for about 25 structural proteins
and also some non structural proteins. The nucleoprotein core is
surrounded by icosahedral sheet and contains 9% phospholipid. The
virus is stable at room temperature for severn I weeks. The virus is
inactivated in 30 minutes at 56 C and survives for months and even
years in refrigerated meat. The virus survives in chilled carcases for 15
weeks or longer and upto 6 months ID processed meat. The virus is
stable over a wide pH range and is inactivated quickly in 2% NaOH.
Unclassified DNA Virus
207
The virus is not inactivated by crystal violet which is used to inactivate

swine fever virus. The virus does not possess the property of
haemagglutination but infected leucocyte cultures haem adsorb pig rbe.
The haemadsorption is inhibited by African Swine fever serum.
Different strains do not cross protect and it is probable that several
anti genic types exist
The virus was fIrst propagated in the yolk sac of chicken embryos.
Most of the strains replicate in the buffy coat cells of swine bone
marrow. The virus produces syncytia and nuclear and cytoplasmic
inclusions. in infected syncytia and nuclear and cytoplasmic inclusions
in infected cell culture. however some strains do not produce CPE.
Epidemiology: Recovered animals develop long resistance to
reinfection with homologous virus type but not against heterologous
types. The virus from the infected animals is excreted in secretions and
transmit the virus by contact or by aerosol route. The wart hogs in
Africa act as reservoirs and transmit the infection to domestic pigs by
indirect contact. The surviving pigs act as life long carriers. lice and
ticks of pigs transmit the infection. Since the virus is resistant to
atmospheric conditions and persists in the tissues of the carcases of
carrier animals. the feeding of swill containing uncooked pork scraps is
responsible for secondary epidemics and virus outbreaks. The outbreak
in Portugal. Brazil etc. were due to feeding of untreated swill. The
swine fever vaccine is also responsible for spread of outbreaks. In
Brazil swine fever vaccine was contaminated with ASFV. In the
inactivated crystal violet swine fever vaccine. the virus of ASFV
survived and caused outbreaks.
Diagnosis: African swine fever is suspected when there is acute or
paracute haemorrhagic disease with high mortality is encountered in SF
vaccinated pigs. The provisional diagnosis can be made on clinical and
postmortem lesions. A confirmation can be made by inoculating pigs
susceptible and immune to ASF with the suspected material. The virus
isolation can be attempted in cell culture from blood. splcen lymph
nodes of infected animals. The infected leucocyte cultures haemadsorb
pigs rbe and this property is not shown by SFV. The test based on this
property of ASFV is applied for diagnosis. Complement fixation and
gel diffusion tests are applied for detection of ASFV antibodies.
Fluorescent antibody is also useful in the diagnosis.
Contrpl: Vaccination with killed as well as with live virus has not
208
been effective. The virus appears to be a poor antigen. Pigs recovered

from the disease are immune to homologous strain but not heterologous
strains. The effective method of eradication is by slaughter and strict
import regulations for meat products as well as live animals.
Reference
COOGlNS, L., 1974. African swine fever virus. Pathogenesis: Progress in medical
Virology. 18: 48-63.
DETRAY, D.E., 1964. African swine fever virus. Advances in Veterinary Science
8: 299-333
W.A. and HAY, D., 1960. Haemodsorption and cytopathic effect

produced by African Swine fever virus in swine bone marrow and
buffy coat cultures. American Journal of Veterinary Research 21,
104-108.
MALlNQUlST,
MAINER, F.D., 1975. African swine fever. In Diseases of swine, 4th Edition,
Edited by H.W.Dunne and A.D. Lemann, Iowa, Iowa State
University, Ames, Iowa.
RuSSELL, P.H. and EDlNGTON, N., 1985. Veterinary viruses. The Burlington
Pr~ss (Cambirdge) Ltd. Foxton, Cambridge
PARTII
SYSTEMATIC
VIROLOGY
R.N.A. VIRUSES
Chapter 19
Picornaviridae
Picorna name was introduced in 1962 to describe a group of very

small RNA viruses. The family contains four genera namely
Enterovirus, Rhinovirus, Cardiovirus and Aplhovirus. The viruses
included in this family are small icosahedral nonenvcloped measuring
22-30 nm in diameter. The capsid appears smooth and round in outline
and made up to 60 capsomeres each consisting of onc molecule of each
of the four major polypeptides: VPl, 2,3 each of about 30K and VP4
of about 1OK. The picorna virus capsid contains one moleucle of
infectious single stranded RNA of positive polarity with a molecular
weight of about 2.5 x 1()6. A convalentIy linked protein VPg is present
at 5' terminus of the RNA. Four major polypeptides participate in the
construction of the capsid. Purified VPl elicit neutralising antibody,
though less immunogenic than intact inactivated virus particle. This
discovery has provided a major impetus fOr the development of
recombinant DNA and synthetic peptide vaccines for foot and mouth
disease.
The most important difference between the physiochemical
properties of the virions of 4 major genera are the their pH stability.
The apthovirus are unstable below pH 7 the rhinoviruses lose activity
below pH 5 and entero and cardioviruses are stable at pH 4. The
cardioviruses can be distinguished from enteroviruses by their
biophasic pH stability in the presence of O.lM halide ions. Another
important difference is the presence of polycytidylic acid tract of
unknown function in the genome of aptho and cardioviruses but not in
entero and rhinoviruses. If protected by mucus and strong sunlight,
212
picornaviruses are relatively heat stable. Replication is entirely in the

cytoplasm in <;lose association with the membranes. Partially double
stranded replicative intermediates are formed. both strands of which are
used as. templates. The functional proteins are found by
posttranslational cleavage from large precursor polyproteins. During
replication the metabolism of cell is stopped and cells seen show signs
of degeneration. This degeneration of cells facilitates the release of
progeny.
APTHOVIRUS
Apthovirus (Foot and Mouth Disease virus): Foot and mouth

disease is a highly infectious disease of cattle. sheep. goats and pigs as
well as of a number of wild animals like buffalo. deer, wild ungulates
and hedgehogs. Man is occasionally infected. The disease is
characterised by the formation of vesicles on the mucosa of mouth and
sometimes alimentary tract and on the skin of the teats and udder.
There is a sudden death in young animals due to heart failure. The
disease has occurred at one time or other in all parts of the world. The
earliest record of the disease comes from Northern Italy in 1514. The
geographical distribution of the disease is world wide. The disease is
prevalent in Europe, Asia, Africa and South America. It has not been
reported from Newzealand and Iceland. Australia had its last outbreak
in 1872. Canada had only one outbreak in 1952. The USA is free since
1929. In India the disease is widespread throughout the country. In
England the disease was ftrst recorded in 1839. Subsequent serious
outbreaks occured in 1871. 1922-24. 1952 and 1967-68.
Properties of the virus: Loeffler and Frosch (1898) reported the
ftrst ftltrable agent responsible for causing foot and mouth disease. It is
one of the smallest and earliest recognised animal virus. The intact
virus particles of 25 nm and 7nm have been found. The viral genome is
single stranded RNA with 8000 bases and the molecular weight of
genome is 2.6 x 1()6 daltons. There is no cap at 5' end of RNA but
instead a protein called VPg. The capsid has 4 polypeptides- VPl. VP2.
VP3 and VP4. Traces of VPO and RNA polymerase is also found.
There are 60 copies of 4 major polypeptides in each particle.
The foot and mouth disease virion is resistant to ether. chlorofonn.
bile salts and detergents. The virus is acid-~nsitive and inactivated at
pH 5.0 but is stable in the presence of magnesium chloride. It is stable
Picornaviridae
213
at pH 7.4-7.6 and at pH 3. The virus is resistant to drying, especially if

it occurs quickly in the presence of protein. The virus persists on hay
and straw for about 15 weeks and on hides for much longer time. It is
stable at 4C or when kept in frozen state but the virus does not survive
in the muscles due to the production of acid during setting of meat. The
virus is readily inactivated by heat but some strains survive higher
temperature. Most strains lose infectivity within 48 hours at 37C. It
remains viable at 4-7C for many months. The virus of FMD is more
resistant to common disinfectants especially when the virus is mixed
with organic material. Phenolic type disinfectants, alcohol, acetone,
detergents and other organic solvents have little affect on the virus.
Formalin, potassium permagnate, lactic acid, ethylene oxide, sublimate
of mercury, 0.4% Beta propiolactone and 1-2% sodium carbonate is
effective in field conditions.
The antigens produced during the infectious cycle of foot and
mouth disease virus in tissue culture are given in Table 19.1.
Table 19.1
DESCRIPIlON OF MAL'! FMD VIRUS ANTIGENS (CROWI1IER,
1986)
Component
Sedimentation
Coefficient in
Sucrose gradients
Description
Whole particles
146S
Empty particles
75S
Subunit
12S
VIA
3.58
Contains a molecule of ss RNA

(2.6 million m.wt) 60 copies
of VP1, VP2, VP3, VP4
m.wts VP1-3 =24000
VP4 =8000
No RNA
60 copies VPO (uncleaved)
VP2& VP4
60 copies of VPl, VP3
Pentamer of VPl, 2 and 3
(5 copies of each)
Virus infection associated
antigen, RNA polymerase
associated m.wt. 56,000
All the components are antigenic. The integrity of whole particle
214
(l46S) is essential for formulation of successful inactivated vaccines.

The subunits are antigenic but do not produce immunogenic response.
The virus infection associated antigen (VIA) is produced in infected
cells and animals and is antigenic. Most studies have implicated that
VPl protein in responsible for antigenic variation. The FMDV was the
fIrst virus in which anti genic differences between strains were
recognised. Vallee and Carre in 1922 observed that French cattle which
had recovered from disease were reinfected by sick animals imported
from Germany. The two strains were named as Oise(O) and
Allemagne(A) from the areas of origin. In 1926 Waldmann and
Trautwein described 3rd type and named as type C. In 1930 there was
an evidence that strains isolated from Africa do not fIt in the framework
of 0, A and C. Galloway and others in 1948 while working at Pirbright
confIrmed the presence of 3 more types and designated them as SAT!,
SAT1 and SAT)JIn 1954 a new strain isolated from Pakistan was
identifIed at Pirbright. The same strain was also confrrmed from
material received from India, Thailand and Honkong and named as
Asia 1. The 0, A and C types are widespread in world while Asia 1
occurs only in Asiatic countries. The SAT types were confined in
African countries but in 1962 SAT! spread through Bahrain to the
countries of Middle East to Turkey and USSR and strain Au in the
same area in 1964-65. Within each serotype there are subtypes which
may be suffIciently different to give very poor protection against other
viruses of the same serotype. There appears to be continual antigenic
drift in enzootic situations. The subtypes are defIned by
crossneutralization tests. If one isolate is neutralized three times more
slowly than another by antiserum to one of the two isolates it is defIned
as different sUbtype and vaccinal cross-immunity is likely to be poor.
The individual subtypes are indicated by numerical subscript. The
number of subtypes in each serotype is A, 32; 0, 11; C, 5; Asia-I, 3;
SAT!, 7; SAT1 , 3; SAT),4.
Cultivation: The virus multiplies in cytoplasm of the cells. After
attachment the virus particle enters the cell by phagocytosis where
uncoating takes place. The virion RNA itself acts as mRNA. The
replication of fresh RNA (virion RNA or mRNA) is via replicative
intermediate shortly after the entry of the virus into the cell and it
involves the translation of RNA into a polypeptide called polyprotein.
This polyprotein is split into primary polypeptides or primary products
Picornaviridae
215
which in turn are split by virus specified proteases into polypeptides.

The polypeptides are finally split into VP4 VPz VP, and VPt The
assembly of polypeptide takes place and RNA is inserted into the
particle which is then released by cell lysis. Intermediate products are
also released on the lysis of cell.
The virus can be grown in laboratory animals. Guineapigs have
long been used as experimental hosts, the route of inoculation being the
hind footpads. Generalization takes place and vesicles are found on the
other pads and tongue. Skinner(1951) used unweaned mice for
cultivation of virus by intramuscular or intraperitoneal route. The virus
multiplies in high titres in the heart and skeletal muscles. The virus can
also be adapted to grow in older mice giving rise to paralysis. Young
hamsters and new born rabbits can be readily infected with the
production of paralysis and death. The virus has also been adapted to
grow in chicks and chick embryos. The most sensitive assay system is
multiple intradermolingual inoculation of cattle. Frenkel(1950) reported
the multiplication of virus in surviving fragments of bovine tongue
epithelium. The virus also grows in monolayers of bovine. porcine.
lamb and goat epithelial cells, secondary bovine thyroid cells and
continuous baby hamster kidney cell lines. Virus growth is detected by
focal cell degeneration.
Epidemiology: The epidemiology of FMD focusses around its
wide geographic distribution, its high transmissbility, the multiplicity
of virus types and subtypes. The susceptibility of wide range of
domestic as well as wild animals, the variety of methods of spread and
the rapidity with which the virus spreads in animals, herds and
countries and persistent infection in some species are the factors which
complicate the epidemiolo~y of the disease.
There are large areas of the world in which one or more FMDV
types are endemic. The disease is enzootic in Asia. Africa, and parts of
Europe. The countries contiguous to infected areas or which import
animal products from infected areas are at high risk. In countries where
the disease in not present, the epizootic develops rapidly from
introquction of disease to one farm. The outbreak spreads to many
farms very rapidly due to highly infectious nature of virus. aerosol
spread and short incubation period, which varies form 12 hours to 14
days. but usually is 2-6 days. The direct contact usually occurs from
animal to animal by droplet infection or when virus contacts the
216
mucosa of the mouth, nose, conjunctiva or abraded skin. Infection may

be indirect through contact with contaminated objects. The spread of
virus may occur during the incubation period or at the time of disease
in an animal. When an animal is slaughtered at a later stage of
incubation' or in the early stage or development of lesions, the virus is
present in the tissues. In muscles the virus is inactivated due to acid
formation but it survives in lymph nodes, bone marrow and offal. The
susceptible animals when come in contact with such material may
develop disease. The virus is excreted in milk in later stages of
incubation period or early states of disease. Similarly, semen contains
virus just before the lesions appear.
The most important route of infection for cattle, sheep and goats
appears to be respiratory tract by inhalation. The cattle is most
susceptible as they breathe greater volu~e of air and the infectious dose
is small. The air borne transmission is dependent on wind direction and
speed and is favoured by low temperature, high humidity and overcast
skies. The long distance airborne transmission was only realised in
1967-1968 in England which caused direct and indirect losses
estimated to $200,000,000 and approximately 634,000 animals were
slaughtered. Most spread over land is within 10 kms but depending
upon topography, the spread may be 60 kms or more over land and 250
kIns over sea. The criteria for spread are high virus output and survival,
low virus dispersi~n and large number of susceptible livestock. The
airborne spread has been found mainly in temparate climate, where
there is high humidity (60 percent) and density of livestock population
is high. In tropical and subtropical countries airborne spread takes place
over shorter distances especially during night.
The virus may survive longer in the pharyngeal region of
recovered cattle due to which it is maintained for longer periods of
time. Though the spread of virus from such animals to susceptible
animals is difficult. The pharyngeal secretions may also contain
antibodies which neutralise the virus. It could be possible that new
antigenic variants could arise in such immune animals.
In South America the Virus is maintained by continual spread
among the susceptible population of domestic animals. While in Africa
the wild game population plays a role in spreading the infection to
domestic livestock where there is extensive contact between wild game
animals and cattle. The role of sheep and goat varies in different parls
Picornaviridae
217
of world. In Middle East sheep and goat form major pathway of

infection.
In India three major factors of spread of infection have been
delineated, (i) movement of affected animals form one place to another,
(ii) intermixing of affected and healthy livestock at common grazing
ground and (iii) cattle fairs. The problem of the disease is of very
complex in nature. India has approximately 400 million susceptible
livestock apart from wild animal species. The presence of three main
types, i.e A,O,C and their respective variants was reported by
Seetharaman. and Datt (1951). These typing results were based upon the
cross immunity tests in guinea pigs. Dhanda et al. (1957) reported a
fresh isolation of an atypical strain, which was later confirmed from the
World Reference Laboratory belonging to type Asia l. An analysis of
reported outbreaks of the disease among cattle and buffaloes during the
period 1943 to 1973 has shown an average of 5000 outbreaks involving
as many as 2,75,000 animals each year. The accumulated data of 42
years (1943 to 1984) show the following prevalence of different types
of FMD virus in India (Anon, 1984-85)
F & M virus serolypes
No.of isolates recorded.
Percentage
Asia-1
3972
54.8
916
12.6
903
12.5
1463
20.2
The percentage figure given above for type A is inclusive of both

A5/AlO and A22 strains. In India only four FMD virus types O,A, C
and Asia-l are known to occur. The virus types have a regional
pref(1rence. During 15 years period type
has emerged as dominant
type in the 8 states of Jammu and Kashmir, Punjab, Himachal Pradesh,
Haryana, Uttar Pradesh, Bihar, Sikkim and Kerala. Type Asia 1 was
dominant in Madhya Pradesh, Gujrat and Karnataka. Type A was
dominant in Andhra Pradesh and Karnak1ka. Virus type C maintained a
low profile, the outbreaks occured more in Tamil Nadu, Kerala and
Maharastra. Sporadic occurrence was in Himachal Pradesh, Punjab,
Uttar Pradesh, Rajasthnn, Madhya Pradesh and Bihar.
Palhogenesis: The incubation period is usually 2-5 days but may
be extend~ upto 2-3 weeks. The most severe disease is seen among
cattle and pigs. The virus infects the epithelium of upper respiratory
tract or alimentary tract by inspiration or ingestion. The virus multiplies
218
at the site of infection where primary vesicles are fonned in 1-4 days.
Mter primary vescile fonnation the virus initates viraemia associated
with fever. The virus localises in distant epithelia and secondary
vesicles appear. Vesicles occur in stratified squamous epithelia or
mucous membrane. The vesicles appear on the mucous membrane of
tongue, lips, gums, cheeks, dental pad and on the skin of interdigital
space, on the coronary bands of feet, at the bulbs of heels and on teats
and udder. The lesions in the mouth cause excessive salivation,
smacking of lips and anorexia Vesicles and erosions on teats areassociated with rapid drop in milk yield and may be followed by
mastitis. Internally lesions may be found in oesophagus and fore
stomachs. Sheep and goats may only show lameness without mouth
lesions. In pigs lesions may be seen on snout as well as on the feet. In
young animals the virus localizes in the heart and produce myocarditis.
The disease is generally mild in adult cattle, but pregnant cows may
abort. Although morbidity is high, mortality is low but mortality may
be high in young calves and lambs. The importance of FMD lie in
heavy economic losses. In India it causes an estimated annual loss of
US$ 400 million in milk and US$ 10 million in impaired' working
capacity of bullocks.
Immune reaction: Humoral antibodies can be detected after about
4 days of infection. The early antibodies are of IgM type and reach a
peak titre between 7-14 days and declines within 30 days. These early
antibodies of IgM class neutralize and precipitate both homologus and
heterologus virus. The IgG class antibodies appear after 10-14 days and
reach peak at about 28 days. These antibodies are more type specific,
neutralize and precipitate virus and fix complement. The cattle after
recovery remain totally immune for 3-4 months. The partial immunity
can persist for 4-5 years. The rate at which immunity declines depends
upon age, level of nutrition, physiological sUite and breed of animals.
The antibodies are transferred to calves through colostrum. These
antibodies in calves interfere with vaccination for 3-6 months and
afford protection for about 6 weeks of age. Immunity following
vaccination is shorter. A single inoculation of inactivated vaccine
protects the animal to challenge for 3-6 months.
Diagnosis: The highly contagious nature of disease and presence
of salivation, typical raised vesicles with blanched covering epithelium
filled with straw coloured fluid, lameness are usually p'1thogenomonic
of FMD. The laboratory tests are always essential to establish type and
Picornaviridae
219
subtype and also to differentiate FMD from other vesicular diseases

like vesicular stomatis, vesicular exanthema and swine vesicular
exanthma.
Table 19.2
DIFFERENTIAL DIAGNOSIS OF VESICULAR DISEASE
Animal species
Route of inoculation
Natural infection
Cattle
Pig
Sheep & goat
Horse
Experimental Infection
Cattle
Intradermal in tongue, gums
and lips
Intramuscular
Pig
Intradermal in snout, lips
intramuscular
Sheep &
Intradermal
goats
Intradermal in tongue
Horse
FMD
VS
+
+
+
+
+
+
VE
SVE
no lesions
(+)
no lesions
+
(some
strains)
Guinea pig
Intradermal in foot pad
Unweaned mice
Adult chicken Intradermal in tongue
Growth in tissue culture
Calf thyroid
BHK
IB-RS-2
Chick embryo cells
Morphology (electron Microscope)
FMD Foot arId mouth disease

VE
Vesicular exanthema
+
+
+
+
+
+
+
+
Sphe- Bul- Sphe- Spherical

let
rical rical
shaped dark 30 nm
175x65 stainnm ing areas
35--40 nm
VS Vesicular stomatitis
SVE Swine vesicular exanthema
In India vesicular disease except FMD have not been reported. It is
220
worth while to summarise the differential diagnosis in a table 19.2. The

specimens for diagnosis should be collected from those animals that
developed clinical signs recently i.e. when the animals have
temperature., nasal discharge and the vesicles appear in the mouth and!
or the feet. Samples from at least 2 animals should be collected. The
samples include blood in anticoagulant. serum pharyngeal and
oesophageal fluid collected with cup Probang. The quantity of
epithelium available varies from animal to animal but a piece of 2 x 2
cms is appropriate. From dead animals samples may be collected from
lymph nodes, thyroid and heart. The samples are frozen and despatched
to the laboratory in frozen state. Where it is not possible to send
samples in frozen state, the samples are transported in glycerol buffer
pH 7.6.
Virus isolation: Cattle, guinea pigs, suckling mice and cell culture
are used for isolation of virus when the concentration of the virus in the
" samples is low. The isolated virus is identified by complement fixation
or neutralization test.
Cattle: The cattle are most susceptible but are seldom used for
routine investigations because of cost and other difficulties of keeping
the animals in strict isolation. The virus suspension is inoculated
dermolingually into the dorsum of tongue by making several
inoculations. About I ml of inoculum is used for injection. The
temperature, vesicles and other sysmptoms usually develop between
24-72 hours of inoculation. The vesicular epithelium is collected for
carrying out serological test.
Mice: The unweaned mice(6-8 days old) are highly susceptible to
experimental infection and can be used for isolation of virus from field
samples instead of cattle. The suspected material is inoculated in all th~
animals of the litter except one animal, which is kept as control. If any
animal dies between 1-7 days its carcases are used as source of antigen.
The mice after 3-4 days of inoculation show paralytic syptoms and die.
Guinea pigs: Healthy guinea pigs weighing about 400 to 500g
body weight are used for inoculation by intradermal route into the
planter pads by making tunnels. The pads become red and vesicles
appear usually by 3rd day of inoculation. The tissues from the lesions
are collected and llSed as source of antigen.
Cell cultures: Bovine thyroid, BHK-21, IB-RS-2 cells are used for
isolation of virus. Two or three tubes are inoculated with the suspect
Picornaviridae
221
material and incubated at 37C. A marked degenerative type of

cytopatbic effect is observed within 24-48 hrs. The cell culture fluid is
used as antigen. In case cytopathic effect is not visible 2-3 passages
more may be required.
Serological tests: The serological tests are applied directly to the
field material or the material is passaged in experimental animals or in
cell culture and material collected is used as antigen.
Complement fIXation text: It is one of the most commonly used
and very important for identifying the virus type from the suspected
samples of FMD. The antigen used in this test obtained from original
suspected material or from cell culture of tissues from the experimental
inoculated animals. The hyperimmune antisera against known types is
prepared in guinea pigs.
The complement present in the test combines with homologous
antigen-antibody complexes. The amount of complement fixed to the
extent of 50% by type specific serum and unknown antigen are
assessed. The serum antigen complex giving the highest fix.ation is
described as homologus. In this way the unknown antigen is identified
as belonging to particular type. The method described by Brooksby
(1952) is followed in most of the laboratories. The method is quick and
results can be obtained within 2-3 hours. The quantity of complement
fixed is estimated visually, semiquantitatively, or quantitatively and the
exact amount required for giving 50% fixation is ascertained. The
micro-complement fixation test is preferred because it conserves the
reagents, is simple to perform and large number of samples can be
processed at one time.
Neutralization text: This test is used to detect specific antibodies
in the sera of recovered animals or for identification of virus with
known hyperimmune sera. The sub types are defined by cross
neutralization test. The tests are performed by constant virus and
varying dilution of sera or vice versa. In cross neutralization test if one
isolate is neutralized three times more slowly than another by antiserum
to onc of the 2 isolates it is defined as a different subtype and vaccinal
cross immunity is likely to be poor.
Gel diffusion lest: The gel diffusion test in agar can also be used
for identifying unknown FMD virus strains. The test can be carried out
in slides. The test is not very sensitive.
Radial immuno haemolysis lest: This test was applied by several
222
workers to type and subtype strains of FMD virus and a positive

correlation of the results with complement fixation test has been
reported. The virus antigen is coupled with sheep erythrocytes in
presence of chromium chloride and is used for detection of specific
antibodies ill the serum.
Immunofluorescence test: The immunofluorescence test is used
for detection of antigen in tongue epithelium, oesopharyngeal fluid and
various organs of dead animals suspected to have died of FMD. It gives
a quick diagnosis in the face of outbreak where the virus cannot be
isolated from the samples or for screening of large number of carrier
animals by examining the oesopharyngeal fluid. The test can also be
used for typing of FMD virus strains but it is a very lengthy procedure
and cannot be used as a rountine test.
Immunoperoxidase test: The enzyme horse redish peroxidase is
attached with antibody. The peroxidase labelled antibody is reacted to
viral antigen (intracellular or extracellular). The reaction is observed by
use of substrate which reacts with peroxidase and produces a colour.
The test can also be applied for typing of FMD strains where
confirmation of complement fixation test is required. In routine, the test
is not applied forvirus typing.
Enzyme linked immunosorbent assay (ELISA). The test is hundred
fold more sensitive and is likely to replace complement fixation test in
FMD virus diagnosis in future. The test is used for serotyping of FMD
viruses using ELISA has a. potential to compare viruses according to
their immunogenic properties so that in future, it may be possible to
predict the protective qualities of vaccine strains against field isolates.
Recently electrofocussing and finger printing of ribonuclease T]
oliogonucleotides have been used in FMD virus diagnosis and typing of
strains.
Control: The recovered animals produce VN and CF antibodies
and resist reinfection by the same subtype of virus for upto one year or
more. A modified disease may result when animals immune to one
sUbtype are exposed to a second sUbtype of the original serotype. If
exposure is to a second serotype there is no resistance to disease.
Various methods for dealing with FMD outbreaks are used in
different countries. The stamping out or slaughter method of
eradication has been used in USA and other countries. In our country
where the disease is enzootic the vaccination is practised. The widely
Picornaviridae
223
used vaccines are inactivated and include the viral types and subtypes
that prevail in the field. Some of the current killed vaccines are made
from virus grown in BHK cells, Bovine Kidney and PK-15 cell lines.
The adjuvants used to enhance the immune response are aluminium
hydroxide and oil _adjuvant. For- inactivation acetyl ethylineimine or
formalin is used. Vaccination is usually repeated after 6-12 months.
Live vaccines are used to a limited extent only. Research is in progress
to produce a subunit vaccine. The following methods are being
investigated.
a. A DNA copy is made of the fragment of RNA which codes for
VP-l. This DNA is ligated to an erythromycin resistant phage of E.coli.
Selective medium is used to select for E.coli containing the phage
vector and some such E.coli produce VP-l. This can be produced in
large quantities.
b. Peptide synthesizer is used to make peptide sequences of VP-l,
10-15 amino acids in length. These peptides have been tested and some
are protective in guinea pigs. This may be the future hope.
ENTEROVIRUS
Enteroviruses are resistant to ether and chloroform and can be
distinguished from rhinoviruses by their resistance to acids. These
viruses are readily isolated from faeces and other excretions and rapidly
produce CPE in a variety of cell culture.
Swine Enteroviruses
The porcine enteroviruses (PEV) are ubiquitous in nature. There
are possibly 11 serotypes. Procine enterovirus I cause polioencephalitis
or Teschen disease while PEV9 induces swine vesicular disease. The
other serotype do not appear to be pathogenic.
Porcine Enterovirus-l (Teschan virus, Talran disease virus, Polio

encephalomyelitis virus)
The domestic and wild pigs are susceptible to Teschen virus. The
disease was reported in 1930 from Czechoslovakia. The disease occurs
in milder form in Denmark and England. The disease has been reported
from several European and African countries. The disease may also be
present in USA, Canada and Australia in a milder form.
Properties of the virus: The porcine enteroviruses are larger than
foot and mouth disease virus, measuring between 25-30 om in
224
diameter. They are non-enveloped with 32 capsomeres. They are ether

and chloroform resistant; the change in pH has little effect. The virus
can be preserved at 4C in glycerine saline or -50C but lose infectivity
if preserved by freeze drying.
Cultivation: The virus does not grow in chicken embryo or other
laboratory animals. The virus can be cultivated in pig kidney cell
cultures.
Epidemiology: The disease occurs mainly in young animals aged
5-8 weeks. Faeces are the main source of contamination but pharyngeal
secretions make aerosal spread possible. Carrier animals are thought to
exist; the virus persist in environment and can be transmitted by
fomites.
Palhogenesis: The disease among pigs may be acute, mild,
inapparent or chronic. Most of the outbreaks are sporadic and
inapparent while the disease may effect the entire herd sometime. The
incubation period ranges from 4 to 20 days. The clinical symptoms are
fever, anorexia and depression. There may be tremors and
incoordination of hind legs with stiffness, prostration and coma leading
to death. Paralysis of hind legs is a dominant symptom. There is diffuse
encephalomyelitis with degeneration of neurons and perivascular
cuffing. The mortality ranges from 50 to 75 percent.
Diagnosis: Isolation and identification of virus from the brain, the
intestinal tract and faeces should be carried out. The FAT may be
applied to the sections of brain.
COlltrol: Live attenuated or formalin inactivated vaccines are in
use in Europe with some success.
Porcine enterovirus-9 (PEV 9) (Swine Vesicular disease virus)

Swine vesicular disease (SVD) is clinically indistinguishable from
FMD, Vesicular stomatitis (VS) and Vesicular Exanthema (VE) of
swine. The disease was rust reported from Italy in 1966. It has been
reported from Hongkong, Japan, UK and C?ther parts of Europe. The
disease is only restricted to pigs.
Properties of the virus: The virus measures 30 nm in diameter.
The density of the virus is 1.34 glml with a sedimentation coefficient of
1508. The virus is stable over a wide pH range of 2-12 and protected
against inactivation at 50C by IM MgCl z Minor anti genic variation in
different strains have been observed. The virus is antigenically related
to coxsackie-B-5 virus of man.
Picornaviridae
225
Cultivation: The virus does not grown in chicken embryos but

readily grows in primary PK cells and in PK-15 cell lines. It is highly
cytopathic, CPE is detected as early as 6 hours after inoculation and
cell destruction is completed in 24 hours. Newborn mice are readily
infected by intracerebral or intraperitoneal inoculation but older mice
are refractory.
Epidemiology: Transmission of the virus is by direct and indirect
contact and ,by feeding pigs with uncooked garbage. Feeding of swill
has remained the primary source of outbreak:. The virus is very resistant
and therefore persists in the environment and carcases.
Pathogenesis: The incubation period is 2-7 days. The clinical signs
in pigs are high temperature (41-42C) and development of vesicular
lesions on snout, coronary bands, bulbs of heels and interdigital spl!ce.
Healing is quick and animals recover completely. Subclinical disease
can occur. Laboratory workers get the infection and show symptoms of
malaise and mild meningoencephalitis.
Diagnosis: Vasicular fluid is a rich source of antigen for CFT and
counter immunoclectrophoresis (CIE). The CIE is rapid and very little
quantity of antigen is required. Animal susceptibility tests should be
used to differentiate from FMD~ VS and VE. Day old mice are
susceptible and die within 5-10 days of inoculation but 7 days old mice
and guinea pigs are susceptible only to FMDV. Virus isolation can be
done in pig kidney cells or PK 15 cell lines.
Control: No vaccine is available. Quarantine and slaughter
policies are implemented to control the disease. Prohibition of feeding
of uncooked swill must be enforced.
Bovine Enteroviruses
These viruses have been isolated from the intestinal tracts of
healthy cattle as well as from the respIratory and reproductive disease.
Ther are 7 serotypes known. These viruses grow well in cell cultures of
bovine embryonic and adult kidney. They are cytopathic and produce
lysis of cells. Some strains possess the property of haemagglutination.
The role of these viruses in respiratory and reproductive disease in
cattle and nconatal calves is not clear. Some serotypes are reported to
produce catarrhal type vaginitis. Respiratory illness can be produced in
cortisone treated calves; in colostrum deprived calves the virus
produces fever and leukopenia. Experimentally the virus produces
226
abortion and stillbirth in guinea pigs. Dunne et al. 1973 detected BEV
antibodies in bovine aborted foetuses. They consider these viruses as
possible causative agents in bovine abortion either alone or in
combination with bovine parainfluenza 3 virus.
Avian Encephalomyelitis Virus (AEV)
The virus produces an acute disease of young chicks; the infection
in mature birds is mild or inapparent The natural outbreaks occur in
chickens, pheasants and Japanese quail. The disease was reported in
1930 from USA for the fIrst time. Now the disease occurs in almost all
areas of the world where poultry is raised. The virus' is embryo
transmitted and disease spreads via egg or young chicks.
Properties of the virus: The virus has properties similar to other
enteroviruses. All the strains are anti genically similar. There are 15
serotypes of avian enteroviruses; the AEV is particularly a
neuturotropic and exhibit variable pathogenecity. Some strains are
more neturotropic.
Cultivation: The virus can be cultivated in baby chicks, chicken
embryo kidney, chicken fIbroblasts and cultures of neuroglial cells. The
yolk sac route of inoculation in chicken embryos is route of choice.
Epidemiology: The disease is transmitted vertically through
infected eggs or horizontally by ingestion of contaminated feed. The
virus is shed in the droppings. The transmission of infection can occur
in the incubator.
Pathogenesis: The incubation period by experimental inoculation
by various routes is 6-10 days. The disease mainly affects young chicks
during first 6 weeks of age. The disease is characterized by dullness,
progressive ataxia especially of head and neck, loss of condition,
paralysis, prostration and death. The mortality is about 25% while
morbidity is upto 60%. The adult birds only show a drop in egg
production and loose droppings. No gross lesions are found, the
principal microscopic lesions are found in the eNS.
Diagnosis: A tentative diagnosis in young chicks can be arrived at
by observing ataxia and tremors in large number of young chicks. The
diagnosis is diffIcult in adult birds. ConfIrmatory diagnosis can be
made by isolation and identifIcation of the virus. The brain from the
suspected chicks is inoculated via yolk sac in 5-7 days old chick
embryos. The chicks are allowed to hatch and observed for 10 days for
Picornaviridae
227
symptoms. The clinical material can also be inoculated intracerebrally

in day old chicks. The FAT is used for detection of antigen in the CNS.
Control: Recovered birds are immune. Attenuated egg adapted
vaccines are given in drinking water to chickens 10-20 weeks old. The
adult birds can be vaccinated by wing web method as well. Inactivated
vaccines have been used in laying flocks in some countries.
Immunization of breeding flocks prevents the transmission of virus via
egg. The chicks which survive the attack remain unthrifty or become
blind.
Duck Hepatitis Virus (DHV)
The virus causes a fatal disease of young ducklings, affecting those
under 1 month of age. The disease is prevalent all over the world
including India.
Properties of the virus: The virus measures 20-40 nm in diameter
with a core of ~ingle stranded RNA. The virus is resistant; it survives
for 10 weeks in brooders and 5 weeks in droppings. The virus is
resistant to 2% lysol at 37C for 60 .minutes and 0.1 % formalin for 8
hours at 37C. It does not possess the property of haemagglutination.
Cultivation: The virus can be cultivated in chicken embryos via
allantoic cavity route and kills 60% of embryos after 5-6 days of
inoculation. The embryos are stunted and oedematous. After 20-30
serial passages, the virus becomes attenuated for duckling. The virus
replicates in chicken embryo tissues without showing any CPE.
Epidemiology: The virus is present in the blood. Transmission is
by contact. Recovered ducks shed the virus upto 8 weeks in the faeces.
Both vaccinated and affected birds excrete the virus in faeces for
months.
Pathogentsis: Duckling upto 3 weeks are naturally affected. The
adult ducks, chickens and turkeys are not affected. The incubation
period is 18-24 hrs but may range from 1-5 days. The disease takes
rapid course and mortality goes upto 90-95%. The ducklings stop
moving, fall on their sides, kick spasmodically and die within 3-4 days.
The characteristic lesions are found in the liver, which is enlarged,
oedematous and mottled with haemorrhages. The spleen and kidneys
may be swollen. Microscopically' there is necrosis of hepatic cells and
proliferation of bile duct epithelium.
Diagnosis: The history of fatal rapidly spreading illness among
ducklings and presence of gross lesions in the liver are helpful in the
228
diagnosis of the disease. The virus can be isolated from liver or blood
of affected birds in the chicken embryos via allantoic cavity roule. The
FAT is used for diagnosis.
Control: Recovered birds are immune. An egg adapted attenuated
vaccine is used to vaccinate day old ducklings via foot web or
intramuscular or intranasal route of inoculation. The vaccine can be
given to breeding stock to transfer passive immunity giving two doses
at 6 weeks apart.
RHINOVIRUS
The rhinoviruses are small spherical particles measuring 20-30 nm
in diameter with a single stranded RNA with molecular weight of 2.8 x
1()6 daltons. Unlike human rhinoviruses of which more than 110
serotypes are recognised, bovine and equine rhinoviruses are in a
narrower antigenic group with only 2 serotypes in each group.
Bovine Rhinovirus-l (BRV-l)
Bovine rhinoviruses are known since 1962. They are widely
distributed among cattle population throughout the world but their
pathogenic potential remains to be elaborated.
Properties of the virus: They are small in size measuring about 30
nm in diameter. They are sensitive to mild acid and resistant to sodium
dodecyl sulphate and are not stablized in MgClz at 50C.
Cultivation: The virus replicates in cell culture of bovine origin.
Primary bovine embryo kidney, bovine kidney and bovine tracheal
organ cultures are used for propagating the virus. The
cytopathogenicity may be meagre on primary isolation but becomes
pronounced after adaptation in kidney cells. The virus grows better at
30-33C rather than at 37C, this may reflect to their adaptation to the
lower temperature of their natural habitat in the nasal passages.
Epidemiology: The disease spreads by close contact. Nasal
secretions are probably source of infection. The cattle acts as a
reservoir host.
Pathogenesis: The pathogenecity of different strains vary. These
viruses have been isolated from cattle with respiratory diseases from
USA, England, Germany and Japan. They have been isolated from the
epizootics of shipping fever. In Japan these viruses have been isolated
from calves suffering from non-fatal but rapidly spreading respiratory
229
Picornaviridae
disease. Experimental infections have been mild and a symptomatic,

however, a transient rise in temperature and pneumonia may occur.
Gross lesions are mainly confmed to respiratory tract Areas of
pulmonary consolidation, collapse and emphysema are present.
Diagnosis: Isolation of virus from cattle with acute respiratory
disease alongwith increase of four fold SN antibody titre confIrms the
diagnosis.
Control: There is no vaccine available.
Equine Rbinoviruses 1 & 2
Two distinct serotypes are known. The 2 serotypes differ from one
another in physical and chemical characters. These viruses grow and
produce CPE in equine foetal kidney cell cultures and in rabbit kidney
cell lines. These viruses cause a disease of upper respiratory tract. The
disease is mild or inapparent in uncomplicated cases. The incubation
period is 3-9 days. The affected animals show fever, cough, nasal
discharge and pharyngitis. The morbidity is high. Transmission is by
direct and indirect contact with nasal secretions of affected horses.
Diagnosis is confined by isolation and identiflcation of viruses by VN
and CF tests. There is no vaccine currently available.
References
ADLAKHA,
S.C., 1985. National overview. India: In Veterinary viral diseases.

