1.

GENERAL VIROLOGY
1.1. HISTORY AND DEVELOPMENT OF VIROLOGY
The history of human development has been shaped by at least three major recurring elements.
These are:-
 Environmental changes
 Human conflicts
 Infectious diseases
With regard to infectious diseases the impact has been not only directly on the human population but
also on the food supply. The origins of veterinary medicine are rooted in efforts to maintain the
health of animals for food, fiber production and for essential work by animals’ force. Control of
animal disease outbreaks that were caused by bacteria and viruses was not possible until the
pioneering work of the late 19th century that linked microbes to specific diseases of plants and
animals. The beginning of virology is linked with the work of Ivanovsky and Beijerink (1892 –
1898).They discovered the transmission and filterable nature of the agent that cause tobacco mosaic
disease.This transmissible and filterable agent is a tobacco mosaic virus. Both scientists were able to
show the transmission of the agent causing disease in tobacco plants using fluids that passed through
filters that retained bacteria. Beijerink also noted that the filterable agent could regain its “strength”
from diluted material only if it was put back into the tobacco plants. The initial studies on tobacco
mosaic virus led to further understanding of “filterable agents”—namely viruses. Specifically, the
high concentration of virus produced in infected tobacco plants permitted the chemical and physical
characterization of the infectious material.
Loeffler and Frosch (1898) discovered the filtration criteria of the mycoplasmas. Some agents
were inactivated with organic solvents like ether and ester whereas others are resistant. For research
on animal diseases, early workers were restricted to use animal inoculation in order to assess the
impact of any treatment on any putative disease causing agent. A further advance in animal virology
was the use of embryonated eggs for culturing virus in 1931. In the same year, Shope identified
influenza virus in swine; and in 1933, influenza virus was isolated from human cases of the
infection. The identification of the strain H1N1 in swine might be considered the first “emerging”
disease in animals that is a virus crossing a species barrier and maintaining itself as an agent of
disease in the new species. Attempts were done to move away from large animal experimentation to
study different viruses in laboratory; as the result mice and rats became important tools for studying
animal viruses. The era was the era of the birth of laboratory animals when they became essential
back bone for medical viral research works.
In 1938 – 1948 research works with bacteriophages those were done by Ellis, Delbruck and Luria
made advance in understanding the properties of viruses much more rapidly because the work could
be done in artificial media without the need of living plants or animals. The bacterial viruses
(bacteriophages) assisted in defining some of the basic principles of genetics through the study of
mutations and the inheritance of phenotypic changes. In 1946 Bovine Viral Diarrhea Virus
(BVDV) was identified in USA as a new disease causing agent of Bovine Viral Diarrhea in cattle.
In 1948 – 1955, animal virus studies made a dramatic shift in emphasis with development of
reliable “in vitro” animal cell cultures; as the result: -
 Single cell culture procedures were defined.
 Cell culture media were standardized.
 Human cell lines were developed.
 The growth of polio virus in non – neuronal cell was demonstrated.
Cell culture procedures permitted isolation of the viruses in the early of 1960s.So that “in
vitro”replication of viruses in cell cultures helped:-

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  To isolate and to identify the new viral gents
 Soon enabled them in the production of effective vaccines for many viral diseases.
In 1961 influenza virus was detected for the first time in wild birds. This led to the identification of
fowls and shore birds as natural reservoir of Avian Influenza Virus (AIV).The beginnings of the
molecular era of virology reside in the late 1970s and early 1980s.Although molecular biology is not
related directly to virology, the development of the Polymerase Chain Reaction (PCR) in 1983
had an impact on virology cloning of nucleic acid sequences and this led to:-
 To have the first infectious molecular clone of a virus (poliovirus) in 1981.
 To use molecular techniques for diagnostic purpose in identification of viruses by molecular
technique without isolation of the virus from specimens (“in-vitro” culture of the virus).
 To identify some viruses those could not replicate even upto nowadays “in vitro” cell
culturing technique like:-
• Papillomaviruses
• Noroviruses
• Rotaviruses
• Certain nidoviruses
Viruses that could not be easily cultured “in vitro”could now be characterized and routinely
detected by tests at the molecular level. The first virus that was identified by molecular technique
was hepatitis C virus. Then after, as analytical chemical procedures developed, it was shown that
viruses contained nucleic acids. When Watson and Crick defined the structure of DNA; it helped
them to understand that: -
 Viruses became key players in defining the role of nucleic acids as the database for life( that
means viruses became tools with which to probe the basic biochemical processes of cells
including gene transcription and translation).
 Some of scientists believed that the future value of viruses would only simply be as tools for
studying cellular processes no more.
However, the unpredictable emergence of new viruses such as: -
 HIV virus
 Hepatitis C virus
 Nipah and Hendra viruses
 The high-pathogenic H5N1 influenza virus
Together with the expansion of individual viruses into previously free areas such as: -
• West Nile virus into North America
• Bluetongue virus into Europe
It made clearly to confirm that much has yet to be learned about this class of infectious agents and
the diseases that they cause. Viruses have traditionally been viewed in negative context because they
are considered only as diseases producing agents in human beings, animals and plants; that is why
we understand that all viruses must be controlled and eliminated. However, a few viruses have some
beneficial properties that can be exploited by us for useful purposes and some of the useful
properties of a few species of viruses are: -
1. Viruses have been engineered to express proteins for production of non virus proteins; example
baculovirus.
2. A few species of viruses are used to prepare vectored vaccines for immunization purpose because
they can express viral proteins of other species; example attenuated vaccinia and adenoviruses are
used as vector to prepare recombinant vector vaccine of small pox vaccine because the attenuated
organisms serve as a vector, replicating within the host and expressing the gene product of the
pathogen.

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3. Lent viruses have been modified for the purpose of inserting new genetic information into cells
for research and gene therapy purposes.
4. Bacteriophages are being considered useful for controlling certain bacterial infections.
5. Some viruses are used to control some plant pests; example baculovirus infest plant pest like
insects.
6. Hypothetically, it is considered as viruses may have potential to be vectors that selectively target
tumor cells for controlling cancers.
In broader context of our Earth’s ecosystems, viruses are now viewed not only in negative sense but
also in positive sense because they may be a component of population control and perhaps a force in
the evolution of species in our ecosystem. Therefore, the ability of viruses to restrict the population
of human beings, animals and plants is viewed as negative from the human perspective but the
ecosystem might benefit from the reduction of some species in its success is at expense of others.
Generally, there are many species of viruses which are harmful to animals, human beings and plants
but there are a few species of viruses which are useful. It is known that, we are now fully
comfortable with the concept of the presence of beneficial viruses in our ecosystem.
Do we need to start to consider that viruses that have evolved with the species may also have
beneficial properties?
1.2. VIROLOGY AND ITS SUBDISPLINES
Virology is a branch of microbiology which studies viruses. Scientists who study viruses are called
virologist. Virology has many branches and some of the common branches of virology are:-
 Veterinary virology
 Medical virology
 Plant virology
 Entemovirology
Veterinary virology began as a discipline focusing on the effects of viral infections on animals of
agricultural significance. Veterinary virology focuses on:-
 Understanding the viral diseases of animals
 In the characterization of the viruses of veterinary importance
 In the development of the fields of immunology to study the viral infection process.
 Improvement of diagnostic technologies to diagnose viral animal diseases
 Working on the methods of controlling of viral animal and zoonotic viral diseases

1.3. MICROSCOPY OF VIRUSES; TYPES OF ELECTRONMICROSCOPE

AND ELECTRONMICROSCOPY TECHNIQUES
We know that the resolving power of light microscope unable us to demonstrate most of viruses, so
that to demonstrate viruses needs special microscope which has a resolving power lower than light
microscope. An instrument which is used to demonstrate viruses is called electronmicroscope.
Demonstrating of viruses using electronmicroscope is called electronmicroscopy. All viruses are
visible only under electron microscope; however, poxvirus which is the largest virus can be seen
under light microscope as its size is as large as mycoplasmas.The first electronmicroscope was a
transmission electronmicroscope; and it was constructed by the German engineers Max Knoll and
Ernst Ruska in 1931; however its magnification power was lower than the light microscope. Two
years later, Knoll and Ruska successfully proved the design and were possible to construct the first
electronmicroscope with magnification power higher than light microscope. Electron microscope is
a type of microscope that uses a beam of electrons to create an image of the specimen and as a
source of electrons; the electron microscopes are commonly fitted with a tungsten filament

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cathode (electron gun) while phosphorus sheet is used as deflector of beam of electrons; so that
electron transparent regions appear brighter while denser region scatters electrons and appear darker.
Electronmicroscope has: -
 Higher magnification power than light microscope
 Greater resolving power than a light microscope
These properties of electronmicroscope allow us to see much smaller objects in finer detail. An
electron microscope (EM) has greater resolving power than a light-powered optical microscope
because electrons that are used in electronmicroscopes have a wavelength which is 100,000 times
shorter than visible light (photons). The resolving power of a microscope is indirectly related to the
wavelength of the irradiation used to form an image but it is directly related to the speed of light
used so that beam of electrons have shorter wavelength and travel faster than light, and has higher
resolution power; that is why electronmicroscopes have a resolving power of 0.5 nm; while light
microscopes have a resolving power of 0.2 μm. Electronmicroscope is used to observe objects
smaller than 0.2μm and has an ability to magnify objects even down up to 0.05 nanometers like: -
 Viruses
 Internal structures of living cells (nucleus, mitochondria, ribosome and endoplasmic
reticulum etc)
 Ultra internal and external structures of bacteria and other microorganisms
All electronmicroscopes use electromagnetic and/or electrostatic lenses to control the path of
electrons. The maximum magnification power electron microscopes may reach upto 1000000X.
However, electronmicroscope differed from light microscope in that:-
 They are large in size
 They are expensive pieces of equipment, generally standing alone in a small, specially
designed room.
 They require special trained personnel to operate them.
Some important parts of electronmicroscope

1.3.1. Types of electronmicroscope

Due to their difference in the way of image formation by beam of electrons, there are four types of
electron microscope.
 1.Transmission Electron Microscope (TEM)
 2.Scanning Electron Microscope (SEM)
 3. Scanning Transmission Electron Microscope (STEM)
 4. Reflection Electron Microscope (REM)
Among them the most widely used are TEM and SEM.
1. Transmission ElectronMicroscope (TEM)
Transmission electronmicroscopes produce images by recording the electron beam after it has
passed through a thin slice of specimen. The specimen is placed on a copper wire grid and subjected
to an electron beam, normally generated by running high voltage across a tungsten filament. The

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electron beam travels through a condenser lens, strikes the specimen and image is formed on
fluorescent screen which has camera.
2. Scanning ElectronMicroscope (SEM)
Scanning electron microscopes, along with transmission electron microscopes, are the most widely
used. Unlike the TEM, scanning electronmicroscope (SEM) produces images by collecting the
secondary or in elastically scattered electrons that bounce off the surface of a specimen. The primary
electron beam travels through several condenser lenses, scan coils and an objective lens before
striking the surface of the specimen. The electron beam is scattered upon hitting the specimen and a
secondary electron detector collects the scattered electrons. The electron data is then raster-scanned
to produce surface images with considerable depth of field.No sectioning is required and produces a
3-dimensional image of specimen’s surface features.
Magnification ranges of light and electron microscopes

1.3.2. Electronmicroscopy techniques

The following techniques are commonly used to reveal the different structural details of viruses by
using electron microscopy.
 1. Negative staining
 2. Positive staining
 3. Combined staining
 4. Thin sectioning
1. Negative staining
The viral particles are mixed with a solution of a salt which has high opaque to electrons. The usual
solution which is used in negative staining of viral particle is sodium phosphotungstate. The
mixture is then spread on thin layer on film and dried. The salt solution surrounding the viral
particles penetrates between viral particles and the film then when you examine under electron
microscope, the parts of viral particles that are not penetrated by the salt stand out as electron –
lucent areas on an opaque back ground; as the result, surface projections where viral particles are
found as transparent areas on an opaque back ground.
2. Positive staining
Certain components of viruses can be stained by salts which become selectively absorbed by viral
particles. The usual salt which stains the viral nucleic acid and other components of virus particles is
uranyl acetate. Antibody conjugated to electron – opaque molecules like ferritin can be also used
as positive stains as it stains the proteins of the viruses. However, this method of staining has
specificity to particular virus strains.
3. Combined staining

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This method of staining uses both negative and positive staining methods in combined. Combined
staining method has a better result in demonstration of viral particles than negative and positive
staining alone.

4. Thin sectioning
This method is used to study viral particles in cells or centrifugal pellets. The size of viral particles
in thin sectioning will be smaller if we use negative staining with sodium phosphotungstate after
thin sectioning of tissues because the action of knife tends to collapse the viral particles. What you
must understand is that the size of viral particles obtained by negative and positive stainings of viral
particles or by thin sectioning is smaller than the size of viral particles in water because as drying is
required for electron microscopic examination which causes shrinkage of as much as 30% in linear
dimensions.

1.4. VIRUSES VERSUS UNICELLULAR ORGANISMS

Initial operational definition of viruses was filterable agents and this definition of viruses was made
to identify viruses from other microorganisms. However, there are some bacteria those are filterable
through bacterial filter like mycoplasmas and chlamydiaes and there are also some DNA viruses in
the family of Poxviridae and Iridoviridae which are retained by conventional bacterial filter with the
size of 300 nm. Even in the earliest time, it was known that the filterable agents called viruses were
not grow in/on artificial media and were considered the only obligate intercellular parasites;
however, all obligate intercellular parasites are not only viruses because members of certain
bacterial genera like Chlamydia and most Rickettsia are also unable to replicate outside the host cell.
This “degenerate” that some bacteria lack key metabolic path ways, the products of which must be
provided by host cell. Viruses, by contrast, lack all metabolic capabilities necessary to reproduce,
including energy production and the processes necessary for protein synthesis. Viruses do not have
cellular organelles like mitochondria, chloroplast, Golgi, endoplasmic reticulum with associated
ribosomes. However, certain bacteriophages have genomes that encode certain enzymes involved in
nucleotide biosynthetic path ways and it is known that viruses are inert outside living cell but inside
living cells, viruses replicate by using living cell processes to synthesis their proteins and nucleic
acids. Other inviolate characteristics of viruses are that they do not reproduce by binary fission,
asexual reproduction in which a pre – existing cell splits into two identical daughter cells. However,
viruses replication resembles as assemble line in which:-
 The virus attach to susceptible host cell
 Then the virus enters to the cell
 In the cell the intact virus particle ceases to exist in the cell.
 The virus genome directs the synthesis of new viral macromolecules.
 Finally the genome and protein of virus are assembled and results in ultimate re – emergency
intact many progeny of viral particles.
The period of time between penetration of virus particle into the host cell and production of the new
virus particle is called eclipse period. Duration of eclipse period varies with each virus family.
Disrupting cells during the eclipse period will not release significant numbers of infectious virus
particles; so that uninterrupted, a single infectious particle can replicate within a single susceptible
cell to produce thousands of progeny virus particle. In general viruses contain one type of nucleic
acid; either DNA or RNA that carries information for replicating viruses.
One-step growth curve of viruses

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The eclipse period is the period during which no intracellular infectious virus can be recovered but
infectious nucleic acid might be recoverable in some cases. The burst size is the average yield of
infectious virus per cell.
Properties of unicellular microorganisms and viruses
Properties Bacteria Mycoplasmas Rickettsiaes Chlamydiaes Viruses
Relative Complex Complex Complex Complex Simple
organization
Canbe Obligate
Relation to Obligate Obligate
extracellular Extra cellular intracellular
host cell intracellular intracellular
and facultative except one
intracellular genus
Size > 300 + + + + -
nm diameter
Growth
on/in non – + + - - -
living
medium
Binary + + + +
-
fission
DNA and
+ + + + _
RNA
Functional
+ + + + -
ribosomes
Metabolism + + + + _
Susceptibilit
y to most + + + + _
antibiotics
NB.
 Some mycoplasmas and chlamydiaes are less than 300 nm in diameter and few viruses
like poxvirus and mimiviruses are greater than 300 nm in diameter.
 All rickettsiaes except Rachalimaea quintana never grow in/on conventional media.
 But Rachalimaea quintana which is a pathogen for human being grows in/on artificial
medium.

1.5. ORIGIN AND EVOLUTION OF VIRUSES

The origin and evolution of viruses were suggested by different scientists. Among them the most
common theories are:-
 1. Regressive theory
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  2. Progressive theory
 3. Co - evolution theory
1. Regressive theory
Regressive theory suggested that viruses are the degenerate forms of intracellular parasites.
2. Progressive theory
Progressive theory suggested that viruses are originated from normal cellular nucleic acids that
gained the ability to replicate autonomously.
 DNA viruses came from plasmids or transposable elements.
 Retroviruses derived from retrotransposons
 RNA virus from mRNA
3. Co - evolution theory
Co - evolution theory suggested that viruses were co - evolved with life.
All could be correct!!

1.6. REASON FOR STUDYING VIRUSES

Some of important reasons why we are studying viruses is that there are:-
 1. Some viruses that are pathogenic
 2. Some viruses that are useful
 3. Viruses studies have contributed to knowledge
1. Pathogenic viruses
Some viruses are pathogenic; cause diseases in human being, animals and plants. Viruses are
important agents of many human diseases ranging from common cold upto lethal disease like rabies.
Viruses are also play role in development of many cancers in human being; so that virus diseases
affect the well – being of the societies. For example small pox had a great impact in the past and
HIV - AIDS is having a great impact today. Veterinary virology and plant virology are also
important; because:-
 Many viral diseases of domestic, wild animals and plants have an economic impact. For
example foot and mouth disease virus and rice yellow mottle viruses.
 Viruses can cause economic damage in dairy industry because phages can infect the lactic
acid bacteria that are responsible for fermentation of lactose in the milk to produce cheese,
yogurt and other milk products in dairy industry.
Therefore, detail study of pathogenic viruses is crucial to understand: -
 The nature of pathogenic viruses
 How they replicate?
 How they cause diseases in human beings, animals and plant?
This knowledge permits us the development of effective means of:-
 Prevention of viral diseases
 Diagnosis of viral diseases
 Treatment of viral diseases
These can be achieved through:-
 Production of effective viral vaccines
 Development of effective diagnostic methods and techniques
 Development of effective antiviral drugs
2. Useful viruses
Some viruses are studied because they are useful and some of the uses of useful viruses are:-
 A. Phage typing of some pathogenic bacteria
 B. Sources of enzymes