Edited by Antony J. Dell-Porte, New York,
1984-1985. Report of the task force on foot and mouth disease.
Dept1. of Agriculture and Cooperation Minsitry of Agriculture and
Rural Development, Gov1. of India.
ANoNYMOUS
1952. The technique of complement [vcation infoot and mouth

disease research. Ser. No.12, London Her Majesty's Stationery
Office, London.
BROOKSBY, lB.,
BUXTON, A. and FRASER, G., 1977. Animal Microbiology Vol. 2. Oxford,

lR., 1986. Antigenic structure of foot and mouth disease virus.
Rev. Sci. Tech. Off. Int. Epiz.5, 299-314.
CROWTHER,
DeuA,
A.l., 1983. Current status of foot and mouth disease vaccines

including the use of genetic engineering. Aust. Vet. 190: 129-35.
PORTB,
230
DHANDA, M.R.; GoPALKRISHNAN, V.R. and DHlLLON, M.S., 1957. Studies onfoot
and l'IIQuth disease vaccination in IndiaJI. A concentrated crystal
violet vaccine. Indian J. Vet. Sci. 27: 127-132.
DIN'rnR, Z. and MORBlN, B., 1990. Virus infections of ruminants. Elsevier

Science Publishers B.V.Amsterdam.
DUNNE, H.W. and lEMAN, A.D., 1975. Diseases of swine, 4th Ed. Ames, Iowa,
Iowa State University Press.
FRENKEL, H.S., 1950. Research onfoot and mouth disease. 11. The cultivation of
the virus in explantations of tongue epithelium of bovinne animals.
AmerJ. vet. Res.ll~ 371-373.
GALLOWAY, lA.; HENnPJtSON, W.M. and BROOKSBY, lB., 1948. Strains of the
virus offoot and mouth disease recovered from outbreaks in Mexico.
Proc. Sco. Exp. BioI. Med. 67, 57-63.
HOFSTAD, M.S.; CALNEK, B.W.; HELMBOLDT, C.F.; REID, W.M. and YODER, JR.
H.W., 1975. Diseases of poultry . Oxford Publishing Company,
Bombay.
LoEfR.ER and FROSCH., 1889. Cited by HENNJNG W.M., 1955. Foot and Mouth
Disease. Page. 895. South Africa, Central News Agency Ltd.
Oonderstepoort, South Africa.
MOHANTY, S.B and DU1TA, S.K., 1981. Veteriary Virology, Lea and Febiger,
Philadelphia.
MowAT, G.N., 1986. Epidemiology offoot and mouth disease in Europe. Rev.
Sci. Tech. Off. Int. Epiz. 5,271-78.
PEREIRA, H.G., 1981. Foot and Mouth disease in virus diseases of Food
animals. Edited by E.P.J. Gibbs, VoI.II. New York Academic Press.
PHnJp,
J.I.H. and DARBYSHIRE, I.H., 1974. Respiratory viruses of cattle. In

advances of Veterinary Science and comparative Medicine. Edited,by
C.A. Brandly and C.E. Comellius Vol. 15, New York Academic
Press
RusSllLL, P.H. and EDINGTON, N., 1985. Veterinary viruses. The Burlington
Press (Camb) Ltd. Foxton, Cambridge.
SEETHRAMAN, C. and DATT, N.S., 1951. Frequency of occurrence of foot and
mouth disease virus in India. Indian 1. Vet. Sci. 21, 251~255.
WALDMAN, O. and TRAUTWEIN, K., 1926. Experimentalli unlerauchungen uber
Maul-and Klauenzeche virus. Berl.tierarztle Wochebschr 42, 569571.
Chapter 20
Calciviridae
The members of this family are isometric nonenveloped particles

measuring about 35-40 nm in diameter. They have a icosahedral
symmetry with 32 capsomeres. The surface of the capsid is composed
of a number of dark hollow cup shaped structure from which the
generic name is derived They are ether and heat resistant, stable at pH
5 but not at pH 3. The genome is (+) sense ss RNA and is infectious.
During replication at least 3 RNA species are transcribed from a (-)
sense RNA template. One of these is of genomic size while two are
subgenomic size. Out of the two subgenomic size RNA the larger one
codes for large capsid polypeptide. Other polypeptides are found in the
cytoplasm of infected cells. The posttranslational cleavage of a
polyprotein does not occur. Calciviruses replicate in the cytoplasm and
are rapidly cytopathogenic. They can be cultivated easily in the
embryonic kidney cells from the species of origin. Two members of
this family viz. vesicular exanthema virus (VEV) and feline calcivirus
(FCV) cause serious animal diseases.
Vesicular Exanthema Virus (VEV)
The virus causes an acute disease of swine characterised by
formation of vesicles on the snout, mouth and feet. The only natural
host is pig but dogs and horses can be infected experimentally. The
disease has only been reported from California coast.
Properties of the virus: The virus particles are spherical in shape
and measure between 30-40 nm. The virus contain 20-24% RNA with a
sedimentation rate of 207S. The virus is heat resistant and survives at
232
60C for 1 hour. The virus is also resistant to ether, chloroform,

deoxycholate but is readily inactivated by 2 percent NaOH. There are
18 serotypes of the virus known which do not cross protect.
The virus can be easily cultivated in pig kidney, lung, liver, testis
but to a lesser extent in cell cultures of other hosts like horses, dogs and
cats. The virus does not grow in chicken embryos.
Epidemiology: The disease spreads by contact with infected pigs,
unboiled swill and carrier animals. Virus is present in almost all the
tissues of infected swine. Small amounts of virus may be excreted in
the urine and faeces. During outbreak, transmission occurs by direct
contact from pig to pig.
Pathogenicity: YE is a disease of swine. Swine of all ages are
susceptible. The mortality rate in pigs is about 5 percent but morbidity
in high. There is weight loss in affected animals and abortions.
Incubation period is 1-4 days but may be extended to 12 days. The
disease is characterized by fever and vesicles in the mouth, snout, teats,
udders and on the coronary band of feeL The primary lesions rupture
and secondary lesions may develop.
Diagnosis: Since YE closely resembles, FMD, vesicular stomatis,
a quick diagnosis is necessary. Animal inoculation test should be
carried out to distinguish from other disease with vesicular lesions.
Virus isolation form vesiculaP fluid can be made and virus identified by
VN and CF tests.
Control: The recovered animals are immune .to the same serotype
for at least 6 months. Vaccination is not attempted.
Feline Calcivirus (FCV)
Feline calcivirus is associated with respiratory illness of cats. The
virus is moderately stable at pH 5 and is inactivated at 50C in 30
minutes. There are numerous strains with serologic differences by VN
test. All the strains are regarded as serologic variants of one serotype.
The virus multiples in feline cell cultures and produce CPE. The
incubation period in cat is 2-3 days followed by epithelial necrosis with
vesicles or ulceration of external nares and of oral and lingual mucosa.
Virulent strains may produce pneumonia. The clinical signs are fever,
nasal and ocular discharge, dysponea, sneezing and rales. The most
virulent form causes 30% mortality among young animals. The
recovered cats develop long lasting immunity but few cats become
Calciviridae
233
oropharyngeal carriers and excrete virus for a long time. Spread of the
virus is rapid by aerosal and saliva. For diagnosis the virus is isolated in
cell culture and identified by VN test.
Control: Live and inactivated vacines are commercially available.
The vaccination may be done by intranasal route in 9-12 week old
animals.
Rererences
GILLESPIE, I.H. and Scorr, F.W., 1973. Feline virus infections. Advances in
Veterinary Science and comparative Medicine. 17,163-224.
Chapter 21
Togaviridae
The viruses of this family are small enveloped viruses. Togaviruses

owe their name to the envelope (latin-Toga cloak). Some members of
the family are transmitted by arthropods. The non-arthropodbome
agents show a predilection for recticulo-endothelial system, cause
arteritis and _pass through placenta. The togaviridae family have four
genera: (i) Alphavirus, (ii) Rubivirus, (iii) Pestivirus and (iv)
Arterivirus. The flaviviruses have been assigned an independent family
status because of the differences in properties as given below:
Table 21.1
DIFFERENCES IN PROPERTIES
Togaviridae
Flavivi,idae
1.
Spherical virion, envelopd,

40-50 nm in diameter.
Probable icosahedral 25-30 run.
One envelope glycoprotein
contains epitopes for neutralizing antibodies one nonglycosylated envelope protein, one
capsid protein.
Linear (+) sense ss RNA genome,
5' end capped, 3' end not poly adenylated, genes for structural
proteins located at 5' end of
genome.
Cytoplasmic replication,
maturation within cytoplasmic
vesicles.
2.
3.
4.
5.
Spherical virion, enveloped,

60-70 run in diameter.
lcosahedral capsid, 28-35 run.
Two envelope glycoproteins
contain epitopes for neutralizing
antibodies and alphavirus
serogroup and subgroup specificity,
one capsid protein.
Linear (+) sense ss RNA genome,
5' end capped, 3' end polyadenylated, genes for nonstructural
proteins located at 5' end of
genome.
Cytoplasmic replication,
budding from plasma membrane.
To gaviridae
235
The alphavirus genus has 25 members. The pestivirus has mucosal

disease/bovine virus diarrhoea and a closely related border disease
virus of sheep and hog cholera virus. The Rubivirus and Arterivirus
genera has one virus each, rubella virus and equine arteritis virus
respectively.
The virus particles are small enveloped, RNA viruses measuring
40-74 nm in diameter. The nucleocapsid measuring about 25-37 nm in
diameter is tightly surrounded by envelope. The nucleocapsid in
alphaviruses is icosahedral in symmetry with 32 capsomeres. The
envelope has spikes with HA activity. The nucleic acid is a single
molecule of positive sense, single stranded RNA with a molecular
weight of about 4 x 1()6. The virions contains 3 or 4 polypeptides, one
or more of which are glycosylated. The viruses are ether resistant :md
show variable resistance to trypsin. Th? viruses replicate in the
cytoplasm and mature by budding of either preassembled or assembling
nucleocapsids through the plasma membrane. During replication of
aJphaviruses and rubiviruses a sUbgenomic 26SRNA is synthesised
which contains information for the virion structural proteins.
Arteriviruses produce five sUbgcnomic mRNA as a"nested set.
With recent data on genomic organisation and transcription
strategies, it appears that pcstiviruses are more related to flaviviruses
than to togaviruses. The coronavirus-like transcription strategy of
arteriviruses haS a reason to eliminate from their present taxonomic
cluster.
Alphaviruses agglutinate chicken and goose rbe. The alphaviruses
are capable of infecting wide range of species. The natural cycle may
be in non mammalian hosts while mammals are accidental or dead end
hosts. These viruses show a visceral localisation which is not fatal
except in the pregnant animals or neonates. These viruses initiate a
secondary encepahlitis which is often fatal. Pastiviruses replicate in
alimentary tract epithelium and show a predilection for
reticuloendothelial system. These viruses pass through the placenta and
infect the foetus and produce infertility, abortion, foetal abnormalities.
ALPHA VIRUSES
Equine Encephalomyelits Virus
There are three antigenically distinct viruses which cause
encephalitis in horses, other vertebrate animals, birds and man. Eastern
236
Table 21.2
FAMILY: TOOAVIRlDAE
Gerws
i.
Alphavirus (Mosquito
transmitted)
ii.
Pestivirus (non
arthropod borne)
iiL Arterivirus (non

iv.
arthropod borne)
Rubivirus (non
arthropod borne)
Virus
Disease
Eastern equine encephalitis virus

Western equine
encephalitis virus
Venezuelan equine
encephalitis virus
Encephalitis Horse
(Man) Encephalitis
Encephalitis
Horse (Man)
Encephalitis
and Febrile disease
Horse (human)
Febrile disease Horse
Generalised infection
cattle.
pig.
and abortion Horse
Man
Getab virus
Bovine virus
diarrhoea virus
Hogcholera virus
Equine arteritis virus
Rubella virus
equine
encephalomyelitis
(EEEV)
and
Western
equine
encephalomyelitis (WEE V) virus were fIrst found in Eastern and
Western States of USA. The third virus Venezuelan encephalitis virus
(VEEV) was found in Venezuela. These viruses cause encephalitis in
horses in America. The natural hosts are equlnes, man, mosquitoes and
reservoir hosts like birds, small rodents, leopard frog etc. The disease is
known t.o occur for many years in the USA, Southern Canada and
South America. In India the sporadic cases characterized by
incoordination of movement and paraplegia, have been frequently
reported. The encephalomyelitis cases have been suspected to be due to
a virus. There is no report of virus isolation from India.
Properties 0/ the virus: The morphology is typical of the family.
The virion measures 20-30 nm in diameter and contains a single
stranded RNA. They haemagglutinate chicken rbe in a narrow range of
pH 6.2 to 6.4. The three distinct serotypes occur with geographical
restriction. SUbtypes of EEEV and WEEV have not been identified but
minor serologic differences among their strains have been noticed. Four
major subtypes have been reported in VEEV. A few antigenically
Togaviridae
237
distinct strains of VEEV subtype 1 are assoicated with equine

epizootics, whereas majority of viral subtypes are associated in wild
rodents and do not cause disease in horses. These viruses grow well and
produce cytopathic effects in tissue culture derived from chick embryo,
mouse hamster, monkey, guinea pigs and other species. The virus can
also be propagated in embryonating chicken eggs causing
haemorrhages and deaths of embryos in 24-48 hours. Equine epizootic
strains make small discrete plaques in Vero cells and cause
haemagglutination at pH6, indicative of virulence for equine species.
The non epizootic strains us~ally produce large plaques with less
distinct border and exhibit HA over a broad pH range. The VN test
differentiates, EEEV, WEEV and VEEV and identifies the strains. The
CF test detects common antigen shared by all viruses. The HI test also
differentiates the 3 viruses.
Epidemiology: The EEEV and VEEV spread naturally in birds and
small rodents and produce harmless and symptomless infections. The
virus is transmitted chiefly by culicine mosquitoes in which the virus
propagates and persists throughout life. The horses are accidental dead
end hosts. Contact transmission in horses does not take place. Young
cattle and pigs can be naturally infected.
Pathogenicity: The clinical features of WEEV and EEEV are
indistinguishable. The affected horses show fever, sommolence,
paralysis of lips and pharynx and incoordination of movement. Death
occurs in 3-8 days after onset of clinical signs. In VEEV, a fatal
fulminating form of disease is seen in which generalized, acute febrile
disease predominates. The disease is diphasic. In the frrst stage the
virus multiplies in viscera giving rise to viraemia between 2-5 days of
illness. In the second phase, virus multiplies in eNS and causes nerve
cell degeneration in many parts of brain and spinal cord. The affected
areas show mononuclear infiltration and perivascular cuffing. In the
Eastern and Western form, the main lesions are found in the brains
stem while in VEEE the virus is viscerotropic and gives rise to necrotic
foci in spleen and lymph nodes. There are no characteristic gross
changes in the organs.
Diagnosis: The diagnosis is arrived at by virus isolation or by
serological methods. Detection of rise of antibody titre in animals that
survive long is carried out by VN test in mice, egg embryos and cell
-culture. The HI test is simple and reliable diagnostic procedure. The HI
238
test recognises group rather than type characters. In EEEV and WEEV
brain tissue is the best source of material for virus isolation while in
VEEV the blood plasma or serum of febrile animals contains the high
concentration of virus and is suitable for virus isolation. Intracerebral
inoculation of mice or wet chicks produces viral encephalitis. Cell
cultures or chicken embryos are suitable for virus isolation.
Control: Formalin inactivated bivalent or trivalent vaccine are
used for immunization. Minimizing contact with vector is important
either by spraying or mosquito proof accommodation.
PESTIVIRUS
Bovine viral diarrhoea viruslMucosal disease virus (BVDVIMDV)
Bovine viral diarrhoea is caused by bovine virus diarrhoea
virus(BVDV), a Ubiquitous, easily transmitted virus. Cattle are
naturally infected but outbreaks have been reported in deer. The disease
was fIrst recognised in USA in 1946 and the virus was isolated in 1957.
The virus is prevalent worldwide in cattle population. The presence of
BVD has long been suspected in this country. Pande & Murty(1960)
recognised mucosal disease like syndrome among young cattle and
buffaloes. The virus isolation has not been reponed so far from this
country but there is serological evidence of presence of this disease.
Based on serological studies Nyak(1982) reported abortions in cows at
Orissa due to BVDV.
Properties: The morphology is typical of the group but variable
sizes are recorded. The virus particles are sensitive to low pH, ether,
chloroform and other lipid solvents. The virus is readily inactivated at
56C. Few serotypes of the virus haemagglutinate rhesus monkey, pig,
sheep and chicken red blood cells. Variation in antigenicity and cross
protection occur. There are virulent and avirulent strains of the virus.
The neutralization and immunofluorescence tests suggest that the non
pathogenic strains are more closely related to one another than the
cytopathic strains. There is a close antigenic relationship with Border
disease and swine fever viruses. The virus grows in cell cultures of
bovine and ovine origin. Several strains do not produce CPE and are
diffIcult 10 detect by an interference test using cytopathic strains or by
FA technique, as well as by enhancement of CPE of Newcastle disease
virus in bovine testicular cell cult'lfes. There are reports that certain
serotypes can grow in yolk sac of chicken embryo.
Togaviridae
239
Epidemiology: The host range of BVDV is restricted to domestic

and wild ruminants and pigs. These species can be infected in the field
as judged from the detection of antibodies specific for BVD as reported
by many workers. No reservoir host exist The cattle are persistently
infected. Vectors among invertebrate hosts have not been reported so
far. The BVDV has a global distribution but from China there is
absence of neutralising antibodies in the serum of cattle population
against this virus. The spread of virus cannot be properly determined, in
most cases it occurs unnoticed. Over 90% of infections are inapparent.
The virus may spread from infected to susceptible animals by direct or
indirect contact. The calves are infected in utero by transplacental virus
transmission.
Pathogenesis: The disease principally affects cattle but also has
been reported in deer. A variety of clinical disease pattern is attributed
of BVDV. The disease may be acute, mild or chronic. The most
common form of BVD is subclinical probably due to widespread SN
antibodies to the virus. The bovine viral diarrhoea syndrome is seen
predominantly in 6-18 months old cattle as a primary infection, which
is via oropharyngeal route. The primary replication of the virus takes
place in the oropharynx followed by rapid-uptake of virus in the
draining lymph nodes. Viraemia leads to infection of lymphocytes
giving rise to leucopenia and spread to other lymphoid tissues
particulalry Peyer's patches. There is concurrent replication in the
epithelium of alimentary tract which results in discrete erosions and
induces diarrhoea. Lesions in the nasal cavity and conjunctiva may also
appear. Concurrent respiratory disease occurs.
The majority of animals recover since disease has high morbidity
and low mortality. The primary infection of pregnant cows results in
transplacental infection and symptoms appear according to the stage of
pregnancy. In early pregnancy there may be infertility and embryonic
death. In mid pregnancy congenital anomalies like cerebraller hypolasia
or abortion may result in congenital infection of apparently normal
calves. These animals frequently excrete virus and are source of
infection and may also develop mucosal disease. The mucosal disease
seems to occur in congenitally infected animal which are
immunotolerent and harbour the virus without showing symptoms. The
mucosal disease is usually sporadic, progressive and fatal. When these
animals get infected with the second strain .the animals develop
240
TexJbook o/Veterinary Virology
extensive erosions throughout the alimentary tract. The outcome may

be fatal or chronic progressive disease develops with animals remaining
antibody negative. Lesions similar to those produced by rinderpest
virus primarily occur in the alimentary tract and lymphatic system. The
lesions may also be present in the upper respiratory tract and consist of
congestion, haemorrahge, oedema and erosions. The oral lesions are
small, shallow and irregular and may be found on the muzzle, tongue,
dental pad, oesophagus and pharynx. In rumen, haemorrhages and
erosions are often seen while the abomasum is inflammed and
oedematous. Oedema, congestion and erosions are seen in the intestin~.
There is damage to immunocompetent cells. Lymphnodes and Peyer's
patches are enlarged and may be devoid of lymp~oid tissues. In
congenitally defective calves, there may be cerebellar hyperpIesia,
cataract, retinal degeneration etc.
MVDV usually induces high SN antibody titres that persist for
long time. Due to immunological tolerance or due to destruction of
immunocompetent cells the virus persists with circulating antibodies.
The fatally infected cattle frequently do not produce detectable
antibodies against BVDV. It is persumed that it is due to immunologic
tolerance as a result of antigenic experience at or before birth. The
cause of fatal BVDV infections and the role of immunologic tolerance
remained obscure.
Immune reaction: BVDV usually produces life long immunity.
The presence of neutralizing antibody in ~he serum reflects immunity.
The disease picture of BVD is of immune pathogenetic phenomenon,
especially of immunologic tolerence in persistently infected animals to
a particular variant of virus. In calves failure to develop antibodies has
been attributed to immune tolerance, immune paralysis or immune'
suppression.
Diagnosis: It is difficult to diagnose the disease clinically. Paired
serum samples indicate increase in neutralizing antibody. A definitive
diagnosis is made by virus isolation and identification. Virus isolation
can be attempted from nasal secretions, faeces, blood, lymphnodes and
intestines. The fluorescent antibody test is very useful in detecting the
virus. Agar gel diffusion test using tissues form infected animals is also
useful.
Control: Recovery from natural infection confers a desirable
immunity. Inactivated and modified live virus vaccines are available in
Togaviridae
241
many countries. The inactivated vaccines are safe for use in pregnant
animals. The attenuated vaccine either alone or combined with IBR and
PI) provide satisfactory immunity. Colostral antibodies appear in calves
and last for 4-6 months.
Border Disease Virus

Border disease or hairy shaker disease is a congenital condition of
lambs characterized by excessive hairiness of birth coat, poor growth
and nervous abnormalities. The disease has been reported from U.K.
Australia and Newzealand.
Properties of the virus: The virus of Border disease (BDV) is
morphologically similar to BVDV and HCV and has a size of 45-54 nm
with a core of 30 nm. The physical properties are also similar to other
members of pestiviruses. The virus have both cytopathic and non
cytopathic variants and have been grown in cells or foetal lamb kidney,
procine kidney, sheep choroid plexus and bovine testis where it
produces CPE. The virus of BDV is inactivated at 56C for 30 min;
lipid solvents, U.V light, desicalion and common disicfectants.
There are strain differences with BDV, in terms of pathogenicity
for different breeds of sheep. Moreover, there is varying ability of BDV
antibodies to neutralize or protect against different strains of virus both
in vitro and in vivo indicating appreciable antigenic differences.
Epidemiology: It is a disease of sheep, although goats, cattle and
swine can be experimentally infected. It has been shown
experimentally that contact infection of BDV from sheep to cattle and
of BVDV from cattle to sheep readily occurs. There is serological
evidence of BDV infection in several species of wild deer, the wild
ungulates may serve as a reservoir and source of infection for sheep.
The infection is acquired by inhalation or ingestion. The infection
in adults is usually subclinical and short lived. In pregnant ewes, the
foetus also is infected. The infected foetus may be expelled or born
alive at full term. either immune or as persistently infected animals.
The virus is present in the aborted foetus and its membranes and in the
secretions and excretions of persistently infected animals. Outbreaks of
BD characterized by birth of 'hairy shaker' lambs, clinically- vary
greatly in severity.
Pathogenesis: In nonpregnant sheep, infection with BDV is
subclinical, although pyrexia and transient leukopenia has been
242
encountered 6-11 days pI. In pregnant animals the virus crosses the
placenta. The effect on foetus depend upon the stage of pregnancy.
The virus replicates in the lymphoid tissues of the oropharynx
followed ,by viraemia. In adults no clinical signs are exhibited. The
virus passes the placenta and cause abortion associated focal
necrotising placentitis. Infection may result in foetal developmental
abnormalities which are: abnormal skin follicle development giving
rise to hair rather than wool in the coat, the lambs appear hairy; hypomyelinogensis-infection of myelin forming cells leading to lack of
myelin, resulting in muscle tremors and incoordinate gait which is
described as 'Shakers'. There is poor growth rate of foetuses and
hydrocephalus
occurs
in
foetuses.
Congenitally
infected
immunotolerant foetuses excrete virus but lack antibodies. Most
clinically abnormal animal die before weaning, but in some animals
locomotor disorders progressively improve with age. The brain and
spinal cord show varying degree of deficiency of myelin and
hypercellularity of white matter with many glial cells of abnormal
appearance.
Diagnosis: The best material for diagnosis is the newborn affected
lamb, alive and unsuckled along with blood samples from the mother
and from other animals of the affected flock. If examination of cryostat
sections, neuropathology and serology is carried out on the suspected
material.
The clinical symptoms, particularly the hairy coat and locomotor
abnormalities, are suggestive of the disease. Virus can be isolated from
infected foetuses and nasal swabs of excretors in cell culture.
Neutralizing antibodies in ewes can be detected. Immunofluorescence,
virus neutralization, Cfl, and immunodiffusion tests are used to detect
the presence of antibodies.
Control: No commercial vaccines are currently available. The
disease may be controlled by eliminating the infected progeny,
although symptomless excretors are difficult to detect. The excretion
can be detected from seronegative animals.
Swine Fever or Hog Cholera

Swine fever is a highly infectious virus disease of pigs which on
occasion can cause high mortality. The natural disease occurs in swine
only. The virus is world wide in distribution. It was described for the
Togaviridae
243
flfSt time at Ohio(USA) in 1833. The disease has been eradicated from
Australia, Denmark, Newzealand, UK and USA. In this country the
disease was recorded for the ftrst time in Punjab in 1961 and
subseqently form Maharastra and UP in 1962. During the past few
years the outbreaks of swine fever have been recorded from the states
ofNagaland, Manipur, Tripura, Meghalaya etc. However, the disease is
also occasionally reported from other parts of the country.
Properties: The virus particles are 40-60 nm in diameter. The
envelope is easily distrupted to reveal nucleocapsid which has
icosahedral symmetry. The virus contains three structural proteins, two
of them being envelope glycoproteins and a third non-glycosylated
protein. It is a stable virus under laboratory conditions but is inactivated
when exposed in the field. It persists for a long time in pork or garbage.
The virus is resistant to most of the ordinary disinfectants. Strains of
varying virulence and antigenicity have been reported to cause vaccine
break down but there appears to be only one serotype. It is antigenically
related to bovine virus diarrhoea.
The virus replicates in primary pig kidney, spleen, bone marrow,
testicle. lymph node, leucocytes and in PK-15 cell lines. Most of the
strains produce little or no CPE. These strains are dctectcd by FA test
and by exaltation of virulence of Newcastle disease virus (NDV)
method. Mild noncytopathic strains enhance CPE of Newcastle disease
virus in pig testicular cells. The virus has been adapted in chicken and
duck embryos.
Epidemiology: The virus is found in saliva, blood, nasal and
conjunctival secretions, faeces and urine. Transmission occurs by
droplet. fomites and infective swill. The virus. survives for several
months in fomites at 20C. Marketing of pigs is a common source of
dissemination. The congenitally infected excreting animal is important
particularly as carrier sows. It has been mooted that flying insects and
birds may act as vectors.
Pathogenesis: Domestic and wild pigs only are susceptible to
swine fever. The virus enters the body via oropharyngeal route and
primary multiplication takes place in lymphocytes and macrophages of
tonsils. The virus has a predilection for RE system and alimentary
epithelium. The incubation period of the natural disease is 3-8 days.
The morbidity is 95-100% and the mortality is as high. The disease
may be acute or chronic. In acute cases the mortality i~ 100%. The
244
affected pigs have high temperature(41.0C) and are lethergic. This is

followed by hyperaemia of skin, particularly of ears, conjunctivitis
nasal discharge and vomiting. Sometimes convulsion may be observed.
In peracute cases, death takes place as early as 5 days after exposure to
virus, while in acute cases death takes place between 10-20 days. In
chronic cases the death takes place after 30 days of exposure. The pigs
have persistent viraemia and have an imparied antibody response.
Transplacental transmission follows a mild infection is which no
clinical symptoms are noticed. These mild infections are due to virus
strains of low virulence. The virus produces embryonic death,
malformation, mummification. still birth. or perinatal death. Life long
persistent infections acquired in uterus have been reported.
The replication in lymphoid cells produces lymphadenitis and
leucopenia. Peyers patches are extensively affected. In the caecum
healing ulcers, sometimes associated with concurrent bacterial infection
over lymphoid foci are described as button ulcers. Tertiary
dissemination to eNS results in a widespread encephalomyelitis and
symptoms of tremors, incordination and paralysis are observed. Gross
changes are usually absent in peracute cases. Secondary pneumonia and
enteritis may accompany the primary lesions. There are petechial and
echymotic haemorrhages on all serous surfaces. There is haemorrhagic
lymphadenitis, petechial haemorrhages in kidney, infection in the
spleen and ulcers in the colon. The histotological changes are hydropic
degeneration and proliferation of endothelial cells which occlude blood
vessels.
Diagnosis: The clinical symptoms and post-mortem examination is
helpful. Viral antigen can be detected by gel diffusion test using
pancreas as a source of antigen or by fluorescent antibody test on the
cryostat sections of spleen, lymph nodes and tonsils. The virus isolation
can be made from spleen. tonsils lymph nodes and blood in cell culture.
Control: 111ree methods of vaccinations are used. The killed
vaccines are safe but are not very effective. The crystal violet vaccine
was used in Europe before 1960. Simultaneous vaccination with
virulent virus and antiserum is still used in certain countries. Modified
live vaccines made by serial passages in rabbits, swine or tissue
cultures are used for immunization. The live vaccines are not safe
because transplacental infection can occur and the modified virus may
spread in the herd, thus complicating diagnosis of the disease. The live
attenuated vaccines are recommended in those herds where incidence
of the disease is high, strict quarantine and enforcement of garbage
245
Togaviridae
cooking should be enforced. Heterotypic vaccines using BVDV do not

provide complete protection against swine fever.
ARTERlVIRUS
Equine Arteritis Virus (EAV)
It is an acute infectious disease of horses characterised by fever,
depression, rhinitis and oedema of conjunctiva, trunk, limbs and
external genitalia. The disease occurs mainly in USA and Northern
Europe. Neutralizing antibodies against EAV has been reported from
India recently but requires further confIrmation.
Properties: The virus measures 50-70 nm in diameter. It is
sensitive to ether, heat, and low pH. The virus does not grow in
embryonating chicken eggs. The virus can be propagated in equine,
rabbit and baby hamster kidney cells. The isolates require repeated
passage before producing CPE in equine monolayers.
Epulemio!ogy: The virus is transmitted through respiratory tract
particularly in foals. The virus persists for a long time in the kidneys of
infected horses and disease may spread by the infectious virus in urine.
The carrier horses are important source of spread.
Pathogenicity: The oral or aerosal infection results in replication
in the lymphoid tissue of nasopharynx and then viraemia occurs.
Following incubation period of about 5 days there is pyrexia, anorexia
and leucopenia. The predilection sile is medium sized blood vessels;
virus replicates in the endothelium of blood vessels resulting in necrotic
lesions in the muscularis of blood vessels. This leads to haemorrhages,
oedema and sometimes thrombosis in the muscularis of blood vessles.
There is conjunctivitis and palpebral oedema which give the name
'Pink eye'. Oedema may also appear in the leg and lower abdomen.
The infection of respiratory and alimentary tracts leads to nasal
dis_charge, coughing, dyspenea, diarrhoea and colic. In about 50% of
pregnant mares, abortions takes place after 10-30 days of infection.
Congenital infection can also occur and some animals seem to be
consitently infected. The gross lesions are oedema, congestion and
haemorrhages of the subcutaneus tissue, lymph nodes, and viscera of
peritoneal and pleural cavities. There is degeneration and necrosis of
arteries.
Diagnosis: The tentative diagnosis is made from the clinical signs
and characteristic lesions in the arteries. The CF test is useful in
diagnosing the recent infection. Virus isolation from fIeld cases is often
diffIcult. The specimens taken from conjunctival sac and nostril of
246
infected horses and blood, liver and spleen of aborted foetuses is used
for virus isolation in cell culture of equine origin.
Control: An attenuated live virus vaccine is available which is
protective against abortion but it should 110t be given to mares in late
pregnancy.
References
BUXTON, A. and FRASER, G., 1977. Animal Microbiolgoy, Vo1.2, Oxford,
BARLow, R.M., 1982. Epidemiology. In: R.M. Barlow and D.S.P. Patterson
(Editors), Border Disease of sheep: A virus induced Terqtogenic
Disorder. Paul, Parey, Berlin Hamburg pp. 70-71.
DUNNE, H.W., 1975. HOG CHOLERA. In Diseases of Swine, 4th Ed. Edited by
H.W. Dunn and A.D. Leman, Ames, Iowa. Iowa State University
fress.
KAIIRS, R.F., 1985. Viral diseases of cattle, Kalyani Publishers Ludhiana.
MALMQUIST, W.A., 1968. Bovine viral diarrhoea-mucosal diseases: Etiology,
pathogenesis and applied immunity. Journal Veterinary Medical
Association 152: 763-68.
MOIlANTY, S.B. and Durrs, S.K., 1981. Veterinary Virology. Lea and Febiger,
Philadelphia.
MURTHY, D.K. and AoLAKHA, S.C., 1962. Preliminary studies on outbreaks of
swine fever. Indian Veterinary Journal. 39: 406-19.
NAYAK, B.D; PANDA, S.N.; MISRA, D.B.; KAR, B.C. and DAS, B.C., 1982. Note
on serological evidence of viral abortion in cattle in Orissa. Indian
Journal Animal Sciences, 52 102-103.
PANDE, P.G. and MURTY, D.K., 1960. Incidence and pathology of some recently
recognised disease-like syndromes amongst cattle and buffaloes in
India. 9th Animal Disease conference Bhubneswar, Orissa.
RussELL, P.H. and EOINGTON, 1985. Veterinary viruses. The Burlington Press
(Cambridge) Foxten, Cambridge.
S'jCWARO, W.C., CARBREY, E.A and KRESSE, 1.1., 1972. Transplacental hog
cholera in immune sows. American Journal of Veterinary Research,
33,791-98.
UPPAL, P.K., SiNGH, B.K and YAOAV, M.P., 1990. An outbreak of abortion in
mares due to equine herpes virus with possible concurrent infection
of equine arteri/is virus. Virus information Exchange letter. 7, 95.
Chapter 22
Flaviviridae
Flaviviridae is a recently established family which has only one

genus-Flaviviruses.
The virions arc enveloped measuring about 40-50 nm in diameter
and consist of core particle surrounded by a projection bearing
membrane. The virus particle contains a single molecule of single
stranded RNA with a moleular weight of 4-4.6 x 1()6. The structural
polypeplides conslilue a nucleocapsid protein and a small additional
polypeptide as well as one glycosylated envelope protein. Flaviviruses
replicate in the cytoplasm and maturation occurs by budding through
intracytoplasmic membranes. Subgenomic mRNAs do not occur and
posttranslational processing of one large precursor molecule is the
mechanism involved.
Yellow fever virus is the type species of this family. The
flaviviruses of veterinary importance are: the louping ill virus and
WesselsbOrn disease virus, Encephalitis viruses and Dengue viruses.
Table 22.1
FAMILY: FLAVIVijUDAE
Genus
Virus
Disease
Flavivirus
Louping ill virus

(tick borne)
Wesselsbom disease
virus (Mosquito
borne)
Japanese encephalitis
virus (Mosquito borne)
Encephalitis-sheep
Generalised infection,
abortion sheep
Encephalitis, abortion pig
248
Japanese B Encephalitis Virus

Japanese B encephalitis virus produces an acute arthropod borne
disease among animals and human being. Japanese encephalitisOE) is
widespread throughout Asia but does not exist in other parts of the
world. Th~re is serological evidence of existence of JE in animals in
India. In a serological survey conducted by Mall and Natarajan (1981)
in India, the highest percentage of positive sera was reported in horses
(66.17%), followed by pigs(62.5%), buffaloes (13.35%) and dogs
(9.0%). Cattle, sheep, goat and mule sera were negative. Horses, pigs,
man and cattle are susceptible.
Properties of the. virus: The virus is typical of the family. It
contains a large amount of lipid and is sensitive to ether and
deoxycholate. It is inactivated at 56C in 30 min. The virus
haemagglutinates chicken, pigeon and goose rbc. The virus has a wide
natural host range and can infect a variety of laboratory animals like
monkeys, hamsters, guinea pigs, rabbits, chickens etc. Suckling mice
are highly susceptible to intracerebral inoculation, which produces
encephalitis and death. The virus readily grows and produces CPE in
various cell cultures. JEV also grows in chicken embryos by yolk sac
route of inoculation and causes death of embryos. There is some
antigenic cross reaction with other flaviviruses. Some antigenic
modifications have been reported. The infected animals produce SN,
CF and HI antibodies. The SN and HI antibodies appear in 1-2 weeks
and persist for several years while CF antibodies appear later and
disappear sooner.
Epidemiology: Japanese B encephalitis virus is probably a primary
parasite of wild birds. Night herons is probably a primary parasite of
wild birds. Night herons is the principal reservoir host. Pigs act as
amplifiers of the infection in nature. Mosquitoes are the natural vectors
and the infection maintained mosquito-heron cycle or mosquite-pig
cycle. Humans and horses are victoms and appear to be dead-end hosts.
Pathogenecity: The incubation period is 8-10 days. In mild
infections in horses, clinical signs are fever, slow movement and
sometime jaundice. In several cases in horses the course of infection
resembles that of WEE, with facial paralysis, encephalitis following an
initial febrile phase. Mortality ranges from 40-70%. The virus is less
pathogenic for pigs; most of the pigs recover after viraemia while a
very low percentage show encephalitis. Stillbirths in pregnant sows
Flaviviridae
249
occur in high percentage of cases. Persons working with infected horses

or pigs may develop fatal encephalitis. Pathologic lesions in the
animals are restricted to haemorrhage, oedema and congestion. In the
brain, lesions of encephalitis are seen in pigs.
Diagnosis: Tentative diagnosis can be made on the clinical
symptoms and histopathological lesions. The- confrrmatory diagnosis
can be arrived at by isolation of the virus from infected brain and by
serological tests like, CF, HI and VN with the paired serum samples.
Control: Formalised virus grown in chick or mouse brain is
effective as vaccine in horses. Contact of animals with mosquitoes
should be minimized.
Wesselsborn Virus
A mild but sometimes acute febrile disease giving rise to abortions
among sheep and death of newborn lambs and pregnant ewes is
enzootic in certain areas of South Africa and Zimbabwe. Humans, adult
sheep and other ruminants get inappar~nt infection. The transmission of
the virus takes place by culicines. The virus can be readily propagated
in chicken embryos by yolk sac route and grows in cell cultures of
lambs kidney. Un weaned mice are highly susceptible to intraperitoneal,
intramuscular and intracerebral route of inoculation. The adult mice is
susceptible to intrflcerebral route only. A formalized vaccine is used to
control the disease.
Louping III Airus
The ,;:irus causes ovine encephalomyelitis, a tick borne disease in
sheep and cattle. Occasionally the disease has been reported in dogs.
Human infection has been reported' among laboratory workers,
veterinarians and shepherds. There is evidence that 32 vertebmte
species can be infected. Most infections are accompanied by negligible
clinical response. The disease is prevalent in Scotland, England and
other parts of Europe. Antigenic variants of this' virus with similar
epidemiology are: Central Europea'n encephalitis; Russian spring
summer encephalitis and Far Eastern Russian encephalitis.
Properties: The virus has a typical flavivirus morphl.!~gy. In
glycerine-saline the virus survives at room temperature for 3 months.
The virus is inactivated at 60C in 5 minutes and by 2% phenol and 1%
formalin. The virus haem agglutinates chicken (newly hatched) and
goose rbe. Th(1 virus can l;>e readily propagated in chicken embryo by
250
CAM or yolk sac route of inoculation. On CAM the virus produces

discrete pock marks. The virus can grow in cell culture of various
mammalian and avian species and produce cytopathic effec~
Pathogenecity: Two distinct phases follow in sheep, the first phase
is charaGterized by fever upto 42C and viraemia. The animal returns to
nonnal and may recover. After about 5 days a second phase of pyrexia
may occur associated with incoordination and ataxia followed by
progressive paralysis and death. The incubation period is 7 to 18 days.
Natural infection can occur in various domestic animals. Gross lesions
are not seen in infected sheep. Microscopic lesions are around the
Purkinje cells of the cerebellum in the motor nuclei of the brain stem
and ventral horns of spinal cord. In man influenza like symptoms occur.
Epidemiology: Tick activity is optimal from April to June and in
September, so is the disease. The transmission occurs through tick bite.
Transoviral spread of virus occurs. Animal to animal transmission is
rare but laboratory workers are infected via respiratory or conjunctival
routes..
Diagnosis: The diagnosis can be confirmed by isolation of virus
from the brain and spinal cord in cell cultures or by intra-cerebral
inoculation in mice. HI and VN tests are used to detect presence of
antibodies and rise of antibody titre in the sera samples of sheep.
Control: Formalized sheep spleen or brain tissue was formerly
used which has now been replaced by sheep kidney tissue culture
methanol inactivated vaccine. Regular dipping of sheep should be
carried out to control the vector.
References
BARNARD, B.J.H., 1990. Virus infections of ruminants In: Z. Dinter and B.
Morein (Editors), Wessels-bron disease Virus. Elsvier Science
Publishers B.V.pp. 291-294.
MALL, M.P., and NATARAJAN, C., 1981. Paper presented at XV annual
convention of Indian Allergy and applied immunology, Indian
Veterinary Research Institute, Izatnagar, Nov. 9-13.
REID, H.W., 1990. Virus infections of ruminants. In: Z. Dinter and B. Morein
(Editors) Louping ill virus. Elsvier Science Publishers B.V. pp. 279289.
Chapter 23
Reoviridae
Reo is an acronym derived from 'respiratory enteric orphan'.