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  C. Pesticides
 D. Antibacterial agent
 E. Anticancer agent
 F. Gene vectors for protein production
 G. Gene vectors for treatment of genetic diseases
A. Phage typing of some pathogenic bacteria
Some bacteria like Salmonella species are classified into strains on the basis of the spectrum of
phage to which they are susceptible; so that identification of phage types of isolates can provide
useful epidemiological information during outbreaks of the disease by these different strains
bacteria.
B. Sources of enzymes
Many enzymes that are used in molecular biology are from viruses.
 Reverse transcriptase is from retroviruses
 RNA polymerase is from phages.
C. Pesticides
Some insect pests can be controlled by viruses. For example baculovirus used to control many pests.
D. Antibacterial agent
In the mid – 20 th century phages were used to treat some bacterial diseases of human beings.
However, the use of phages to treat bacterial infections was limited because of the discovery of
some antibiotics. Nowadays the use of some phages to treat bacterial infections has been renewed
with emergency of antibiotic resistant strain of bacteria.
E. Anticancer agent
Genetically modified strains of viruses are being investigated for treatment of cancer because these
strains have been modified so that they are able to infect cancer cells selectively but not normal
cells. Some strains of viruses that are modified to treat cancer are:-
 Herpes simplex virus
 Vaccinia virus
F. Gene vectors for protein production
Some viruses are used as vectors to insert genes into animal cell cultures; growing in laboratory.
This technology is used to insert genes of some pathogenic microorganisms which encode proteins
of interest; so that when the cells grow produce the protein of pathogenic microorganisms. It is used
to prepare vaccine. Some viruses that are used as vectors to insert genes of interest into animals’ cell
culture are:-
 Baculovirus
 Adenoviruses
G. Gene vectors for treatment of genetic diseases
Some viruses are used to treat genetic diseases. It is done by using some viruses as vector to replace
the gen which is responsible for a disease. For example sever immunodeficiency disease of children
was successfully treated by using retroviruses as vector to introduce in to their stem cells a non –
mutated copy of mutated gene responsible for the disease.
3. Viruses studies have contributed to knowledge
Much of the basic knowledge of molecular biology, cell biology and cancer has been derived from
studies with viruses. For example strong evidence which showed that gen is composed of DNA was
known by study that was done using phages and E. coli.

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1.7. GENERAL CHARACTERISTICS OF VIRUSES
Now you know that the evidence for the existence of very small infectious agents was first provided
in the late 19 th century by two scientists working independently. These two scientists were:-
 Martinus Beijerinck in Holland
 Dimitri Ivanovskii in Russia
They made extracts from diseased plants which we now know were infected with tobacco mosaic
virus. They passed the extracts through bacterial filters and the agents were passed through the filter.
The filtrates contained an agent that was able to infect new plants but they were not bacteria because
the filtrates were not growing on/in conventional inert bacterial media but the agent remained
infective through several transfers to new plants, eliminating the possibility of bacterial toxins and
Beijerinck called the agent ‘virus’ and the term has been in use now. At around the same time,
Freidrich Loeffler and Paul Frosch transmitted foot and mouth disease virus (FMDV) from
infected animal to susceptible animal inoculum that had been highly diluted. A few years later
Walter Reed and James Carroll demonstrated that the causative agent of yellow fever is filterable
agent. So that viruses are a unique class of infectious agents and they were originally distinguish
from other infectious agents in that:-
 They are smaller than other infectious agents that are why the original term” filterable
infectious agent” was used to describe them.
 They are obligatory intercellular parasites.
However these properties of viruses are shared by some bacteria like mycoplasmas, rickettsiaes and
chlamydiaes. This is because: -
 The larger virus like vaccinia virus (poxviruses) is as large as certain small bacteria like
mycoplasmas, rickettsiaes and chlamydiaes.
Viruses like prokaryotic cells do not possess standard cellular organelles like: -
 Mitochondria
 Chloroplasts
 Golgi apparatus
 Endoplasmic reticulum
 Ribosomes( bacteria have non membrane bounded ribosomes)
So that the truly distinctive features of viruses are known to be: -
 1. Their simple organization and genomic content
 2. Their mode of replication
 3. Non – susceptibility to many antibiotics
1. Simple organization and genomic content of viruses
The complete virus particle which is called virion differed from other cells in that: -
 It has either DNA or RNA but not both
 It has a simple organization
Virion has a simple organization in that they have: -
 Nucleic acid either DNA or RNA
 The protein called capsid which surrounds the nucleic acid.
 Sometimes additional membrane (not all viruses) called envelope which surrounds the
nucleocapsid.
All complete virions have nucleic acid and capsid. The nucleic acid and capsid of virions are called
nucleocapsid (core – protein).
2. Mode of replication of viruses
Unlike many cells, viruses do not grow in size and do not divide by themselves out side living cells
because they lack all metabolic capabilities like: -
 Energy production (ATP synthesis)
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  Protein synthesis
So that viruses multiply only in living cells by synthesis of their separate components followed by
assembly. This means the viruses after shedding their coats inside living cells; they come contact
with appropriate cell machinery where it specifies the synthesis of proteins required for viral
replication. This results in replication of viral nucleic acids through the use of both viral and cellular
enzymes followed by formation of viral coats. Finally the components of viruses are assembled and
form many matured virions.
1.7.1. Host range in viruses
Viruses infect all cellular life forms including:-
 Eukaryotes (vertebrate animals, invertebrate animals, plants and fungi).
 Prokaryotes(Eu - bacteria and archaea bacteria)
The viruses that infect prokaryotes are often referred to us bacteriophages or phage in short. It is
known that each virus within each classes of viruses infect only a certain species of cells. The host
range of viruses is determined by specificity of attachment to the cells and by cellular factors
required for viral replication. This means the host range of viruses depends on: -
 The properties of both the virion’s coat and specific receptors on the cell surface
 The availability of cellular factors required for viral replication
However, the importance of specificity of attachment to the cells disappears when there is trans –
infection of different species of animals by one species of virus because transinfection is carried out
by the naked viral nucleic acid whose entry does not depend on virus – specific receptors; so that the
host range of naked viruses and virions is affected not only by specific receptors on cell surfaces and
virions but also by availability of cellular factors required for viral replication. Depending on their
host ranges, all viruses are subdivided into:-
 Animal viruses including human being
 Plant viruses
 Bacterial viruses
 Fungal viruses
1.7.2. Are viruses alive?
You know that life is a complex set of processes resulting from the actuation of the instructions
encoded in nucleic acids (DNA and RNA). In the nucleic acids of all living cells the instructions
encoded in nucleic acids are actuated all the time. However, in viruses, they are actuated only when
the viral nucleic acid (DNA or RNA) enters into susceptible host cells and synthesis virus specific
proteins; so that viruses are “alive” when they replicate in living cells. Outside living cells virus
particles are metabolically inert. It is possible to say virus particles outside living cells are no more
alive than fragments of DNA as DNA used in bacterial transformation or RNA.
1.7. 3. Sensitivity of viruses to physical and chemical agents
Sensitivity of viruses is different for different physical and chemical agents. Sensitivity of viruses
can be:-
 Heat sensitivity
 PH sensitivity
 Sensitivity to lipid solvents
 Sensitivity to different chemicals
 Sensitivity to radiation and ultraviolent light
Heat sensitivity of viruses is relative to thermal temperature and time of exposure. In general at high
temperature viruses lose infectivity rapidly because of denaturation of viral proteins. For example
most viruses lose infectivity rapidly at 60 0C in few minutes; but the rate of inactivation of most
viruses is slower (hours to days) at room temperature. In general enveloped viruses are more heat –

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labile than naked viruses. Low temperature (freezing) in general preserves viruses. Viruses can be
stored for months to years at – 60 0C to – 90 0 C and at - 196 0C in liquid nitrogen. Viruses can be
preserved for a long time if stabilizing proteins or cryoprotective agents like glycerol are added.
Most viruses can be best preserved for a long lime in lyophilized form. Lyophilization is the
process of preservation of biological agents in freezed dried form in vacuum.

Viruses are also sensitive to PH and extremes of basic and acidic PH are destructive for most
viruses. However, neutral PH is ideal for most viruses. Sensitivity of viruses to lipid solvents like
ether and chloroform is different. Most enveloped viruses especially lipid enveloped viruses are
readily inactivated by lipid solvents like ether and chloroform. Chemical inactivation of viruses is
also different. Most chemicals like formaldyde slowly inactivate most viruses by reacting with
amino groups in nucleic acid and protein. Most disinfectants inactivate viruses by reacting with their
nucleic acids and proteins. In general enveloped viruses are sensitive for many disinfectants than
naked viruses. Ultraviolet light and radiation inactivate viruses by damaging their nucleic acids.
1.7.4. Viruses as infectious agents
Two properties of viruses explain why viruses are infectious, pathogenic agents.
 Virions produced in one cell can invade other cells and thus cause a spreading infection.
 Viruses cause important functional alterations of the invaded cells, often resulting in their
death.
1.7.5. Other virus related genomes
There are many genomes in different cells which are not viruses but they are naked genomes. Some
of the common genomes which are similar to the naked viral genome are:-
 1.Bacterial plasmids
 2. Viroids
1. Bacterial plasmids
Bacterial plasmids like viruses are present in bacterial cells as naked genomes; however, plasmids
differ from virus in that:-
 Plasmids do not form a protein coat
 They are transmitted from one bacterium to another by conjugation rather than as free
extracellular particles.
2. Viroids
Viroids are small naked infectious molecules of circular single – stranded RNA. They cause diseases
in plants. The RNA of viroids is tightly folded and it is closed circle presumably. This structure of
viroids protects them from extracellular nucleases.