Reoviridae was once part of picomaviridae but were reclassified on the
basis of being larger in size and less cytopathic. The family comprises
of the following genera:
Reovirus - Respirarory enteric Orphan viruses
Orbiviruses - Orbis means ring shaped appearence
Rotavirus - Rota means wheel like arrangement of capsomeres
PhYloreovirus - Plant viruses
Fijivirus - Plant viruses
Only the first three genera are of Veterinary importance.
The viruses of this family are spherical particles with diameter of
60-80 nm with icosahedral symmetry and double shelled capsid. The
outer shell is digested by chymotrypsin. The viruses do not possess the
envelope. The genome of rotaviridae family is segmented and consists
of ten (Reovirus, Orbivirus) or eleven (Rotavirus) fragments. The
nucleic acid molecules arc linear and double stranded with a mol. wt.
between 0.7 and 7 x 106 There are 6-10 polypeptides in the virions
including RNA dependent RNA polymerase. The removal of outer
shell by proteolytic enzymes is required for activation of RNA
dependent RNA polymerase. Replication of reoviruses occurs in the
cytoplasm. The infecting virion genome of rotavirus is not completely
uncoated. Virion transcriptase synthesize first positive strands and
replicase enzyme makes the negative strands, thereby forming the
double stranded progeny RNA and excess viral antigens accumulate in
larger masses in the cytoplasm, forming the characteristic inclusions.
252
The three genera can be distinguished morphologically. The

characteristic of orbiviruses being ring shaped capsomeres of the inner
capsid and rotaviruses a wheel like capsid appearance with a wide hub,
short spokes and well defined rim. The outer capsid layer of rotaviruses
is easily l.ost, which results in incomplete virus particles of 58-62 nm in
size.
REOVIRUS
Three mammalian serotypes have been recognised. The viruses of
animal origin are indistinguishable form those of human origin.
Antibodies against reoviruses have been found in cattle and other
animals in many countries. This indicates that animal reoviruses like
those in man are ubiquitous in distribution. The rcoviruses have been
isolated from respimtory and intestinal tmcts of cattle and are
antigenically similar to human serotypes.
Bovine Reoviruses (type-I-type 3)

Antibodies to all the 3 bovine reoviruses have been found in cattle
serum. In India reoviruses have been isolated from call1e. Naturally
occuring infection in cattle was first reported in 1960. It has been
postulated that rcoviruses of cattle are less important in etiology of
respiratory disease than the number of other viruses that have been
implicated as primary pathogens. In the case of alimentary tract, their
role as pathogens is even less defined.
Reoviruses have also been recovered from sheep showing signs of
concurrent signs of respiratory and alimentary tract disease but the
evidence of pathogenic role is limited. It appears that behaviour of
reoviruses as pathogens of sheep and cattle depends upon the secondary
agents which precipitate the disease.
Virus properties: When the virus is heated at 55C in the presence
of magnesium ions the virus is not inactivated but its infectivity is
enhanced. The infectivity is also enhapced when the virus is treated
with the proteolytic enzymes. The virus resists the effect of 1% phenol
for 1 hour but is inactivated by 70% ethanol or 3% formalin at 56C. It
is stable between pH 2.2-8.0 and resists the action of ether and
chloroform.
The mammalian reoviruses comprise of three serotypes. All the 3
serotypes occur in cattle, sheep and man and possess two group specific
Reoviridae
253
antigens in common. By immunoelectrophoretic analysis it has been

found lhat lhere are 4 group specific and two type specific antigens.
The reoviruses agglutinate human'O' rbe. For virus isolation
monkey kidney cells are superior for bovine strains in comparison to
cells of bovine origin. Eosinophilic inclusions are produced in the
cytoplasm of infected cells.
Epidemiology: The host range of three mammalian serotypes is
very wide. They are found in most domesticated and wild animals. The
reoviruses have also been isolated from culicine mosquitoes and may
act as live vectors or merely mechanical carriers. The bovine reoviruses
have been isolated from North America, Europe, Africa and Far East
including India. The viruscs spread easily in callle and sheep
population. The infection is mild.
Pathogenesis: The rcoviruses initiate infection in the rcspiratory
and alimentary tract of both callle and sheep. In experimental infection
the latent period is of less than 24 hours. This is followed by viraemia
which lasts upto onc week. The virus can be recovcred from nasal
discharges faeces and conjunctival sac up to 2 weeks. Most of the virus
strains have tropism for respiratory tract, nasal turbinate mucosa,
tonsils, trachea, lungs and mediastinal lymph nodcs. The virus may also
be demonstrated in the spleen and kidney and throughout alimentary
tract and mesentric lymphnodes. Chlamydia may act synergistically
wilh reoviruses to induce pneumopathy lhan either agent alone. The
clinical response to infection is more pronounced in sheep than in
cattle. After an incubation period of 4-6 days the lambs show pyrexia
nnd ocular and nasal discharge, there was evidence of pneumonia
accompained by diarrhoea. In lambs only reovirus type-l is capablc of
producing disease whereas type 2 and 3 are not. The experimentally
infected calves with type 1 and 2 show rise of tcmparaturc only. In
Natural outbreaks in sheep mild respiratory and enteric signs are noted.
Diagnosis: The diagnosis can be made by virus isolation in
monkey kidney cells or pig or lamb kidney. Paired serum samples arc
collected for demonstration of HI and SN antibodies.
Control: Limited number of attempts have been made to evaluate
vaccines against reoviruses in cattle. Both inactivated and multifactoral
vaccines have been tried but results are not encouraging.
The revorises have also been isolated from horses (type 2 and 3),
pigs (type 1 and 3), dogs (type 1) and goaLS (1 and 3).
254
Avian Reovirus type 1 to 5

Numerous reoviruses have been isolated from chicken with
arthritis (tenosynovitis), respiratory signs, diarrhoea retarded growth
and healthy chickens. The presence of avian reovirus is world wide. In
India' the virus has been isolated from arthritis cases and presence of
precipitins to avian reovirus has been reported. The avian reoviruses
share a group specific antigen. Five serotypes have been reported.
Avian reoviruses differ antigenically from other mammalian reoviruses.
They grow readily in yolk sac or CAM of chicken embryos. The
chicken embryos die between 3-8 days of infection. Small white.
discrete pocks are found on the CAM of chicken embryos. The virus
also multiplies in the cell culture of chicken embryos and vero cells. In
CK cells syncytia are produced. Avian reovirus type 1 is associated
with viral arthritis in chicken. Avian reovirus type 1 is associated with
viral arthritis in chicken. It is likely that other serotypes may be
involved. The viral arthritis is common among 7 week old chicken.
After an incubation period of 1-11 days, the chicks develop inapparent
infection in most cases. In the acute cases the birds become lame and
may also he stunted. Lamencss becomes more pronounced in chronic
cases. The morbidity is 100% in broilcrs and mortality is about 1%.
Growth ratar~ation is prominent sign. Gross lesions involve swelling of
digital flexor or metatarsal extensor tendons. A small. amount of
exudate is found in hock or elbow joint Myocarditis is a constant
feature. A carrier state is known to exist. A definite diagnosis can only
be made on isolation of virus. An attenuated vaccine has been
developed for the control of the disease.
ROTAVIRUS
Rotaviruses is responsible for neonatal diarrhoea in man and farm
animals. Virus specific antibodies are widespread in adult animals. In
India there is a serological evidence of presence of rotavirus antibodies
among cattle and there is a report of isolation of rotavirus from a calf.
Bovine Rotavirus
Properties: The complete virus particle measures about 70-75nm

and consist of a central core surrounded by two layers of virus specific
polypeptides. The genome consists of 11 double stranded RNA
segments. All rotaviruses share a common antigen associated with the
Reoviridae
255
inner capsid layer. Rotaviruses of different animal species can be

distinguished by VN tests. Gel electrophoresis of the genome segments
have revealed differences in RNA patterns for rota viruses obtained
from different species as well for different isolates from the same
species. Inspite of large number of virus particles found in the faeces,
rotaviruses are difficult to propagate in cell culture. Bovine rotaviruses
have been adapted to grow in bovine and simian kidney cultures.
Pretreatment of faecal samples with trypsin and pancreatin facilitate
isolation and cultivation. The enzymes cleaves the outer capsid and this
cleavage renders virus susceptible to uncoating wiLhin the cell. This
uncoating is a necessary step in converting n'on infectious virus to
infectious virus.
The bovine rotaviruses remain viable for six months in faecal
material at room temperature. Sodium hypochlorite (3%), lysoI"(5%),
formaline (10%) had little effect on lamb rotavirus in the intestinal
contents. Lysol .(5%) and formaline were effective when intestinal
contents were exposed for 2 hours.
Epidemiology: The rotavirus infections have been reported from
many species of domestic and zoo animals including man.
Experimentally human infant rotavirus caused diarrhoea in calves but
serverity' of illness was less than caused by infection of bovine
rotavirus.
There is no evidence under field conditions Lhat cross-species
infection is important but experimentally cross-species infection has
been reported.
The high antibody prevalence among cattle and sheep and usual
onset of disease between 3-10 days of age suggests widespread
distribution of virus, carrrier state and age susceptibility. In different
surveys rotavirus has been found to be assoicated wiLh 41-48% of cases
of neonatal calf diarrhoea. Animals confined to sheds and barns have a
high incidence of disease because of exposure to accumulated viruses
and bacteria. The rotavirus diarrhoea is highly contagious. An animal in
early stage of diarrhoea excretes IOS - 1()9 virions per ml of faeces. The
rotavirus diarrhoea develops a pattern at certain age. In one herd age
may be 3-5 days and in another 14 day. No age resistance has been
reported. The climatic conditions cause stress in the occurrence of
disease. Mortality depends upon the secondary infection. In
uncomplicated cases most of the animals recover but mortality is high
256
when there are complicating bacterial infections. Certain animals

species like dogs and cats may play a role in dissemination of bovine
rotavirus. In dogs and cats bovine rotavirus mUltiply asmyptomatically
by experimental infection and rotavirus of bovine origin has been
isolated from faeces of dogs.
Pathogenesis: The portal of entry is mouth. The primary site of
replication of virus is the mature villous epithelial cells of small
intestine but in lambs replication has been found to occur in the
epithelium of caecum and colon. There is absence of viraemia in
rotavirus infection. The virus is shed in the faeces. The titre of virus is
very high about 108 _109 per ml in faeces in ftrst few hours of
diarrhoea. There is circumstantial evidence that healthy calves and
adult cattle act as carriers. Persistant infection has also been suggested.
In experimental infection the incubation period varies and is about 12.5
hours to 36 hours. The clinical signs arc anorexia, depression, drooling
of thick saliva and diarrhoea. The calves generally suffer at 3-10 days
of age. The columnar villous epithelial cells are destroyed by infection
and replaced by cuboidal squamous cells.
Immune reaction: In both calves and lambs circulating rotavirus
antibody has little effect in preventing enteritis. The passive protection
results from the presence of rotavirus immunoglobulin in the lumen of
intestine. Susceptible calves are resistant to rotavirus infection just 48
hours of inoculation with attenuated rotavirus.
Diagnosis: Immunofluorescence is used to detect rotavirus antigen
in faecal smears or in sections of infected small intestine collected early
in the course of infection. Electron microscopic examination of faeces
sometimes permits recognition of virus particles. Immunoelectron
microscope is more speciftc for identification of virus particles. Virus
isolation in cell culture can also be used. The clinical specimen of
choice are faeces from early cases of diarrhoea or section of intestine
and these yield satisfactory results. Number of other serological tests
like ELISA reverse passive haemagglutination are useful for detection
of viral antigens in faecal samples.
Control: An attenuated vaccine is available for oral immunization
for newborn calves. Inoculation of cows with inactivated vaccine has
been reported to reduce the incidence of diarrhoea is new approach for
use on pregnant cattle. A combined rotavirus and E.coli antigen as a
vaccine is also found to be effective for immunization of pregnant cows
and newborn calves.
Reoviridae
257
Rotavirus infection in other animals

Rotavirus gastroenteritis has been reported in foal usually during
ftrst 2 weeks of life. The equine rotavirus can infect piglets. A bovine
rotavirus vaccine is used in foals to control the disease. Ovine rotavirus
is anti genically related to bovine rotavirus. The virus causes
gastroenteritis in 1-2 week old lambs. Rotavirus has also been isolated
from goal<;. ~ota-like viruses have been observed in faecal samples of
pups with diarrhoea but their etiologic role has not been ftrmly
established. Naturally occuring rotavirual diarrhoea has been observed
in pigs 10-28 days of age. The rotaviruses have also been isolated from
turkey and broiler chIcken with and without diarhoea.
ORBIVIRUS
The virus particles of this genus have a double protein shell, the
outer being skin-like; the inner shell has 32 ring shaped capsomeres
with icosahedral symmetry. The virus consists of 10 segements of
double stranded RNA.
Blue Tongue Virus (BTV)
Blue tongue (BT) is an insect borne infection and non-contagious
disease of domestic and wild ruminants. It is prImarily a disease of
sheep but cases of inapparent infection in cattle and goats take place
and these animals may act as reservoir of virus for sheep and wild
ruminants. The disease was recognised in Africa in 1652. It is
widespread in African continent. The disease was identified outside
Africa in Cyprus if! 1943. The disease was subsequently reported from
Palestine, Turkey, Syria. The disease has been reported from USA and
certain European countries like Portugal and Spain. Recently evidence
of the presence of the disease has been reported from Latin America,
Caribbean, South East Asia and Australia. In India the disease was first
reported by Sapre (1964) from Maharasthra state. Uppal and
Vasudeven (1980) reported the occurrence of disease in this country
and virus was isolated and identified as serotype 3,4,9,16 and 17 from
Himachal Pradesh and serotype 1,4 at Haryana. Subsequently Kulkarni
and Kulkarni (1984) reported isolation of virus serotypes 9 and 18 from
Maharasthra.
Properties of the virus: The virus particle is spherical and about 69
nm in diameter with 32 capsomeres. The capsid is double layered. The
258
genome is of 10 segments double stranded RNA. The capsid contains

fou/ major and three minor polypeptides. The virus particle is resistant
to ether, chloroform and deoxycholate and gets inactivated below pH 6.
The virio,n is stable at 4C and -70C but losses infectivity rapidly
when frozen at -20C. No haemagglutinin has been reported so far.
There are 22 serotypes in BTV as revealed by virus neutralization test.
Group specific antigens are revealed by complement flxation agar
immuno-diffusion and -immunofluorescence tests.
Cultivation: The virus grows readily in fertile eggs after
inoculation of embryos 10-11 da,Ys old by intravenous route or 6-8 days
old embryos by yolk sac route. The intravenous route is more sensitive.
The embryos die by both the methods of inoculation with multiple
haemorrhages and are cherry red in colour. The optimium temperature
of incubation of chicken embryos is 33.5C. The virus can also be
cultivated in 1-4 days old mice by intra-cerebral route of inoculation.
After one or more passages in chicken embryos the virus grows in cell
cultures like BHK21 , Vero and L cells with production of CPE. Lamb
kidney monolayer cultures are less readily infected and the CPE
production is less extensive.
Epidemiology: The blue tongue (B1) disease is an African disease
but now it has been reported from almost all the countries of the world.
The disease occurs in USA, in the near Middle and Far East, Japan,
Indonesia, India and Northern region of Australia. Europe appears to be
free at present. BTY has a wide host range including sheep, cattle,
goats, deer, most Mrican antelopes and various other artiooacytles. The
disease may be fatal and inapparent. Mostly the inapparent infection
exists in most species. The infection progresses in cattle midge cycle
and once a certain level of infection is attained the infection spills over
to sheep. Sheep are apparently involved in a secondary cycle. Midges
of the genus culicoides act as biological vectors of BTV. Midges
become infected after feading an infected animal and the virus
replicates in the salivary glands where it reaches a maximum titre in 68 days. The infected midge is infective for life. Transovarial
transmission does not take place in midges. Overwintering of BTY is
due to survival of infected midges as well as latently infected cattle. At
least 22 species of culicoides transmit the disease. The BTY is not
contagious. The transmission occurs only when infective midges bite
the susceptible host or infective blood or tissue suspension arc
Reoviridae
259
inoculated parenterly. The excretions and secretions from infected

animals contain minimal concentration of virus and therefore, regarded
as harmless from epidemiological point of view. The semen from
infected bulls during viraemic stage may be infective and cows
inseminated with such semen become infected. The embryo transfer in
cattle appears to be safe from risk of transmitting BTY, even when the
embryo donors are viraemic at the time of transfer. However, sheep
embryos collected from infected ewes occasionally may give rise to
BTY infection in recipient ewes.
Palh~genesis: Following infection the virus replicates in regional
lymphnodes and thereafter in other lymphnodes and lymphoreticular
tissues and in endothelium, periendothelial cells and pericytes of small
blood vessels. This leads to degenerative changes and necrosis leading
to vascular occulusion, stasis and exudation. Once replication in target
cells take place, virus appears in the blood stream and spreads to entire
body. Virus is detected in blood from about 3-6 days PI and viraemia
reaches its peak in about 7-8 days and then declines rapidly. After 14
days the virus is not detected in the blood of sheep but in cattle
viraemia persists for a longer period. The virus is cell associated
involving blood cells. A panleukopenia proceeds the appearance of
viraemia. In cattle there is evidence that expression of clinical discase
is due to IgE-mediated hypersensitivity reaction induced by previous
exposure of BTV oc related viruses, however, it does not happen in
sheep.
In sheep the incubation period is 6-7 days. The virus causes
viraemia and is easily isolated from the blood during febrile stage. The
affected sheep have a marked fever, oedema of muzzle, oedema and
hyperaemia of the lips, buccal, nasal mucosa of eyelids. There is nasal
discharge which may become mucopurulent. The saliva drops from the
lips and muzzle becomes encrusted with discharge from nose and
mouth. There is swelling and hyperaemia of the mucosa of mouth and
ulceration of tongue, dental pad and lips. The tongue becomes swollen,
eynotic and purple blue. Swelling and tenderness of coronary band and
sensitive lamina of hoof result in lameness. Pregnant ewes may abort.
Mortality in sheep may be high.
Experimental disease in cattle is subclinical. In natural infection,
laminitis, stiffness, ulcers in the mouth, nose and muzzle and salivation
are seen. In affected pregnant cows the foetus may die and be
260
reabsorbed, aborted or still born. The BTV has teratogenic properties.

Affected newborn lambs or calves are blind ataxic, undersized and have
congenital defects.
lmmu",e reaction: In immunocompetent animals the group
specific and type specific antibodies appear within 7-10 days of
infection. The type specific neutralizing antibodies persist for more
than 3 years, while the group specific antibodies persist for 6-18
months only. The protective immunity is generally associated with
neutralizing antibodies but sometimes animal resist infection of BTV
w.ith no demonstrable neutralizing antibodies. The CMI responses to
BTV infection in sheep are also protective and may be less type
specific. Lambs born to BTV immune animals are passively immune
and remain protected upto 6 months.
Diagnosis: In sheep, tentative diagnosis is made on the clinical
signs. Confirmation is made by positive disease transmission to
susceptible sheep or by viral isolation from blood, spleen, lymph nodes
etc in chicken embryos. The blood should be collected from febrile
animals in the early stage in EDTA or citrate. Occasionally isolations
can also be made by intracerebral inoculation of newborn mice or
inoculation of cell cultures, BHKzI' Vero or Aedes albopicuts cells. The
embryos die within 6 days of inoculation with haemorrhages. Infected
CAM is used as a source of virus in virus neutralization or complement
fixation test, ELISA etc.
Control: Live egg adapted polyvalent vaccines are widely used
and are effective. The live vaccines are not recommended to be used
because the live virus may cause some abnormalities in foetus.
Promising results are being obtained with inactivated vaccines. Import
controls is practised in certain countries like Australia.
African Horse Sickness Virus
African horse sickness (AHS) is an insect borne virus disease of
solipeds. Horses are more susceptible as compared to donkeys. The
disease is prevalent in Africa and Middle East. The epidemic of 1959
spread over a large area reaching on one side upto Turkey and on the
other side upto India. In India the frrst outbreak was reported in April,
1960. Approximately about 90% of affected animals died. It is
suspected that movement of affected animals in the adjoining areas of
Pakistan was responsible for the spread of the disease. Since 1963, no
new case of the disease has been reported from India.
Reoviridae
261
Properties of the virus: The virus has an icosahedral symmetry

with 32 capsomeres and measures about 50 nm in diameter. The virus
is stable between pH 6-10 but is labile at pH 3. The virus is stable at
4C but is inactivated at 36C when kept for 40 days. The infected
blood can be preserved in oxalate-carbol-glycerine solution for years.
The virus haemagglutinates equine rbe. Nine anti genically distinct
serotypes of the virus have been identified. A common group specific
antigen is shown by complement fixation test in different strains of
virus. By virus neutralisation and haemagglutination inhibition the
virus strains can be distinguished. Strains within each serotype vary in
virulence.
Cultivation: The virus can be cultivated by intracerebral route in
mice, rats, guinea pigs and other laboratory rodents. Viscerotropic field
strains become neurotropic by serial intracerebral passages. The virus
become attenuated for horses without loosing antigenicity. Ferrets can
be infected by intravenous route. Neurotropic strains can be grown by
CAM route of inoculation in chicken embryos. The small plaque
variant is usually more virulent than the large one.
Epidemiology: The AHS is the disease of horses, mules and zebras
are less susceptible but donkeys are generally resistant. Contact
transmission does not occur among horses. A number of blood sucking
insects have bcen circumstantially incriminated in the spread of virus.
The culiocoides transmit the disease but exact role has not yet been
determined. Trransmission is primarily through the mosquitoes.
Lymphnodes, lungs and spleen have the highest concentration of virus.
Pathogenesis: Under natural conditions, the incubation period is
less then 9 days while experimentally the incubation period range from
2-21 days. There are four different forms of disease. African horsesickness fever, acute pulmonary form, cardiac form and mixed form.
There is no relationship between the virus type involved and the kind of
clinical disease produced.
Mixed form is a common form where combination of pulmonary
and cardiac form symptoms are observed. The mortality rate varies:
sometime it may be 90% and in certain outbreaks it may be 25%. The
pathologic lesions depend on the clinical form of the disease. In
pulmonary form the changes are oedema of lungs and hydrothorax. In
the cardiac form the lesions are oedematous infiltration of
subcutaneous, subserous and intramuscular tissues and lymphnodes.
Hydropericardium is a constant 'feature.
262
Diagnosis: During outbreak of disease tentative diagnosis can be

made fonn the clinical symptoms and gross pathologic lesions. The
virus isolation is done from the blood collected at an early stage of
disease or from the spleen extracts when inoculated intracerebrally into
suckling mice, ferrets, embryonating chicken eggs and cell culture.
Infected mice die within 5-15 days of inoculation. The virus is
identified by virus neutralization test conducted in mice or cell culture
system. Antibodies in the convalescent serum are detected by virus
neutralization, complement fixation, haemagglutination inhibition and
immunodiffusion tests.
Control: Polyvalent vaccines are used for immunization. There is
some cross immunity among certain serotypes. Mouse brain adapted or
cell culture vaccines are used for animal vaccination. Antigenic drift or
vaccinal break-down can occur. Therefore, horses in enzootic areas
should be protected from flies during outbreaks.
References
BARBER, T.L. and IOCHlM. M.M., 1985. Bluetongue and related orbiviruses
(progress in clinical and Biowgical Research, Vol. 178) Alan R. Liss,
New York. 746 pp.
BUXTON, A. and FRASER, G., 1977. Animal Microbiology Vol. 2. Oxford,
CAMPBEU., C.H. and GRUBMAN, M.I., 1985. Current knowledge on the
biochemistry and immunology of bluetongue. In R. Pandey (Editor),
Progress in Veterinary Microbiology and Immunology, 1, 58-79.
GIBBS, E.P.l, 1981. Virus diseases offood animals. Vol. IT. Academic Press,
London.
GORMAN, B.M.; TAYLOR, I. and WALKER, P.I., 1983. Orbiviruses In: W.K. Joklik
(Editor). The Reoviridae.Plenum New YQrk. pp. 287-357.
HOUSE, lA., 1978. Economic impact of rotavirus and other neonatal disease
agents of animals. 1 Am. Vet. Med. Assoc. 173: 573-576.
KAHRs, R.F.,1985. Viral diseases ofcattle. Iowa State Press.

KULKARNI, 0.0. and KULKARNI, M.N., 1984. Isolation of bluetongue virus from
sheep. Indian lournal Comparative microbiology Immunology and
Infections Diseases 5, 125.
Reoviri dae
263
KUMAII., S., 1976. The African horse sickness. Indian Council of Agricult
ural
Research, New Delhi.
MRSOLAIS, G.; AssAF, R.; MoNTPe1TJ', C. and MAROIS, P., 1978. Diagnos
is of
viral agenls associated with neOTUJlai calf diarrhoea. Canadia n
Journal Compar ative Medicine. 42, 168-71.
MARtEL, J.L. and PERRIN, B., 1981. Infectious etiology of diarrhoea
in newborn
calves. Prevalen ce in France of E.coli Kgg + and rotavirus. Practcical
diagnosis techniques. Bull. Group Tech.Vet., 4,45-54 .
OLsBN, N.O., 1978. Reovirus infection. In diseases of poultry, 7th ed.
By M.S.
Hofsted et al. Ames. Iowa. Iowa State University.
PBNsAERT, M.B., 1984. Viral gastroenleritis in suckling pigs. Rev.
Sci. Tech.
Off. INt. Epiz. 3,809-1 8.
RusSEl.L, P.H. and EDINGTON, N., 1985. Veterinery Viruses. The
Burlingt on
Press (Cambri dge ltd) Foxton, Cambridge.
SAPRE, S.N., 1964. An outbreak of bluetongue in goats and sheep in
Maharas tra
State India. Veterinary Review (M&B) 15, 69-71.
TAYLOR, W.P., 1986. The epidemiology of bluetongue. Rev. Sci.
Off. Int.
Epizootics 5, 351-56.
UPPAL, P.K. and V ASUDEVA1~, B., 1980. Occurrence of bluetongue
in India.
Indian Journal comparative microbiology, Immuno logy and
Infectious Diseases 1, 18-20.
VERWOERD, D.W.; HUlSMANS, H. and ERASINUS, BJ., 1979. Orbiviru
ses. In H.
Fraenkel-Conrat and R.R. Wagner (Editors), Comprehensive
Virology, 14,285- 345.
Chapter 24
Birnaviridae
In 1984 a new family was designated Bimaviridae to include

infectious bursal disease of chickens and infectious pancreatic necrosis
virus of fish.
The viruses in this family are icosahedral having a size of 60 nm in
diameter with 32 capsomeres. The virions are relatively heat stable and
resistant to ether, chloroform and at pH 3. There are four structural
nonglycosylated polypeptides. The genome consists of 2 segments of
ds RNA. The viruses replicate in the cytoplasm without depressing
cellular RNA or protein synthesis to great extent. The infectious bursal
disease virus (IBDV) replicates in both chicken and mammalian cells
and produce CPE in 3-4 days.
The family comprises of IBDV, infections pancreatic necrosis
virus of fish and viruses affecting insects and molluscous.
Infectious Bursal Disease Virus (IBDV)
Infectious bursal disease virus is no longer considered to be in
reovirus group. The virus has one layer of capsid protein and genome
has two segments instead of 10-12 in reoviridae.
The virus causes an active infectious disease of young chickens.
The virus has two synonyms; avian nephrosis and Gumboro disease
after the name of a district in Delaware (USA), where the virus was
flfst encountered, in 1950. Now the virus has been reported from all
major poUltry producing-areas of USA, Great Britain, Italy, Israel,
West Germany, the Netherlands, Japan, Iraq as well as South American
countries. The disease is prevalent in India as reported by Mohanty et
Birnaviridae
265
al. (1971) and other workers. The virus infects chickens and turkeys but
not ducks. The disease incidence is greatest in 3-6 weeks old chicks but
outbreaks have been reported in chicks 11 days old as well as 12 weeks
old. The virus destroys immunological organ namely bursa and leads to
breakdown of imunity breading the flock susceptible to many infections
and there are also cases of vaccine failures.
Properties: The virus particle measures 50-60 nm in diameter. The
genome is double stranded RNA with 2 segments. The virus is
comparatively heat resistant. It survives for 5 hours at 56C. The virus
is inactivated by formalin and iodophores but is resistant to ether,
chloroform, phenol, thiomersal etc. The virus grows on embryonating
chicken eggs. The CAM route of inoculation is more sensitive then
yolk sac and allantoic cavity route. Lesions produced in chicken
embryo are subcutaneous oedema, haemorrhages, dwarfing, liver
necrosis and death. Serial passages in chick embryo reduces virulence
for chicks. IBDV also grows in chicken embryo kidney cell cultures
and produce CPE in 3-5 days. No CPE is produced in pig, hamster, calf
kidney, HeLa and amnion cell lines.
Epidemiology: IBDV affects chicken only. The chickens 3-6
weeks of age are most susceptible but older chickens also catch
infection. Laying birds as well as chicks 1-14 days of age do not show
disease. The yirus spreads directly by contact. It is highly contagious
disease. The specific mode of transmission appeared to be
contaminated feed and water. IBDV remains viable for atleast six
months in dry litter and in unused houses for more than one year. The
virus has also been isolated from insects and mosquitoes which
indicates that insects may be involved in the spread of virus.
Pathogenesis: IBD has a short incubation period of 2-3 days.
Lesions normally appear in bursa before the onset of clinical signs.
Chickens between 3-6 weeks are most susceptible but older chickens
have been reported to sOffCJ. The clinical signs are ruffled feathers,
anorexia, depression, diarrhoea with soiled vent. The affected birds
become dehydrated and die. The mortality is 20-30% but morbidity
reaches 100%. The lesions are dehydration, haemorrhages in leg and
thigh muscle, hepatic infarction, enlarged kidneys; the bursa is usually
enlarged, oedematous and yellowish after 2-3 days of infection. It
returns to its normal weight by 5th day and continues to atrophy
rapidly. It is necrotic in many birds. The histological changes include
266
fecal necrosis and hyperplasia of epithelial and reticular cells of bursa,

spleen and thymus. There is suppression of cell mediated immunity by
IBOV infection.
Diagnosis: The clinical signs, gross lesions and histopathological
changes in. the bursa are characteristic and helpful to arrive at a
tentative diagnosis. Other factors like, age, history of birds, clinical
signs and mortality pattern are important in arriving at tentative
diagnosis. Confirmation of the disease is based on isolation and
identification of the virus. The isolation can be made from the kidneys,
spleen or bursa of the affected birds in the eggs. The virus can be
identified by VN in chicken embryos or cell cultures. The FAT and
agar gel precipitation test is applied to affected bursal tissue of the
birds.
Control: A solid immunity results in 10-16 weeks of age. Chicken
embryo or cell culture vaccines are available in certain countries for
oral or ocular administration. These vaccines produce about 1%
mortality and bursal lesions. The attenuated vaccine prepared in Vero
cells is giving good results.
References
BESHT,
H., 1980. Infeclious bursal disease virus. Curr. Top. Microbiol.

Immunol. 90, 107.
FARAGHER,
J.T., 1972.lnfeclious bursal disease of chickens. Vet. Bull. 42, 361.
HITCHNER, S.B., 1978.lnfeclious bursal disease. In diseases of poUltry. 7th Ed.

Edited by M.S. Hofstad el al. Ames Iowa State Univeristy, Press.
OKOYE, J.O.A., 1984. Infeclious bursal disease. In diseases of Poultry. 7th Ed.
Edited by M.S. Hofstad et a1. Ames Iowa State University, Press.
Chapter 25
Coronaviridae
The characteristic club-shaped surface projections studding the

virion envelope (Latin corona-crown wreath) have given the family its
name.
The viruses of this family are pleomorphic, enveloped, measure
about 100-120 nm in diameter with unique club shaped peplomers
projecting from envelope, which measure about 20 nm in length. They
are widely spaced then in myxoviruses and resemble a coronet The
capsid is of helical symmetry. The genome consists of one molecule
infectious RNA of about 7 x 1()6 mol wt. The virions contain three
major classes of structural proteins, the nucleocapsid protein, the matrix
or envelope protein and the surface or peplomer protein. The capsid
protein is phosphorylated.
Coronaviruses replicate in the cytoplasm. There is production of 3'
coterminal sUbgenomic RNA with unique sequences extending in S'
direction. The virions bud into cisternae acquiring lipid membrane from
the cell and subsequently transported to and accumulated in Golgi
vesicles. The budding from plasma membrane does not take place.
Coronaviruses do not grow well in continuous cell lines until they are
adapted in tissue culture. The viruses can be isolated in primary culture
of the appropriate species. The CPE may be minimal with some
vacuolation.
There are several coronaviruses of veterinary importance such as:
Chickens: Avian infections bronchitis virus (IBV)
Pigs: Transmissible gastroenteritis (TGE) virus, haemagglutinating
encephalitis virus and epidemic diarrhoea virus.
268
Dogs: Canine coronaviruses

Cats: Feline infectious peritonitis virus (FlPV)
Mice: Mouse hepatitis virus
Cattle: Bovine coronavirus causing diarrhoea in calves and
occassioI1ally precipitates in .respiratory tract infections. In adult cattle
the bovine coronavirus may be responsible for a disease ca\1ed winter
dystentry. All isolates belong to one serotype.
Bovine Coronavirus
Bovine coronavirus produces neonatal calf diarrhoea in calves
day to 3 weeks Or" more of age. The virus has also been isolated as a
member of mixed infection from trachea and lungs of calves with
respiratory disease. A virus similar with morphological, physicochemical and antigenic properties to coronaviruses was identified in
faeces of calves suffering from winter dystentry. The young mature
dairy cattle aged 2-3 years are infected during winter in crowded barns.
The bovine coronavirus is also called the Nebraska calf diarrhoea
coronavirus.
Properties of the virus: In negatively stained preparations the virus
measures 120 nm in diameter and covered by petal shaped projections
of 20 nm long. The virus haem agglutinates and haemadsorbs red blood
cells of hamster, mice and rats. The virus is stable at pH 3 and
thermostabilized by IM MgCI2 The virus 'is sensitive to ether,
chloroform, deoxycholate, and exposure to 50C for 1 hour. Formalin
0.02% at 37C completely inactivates the virus in cell culture fluid in
24 hours. The virus can be isolated in BEK cells and vero cells. The
CPE is produced after few passages and is characterised by syncytium.
Some bovine strains have not been grown in cell culture and are
maintained by oral inoculation of calves. Only one serotype has been
identified so far.
The virus strains isolated from cases of winter dystenlry in adult
cattle have properties similar to bovine corona virus. Morphological,
physical and chemical properties, anti genic properties are similar to
those of coronaviruses.
Epidemiology: The epidemiology is not completely known. It
appears that carrier and infected cows are the reservoir animals and
source of infection. The virus is excreted in faeces and ingestion is the
source of infection. The virus appears to be ubiquitous in cattle
Coronaviridae
269
population. The occurrence of coronavirus is suspected in India.