1.8. CHEMICAL COMPOSITION OF VIRIONS

The chemical composition of virus particles varies. It is different in different virus particles that are
found in different families of viruses. The simplest virus, the virions are composed of:-
 Structural proteins
 DNA
Other simple virus, the virions are composed of:-
 Viral protein
 RNA
The chemical composition of virions becomes more complex in enveloped viruses. Enveloped
viruses are those viruses which mature by budding through different cellular membranes. The
cellular membranes of the host cells are modified by insertion of viral proteins. Host cell proteins
are not a significant component of viruses; however, minor amount of cellular proteins can be

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identified in viral membranes and in the interior of the virus particles. For example ribosomal RNA
of the host cell can be found in virions but there is no any evidence its functional role in virus
replication. In enveloped viruses, the major chemicals that can be found are: -
 Glycoprotein or lipid which are located to the exterior of the membrane is either
 Matrix protein which is located to interior to the capsid.
The presence of a lipid envelope provides virologists to separate viruses into two distinct classes.
 1. Those that are inactivated by organic solvents like ester, ether and chloroform called
enveloped viruses.
 2. Those that are resistant to organic solvents like ester, ether and chloroform called non –
enveloped viruses.
Viruses may have the following components:-
 1. Viral nucleic acid
 2. Viral proteins
 3. Viral envelopes
 4. Viral enzymes
 5. Other virion components
1. Viral nucleic acid
Viruses contain genomes. It is known that viruses exhibit a remarkable variety of strategies to:-
 Express their genes
 Replicate their genomes
The type of nucleic acid and the genomic structure of the nucleic acids are used to classify viruses.
Viruses contain only one nucleic acid type to transmit genetic information that is why the virus
world can simply be divided into:-
 RNA viruses
 DNA viruses
The proportion of nucleic acid to the total virion particle in the viruses varies. It may have: -
 About 1% in influenza virus
 Upto 50% in some bacteriophages
The amount of genetic information per virion also varies from 3 kilobases to 300 kilobases (Kb) per
strand. If 1 kb is taken as the size of an average gene, so that small viruses contains 3 to 4 genes and
large viruses contains several hundred genes. Generally, virion genomes are smaller than other cell
genomes. The size of the genome certainly reflects on the protein coding capacity of the virus. The
question arises if viruses have small genome, how they encode all their requirements with these
small genomes. Viruses achieve this requirement in a number of ways and some of the common
ways are:-
 1. Viruses use host cell proteins
 2. Viruses code efficiently
 3. Many viruses proteins are multi - functional
1. Viruses use host cell proteins
The genomes of large viruses duplicate some of the functions of the host cell but the small viruses
rely very heavily on function of the host cell except small RNA viruses because cells do not encode
enzymes that can replicate RNA viruses; so that small RNA viruses must encode all requirements,
no matter how small their genome. This function is the function of RNA polymerase enzyme.
2. Viruses code efficiently
There may be overlapping genes and genes encoded within genes.

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Genome sizes of some DNA viruses and other prokaryotic and eukaryotic cells

1. Double stranded viral DNA

In double stranded DNA viruses, the DNA is a single molecule of double stranded (ds). Double
stranded (ds DNA) genomes of viruses have different structural morphologies; and it can be:-
 Linear
 Cyclic
The linear ds DNA genome of viruses has also wide range of nucleic acid structure which can be:-
 Linear double strand without chain breaks; example herpesviruses and adenoviruses etc.
 Linear double strand with chain breaks; example T5 coliphages.
 Linear double strand with cross – linked ends; example vaccinia virus.
However, cyclic ds DNA viruses have closed circular double strand DNA; example papovaviruses
(polyomaviruses) and papillomaviruses. Linear and cyclic ds DNA viruses have different molecular
weight and some of the examples are:-
 Viruses that are found in the family of Polyomaviridae and Papillomaviridae have super-
coiled cyclic genome with molecular weight of 5 – 8 Kbp.
 Viruses of the subfamily Herpesvirinae have linear genome with molecular weight 125 –
235 Kbp.
Structure of viral DNA

 ss: single-stranded
 ds: double-stranded
2. Single stranded viral DNA
This group of viruses has also simple structure which is composed of a single molecule of single
stranded DNA. Morphologically, the genomes of ss DNA can be:-
 Linear single strand; example viruses in the family of Parvoviridae
 Circular single strand; example viruses in the family of Circoviridae
Viruses with ss DNA genome have molecular weight ranging from 2.8 to 5 kb.
Nucleic acid structure of RNA viruses
RNA viruses can be:-
 1. Double stranded RNA
 2. Single stranded RNA

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Distinct features of RNA viruses are that they can be:-
 Positive sense RNA viruses or
 Negative sense RNA viruses
 Segmented RNA viruses
Positive sense RNA viruses are those RNA viruses in which the RNA base sequence may be
identical to that of viral mRNA; so that it is directly capable of translation to protein. Negative
sense RNA viruses are those RNA viruses in which the RNA base sequence may instead have a
sequence complementary to that of viral mRNA; as the result it requires transcription of the genome
to generate mRNA equivalent. The size of animal RNA viral genome varies and it can be:-
 Less than 2 kbp in deltaviruses.
 More than 30 Kbp in the largest RNA viruses in the family of Coronaviridae
1. Double stranded viral RNA
A few viruses have double stranded RNA (ds RNA) genomes and all appear to be segmented. Some,
such as reoviruses have 10 to 12 segments.
2. Single stranded viral RNA
The majority of RNA viruses employ single – stranded RNA (ss RNA) as their genetic material.
However, the RNA base sequence is different and can be:-
 Negative sense
 Positive sense
If the RNA base sequence is identical to that of viral mRNA, in which case the RNA strand is called
the plus strand or positive strand. That is why viral mRNA is defined as plus or positive RNA.
However, the viral RNA genome may instead have a sequence complementary to that of viral
mRNA, and in this case it is referred to as a minus or negative strand. By their nucleic acid
structure, single stranded RNA viruses can be:-
 Linear ssRNA positive sense; examples, picornaviruses (poliovirus), togaviruses and RNA
bacteriophages.
 Linear ssRNA negative sense; example, rhabdoviruses(rabies) and paramyxoviruses(mumps
and measles)
 Linear ssRNA segmented; diploid( two identical single strands) positive sense; example
retroviruses
 Linear ssRNA segmented, negative sense; example paramyxoviruses and orthomyxoviruses
(influenzaviruses).
 Linear circular ssRNA

 ss: single-stranded
 ds: double-stranded
NB. There are no viruses known with circular dsRNA genomes

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Nucleic acid structure and size of DNA viruses in define virus families
Genome size in
Virus families Nature of genome Genome configuration
Kbp
Poxviridae dsDNA 1 - linear 130–375
Iridoviridae dsDNA 1 - linear 135–303
Asfarviridae dsDNA 1 - linear 170–190
Herpesviridae dsDNA 1 - linear 125–240
Adenoviridae dsDNA 1 - linear 26–45
Polyomaviridae dsDNA 1- circular 5
Papillomaviridae dsDNA 1 - circular 7–8
Hepadnaviridae dsDNA-RT 1- linear 3–4
Circoviridae ssDNA 1 – or +/ - circular 2
Parvoviridae ssDNA 1 - +/ - linear 4–6
Anellovirus(unassigned) ssDNA 1 - circular 3–4
dsDNA = double stranded DNA Kbp = kilobase pairs
SSDNA = single stranded DNA

Nucleic acid structure and size of RNA viruses in define virus families
Virus families Nature of genome Genome configuration Genome size in Kbp
Retroviridae ssRNA-RT 1 + (dimer) 7–13
Reoviridae dsRNA 10–12 - segments 19–32
Birnaviridae dsRNA 2 - segments 5–6
Paramyxoviridae NssRNA 1 - segment 13–18
Rhabdoviridae NssRNA 1- segment 11–15
Filoviridae NssRNA 1 - segment ≈19
Bornaviridae NssRNA 1 - segment 9
Orthomyxoviridae NssRNA 6–8 - segments 10–15
Bunyaviridae NssRNA 3 – or +/ - segments 10–15
Arenaviridae NssRNA 2 +/ - segments 11
Coronaviridae ssRNA 1 +segment 31–38
Arteriviridae ssRNA 1 + segment 13–16
Picornaviridae ssRNA 1 +segment 7–9
Caliciviridae ssRNA 1 + segment 7–8
Astroviridae ssRNA 1 + segment 6–7
Togaviridae ssRNA 1 + segment 10–12
Flaviviridae ssRNA 1 + segment 10–12
Hepevirus ssRNA 1 + segment 7
(unassigned)
dsRNA= double-stranded RNA; kbp = kilobase pairs; NssRNA= negative sense single-stranded
RNA; RT = reverse transcription; ssRNA = single-stranded RNA.