Morbidity in an outbreak in calves in high but mortality is influenced
by the age of the calf, management and type of secondary infections.
The winter dysentry in cattle has been reported from USA, Great
Britain, Sweedan, Germany, France, Belgium, Israel, Japan, Australia
and Newzealand. Animals frequently affected are of 2-3 years of age
and are infected during winter in crowded barns especially during post
partum. The young calves under 4 months resist the disease or are
affected with a milder form. The mortality does not exceed 10% but
morbidity is 100%. The economic consequences concern milk
production, which is reduced by 25-95% for 1-2 weeks and afterwards
it does not come to the intiallevel. The faeces of the effected cattle is
the source of infection and virus is transmilted by oral route.
Pathogens is: Portal of entry is the mouth through contact with
feed or fomites contaminated with infected faeces. The primary site of
viral replication are the mature epithelial cells on the small intestinal
villi and surface epithelial cells of the colon.
There is no precise data available of the winter dysentry. It is
persumcd that (i) coronavirus isolated from cases of winter dysentry
may be of a single serotype causing diarrhoea in cattle regardless of
their age, (ii) the campylobacter acts as opportunistic agent due to their
invading property and colonize the crypts in the same region, (iii)
lipopolysaccharides or endotoxins of bacteria induce a phenomenon of
localised and/or disseminated intravascular coagulation, the source of
dysentry.
The incubation period in gnotobiotic calves by oral inoculation is
20 hours. Initially the calves are depressed, eat slowly and have a liquid
yellowish diarrhoea. The diarrhoea is acute and faeces may contain
coagulated milk or mucus. There may be excess salivation, weakness,
lethargy and severe dehydration and shock. The diarrhoea may persist
for 5-6 days. The diarrhoea probably results from villous atrophy in
small intestinal mucosa and consequent maladsorption and loss of
enzymatic activity. Severely shortened villi and occasional fusion of
adjacent villi are seen in the intestine. Immunodeficiency, failure to
ingest colostrum, secondary bacterial infection and multiple viral
infections all contribute to the clinical signs observed. Except for
distended bowel and small intestine with liquid faeces, no gross lesions
are observed. There may be pulmonary congestion and pneumonia. In
270
winter dysentry the incubation period is between 3-7 d.ays. The

symptoms start all of a sudden with brief period of temperature (39.540.5C). anorexia and apathy. Sometimes there is cough and nasal
discharge. Milk production is reduced drastically. In most cases the
diarrhoea lasts for several days about 2 weeks on an average.
Immune reaction: Circulating coronavirus antibodies do not
prevent infection but cause attenuation of infection in neonatal calf
diarrhoea. Regular ingestion of colostrum from immune dams can be
protective. The resistance to coronavirus infection is primarily due to
presence of colostral and milk antibody or actively produced IgM and!
or IgA in the intestinal lumen.
.
In winter dysentry. immunity is formed after a herd epizootic. The
protection ranges from 6 month to 3 years. The greater susceptibility in
young adults may be due to loss of colostral antibodies and increasing
stress.
Diagnosis: The diagnosis can be made by demonstration of virus
particles in faecal samples collected at early stages of virus diarrhoea.
The IF staining of spiral colon sections collected from a calf killed
within 4 days of onset of diarrhoea are of diagnostic value. In faecal
samples coronaviral antigen can be detected by immunoelectrophoresis.
reversed passive HA and ELISA. The virus can be cultivated in
primary bovine embryonic kidney cells. BEK-l cell lines. The
replication is enhanced by the addition of trypsin.
Control: A live attenuated vaccine is available and is effective if
given to newborn calves soon after birth. A combined attenuated rotacorona virus vaccine is also available which can be given to newborn
calves as well as to pregnant cows.
Canine Coronav!rus (CCV)
In 1971 CCV was isolated in United States from military dogs with
diarrhoea! disease. The significance of CCV infection in dogs was
recognised in 1978 when outbreak of corona associated diarrhoea
occured in dogs in USA. The infectious range in severity from
inapparent to fatal. younger dogs are mpre severely affected.
Experimental infection of neonatal dogs causes gastroenteritis with
diarrhoea and virus is excreted in faeces for 6-9 days. The corona viral
infections occur concurrently with other viruses. parasites or
pathogenic bacteria. In natural infections the majority of dogs recover
Coronaviridae
271
in 7-10 days but death in few dogs takes place within 24-36 hours of
onset of symptoms. The morbidity rate is variable and mortality rate is
low. The CCV produces CPE in several primary and continuous dog
cell cultures. Primary dog kidney and thymus cells, thymus cell lines
and other dog cell lines support the growth of virus. Infected cells
become enlarged and bizzare shaped. At present no vaccine is
available. The anti genic differences observed among CCV isolates may
complicate the development of a vaccine.
Feline Infectious Peritonitis Virus (FIPV)

Feline infectious peritonitis is a disease of wild and domestic cats.
Two strains of virus are reported. One strain causes clinical disease in
domestic and wild cats and second one is less virulent and causes
diarrhoea in young animals. The virus is similar in morphology to other
corona viruses and has similar physical properties. The virus can be
propagated in organ cultures of feline tissues. Virus can sometimes be
isolated by co-cultivating peritoneal macrophages with susceptible cat
cell lines. The virus causes rounding of few foci of cells and syncyti
a
and replicates by intracerebral inoculation in suckling mice. The
replication of virus in the brain of suckling mice or in cell culture can
be detected by IF test. FIP occurs in cats of all ages. The disease
is
inapparent in most cases but is fatal once the symptoms appear. The
incubation period is at least 4 months. The initial symptoms are
anorexia, depression and biphasic fever causing weakness. In effusive
form of disease there is abdominal distension due to peritonitis. The
cats die within 1-8 weeks after showing the clinical signs. The CNS,
eyes and visceral organs are also involved. There is moderate to
marked leucocytosis. Hyperproteinemia is a constant feature due to
elevation of gamma globulin. The characteristic post mortem lesions is
parietal fibrinous peritonitis and/or pleuritis with fluid in body cavities
.
Inflammation or necrosis of visceral organs occur. Mesenteric and
caecal lymph nodes are enlarged. The exudate layer adhering to the
peritoneum is composed of fibrin. The mode of the transmission of
disease is not known but close contact allows transmission. The disease
results from Arthus type of immune complex vasculitis. The tentative
diagnosis can be made from clinical signs. Antibodies can be detected
by IF test. The virus isola,tion can also be attempted. Presently there is
no vaccine availab~ and incidence is sporadic.
272
Porcine Coronaviruses
Transmissible gastroenteritis virus(TGEV), Haemagglutinating
encephalitis virus (HEV) and Epidemic diarrhoea virus.
Transmlssble
gastroent~ritis
virus (TGEV)
The virus produces highly contagious disease of swine

characterized by profuse diarrhoea, vomiting, dehydration and high
mortality in young piglets. Only pigs suffer from natural disease. Mice
and dogs can be experimentally infected and subsequently shed virus in
their urine. The disease was flrst reported in United States in 1946 and
subsequently reported from Canada, Taiwan, Britain, Japan, Soviet
Union and many European countries.
Properties of the }'irus: The virus is mostly circular in shape but
pleomorphic forms are also present. The size of the virus is between
l)()-200 nm. The morphology is typical of the group. The virus survives
several weeks in frozen tissues. TGEV is inactivated at 56C within 30
min., but survives for 3 days at room temperature. The virus is stable at
pH 3 and is not affected by 0.5% trypsin. It is inactivated by detergents,
ether and other lipid solvents. The virus does not show
haemagglutination. All strains belong to one antigenit type.
TGEV can be grown in primary pig kidney, thyroid, testis and
salivary gland cell cultures. Some fleld strains may produce transient or
negligible CPE in initial passages. Growth in dog kidney cells and
chicken embryos has been reported.
Epidemiology: The route of infection is by ingestion and probably
by respiratory route. The virus multiplies in the intestine. The virus is
excreted in the faeces and this continues upto 8 weeks or longer in
recovered pigs. Non lactating sows have mild disease but act as
carriers. The persislance of virus in farm buildings, swill and recovered
carrier sows play a major role in the spread of the disease.
Pathogenecity: The severity of the disease is related to the age of
pigs but swine of all ages are susceptible. The disease is fatal 10 piglets
under 1 week of age but mortality halves with each extra week of age.
The mortality in pigs over 5 weeks of age is low. The majority of
outbreaks occur from mid winter 10 early spring. The incubation period
is 18 hours to 3 days. The clinical signs are vomiting, profuse
diarrhoea, thirst, loss of body weight with high morbidity and mortality
in pigs under 2 weeks of age. In older pigs the diset\ses runs a chronic
Coronaviridae
273
course and often no symptoms are observed except failure to gain

weight The post mortem examination of young piglets show the
stomach filled with coagulated milk and intestines distended with
yellow foamy fluid. There is acute enteritis and severe ulceration. In the
terminal stages of the disease the intestinal tract becomes thin and
transparent There is extensive atrophy of intestinal villi in jejunum and
ileum. Degenerative lesions and haemorrhages may be found in the
spleen, kidney, urinary bladder and heart. There may be some
congestion and encephalitis in the central nervous system. Excessive
villous atrophy of jejunum and ileum and absence of chyle in mesentric
lymphatics is characteristic ofTGE.
Diagnosis: A presumptive diagnosis is possible on clinical and
epidemiological features of the disease. Villous atrophy is suggestive of
TGE. The FA test is applied to demonstrate the presence and viral
antigen in colonic lesions and faecal smears. The virus particles can be
demonstrated in the faeces or small intestine by electron microscopy or
immune electron microscopy. Four fold increase of neutralizing
antibodies can be demonstrated in paired serum samples. The virus
isolations can be done in cell cultures and identified by neutralization
test or FA test.
Control: The control measures are usually inadequate. Available
vaccines are unsatisfactory because they fail to elicit adequate
lactogenic immunity.
Haemagglutinating-Encephalomyelitis Virus (HEV)
Vomiting/wasting disease
The virus produces an acute highly infectious disease of young

pigs under 3 weeks of age. The disease was reported from Canada in
1958 and the virus was isolated in 1962 in Canada from the brains of
suckling pigs with fatal encephalomyelitis. HEV is prevalent in
Canada, England and USA. All strains of the virus are believed to be
anligenically homogenous.
The virus haemagglutinates and haemadsorbs chicken, turkey,
sheep, rabbit, mouse and hamster rbe but does not possess
neuraminidase. The virus can be cultivated in pig kidney cell cultures
and produces syncytia. The vomiting and wasting syndrome appears in
pigs 1-3 weeks old. There is vomiting, depression, anorexia and
constipation. Some- piglets may have respiratory signs, including
274
sneezing and coughing. The piglets become emaciated and die of

starvation; recovered pigs are unprofitable. In the encephalitis form
there is depression, anorexia and loss of oondition. Hyperesthesia,
incoord\nation and tremor with occasional vomiting for 2-3 days is
followed by acute encephalitis, convulsions and death. The mortality
rate goes upto 100%. There are no gross lesions on post mortem
examination. Mild lesions may be seen in eNS, a presumptive
diagnosis can be made from the clinical history. The definite diagnosis
is arrived at by isolating the virus from the respiratory tract and brain.
The FA techniques can be applied to demonstrate the viral anti~en in
tissues.
Avian Infectious Bronchitis Virus (IllV)

Avian infectious bronchitis virus (lBV) causes an acute, highly
contagious respiratory disease of chickens. The disease probably occurs
in most parts of the world where poultry is raised. The disease was first
described in United States in 1931. The presence of disease in India
was established in 1969. There is quite high incidence of the disease in
this country. The chicken is the only natural host of IBV.
Properties: The virion are approximately spherical in shape and
measure about 80-120 nm in diameter. The virus particle has club
shaped projections distributed on the surface. These are approximately
20 nm in length. The virion contains a central core of single stranded
RNA and is enclosed within a capsid. Approximately 16 polypeptides
have been found. Some IBY types after treatment with phospholipase C
agglutinate chicken rbe. This is inhibited by IBY antiserum. The virus
is sensitive to treatment with ether and other lipid solvents:The thermal
stability varies from one strain to another. Newly isolated strains are
inactivated at 56C within 15-30 mins whilechicken embryo adapted
strains survive upto 3 hours at this temperature. Infectious bronchitis
strains vary in antigenicity. By neutralization test, two antigenic
variants have been recognised-Connecticut and Massachusetts. There
are reports that other serotypes also occur. By immunodiffusion test
three soluble antigens have been recognised. One of the antigens
resides in the virus particles while other two are distributed over the
surface of virus.
Cultivation: The virus grows well. in 9-12 days old embryo by
allantoic cavity inoculation. The yolk sac route is unreliable because of
Coronaviridae
275
the possibility of maternal antibodies in yolk. The virus does not

produce pock marks on the CAM but there is characteristic dwarfing
and curling of embryos. Serial passages in egg embryos result in
decreased pathogenicity, antigenicity and immunogenecity with a
progressive increase in embryo lethality. A genotypic modification
probably occurs after serial passage3 in chicken embryos. A few strains
can also be grown by intracerebral inoculation in unweaned mice,
producing ascending prsalysis and death after 3-4 days of inoculation.
The virus can be propagated in chicken embryo kidney, lung and liver
cells, in CK and Vero cells and chicken ~acheal organ cultures. The
virus produces syncytia in affected cell cultures. New isolates or low
embryo isolates grow poorly or not at all in cell cultures; tracheal organ
cultures are suitable for this purpose.
Epidemiology: The virus is highly infectious and may be shed in
the respiratory discharge for 4 weeks and faeces 3 .weeks after
infection. Virus may be present in the fertile eggs laid during illness but
chicken hatched out of these eggs are not infected. In some cases the
virus is shed upto 7 weeks. The virus spreads by droplet infection but
indirect transmission may also occur through feed racks, water troughs,
clothing and equipment.
Pathogenicity: Chicken is the only natural host. Birds of all ages
are susceptible; however, chicks of 1-4 weeks of age are most severely
affected. The incubation period is i8-36 hours. The severity of clinical
symptoms vary considera!)ly and may go unnoticed in growing and
laying birds. Symptoms are severe in younger birds where mortality is
between 20-90% particularly with secondary infection by mycoplasma
or septicaemic E.coli. The clinical symptoms are depression, coughing,
gasping, tracheal roles, watery eyes and nasal discharge. The virus
replicates in respiratory and urogenital tract epithelium 1-8 days post
infection. Urogenital tract infections results in nepharitis with tubular
damage and interstitial infiltration of lymphoid cells and its epithelium
cells. The laying flocks have lowered egg production and eggs may be
misshapen, rough; soft shelled and of poor internal quality. Necropsy
reveals sinusitis, catarrhal tracheitis; bronchitis, congestion and oedema
of lungs. The caseous plugs may be found in the lower trachea and the
bronchi.
Immune reaction: The virus induces S.N, C.F and precipitating
antibodies in infected chickens. IgA type antibodies have also been
276
demonstrated The chickens which recover from natural infection are

resistant to intratracheal inoculations with homologous strains. Some
degree of immunity and antibodies can be demonstrated for at least one
year after natural infection. A high level of antibodies appear 3 weeks
after exposure.
Diagnosis: A presumptive diagnosis of IB is possible on the basis
of clinical signs and highly contagious nature of the disease.
Confirmation is based on isolation of IB V in chicken embryos from
trachea, bronchi and lungs. The tissue cultures are not sensitive for
isolation of the virus but tracheal organ cultures can be used.
Antibodies in the infected flocks can be detected by HI tests using
phospholipase with treated virus. The FA test on tracheal scrapings
provides a rapid diagnosis. A blot-dot hybridisation test has been
developed which identifies all isolates of IBV because their RNA will
hybridise with DNA probes from the matrix protein gene of a standard
strain.
Control: Recovered chicks are immune after natural attack for
atleast one year. Local immunity in the respiratory tract is important in
protection against the disease. Vaccination with attenuated strains is
widely practiced. The vaccination is done with attenuated virus
Massachusetts(1924) by aerosal spray, intranasal or intraocular in 1 day
old chicks or in the drinking water 2-3 weeks old. Administration by
aerosal or spray is more effective as compared to drinking water.
References
BUXTON, A. and FRASER, G., 1977. Animal Microbiology. Vo1.2 Oxford,
CARMICHAEL, L.E. and BlNN, L.N., 1981. New enteric viruses in dogs. Advances
in Veterinary science and comparative Medicine. 25,1-37.
DURHAM, P.J.; SlEVENSON, DJ. and FARQUIIARSON, B.C., 1979. Rotavirus and
coronavirus associated diarrhoea in domestic animals. N.z. Vet. 1.
27,3()"32.
GRElG,
A.S., 1975. Haemagglutinating encephalomyelitis virus infection. In

disease of Swine 4th Ed. Edited by H.W. Dunne and A.D.Leman,
Ames, Iowa, Iowa State University, Press.
M.S., 1975. Immune response to infectious bronchitis virus. Am. 1.

Vet Res. 36, 520.
HOSFSTAD,
Coronaviridae
277
HOFSTAD, M.S., 1978. Avian injections bronchitis virus. In diseases of poultry,

7th ed. Edited by M.S. Hofstad at al. Ames, Iowa, Iowa State
University Press
KAIIRS, R.F., 1985. Viral diseases of cattle. Iowa State University Press.
MAC
lNToSH, K., 1974. Coronaviruses: a comparative review. Curr. Top.

Microbiol. InunW1ol. 63: 85-139.
MEBUS, C.A.;- STAIR, E.L.; RHODES, M.B. and TWICHAus, MJ., 1973. Neonalal
calf diarrhoea! Propagation, attenuation and characteristics oJ-a
coronavirus like agent. Am. J. VeL Res. 34,145-150.
RUSSELL, P.H. and EDINGTON, N., 1985. Veterinary viruses. The Burlington
Press (Cambridge) Ltd.Foxton, Cambridge.
SIDDELL, S.; WEGE, HaMUT and MEULEN,VoLKER,TER; 1983. The biology of
corona viruses. J. General Virology.64: 761-776.
TAKAHASHI, E.; lNABA,Y.; SATO, K.; ITo, Y.; KUROGJ, H.; AKAsm, H.; SATODA, K.
and OMORJ, T., 1980. Epizootic diarrhoea of adult cattle associated
with a coronavirus-like agent. Vet. Microbiol. 5, 151-154.
Chapter 26
Orthomyxoviridae
The virus family orthomyxoviridae consists of one genus made up

of influenza A, influenza B and influenza C on the basis of shared
antigens by complement fixation and immunodiffusion tests. Type A
influenza viruses include all pathogenic veterinary viruses. However,
type C has been isolated from pigs in China during abaLloir surveys.
The influenza A viruses consist of roughly spherical or filamentous
enveloped particles which measure 80-120 nm is diameter. The
nucleocapsid is helical and is enclosed within a protein matrix. The
protein matrix is enclosed by a lipid membrane which is covered by
two types of glycoprotein spikes with which haemagglutinin and
neuraminidase activities are assoicated. The virus genome is a single
stranded RNA in eight segments with a moleuclar weight of about 4 x
1()6 daltons. Recombination occurs with high frequency within a
species but not among species. The influenza viruses are divided into
types, A, B and C on the basis of anti genic character of internal
nucleoprotein antigen. The influenza A viruses are further divided into
sUbtypes on the basis of haemagglutinin (H) and neuraminidase (N)
antigens. Influenza viruses are sensitive to heat, drying, detergents and
disinfectants and do not survive in external environmenL The viruses
can be grown in chicken embryos in amniotic and allantoic cavity of 10
day old fertile eggs. Some isolates grow in cell cultures; the CPE
produced is rounding and detachment of cells. Influenza viruses are
usually limited to their original host species.
Strain differences within the subtypes take place with altered H &
N antigens. The alteration is the res.ult of residual virus infection in
Orthomyxoviridae
279
layer
Lipid layer
----_.--'
Fig. 26.1 Schematic Representation or the Structure ofInnuen:l.a Virus.
animals whose antibodies favour the selection of mutants with altered

Hand N epitopes. A change of subtype within species is known as
antigenic shift The antigenic shift may result from, (i) mutation, (ii)
genetic reasortment, (iii) adaptation of influenza viruses from one
species 10 another.
Equine Influenza Virus subtypes 1 and 2
The virus causes an acute respiratory illness in horses. The disease
is world wide in occurrence. There was a disease outbreak is 1987 in
Northern India.
Properties of the virus: The virus of equine influenza has a
morphology typical of the orthomyxoviridae family. The virus
agglutinates horse, calf, pig, rhesus monkey, fowl, pigeon and human
rbe. The viruses belong 10 type A on the basis of their type specific
internal soluble antigen as detected by CF test. There are two distinct
serotypes. The virus type 1 carries I~N7 antigen while type 2 isolates
carry ~. antigens.
The virus can be cultivated in embryonating chicken eggs and in
cell cultures of equine, bovine and monkey kidney celIs. The CPE is
characterised by an early development of syncytia and formation of
multiple eosinophilic intracytoplasmic inclusions. The infected cell
cultures haemadsorb guinea pig red blood cells.
280
Epidemiology: The horses excrete virus upto 10 days via nasal

secretions. Spread is by droplet infection. The disease spreads rapidly
due to frequently violent cough and partly due to short incubation
period.. The infection is transmitted exclusively form horse to horse by
aerosal infection.
Pathogenesis: Equine influenza is an acute disease of lower
respiratory tract of horses that affects all ages. The serotype 2 is more
pathogenic than serotype 1. The incubation period is 1-3 days. The
morbidity rate is 95-98% while mortality rate in uncomplicated cases is
low. In foals the disease is more severe. The Symptoms are rise in body
temperature, loss of appetite, depression, increased respiiatory and
pulse rate with nasal discharge and cough. Pneumonia occurs
frequently in foals. The typical pathogenic lesion is bronchiolitis in
which bronchioles are filled first with serous and then with mucoid
discharge.
Diagnosis: The diagnosis can be made from the symptoms and
rapid spread of disease. The virus can be isolated in embryonating
chicken eggs and in primary cultures of equine or monkey kidney cells
from nasopharyngeal swabs during 48-72 hours of illness. The HA test
is applied for detection and HI test for identification of virus. A
serologic diagnosis can be made by detection of four fold increase in
antibody titres between acute and convalescent sera by CF and HI tests.
Control: After infection the immunity to homologous virus may
persist upto 2 years. Inactivated virus vaccines containing both
serotypes mixed with oil adjuvant are available. Foals at 3-4 months are
vaccinated and second vaccination is given after 2-6 weeks followed by
a booster at 1 year of age. More frequent vaccination is advocated for
animals at risk.
Swine Influenza Virus
The swine influenza is endemic throughout the world. The first
outbreak was reported in 1918 from USA when most serious human
influenza pandemic of the century was going on. The swine influenza
and human influenza of 1918 share the same haemagglutining
antigen<Rt). The pig pandemic of influenza might have resulted from
the host adaptation by the human HI virus. There are evidences of
intercbange of HI viruses betwen turkeys and pigs in France. The
zoonotic nature of swine influenza has been confmned when influenaza
Orthomyxoviridae
281
virus in 1976 was simultaneouly isolated from infected pigs and from a
man in USA with clinical signs of influenza who had been attending
the pigs.
Swine influenza is 'A' type virus. The swine influenza has its own
haemagglutinin but its neuraminidase is of human subtype (HI NI).
Like all influenza viruses the swine influenza virus undergoes antigenic
changes. The virus readily grows in chicken embryos and can be
propagated in pig embryo kidney besides cell lines of canine and
human origin. The virus haemagglutinates and haemadsorbs the red
blood cells of several species.
The disease occurs naturally as a herd disease during autumn,
winter and early spring. The incllbation period is 2-7 days. The
mortality rate is 1-2 per cent but morbidity rate is high. The virus
causes a'mild upper respiratory tract disease but the disease is severe in
association with secondary invaders like Haemophilus suis or other
bacteria. The animals generally recover in 4-6 days but with a
considerable loss of weight. The virus is transmitted by droplet
infection. The idea that pigs acquire infection by eating earthworms
containing virus infected larvae of pig lung worm appears to be
questionable. It appears that virus may be latent in some pigs. The
disease can be diagnosed by isolating the virus from diseased pigs. The
vaccination is not practised.
Avian Influenza Virus
Numerous type A influenza viruses with HI_13 N I_, have been
isolated from the faeces of wild and domestic birds. Isolates of the
avian influeza virus vary in pathogenicity from rapidly fatal disease of
fowl plaque to mild air sac disease and drop in egg production. Acute
disease of influenza is termed as fowl plague. The role of avian
influenza in public health is undefined. There is a possibility that
recombinational events could occur among many of the viru~s
circulating in brids. This mixture could result in emergence of viral
hybrids infectious for avian and mammalian species.
Fowl Plague Virus
The virus causes an acute fatal disease in several avian species like
chickens, turkeys, pheasants, terns and probably other wild birds. The
disease is enzootic in United Arab Republic, North Africa and some
282
countires of Europe. The disease has not been reported from India.
Properties of the virus: The disease is usually caused by the
viruses carrying H, or H, antigens. Viruses are thought to be introduced
into domestic flocks by wild brids e.g. migrating ducks, pheasants,
starlings or terns. These viruses increase in virulence during chicken to
chicken or turkey to turkey passage.
The fowl plaque virus can be cultivated in chicken embryos and
chicken embryo fibroblasts. Some of the strains replicate in chicken
and monkey kidney cells.
Epidemiology: The virus excretion ~curs prior to clinical disease
in birds. Virus survives in fomites and in dead birds upto 15 weeks at
4C. Spread within farm is direct while between farms is indirect via
infected fomites. Wild birds are considered to introduce viruses into
poultry flocks. The spread in wild birds is aided by water or by clonial
testing of terns.
Pathogenesis: The virus enters via nasal epithelium and replicates
to high titre in every tissue of body with resultant necrosis and
haemorrhages. The incubation period is 3-7 days. The disease has a
rapid onset and runs a rapid course. The affected birds are lethargic
with nasal discharges, oedema of head and neck, respiratory signs,
gasping and diarrhoea. The combs :md wattles become cynotic. The
post mortem lesions may be similar to Newcastle disease which include
haemorrhages on proventriculis, haemorrhages in serosal surfaces,
swollen kidneys and caseous bronchial plugs. The mortality rate varies
from 40-100%. The mortality among younger birds is higher.
Diagnosis: The confirmation of diagnosis can be done by isolating
the virus in the allantoic cavity of embryonated eggs. HI and VN tests
are applied for identification of the virus.
Control: Slaughter and disinfection of premises should be done
Inactivated vaccines are not satisfactory, chicken embryo attenuated
vaccines have been used with sQme success.
References
B.c., 1975. Swine influenza. In Diseases of swine, 4th Ed. Edited
by H.W. Dunne and A.D. Leman, Ames, Iowa, Iowa State University
Press, 1975
EASTERDAY,
EASlF..RDAY,
B.C. and TUMOVA, B. 1978. Avian influenza. In Diseases of poulJ.--y.
Orthomyxoviridae
283
7th Ed. Editl!d by M.S. Hofstad et. a!. Ames, Iowa, Iowa State
University Press
S.B. and DlTITA, S.K., 1981. Veterinary Virology Philadelphia, Lea
and Febiger.
MOIIAL'iTY,
RUSSELL,
P.H. and EDINGTON, N., 1985. Veterinary viruses. Cambirdge, Foxton,

Burlington Press.
Chapter 27
Paramyxoviridae
The viruses in this family are usually spherical with 100-150 nm in
diameter. The filamentons forms of several micrometers in length also
occur. There is lipid bilayer which contains matrix proteins. It is
trnnsversed by glycoprotein spikes. The envelope has two glycoproteins
which comprise separate spikes, the haemagglutinatioin (H) and fusion
(F) proteins. The genome is single stranded unsegemented RNA with a
molecular wight of 5.8 x 1()6 daltons. The RNA is negative sense. The
viruses are relatively heat stable and pH st.'lble. The viruses enter the
cell by fusion of their envelope with the cell surface membrane. The
neucleocapsid is the functional tempelate for transcription of
complementary viral mRNA and for RNA replication. The assembled
nucleocapsids are enveloped at the cell surface at sites at which the
viral surface proteins (envelope) have been inserted. Virions leave the
cell by budding. The viruses grow in chicken embryos and tissue
culture. The CPE is characterised by syncytial formation and inclusion
bodies, death of the cells and detachment. The paramyxoviridae family
contains the following 3 genera:
1. Para myxovirus- Virions are pleomorphic but usually
spherical with about 150 nm in diameter. The envelope is derived from
cellular lipids and contains viral glycoproteins at the outer surface and
nonglycosylated inner protein. The viral envelope bears two types of
projections, the larger one (HN) responsible for haemagglutinating and
neuraminidase activities while the smaller type (F) causes cell fusion
and haemolysis.
Paramyxoviridae
285
2. Morbilivirus- The envelope contains haemagglutinin but not

neuraminidase activity.
3. Pneumovirus- The virions contain neither haemagglutinin nor
neuraminidase.
Avian Paramyxoviruses
There are nine serotypes of avian paramyxoviruses of which
paramyxovirus 1 or Newcastle disease virus is most important.
However, paramyxovirus 3 also causes clinical symptoms and drop in
egg production in pOUltry.
Avian Paramyxovirus or Newcastle Disease Virus (NDV)
Newcastle disease is also known as Ranikhet disease in India.
Virus causes a world wide disease of birds e.g. chickens turkeys,
guinea fowl, pheasant'> and pigeons. Man is susceptible and suffers
from self limiting conjunctivitis. Different isolates of virus cause a
spectrum of disease ranging from a highly lethal disease to subclinical
respiratory tract infection. This depends upon the virulence of the virus
strain. The original description of the disease by Kraneveld (1926) and
Doyle (1927) related to the virulent form of disease while Beach (1944)
reported that a mild strain of the virus causes avian
pneumoencephalitis. The disease was recognised for the first time in
Batavia (now Djakerta), Indonesia (Kraneveld, 1926). In the same year
in autumn the disease was reported by Doyle (1927) at Newcastle
upon-Tyne, England. The disease in India was reported in 1927 at
Ranikhet Kumaon hills by Edwards (1927). Today the disease is'almost
world wide in distribution except in the Scandinavian countries of
Denmark, Norway, Sweden and Finland.
Properties of the virus: The mature virus particles are relatively
large, 100-250 nm. The envelope of NDV has haemagglutinin (H),
neuraminidase (N) and fusion protein (F) spkies. The' virus is
inactivated at 55C within 45 minutes and at 100C within 1 minute.
The sunlight inactivates the virus within 30 min. The infectivity
remains unchanged at 4C for a few days. The virus survives jn frozen
poUltry carcases for more than 2 years. NDV is more su.sccptible 10 the
action of alkali than acid. A solution of sodium hydroxide (2-5%) is
used for cleaning the buildings and for disinfcclion of wooden poUltry
crates or similar equipment. All strains of the virus agglutinate red
286
blood cells of a number of mammalian and avian species but chicken,

guinea pig and human'O' erythrocytes are most frequently used. After
haemagglutination the elution of red cells takes place. The virus also
possess haemolysin which haemolyses fowl red blood cells. The
property of haemadsorption is also shown by the infected celis with the
virus.
The
specific
antiserum
inhibits haemagglutination,
haemadsorption and haemolysis.
Cultivation: The virus is readily cultivated in chicken embryos.
The most common route of inoculation is by "allantoic cavity route.
Most of the strains kill embryos between 24-72 hours causing
heamorrhagic lesions and encephalitis in chicken embryos. The
maximum titre of about 1O-9EIDso per 0.1 ml is obtained belween 24-36
hours of inoculation. The virus also replicates in many cell culture
systems like chicken embryo fibroblast, chicken embryo kidney,
chicken, rabbit, pig, calf and monkey kidney and BHK-21 and HeLa
cells. Polykaryocytes are produced in infected cells which may become
chronically infected. The NDV is a potent inducer of interferon.
Epidemiology: Transmission is by droplet, direct contact, fomites
or the ingestion of excreted virus. Excretion can occur for at least two
months following infection of partially immune birds. The disease
spreads between farms or countries because virus can survive for
several months in either poultry carcases and eggs in dried material.
The virus has been isolated from variety of wild birds. The caged birds
have been incriminated in the introduction of exotic strains of NDV.
Migratory birds and scavenger birds appear to be of little importance in
the spread of ND. It is probable that wild birds act as mechanical
carriers. Dried material may be transmitted on dothing or as wind
borne dust. There is minor serologi.i:al variation between isolates
obtained from different outbreaks as shown by serum neturalizatipn,
haemagglutination inhibition and neuraminidase inhibition tests, one of
these differences indicate a significant antigenic variation which should
have a bearing on practical immunology. Therefore, there is no
evidence to date to indicate that the field virus is not immunologically
different from vaccine strains of virus.
Pathogenecity: The virus mainly infects chickens and turkeys but
is capable of infecting a variety of wild and domestic brids. The
average incubation period is 5-6 days but it may vary from 2-15 days.
In severe outbreaks the symptoms appe.ar within 3 days. The
Paramyxoviridae
287
pathogenicity is largely detennined by the viral strains and age of

chickens. According to pathogenicity three types of strains have been
described velogenic (highly virulent), mesogenic (moderately virulent)
and lentogenic (avirulent). The classification of three types of strains is
based on the three techniques.
1. The 'Mean Death Time' of infected 10 day old embryonated
eggs.
2. Intracerebral pathogenicity index in day old chicks.
3. Intravenous pathogenicity index in 6 week old chicks.
A large number of comparative studies have been carried out to
find strain differences in relation to virulenc~. Two parameters namely,
the capacity to induce cell fusion and to form plaques emerged as
factors related to virulence. This led to search systematically for
structural and functional differences in fusion glycoproteins between
virulent and avirulent strains.
Four distinct form of disease have been described 1. Doyle's (Asiatic) form: This is an acute fatal disease of chickens
of all ages casued by velogenic viscerotropic strains. The clinical signs
are hypcrpnea, blood-stained diarrhoea, dehydration, tremors, torticillis
and paralysis of legs or wings. Haemorrhagic lesions are prominent in
the digestive tract. The mortality is close to 90%.
2. Beach's (Pneumoellcephalitis) form: An acute infection caused
by velogenic, neutrotropic strains. The clinical signs are respiratory
distress, coughing, gasping, lowered egg production and paralysis.
Haemorrhages are absent from digestive tract but lesions arc present in
respiratory tract and eNS. The mortality is 10-50% but may be higher
upto 90% in young chickens.
3. Beaudelle's form: It is an acute respiratory and occasionally
lethal nervous infection of young chickens caused by mesogenic
strains. The clinical signs are coughing, anorexia and lowered egg
production but mortality is rare.
4. Hitcher's form: A mild inapparent respiratory infection caused
by lentogenic strains.
The virus causes mild influenza like signs and conjunctivitis in
human beings. In this country the disease produced is by velogenic
strains and is of Doyle's form.
Diagnosis: A presumptive diagnosis is possible on the basis of
288
clinical signs in severe fonns of ND but milder fonns may be confused

with other respiratory disease. The conftnnatory diagnosis can be
arrived at by isolating the virus from the blood, brain, spleen, lungs and
other tissues of diseased birds. The virus is easily isolated in 9-12 days
old chicken embryos by allantoic cavity route. The chicken embryo
ftbroblast and kidney cell cultures have also been used. The harvested
material which shows haemagglutination is conftrmed by
haemagglutination inhibition or serum neutralization test The sera of
recovered birds reveal antibodies in the haemagglutination inhibition
test.
Control: In countries where the disease is unknown, the slaughter
policy is followed. In Australia the slaughter policy has proved
effective. Most countries where disease occurs have adopted a
vaccination policy as being the most practical method of control. Live
and inactivated vaccines are used.
Inactivated vaccines: The virulent virus strains are used to
produce inactivated vaccincs~ The virus is inactivated without
destroying its antigenecity. The inactivating agents used are ultraviolet
radiation, formalin, chloroform of ~-propiolactone. The inactivating
agent not only inactivates NDV but also other viruses pathogenic for
poultry. The inactivated vaccine is safe and is not capable of initiating
infection or spread of virus. It is suitable for young chicks as well as
laying birds and chicks in poor health. The duration of immunity is
short and is usually 6 months. The adjuvants like aluminium hydroxide,
mineral oil or vegetable oil emulsion are incorporated to prolong
immunity. The standard programme of vaccination is ftrst dose at 21
days of age; second one at 8-10 week; third one at 16-20 weeks
followed by another at 5 months.
Live Vaccines
Live Lentogenic strains: The known strains F., Hitchner Bp La

Sota are used. The optimum dose is 1()6-' to 107EID~ per bird.
Live mesogenic strains: The known strains used arc Roakin,
Komorov, Hertfordshire, Mukteswar (R1B). The optimum dose is about
100EID~. The mesogenic strains produce a long lasting immunity but
cannot be administered in chicks below 6-8 weekS old. The lentogenic
strains are usually administered by instilling a drop of vaccine either in
eye or in nostril or as aerosal or through drinking water in day old
Paramyxoviridae
289
chicks. This is followed by a mesogenic strain. The mesogenic strain is

given by intramuscular route or by wing web method. In India
lentogenic F strain is given in day old chicks by intranasal or
intraocular route followed by mesogenic strain Mukteswar (R1B) at 6-8
weeks old by intramuscular or wing web method. The necessity to
revaccinate the birds again does not arise a~ the vaccine gives sufficient
immunity to birds in productive life. Although the live vaccines
especially the mesogenic strains give a long lasting immunity but it
also perpetuates the presence of Newcastle disease virus and is
considered to be responsible for the higher incidence of chronic
respiratory disease and other respiratory disorders activating potential
pathogens present in the flock at vaccination time.
Avian Paramyxovirus-3
This virus is prevalent in Europe & USA and causes drop in egg
production, respiratory disease and stunting of turkeys. The virus
evokes a low level of cross-protection and cross reactive HI antibodies
toNDV.
Mammalian Parainnuenza Viruses
The parainOuenza viruses generally replicate. in the upper
respiratory tract and produce mild disease. The immune response is
poor and reinfection usually follows. The parainfluenza virus (PI) has 5
serotypes.
Parainnueza-l (Sendai Virus)
The virus is associated with the upper respiratory tract infection
and results is chronic ill health and breeding problems in most of the
mouse colonies. The virus haemagglutinates and haemadsorbs a variety
of rbc and possesses a haemolysin active against chickens and guinea
pig rbc. The virus can be isolated in a variety of cell cultures and
embryonating chicken eggs from the lungs of infected mouse.
Experimental mouse to mouse passage of virus increases its virulence
which can cause fatal pneumonia in young mice.
Parainnuenza-3 Virus in Cattle
The infection of the Parainflueza-3 (PI-3) is widespread in cattle
population throughout the world. About 80-90% of cattle have HI
antibody. Repeated subclinical infections occur when the virus is
290
associated with bacteria and stress. PI3 has also been associated with
pleural lesions of sheep.
Properties 0/ the virus: The structure, physical and chemical
properties pf bovine parainflueza types 3 virus are the same as other
members of this genus. The important viral proteins are HN and F
proteins. Various parainfluenza virus type 3 (pI3) strains differ in
neuraminidase activity, some strains being strong while others weak in
neuraminidase activity. PI-3 haemagglutinates and haemadsorbs rbe of
certain species of animals including bovine rbc. The PI3 virus strains
grow in cells of bovine porcine and human origin. The ePE is
characterised by plaque formation, syncytia and eosinophilic inclusion
body formation. Variations in cytopathogenicity, plaque morphology
and haemagglutinating activity and syncytium activity have been
reported with various PI3 virus strains. No anti genic differences have
been reported between different strains isolated from different
countries. With monoclonal antibodies variation in the structure of viral
envelope proteins have been observed in some bovine PI3 virus strains
and these differences might be related to viral pathogenicity.
Epidemiology: PI3 virus was first isolated from nasal mucus of
cattle suffering from clinical signs of shipping fever in 1959. The
distribution of PI3 is now world wide. Most reports of PI3 association
with respiratory disease are from young cattle while reports from adult
cattle have been much less. The bovine PI3 infections are not always
associated with disease. The subclinical infections often take place and
are common during winter months. Most calves are infected by
respiratory route. The virus multiplies to a high titre in the upper and
lower respiratory tract and large quantities of virus arc excreted in nasal
mucous, ocular secretions and in droplets. The virus is very stable in
aerosals of nasal secretions when the temperature is low. The infection
has been demonstrated in the respiratory tract of calves aged few days,
removed from dams shortly after their birth and reared in isolation. It is
thought- that the source of this early caltbood infection may have
occured in utero or shortly after birth. Experimentally it is possible to
establish infection of bovine foetus by direct intrafetal inoculation. The
possibility of cross-infections between cattle and sheep exists in nature.
The cross infection between calves and man is not considered
important. The PI3 once establishes infection, spreads quickly in
Paramyxoviridae
291
susceptible young cattle in close contact in poorly ventilated houses.

These conditions are frequently encountered in modem intensive calf
rearing systems.
Pathogenecity: PI3 virus enters the animal body via the respiratory
tract. Experimentally cattle of all ages can be infected. The infection
cannot be established with neuraminidase weak strains of the virus.
Once the infection is established in the respiratory tract, viraemia occur
with transient location of virus outside respiratory tract. Occassionally
virus infection has ~n associated with systemic lesions such as
splenitis, or enteritis. The virus, however, is a primary respiratory
pathogen and produce severe effect on respiratory tract epithelial cells.
The glycoproteins of mucus secretions of the respiratory tract are rich
in N-actyl-neuraminic acid (NANA) for which paramyxoviruses have
affinity. The mucus gJycoproteins thus act as receptors for PI3 virus.
After viral adsorption to rcceptor site, viral neuraminidase splits off
NANA from the mucous glycoproteins and virus free to attach to the
next rcceptor bound NANA. The virus replicates in the epithelial cells
of upper and lower respiratory tract and in alveolar macrophages. The
replication of PI3 virus r;auses epithelial changes, including hyperplasia
and hyperplasia of'type II pneumocytes. This leads to thickening of the
respiratory barrier by cuboidal epithelization of alveolar walls. -The
amount of damage caused on the respiratory tract by virus depends on
the virulence of the infecting strain, the immune status of animal and on
environmental stress factors.
Primary infection of seronegative calves results in virus shedding.
Calves with maternal antibody also shed virus. The shedding is usually
for 8-10 days. In natural infection the virus has been found in the
respiratory tract of calves over a period of months. In natural outbreak,
the PI3 virus infection is accompanied by other microorganisms
including other respiratory viruses, mycoplasma and bacteria. The most
important respiratory disease of PI3 virus is enzootic pneumonia in
calves and shipping fever. In enzootic pneumonia in calves there is
high morbidity and a mortality approaching 10% has been recorded.
The survivors have a permanent lung damage with less food conversion
efficiency and light weight. In shipping fever there is depression,
anorexia, fever rhinitis and nasal discharge, diarrhoea occurs in few
animals. Morbidity rate varies from 444% and mortality upto 20%.
292
Immune reactions: The seronegative calves develop humoral and

local virus neutralizing antibody to PI3 infection. IgM and IgG are
predominatly the serum antibodies while IgA resides mainly in the
nasal tract. The serum antibodies appears 6 days post infection and
persist at high titre upto 3-5 months. The maternal antibodies in calves
persist upto 10 week~ of age. CMI also participates in the immune
response. The relative importance of CMI compared with antibody
mediated immunity in protection against PI3 disease virus is not
known.
Diagnosis: The diagnosis can be established by taking nasal
samples for antibody detection and necropsy material for
histopathological examination. The nasal swabs should be collected at
an early stage of disease. The nasal swabs is an excellent material-for
viral isolation as well as for rapid diagnosis by IF because nasal mucus
samples provide excellent material for IF. In PI3 infection outbreak a
four fold or greater rise of serum antibody titre is detected. The
commonly used serological tests are HI, SN, indirect IF and ELISA. In
the histopathological examination eosinophilic intracytoplasmic
inclusion bodies~ presence of epithelial. syncytia on bronchiolar and
alveolar walls is often accompanied by hyperplasia of alveolar
epithelium. The viral antigen in lung tissue can be detected by direct or
indirect IF.
Control: Overcrowding should be avoided. Both inactivated and
modified live vaccines are available. Inactivated vaccine stimulate
systemic and local antibody production and reduce virus excretion on
challenge. The modified live vaccines are given by intranasal or
intramuscular route, also result in the production of systemic and local
secretory antibody. Vaccinal virus may be shed from nasal passages.
Inactivated vaccines are frequently combined with inactivated IBR
vaccine and Pasturella bacterins. Two injections are usually
recommended.
Parainfluenza-3 Virus in Sheep
Only one serotype Qf PI3 virus has been recognised. The virus is
antigenically related to both bovine and human PI3 but can be
distinghished by serological methods. Infections of sheep with PI3
virus are common. The antibodies in sera of sheep are prevalent in
several countries throughout the world and percentage of prevalence is
293
Paramyxoviridae
over 70%. The maternal antibodies in lambs prevent the infection. The
maternal antibodies wane rapidly and animals become susceptible
within a year of age. Transmission of virus between animals is due 10
respiratory excretion. It is not clear as to how the virus is maintained in
a flock, it may be due 10 viral persistence. It is known that majority of
infection are mild in nature but severe outbreaks have also been
recorded. The role of PI3 virus in these outbreaks is confounded by the
dominating role of Pasteurella haemolytica. Experimentally it has been
observed that virus may initiate the disease process and predisposes 10
Past. haemolytica.
The infected lambs killed at different intervals show the presence
of viral antigen in the bronchioles and surrounding alveoli. The lesions
are formation of inclusion bodies and bronchiolar epithelial hyperplasia
followed by necrosis and desquamation. The development of these
lesions is associated with appearance of illness in the affected animals.
The clinical signs regress between 6-7 days of PI when the antibodies
appear. The epithelial destruction and accumulation of debris in the
lower respiratory tract provides a focus in which secondary bacteria
localize and proliferate. The PI3 infection predisposes to lambs 10
severe pneumonia caused by several serotypes of P. haemolytica
biotype A.
Most PI3 virus infections are subclinical but occasionaly acute
outbreaks of disease occur.
Immunity to PI3 virus can be produced by local or parenteral
administration of PI3 antigens, which prevent virus replication and
development of lesions.
Parainfluenza-5
Parainluenza-5 (PI-5) is associated with Kennel cough. The virus
in severe cases is associated with canine distemper virus or Bordetella
bronchiseptica. Most dogs have antibodies 10 PI5 without having
clinical disease.
MORBILIVIRUS
The genus includes the triad measles, distemper and rinderpest
viruses. All these viruses possess 'H' antigens but measles virus
haemagglutinates rbc, none of three viruses possess neuraminidase
antigens. These viruses share some (F) antigens which may be cross
294
protective. Antigens differ in three viruses. The H and F antigens occur

on separate peplomers.
Canine Distemper Virus (CDV) Hard pad disease

The canine distemper is a highly contagious disease of dogs with a
heavy death rate in young susceptible dogs. Prior to the development of
vaccination this was the major pathogen of dogs. The disease occurs
naturally in dogs (Canidae family) mink, ferrets and skunks. The
members of family felidae (cat, lions and tigers) are not susceptible.
The disease is world wide in distribution. Laidlaw and Dunkin in 1926
confinned the viral aetiology of the disease. Till then, Bordelella
bronchiseplica was considered to be the primary aetiological agent.
Properties 0/ the virus: The morphology is similar to other
members of the genus. The virus particle measures 90-250 nm in
diameter. Thermal inactivation of the virus is rapid. The virus is rapidly
destroyed if stored above OC. But survives for months at -lOoC. It is
inactivated by ether, 0.1 % formalin and 1% lysol. An irregular partial
haemagglutination of chicken and guinea pig rbe has been reported in
high concentration of egg adapted virus but this property is variable.
The CDY forms are triad with measles and rinderpest virus, the last two
viruses provide protection against CDV. Currently, only one antigenic
strain is recognised although it is well established that strains vary in
virulence. The pneumo, neutrotropic and epidermal strains (causing
Hard Pad) have been recognised. The variation in virulence exists in
measles and rinderpest viruses as well. In measles virus, antigenic
variation between haemagglutinins of different isolates have been
detected by monoclonal antibodies. It is thus likely that antigenic
variation also exists between isolates of distemper virus while the
immunodominant epitopes remain cross protective.
The virus can be adapted to grow in un weaned mice, baby
hamsters and rabbits. Green (1939) by successive passages of virus in
ferrets discovered that the virus was reduced in virulence for dogs
while the virulence for ferrets was enhanced. This led to the discovery
of live modified vaccine against distemper in dogs. The ferret adapted
strains can be further adapted in chicken embryos. After about ten
passages the virus produces thickening of the CAM. After about ioo
passages in chicken embryos, the virus becomes attenuated for dogs
and ferrets and can be used as vaccines. The virus replicates in primary
Paramyxoviridae
295
and continuous dog and ferret kidney cells and in chicken embryo
fibroblasts. The CPE is typical of the group which includes granular
degeneration and vaculation of cells, formation of giant cells and
syncytia with cytoplasmic and sometimes nuclear inclusion bodies.
Attenuation of the virus occurs after repeated passages.
Epidemiology: Distemper is a highly contagious disease. 'The
portal of entry is the respiratory tract. Transmission is both by droplet
infection and direct contact. The virus is shed in excretions and
secretions. The dog is the principal reservoir of the virus. The virus
appears in the urine upto 8th week post inoculation. Mortality is
variable according to the virulence of the strain (average is 20%),
sometimes mortality reaches 90%. Maternal antibody is transferred by
colostrum to the puppies and prevents infection usually upto 8 weeks.
Pathogenesis: The disease in nature occurs in dogs, wolves, foxes,
coyotes, ferrets, raccoons; minks, weasels and dingos. It primarily
attacks puppies and seldom older dogs, because of natural immunity.
The incubation period is 3-8 days. The clinical signs are diphasic fever,
coryza conjunctivitis, viraemia, leukopenia, followed by Icukocytosis,
severe gastroenteritis, respiratory distress, vesicular and pustular
dermatitis and hardened foot pads. Some dogs have nervous
manifestations like myalgia, incordination, convulsions and coma. The
nervous signs usually appear after the second febrile phase and are
generally followed shortly by death. The pattern of distemper is largely
dominated by the immune status of dog population in a given locality at
one time.
Haemorrhagic enteritis is common in puppies under 8 weeks of
age. The epithelium of bladder and pelvis is congested and large
intestine has excessive mucus. The lungs are generally congested and
pneumonic. The spleen may be enlarged and necrotic areas seen in
liver. Perivascular cuffing, neuronal degeneration and demyelination
occur in dogs with neurologic signs. Intracytoplasmic and sometimes
intranuclear inclusion bodies may be found in the bladder, renal pelvis,
epithelial cells of respiratory tract, intestine and brain.
Diagnosis: A provisional diagnosis can be made from the clinical
symptoms which include respiratory symptoms, diarrhoea, catarrhal
discharges from eyes and nose, hyperkeratosis of foot pad and nervous
signs. Intracytoplasmic inclusions may be found in stained conjunctival
smears. On postmortem examination inclusion bodies may be seen in
296
bronchial epithelium. bladder epithelium and lung macrophages. A

confmnatory diagnosis is made by isolation of virus from blood.
excretions and secretions. lymphnodes. liver. spleen. lungs and brain of
dogs with nervous mainfestations. Susceptible dogs and ferrets are used
for isolations. Virus isolation in canine or ferret kidneys can be made.
Detection of specific distemper antigens using immunofluorescence.
complement fixation or gel diffusion test with hyperimmune
antidistemper or antirinderpest sera.
Control: In most animals long lasting immunity develops after
recovery from natural infection. Maternal antibody is transferred to
puppies via colostrUm and usually prevents infection upto 8 weeks or
occasionally upto 12 weeks. The maternal antibodies interfere with
active immunisation . .The puppies cannot be immunized until they
loose maternal antibodies. Earlier inactivated vaccines were used
followed by virulent virus or by simulataneous antiserum and virus or
by ferret attenuated live virus. The current vaccines arc attenualCd egg
passaged virus and then grown in cell culture. These vaccines evoke
antibodies both against F&H antigens and give strong immunity. These
vaccines are usually given in puppies more than 12 weeks of age.
Combined CDV and infectious canine hepatitis vaccines arc also
available. The attenuated measles vaccine has been used in puppies of
3-6 weeks of age. The measles virus stops symptoms of distemper to
develop but may not prevent the infection of CDV.
Rinderpest Virus
Rinderpest generally known as cattle plague is one of the most
dreaded disease of ruminants (mainly cattle and buffaloes) and of the
swine characterized by ulceration of mucous membranes of the mouth.
erosion and necrosis of the mucous membranes of the digestive tract
with diarrhoea and lachrymal discharges.
Rinderpest is believed to have originated from China in pre-Roman
times. The disease was spread to Near East and Europe with the stock
brought in by invading forces. The disease was introduced in Africa in
1842 with cattle imported to Egypt from Romania. Towards the end of
19th century. the disease was introduced from India through the horn of
Africa and within few years swept through 90% of cattle population
south of Sahara. The economic importance of this disease was
responsible for establishment of veterinary services by colonial powers
Paramyxoviridae
297
at home and in their colonies, including first Veterinary school at Lyon

in 1764 and the international office of Epizootics in Paris. In India the
disease must have been prevalent through centuries. In view of serious
losses a cattle plague commission was appointed by the Government of
India in 1869. On the recommendations of the commission the Imperial
Bacteriological Laboratory (now Indian Veterinary Research Institute)
was set up in 1889 at Mukteswar, Kumaon.
The disease has been eradicated by slaughter policy in all
European countries, Southern Africa, Australia and Brazil. Eradication
plans were undertaken in India and in Africa and were successful in
bringing the disease under control. Before the eradication plan 'in India
there used to be 8000 outbreaks and 2,00,000 deaths in cattle which has
come down to 1200 deaths in 1983 and 147 outbreaks. In spite of the
concrete efforts the disease still occurs in certain countries of Africa
and Asia in cattle, sheep and goats and in wild life. Th~ countries where
the disease is still prevalent are India, Mali, Upper Volta, Niger, Benin,
Nigeria, Cameroons, Chad, Sudan, Egypt, Ethopia, Uganda, Kenya,
Tanzania, Turkey, Syria, Iran, Saudi Arabia and Kuwait.
Properties: The ultramicroscopic nature of rinderpest virus was
demonstrated in 1902 by Curasson when cattle were infected with
bacteria-free filtrate. The virus particle is spherical or ovoid in
morphology and measures 90-250 nm in diamater, some filamentous
and elongated forms vary upto 500-100 nm. Most viral particles are
bound externally by a membrane with large projections. The virus is
relatively fragile. The infectivity of cell culture preparations has a half
life of 1-3 hours at 37C and only few minutes at 56C. The virus is
stable between pH 7.2 and 7.9 but can survive between pH 4 to pH
10.2. The virus preparations are best preserved by lympholization or by
addition of 2% dimethyl sulphoxide and stored at 4C. The virus is
sensitive to ether, chloroform and trypsin. Various chemicals like-f3propiolactone, hydroxylamine, phenol, formalin inactivate the virus.
The virus also gets inactivated with prolonged treatment with glycerine.
The virus is rapidly inactivated by putrefaction. There are reportS that
rinderpest virus haemagglutinates rbc of rodents, rabbits and monkeys.
DK strain of rinderpest virus treated with Tween 80 and ether
agglutinated red cells from rodents, rabbits and monkeys. The titres
were low but reaction was inhibited by hyperimmune rinderpest
antiserum. The haemadosorption with rinderpest infected cells has not
been reported so far.
298
Texlbook o/Veterinary Virology
Strains of rinderpest virus are considered to be immunologically

homogenous. The attenuated vaccine strains like caprinised, lapinised,
avianised and tissue culture adapted strains protect the animals against
natural outbreaks as well as experimental challenge with any virulent
strain of.rinderpest virus. The rinderpest virus is antigenically related to
distemper and measles virus. It has been reported that the three viruses
have immunologically identical nucleocapsids and share envelope
antigens. The serological relationship between peste des petits (PPR)
and rinderpest viruses is close but the viruses are not identical. The
cross neutralization tesl~ diffe.renliate them. PPR virus protects cattle
against rinderpest and rinderpest virus protects sheep and goats against
PPR. There are two soluble antigens apart from infectious rinderpest
virus particle. The soluble antigens are complement fixing and
precipitinogen. The complement fixing antigen survives boiling for 30
mins. Strains of. rinderpest virus vary in their virulence for particular
hosts. The strain 01 isolated from sick elands is avirulent for cattle
while a strain isolated from sick giraffe kills upto 84% of inoculated
cattle.
Cu/timlion: The virus has been adapted to grow in sheep, goats,
rabbits, hamsters and mice. The virus has been adapted to grow in
chicken embryos by CAM, intravenous and yolk sac route of
inoculation, some strains become attenuated for cattle after 20
passages. The virus grows well in primary or continuous cell cultures
and produce a CPE. The susceptible cells are from cattle, sheep, goats,
chicken embryos, pigs hamsters dog and man. The CPE is characterised
by multinucleated giant cells or syncytia with intranuclear inclusions.
Epidemiology: Natural infections occur in even-toed ungulates
belonging to the order Artiodectyla. Cattle and buffaloes are most
commonly attached and perpetuate the disease. Epizootics in goats and
sheep have been reported from Africa, Asia and Europe and regularly
reported from India mostly from the South India. Asiatic domestic pigs
frequently suffer and die to rinderpest while pigs of European origin do
not suffer although the virus multiplies and is excreted. Free living wild
life also suffer and losses are highest in buffalo, eland, giraffe, kudu,
warthog, wildbeest in Africa; blackbuck, gaur nilgai and sambhar in
Asia. It is possible thal low pathogenic strains of rinderpest virus are
being maintained in wild East African Ungulates.
The role of vector in the epidemiology of rinderpest is not
Paramyxoviridae
299
important. The virus may survive for sometime in horse flies, house
flies, stable flies, tsetse flies, mosquitoes lice, ticks, leeches and
vultures.
Animals of all ages are attacked in virgin-soil epizootics while the
disease in enzootic area is characterized by age incidence. The adults
and very young calves do not suffer as adults are immune either
through natural attack of virus or vaccination, young are immune due to
colostrum antibody. Innate resistance varies widely within and between
species. In India local plain cattle are less susceptible in comparison to
exotic breeds, cross breed caule and hill cattle. The Asiatic pigs have
severe clinical reaction and often die, while European pigs, undergo
inapparent infection. Species differences have also been observed in
cohabitating wild life. The morbidity and mortality are profoundly
influenced by innate resistance of the infected host and the virulence of
strain. The degree of virulence also varies for particular host. ~Jne
attenuated vaccine strains for cattle and buffaloes have proved to be
virulent' for wild life. In enzootic areas the mortality rate is around 3%
but when exotic animals arc exposed to the same strain the mortlity rate
exceeds 80 to 90%.
Most of the strains are catholic and spread rapidly between cattle
and buffaloes while a few of the strains are remarkably selective. The
rinderpest outbreak may not spread to all susceptible animals in
aerosals and contact has to be close as infectious droplets arc large and
short lived. Air borne transmission is possible at night at high or low
humidities. Transmission through ingestion of contaminated food and
water is rare but pigs acquire infection by eating contaminated meat
scraps.
Palhogenesis: The virus enters the animal body through mucous
membranes of upper respiratory tract The primary multipication occurs
in the palatal tonsil or in the pharyngeal or mandibular lymph nodes.
After primary multiplication the virus enters in the blood circulation
where it attaches to mononuclear cells. The viraemia is detectable -1-2
days before the symptoms appear. Secondary multiplication of virus
takes place in all the lymphoid tissues, in mucous membranes of the
alimentary and respiratory tracts and in the lungs. The virus does nOl
multiply in the brain, kidneys or myocardium. The virus titre is at the
peak during prodromal fever and remains in the circulation upto erosive
mucosal phase and then falls slowly till the antibodies appear, 4-5 day
300
after the onset of fever. The duration of viraemia is influenced by the

strain of the virus and innate resistance of the host. The virus is shed in
all the secretions and excretions of affected animals, with ocular and
nasal secrytions and faeces. The virus is shed throughout the fever
period and few days after fever regresses. Recovered animals are not
considered as carriers as clear evidence of latency has not been found.
The persistent infection of rinderpest virus has been established in Vero
cells. This has led to controversy over the possible resistance of carriers
in rinderpest virus infection.
The classic rinde~est syndrome is characterized by a short sharp
fever, erosive stomatitis, gastroenteritis, dehydration and death. The
disease progresses through five distinct clinical phases. The incubation
period, the prodromal phase, mucosal or erosive phase, diarrhoeic
phase and convalesrence.
The incubation period ranges from 3-15 days depending upon the
innate resistance of animals. It is longest in animals having high innate
resistance. A sudden onset of fever marks the end of incubation period
and start of prodromal phase in which the clinical signs are absent,
within 48 hours the signs of fever are mainfested by depression and
restlessness; dry muzzle staring coat, serous nasal and lachrymal
discharges, congested mucous membranes, anorexia and constipation.
There is leucopenia. The mucosal phase begins 2-5 days after the onset
of prodromal fever with necrotic pinheads on the mucosa of mouth
nostrils and urogenital tract. The necrotic foci are readily abraded
leaving erosions with red raw floors. As the disease progresses the
erosions enlarge and coalesce, profuse salivation follows and lachrymal
discharges. the infected animal becomes restless, drinks copiously but
does not eat and passes soft faeces. The diarrhoeic phase starts when
the fever falls after two to three days after the appearance of mucosal
erosions. The faeces are dark and fetid with mucus and streaks of
blood. The profuse diarrhoea causes dehydration; emaciation and
prostration. Most of the deaths occur in diarrhoeic phase. The onset of
convalescence is ill defined. The recovery to full health takes many
weeks. Pregnant aniI1lals abort within six weeks of illness.
Abbarrant clinical forms are not uncommon and range from
peracute encephalitis and sudden death to transient subacute diarrh,oea.
The clinical picture in buffaloes simulate those in cattle. In pigs after an
incubation period of 3-5 days the symptoms are manifested by fever,
,"'"
Paramyxoviridae
301
inappetance and depression. Within 48 hours the animal starts shivering

and becomes prostrate. Vesicles appear around the anus, vomiting and
epistaxis takes place. Diarrhoea begins within 48 hours of illness and
persists until death or 10-12 days in survivors. In sheep and goats the
incubation period is 3-5 days. The clinical symptoms are manifested by
sharp fever, there is loss of appetite. :Qiarrhoea begins after 2 days of
onset of fever. Prostration and subnormal temperature precede death.
Lesions are moslty found in the digestive tract Erosive lesions are
found in the mouth the upper respiratory tract and upper part of
oesophagus. In abomasum similar type of lesions also occur in ~yer' s
patches. There is a massive destruction of lymphocytes especially in
lymphnodes. Congestion, necrosis ar.d erosion occur in the ilea-caecal
valve, caecocolic junction and rectum.
Immune reaction: The strains of rinderpest virus are
immunologically homogenous and there is complete cross protection.
The animals that survive the infection are life long protected although
in few animal there may be transient multiplication of virus in tonsillarpharyngeal tissues when exposed to virulent virus. The humoral
antibodies, IgM appear after 5-7 days of illness. Within 2 weeks IgM
decline and IgG class appear. The IgG antibodies reach peak within one
week and then the titre fall back to a threshold level and persist. The
reexposure results in anamnestic response. IgA antibodies appear in the
respiratory mucus in naturally infected cattle or vaccinated by
intranasal route. From immune catlle the calves get the maternal
antibodies in the colostrum. The passive protection in the calves in
maintained from 4-8 months or sometimes longer. The role of CM! in
cattle against rinderpest virus is not understood.
The rinderpest virus is immunosuppressive because it destroys T
and B lymphocytes. Due to immunosuppression in rinderpest virus
infection the latent infections are activated, which complicates the
clinical picture and causes diagnositc confusion. The virus suppresses
both humoral and cell mediated mechanism but production of memory
cells is not impaired. In rabbits. the destruction of lymphoid tissues by
virus triggers autoimmunity. Antinuclear and cold haemagglutining
antibodies are produced.
Diagnosis: The clinical symptoms, pattern of the disease and post
mortem examination generally lead to strong suspicion of rinderpest.
The examination of stained impression smears prepared from the
302
epithelia of tonsils and other lymphoid tissue reveals the presence of

syncytia and acidophilic ~nclusion bodies. The method is useful but
cannot be used as a routine diagnostic procedure. For conftrmatory
diagnosis the laboratory procedures employed are 1) isolation of the
virus 2) detection of rinderpest speciftc antigen and 3) demonstration of
the development of rinderpest antibodies.
For isolation of virus whole blood in EDTA, lymphnodes, spleen
and other tissues are employed. These clinical material is obtained
preferably during the prodromal or early mucoid phase of the disease.
The material is preferably inoculated in susceptible and known immune
cattle. The susceptible cattle will react in 2-10 days time. The material
is preferably inoculated in bovine kidney cells cultures. If the virus is
present, characteristic CPE develops within 3-12 days, the spccificity of
the reaction is conftrmed by means of neutralization tests.
The soluble antigens of rinderpest can be detected by complement
ftxation and agar gel diffusion tests. The tissue of choice are fresh
carcase or visceral lymph nodes or spleen taken at 3rd to 6th day of
pyrexia.
The speciftc neutralizing complement fixing antibodies develop in
the sera of canle which develop upto 4th day of the disease or later.
Paired serum samples are detected for the presence of antibody. The
serum neutralization test is carried out in cell culture or in chicken
embryos.
Other tests which are more sensitive and rapid have also been
developed like single radial haemolysis test, reverse passive
haemagglutination
test,
fluorescent
antibody
technique,
immunoperoxidase test, enzyme immunosorbent assay etc.
Control: Recovered animals develop a long lasting immunity but it
may not be life "long. The rinderpest virus is always introduced into
disease free terrilOries by animals having mild infection. The rinderpest
free countries ensure the freedom from disease by imposing a complete
embargo on the importation of live animals and frozen animal products
from countries where disease is prevalent. Virgin outbreaks in disease
free countries can be controlled by slaughter of infected and in contact
ruminants and swine. Countries where the disease is endemic, regular
vaccination is carried out to limit the losses.
The vaccine that revolutionized the control of rinderpest was
attenuated goat adapted virus. The vaccine was developed at Indian
Paramyxoviridae
303
Veterinary Research Institue, Mukteswar by Edward in 1928. The

vaccine is still being used in India and certain African countries. The
goat adapted virus is not attenuated enough and produces severe
reaction in susceptible breeds like exotic cattle, hill cattle and certain
Japanese breeds. The tissue culture adapted virus vaccine developed by
Plowright and Ferris is suitable for all breeds of cattle and other species
of animals. The immunity produced by attenuated vaccines are of long
duration lasting for about 10 years or more. The efficacy of vaccination
is dependent on the variable dUJation of the passive protection
conferred by the ingestion of antibodies in the clostrum.
Peste-des-Petits-Ruminants Virus
In early 1940's, a fatal disease of goats with high mortality was
described as peste-des-petits-ruminants (PPR) in the Ivory coast in
Africa. Momet and his colleagues in 1956 rediscovered the disease and
observed that PPR was a strain of rinderpest virus since it had a
serologial relationship with rinderpest virus. PPR has since been shown
to be a distinct entity having antigenic re,lationship with rinderpest,
canine distemper and measles viruses. The disease has synonyms like
'goat plague', erosive stomatitis and enteritis of goats, goat catarrhal
fever, 'Kata' and stomatitis peneumonitis complex.
The virus looses its infectivity within 30 min at 50C. The virus is
sensitive to lipid solvents, low pH etc. but in lymph nodes the virus is
protected from pH changes after death. The virus does not
haemagglutinate rbe but PPR antibodies inhibit haemagglutination of
monkey rbe by measles virus. The strains isolated from different parts
of Africa are homogenous. Cross-protection has been observed, PPR
virus protects cattle against rinderpest virus and rinderpest vaccines
protect against PPR.
Natural disease is prevalent in goats and much less in sheep. Cattle
in contact with sick goats do not suffer, Experimentally deer and pigs
support virus mulliplication but their role in epidemiology of disease is
not important. The PPR virus is prevalent in African continent south of
Sahara and in Arabian peninsula. The disease outbreaks mostly occurs
in West Africa but disease has been reported from Sudan. The disease
has also been reporte,d from sheep and goats in Saudi Arabia. Morbidity
in goats is high while mortality in clinically affected goats range from
70-80%. The animals get the infection from the sick animal when the
304
Textbook ojVeterinary Virology
animals are concentrated during rain etc. The infection is aerosal in

nature. The virus after entry in the upper respiratory tract is
disseminated before the onset of clinical signs and virus is shed in nasal
secretions, tears, saliva and urine.
After an incubation period of 6 days, sheep and less frequently
goats develop subacute infection. The illness is manifested by low
glade fever, nasal catarrh, mucosal erosion and intermittent diarrhoea.
After 10-14 days the animals usually recover. The acute reactions are
observed in goats after incubation period of 3-4 days. Fever and
diarrhoea appear but the animals look normal but after few hours the
animals show depression. Erosions develop in the mucous membranes
lining the upper respiratory, upper alimentary and urogenital tracts after
1-2 days after onset of fever. There is profuse salivation with protruded
tongue. There is lot of mucous secretion from the nostrils which often
block the nostrils. The mucosal erosions coalesce and produce
extensive lesions. Diarrhoea and pneumonia then supervenes. Most of
the animals die within 10 days. The incubation period is about 2 days in
paracute cases. There is profuse nasal catarrh, high fever, dyspnea,
anorexia and constipation. The mucous surfaces are congested and
eroded. The diarrhoea starts after about 3 days of onset of clinical
symptoms and animal die within 4-6 days after iJIness. Paseurella
pneumonia invariably supervenes in actue cases. With the onset of
fever there is severe leukopenia. On histopathological examination
multinucleated giant cells containing intranuclear and intracytoplasmic
inclusions, the syncytia in lung parenchyma is conspicuous.
Surviving goats develop immunity and resist infection and
immunity is lifelong. The humoral antibodies can be detected by CF,
AGID, measles HI and neutralization test. Clostral immunity protects
kids and lambs until they are weaned.
The diagnosis of PPR outbreak is confirmed by detection of
antigen in the tissues and isolation of the virus. The histopathology is
also useful in the diagnosis. The lymph nodes and tonsils collected
from animals killed in the early stage of disease or even after death of
animals are used in AGID test or CIEP test against PPR hyperimmune
serum. Blood collec~ed over EDTA during febrile stage of the disease
or lungs, lymph nodes and tonsils from killed animals during febrile
stage are useful for virus isolation. The tissue suspensions are
inoculated into primary lamb kidney cells or in known susceptible and
Paramyxoviridae
305
immune goats. The animal inoculation test is quicker. A

histopathological examination of early mucosal le$ions, lymphoid
tissues and lungs reveal multinucleated giant cells with incusions. The
convalesent sera of sheep and goats is used to detect antibodies by
CIEP, AGID or neutralization tests. It is difficult to distinguish PPR
form rinderpest without laboratory tests or animal inoculations. PPR
has been reported from India by Shaila et al. (1989). The rinderpest
virus infection is also common in sheep and goalS in India. The
importance of laboratory diagnosis in these animals becomes all the
more important.
Virulent cell culture passage PPR virus when ~dministered
simultaneously with hyperimmune serum gives durable immunity. An
attenuated PPR virus strain has been developed but it is not yet
available for vaccination purposes. The cell cultured rinderpest virus
vaccine gives protection against PPR and is used widely for
immunization but there have been some failures as well.
PNEUMOVIRUS
Hovine Respiratory Syncytial Virus (BRSY)
The antibody to bovine respiratory syncytial virus is widespread in

bovine population. The epizootics among bovines of acute viral
infections have been reported form Europe, Japan, United States,
Australia and North Africa.
Properties: The-morphology is that of paramyxovirus. The virus
shows great pleomorphism with a diameter of 80-450 nm. The genome
codes for ten proteins. The virus docs not possess active
haemagglutinin or neuraminidase and antigenically distinct form
morbiliviruses. Bovine RSV is antigenicaIIy related to human RSV but
it is not serologically identical. The virus is fragile and can be
recovered from clinical specimens in cell culture within one hour of
collection of the specimen. The virus grows in the cell culture of bovine
origin. Monolayers of bovine testis are very sensitive for virus
isolation. The CPE produced ;lre numt:rous syncytia and eosinophilic
cytoplasmic inclusions. The virus does not grow in embryonating eggs.
Epidemiology: The presence of virus carriers has not been proven
but the disease generally. appears in BRSV free country after
introduction of infected cattle. The velerinarians and animal handlers
play a role in virus transmission. The disease rapidly spreads from farm
306
to farm. The animals especially beef cattle are most susceptible

between 3-9 months of age, the younger calves of few weeks can also
be affected.
Pathogenesis: Experimental infection with BRSV produces a mild
infection, The concomirant presence of BVDV as well as sudden fall in
atmospheric pressure or a drop of temperature seem to aggravate the
disease. The incubation period is 5-7 days. Pyrexia occurs four days
after exposure to virus and is associated with the replication of virus
throughout the upper respiratory tract Syncytia formation occurs in the
epithelial lining and intracytoplasmic inclusions occur in the infected
cells. The mild rhinitis and tracheitis is associated with cough but
extensive bronchopneumonia occurs only when secondary bacterial
infection takes place. The animals recover within 2 weeks of infection.
There are reports ihat BRSV infection can occur in the presence of
circulating antibodies, however, the protective effect of nasal
neutralizing antibodies against infection has been shown.
The mortality goes upto 20%. The lossess are high among the
calves. Surviving calves recover after few days of illness.
Diagnosis: A 4 fold increase in CF or VN antibody titre in paired
scrum samples is diagnostic of RSV infections. The virus can be
isolated from the early cases. The nasal secretions are the source of
virus. Monolayers of bovine testis are incubated with the fresh material.
1be isolation in cell culture is difficult because of late appearance of
CPE which ranges from 20 days to 50 days. The earliest CPE noted are
small areas where four or five cells are balloolJed with shrinkage of
cytoplasm followed by syncytia formation and then become opaque and
holes appear in the monolayer. The viral antigen can be detected in
nasal mucus and lung tissue by direct IF. FLISA is now applied more
extensively for detection of viral antigens.
Control: The management of calves is important. Live vaccine
administered parenterally are available. The vaccines do not prevent
infection but decrease the severity.
References
ADLAKlIA, S.C., 1982. Rinderpest. Indian Council of Agricultural Research,
New Delhi
AI.ExANDER, DJ., 1980. Avian paramyxoviruses. Veterinary Bull. 50: 737-52
Paramyxoviridae
307
Au..AN, W.H; LANCASTER, J.E and Tom, B., 1973. Newcastle disease voccines.
FAO
BANSAL, R.P., 1986. Rinderpest and its eradication from India. National
symposium onAnimal Health at IVRI, Izatnagar June 24 to June 26,
1986.
BANSAL, R.P., 1986. Diagnosis of rinderpest with special reference to
application of modern techniques. Indian Vet. Res. Inst. Mukteswar,
55 pp.
BETfS, AP.; JENNINGS, AR.; OMAR, AR; PAGE, Z.E.; SPENCE, J.B. and WALKER,
R.G., 1964. Pneumonia in calves by parainJluenza virus type3. Vet.
Rec. 76, 382-384.
BRYSON, D.G.; EVERMANN, J.F.; LIGGIT, D.; FOREYT, W.J. and BREEZE, R.G.,
1988. Studies on patfwgenesis and interspecies transmission of
respiratory syncytial virus isolated from sheep.Am. J.Vet. Res. 49:
1424-1430.
BUXTON, .A. and FRASER,. G., 1977. Animal Microbiology Vol.2. Oxford,
CURASSON, G., 1932. Le Peste Bovine. Vigot Freres, Paris, pp.344
DAVIES, D.H; DUNGWORTII, D.L; HUMPIIREYS, S. and JOIINSON, AJ., 1977.
Concurrent infection of lambs with parainJluenza virus type 3 and
Pasteurella haemolytica. N.Z. Vet. J. 25,263-265.
FRANK, G.H. and MARSIIALL, R.C., 1973. ParainJluema-3 viruses infection of
cattle. J.A Vet: Med. Assoc.163: 858-60.
GILLESPIE, J.H. and CJ\R.\UCHAEL, L.E., 1968. Distemper. In Canine Medicine,
Edited by E.J. Contocott, Wheaton, III. American Veterinary
Publications.
LANCASTER, J.E., 1981. Newcastle disease. In virus disease of Food Animals.
Vol.Il. Edited by E.P.J. Gibbs, Academic Press, London.
McFERRAN, J.B. and McNULTY, M.S., 1981. Aids to diagnosis of virological
disease. Br.Vet. J.137, 455-463.
MOHANTY, S.B. and DUTIA, S.K., 1981. Veterinary Virology, Lea and Fibger,
Philadelphia.
MOHANTY, S.B. ,1978. Bovine respiratory. viruses. Adv. Vet. Sci. Comp. Med.
22,83-109.
308
NAOAl, Y. and TOYODA, T., 1987. MolecularlJiology ofNewcastle disease virw.

Prog.Vet. Microbiol. Immunol. 5,16-64.
NAWA1lIB, D.R. and Lm.toRDA, A.G., 1983. Towards global eradication of
rinderpest. Rev. Sci. Tech. Off. Int. Epiz 2,998-1012.
PLOWRIGHT, W., 1968. Rinderpest virus. Virol. Monogr. 3,25-110.
RusSEU.. P.H. and EDINOTON, N., 1985. Veterinary viruses. The Burlington
Press (Cambridge) Led.Foxton. Cambridge.
REISINGER, R.C.; HEnDLBSTON, K.L. and MANI'HER, C.A., 1959. A myxovirus (SF4) associated with shipping fever of cattle. J. Am. Vet. Med. Assoc.
135, 147-152:
RYDBIlCK, R; LoVEL, A.; ORVELL, C. and NORBY, E., 1987. Anligenic Analysis of
human and bovine parainJluenza type 3 strains with monoclonal
antibodies. J. Gen. Viral. 68: 2153-2160.
SCOlT, G.R., 1990. Rinderpest virus. In Z. Dinter and B. Morain (Editors).
Virus infections of ruminants. Elsevier Science publishers B.V.
Amstardam pp. 341-354.
SCOlT, G.R., 1985. Rinderpest in 1980. In Prog. vet.Microbiol and
ImmunoI. Vol. 1 pp. 145-74 Frager, Basel.
SCOlT, G.R., 1981 Rinderpest and peste despetits ruminants. In virus diseases
of food animals. Edited by E.P-Gibbs Vol.ll. Academic Press,
London.
SHAll-A, M.S ; PRUSHOlTAMMA, V.; BHAvASAR, D.; VENUGOPAL, K. and
VENKA'IESAN R.A., (1989). Peste des petits ruminants of sheep in
India. Vet. Record 125: 602.
TAYLOR, W.P., 1984. The distribution and epidemiology
ruminants. Prev. Vet. Med. 2,157-166.
of pestes-des-petits
G., 1977. Laboratory diagnosis, methods for bovine respiratory

syncytial virus. Vet. Sci. Commun.l, 179-189.
WELLEMENS,
BEACH,
J.R., 1944. The neutralization in vitro of avian pneumoencephalitis

virus by Newcastle disease immune serum. Science 100: 361-362.
DoYLE,
T.M., 1927. A hitherto unrecorded disease of fowls due to a fillerpassing virus. 1. comp. Path. 40, 144-169.
EDWARDS, J.T., 1928. A new fowl disease. Ann. Rep.lmp, Inst. Vet. Res.
Mukteswar, 1928. p. 14-15.
KRANEVELD, F.C., 1926. A poultry disease in Dutch East Indies (Dutch). Ned.
Ind. B I , Diergeneesk. 38,448-451.
Chapter 28
Rhabdovjridae
This is a remarkable family of animal viruses for its characteristic

morphology and for the wide range of forms of life that are infected
from plants to mammals by the members of this family.
Animal rhabdoviruses (from Greek rhabdos means rod) are oblong
particles with one rounded and one planar end which gives the virus
particle the shape of a bullet. The virus particles are of varying sizes,
length 130-380 nm and diameter between 50-95 nm. The lipo-protein
envelope contains virus specific peplomeres and has projecting
glycoprotein spikes which determine cell tropism and virulence. These
projections are 5-10 nm in length and 3 nm in diameter. The
nucleocapsid measures about 50 nm in diameter and consists of helical
structure (20 x 700 nm) rightly coiled into a cylinder. This gives a
characteristic striations of the particle in electron microscopic
preparations. The genome consists of one molecule of non infectious
linear single stranded RNA of negative polarity with a molecular
weight between 3.5 to 4.6 x 1()6 daltons. Virion proteins are associated
with the membrane and the nucleocapsid. Five major proteins are
possessed by the virus particle. Protein G forms the glycoprotein spikes
which determine cell tropism and virulence. Protein M unites with host
lipid to form envelope. N and NS proteins form structural components
for RNA helix, while L protein is associated with transcriptase. There is
also a protein in the envelope associated with haemagglutination of
goose erythrocytes. Thin ouier !ay~r is usually derived from host cell
membrane either by budding out through p!~ma membrane or into
cytoplasmic vesicles. The virus particles are sensitive to UV light but
310
TeXJbook o/Veterinary Virology
survive in dark place at lower temperature. The virions are sensitive to

lipid solvents, moderately resistant to heat, acid pH and deter~ts.
Members of the family grow in wide range of tissues and prod lice
rounding and retraction of cells. Viral replication is entirely
cytoplasn:tic with formation of inclusion bodies. The virion
transcriptase copies the viral RNA into several positive mRNA which
are encountered in polysome complexeS. Virion RNA replication
involves an RNA nucleoprotein intermediate. Morphogenesis is by
budding from intracytoplasmic or peripheral membranes.
The family of rhabdoviruses is d~vided into two antigenically
distinct genera. 1. Vesiculovirus which includes vesicular stomatitis
virus and is the only virus in this genus. 2. Lyssavirus-Rabies Virus is
the only member of vaterinary importance. The other animal pathogens
in this family do not have an established group. The important viruses
of veterinary importance are Ephemeral fever virus of cattle and
Marburg virus of man and monkeys.
Vesicular Stomatis Virus (VS V)
The virus causes a benign contagious vesicular disease in cattle,
pigs, horses, deer and occasionally in man. The disease primarily
occurs in Western Hemisphere and is enzootic in parts of North,
Central and South America. Occassional outbreaks have been reported
in Europe.
Properties 0/ the virus: The virus particles are cylindrical, bullet
shaped rods with a diameter of 150-180 x 50-70 nm. The envelope is
covered by short spikes about 10 nm long. Truncated virus particles are
also present in addition to bullet shaped particles. The virus
agglutinates goose rbe at 0-4C. The virus is resistant to physical and
chemical agents. At 4C in soil it is infective for many days. There are
two immuno\ogicaIly distinct types, New Jersey and Indiana identified
by neutralization and and complement fixation tests.
There are three subtypes of Indiana VSV. Indiana virus is
antigenically diverse. New Jersey virus isolates are antigenically
similar irrespective of the host species or the geographic location.
Hybridization studies conducted on several antigenically similar New
Jersey isolates RNA has shown that New Jersey isolates can be
differentiated into two groups having less than 25% homology. More
genetic difference exists between two antigenically similar New Jersey
Rhabdoviridae
311
groups than three antigenic subtypes of Indiana Virus. Isolates of both