1.8.1. 1. Defective viral nucleic acids

In rare cases stocks of either DNA or RNA viruses often have defective virions. That means:-
 Some may contain only a fraction of their normal nucleic acid complement

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  Others may contain nucleic acid nearly normal length but made up of repetition of a limited
viral sequence or even cellular nucleic acid.
These virions are not infectious but in most cases their nucleic acids can replicate in cells coinfected
by regular viruses. These coinfected regular viruses that help defective virions to replicate in cells
are called helpers. Defective virions in the presence of infective helper viruses replicate rapidly than
infective helper viruses. This can be because of:-
 Their shorter nucleic acid or
 Having several replication origins
So that some defective virions:-
 Retaining viral functions and can cause cell transformations.
 Others inhibit the replication of regular virus infecting the same cells.
That is why they have another name called defective interfering particles.
1.8.2. Viral proteins
Depending on their genome size, animal viruses encode from as few as one protein to more than 100
proteins. All viral proteins can be classified into:-
 Structural viral proteins
 Non – structural viral proteins
Structural viral proteins are those proteins that are present in virions (mature virus particles).
Structural proteins that are found in matured virions are:-
 Capsids
 Glycoproteins in the envelope of enveloped viruses
Proteins of viruses that are mostly viral enzymes which are involved in:-
 Replication of viral genomes
 Assembly of the particle
 Modification of the host innate response to infection
These viral proteins are referred to as non-structural viral proteins. However, a few viral enzymes
like RNA polymerase in Nss RNA viruses like Paramyxoviridae and Rhabdoviridae etc. are
essential in the initial stages of virus replication. Since RNA polymerase of Nss RNA viruses
participates in transcription of the viral genome in the first step of their replication cycle as soon as
their nucleocapsid enters in the cytoplasm of infected cells, it is impossible to categorize as non –
structural viral protein. That is why upto now whether the RNA polymerase of Nss RNA viruses has
a structural role in the mature particle in addition to its transcription activity remains unresolved.
Numerous other viral proteins that occur within the virions of complex viruses like Poxviridae,
Herpesviridae and Asfarviridae have no apparent structural role so that they are non – structural
viral proteins. Depending whether the viral proteins are chemically changeable or not when they
replicate in infected cells, virion proteins fall into two general classes.
 Modified viral proteins
 Un modified viral proteins
The capsids of the non-enveloped viruses are composed of proteins with few modifications because
there is the interaction of viral capsid protein with protein of infected cells during assembly. Since
proteolytic cleavage of precursor proteins in the nascent capsid is common in the final steps of
assembly of the mature capsid proteins.
These types of viral proteins with few modifications in matured virions are called modified viral
protein. It is clear that glycoproteins are predominantly found in those viruses that contain a viral
membrane (enveloped viruses). Glycoproteins of enveloped viruses are also structural viral proteins.
These structural viral proteins can be: -
 Type I integral membrane protein (amino terminus exterior) (e.g., hemagglutinin [HA] of
influenza virus) or
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  Type II (carboxyl terminus exterior) (e.g., neuraminidase (NA) of influenza virus).
Hemagglutinin and neuraminidase are the virulence factor and the antigens of many pathogenic
viruses like viruses in the family of Orthomyxoviridae (highly pathogenic avian influenza virus
(AIV)). So that capsids and envelopes of viruses are structural and modified viral proteins
respectively; while many enzymes in viruses are non – structural and unmodified viral proteins.
Structural proteins in the infectious virus particle have numbers of key functions and some of them
are:-
 Protecting the genomic nucleic acid and associated enzymes from inactivation
 Providing receptor- binding sites for initiation of infection
 Initiating or facilitating the penetration of the viral genome into the correct compartment of
the cell for replication
1.8.3. Viral envelope
Many animal viruses, some plant viruses and at least one bacterial virus are bounded by an outer
membranous layer called envelope. Animal viruses that mature by budding through a cellular
membrane, a major constituent of the virion are phospholipids that form the structural basis of the
viral membrane. The maturation site for animal viruses can be:-
 Plasma membrane
 Nuclear membrane
 Golgi apparatus
 Endoplasmic reticulum
So that it is possible to know where budding of enveloped animal viruses take place. It can be
known by identification of the chemical composition of the envelope of enveloped viruses.
 For those viruses budding from the plasma membrane, cholesterol is a constituent of the viral
membrane
 Whereas the envelopes of those viruses that bud from internal membranes (nuclear
membrane, Golgi and endoplasmic reticulum) lack cholesterol.
Animal viruses’ envelopes usually arise from host cell nuclear or plasma membranes; so that the
lipids and carbohydrates in envelope are the normal host constituents. In contrast, proteins in the
envelope are coded by virus genes. Generally, there are two kinds of membrane proteins in the
envelope of enveloped viruses. These are:-
 A. Glycoprotein
 B. Matrix protein
A. Glycoprotein
Glycoproteins of envelope are exposed at the outer surface of virions. Glycoprotein of the envelope
has two parts.
Glycosylated hydrophilic end which protruding into the medium
Glycosylated hydrophobic end which protruding into the lipid bi – layer.
However, some glycoproteins span the membrane and are connected to the underlying nucleocapsid
or matrix proteins. Proteins in envelope of enveloped viruses may even project from the envelope
surface as spikes which are often recognizable by electronmicroscopy called peplomers (influenza
viruses).
Enveloped viruses with peplomers

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Since peplomers are the protein part of envelope and is coded by virus genes, it is specific for each
species of enveloped viruses. So that peplomers are different among different species of enveloped
viruses. Peplomers are used:-
 For attachment to host cell surface
 To identify some peplomer viruses
In some enveloped viruses, the envelopes are disrupted by solvents like “ether” as the result in some
extent that lipid – mediated activities are blocked and envelope proteins are denatured and rendered
inactive.This group of viruses are said to be “ether sensitive” viruses; while other group of non –
enveloped viruses are not sensitive to “ether “ are called “ ether non – sensitive” viruses.
B. Matrix protein
It is the second type of a non - glycosylated envelope protein. Matrix protein forms a layer at the
inner surface of the envelope to the capsid of enveloped viruses. This protein appears to establish
connection between the envelope and the capsid of enveloped viruses. The proteins of the envelope
like those of the capsid are specified by viral genes; that is why they contribute the major antigenic
specificities to the virions. However, the lipids and the carbohydrate moieties of glycoproteins of
envelope depend on the host cell and they are often different in the same virus grown in different
cells. So that the virion surface may contain polysaccharide which expresses the cellular antigens in
which they grow.
1.8.4. Viral enzymes
It was originally thought that virions had only structural capsid proteins and lacked enzymes.
Viruses lack true metabolism and cannot replicate independently of living cells. However, one or
more numbers of capsid – associated enzymes have been discovered in many viruses which are
essential for the completion of their life cycles.
These virus enzymes are found very often in enveloped animal viruses. Many of these virus
enzymes are involved in nucleic acid replication.
Some common viral enzymes and their functions
Enzyme Function or product Virus having the enzyme
I. Enzymes affecting
interacting of virions with
host cell surface
1.1. Neuraminidase Splits off NANA( N - Orthomyxoviruses and
acetylneuraminic acid paramyxoviruses
1.2. Endoglycosidase Breaks down surface E. coli K bacteriophages
polysaccharides
1.3. Fusion factor Alters lipid bilayer Paramyxoviruses
II. Enzymes transcribing
the viral genome into
messenger RNA
2.1. DNA - dependent RNA Single – stranded mRNA Poxviruses and bacteriophages N4
polymerase etc.
2.2. Double - stranded RNA Single – stranded mRNA Poxviruses and bacteriophages N4
transcriptase etc.
2.3. Single – stranded RNA Single – stranded Viruses with single – stranded
transcriptase mRNA(positive strand) RNA( Nss RNA)

III. Enzymes involved in

copying virion RNA into

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 DNA
3.1. RNA dependent DNA DNA – DNA hybrids, Retroviruses
polymerase double stranded DNA
3.2. Rnase H( in association Breaks down RNA strand in Retroviruses
with reverse transcriptase RNA – DNA hybrids
3. 3. Polynucleotide ligase Closes single – strand breaks Retroviruses
in double stranded DNA
IV. Enzymes for nucleic acid
replication or processing
4.1. RNA – dependent DNA Synthesizes double stranded Hepatitis B
polymerase DNA
4.2. Deoxyribonucleases(exo Breaks DNA chains and Hepatitis B
– and endo- ) cross links
4.3. Endoribonuclease Processing of mRNA Viruses with single – stranded
mRNA(e.g; poxvirus
V. Other enzymes
5.1. Protein kinase Phosphorylate proteins Retrovirus, orthomyxovirus,
paramyxovirus and adenovirus
1.8.5. Other virion components
In addition to the coat proteins, virions contain some internal proteins. These proteins are bound to
the nucleic acid in the core. Some of internal proteins of virions are:-
 Histones
 Histonelike proteins
 Small peptides and polyamines
 Small oligonucleotides
Histones are proteins found in papovaviruses and are not specific; while in adenoviruses they are
histonelike and are virus specific. In both cases they form with viral DNA complexes similar to the
chromatin of animal cells. Small peptides and polyamines are also present in bacteriophages. These
polycationic compounds help the folding of the nucleic acids by linking together in different loops.
Each reovirus virion also contains about 200 small oligonucleotides of unknown function.