Indiana and New Jersey viruses differ in their physical, biological and
genetic properties. The virus grows in the chicken embryos by
amniotic, allantoic and yolk sac routes and produce pocks on the
chorioallantoic membrane. VSV can be propagated in a wide variety of
cell cultures showing rapid CPE. The virus can be adapted to
mosquitoes and nearly all mammalian species can be infected.
Epidemiology: The epidemiology of VSV is complex, due to two
antigenically distinct viruses, Indiana and New Jersey. Indiana virus
has further three distinguishable serotypes. These viruses induce
vesicular lesions In mouth and on the feet of cattle, horses and swine.
With the exception of white tailed deer, there is no evidence of clinical
disease occuring in wild mammals. Disease in man has been reported in
individuals working in virus laboratories, clinicians and livestock
handlers. The antibodics are prevalent in these persons.
The manner in which thc virus is transmitted form one animal to
another is controversial. From experimental studies it is known that
virus can be transmiLlcd from infected to susceptible animal by
contaminating an abmsion in mucosa of gum or tongue, skin of teat
orifice or skin bordering the hoof with saliva or any exudate from a
lesion. The infection through intact skin is not established. Insects may
introduce vesicular stomatitis virus either as mechanical or biological
vectors into susceptible sites of the skin at the teat orifice or within the
narrow band of skin adjacent to hoof of cattle. Mouth lesions may be
produced as a result of generalization and viraemia.
The virus has been isolated from Aedes and Phelbotomus
mosquitoes as they support viral replication and transovariaI
transmission occurs in these flies. Apart from insect vectors other
modes of transmission may occur.
The natural reservoir is still not known but virus probably persists
in one or more unknown hosts. Antibodies against this virus are found
in wide variety of wild animals like feral swine, white tailed deer and
wild turkeys. The virus may be present in pastures and associated with
forage. VSV has been isolated from phlebotomus and culex species of
mosquitoes. The mode of transmission vary with different
circumstances. The virus is transmitted mechanically by milking
machines. The abrasions may be required for contact transmission.
Pathogenecity: VSV produces primarily a disease in horses but is
312
also important in cattle and pigs because the disease resembles FMD.
After a short incubation period of 1-3 days, the animals become febrile
and viraemic. Vesicles on the tongue, dental pad and buccal mucosa
appear with 3 and 5 days on coronary band but teat lesions are rare.
Milking cows may get teat and udder lesions which may lead to the
development of mastitis. The vesicles rupture quickly and erode the
surface epithelium. The ruputure of vesciles is associated with anorexia
and excessive salivation. In older animals, more extensive leSions
develop. The lesions heal if secondary infection is not serious. There is
no mortality but morbidity may be 5-10%. A mild influenza like
syndrome has been reported in humans.
Immune reaction: Neutralizing and CF antibodies are detectable
in 10-14 days PI. The titres rise upto 4 or 5 weeks and then remain at
higher levels fot several months and then decline gradually. Recovered
cattle are immune for one year.
Diagnosis: For differentiation of VSV from other vesicular
diseases and particularly from FMD, animal inoculation as detailed
below is useful.
Table 28.1
ANIMAL INOCUlAnON
Animal
Ro.uJe of
inoculation
Cattle
Horse
Intradermal tongue
Intradermal
tongue
Intramuscular
Intradermal
foot pad
Swine
Guinea
pig
FMD
+
+
Vesicular Vesicular
stomatitis Exanthema
+
+
+
+
Swine
vescular
disease
The virus can be isolated from vesicular fluid or affected tissues in

chicken embryos and cell culture; serologic diagnosis can be made by
CF, VN and agar gel diffusion test
Control: Vaccines from vesicular stomatitis virus are primarily
used in dairy cattle. Live embryo-attenuated vaccines are used in large
scales in certain South American countries and a significant reduction
Rhabdoviridae
313
in field cases has been observed. The same vaccine when used in
swines, vesciular lesions developed and there was shedding of virus.
Rabies virus
Rabies is one of the oldest disease known to mankind. The disease
existed as early as 500 BC. The infectious nature of saliva from
infected dogs was recognised by Zinke in 1804. Pasteur in 1881
demonstrated the neurotropism of the virus. In 1885 Pasteur used
antirabic vaccine successfully. Remlinger in 1903 demonstrated the
fitrability of rabies virus and in the same year Negri demonstrated
acidophilic intracytoplasmic inclusions called Negri bodies -in the
neuron cells of dogs, cat and rabbits experimentally infected with rabies
virus. The disease is prevalent throughout the World except for rabies
free areas like Britain, Newzealand, Australia, Hawaii and few other
islands. Rabies is an acute, bite transmitted fatal encephalomyelitis of
all mammals. The birds can be experimentally infected. The most
comtnon reservoir hosts are fox, dogs, wolf, jackal, skunk bats etc.,
occasional hosts are man, cats, catlle and horse. The bats are the only
species in which the virus is not pathogenic. In India rabies problem in
man and animals is quite serious. It has been generally observed that
3,00,000 people undergo antirabies treatment annually and 15,000 to
20,000 die. The problem among animals is eSlimated to be 100 times
more than in man.
Properties of the virus: Morphology is typical of the family. The
virion is bullet shaped measuring 140-180 nm x 75-80 nm in diameter.
In some preparations particles are cone shaped. The virus consists of an
internal helical, structure called ribonucleoprotein which is surrounded
by a, closely associated protein layers called the matrix. The
ribonucleoprotein and matrix form the core of the virus particle. The
core is surrounded by a membranous envelope which is derived from
the host cell. The ribonucleoprotein has 30-35 coils in the form of a
cylinder. The rabies virus particles contain about 2-3% RNA, 15-25%
lipid and a small amount of carbohydrate, the rest being made up of
four major proteins. The proteins are designated as G, N, M and ~
with molecular weight of 80,000, 62,000, 40,000 and 25,000
respectively. There is also a minor protein L of molecular weight
150,000. The protein N is the major protein associated with
nucleocapsid, while the spikes or the surface projections consist of
'.
314
glycoprotein G. The exact positions of ~ and M, proteins are not

known. The protein L is probably associated with ribonucleoprotein.
The N protein is present in large quantities in cytoplasmic inclusions.
The surface projections are important in the attachment of the virus to
susceptible cells. They also carry the virus specific antigen which
elicits the production of neutralising antibody when injected into the
animal. The G protein can fix complement and is responsible for
hamagglutinating activity. Like most other enveloped viruses, rabies
virus is fragile. It is readily inactivated by organic solvents and
detergents and media having pH below 4 or pH above 10. It is
sensitive to DV light and heat. Temperature above 56C destroys the
infectivity within minutes. Rabies virus is inactivated by most
disinfectant but it is partially resistant to phenol.
Although minor antigenic differences between isolates have been
reported, rabies virus was regarded a single serological entity
embracing classical virus strains. Derriengue (Vampire bat rabies)
which causes a paralytic dumb form of rabies in cattle, Oulofato, a nonfatal encephalitis of dogs in West Africa and Arctic rabies, Nigenain
horse virus associated with clinical condition known as 'Staggers' and
viruses isolated from shrews in Czechslovakia and German Federal
Republic are ronsidered to be rabies virus. Shope and his assoicates
have described several new viruses which have a distant serologic
relatedness to rabies virus and to each other. None of these viruses
appears to be of a major epidemiological significance. Recent work
with monoclonal antibodies has shown that aminoacid changes in 'G'
prote,in alter both antigenicity and virulence. Thus antigenic variation
and antigenic drift may be responsible for vaccine failure. Monoclonal
antibodies clearly distinguish between strains with different passage
histories and of different geographic origin and between rabies viruses
circulating in different host populations.
Rabies virus isolates from clinical cases are referred to as 'Street'
viruses. The street viruses by intracerebral inoculation in the mammals
produce a laboratory or fixed virus. The fixed virus has a diminished
capacity of infection by peripheral routes and is consistent in
behaviour, when inoculated intracerebrally, usually killing the animals
in 5-7 days in contrast to ematic reaction produced by street virus
where death takes place between 7-8 days. Origninally dogs and rabbits
were the species used to obtain fixed virus. Fixed viruses are virulent
Rhabdoviridae
315
even after peripheral inoculation. The attenuated viruses of LEP (Iow

egg passage), HEP (high egg passage) and street Albama Dufferin
(SAD) immunize after peripheral inoculation of most species but SAD
and LEP strains kill when given intracerebrally. Nowadays, suckling
mice or embryonated eggs are commonly used. The virus can be grown
in laboratory animals by intracerebral method of inoculation. Strains of
virus isolates from natural cases of disease are difficult to cultivate in
embryonated eggs and tissue culture. Embryonated chicken eggs 5-6
days old are suitable for cultivation of a number of rabies virus strains.
Tissue cultures have been used for the growth of rabies virus but in
most cultures the CPE is poor, a few cells are infected and virus yield is
low. In human diploid cell strains almost all cells are infected and there
is marked CPE. The yield of virus may be increased by incubating the
infected baby hamster kidney cells at 35C for 5-7 days. Cell infected
with rabies virus can also be maintained and passaged in culLures for
extended periods.
Epidemiology: The rabies virus is prevalent in all continents
except Australia, Antractic, Great Britain, the Netherlands, Japan and
Jamaica. One can easily distinguish the areas where the rabies is
maintained by dogs and where it is maintained by wild animals or
vampire bats, like in case of latin America.
The transmission through the broken skin is invariably caused by
bite wounds. The virus can also be introduced by inhalation and
through mucous membranes of eyes and mouth. In India and many
other countries of the world dog has been the major reservoir of rabies.
Dog bite cases are maximum in this country (93%). The importance of
other species like jackals, wolf, fox, mongoose cannot be properly
assessed until the data from the total dog bite cases is segregated.
About 2000 human beings die every year from rabies while the number
of herbivores dying of rabies infection is not fully known. There is
definite evidence of rabies in mongoose. This suggests the possibility
of transmission of rabies from mongoose to other rodents like rats.
There is no evidence of rabies infection in the various species of bats in
India. Vampire bats are the reservoir and usual source of infection for
paralytic bovine rabies in latin America. The disease with such bats kill
about 50,000 to 1,00,000 cattle each year from bat-transmiued rabics.
Vampire and insectivorous bats in South America infect cattle and
cause paralytic form of rabies. Asymptomatic salivary gland infection
316
TeXlbook o/Veterinary Virology
occurs in bats and results in a prolonged viraemia. Rabies is frequently

enzootic in wild animals and bats. In India about 3-4 lakh people take
rabies prophylexis and 2000 people die annually.
The virus is not always present in the saliva of rabid dogs, but in
other cases it may be found in saliva 3-7 days before signs appear in
dog. In blOOd, urine, milk and other secretions, its prescence is rare.
Pathogenesis: The rabies virus primarily affects dogs, cats and
other carnivores but all warm blooded mammals including man are
susceptible. The fowl can be experimentally infected. The transmission
is usually by bite or an accidental break in the skin, though it can be
introduced by inhalation and through the mucous membranes of eyes
and mouth. The infection does not take place by feeding the infected
carcase. The primary replication occ~s in the muscle fibre at the site of
inoculation, the virus then aggregates around the proprio receptor nerve
endings of their acetylcholine receptors, and migrates along the
exoplasm, the centripetal migration takes place. When the virus enters
centrol nervous system, usually the spinal cord, its migration to brain is
rapid. Although the ultimate target of the virus is eNS, it also infects
salivary glands by centrifugal movement along the exoplasmic route.
And thus the virus is secreted in saliva. The incubation period which
may vary from 10 days to 12 months depends upon the distance of bite_
from the eNS. Early clinical signs in the prodromal stage include fev,*,
slight changes in temperament and dilatation of pupils. The disease is
manifested in one of the two main forms.
a Dumb or paralytic: Initially there is change in voice which is
howling or bellowing. There is short period of excitement followed by
incoordination, paralysis, dropped lower jaw, dehydration, loss of
condition and death. In cattle there is rumina 1 tympany, tenesmus and
sometimes diarr~oea. Tremors and progressive paralysis follow leading
to coma and death. The rabies is not always fatal in bats.
b. Furious: Animals attack without provocation. Incoordination,
convulsions, coma and death follow within 3-10 days of showing signs.
The rabies is not always fatal in bats, there is inapparent infection
and they are consistently viraemic. Skunks and foxes may recover from
infection. Dogs rarely show mild symptoms and J;ccover but remain as
excretors of virus.
The average incubation period observed in cattle bitten by vampire
bats was 60-70 days, while after skunk or fox bites it was 35-45 days.
Rhabdoviridae
317
This may be due to amount of virus introduced and site of the bite.
Skunks and foxes bite mostly on the head or nose while vampire bats
bite mainly neck, ears, fetlocks and vulva. Cattle also excrete virus in
saliva when ill, like the dogs. The virus in caule is mainly distributed in
salivary glands and brain. The virus has never been isolated from meat.
Virus in the milk or rabid cows is absent or is present at low levels,
since there are few direct glandular nerve connections to the udder and
virus in milk is highly diluted.
The clinical signs of rabies in cattle include a short premonitory
period which lasts from few hours to few days, in which there is mild
temperature, malasie, anorexia and cessation of milk. This is followed
by salivation (92%), aggressiveness (47%), bellowing (69%),
paralysis(30%) and straining (12%). Most human cases occur during
early period when the veterinarian or the owner suspects choke and
searches for some object in the throat of affected cattle. In vampire bat
rabies the predominant signs in cattle is paralysis.
On histopathological examination the brain show lesions of
encephalitis with neuronal degeneration in the midbrain and medulla.
Negri bodies in the neurons of hippocampus and cerabellum are
pathogenomonic.
Diagllosis: Early clinical diagnosis of rabies is very critical since
there is probability of human exposure. The clinkal signs are helpful
but are not confirmatory. If the animal dies within 2-3 days after
showing symptoms or if the animal dies in the quarantine or the animal
has the symptoms, tissues like brain preserved in glycerine saline
should be sent to laboratory for diagnosis. The laboratory diagnosis
includes the following:
1. Search for Negri bodies in sections or impression smears of the .
cerebellum stained by selle~'s or Giemsa stain.
2. If Negri bodies are not found, weaned mice are inoculated
intracerebrally. The mice show symptoms within 6-15 days but the
symptoms may be delayed upto 21 days and the mice usually die within
1-2 days after that. The mouse brains are examined for presence of
Negri bodies from dead as well as from those which survive 5-6 days of
inoculation.
3. The fluorescent antibody test is the preferred 'method of
diagnosis and is about 100% correct. Smears from hippocampus
medulla and cerebrellum are used.
318
Serum neutralization test can be used to detennine the antibody

response of vaccinated individuals and antibody titre of antiserum used
for post-exposure treatment The serum neutralization test is performed
in mice or in susceptible cell culture.
Control: The immunization in dogs and cats is generally
prophylactic whereas in man and cattle the vaccination is usually
carried out after exposure. Prophylactic vaccination is recommended
for veterinarians and other persons with a high risk. Hyperimmune
serum or immune gammaglobulin obtained from horses or sheep is
administered within 48 hours especially in cases of severe bites. This is
followed by vaccination after 24 hours. Various types of vaccines have
been used since Pasteur vaccine. Previously inactivated vaccines
derived from brain were in use, which are still being used in India and
some other developing countries. Heat, phenol, UV irradiation and P
propiolactone have also been used as inactivating agents. Since a series
of injections of vaccine are given in animals and man, the brain tissue
somtimes results in allergic encephalitis. These vaccines in man are
now replaced with tissue culture inactivated vaccinc. The commonly
used vaccine is prepared in human diploid cells. Efforts arc bcing made
to prcpare a sub unit vaccine for human use to over come harmful side
effects.
The attenuated vaccines used for animals are low egg passaged
(40-50 times) and high egg pass aged (180 times) Flury strain. The low
egg passaged (LEP) vaccine is less attcnuated and is given to adult
dogs only. The high egg passaged (REP) is highly attenuated and is
given to puppies, cats and cattle. The inactivatcd and attenuated
vaccines have been prepared in cell cultures from hamster kidney etc.
The attenutated tissue culture vaccines provide immunity for 3 years.
In India the dog is considered to be the chief animal reservoir for
the disease. It is estimated that there are about 10-30 million of dogs in
the country. Most of these are stray dogs. All domesticated dogs must
be licensed and immunized. In Latin America efforts have been made
to reduce the Vampire bat population by inoculating cattle with an
anticoagulant (Warfarin) in dosage harmless to cattle but lethal to blood
sucking bats. In Switzerland and Bavaria chicken heads inoculated with
live attenuated virus have been dropped in mountainous areas in an
attempt to vaccinate the reservoir host-foxes.
Rhabdoviridae
319
Bovine Ephemeral Fever Virus (BEFV)

Ephemeral fever is an acute insect transmitted disease of cattle.
Sheep and other domestic animals do not suffer. The disease was fIrst
reported from central Africa in 1867. The disease is now enzootic in
parts of Africa, most of Asia, including India, Japan and parts of
Australia.
Properties of the virus: In electronmicroscopic preparation of
BEFV three different shapes are found. The longest virions are bullet
shaped with a length of about 183 nm and coiled helical nucleocapsid.
Particles are also found which have. a truncated bullet shape, while
others appear as blunt cones. The length of short particles varies from
70-140 nm and these are considered to be defective and cause
interference with the growth of BEFV in tissue cultures. The genome is
a single strJndcd RNA and not double stranded as reported by earlier
workers. The virus is inactivated within 10 min at 56C. Citrated blood
from affcctcd cattle remains infective for 8 days at 4C. The virus can
be adapted to grow in the brain of unweened mice or hamster which
leads to rapid loss of pathogenicity for calves. BEFV can also be
adepted to grow in BHK 21 cells, monkey kidney cell line and bovine
embryonic kidney.
Epidemiology: It appears that disease maintains a state of
endemicity with sporadic individual cases where ideal ecologic
conditions prevail. Periodically, after development of susceptible
populations, the infection overflows during arthropod season. The usual
source of infection is believed to be arthropod vectors. The virus has
been isolated from mosquitoes and midges. The role of wild life as
perpetuating or amplifying hosts needs to be explored.
Pathogenicity: The incubation period of BEF is 2-10 days. The
affected animals show clinical symptoms characterized by a sudden
onset of high fever, muscular shivering, lameness, stiffness of joints
and enlargement of peripheral lymphnodes. The fever lasts 2-3 days
during which time the animals remain pnostrate. There is hyperpnea
and dyspnea, excessive salivation, nasal and ocular discharge, anorexia
and sharp fall in milk yield. In most of the cases recovery takes place in
3-5 dyas. The lymph nodes are usually enlarged and oedematous. There
is congestion of mucous membranes in the nasal cavities, abomasum
and small intestine. Degenerative changes in the synovial membrane
and increase in synovial fluid are seen.
320
Diagnosis: The sudden onset of disease lasting 2-3 days with

recovery is clinically diagnostic. Virus isolation is accomplished by
inoculation of buffy coat of caule undergoing ~cute febrile disease in
the suckling mouse brain. Serologic diagnosis can also be made by
complement flxation and virus neutralisation tests.
Control: Both mouse attenuated or inactivated vaccines mixed
with adjuvant are available in certain countries. The inactivating agent
is either P propiolactone or formalin. Both the vaccines evoke a
protective response.
Marburg Virus (Varet monkey disease)
The virus produces a febrile infectious illness of man handling
infected monkey tissues or cell cultures. African green monkey may be
the natural host and rarely exhibit clinical symptoms.
References
BURGESS, G.W., 1971. Bovine ephemeral fever: A review. Veterinary Bulletin
41,887-95.
BUXTON, A. and FRASER, G., 1977. Microbiology Voi.2. Oxford Blackwell
Scientific Publications.
COMBS,
G.P., 1978. Bovine ephemeral fever. Proc. U.S. Animal Health.

Assoc.82, 29-35.
Consultation on the WHO!FAO programme in comparative Virology. Report of

the rhabdovirus study group Rome. Sept. 25-27, 1979.
HANSON,
R.P., 1952. The natural history and vesicular stomaJitis.

Bacterial.Review 16, 179.
HENDERSON, W., 1960. Foot and mouth disease and related vesicular disease.
Veterinarian, 26 726-730.
KAHRS, R.F., 1985. Viral diseases of cattle. Iowe State University Press, Iowa
KURVERT, E.; MERIEUX, C.; KOPROWSKI, H. and BooET, K., 1985. Rabies in the
Tropics, Springer-Verlag. Berlin.
MOHANTY, S.B. and DurrA, S.K., 1981. Veterinary Virology. Lee and Febiger,
Philadelphia.
RusSELl., P.H. and EDINGI'ON, N., 1985, Veterinary Viruses. The Burlington
Press(Cambridge) Ltd. Foxton, Cambridge.
Rhabdoviridae
321
'"
P.B., 1977. Vaccines against bovine ephermeral fever.

Aust.Veterinary Journal. 53,351-52.
SPRADBROW.
SCHNEIDfR, L.G., 1983. Rabies Bulletin Europe/Information Surveillance

Research, Federal Research Institute for Animal Virus Diseases,
Tubingen, Germany.
WHO expert Committee on rabies. Sixth Report WHO Technical Report.
Service, No. 523, 1973.
Wno document: Guidelinesfor rabies control VPH? 83-43. 1984.
Chapter 29
Retroviridae
The family retroviridae derives its name from the enzyme reverse
transcriptase (RNA-dependent DNA-polymerase) which transcribes a
DNA copy from viral RNA genome. The Latin word retro means
backwards. The family consists of three subfamilies: oncovirinae,
lentivirinae and spumavirinae. Oncovirinae subfamily contains
tumorigenic viruses, the oncoviruses. The lentivirinae cause chronic
diseases in sheep, goats, horses and human acquired immunodeficiency
virus of man. The spumaviruses are not pathogenic.
The subfamily oncovirinae includes three genera namely type C,
type B and type D oncovirus groups. Many members of these genera
cause neoplastic disease of animals and birds. Type C oncovirus group
includes number of import.'mt species such as bovine type C oncovirus,
feline leukemia and sarcoma viruses, murine leukemia and sarcoma
viruses, avian leukosis and sarcoma viruses and avian
reticuloendotheliosis viruses. Type B oncovirus group includes the
species such as murine (mouse/guinea pig) mammary tumour virus.
Type D oncovirus group do not have species important for domestic
animals and birds.
The retrovirions are spherical enveloped particles measuring 80100 nm in diameter and possessing glycoprotein surface projections of
approximately. 8 nm. The particles have a three layered structure. The
innermost is the genome-nucleoprotein complex, which includes 30
molecules of reverse transcriptase. This is enclosed within an
icosehedral capsid which in turn is surrounded by host cell membrane
derived envelope. The genome of retroviruses is an inverted dimer of
Retroviridae
323
linear single stranded RNA with a positive polarity. The monomers are
held together at 5' ends by hydrogen bonds probably by base pairing. A
cellular tRNA serves as a primer for reverse transcription and is bound
to specific primer attachement site about 100 residues from 5' end of
the genome. The genetic information is contained in the genes gag
(coding for the nonglycosylated internal proteins), pol (coding for
-reverse transcriptase) and env (coding for envelope glycoproteins).
Some retroviruses incorporate genetic information for nonstructural
proteins from the host cell which are important for pathogenesis in
oncogenicity. The RNA of ral?idly transforming oncoviruses contain a
fourth gene, the viral oncogene (V-onc). The presence of V-onc gene is
usually assoicated with deletion of some of env base sequences and this
renders the virus as replication defective and depend upon transforming
helper virus for the production of infectious progeny. After entry of the
virus into the cell and uncoating, replication starts with the reverse
transcription of virion RNA into DNA, which is made double stranded.
This double stranded DNA is integrated into chromosomal DNA of the
host cell at nonspccified sites. The virus can replicate when DNA is
integrated. Cellular RNA polymerase II transcribes the integrated
provirus into virion RNA, mRNA. Virus is released from the cells by
budding.
ONCOVIRUSES
Bovine Leukaemia Virus (BL V)
Bovine leukemia virus (BLV) is the causative agent of bovine
leukemia (lymphosarcoma, leukemia) a lymphoproliferative disease of
catLIe.
Properties of the virus: BLV is classified as a type-C oncovirus in
the retroviridae family and shares characteristics with leukaemia
viruses of other species. The mature virus particle is 90-120 nm
diameter in size. The virions possess a major nucleoprotein antigen
with molecular weight of 24,000 daltons and an envelope glycoprotein
antigen of 51,000 daltons molecular weight. The virus is fragile and
looses its infectivity by routine manipulations. It is readily destroyed at
56C within 30 minutes. The virus is inactivated at pasteurization
temperatures. Binary ethylenemine and N-acetylethylcnemine are
effective inactivating agents.
The BLV isolates from various parts of the world do not show
324
antigenic differences. There is no antigenic relationship between BLV

and leukemia virus of other domestic animal species. There is some
evidence that BLV shares structural and functional properties and even
minor antigenic determinants with human-T cellleukemia virus.
The. virus replicates in some primary cell cultures of bovine or
ovine origin and causes syncytia formation in various transformed and
non-transformed cell cultures.
Epidemiology: BLV infection among cattle are world wide in
distribution but certain countries like the Netherlands, Austria' and
British Isles have a low incidence. The cattle are considered to be
primary and important host of BLV. Although sheep have been found
to be infected and deaths due to lymphosarcoma have taken place, but
these instances are rare and are regarded as due to accidental
introduction of BLV rather than considered that sheep are significant
reservoir of the virus. There is report that water buffalo and a large
rodent. capybara in South America has antibodies against BLV but the
percentage of positive sera prevalence is well below that of cattle. The
information supports the hypothesis that BLV is propagated and
maintained primarily in caule population and infections in other species
are accidental introductions. The frequency with which the
transmission occur may be related to the vectors capable of
transmission to callle as well as to other species. Ticks and biting flies
have been incriminated but other biting insects may also be involved.
The available evidence indicates that arthropods are only transport
vectors and not true biological vectors.
There is no evidence of human infection with BLV. However,
there is an evidence of distinct antigenic relationship with T-cell
leukemia viruses. This results into appearance of antibodies which react
with BLV in few human subjects known to be infected with human Tcellleukemia virus.
The economic losses arise from deaths and restrictions on the
movement of BLV infected animals or their products (semen, embryo)
in international trade. The virus is transmitted horizontally and does not
gain enterance to the germ line and there is no vertical transmission but
the transmission in utero takes place. The in utero infection perpatuates
the virus form one generation to the next. Calves which are not infected
in utero but born to infected cows are occasionally infected b)'
ingesting of colostrum or milk that contains infected lymphocytes.
Relroviridae
325
Contact virus transmission may occur among young animals. The

calves remain protected from contact infection until the colostral
antibody wanes, usually 4-6 months of age.
The factors which influence epidemiological pattern have not been
identifIed but the prevalence of infection is more among dairy animals
than beef cattle. The genetic factors may be responsible for this. The
development of tumors in infected animals is influenced by virologic,
genetic and immunologic factors but these are not understood as yet.
The genetic factors influence the development of tumors because
numerous tumor cases have been recorded within certain cow families
and sire groups.
Palhogenesis: The susceptible host gets probably BLV infection
through infected lymphocytes. Experimental inoculation with infected
lymphocytes by parenteral route, particularly intracutaneous
inoculation is most successful route of establishing infection. The oral,
intranasal and intauterine instillation may establish infection in
susceptible host not in all the animals, when large quantities of
infectious material is inoculated. It indicates that blood sucking
parasites may play a role in the transmission of virus. Once the infected
lymphocytes enter the host, a cycle of virus replication starts in the
infected lympnocytes. The virus produce infection in susceptible cells,
probabl,y of lymphoid or stem cell origin and further replication goes
on. The plasma of infected animals may contain virus on occassion but
not regularly. The B,LV infection is established in lymphoid tissues in
the first few weeks of infection and virus can be isolated from
lymphocytes 3-5 weeks of inoculation. No cell free virus is produced in
the tissues. Blood is the potent source of infectivity. Saliva, urine,
semen, milk may contain infectivity due to their lymphocyte content
The molecular mechanism involved in cell transformation and
tumor development is not known. BLV is integrated at various sites in
the cell genome, one of the BLV gene products is capable of activating
cellular genes and brings about malignant transformation.
The BLV infection is associated with lymphocytosis. About 2985% animals develop lymphocytosis. The development of
lymphocytosis and tumor formation are determined by separate genetic
factors. The development of lymphosarcoma is a rare result of BLV
infection. There is no tissue predilection for tumor development and the
clinical signs of disease may be extremely varied. There may be
326
Textbook ofVelerinary Virology
clinical signs related to organ involvement such as cardiac distress,

digestive disturbances or neurological signs. In some cases there may
be vague clinical signs like decreased production, inappetance, weight
loss, weakness and debility.
. Immune Reaction: TIle humoral antibodies appear within 3-9
weeks of infection. The neutralizing antibodies do not lead to
elimination of BLV because of the mechanism of virus persistence. The
constant presence of viral genome is accompanied by continuous
presence of antigen, with the result that antibody titres usually remain
high throughout the life span of the infected animal. There is no
evidence of CMI response to BLV infection.
Diagnosis: The diagnosis is aimed at detection of BLV antibodies
and diagnosis of lymphosarcoma.
For BLV infection serological tests are carried oul. A complete
herd of all cattle over 6 months are tested. The RIA, ELISA and VN
test have the greatest sensitivity but AGID test is widely used because
of its practicality.
The diagnosis of clinical cases (lymphosarcoma) requires a
pathological investigation. The tissues may be from necropsy or biopsy.
The effect of BLV infection on the haematologic values in cattle is
important from diagnostic point of view. Many animals develop
persistent lymphocytosis. The examination of blood smears reveal the
presence of abnormallymphocytes.
Prevention and Control: No vaccines are available. The
management practices such as sanitation and good nursing care are
valuable for short period of time. Common use of needles and surgical
equipment should be avoided. Insect control, prevention of the
introduction of cattle from positive reactor herds and slaughter of
infected heards are important.
Feline Leukemia/Sarcoma virus
The
feline
leukemia/sarcoma virus
causes
Ieukemia/
lymphosarcoma in cat and is manifested by malignant, proliferation and
degenerative changes of haematopoietic tissues.
Properties of the virus: The virus particle resembles C type
particle. They are ciruclar or oval in shape with 100-110 nm external
diameter. Two kinds 9f viral structural antigens such as glycoprotein
envelope antigen which has a MW of 70,000 and a major internal
Relroviridae
327
.protein antigen with a MW of 30,000 are present. Three subgroups (A,

B, C) of feline leukemia virus were recognised. All subgroups of feline
leukemia and sarcoma virus have an identical major internal antigen. A
cell surface antigen called feline oncovirus associated ceU membrane
antigen has been detected in lymphoid cells of cats with
lymphosarcoma. The feline leukemia/sarcoma virus can be propagated
in normal embryonic feline cell cultures inoculated with extracts' of
tumour tissues. The virus can also reproduce in cultured cells derived
from canine, procine and human sources but fails to replicate~
chicken and bovine cell cultures. Feline leukemia virus can activate
defective mouse sarcoma viruses which can infect kitten cells an'd the
'rescued hybrid' experimentally can produce sarcoma in kittens.
Epidemiology: Most infected cats excrete the virus in saliva, urine,
faeces, milk and nasal secretions. The virus can be transmitted
vertically from infected mother to off spring in utero and also through
the milk from infected dams to the kittens. There is a report that the
virus is present in the salivary glands and the virus can be transmitted
from cat to cat by biting and scratching.
Pathogenesis: Young cats under 4 months of age are
comparatively more susceptible and the susceptibility decreases
following increase of age. The incubation period of the disease is
variable. On experimental infection, virus can be demonstrnted in
tissues within 3 to 4 weeks and lymphosarcoma appaears at about 6 to
12 months of age. Early symptoms of anaemia, leukopenia,
. thrombocytopenia develop few weeks or months prior to the
development of acute leukemia. Lymphosarcomas commonly originate
from lymphnodes, kidney or intestine and from these sites the disease
may spread to other organs and to the bone marrow. The thymic form
of feline lymphosarcoma is most common and is characterised by the
presence of tumor mass in the anterior mediastinum.
Experimentally feline lymphosarcoma can be produced in kittens
under 12 hours old by the inoculation of cell-free extracts of
spontaneous and induced tumor tissues.
Diagnosis: Provisional diagnosis can be made from the gross
lesions, clinical and haematological findings. Confirmatory diagi}osis is
achieved by isolation of virus from the plasma of infected animals or
tumour tissues, electron microscopical examination of affected tissues
and serological tests. Virus specific antigen can be detected in
328
ciruclating leukocytes of affected animal by immunoflorescence.

Immunodifussion .test is suitable for detecting viral antigen in the
tumour mass. Indirect haemagglutination and virus neutralization test
can also be used for detection of virus specific antibody.
Control: A live virus vaccine derived from the virulent virus types
has been' shown protective against the disease. Immunization of
pregnant mothers with inactivated virus provides 3-4 months protective
passive immunity to the new born. Kittens should be vaccinated at
abOut 3-4 months of age.
Feline Leukemia is an excellent model for the study of viral
oncogenesis in man.
Murine Leukemia/Sarcoma Viruses
The murine leukemia (MuLV) and sarcoma (MSV) viruses are
assoicated with the production of a vareity of morphologic tumours,
leukemias and sarcomas in a number of host species.
Properties of the virus: First MuLV was discovered by Gross in
1951. Subsequently founeen different leukaemogenic RNA murine
viruses have been isolated. It is probable that the majority of murine
leukemia viruses do not bear any etiological relationship to the tumour
in different host species. All MSV exist as a mixture with MuL V and
bear the antigenicity of assoicated MuLV. An envelope glycoprotein
antigen with a MW at about 70,000 has been recognised and this
antigen is common to mouse leukemia/sarcoma viruses, but does not
cross react with the glycoprotein antigens of other mammalian or avian .
oncoviruses. The MSV and Rous sarcoma virus of chicken and the
avian leukosis virus and MuL V are similar in their nueleo protein
antigen. The MuLV and MSV possess a major internal antigen with a
MW of 30,000. MuLV can mUltiply in cell cultures of mouse and rat
without producing CPE. MSV are defective but can cause
transformation of cell cultures derived from mouse, rat, hamster or
human embryonic tissues.
Palhogenesis: MuL V and MSV are widespread in nature. Viruses
can be transmitted vertically as well as horizontally through milk and
sperm of infected animals. Most often these viruses remain in latent
form and the activation of viruses leading to formation of leukemia/
sarcoma occurs by an unknown mechanism. Different strains of mice
show considerable variation in susceptibility to one virus.
Relroviridae
329
Experimentally tumor formation takes place following inoculation of

the virus to the newborn mice of high susceptible strain. Most MuLV
also produce leukemia in rats. The gross's MuLV may give rise to
almost all known types of leukemia including lymphatic stem cell,
myeloid and monocytic leukemia, erythroblastosis, lymphosarcoma and
reticulum cell sarcoma.
Diagnosis: Diagnosis is based on the gross lesions, demonstration
of virus specific antigen by radioimmunoassay, complement fixation
(COMUL), immunofluoreScence and immunodiffusion test
Control: Vaccines have been prepared and are in the experimental
trials.
Avian Leukosis/Sarcoma viruses
Avian Ieukemia/sarcoma viruses cause a variety of transmissible
benign and malignant ncoplasma in chickens and to a lesser extent in
other avian species. The neoplastic conditions are lymphoid leukosis,
erythroblastosis, myeloblastosis, myelocytomastosis, endothelioma,
nephroblastoma, hepatocarcinoma, fibrosarcoma and oteopetrosis. Of
these ncoplastic conditions, lymphoid leukosis is most common. Avian
leukosis virus predominately affects the haematopoietic cells and the
sarcoma virus affects the fibroblastic cells.
Properties of the virus: Avian leukosis/sarcoma viruses are
grouped together because they possess may properties in common.
Viruses of the avian leukosis/sarcoma group cannot be distinguished on
the basis of their ultrastructural characteristi.s such as size, shape etc.
The virus has a MW of 10 x 1()6 daltons and 60 to 70s sedimentation
coefficient. Avian lcukosis/sarcoma viruses are divided into subgroups
(A to G) on the basis of host range in genetically different chick
embryo fibroblast cultures, interference ~ith members of the same
subgroup and viral envelope glycoprotein antig<!ns. Genetic host range
or cellular genetic resistance to infection by viruses of a particular
subgroup is controlled by an autosomal locus called the avian tumour
virus locus and cells resistant to any viral subgroup are designated as Cl
subgroup and cells susceptible to all subgroups are desiggnated as C/o.
Interference between leukosis and sarcoma viruses has been well
characterised. Chicken embryo fibroblast cell cultures infected with
avian Ieukosis virus become resistent to superinfection by sarcoma
virus of the same subgroup and this is the basis of the resistance
inducing factor (RIF) test.
330
The avian leukosis/sarcoma viruses can be inactivated by heating

at 56C for 30 minutes. Inactivation can also be achieved by
formaldehyde and by common disinfectants such as 0.5% phenol.
Viability of virus preparations can be maintained for long periods at 70C or in 50% buffered glycerol saline.
The, oncogenic leukosis viruses are exogenous in nature but the
endogenous leukosis viruses are non oncogenic. All chicken cells can
carry an endogenous viral genome required to produce a complete
leukosis virus. Most of the avian Leukosis viruses on the other hand
infect genetically susceptible chicken cell cultures and produce virus
continuously. 'The avian leukosis/sarcoma viruses can be grown in
embryonated chicken eggs of susceptible lines. Avian sarcoma viruses
produce pocks on the CAM of embryonated eggs and are capable of
producing tumors in embryos when inoculated by the intravenous and
yolk sac routes of inoculation. Avian leukosis virus when inoculated by
intravenous route to chicken embryos cause death of chicks 1 to 2
weeks after hatching.
Epidemiology: The natural host for avian leukosis/sarcoma viruses
is the chicken. Experimentally, wide range of hosts may be infected.
The viruses can be transmitted vertically form parent to off spring or
horizontally form bird to bird. The transmission of the viruses through
egg is of utmost importance in the perpetuation of virus infection.
Chicks that are developed from congenitally infected embryos are
immunologically tolerant and have large quantities of infectious virus
in their blood and tissues. Large amouts of virus are also shed in their
droppings and saliva.
Palhogenesis: The incubation period of the disease in natural
conditions is not clearly known. Natural disease usually occur after 14
weeks of age and the incidence is highest at about sexual maturity.
Experimentally when day old chicks were inoculated intra abdominally
with leukosis virus, the lymphoid leukosis develops within 5 to 8
months and those that receive sarcoma virus intramuscularly or
intradermally the sarcoma develops with in 3 to 35 days. The clineal
signs of lymphoid leukosis are not specific. There may be paleness of
the comb and cyanosis, inappetance, emaciation and weakness occur.
Abdomen is often enlarged due to the presence of tumours.
Enlargement of liver, bursa of fabricious and kidney can often be
detected on palpation. It is suggested that the lymphoid leukosis is a
Retroviridae
331
malignancy of the bursa dependent lymphoid system. There is also

evidence that the surgical or hormonal removal of the bursa from
chickens reduces the incidence of malignancy from leukosis virus.
Similarly hormonal atrophy of bursa resulted in similar effect. Grossly,
however, visible tumours in natural conditions can be seen in liver,
spleen and bursa of fabricious. Many other organs such as kidney,
lungs, gonads, heart, bone marrow and mesentary may also be affected.
The tumors are soft, smooth and glistening and the cut surface had
greyish or creamy white appearance. Granular or miliary form of
leukosis is usually seen in liver and distributed throughout the
parenchyma. Microscopically tumours consist of aggregates of large
immature lymphoid cells. In erythroblastosis the prominant clinical
signs are weekness, cynosis of the comb, anaemia and the presence of
erythroblasts in the blood. There may be cherry red discolouration ..md
diffuse enlargement of the liver and spleen due to accumulation of
erythroblasts in the blood sinusoids and capillaries. In myeloblastotis
signs are similar to that of erythroblastosis and massive accumulation
of myeloblast can be seen both intravascularly and extravascularly. In
osteopetrosis thickening of long bones of the limbs is a common
feature.
Diagnosis: Provisional diagnosis can be made from the history,
clinical signs and the histopathological lesions. Confirmatory dagnosis
tests is achieved through isolation of the virus and perological tests.
Virus can be isolated from plasma, serum or tumour tissues of infected
birds by inoculating into cell cultures. Virus specific antigen can be
detected by radio immunossay, complement fixation test (COFAL),
immunoflourescence and the EpSA. Presence of virus in infected cell
cultures can be detected by reverse tianscriptaseassay. Virus specific
antibodies can be detected in serum and in egg yolk of infected chicken
by VN test using sarcoma virus pseudotypes. Differential diagnosis
should be made from Marek's disease, a herpes virus disease which
usually occurs in chickens at lower age. Le. less than 14 weeks old.
Prevention and control: No vaccines are available. Control is only
by flock managment and genetic selection of chickens for resistance to
avian leukosis/Sarcoma viruses.
Avian Reticuloendotheliosis Viruses (REV)
This group of virus causes tumours of the reticuloendothelial
332
system. This group of virus comprise of turkey REV, chicken syncytial

virus, duck infectious anaemia virus and spleen necrosis virus. All
these viruses are grouped together and termed as strains of REV.
Properties of the virus: The virus belongs to the subfamily
oncovirinae and genus oncovirus type C of the Retroviridae family. The
mature virus particles measure 85 to 110 nm in diameter, possess a
diffuse, relatively dense nucleoid and a limiting membrane and can be
distinguished from leukosis viruses. The virus particles consist of 2
distinct nucleoid component and the major internal antigen had a M.W.
of 30,000 daltons and the M.W. of envelope glycoprotein antigen is
73,000 daltons. The v'iruses are thermolabile, viral activity is destroyed
at 56C in 30 minutes and after exposure to 25 to 50% ether. Cell free
virus retained activity at -70C for 4 months and at 4C for 12 hrs.
Virus replicates in cultures of chicken, quail and duck embryo
fibroblasts and can persist for long periods without marked changes in
the infected cell. Chicken emrbyos are also infected when inoculated by
yolk sac and CAM routes.
Pathogenesis: Natural infection is only reported in turkey flocks.
Experimentally, chicken, turkey, Japanese quail and ducks are infected.
Turkey poults are most susceptible. The viruses may induce a variety of
syndrome such as spleen necrosis, anaemia, lymph nerve lesions etc.
The affected birds manifest enlarged livers and spleens and infiltration
of subcapsular nodules. There may be splenomegaly, liver may be
yellowish brown or pale. Distended gall bladder is characteristic.
Nodular lesions may be seen in other visceral organs including gonads,
heart, kidneys and pancreas. Enlargement of peripheral nerves
particularly the cervical portion of the vagus nerve can be seen.
Histologically most characteristics findings are rapid proliferation of
mononuclear cells of the reticulo endothelial system around blood
vessals and in lymphoid follicles. Mitotic figures are common.
Proliferation is marked in liver and spleen. Sometimes there may be
diffuse infiltration of lymphocytes and plasma cells in peripheral
nerves.
Diagnosis: Provisional diagnosis can be made from gross and
microscopic changes. Confirmatory diagnosis can be obtained by
isolation-of the virus from cell free tumour homogenates, spleen, whole
blood or plasma in susceptible cells culture or in chicken embryos by
yolk sac route. Virus specific antigen can be detected by IF and VN test
Retroviridae
333
in infected cell cuture. Experimentally disease can be reproduced by

inoculations of day old chicks, turkey poults and Japanese quail.
Differential diagnosis should be made from lymphoid leukosis and
Marek's disease.
Murine Mammary Tumour Virus (MMTV)
Murine mammary tum~)Ur virus (MMfV) produces mammary
carcinoma in certain strains of mice and in guinea pigs.
'Bittner(1936) observed that newborn mice of high incidence
strains showed low incidence when nursed on low incidence foster
mothers and low incidence strains showed a high incidence when
fostered on high incidence mice. He regarded this phenomenon as a
milk borne factor in this disease, but susbsequently causative virus has
been discovered.
The MMfV is transmitted horizontally to new borns, Some strains
of MMTV are also transmitted vertically also. The MMTV strains
showed variability in inducing tumors. Hormonal factors or host
genetic factors play important role in the production of the disease.
The MMfV particles in milk or extracts of infected mouse tissue
can be inactivated by trypsin and by heating at 56C for 30 minutes.
The virus cannot be propagated by conventional tissue culture methods.
Several strains of MMTV have been recognised and are antigenically
related.
The MMTV produce carcinoma of the mammary gland after a
latent period of 6 to 12 months, high concentration of the virus is found
in the milk and is transmitted to the newborns through the milk: Virus
is also present in the sperm of male mice.
No antibody against the virus has bren found in infected mice
serum but sera from guincapigs, rabbits and rats immunized with
MMfV when mixed with the virus and injected into susceptible mice
results in the failure of the production of carcinoma.
SPUMA VIRUSES
Bovine Syncytial Virus (BSV)
Bovine syncytial virus (BSV) is associated with lymphosarcoma of
cattle and was first isolated in 1969 in the United States. The virus has
been isolated in different countries not only from the diseased cattle but
also from apparently normal cattle. It is believed that approximately
334
25% of nonna! caule remain as carriers of BSV.