1.9. VIRION SIZE, MORPHOLOGY OF CAPSID AND VIRIONS

1.9.1. Virion size
Virions range in size from about 10 to 300 or 400 nm in diameter. The smallest viruses are little
larger than ribosomes; whereas the poxviruses, like vaccinia and mimiviruses of amoeba are about
the same size as the smallest bacteria (Mycoplasma, Chlamydia and Rickettsia) and can be seen
under light microscope. However, most viruses are too small to be visible under light microscope
and must be viewed with scanning and transmission electronmicroscopes.
1.9.2. Viral capsid morphology
It is well known that; early attempts to characterize viruses were hampered by the lack of
appropriate technology. Filtration and sensitivity to chemical agents were two standard tests that
were applied to new known infectious agents (viruses) for 40 years. Work with tobacco mosaic virus
in the 1930s strongly suggested that the virus was composed of repeating protein subunits.
Crystallization of the virus in 1935 supported this notion; however, it was not possible to understand
the general structural properties of viruses until 1939 that a virus was visualized using an electron
microscope. Under electronmicroscopy, tobacco mosaic virus appeared as a rod-shaped particle,

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confirming the particulate nature of viruses. A major advance in determining virus morphology was
the development of negative staining in electronmicroscopy in 1958. In this procedure, electron
dense stains were used to coat virus particles and produce a negative image of the virus with
enhanced resolution. Advances in determining virus morphology down to the atomic level came
from studies initially using X-ray crystallography and then combining this technique with other
structural techniques such as electron cryo –Electron Microscopy (cryo-EM). In this process,
samples are snap frozen and examined at temperatures of liquid nitrogen (- 1960C) or liquid Cryo-
EM. These techniques offered the advantage that the samples are not damaged or distorted in the
process of analyzing the structure, as occurs with negative-stain electronmicroscopy and X-ray
crystallography. Critical to these analyses were the developments in computer hard ware and
software that were able to capture, analyze, and construct the three-dimensional images of viruses
which is applicable nowadays. Nowadays, it is known that all virions, even if they possess other
constituents, they are constructed around nucleocapsid core. The capsid accounts for most of the
virion mass, especially in small viruses. Some viruses consists indeed only nucleocapsid and are
called naked viruses. In naked virions the capsid:-
 Protects the nucleic acid from nucleases in biological fluids
 Promotes the attachment of virions to susceptible cells
The nucleocapsid is composed of a nucleic acid, either DNA or RNA, held within a protein coat
called capsid. Capsid of virions in naked or enveloped viruses protects viral genetic material which
aids in transferring the genetic materials between host cells. Capsids usually consist of a single or
several type of proteins unit called protomers. Protomers of helical capsids usually consist a single
type of proteins; while protomers of icosahedral capsids may have one or several type’s proteins.
The protein units of the capsid called protomers are specified by viral genes and their aminoacids
sequence can be altered by some viral mutations. The protomers forming the capsid must be
connected by bonds between suitable chemical groups on their surfaces. The shape and dimensions
of the capsid depend on:-
 The characteristics of its protomers
 The length of nucleic acid for helical capsids
Some naked viruses

Some enveloped viruses

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There are three general morphologies of virion capsid which is also called symmetry of capsid.
These are:-
 1. Helical viral capsids
 2. Icosahedral viral capsids
 3. Conical and rod-shaped capsids
 4. Complex viral capsids
Different capsid morphologies of viruses

Virions with complex symmetry

NB. All these types of symmetry are seen amongst plant and animal viruses; however, the most
common are helical and icosahedral symmetries.
1. Helical viral capsids
Some virions form long rods and shaped like hollow protein cylinders. Helical capsids are shaped
much like hollow tubes with protein walls; that means the nucleic acid is surrounded by a cylindrical
capsid. In helical capsid a single type of protomer associates together in a helical or spiral
arrangement to produce a long, rigid tube, 15 – 18 nm in diameter by 300 nm long. Viruses with this
type of capsid structure are called viruses with capsids of helical symmetry. In helical capsid, the
genetic material is wound in a spiral and positioned toward the inside of the capsid where it lies
within a groove formed by the protein subunits. Helical capsids can be:-
 Rigid
 Flexible
In rigid helical capsid virions, the capsids are very tight that gives rigidity of the virion in its
morphological features and these virions with such morphological feature are called naked helical
viruses. Tobacco Mosaic Virus (TMV) is one example of virion with helical capsid and naked virus;
that is why TMV is rigid naked helical virus.
Viruses with capsids of helical symmetry

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(a) Structure of a capsid with helical symmetry. The ssRNA coils are coated with repeated copies of
a protein.
(b) Part of measles virus nucleocapsid. The complete nucleocapsid is folded and enclosed within an
envelope.
(c) Virus with helical symmetry under negative staining
Negative-contrast electron micrograph of TMV particle stained with uranyl acetate (Naked
helical virus)

In contrast, the capsids of enveloped helical viruses are very flexible because they have to coil
within the envelope; even in some viruses, they form rings, although the nucleic acid is not cyclic.
However, the helical structure of the capsids in flexible virions can be demonstrated by electron
micrograph; so that the loose structure of enveloped helical capsid virions is the envelope rather than
the capsid; that may provide the required barrier to nuclease. A good example of flexible enveloped
virus with helical capsid is influenza virus. In influenza virus the RNAs are enclosed in thin, flexible
helical capsids folded within an envelope. The diameter of the helical capsid is determined by the
characteristics of its protomers; while the length of helical capsid viruses is determined by the length
of nucleic acid it encloses because the capsid does not seem to extend much beyond the end of the
DNA or RNA.
Some examples of helical capsid viruses are:-
 Tobacco Mosaic Virus(TMV)
 Bacteriophages M13, fd, f1Influenza virus
Helical naked and helical enveloped viruses

2. Icosahedral viral capsids

When icosahedral viruses are negatively stained and viewed through transmission electron
microscope, a complex capsid structure is revealed. Icosahedron means a regular polyhedron with
20 triangular faces, 12 corners and 30 edges, at each of which the sides of two triangles meet. An
icosahedron has five-, three- and two-fold axes of rotational symmetry; so that viruses with this type
of capsid structure are called viruses with capsids of icosahedral symmetry.
The three axes of symmetry of an icosahedron

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The capsids are constructed from ring or knob – shaped units called capsomers. Each capsomers is
made of five or six protomers. The five protomer are called pentamers (pentons).So that pentamers
(pentons) have five subunits of protomers; while the six protomers are called hexamers (hexons).So
that hexamers (hexons) have six subunits of protomers. Pentamers (pentons) are located at the
vertices of the icosahedron capsids; whereas hexamers (hexons) form the edge and triangular faces
of the icosahedron capsid. Protomers in capsomers and capsomers in icosahedron capsids are held
together by noncovalent bonds. The bonds between protomers are usually stronger than between
capsomers. The number of pentons, hexons and types of their subunits are different in different virus
families with icosahedron capsids; that is why viruses with icosahedron capsids have different size.
Larger icosahedron capsid viruses are made if more hexamers are used to form the edge and the
faces of the triangle and have several types of protein subunits. Mostly plant viruses and RNA
bacteriophages with icosahedron capsid are made up of both pentons and hexons with only one type
of protein subunits; however animal viruses with icosahedral capsids usually made of both pentons
and hexons with different protein subunits. For example bacteriophages f2 and MS2 are the smallest
naked icosahedron capsid virions which have twelve pentons without hexons and are made from
single protein subunits. However, adenoviruses are animal viruses made up of 252 capsomers of
both pentons and hexons with different protein subunits. Some examples viruses with icosahedral
capsid are:-
 Picornaviruses
 Adenoviruses
 Herpesviruses
 Polyomaviruses
 Papillomaviruses
 Bacteriophages f2, MS2 and øX 174 etc
Some common viruses with icosahedral capsids

Model of adenovirus
In adenovirus virion; at each of the 12 vertices of the virion there is a penton, and attached to each
penton there is a protein fiber with a knob at the end. The rest of the capsid is constructed from
hexons.
3. Conical and rod-shaped capsids
HIV-1 and baculoviruses have capsids that are conical and rod shaped, respectively. Inside each
capsid is a copy of the virus genome coated in a highly basic protein. Both of these viruses have
enveloped virions.
4. Complex viral capsids
The majority of viruses have either icosahedral or helical capsids structure but there are many
viruses that do not fit into either of the two categories; because they have:-

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  The combination of the two capsids structures (helical and icosahedral) with addition of
some sort of structures in their capsids. or
 Neither of the two structure but they have an exceptional complex internal structure.
Viruses with either of the two capsid structures are called viruses with capsids of complex
symmetry. Some of the examples of viruses with complex capsid symmetry are:-
 Poxviruses
 Large bacteriophages
Poxviruses are the largest of the animal viruses with the size of about 400× 240 × 200 nm. This
virus can be even seen with phase – contrast microscope or in bright field microscopy with special
staining. The viruses posses an exceptionally complex internal structure with an ovoid to brick -
shaped exterior. The double – stranded DNA is associated with proteins and contained in nucleoid.
The nucleoid is the central structural component of the virus and has biconcave disk shape
surrounded by two membranous layers. Two elliptical bodies lie between the nucleoid and its outer
coat. The thicker elliptical body of the virus is covered by an array of tubules and fibers. Large
bacteriophages especially bacteriophages of E.coli which are called coli – phages; and lambda
phages have complex capsid structure because, the majority of large bacteriophages have two parts.
 Head
 Tail
Coliphages are designated by letter T and numbers; and areT1, T2, T3, T4, T5, T6 and T7. They are
divided into two based on the difference in capsid structures.
 T – even coliphages which includes T2, T4 and T6
 T – odd coliphages which includes T3, T5 and T7
The T – even coliphages (T2, T4 and T6) have the following capsid structures:-
 The head
 The tail
The head of T – even coliphages resembles an icosahedron elongated by one or two rows of
hexamers in the middle and contain the DNA genome. The tail of T – even coliphages is composed
of:-
 A collar joining the tail to the head
 A central tube with a helical sheath which covers the central tube
 A complex base plate
In T – even coliphages, the base plate is hexagonal and has a pin and a joined tail fiber at each
corner. The tail fibers are responsible for virus attachment to the proper site on the bacterial surface.
The T – odd coliphages have different capsid structures. T1 and T5 coliphages have:-
 True icosahedral heads
 Sheathless tail that lacks a base plate and terminates rudimentary tail fibers
However, coliphages T3 and T7 have:-
 True icosahedral head
 Short and noncontractile tails without tail fibers.
Lambda (λ) phages have: -
 True icosahedral head
 Sheathless tail that lacks a base plate and terminates with rudimentary tail fibers
So that T – even coliphages and lambda (λ) phages are large bacteriophages with complex virion
structure. Those T – odd coliphages that do not have a tail (T3 andT7) are not complex viruses.
However T1 and T5 are complex viruses as they have helical capsid tail with icosahedral head.
What you must understand is that there is considerable variation in structure of capsid among the
large bacteriophages, even those infecting a single bacterial host.
Virion with complex capsids in the family of Poxviridae
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Model of virions with complex capsid structure Coli phages T4