Properties of the virus: The virus belongs to the subfamily
spumavirinae under the family Retroviridae. Electromicroscopy
revealed that the double mambrane bound virions had approximately
125 nm in diameter. The virus is extremely cell assoicated. It is
sensitive to chlorofonn, heat and freezing. The viral replication can be
inhibited by actinomycin D and 5-bromouridine. The virus can be
propagated with production of syncytia in a number of cell systems
such as bovine embryonic spleen cells, rabbit cornea cells, cell lines of
vero and BHK-21 cells. Bovine embryonic cells appear to be most
sensitive.
Pathogellesis: The virus can be recovered from the buffy coat cells
of diseased and apparently normal cattle. Therefore blood transfusion
or use of contaminated needles etc. may be possible source of infection.
The virus has been recovered from milk of infected cattle. The role of
the virus in disease production is not clearly understood. However virus
has been recovered from the spleen, lungs, lymph nodes and uterus and
foetuses of infected animals. Virus has also been isolated from tissues
of cattle suffering from lymphosarcoma.
Diagnosis: Virus can be isolated from the buffy coat cells and
from other infected tissues by inoculating into the sensitive cell
systems. Virus specific antibodies can be detected by immunodiffusion
and 1FT.
Control: Young calves acquired colostral immunity for a period of
3 to 4 months. No vaccines are available.
LENTIVIRUSES
Equine Infectious Anaemia Virus (EIA V)

Equine infectious anaemia virus (EIA V) is responsible for the
production of a progressive disease characterised by severe anaemia in
horses. The disease casued by EIA V is also known as Swamp fever.
The disease was first recognised in France in 1843, and its viral
etiology was detected in 1904 by Vallee and Carre. Disease has been
reported from the different parts of the world. In India the disease was
first reported by Uppal, Yadav and Ahmed in 1987 from the National
Research Centre on Equines, Hisar, India. Horses are the only animals,
affected. However, the outbreaks have been recorded in donkeys and
mules. Human beings are also susceptible.
Retroviridae
335
Properties of the virus: The virus belongs to the family

Retroviridae and subfamily Lentivirinae. Nucleic acid of the virus is
RNA and the virions possess RNA dependent DNA polymerase.
Virions possess surface knobs and a tubularly shaped core. The virion
has a MW of 4.8 x IOS dalton and measures 90 to 140 nm in diameter
and the sedimentation coefficient is 110 to 120s. The virusis relatively
.resistant to heating, freezing and drying. It is inactivated by heating at
58C for 30 minutes and by a variety of chemical disinfectants such as
2% phenol, 0.022% mercury chloride, 4% formalin, sodium hydroxide
and sodium hypochloride. The virus particles have a major group
specific antigen and also bear surface antigens which may vary. The
virus has a number of strains or variants that are immunologically
distinct by their surface glycoprotein antigens. The virus can be
propagated in equine cell cultures and in horse leukocyte cultl!res.
Epidemiology: The disease has a seasonal incidence and is not
prevalent during the late summer and autumn. The commonest vectors
are the blood sucking insects. Mosquitoes such as Aedes, Anopheles
and Psorophora and biting flies of the family Tabanidae and Stomaxys
calcitrans play important role in natural transmission of the disease.
Mechanical transmission occur following the use of contaminated
surgical instruments and syringe needles. Vertical transmission also
occurs but not all foals from affected mares acquire the disease.
Although horses, asses and mules are only naturally susceptible
animals, pigs may become affected occasionally. Several workers
reported transmission to sheep, rabbits, mice and rats without the
production of any symptoms.
Pathogenesis: The incubation period ofthe disease varies from 1-3
weeks but it may be longer. The disease may occur in acute, subacute,
chronic or inapparent forms.
In acute form there is sudden rise in temperature, which is
intermittent beside depression, anorexia, thirst, serous, ocular and nasal
discharges, progressive weakness and debility. Congestion and
petechial haemorrhages are found in the visible mucous membranes.
Slight albuminuria, anaemia and general weakness may be noticed.
Oedema is most prominant in the subcutis of the ventral wall. The acute
illness lasts for 3-5 days and if "it does not terminate fatally than it
passes to subacute or chronic form. In the subacute form there may be
relapses of temperature, oedematous swelling in the lower trunk or
336
limbs and horses may die unexpectedly. Swelling and pigmentation of

the liver, enlargement of the spleen, lymphnodes, kidney and
hyperplasia of bone marrow are found.
In chronic form there is debility and anaemia. Hypertrophy of the
spleen and pone marrow are characteristic pathologic lesions.
Sometimes there may be inapparent infections and horses may
remain as symptomless carriers for long periods.
The anaemia that appears intermittently in this disease is possibly
due to the destruction of RBC by an immunologically mediated
mechanism. It has been observed that RBC of infected horses are
coated with antiviral antibodies and compliment component. This
binding to the cell surface results in increased osmotic fragility,
shortened half life and erythrophagocytosis. Plasma haemoglobin
increases and serum hepatoglobin levels decrease in infected animals.
This finding suggests that the haemolysis is responsible for the
development of anaemia. Another less important factor in the genesis
of anaemia is the depression of bone marrow during acute episodes.
Glomerulitis was also observed in infectious anaemia and this was
thought to be due to deposition of virus antibody complexes. Similar
immunologic factors are likely to be associated with the development
of lesions in other organs.
Diagnosis: Provisional diagnosis of the disease can be made from
the history, clinical signs and lesions. Confirmatory diagnosis is based
on the isolation of virus from the serum whole blood or leukocytes of
infected horses in equine leukocytes culture, demonstration of virus
specific antibodies using immunodiffusion (Coggins test) and
complement fixation tests and by transmission experiments.
Transmission experiment is carried out by inoculating 20 ml of whole
blood or serum from infected animal into young (12 months) healthy
foals. Recently it has also been observed that the FAT is useful for
detection of virus specific antigen in peripheral leukocytes cultures of
infected animals.
Control and Prevention: There is no satisfactory vaccine against
the disease. The control of the disease depends on the early detection of
infected animals, their separation and slaughter besides close
observation of the incontact animals. Regular quarantine practices and
control of the flies and mosquitoes are also important for the control of
the disease.
Retroviridae
337
VisnaIM aedi Virus

Visna and Maedi are two disease manifestations due to a virus
belonging to lentivirus subgroup of retroviruses. Maedi is a word
derived from Icelandic word which means dyspnea and is used for slow
progressive interstitial pneumonia. While the tenn visna means wasting
is used for slow progressive inflammatory disease of the central
nervous system resulting in paralysis.
Properties of the virus: The maedi-visna virus (MVV) has a
typical morphology of retrovirus. It is about 100 nm in diamete
r
contains about 30-40 nm nucleoid. The virus is readily inactivated
by
ether, chloroform, ethanol , formaldehyde and trypsin. It is stable at
50C and between pH range of 5.1 and 10. The structural proteins
are
similar to lentiviruses except certain differences in the glycoproteins.
A
major virus compon ent (40% of the virion mass) consists of a single
protein of 25-30,000 daltons and is a constituent of the virus core;
a
glycoprotein of 125000 daltons is associated with surface knobs. These
two proteins give two precipitation bands in immunodiffusion tests.
The envelope of MVV contains neuraminic acid. The virus does
not
show haemagglutination property but inhibits haemagglutination
by
influenza virus. Maedi and Visna viruses are antigenically similar.
Antigenic drift of the virus has been observed in infected animals
.
More than 1 strain of virus can exist in the same animal.
The virus replicates in sheep cell cultures with the production of
syncytia and cellular degeneration. Virus can also be propagated
in
BHK-21 cells and primary cell cultures of bovine, porcine, canine and
human choroid plexus cells. The virus does not grow in embryonated
chicken eggs.
Epidemiology: MVV infection are found in sheep only. The virus
can be transmitted to goats by parenteral infection but it is not clear
if
the infection of sheep can be transmitted to goats under natural
conditions. It is not possibl e to infect laboratory animals. There are
no
known reservoir hosts or vectors. There is a widespread geograp
hic
distribution of MVV. The disease is prevalent in USA, Netherlands,
South Mrica, France, Germany, Belgium, Spain, Italy, Greece
,
Hungary, Bulgaria, Switzerland, Israel, Soviety Union, Canada, Kenya,
Peru, India besides Iceland. There is indication that genetic differen
ces
in the virus and/or in sheep population may determine which
manifestation of the infection is dominant.
338
Pathogenesis: Naturally the virus spreads laterally probably by

respiratory route. Where the disease is enzootic the important route of
infection is from ewe to lamb through clostrum. The transmission of
infection in utero is rare under natural conditions. The virus is found in
lungs, CNS, spleen lymphnodes, salivary glands, white blood cells and
mammary gland. There is no evidence of transmission through semen.
In experimental infection there is severe restiriction of virus replication
in various tissues. Many cells contain viral genome in DNA provirus
form. In smaller fraction of cells the DNA is transcribed into RNA. The
production of lesions in visna appears to be immunologically mediated,
perhaps by a CM! response to virus antigen on infected cells. The
pulmonary lesions in maedi may also be due to some
immunopathological process taking place in the lungs. The infected
animals never become free from the virus and remain a constant threat
to any healthy contact animal.
The clinical symptoms of maedi is failure to thrive. The affected
animals show respiratory distress when led to pastures and lag behind
the healthy animals. Later there is increased respiration at rest.
Coughing is not prominent and no discharge from nostrils. The animals
become weak inspite of good appetite. The clinical course lasts for
months or even more then a year. The clinical maedi is rarely seen in
animals younger then 3-4 years of age.
The clinical symtoms of visna are dominated by nervous symtoms.
The animals show lameness of one or both hind legs, weight loss
inspite of good appetite. Paralysis of hind legs progresses but the front
legs are atfected at final stage of disease. There is slight tremor of head
and of the muscles around the face and occasionally blindness. The
course of disease runs for several months or even years and ends in
prostration and death. Maedi and visna may coexist in the same flock or
even in the same individual. Recently arthritis has been observed in
flocks in USA with progressive pneumonia. Chronic indurative mastitis
is also associated with MVV.
In maedi the lesions primarily affect the lungs and maediastinal
lymph nodes. The lungs increase two to three times of their normal size
and do not collapse when the thoracic cavity is opened and exhibit a
dark greyish blue or greyish brown in colour rather than pink colour.
The lungs are compact to touch like a rubber sponge; The
tmcheobronchial and mediastinal lymphnodes are enlarged.
Relroviridae
339
Microscopically lesions consist of diffuse thickening of interalveolar

septa which encroaches upon alveoli. The thickening of septa is caused
by infiltration of lymphocytes, monocytes and/or macrophages with
few plasma cells.
In Visna the cardinal features are chronic inflammation of brain
and spinal cord, lesions are mainly distributed around the ventricular
system affecting both grey and white matter. Subependymal and
perivascular infiltrates of lymphocytes, monocytes and/or macrophages
with plasma celis are typical. The inflammatory changes are sometimes
severe with liquefaction necrosis. Destruction of myelin together with
the axons is seen in necrotic foci.
Immune reaction: The infected sheep develop MVV antibodies
which can be detected by VN test. The VN test has several drawbacks
because it is time consuming and gives false negative results due to
antigenic variation of virus. The neutralizing antibodies appear 2-3
months after experimental infection of sheep. The CF antibodies appear
3-4 months'after allaining a plateau. More positive animals are detected
with CF test rather than VN test. The glycoprotein and core antigen can
be detected by AGID. About 90% of the sheep infected with MVV
show a positive reaction. The ELISA, indirect IF are equally effective
is detecting MVV specific antibodies.
The CMI response is irregular and vary with time. There is
evidence from experimentally infected sheep that CNS lesions are
immunologically mediated in the disease. The pathogenesis of primary
demyelination indicate immune attack on infected oligodendrocytes
may be responsible for the primary demyelination.
Diagnosis: In living animals the useful test for diagnosis are aimed
at detecting the antibodies against the virus. ELISA and AGID tests are
used as herd tests, since individual sera may give false negative results.
At autopsy the histopathology of lungs and brains reveals typical
lesions. The virus isolation is the final test for detection of infection.
The best method of virus isolation are the cultures in plasma clot and
cocultivation of cells from infected animals with susceptible cell strains
such as cultivated plexus choroideus or testis cells. The CPE produced
is rounding of cells, giant cell formation, degeneration and lysis. Some
strains are not strongly cytolytic and do not produce higher titres. The
spleen, lungs, mediastinal lymph nodes and plexl\s choroideus are
organs of choice for virus isolation.
340
Control: No vaccine is available. The only method to control the

disease is slaughter of infected animals. Attempts to clean up the
infected herd by removing lambs at birth from ewes and raising them
artificially in isolation have been successful in Netherlands and USA.
In Iceland the disease has been eradicated by stamping out policy.
Caprine Arthritis-Encephalitis Virus
Caprine arthritis-encephalitis virus (CAEV) produces a disease
complex among domestic goats of all ages. The disease is characterized
by paresis leading to paralysis among young goats while in adults there
is chronic persistent arthritis and mastitis. Rarely the adult goats also
develop progressive fatal pneumonia and encephalitis. The disease is
widely disseminated in goat herds in North America, Europe and
Australia.
Properties of the virus: The CAEV is a nononcogenic member of
lcntiviruses. The polypeptides of the virion are similar to MVV.
Antigenically the virus is related to MVV: The virus can be cultivated
in early subcultures of outgrowths of synovial membranes of goats.
Replication of virus produces CPE, characterised by cell fusion and is
the major CPE produced by the virus.
Epidemiology: The epidemiology of CAE is still being evaluated.
The studies on epidemiology have bcen complicated because a single
virus causes three different diseases in different age groups of the host.
These are (i) rapidly progressive lcukoencephalitis is new born and
young animals (ii) chronic mastitis and arthritis and masti~s in adult
animals (iii) a sporadic slowly progressive pneumonia encephalitis in
adult goats. The n~urological disease in kids occur in age below 4
months and incidence may go upto 20% while the arthritic disease may
have an incidence of 10-20%
Another complication in the evaluation of CAEV epidemiology is
its close relation with MVV in sheep. The question is whether goat
disease could be caused by sheep agent or vice versa. There is recent
demonstration that arthritis and mastitis also occur as a complication of
MVY of sheep. It keeps the issue of cross species infection alive.
The main method of dissemination of virus of CAE may be via
feeding of infected clostrum. Transplacental spread of infection to
foetus by maternal blood does not appear to be major method of virus
spread.
Retroviridae
341
The infection in goats persists often for life and u'1ese animals
become virus shedders either via colostrum or via respiratory secretion.
The infection is subclinical and the virus spread is insidious. The bottle
feeding of infected kids as well as physical contact due to handling of
kids also fosters virus spread.
Pathogenesis: CAEV and MVV leads to persistent infection.
These viruses can sequester themselves in host cells by integrating their
provirial DNA into- host cell DNA and these elude immunologic
elimination. The viruses replicate in macrophages and these viruses do
not usually induce virus neutralizing antibodies. Due to these reasons
the virus replication can thus continue independent of any control by
humoral immune system.
Infection of sheep and goats with their lentiviruses results in
persistent systemic infection during which the virus infects and
replicates in the cells of monocyte-macrophage series. The clinical
cases show low grade systemic infection i.e. non productive infection
in monocytes. The macrophages in organs with lesions may be the sites
for virus replication. Newborn goats are susceptible to infection of CNS
with CAEV but isolation of virus form CNS of persistently infected
adult animals is rare except with encephalitic disease. This also applies
to animals with chronic arthritis and/or pneumonia. Experimental
infection also corroborates the rarity of CNS involvement. Intracerabral
inoculation with virus results in virus replication in brain with acute
leukoencephalitis. Viral antigens produced by these cells elicit CMJ
response which cause the immunopathological consequences.
CAE causes at least 3 disease syndrome in nature. The most
frequently encountered is arthrititic disease in adult goats. The arthritis
is insidious in onset and progresses slowly over a period of months to
years. Joints, bursae and tendon sheaths are targets of disease but the
common and severe localisation is in the carpal (knee) joints. The hock
and stifle joints are involed to a lesser extent. Disease is common in
animals of 2-9 years of age and longer the disease duration the greater
is tissue damage. The affected animals are weak and have long coarse
poor hair. The affected animals are afebrile and maintain good appetite.
The histopathological lesions are characterized by proliferation of
synovial membrane with development of villous projections into the
lumen of joint Later there is necrosis of collagen structures.
In addition to arthritis the lactating animals have a chronic
342
inflammatory lesions in the mammary glands. These lesions consist of

marked lymphoid hyperplasia. The infammatory cells are of the same
type as in arthritis lesions like lymphocytes, plasma cells and
macrophages.
Rapidly progressive neurological disease of young goats of 2-4
months old show this syndrome. The earliest signs are posterior paresis
and ataxia or weakness in hind quarters. It progresses and includes
front legs. The kids become recumbent and die. Goats with
encephalomyelitis have rough hair coats and have muscle atrophy.
Immune reaction: Humoral and CMI response to the virus play
important role in infection but does not produce any benefit to the host.
The cellular immune response in sheep inf~cted with MVV and
goats infected with CAEV is virus specific. The CMI response
correlates with the onset of encephalitis, pneumonia and arthritis and
persists as long as inflammation persists. The sensitized lymphocytes
are important in causation of the inflammatory disease and cannot be
answered directly. Indirectly it has been proved that virus does not
cause primary cytopathic effect in tissues as it does in cell cultures.
Although CAEV and MVV have similar pathogenic mechanism
and share major antigenic determinants, the CAEV is distinct from
MVV. In competitive hybridization assays of radiolabelled RNA of
these two viruses show only 20% homology with each other.
Diagnosis: As the animals are persistently infected after exposure
to virus, the demonstration of antibodies indicates infection. AGID is
the test of choice. ELISA is also being employed. Virus isolation can
be done from 10-20 ml of blood collected over anticoagulant. The
buffy coat cells are separated and cocultivated with normal goat
synovial membrane cultures. The cocuIture is maintained for 2-3 weeks
at 37C and examined for CPE.
Control: The hyperimmunization of animals with live or
inactivated virus preparation fails to protect these animals. Control
measures rest on the identification of infection and elimination of the
positive animals.
Jaagsiekte (Ovine Pulmona~y Adenomatsis) Virus
Jaagsiekte was reported about one hundred years ago in South
Africa but the viral etiology was known recently and identified as
retrovirus. It does not seem to be related to either the known
Retroviridae
343
lentiviruses or oncoviruses. The rust indication of its being retrovirus

was the observation of particles possessing type C morphology in
jaagsiekte lungs and biochemical evidence for the presence of particles
with reverse transcriptase activity in lung extracts as well as
demonstration of morphologically typical retroviruses in cell cutures of
adenomatous lungs.
Properties of the virus: The jaagsiekte retrovirus (JSRV) is about
104 nm in diameter. The mature virus particles are electron dense with
a close fitting envelope. A slightly eccentric nucleoid can sometimes be
seen under electron microscope. The overall morphology is typical of a
retrovirus but can be distinguished in finer details. Morphologically
JSRV seems to be closely related to mouse mammary tumor virus
(MMTV). The JSRV does not show any cross relation with MVV
related lentiviruses.
All attempts to grow JSRV in a variety of cell cultures have not
been successful.
Epidemiology: The domestic sheep is the main host affected by
JSRV but there are reports regarding the disease among goats as well
but these reports are not authentic. Experimentally the infection can be
transferred to newborn kids. The infection cannot be established in
laboratory animals except successful transplantation of cultured tumor
cells into nude mice. Jaagsiekte is widely prevalent. The disease is
endemic in most European, African, Middle East and South American
countries. Sporadic cases have also been diagnosed from USA and
Canada.
The incidence of flock varies from 5-20%. The infection i's
airborne and close contact facilitates the spread of the disease. The
genetic predisposition exists. Certain breeds of sheep in Iceland were
found to be highly susceptible than others. The disease in its natural
form is a slow virus infection as symptoms are rarel y seen in animals
younger than 3-2 years. Once animal shows symptoms, the prOgressive
proliferation of cancerous cells leads to death. 100 martality of the
d~ease cannot be regarded as 100% as certain individuals have the
ability to contain tumorous lesions.
Pathogenesis: The disease can be experimentally uaosmitted by
co-habit3tion, by droplet infection, aerosal spray and by inoculation of
extracts of adenomatous lungs or virus by intrapulmonary, intrapleural,
intratracheal or intranasal routes. Replication of virus takes place in the
344
Textbook ojVelerinary Virology
type IT alveolar epithelial cells, the virus does replicate in other organs
or blood. The infection remains localized in the lung and there is no
viraemia. The type 11 alveolar epithelial cells and other nonciliated
bronchial epithelial cells are transformed to neoplastio cells which
proliferate and eventually fill the alveolar spaces. Normal lung tissue is
replaced by solid tissue. These tumor ceIls are also surfactantproducing secretory cells. The typical jaagsiekte lungs are therefore
oedematous in appearance with copious amount of clear viscous fluid
produced which accumulates in the air passages.
It is not yet known that. JSRV is present as a provirus in the
genome of sheep cells. There is no evidence of vertical transmission or
intra-uterine infection.
The incubation period in natural infection varies from months to
years, while in experimental infection the incubation period can be
reduced to weeks or even days. The natural infection occurs in young
animals. When lesions develop, symptoms are seen and they mainly are
various manifestations of dyspnea. There is increased respiration with
jerky expiratory movement Ultimately animals show acute respiratory
distress. Coughing and moist rales ~e observed. Viscous fluid runs
from the nose. Loss of.appetite and emaciation are terminal signs.
At autopsy the lungs are found double in size or 3-4 times the
normal weight Initially white coloured nodules are found which
expand to form greyish white areas of consolidation. The microscopic
lesions consist of an alveolous lined by cuboidal or columnar epithelial
cells. These cells proliferate, filling the lumen of the alveolous and
forming acinar or papilliform masses.
Immune reaction: There is evidence of acquired resistance to
jaagsiekte. No detectable circulating antibodies develop and there is no
evidence of cellular immunity. The virus derived from lung fluid is
complexed with immunoglobulins predominantly of IgA type. The lung
exudate is also rich in IgA and IgG. It is possible that immune response
to jaagsiekte virus is mainly of local nature and mediated by IgA. It
may be due to absence of viraemia in JSRV infection as the replication
of virus is localised in lung epithelium.
Diagnosis: Clinical signs like dyspnea in absence of fever and
clear viscous lung exudate is indicative of jaagsiekte. Histopathological
examination is method of choice.
345
Retroviridae
Control: Control measures depend upon strict isolation and

elimination ofanimals showing symptoms.
Rererences
BUXTON, A. and FRASER, G., 1977. Animal Virology Vol. 2. BlackweIl Scientific
Publications, Oxford, London, Edinburgh.
CHEBVERS, W.P. and McGuire, T.C., 1988. The lenliviruses: maedivisna,
caprine arthritis-encephalitis and equine infectious anaemia. Adv. Vet.
ResJ4: 189-215.
CRAWFORD, T .B.; ADAMS, D.S.; CHEEVERS, W.P. and CORK, L.C., 1980. Chronic
arthritis in goats caused by a retrovirus. Science, 207, 997-999.
FENNER, I.F., 1980. Bovine lymphosarcoma. Adv. vet Sci. Corn. Med. 24, 168.
HAASE, A.T., 1986. Pathogenesis of lenlivirus infections. Nature, 322, 130-136.
HOFSTAD, M.S., CALNEK, B.W. HELMBOLDT, C.F., RElD, W.M. YADER, H.W. IR.
1972. Diseases of Poultry. 6th Ed. Iowa State University, Press, Ames.
KAHRS, R.F., 1985. Viral diseases of cattle. Kalyani Publishers, New Delhi.
LuCAs, M.H. and ROBERTS, D.H., 1982. Transmission of bovine leulwsis virus
(BLV) Curr. Top. Vet. Med. Vet. Sci.15, 264-266.
MELNICK, I.L., 1982. Taxonomy and Nomenclature of viruses. Progress Medical
Virology. 28: 208-21.
MOHANTI, S.B. and DUITA, S.K., 1981. Veterinary Virology, Lea and Febiger,
Philadelphia.
NARYAN, 0.; Cl'v!E.'ITS, 1.E.; SlRANDBERG, I.D.; CORK, L.C. and GRIFFIN, D.E.,
1980. Biological characterisation of the virus causing leukoencephalitis
and arthritis in goats. I.Gen. Virol. 50, 69-70.
NARYANA, O. and CLEMENTS, 1.E., 1989. Biology and pathogensis of
lentiviruses.1. Gen. Viral. 70: 1617-1639.
NOTKlNs, A.L. and OLDSTONE, M.B.A., 1984. Concepts in viral pathogenesis.
Springer-Verlag.New York, Berlin Heidelberg Tokoyo.
SlRAUB, O.C. 1981. Enzootic bovine leulwsis. In E.P.I. Gibbs (Editors) Virus
Diseases of Food Animals. Vol. I. International perspectives. Academic
Press, New York, NY-pp 693-718.
TUSTIN, R.C., 1969. Ovinejaagsielae, 1.S. Afr. Vet. Med. Assoc.40: 3-23.
TeXlboolc o!Veterinary Virology
346
UPPAL,
P.K. YADAV, M.P. and AHMm, S.N.. 1981. OCCllTrence of equiM

infectious aNlD1Jia in India. Virus information Exchange News letler 4,
45-46.
D.W; PAYNI!, A.L.; MYER, M.M. and YORK, D.F., 1983. IsolatiOfl
and. prelimituJry characterization of jaagsielcJe retrovirus (JSRV).
Ondersteport, 1. VeL Res. 50,309-336.
VERWOI1JtD,
D.W.; TumN, R.e. and PAYNI!, A.L., 1985. Jaagsiekte: An

infections pulmonary adenomatosis of sheep. In O. Olsen Karkowka
and J.R. Blakeslee (Editor) Comparative Pathology of viral diseases.
VERWOARD,
eRe Pr~s, Boca Raton, F.L. pp.53-76.

Virus, Information Exchange Newsletter for South East Asia and the Western
pacifIC. 1987. Vo1.4 No. 2. Australia.
Chapter 30
Bunyaviridae
Bunyamwera is the name of a place in Uganda where type species,

Bunyamwera virus was isolated. Bunyaviruses, with the exception of
hantaviruses, are transmitted by arthropods. The family contains more
than 200 viruses, in five genera: Bunyavirus, Phelbovirus, Nairovirus,
Unkuvirus and Hantavirus. The genera of Bunyavirus and Nairovirus
are organised into various serogroups. The genera of Phelbovirus,
Unkuvirus and Hantavirus at present have a single serogroup. The
viruses is this family are medium sized, spherical or oval measuring 90lOO nm in diameter. They possess a membrane envelope with
projections. The projections are glycoproteins, designated Gt and Gz
The genome consists of three molecules of a single stranded
noninfectious, RNA large (1.), medium (M) and small (S), which are
surrounded by a nucleocapsid protein (N). A minor large protein(L) is
probably a transcriptase. Most of the genome is (-) sense buUt has
been shown with some viruses (phelbovirus) that the 5' end of the S
segment is (+) sense, the term ambisense is used to describe this
genome. Virions contain four major proteins: a transcriptase(L),
nucleoprotein (N) and two glycoproteins (G t and G~).
The virions are sensitive to lipid solvents and detergents. The
viruses replicate in the cytoplasm and mature by budding into smooth
surfaced vesicles in Golgi region or nearby. The viruses readily grow in
many kinds of cell cultures e.g. Vero cells, BHK-21 cells and mosquito
cells. Most of the viruses grow to high titre is suckling mouse brain.
The virus gains entry into host cell by fu!;ion of viral envelope with cell
348
membrane. The replication takes place in the cytoplasm. After

penetration the virion transcriptase is activated and transcribes
subgenomic mRNA from each of virion RNA and a second round of
transcription takes place. The lRNA segment codes for virion
transcriptase and M segment codes for glycoproteins (G 1 and G1). The
sRNA in case of Bunyavirus codes for nucleoprotein and a
nonstructural protein. In case of Phelbovirus, sRNA also codes for two
proteins but it employes an ambisence transcription strategy. The
viruses mature by budding in Golgi vesicles and are released by fusion
of the vesicle membrane with plasma membrane and exocytosis or by
cytolysis.
Rift Valley Fever Virus (RVFV)
Rift valley fever (RVF) is a mosquito borne disease that cause
devastating epidemics among sheep, goats, cattle and human beings.
The disease is characterized by its epizootic nature, short incubation
period, fever and typical focal to diffuse necrosis of the liver. The
disease was first reported from Kenya in 1912 and the virus was
isolated in 1931. RVF is now reported form most of sub-Saharan Africa
and the Nile Valley. RVF has not been reported so far outside African
continent.
Properties of the virus: The virions are spherical in shape bounded
by host cell derived membrane with virus coded glycoprotein spikes,
the diameter of the virion is 85-100 nm. The morphogenesis of
Bunyaviridae is consistent with helical symmetry of the nucleoprotein
core. The virions are formed by budding through smooth endoplasmic
reticulum with many virions in Golgi cisternae which communicate
with extracelluar environment.
All isolates have been found to be identical by using serologic
tests. The difference in strains can be detected by RNA fingerprinting.
RVFV is sensitive to lipid solvents, detergents and low pH. The virus
retains its infectivity at 4C in the presence of protein and at neutral or
alkaline pH for about 4 months and for 6 months in 0.5% phenol. In
serum the infectivity is retained upto 3 hours at 56C. Formalin
inactivates the virus even at low concentration. In human RVFV
vaccine the inactivation is done at 0.3% final concentration for 72
hours at 37C. Betapropiolactone 0.1 % also is used to inactivate the
virus. RVFV contaminated surfaceS are sterilized effectively with
bleaching powder, sodium or calcium hypocholorite.
Bunyaviridae
349
Cultivation: The virus readily grows in the chicken embryos via

yolk sac or by chorioallantoic membrane route. It can also be
propagated in mouse brains, in foetal rhesus monkey lung cells and in
kidney cultures of lamb and vero cell line. Acidophilic intranuclear
inclusions are found in the infected cells.
Epidemiology: In Africa the disease is limited to domestic
ruminants i.e. sheep and cattle. Goats are variable than sheep or cattle
with respect to severe disease. The disease progression and severity are
inversely proportional to age. The adult sheep and cattle suffer 10-30%
mortality while the animals less than 7 days old may have fatality rate
of 100%. Outbreaks are characterized by abortion and rapidly fatal
neonatal disease. Equines and camels develop antibodies in the absence
of disease. The disease is spread by mosquitoes the natural host
reservoirs are unidentified. Mosquitoes of many species of the genera
Aedes, Anopheles, Culex, Eretrnapodites and Mansonia transmit RVFV
under field conditions.
Blood and serum from RVFV infected animals are important
infectious sources for human disease.
Palhogellesis: Arthropod transmission is the main mode of disease
spread, although infections following cOnlac~ with infected tissues or
inhalation of aerosals may occur. The liver is the primary site of
replication. In pregnant animals there is a predilection of virus for
placentomes via haematogenous route. The rapid foetal death is due to
direct viral infection rather than generalized placentitis. There is rapid
progress from mild hepatocellular changes to massive necrosis.
Haemorrhagic diathesis, reduced prothrombin levels and prolonged
clotting time has been observed in experimentally infected animals.
The clinical signs observed are related to species and age of
animals involved. The younger animals are not most susceptible. The
incubation period in sheep and cattle is 4-6 days but is shorter in
experimentally infected animals. The disease appears as paracute,
acute, mild or sometimes inapparent. The paracute form may be seen in
new born and very young lambs which die without showing clinical
symptoms. The acute form is encountered in young lambs, kids and
calves while mild form is seen in adult sheep and goats. The adult cattle
may experience the mild or inapparent form. The most prominent
clinical manifestation is severe icterus and abortion amongst pregnant
animals. In young lambs the disease is characterised by prostration and
350
death. The animals are listless, off feed, pass bloody wine, and exhibit
diarrhoea, dysponea and muscular tremors. There is an elevated
temperature upto about 106F. Abortion is a dominant feature in adult
cattle apart from elevated temperature, buccal erosions, diarrhoea,
necrosis qf skin over udder and scrotum and cessation of milk
production. In human beings there is high fever, icterus, nausea,
epistaxis, headache and muscle pains. Recovery usually takes about 1014 days. The disease may tenninate fatally in babies infected through
the milk of infected mothers.
The most striking features at necropsy is extensive jaundice
because of focal hepatic necoris. There may be oedema, congestion or
hamemorrhange of gall bladder as well as scattered hamorrhages
throughout the body. The kidney are of mottled appearance and show
petechiae on the surface. The gestrointestinal tract mucosa is
hyperaemic, haemorrhagic and lymph nodes are generally enlargcd and
soft Histopathologic examination confIrms the haemorrhagic nature of
lesions. There is focal and diffuse necrosis of the liver with acidophilic
inclusion bodies in the liver ceils.
Immune reaction: After an incubation period of 1-4 days and a
viraemic phase of 1-4 days, humoral antibodies appear and risc mpidly.
The antibodies can be measured by neutmlization, haemagglutination
inhibition, complement fixation, ELISA and immunofluorescence tests.
The antibodies persist in low titre after 1-2 years Post infection. The
maternal antibodies protect the lambs upto 3 months. The
immunogenicity of live attenuated vaccine is superior to inactivated
vaccine. High titre antibodies appear after inoculation with live
vaccine.
In human beings it is speculated that the encephalitic and ocular
clinical RVF syndrome is due to immunopathological component.
Diagnosis: High abortion rate in cows and ewes, increased
mortality among lambs and calves less than 7 days of age, extensive
liver lesions, mosquito season with high mosquito population and
human febrile disease are suggestive of RVF outbreak:. Earlier the
disease had a limited geographical area i.e. sub-Saharan Africa but now
the disease has spread over to Egypt and most countries in Africa.
When the disease in suspected, virus isolation should be attempted in
mouse brain and cell culture. Blood samples and serum collected at the
height of fever, specimens of liver, placenta and foetus are the
Bunyaviridae
351
specimens to be used for virus isolation. Samples from liver, brain,

kidney, heart and spleen, are collected for serological diagnosis.
Histopathological examination of liver reveals characteristic necorisis
of hepatocytes associated with semi demarcated foci of primary
coagulative necrosis. In acute hepatic lesions, haemorrhages are
widespread with no inflammatory response. Eosinophilic intranuclear
inclusion bodies may be seen in degenerating hepatocytes.
Control: Immunization of susceptible animals is the most effective
mechanism of control. Two types of vaccines are used. The modified
live virus 'vaccine is stable w~en properly lyophilized and is highly
effective. It gives a long lasting immunity. The protection is induced
after 6-7 days of administration and the off spring of the vaccinated
animals remain protected upto 5 months of age. The vaccine is not
recommended for pregnant animals as the vaccine is abortogenic and
teratogenic. Formaline inactivated vaccines are effective when multiple
inoculations are done. The immunity produced is not long lasting and
therefore yearly boosting is essential. The vaccine can be administered
in pregnant animals.
Akabane Virus
Akabane virus infection during early pregnancy causes abortion,
premature delivery, stillbirths and a foetal anomaly in cattle, sheep and
goats that is known as arthrogryposis hydranencephaly (AH) syndrome.
The virus causes disease in Japanese cattle. The syndrome in cattle,
sheep and goats has also been reported in Australia and Israel. The
virus was fIrst isolated from mosquitoes in Japan in 1959 (MaLSuyama
et aI, 1960; Oya et ai, 1961) - Akabane is the name of the village
where the virus was fIrst isolated.
Properties of the virus: The virus particles are enveloped roughly
spherical, variable in size, 70-130 nm in diameter. Most of the virions
have a ragged closely adherent envelope with 9 nm peplomers. It is
inactivated by ether, chloroform and sodium deoxycholate. The virus is
readily inactivated ~t pH 3 and trypsin. It is also heat labile loosing
infectivity about 0.3 log per hour at 37C. The virion contains a single
stranded RNA genome of three size classes (L, M and S). There appear
to be five proteins in virus infected cells L, Gp Gz' N and one small
nonstructural protein. The virus shows haemagglutination and the
haemaggluting activity is structurally associated with the virus particle.
352
The haemagglutination is dependent upon the pH as well as NaCI

molarity of the diluent Duck, goose and pigeon erythrocytes are
agglutinated. Cattle, sheep, guinea pigs, day old chicken and human
erythrocytes are not agglutinated. Haemagglutination is specifically
inhibited by antisera. The virus also shows haemolytic activity. It lyses
pigeon erythrocyte at 37C.
Cultivation: The virus replicates in the cytoplasm. For primary
isolation the brain of suckling mouse (1-2 day old) is the most sensitive
system of viral isolation. The virus can also be cultivated in primary
cell cultures and celIlines with the production of CPE. The virus can be
propagated in the chicken embryo by yolk sac inoculation. Inoculated
embryos show dwarfism, cerebral defects, hydroencaphalus, deformed
legs and toes and arthrogrypsis.
Epidemiology: The result of virus isolation and serological tests
carried out in different parts of the world indicate a wide distribution of
Akabane virus among cattle and other domestic animals in many
southeast Asian countries, the Arabian peninsula. the Middle East and
Africa. Apart from cattle, other species found to have antibody are
horses, goats, sheep, pigs and monkeys. The biting midge Culicoides
brevitarsis a principal vector of virus in Australia. The virus has becn
isolated from Aedes vexans. Culex tritaeniorhynchus. and Culicoides
oxystoma in Japan, and form Anopheles /unestus in Kenya. In Japan
the geographical distribution and seasonal occurrence of the disease
and active transmission in summer suggest the involvement of vectors.
However, the information on the vector(s) is lacking and mechanism of
transmission and survival of virus is not yet know.
Pathogenesis: Congenital AH syndrome is caused by intrauterine
infection of foetuses with Akabane virus in pregnant cattle, sheep and
goats. The intrauterine infection of foetus can cause abortion, still
birth, premature birth and deformities but no clinical abnormalities
have been recognised in dams during pregnancy. Arthrogrypotic calves
are often born dead, the musculature of limbs and spinal column is
atrophied and one or more limbs are fixed. The site of inilial infection
is not known, viraemia seems to be a constant feature. The virus does
not produce a persistent infection of the foetus. The histopathological
examination of infected bovine foetus show encephalomyelitis and
polymyositis of skeletal muscles. Immuno fluorescence demonstrates
the viral antigen in skeletal muscles and brain tissue. The lesions in the
Bunyaviridae
353
foetus result in abortion, premature birth, or stillbirth. The foetuses

which survive the infection gradually develop brain lesions and
reduction in the number of motor neurons in the spinal anterior born.
Arthrogryposis may ensue for the damage in the CNS.
An epizootic in cattle may be fIrst noticed by increased incidence
of abortions and premature births. The calves born with
hydranencephly may survive for several months but they never thrive.
The calves are mature but underweight at birth and may show
blindness, nystagmus, deafness, slow suckling, paralysis and
incoordination.
Immune reaction: The infected animals develop neutralizing, HI
and haemolysis-inhibiting antibodies between 7-14 days post infection.
The neutralizing antibodies persist for about 2 years. Foetal calves,
lambs and kids develop antibodies in uterus betwccn 70-96 days of
gestation in cattle and foetal lambs and foetal kids betwccn 30-155 days
of gestation.
Diagnosis: Akabane virus infection is suspected when congenital
AH syndrome appears sporadically or endemically alongwith stillbirths
or abortion. Serum from aborted foetuses, still born calves and dams
can be tested for SN antibodies. The virus can be isolated from foetuses
and still born calves, from blood of cows by intracerebral inoculation of
suckling mice.
Control: There are two approaches of control. The vector control
and vaccination. The vector control measures are not practical as the
knowledge concerning vector is still inadequate. A formalin inactivated
aluminium phosphate gel adsorbed vaccine has been developed in
hamster lung cultures. Two doses at an interval of 4 weeks are
recommended in cattle. The vaccination prevents development of
viraemia and infection of foetus is pregnant cattle and goats after
challenge with virulent virus.
An attenuated strain has also been developed by serial passages in
hamster lung cultures at 30C. The vaccine has been found to be
innocuous in calves but in pregnant ewes it causes intrauterine infection
and viraemia.
Nairobi sheep Disease
It is an acute infectious sheep and goat disease transmitted by ticks.

The disease is prevalent in African continent. The virus withstands a
354
temperature of 500C for 1 hr and is resistant in blood and serum at 4C.