Model of virion with complex capsid and icosahedral isometric head with short, non-
contractile tail (T7 coli phage)

Model of λ(lambda) coliphages with complex capsid structure

1.9. 3. Morphology of virions

Electron microscopy shows that virions belong to several morphologic types. Some virions resemble
small crystals; that means the virion which is the complete virus particle of a simple virus consists:-
 A single molecule of nucleic acid (DNA or RNA)
 Morphological distinct capsids
Capsids which surround the nucleic acid of virions is composed of viral protein subunits; and are
virus encoded polypeptides. The capsid protein subunits can self-assemble into multimer units
(structural units). The structural units may contain one or several polypeptide chains. However, in
rare case capsid structures without the nucleic acid can be detected and are referred to as empty
capsids. In a strict sense, a capsid with its nucleic acid is a nucleocapsid because for some simple
viruses like poliovirus, this structure (capsid and nucleic acid) is also the virion; however, in most
viruses like flaviviruses, the nucleo - capsid (capsid and RNA) is enclosed in a lipid envelope; so
that the nucleocapsid does not represent the complete virion for this group of viruses. For

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paramyxoviruses, the nucleocapsid refers to a structure composed of a single strand of RNA
complexed to a viral protein; then the nucleocapsid assembles into a complete virion by obtaining a
lipid envelope from host cell membranes modified by the insertion of viral proteins. So that the
morphology of virions can be classified into:-
 1.Helical morphology
 2.Icosahedral morphology
 3.Complicated morphology
 4. Complex morphology
1. Virions with helical morphology
These are virions that form long rods and the nucleic acid is surrounded by a cylindrical capsid.
Virions with such morphological features are called helical virions. Some examples of viruses that
have helical morphology are:-
 Filoviruses
 Tobacco Mosaic Virus(TMV)
 Bacteriophage M13
Filovirus with helical morphology under negative staining

2. Virions with icosahedral morphology

These are virions that have an icosahedral protein shell (the capsid) surrounding a core of nucleic
acid and proteins. The capsid and the core form the nucleocapsid; and these virions are called
icosahedral virions. Some viruses with icosahedral morphology are:-
 Picornaviruses
 Adenoviruses
 Papovaviruses
 Calciviruses
 Herpesviruses
 Birnaviruses
 Reoviruses etc.
Model of virion with icosahedral morphology in the family of Picornaviridae

Model structure of virion of adenoviruses with icosahedral morphology

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3. Virions with complicated morphology
These are virions with more complicated morphology and contain lipid. In most cases the
nucleocapsid of virions with complicated morphology can be either helical or icosahedral and
surrounded by loose membrane called envelope. These virions are called enveloped virions (the
opposite of naked virions). The nucleocapsid of these viruses is coiled within the envelope.
Enveloped virions are roughly spherical but highly pleomorphic. Enveloped viruses are
pleomorphic because the envelope is not rigid; due to the following reasons.
 The presence of lipids in a liquid state in the envelopes of many enveloped viruses.
 The absence of connections among the monomers of the envelope proteins.
These two factors prevent the formation of a rigid structure, leading pronounced pleomorphism of
the virions. Pleomorphic viruses have different shapes and this property is called pleomorphism.
In a few enveloped virions containing helical nucleocapsid with loose connection between
nucleocapsid and envelope may assume a bizarre tadpole like shape when dried for electron
microscopy. This is because one end of the nucleocapsid unravels and pushes the envelope out.
However, in other viruses a firm connection between envelope and nucleocapsid; and the closely
adherence the lipid bilayer to the icosahedral nucleocapsid confers on the virion characteristic
shapes. Some of the examples of enveloped virion with a constant morphology are:-
 Rhabdoviruses which have bullet – shaped virions with helical nucleocapsids coiled under
the outer layer.
 Alphaviruses which appear nearly spherical shape.
The presence of lipids makes enveloped viruses sensitive to many disinfectants or to be damaged by
lipid solvents like ether. Some of the examples of enveloped viruses with complicated morphology
with icosahedral capsid viruses are:-
 Herpesviruses
 Togaviruses
 Hepadenaviruses
 Bornaviruses etc.
Some of the examples of enveloped viruses with complicated morphology with helical capsid are:-
 Orthomyxoviruses
 Paramyxoviruses
 Rhabdoviruses etc.
It is clear that viruses with complicated morphology are enveloped; so that their envelopes like
cellular membranes contain lipid bilayer and protein with special functions. There are two kinds of
membrane proteins in the envelopes.
 Glycoproteins
 Matrix proteins
Glycoproteins are glycosylated proteins which are exposed at the outer surface of virions; while
matrix protein is a non – glycosylated protein which forms a layer at the inner surface of the
envelope.Matrix protein appears to establish the connection between the envelope and the capsid
because of the presence of these two proteins makes enveloped virions to have additional structures.
The protein of glycoproteins in the envelope may even project from the envelope as spikes which
are called peplomers. Peplomers are envelope proteins and are coded by virus a gene; that is why
peplomer differ among viruses and are used to identify some viruses. It is thought that peplomers
may be involved in virus attachment to the host cell surface.
1. In orthomyxoviruses like influenza virus
The peplomers are about 10 nm long and 7 – 8 nm apart and are two types.
A. Some binds to RBCs and cause agglutination of RBCs and confers haemagglutination properties
of the virus and this antigen which agglutinates RBCs called haemagglutinins.
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B. The others have neuraminidase activity.
Neuraminidase is an enzyme and commonly referred as Receptor Destroying Enzyme (RDE)
because neuraminidase destroys the receptor activity of RBCs and susceptible host cells for
orthomyxoviruses and paramyxoviruses. Neuraminidase are enzymes that are present on the surface
of orthomyxoviruses and paramyxoviruses which ultimately dissociates the virus from infected
RBCs by splitting NANA(N – AcetylNeuraminic Acid) from receptors; as the result; after the virus
eluted, RBCs cannot be agglutinated again by a new batch of the virus because the RBCs have lost
the receptors. These cells which have lost their receptors and are not agglutinating by a new batch of
the same viruses are called stabilized viruses. However, cells that are stabilized by a given
orthomyxovirus or paramyxovirus can sometimes be agglutinated by another virus of these families.
2. In paramyxoviruses
A single kind of peplomers perform both haemoagglutinating and neuraminidase activities. The
second kind of peplomers causes haemolysis of RBCs and fusion of tissue culture cells to which the
virus is adsorbed.
Model of virion with complicated morphology in the family of Rhabdoviridae

Model of virion with complicated morphology in the family of Paramyxoviridae

Model of virion with complicated morphology in the family of Orthomyxoviridae

4. Virions with complex morphology

They are also called complex viruses because they have capsids of complex symmetry. Viruses
with complex morphology comprise:-
 1. Poxviruses
 2. Large bacteriophages
1. Poxviruses
You know poxviruses are the largest of the animal viruses and can be seen even by phase – contrast
microscope or by light microscope using special stained preparation. Most pox virions are brick
shaped and have the size of about 250×200×200 nm in size except genus Parapoxvirus because
virions in genus Parapoxvirus are oval shaped with size of 250 × 160 × 150 nm.There is no
isometric nucleocapsid conforming to either icosahedral or helical symmetry. So that poxviruses are

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said to have a “complex” structure. Poxviruses are complex viruses because the virions have
complex structure which comprises:-
 Complex internal structure
 Ovoid to brick shape exterior structure
The complex internal structure of poxviruses consists:-
 Biconcave nucleoid with double stranded DNA surrounded by two lipid layers called core
membrane.
 Two elliptical bodies which lie between biconcave nucleoid and the outer layer of the virus.
The two elliptical bodies consist:-
 The inner thicker elliptical body
 The outer thinner elliptical body
The inner thicker elliptical body is found between the outer core membrane of biconcave nucleoid
and the outer thinner elliptical body and it is covered by surface membrane and is thicker than the
outer thinner elliptical body. The inner elliptical body contains:-
 Lateral body
 Tubular structures arranged irregularly except genus Parapoxvirus.
But in genus Parapoxvirus the inner elliptical body is occupied by:-
 Lateral body
 Long thread like surface tubules with spiral arrangements, resembling a ball of yarn.
The outer thinner elliptical body is found between surface membrane of the thicker elliptical body
and envelope (most poxviruses are enveloped viruses). The outer thinner elliptical body is an
elliptical body where soluble antigens of the viruses are found.
Model structure of virions with complex morphology in the family of Poxviridae