The virus can be propagated in sheep and goat kidney cell cultures and
in hamster cell lines in which CPE is produced. Intracytoplasmic
inclusions are seen in infected cell cultures. The virus grows well in
young mice by intracerebral inoculation. The incubation period of the
disease is 4-15 days. The disease is manifested by fever, mucopurulent
nasal discharge and increased respiration. There is haemorrhagic
gastroenteritis and congestion of lymphnodes. Abortion may take place
in pregnant ewes. The mortality ranges between 30-80%. Goats are
susceptible to this disease but cattle are not infected. The disease is
transmitted by tick and is maintained by tick-sheep or tick-goat cycle.
The disease can be diagnosed by isolation of virus from spleen and
infected blood in cell culture a,!d mice. Mouse brain adapted
inactivated vaccine is available. A modified live virus attenuated by
serial mouse brain passage is also available.
References
BUXTON, A. and FRASER, G., 1977. Animal Microbiology, Vol.2, Oxford.
F.G., 1978. Nairobi sheep disease in Kenya. The isolation of virus from
sheep and goats, ticks and possible maintenance hosts. Journal of
DAVIES,
Hygiene Cambridge, 81, 259-66.
DINiER, Z. and MOREIN, B., 1990. Virus infections of ruminants. Elsevier

Science Publishers B.V. Amsterdam.
EASn:.RDAY, B.C., 1965. Rift valley fever, In advances in Veterinary sciences.
Edited by C.A. Brrandly and C.L. Coruelius Vol. 10, New York,
Academic Press.
GWBS, B.PJ., 1981. Virus Diseases of food animals. Academic Press, New
York.
KUROOI, H.; lNABA, Y.; TAKAsHASHI, E.; SATO, K.; <>MORI, T.; MIURA Y.; COTO,
Y.; FUJIWARA, Y; HATANO, Y; KODAMA., FUKUYAMA, S.; SASAKI, N. and
MATIJMOTO, M., 1976. Epizootic congenital arthrogryposishydranencephaly syndrome in cattle. Insolation of Akabane virus from
infected foetuses. Archives of Virology. 51: 57-74.
LUPTON, H.W. and PETERS, CJ., 1984. Rift valley fever. Proc. U.S. Animal
Health Assoc. 87, 279-290.
Bunyaviridae
355
MATSUYAMA, T.; OVA, A.; OoATA, T.; KOBAYASHI, I.; NAKAMURA, T.;TAKAHASHI,
M. and Kitaoka., M., 1960. Isolation of arboviruses from mosquitoes
collected at livestock pens in Gumma Prefecture in 1959. Japn J. Med.
Sci. Bio1.l3, 191-198.
MATIJMOTO, M. and INABA, Y., 1980. Akabane disease and Akabane virus.
Kitasato Arch. Exp. Med. 53, 1-21.
MATIJMOTO, M. and INABA, Y., 1980. Akabane disease and Akabane virus.
Kitasato Arch. Exp. Med. 53, 1-21.
MOHANlY, S.B. and DUITA, S.K., 1981. Veterinary virology, Lea and Febiger,
Philadelphia.
OVA, A., OKuBO,T., OaATA, T., KOBAYASHI, 1. and MATSuyAMA, T., 1960.
Akabane, a new arbovirus isolated in Japan. Jpn. J. Med. Sci. Bio1.14,
101-108.
POR1ERFIELD, J.S. and DELLA-PORTA, A.I., 1981. Bunyaviridae: Infections and
diagnosis. In E.Kurstak and C.Kurstak (Editors) comparative diagnosis
of viral disease. Academic Press, New York, Vol. 4 pp. 479-504.
RUSSELL, P.H. and EDINGTON, N., 1985. The Burlington Press (Cambridge) Ltd.
Foxton, Cambirdge.
The Rift Valley fever, Office International Epizootics Technical Series No.l,
1981.
Chapter 31
Toroviridae
Toroviridae is a newly created family for a group of antigenically

related viruses demonstrated in horses, cattle and men. Two new
viruses detected in fecal material from horse and cattle in 1982 and
1983 with unique morphological (Latin torus-a doughnut shaped ring),
biochemical and serological characters. Toroviruses are enteric viruses
recognised in three different species, namely horse, cattle and human
beings. Evidence of infection has been obtained in pig, sheep, goat, in
lagomorphs and rodents.
Toroviruses are enveloped RNA viruses with peplomer bearing
envelope and an elongated tubular nucleocapsid of helical symmetry.
The nucleocapsid may be bent giving kidney or disk shaped
morphology to the virus particle or the neuclocapsid may be straight
resulting in rod shaped virion. The genome is single stranded RNA
with positive polarity. The moleular weight is about 8 x 106 There are
two major proteins in the virus particle, a phosphorylated nucleocapsid
polypeptide of 20K and a 22K protein the main constituent of envelope.
Additional proteins of 37K and 75-100K range are also present in the
virus particle. Toroviruses replicate in the cytoplasm where four
subgenomic mRNAs are formed. The replication is dependent on some
nuclear function of the host cell.
Breda Virus
In 1972 a virus was isolated from a rectal swab of horse with
diarrhoea in Berne (Switerland). In 1982 Woode et al. described the
isolation of virus from epizootic of neonatal calf diarrhoea in Breda,
Toroviridae
357
Iowa. The resemblence of 'Berne virus' and 'Breda virus' particles was
described some 10 years later. In 1984 particles resembling Berne virus
(BEY) and Breda virus (BRV) in stoqls of adults and children with
diarrhoea, which reacted with antibodies against BEV and BRV were
described.
Properties of the virus: In negatively stained preparations of
intestinal contents, faeces or tissue fluids, the virus particles are either
elongated or kidney shaped with a diameter of 105-140 x 12-40nm or
spherical forms measures 82 nm in diameter; with a pcplomer bearing
envelope. The peplomers may be short (7-9 nm) or long (20 nm). Many
particles have short stubby peplomers which make the virus easily
distinguishable form coronavirus. The jejunal epithelial cells infected
with BRV show brick shaped or elongated particles with dimensions
80-100 x 35-42 nm.
The BRV has two serotypes BRVl and BRV2. This virus is
anligenically related to BEV. BRVl and BRV2 carry common antigens
measurable by IF and ELISA. HI has shown clear differences between
BRVl and BRV2. BRV possess the haemagglutinating activity. Mouse
and rat cells are the only cells agglutinated. The serotype 1 does not
elute from erythrocytes while serotypes 2 does. The BRV looses
infectivity if faeces are stored at 4C for 2-3 weeks. It is probable that
the virus is readily destroyed by disinfectants and heat. The virus does
not grow in cell culture.
Epidemiology: Breda was isolated from acute enteritis of calf aged
5 days. About 56% of calves in the herd developed diarrhoea in ftrst 20
days -of life. Fifteen percent of calves died. The natural spread of virus
takes place by fecal/oral route. The subclinical excretion of rotavirus by
sows at parturition has been conftrmed. The bovine dams are
considered to be source of neonatal virus infections. There are no
reservoir hosts or vectors involved in the spread of virus. The
incubation period is as short as 24 hours, experimentally. The
serological survey carried out in parts of USA, the Netherlands and
Germany by ELISA have shown antibodies against BRV in calf and
cow sera to the extent of 85 to 94%.
Pathogenesis: The virus fed to colostrum deprived calves or
gonotobiotic calves aged 1 hour to 24 days infects the intestine and
reaches faeces within 24-72 hours. Diarrhoea or change in appearance
of faeces conicides with the virus or change in appeearance of faeces
358
conicides with the virus excretion. After recovery the virus can be
excreted for atleast 4 months. In colostrum fed calves from immune
dams the infection cannot be prevented but symptoms are mild.
Viraemia has not been reported.
The calves develop anorexia and depression followed within few
hours by greenish yellow or yellow diarrhoea. Some calves may show
shivering. The signs of dehydration reddening and loss of tone of
intestine are noticed. Villus atrophy takes place due to infection of
crypt and villus epithelial cells. There is focal necoris and moderate
inflammation of the small intestine.
Immune reaction: Following primary infection with BRV, the
animals develop specific IgM and IgG antibodies. The peak titre is
attained 1-2 and 3-4 weeks. The IgA antibodies can also be detected
within 2 weeks. The passively derived antibodies do not protect calves
from infection.
Diagnosis: BRV does not grow in vitro. The EM detection of virus
using 3-4% potassium phosphotungstate, pH 7.0, is used to identify ~
virus. The BRV can be distinguished from coronavirus by !EM. Viral
haemagglutinin titres of infected calves fecal samples vary from 20,000
to 50,000 while the titre in normal calves is 16-32. ELISA test is used
to survey serum antibodies in the herds.
Control: There is no specific control measure available as no
vaccine has been developed so far.
Berne Virus
In 1972 a virus was isolated from rectal swab of horse with
diarrhoea in Berne (Switzerland). Berne virus (BEV) has been cultured
in vitro, in horse kidney cultures. It can also be cultured in other cells
of equine origin. In the presence .of actinomycin D and aminitin the
BEV replication is drastically reduced when drugs are added during the
first 8 hours of infection, indicating that BEV replication depends upon
some nuclear funclion of the host cell. The BEV agglutinates the
erythrocytes of human '0' group, rabbit and guinea pig but not fJf rat
and mouse. The virus shows cross neutralization with Breda virus.
There is wide distribution of Berne virus in Swiss horse population and
antibodies also occur in sera of cattle, sheep, goats, pigs. laboratory
rabbits and two species of wild mice.
359
Toroviridtu
The pathogenic role of BEY has not been proved. It cannot be said
that BEY was responsible for diarrhoea from the horse it was isolated.
References
HOR2JNBX, M.C.; FLEWETI', T.H.; SAIF, L.I.; SPAAN. I.M.; WEISS, M. and WOOD,
O.N., 1987. A new family of vertebrate viruses. Toroviridae
Intervirology.27, 17-24.
Wmss, M.; and HORZINEK, M.C., 1987. Theproposedfamily Toroviridae Agents

of enteric infections. Arch. Virol. 92, 1-15.
Woode. O.N.; Reed, D.E.; RUJUlels, P.L.; Herrig, M.A and Hill, IT., 1982.
Studies with an unclassified virus isolated from dimrhoeic calves.
Veteminary Microbiology 7: 221-224.
WOOD. O.N.; MOHAMMED, K.A.; SAIF, L.J.; WINAND, N.I; QUESADA, M. Kaso,
N.E. and POIll.ENZ, IF., 1983. Diagnostic methods for the newly
discovered 'Breda' group of calf enteritis inducing viruses. In:

Proceedings of the third sympsosium. World Association of
Veterinary Laboratory Diagnosticians pp.533-538.
Chapter 32
Unclassified RNA Virus
Borna Disease Virus

Boma disca<;e virus is a slow viral disease of horses that affects
central nervous system. The disease is prevalent in Germany and othcr
parts of Europe. The name of disease has been derived from the city of
Boma, Gcrmany where large number of horses died in 1899.
The detailed morphology of virus has not been worked out but the
virus is smaller than 100 nm with an envelope. The virus is heat and
acid labile. The virus particles contain RNA. There is only one
antigenic type of boma disease virus. The virus multiplies in many
varieties of cell cultures from many species of animals but monkey
kidney cell lines (MS) are most suitable. The CPE does not appear but
the presence of virus can be detected by IF or by acridine orange
staining. Intranuclear inclusion bodies appear in some cells after
several weeks of incubation. The virus remains closely associated with
cellular structures and persists through several subpassages in cell lines
without losing its infectivity. The virus can also be propagated on the
chorioallantoic membrane of 11 days old chicken embryos at 35C.
.Boma disease is an infectious encephalomyelitis of horses but
natural infection also occurs in sheep. Experimentally most domestic
and laboratory animals can be infected. Hamsters and young rats are
most suitable experimental animals. The incubation period of natural
disease is about 30 days but experimentally the disease in young rabbits
appear between 15-20 days. The affected horses show slight fever,
Unclassified RNA Virus
361
anorexia, excessive salivation, lassitude and constipation. As the

clinical picture develops there is drowsiness followed by restlessness,
biting, kicking, excitability and convulsions. Paralytic signs appear in
early stage of disease and frequently take the form of generalized
paresis. The course of disease is about 1-3 weeks and mostly the
animals do not recover. The mortality rate is about 90%.
The virus is present in saliva and nasal secretions during acute
illness of horses. In the surviving horses the virus can be recovered for
about 6 months after recovery. There are no gross lesions at necropsy
but typical encephalitis affecting brain stem is observed on
histopathological examination of brain. The'intranuclear inclusions can
be seen in the neurons. The spread of infection results from
contamination of food and water, by virus shed in the saliva and nasal
secretions during acute illness of horses. The virus is also excreted, in
the milk and urine. The outbreaks appear in early spring and reach a
peak in warm damp weather and disappear in the autumn. It suggests
that arthropod vectors play an important role in the spread of infection.
The virus has also been isolated from the brains of herons and other
wild birds, as well as from ticks of the genera Hyalomma, dermacentor
and ornithodoros.
The presence of intranuclear inclusion bodies in the nerve cells of
the brain is diagnostic. The virus can also be detected in infected cell
cultures by IF test. The antibodies in the serum and central nervous
system can be detected by IF test.
The recovered animals are immune but the duration of immunity is
not known. Infected rabbit or horse brain tissue inactivated with phenol
or phenol glycerol is used for vaccination. A single dose of vaccine is
administered subcutaneously provides protection for about a year.
Reference
MAYR,
A. and DANNER, K., 1978. Borna-a slow virus disease. Comp. Immunol.
Microbiol. Infect. Dis.l, 3-14.
Chapter 33
Unclassified Agents
Scrapie
Scrapie is an infectious and chronic degenerative condition of thc
CNS of sheep and goats caused by a small and uncharacterized agent.
The disease is endemic in UK but has been exported to Canada,
Australia, USA and India by British shccp.
Properties of the virus: The agent is small and is filterable through
a 40-50 nm filter. It is not known whcther the agent has a small nucleic
acid or no nucleic acid. It is suggested to be a small hydrophobic
complex of protein with an outer layer of protective glycolipid and
inner nucleic acid or a self replicating protease whose enzymic activity
produces more of the agent from precursors found in normal brain. The
agent is extremely resistant to protein and nucleic acid denaturants; e.g.
heat, formalin, propiolactone, ultraviolet light, nucleases, strong acids
and alkalies. The agent kills mice several months after inoculation and
has also been transmitted to rats and primates but no laboratory worker
has so far picked up the infection. Cells from the brains of infected
sheep and mice have been grown in culture and the replication of the
agent has corresponded to the replication of host cells. A continuous
cell line has been established from scrapie infected mouse brain, which
produces the scrapie agent continuously. The scrapie agent has also
been adapted to hamsters.
Pathogenesis: Sheep and goat can be infected by all routes of
inoculation with infected brain tissue. The incubation period in natural
cases is 2-3 years but experimentally it is 6-9 months. The disease is
Unclassified Agents
363
usually seen in sheep and goats, 3 years of age and older. Following
inoculation the agent is first recoverable from lymphoid tissues and 2-3
months later from the medullary region of brain. In the mid brain the
neurons become vacuolated and then shrink and this is accompanied by
the formation of myeloid plaques and proliferation of astrocytes. The
sheep first appear hyperexcitable with an erect bead and high stepping
gait Later on there are muscle tremors, intense pururitis or stuper and
finally ataxia develops. Deaths are most frequent in animals at 2-5
years of age and 10% of the stock may die in endemic area.
The remarkable feature of scrapic agent is the lack of humoral and
CM! responses in the infected animals.
No anitbody or inflammation is produced in the affected animals.
Infection is congenital either via ingestion of amniotic fluid or close
contact Contaminated vaccines may also spread the disease.
Diagnosis: This is' based on clinical symptoms. A definitive
diagnosis can be made by inoculating normal susceptible sheep or goalS
with suspected material and observing the development of clinical
disease as well as the characteristic histopathological lesions in the
central nervous system. There is spongiform degeneration and severe
vacuolation of neuronal cytoplasm. Hypertrophy of astroglial cells is
also characteristic.
Control: There is no vaccine available. The only method available
is to slaughter all the affected animals and animals those had contact
with the affected animals during past 3-4 years.
References
CARP, R.I.; MERz, P.A.; KAsESAK, R.J.; MERG, G.S. and WIsnieioski, H.M.,
1985. Nature of the scrapie agent. Current status and facts and
hypotheses J. Gen. Virol. 66, 1357-1368.
R.H., 1976. Slow virus disease of Animals and Man (Research
Monographs Frontiers of Biology 44) North Holland/Elsevier
Amsterdarn/New York, 264 pp
KIMBERLlN,
PRUSINER, S.B. and HADLOW, WJ., 1979. Slow Transmissible disease of the
Nervous system. Academic Press, New York Voll,472 pp and Vol2,
52 pp.
TYRELL,
D.A.J., 1979. Aspects of slow and Persistent virus Infections (New

Perspectives in clinical Microbiolgoy 2). Martinus Nijhoff, The
Hague, 286 pp.
Inilex
Abinanti, 151
Abortion, 180
Acyclovir, 109
Adcnoviridae virus, 15, 23-24,
164-74
avian adenoviruses, 171-74
bovine adcnoviruses, 165-67
canine adcnoviruses, 169-71
control of, 167, 169, 171, 174
cultivation of, 166, 168, 170, 172
diagnosis, 167, 169-71, 174
effect on egg production, 173-74
epidemiology, 166, 168, 170, 172
equine adenoviruses, 171
irnmunereaction, 167
inclusion body hepatitis, 173
infectious canine laryngotracheitis virus, 171
ovine adenoviruses, 167-69
pathogenesis, 166-70, 173-74
porcine adenoviruses, 171
properties, 165-70, 172
respiratory disease, 173
Adenoviruses, 94, 96,165-74_
African horse sickness virus
(AHSV), 260-62
African swine fever virus (ASFV),
206-08
Agar gel double inununo diffusion
test, 118, 121
Alimentary system, disease of,
180-81
Alphaviruses, 235-38
Anatid herpesvirus, 197-98
Animals,
entry ofviruses in, 68-71
viruses, 20-33,90-92
c1assifaction, 20-33
transmission of, 90-92
Antibiotics, 109
Antigens, in infected cells, 67-68
Anti-idiotype vaccine, 107
Antiviral drugs, 107-08
Arabinofumosyl nucleosides, 109
Arboviruses, 12-13
Arcnaviridae, 17, 29
Arterivirus, 245-46
Astroviruses,19
Attenuated virus vaccine, 102-05
Avian adenoviruses, 171-74
Avian encephalomyelitis virus
(AEV),226-27
Avian infectious bronchitis virus
(lBV),274-76
Avian infectious laryngotracheitis
virus, 194-96
Avian influenza virus, 281
Avian leukosis, 329-33
Avian paramyxovirus-3, 289
Avian paramyxoviruses, 285-89
Avian reovirus, 254
Avian reticu1aendotheliosis
viruses, 331-33
365
Index
B-virus, 193-94
Bacteria, properties of, 4-5
Baudet, A.E.R.F.,197
Beach, I.R. 285
Beijerinck, 3
Benzimidazols.109
Berne virus. 358-59
Birnaviridae viruses. 17, 29,
264-66
Bittner, 333
Blue tongue virus (BTY), 257-60
Border disease virus (BOY), 241-42
Borna disease virus, 360-61
Bovine adenoviruses (BAY), 165-67
Bovine coronavirus, 268-70
Bovine enteroviruses, 225-26
Bovine ephemeral fever virus,
319-20
Bovine herpes virus-l (BHY-l),
177-82
Bovine herpes virus-2 (BHY -2),
182-83
Bovine herpes virus-3 (BHY-3), 187
.Bovine leukemia virus (BLY),
323-26
Bovine papillomavirus, 160-62
Bovine papular stomatitis virus, 143
Bovine parvoviruses (BPOY),
151-53
Bovine reoviruses, 252-53
Bovine respiratory syncytical
virus (BRSY), 305-06
Bovine rhinovirus-I, 228-29
Bovine rotavirus, 254-57
Bovine syncytial virus (BSY).
333-34
Bovine viral diarrhoea virus
(BybY),238-41
Breda virus, 356-58
Brooksby, J.B., 221
Buffalo pox virus, 135-36
Bungaviridae virus, 19,32-33.
347-54
akabane virus, 351-53

control, 351, 353
cultivation, 349, 352
diagnosis, 350-351. 353
epidemiology, 349, 352
immune reaction, 350, 353
Nairobi sheep disease, 353-54
pathogenesis, 349-50, 352-53
properties, 348, 351-52
rift valley fever virus, 348-51
Bumet,47
Calciviridae, 16-17,27,231-33
Camel pox virus, 136-37
Canine adenoviruses, 169-71
Canine coronaviruses (CCY), :UO-71
Canine distemper virus (CDV),
78,294-96
Canine papillomavirus, 162
Canine, parvov:ruses, 156-57
Caprine arthritis-encephalitis
virus (CAEV), 340-42
Carbohydrate, 12
Cell cultures, 49-54
production of, 50-51
virus growth in, 51-53
Cell mediated cytotoxicity, 83
Cell mediated immunity, 80, 83
Cell transformation, 68
Cellular protein, 66
Chase,3
Chicken embryo, 48
Chlamydia properties, 4-5
Chronic infections, 77-78
Clinical virology, 124-25
Cloned viral DNA, 107
Complement fIXation test (CFf).
118,121
Coronaviridae viruses, 17, 30,
267-76
bovine coronavirus. 268-70
canine coronavirus, 27~71
control, 270. 273, 276
366
cultivation. 274-75
diagnosis, 270, 273, 276
epidemiology, 268-69, 272, 275
faline infectious peritonitis
virus, 271
iminune reaction. 270, 275-76
pathogensis, 269-70, 272-73,
275
porcine coronaviruses, 272-76
properties, 268,272,274
Cow-pox virus, 133-35
Creutzfeldt Iakob diSease, 78
Crowther, I.R., 213
Cytopathic effect, 51, 65-67
DNA cloning technology, 122-23
DNA replication, 41
DNA tumor viruses, 94-101
DNA viruses, 14-16,20-26,129-208
adenoviridae, IS, 23-24, 164-74
herpesviridae, 15-!6, 24-25,
176-202
papovaviridae, 15, 22-23, 159-62
parvoviridae, 15,21-22, 150-57
poxviridae, 14-15,20-21, 12948
unclassified, 206-08
Datt, N.S., 217
Dhanda, M.R., 217
d'herelle, 3
Diagnosis of viral diseases, see,
under Viral diseases
Doyle, T.M., 285
Duck hepatitis virus (DHV), 227 -28
Duck herpesvirus-I, 197-98
Duck plaguevirus, 197-98
Duck virus enteritis, 197-98
Dulbecco, 52, 55
Dunne, H.W., 226
Ecthyma (Ort) virus, 14143
Edwards, I.T., 102,285,303
Electron microscopy, 54
TeXlbook of Vclerinary Virology

Enders,49
Enteric viruses, 13
Enveloped viruses, 43
Enzyme linked immunosorbent
assay (EUSA), 118, 120-21
Epithelial polyhedral cell, 50
Equine adcnoviruses, 171
Equine arteritis virus (EAV), 24546
Equine encephalomyelits virus,
235-38
Equine herpes virus-I, 188-90
Equine herpes virus-2, 188
Equine herpes virus-3, 190
Equine infectious anaemia virus
(EIAV), 334-35
Equine influenza virus, 279-80
Equine papilloma virus, 162
Equine rhino pneumonits virus,
188-90
Equine rhinovirus, 229
Experimental animals, 4647
Feline calcivirus (FCV), 232-33
Feline infectious peritonitis virus
(FIPV).271
Feline lentemia virus, 326-28
Feline panleucopenia virus (FPLV),
154-56
Feline parvovirus, 154-56
Fibroblast, 50
Fibroma virus, 145
Filoviridae. 19, 33, 356-59
Aaviviridae, 17,28,247-50
Foetus, damage to, 74-75
Foot and mouth disease virus
(FM DV). 77-78, 107,213-23
Foot and mouth disease virus
antigenes, 213
Fowl herpesvirus-I, 194-96
Fowl plague virus, 281-82
Fox encephalitis virus, 169-7]
Fraenkel-Conrat, 4
Ind~
367
Genome, of animal viruses, 57-59

mapping of 58-59
Glucosamines, 109
Goat pox virus, 140-41
Goodpasture.47
Greig. A.S., 177
185-86, 192-97,200-01
properties, 117-78, 182-84,
188, 191, 193-95, 197-99
Herpesvirus of goats (CHY-2),
187-88
Herpesvirus of sheep (CHY -1), 187
Hershey.3
Hirst, 53
Hog cholera, 242-45
Haemadsorption inhibition test

(HO!), 119, 122
Haemagglutinatingencephalomyelitis virus (HEY),
273-74
Haemagglutination, 52-54
Haemagglutination inhibition (HI)
test, 119, 121-22
Helical symmetry, 7-8
Hen's egg, embryonating, 47-49
Hepadnaviridae, 16,26
Hepatitis B-viruss. 94. 96
Herpesviridae viruses. 15-16. 24-25,
177-202
bovine herpes virus-I, 177-82
bovine herpes virus-2, 182-83
clinical manifestation. 179-81
control, 181-82, 186-87. 190.
192-94,196,198,202
cultivation, 178. 182-84, 188-89,
191. 193-95. 197. 199
diagnosis. 181. 183, 186, 190,
192. 194, 196, 198
epidemiology. 178-79. 183-85.
189, 191-95, 197, 199-200
equine herpesviruses, 188-98
immune reaction. 181. 186,
201-02
malignant catarrhal fever virus,
183-87
Marek's disease virus, 198-202
pathogenesis. 179, 183,
Icosahedral symmtery, 7
Icosahedral virus, 43
Immunity. viral. 81-85
Immuno-electron microscopy, 118,
120
Immunofluorescence. 51
Immunofluoresce'}cil and
immunoperoxidase te~ts, 118-20
Immunopathology, 85
Immunoperoxidase. 51.118-20
Inactivated virus vaccine. 102,
104-06
Inclusion body hepatitis. 173
Infant. damage to. 75
Infestions,
chronic, 77-78
defence mechanism. 80
latent, 76-77
pathogenesis of. 79-80
persistent. 67. 76-80
slow, 78-79
spread of viruses and, 68-72
viral. 81-82. 124-25
Infectious bursal disease virus
(!BOY). 264-66
Infectious canine hepatitis virus
(ICHY).169-71
Infectious canine
laryngotracheitis virus, 171
Influenza virus, 36. 279-82
Interferon, 110-14
Intrnational Committee on
Frenkel, H.S., 215

Frosch, 3, 46
368
Taxonomy of Viruses (lCfV), 14
Intramolecular recombination of
viruses, 59-61
Iridoviridae virus, 16, 25-26
Issacs, 109 '
Ivanovski,3
Jaagsiekte (Ovine pulmonary
adenomatsis) virus, 342-45
Japanese B encephalitis virus,
248-49
Jenner, Edward, 102-03
Kendrick, 177
Kilham,75
Killed vaccine, 104-06
Kranoveld, F-C., 285
Kulkami, 0.0., 257
Kulkami, M.N., 257
Latent infections, 76-77
Lentiviruss, 334-45
Lindenman, 109
Lipid, 12
Live virus vaccine, 103-04
Loeffer 3, 46
Louping III virus, 249-50
Lumpy skin disease, 141
Lwoff,4
Lympho choriomenigitis virus
(LCM), 77, 85
Macrophages, 50, 80
Maedivirus,78,337-40
Malignant catarrhal fever virus
(MCFV), 183-87
Mammalian parainfluenza virus,
289-93
Marburg virus, 320
Marek's associated turnor specific
antigen (MATSA), 201-02
Marek's disease virus, 51, 73,
103,178,198-202
Te~book
o/Veterinary Virology
Marennkikova, 134
Mastitis, 180
Matsuyama, T., 351
Mehrotra, M.L., 169, 177
Messenger RNA production, 38-40
Milker's node virus, 143
Mohanty, S.B., 198,264
Monolayer culures, 50
Morbilivirus, 293-305
Mornet, 303
Mucosal disease virus (MDV),
238-41
Mukerjee, A., 169
Murine leukemia, 328-29
Murine mammary tumour virus
(MMTV),333
Murty, D.K., 238
Mutation, of animal viruses, 56-57
Mycoplasma, properties of, 4-5
Nair,169
New castle disease virus (NO V),
285-89
Nonenveloped virus, 3435
Nucleic acid, 9-10
Nucleic acid hybridisation, 122
Nucleic acid sequency, 123
Nyak, B.O., 238
C>ncogenes, 96-98
C>ncoviruses, 323-33
Organ cultures, 51
Orthomyxoviridae virus, 18,30,
278-82
Ovine adenoviruses (OAV).
167-69
Oya, A_,351
Pande, P.G., 238
Papillomaviruses, 94-96
Papovaviridae viruses, 15, 22-23,
159-62
Parainfluenza-l virus, 289
Index
Parainfluenza-3 virus. 289-93
Parainfluenza-5 virus. 293
Paramyxovirus. 284-93
Parihar. N.S . 183
Paramyxoviridae viruses. 18,31,
284-306
control, 288-89, 292, 296, 302-03,
306
cultivation, 286, 298
diagnosis, 292, 295-96, 301-02,
306
epidemiology, 286, 290-91, 295,
298-99,305-06
Unrnunereaction,292,301
morbilivirus, 285, 293-305
paramyxovirus, 284-93
pathogenesis, 286-88, 291, 295,
299-301,306
pn~ovirus,285,305-06
properties, 285, 290, 294-95,

292-98,305
Parvoviridae virus, 15,21-22,
150-57
bovine parvoviruses, 151-53
canine parvoviruscs, 156-57
control, 153-54, 156
cultivation, 151, 153, 155
diagnosis, 152-54, 156
epidemiology, 151-52, 154-55
feline panleucopenia virus, 154-56
pathogenesis, 152-54, 156
porcrine parvovirus, 153-54
properties of. 151, 153, 155
Pasteur, Louis, 46,102,313
Pathogenesis of viral infections,
65-75
cytocidal infection, 65-67
non cytocidal infection, 65, 67-68
non cytocidal non productive
infection, 65, 67-68
transformation, 66. 68
Persis.tent infections, 67, 76-80
369
Peste-des-petits-ruminants virus,
303-05
Pestivirus, 23845
Picomaviridae virus, 26-27, 213-29
apthovirus, 212-23
control. 222-25, 228-29
cultivation, 214. 224-,28
diagnosis, 218-22, 224-29
enterovirus. 223-28
epidemiology, 215-17, 224-28
irnmunereaction.218
pathogenesis, 217, 224-28
properties of, 212-14,223-24,
226-28
rhinovirus, 228-29
Pigeon herpesvirus, 198
Plaque assay, 52-53
Pneurnovirus,305-06
Pock assay, 53
Polio encephalomyelitis virus,
223-24
Polymerase chain reaction (PCR),
124
Polyomaviruscs, 94-95
Porcine adenoviruscs, 171
Porcine enterovirus-l , 223-24
Porcine enterovirus-9 (PEY -9).
224-25
Porcine herpes virus-I, 191-93
Porcine parvovirus (PPY), 153-54
Pox viruses, see, Poxviridae viruses
Poxviridae viruses, 14-15,20-21,
12948
avipox virus, 130, 14648
capripox virus, 129,13841
control, 135-37, 13941, 14345.
14748
cultivation, 131-33, 136-40. 142,
144, 146
diagnosis, 132-33, 135-37.
13940, 142, 144-47
epidemiology, 132, 134, 136-37,
13940, 142, 144-47
370
Textbook ofVelerinary Virology
family, 129-30
immune reaction, 132, 134,
136-37
1eporipox virus, 129, 144-45
orthopoxvirus, 129-38
parapox virus, 129, 144-43
pathogenesis, 132, 134, 136-37,
139-40,143-45,147
properties, 131, 133, 135-36, 138,
140-42, 144-46
suipox virus, 129, 143-44
Proteins, 10-11
Pseudorabies virus, 191-93
Purines antagonists, 108
Pyrarnirdine antagonists, 108
control, 253, 256, 262

cultivation, 258-59, 261
diagnosis,253,256, 260
epidemiology, 253, 255-56, 261
Fiji virus, 251
immune reaction, 260
orbiviruses, 251. 257-62
pathogensis,253,256, 259-61
phytoreovirus, 251
Quantal assay, 53
RNA viruses,
bimaviridae. 17, 29,264-66
bunyaviridae, 19.33,347-54
calciviridae. 16-17,27.231-33
coronaviridae, 17,30,267-76
flaviviridae. 17.28,234.247-50
orthomyxoviridae, 18,30.278-82
pararnyxoviridae. 18.31,284-306
picomaviridae, 26-27,211-29
reoviridae. 17,28-29.251-62
retroviridae, 19,32-33.322-45
rhabdoviridae, 18,32.309-20
togaviridae, 17,27-28,234-46
toroviridae, 19.33,356-59
unclassified. 360-61
RNaseL,113
Rabbit papillomaviruses. 162
Rabies virus, 313-18
Radio immunoassay (RIA), 118. 120
Reassortment, 60-61
Remlinger, 313
Reoviridae viruses, 17, 28-29.
251-62
~perties,252-55,257-58,261
rovirus, 251-54
rotavirus, 251. 254-57
Reovirus, 252-54
Replication groups, 43-44
Replication of viruses, 34-44
Respi!atory viruses 13
Restriction endonuclease digestion
of DNA, 122
Retroviridae viruses, 18, 32, 322-45
control, 326, 328-29, 331, 334,
336, 340. 342, 345
diagnosis. 326-29, 331-33, 334,
336, 339, 342, 344
epidemiology, 324-25, 327, 330,
335,337,340-41,343
immune reaction. 326,339,342,
344
lentiviruses, 334-45
oncoviruses,323-33
pathogenesis, 325-32, 334-36.
338-39,341-44
properties, 323-24. 326-30,
332,334-35,337,340,343
spumaviruses, 333-34
Retroviruses, 96-98
Rhabdoviridae virus, 18,31,309-21
bovine ephemeral fever virus,
319-20
control, 312-13, 318, 320
diagnosis,312,317-18,320
epidemiology, 311,315-16,319
immunereaction,312
371
Index
marburg virus. 320
pathogenesis. 311-12. 316-17. 319
properties, 310-11. 313-15. 319
rabies virus. 313-18
vesicular stomatis virus, 310-13
Rhinovirus. 228-29
Rickettsia, properties of. 4-5
Rift valley fever virus (RVFV),
348-,51
Rinderpest virus, 296-303
Robbins,49
Rotaviruses. 254-57
Rowe, 164
Rubarth, 169
Sapre, SN., 257
Sarcoma viruses, 326-31
Sorapie, 362-63
Seet.haraman, C.. 217
Sendaivirus,289
Shaila, M.S., 305
Shedding ofvirus, 73-74
Sheep pox virus, 13840
Simian herpes virus-I, 193-94
Singh, 95, 183
Skinner, 215
Slow infection, 78-79
Spwnavirus, 333-34
Stanley, 3
Stoke's law,S
Subacute spongiformviral
encephalopathies, 78-79
Suspension cultures, 50-51
Swine enteroviruses, 223-25
Swine fever, 24245
Swine influenza virus, 280-81
Swine pox virus, 143-44
Swine vesicular disease virus,
224-25
Talf" dis~e, 223-24
Teschan virus 223-24
Thiosemicarbazones, 108
Togaviridae viruses, 17,27-28,
234-46
alphaviruses, 235-38
arterivirus, 245-46
control, 23~, 240-42, 244-46
diagnosis, 237, 240, 242. 24446
epidemiology, 237, 239, 241,
243,245
family of, 236
flaviviridae and, 234
bmununereaction,240
pathogenesis, 237, 23940
pestivirus,23845
properties of, 236-38, 241, 243.
245
Toroviruses,19
Toumier,4
Transformation assay, 53
Transmissble gastroenteritis virus
(l'GEV), 272-73
Trautwein, K., 214
Tubular symmetry, 7-8
Tumor viruses,
effect of interferon on
transformation by, 113-14
families, 94-96
oncogenes, 96-98
retroviruses, 96-101
transformation of genes, 96-98
Twort, 3
Unicellular organism, properties of,
4-5
Vaccinia virus, 131-33
Varet monkey disease, 320
Vasudeven, D.W., 257
Vesicular disease diagnosis, 219
Vesicular exanthema virus
(VEV),231-32
Vesicular stomatis virus (VS V),
310-13
372
Viral disease,
diagnosis of, 115-25
material for, 115-16
molecular biological
techniques, 122-25
serological methods, 118-22
viral nucleic acid detection, 122
virus isolation 117-18
visualisation by election
microscope, 116-17
epidemiology of, 87-92, 124
dissemination of virus, 91-92
environmental factors, 89
host agent, 89
incubation period of vi~s,
91-92
perpetuation of viruses, 89-90
tools, 87-88
transmission of viruses, 90-92
viral agent 88
Viral envelope, 8
Viral genetics, 55-63
genes in viruses, 58
genome, 57-59
marker rescue, 60, 62
mutation, 56-57
reactivation, 60
restricted endonuclease cleavage
maps, 60, 62-63
viruses recombination, 59-60, 63
Viral genome replication, 40-43
Viral immunity, 81-85
antibody effect, 82-83
cell mediated immunity, 83
cytotoxic T cells 83-85
humoral response, 82
infection, 81-82
Viral infectivity assay, 52-53
Viral protein, cytopathic effect of, 66
Viral replication, 34-44
Viral synthesis, 37-38
Viral vaccines,
antiviral drugs, 107-08

attenuated, 102-05
categories, 102-06
inactivated, 102, 104-06
inhibitors, 108-09
interferences, 109-14
biological effects,1l1-13'
clinical use, 114
prod~tion of, 111
tumor virus and,113-14
types of, 110
isolated irnmunogenes, 106-07
Virion assembly, 43
Viroiogy,
divisions of, 3
see Viruses
Virus neutralization test (VN),
118-19
Virus protein synthesis, 40
Viruses,
chemical structure, 8-12
classification, 13-33
cultivation, 44-54
DNA viruses, 14-16,20-26,
129-208
detection and identification,
124-25
diseases, 82-92, 115-25
dissemination, 91-92
entry to animal body, 88-72
genes, 58
genetics, 11-12,55-63
immunity,81.85
incubation period, 91-92
morphology, 5-8
pathogenesis, 65-75
perpetuation of, 89-90
physical structure, 5-8
properties, 4-5
RNA viruses, 16-19,26-33,
211-361
replication, 34-44
see also specific vi.,.ses
373
Index
shedding, 73-74
sized,4-5
sprelld of, 68-72
transmission of, 90-92
tumorogenesis !Lid, 94-101
vaccines, 102-14
Visna virus, 78, 337-40
Waldman, D., 214

Warfield, 151
WeBer, 49
Wesselsbom virus, 249
Woodruff,47
Zinke, 313

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TEXTBOOK

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Tel.: 91-522-2209542,2209543, 2209544,2209545

No part of this publication may be reproduced, stored in a

TeXlbook of Veterinary Virology

Textbook o/Velerinary Virology

Textbook of Veterinary Virology

Textbook of Veterinary Virology

and only DNA was necessary for infection. Fraenkel-Conrat (1956)

Bacteria Mycoplasma Chlamydia

Structure and Composition

The criteria given above clearly distinguish viruses from other

Textbook ojVeterinary Virology

capsomeres consist of one or more molecules of polypeptidcs and are

Fig. 1.1 Schematic Diagram of the Structure of a Virus

The assembly of capsomeres in a virion is defined by the nature of

Fig. 1.2 Schematic Representation of the Structure of a Virus

Structure and Composition

1. lcosahedral Symmetry: An icosahedron means a figure of 12

The lrosahedron. It h~ a Rotational Axes of

2. Helical or tubular symmetry: The nIJcleocapsids of ~ever?l

Textbook o/Veterinary Virology

Structure and Composition

to their considerable purification. The important technique introduced

Textbook o/Veterinary Virology

The genome of all DNA viruses consists of a single molecule,

Structure and Composition

over 100 in ease of poxviruses. There is normally one copy of viral

Structure ofNucleic acid

The surface proteins have an affinity for the specific receptors on

Textbook of Veterinary Virology

3. Lipid and carbohydrate: These constituents are found only in

Textbook o/Veterinary Virology

Poxviridae: Pock means a pustule or ulcer. These are complex

Textbook o/Veterinary Virology

the inner lamellae of nuclear membrane into endoplasmic

Picornaviridae: The name picorna is originally derived from

IcosahedIal with 32 capsomeres and measure about 35-40 nm if.

Textbook of Veterinary Virology

horizontally. The family is subdivided into 3 subfamilies, two of which

F. 1986. The classification and nomenclature of viruses. Summary of

Leporipoxvirus Myxoma virus

Contagious ecthyma virus

Swine pOx virus

Fowl pox virus

Kilham rat virus

Adeno associated virus

Papillomavirus Cotton tail rabbit

Rabbit oral papilloma virus

Mastadenovirus Adenovirus type 2

Herpes simplex virus

Varicella zoster virus

Bovine herpes virus 1,4