2. Large bacteriophages
Some bacteriophages have complex morphology; and the most common large bacteriophages with
complex morphology are:-
 Even numbered coliphages including T2, T4 and T6 and are called T – even coliphages.
 Lambda(λ ) phages
These large bacteriophages have very complex structures including:-
 A head
 A tail
Complex bacterial viruses with both heads and tails are said to have binal symmetry because they
possess a combination of icosahedral (the head) and helical (the tail) symmetry. The head of T –
even coliphages resembles an icosahedron elongated by one or two rows of hexamers in the middle
and contain the DNA genome. While the tail of T – even coliphages is composed of:-
 A collar joining the tail to the head
 A central tube with a helical sheath that covers the central tube
 A complex base plate
In T – even coliphages, the base plate is hexagonal and consists:-
 A pin at each corners
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  A joined tail fiber at each corners
So that there are six pins and six joined tail fiber at hexagonal base plate of T - even coliphages.
Lambda (λ) phages have: -
 True icosahedral head
 Tail without sheath that lacks a base plate and terminates with rudimentary tail fibers
The T – odd coliphages have different capsid structures. T1 and T5 coliphages have:-
 True icosahedral heads
 Sheathless helical tail that lacks a base plate and terminates rudimentary tail fibers
However, coliphages T3 and T7 have:-
 True icosahedral head
 Short and noncontractile tails without tail fibers.
So that among T – odd coliphages only T1 and T5 are complex viruses; and it is clear that the tail
fibers in large bacteriophages are responsible for virus attachment to the proper site on the bacterial
surface.
Model morphology of virion of T- even coli phages

Model morphology of virion of T7 – phage with isometric head

short, non-contractile tail

Model morphology of virion of λ – phage with isometric headlong, non-contractile tail

Size and morphology of virions of different families

Morphology Nature of Virion size Presence of Family of the virus
the genome (diameter in nm) an envelope
Spherical NssRNA 80–100 Enveloped Bornaviridae
Pleomorphic NssRNA 80–120 Enveloped Orthomyxoviridae
Spherical NssRNA 80–120 Enveloped Bunyaviridae
Spherical NssRNA 50–300 Enveloped Arenaviridae

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 Spherical ssRNA 120–160 Enveloped Coronaviridae
Spherical ssRNA 45–60 Enveloped Arteriviridae
Icosahedral ssRNA ≈ 30 Naked Picornaviridae
Icosahedral ssRNA 27–40 Naked Caliciviridae
Icosahedral ssRNA 28 - 30 Naked Astroviridae
Spherical ssRNA ≈70 Envelope Togaviridae
Spherical ssRNA 40–60 Envelope Flaviviridae
Icosahedral ssRNA 27–34 Naked Herpusvirus(unsigned)
Icosahedral ssDNA 30–32 Naked Anellovirus (unassigned)
Filamentous NssRNA 600–800 ×80(L×W) Enveloped Filoviridae
Bullet shaped NssRNA 100–430 × 45– Enveloped Rhabdoviridae
100(L×W)
Pleomorphic NssRNA ~150 Enveloped Paramyxoviridae
Icosahedral dsRNA 60 Naked Birnaviridae
Icosahedral dsRNA 60–80 Naked Reoviridae
Spherical ssRNA-RT 80–100 Enveloped Retroviridae
Icosahedral ssDNA 18–26 Naked Parvoviridae
Icosahedral ssDNA 12–27 Naked Circoviridae
Spherical dsDNA-RT 42–50 Enveloped Hepadnaviridae
Icosahedral dsDNA 55 Naked Papillomaviridae
Icosahedral dsDNA 40–45 Naked Polyomaviridae
Icosahedral dsDNA 80–100 Naked Adenoviridae
Icosahedral dsDNA 150 Enveloped Herpesviridae
Spherical dsDNA 173–215 Enveloped Asfarviridae
Icosahedral dsDNA 130–300 Enveloped Iridoviridae
/naked
Pleomorphic dsDNA 250X200 X 200 Enveloped Poxviridae
Morphology of DNA virions in different families and genera of viruses affecting vertebrates

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1.10. VIRAL TAXONOMY AND NOMENCLATURE
Taxonomy is defined as the science of biological classification. In a broader sense, taxonomy
consists of three separate but interrelated parts. These are:-
 Classification
 Nomenclature
 Identification
Classification is the arrangement of organisms into groups or taxa (singular, taxon) based on mutual
similarity or evolutionary relatedness. Nomenclature is the branch of taxonomy concerned with the
assignment of names to taxonomic groups in agreement with published rules. Microbiologists name
microorganisms by using the binomial system which was used by Swedish botanist called Carolus
Linneaus. The most commonly used taxonomic levels or ranks in biology in ascending order are:-
 Species
 Genera
 Subfamily
 Family
 Order
 Class
 Phylum(division)
 Kingdom
But in virology the most common taxonomic ranks used in descending order are:-
 Order
 Family
 Subfamily
 Genera
 Species
That means virologists are not used kingdom, phylum and class in taxonomic classification of
viruses. Identification is the practical side of taxonomy and it is the process of determining that a
particular isolates belongs to a recognized taxon.
1.10.1. Viral taxonomy and its importance
A basic question that has yet to be addressed is why we should bother with taxonomy at all. This is
due to:-
 For some there seems to be a human need to place things into an ordered system.
 In characterizing an entity and defining a nomenclature, a basic understanding of the subject
under study may be achieved.

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  In a larger context, taxonomy provides a tool for comparing one virus with another or one
virus family with another.
 It also enables one to assign biological properties to a new virus that is provisionally linked
to a given family.
There are two methods that used in classification of viruses.
 1. Early methods of virus classification
 2. Modern methods of virus classification
1. Early methods of classification of viruses
The earliest recognition was that infectious agents were associated with a given spectrum of clinical
outcomes. In the earliest time; it was natural for an agent to take its name based on:-
 The name of the disease with which it was associated or
 The geographic location where it was found
Because there was no other basis for assigning a name of viruses; thus the agent that caused foot-
and-mouth disease in cattle becomes “foot-and-mouth disease virus” and an agent that caused a
febrile disease in the Rift Valley of Africa became “Rift Valley fever virus. As the result different
names might be given to the same virus in different area of the world. As one virus may attack
different species of animals and may have different clinical manifestations on the same species of
animals. Some of the examples are:-
 Different names might be given to the same virus which the agent is causing a disease in a
cow in England and that causing disease in a water buffalo in India.
 Hog cholera virus existed in North America where as, in the rest of the world, it was named
as: classical swine fever virus, not to be confused with African swine fever virus.
 Within the same animal, one had infectious bovine rhinotracheitis (IBR) virus and infectious
bovine pustular vulvovaginitis (IBPV) virus even if both disease entities being caused by
bovine herpesvirus 1.
However, with the development of negative stain electron microscopy as a readily available
technology, the size and shape of viruses became a characteristic for defining them. This, along with
the ability to define the type of nucleic acid in the virus particle, provided the beginnings of a more
rational system of classifying and naming new viruses. Even with a defined shape and a type of
nucleic acid, there were still ambiguities in the classification systems that were being developed.
Some of the examples are:-
 Viruses that were transmitted by insect vectors were loosely defined as arboviruses
(arthropod-borne viruses).
 The group of small RNA viruses collectively known as picornaviruses (small RNA viruses).
However, there were viruses that “looked like” arboviruses that had the same nucleic acid like
togaviruses—viruses with a symmetrical lipid membrane but did not have an insect vector and these
viruses became “non-arthropod-borne” togaviruses. The answer to many of these issues came with
the advances in the ability to determine the nucleotide sequences of these agents. Thus the “non-
arboviruses” togaviruses became members of:-
 Family: Arteriviridae.
 Genera: Rubivirus or Pestivirus
But most of arboviruses nowadays are classified under:-
 The family: Bunyaviridae
 Genera: Phelbovirus or Nairovirus or Hantavirus etc.
Virologists are no different to other scientists in that they find it useful to classify the objects of their
study into groups and sub-groups. Due to a lack of knowledge of the origin and evolutionary history
of viruses in the ancient time, viruses were separated into several large groups based on their host
preferences.
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  Animal viruses
 Plant viruses
 Bacterial viruses( bacteriophages)
 Insect viruses etc.
As more was learnt about the characteristics of virus particles, they were used for virus
classification. Some of the virus characteristics that were used to classify viruses were:-
 Whether the nucleic acid is DNA or RNA
 Whether the nucleic acid is single stranded or double stranded
 Whether genome is segmented or not
 The size of the virion
 Whether the capsid has helical symmetry or icosahedral symmetry
 Whether the virion is naked or enveloped
Unfortunately, virologist working with these groups has been unable to agree on a uniform system
of classification and nomenclature of viruses. To solve this problem:-
 Advanced technology to characterize individual viruses was developed
 Procedure and guidelines for developing a universally acceptable taxonomy for viruses was
prepared.
2. Modern methods of virus classification
In 1966, the International Committee on Taxonomy of Viruses (ICTV) was established and
charged with establishing, refining, and maintaining universal virus taxonomy. Nucleotide sequence
is used to classify viruses. Serological relationships between viruses were investigated, and distinct
strains (serotypes) were distinguished. Serological tests using antisera against purified virions were
used to determine their serotype. Serotypes reflect differences in virus proteins and have been found
for many types of virus, including rotaviruses and foot and mouth disease virus.
International Committee on Taxonomy of Viruses (ICTV) has sub committees. The subcommittees
and study groups meet periodically to assess:-
 The new data submitted from the research community to refine the classification system
 The places of new viruses in their most logical position in the taxonomy scheme.
It was not until (2000) that the concept of virus species as the lowest group in the viral taxa was
accepted. Higher taxonomic groupings, such as kingdom, phylum and class are not used in virus
classification. Only some virus families are divided into sub families; so that the following rules are
used in virus classification.
 To be a member of the taxa higher than species, a virus must have all properties defining the
classification.
 In contrast, species are considered a polythetic class, in which members have several
properties in common but all do not have to share a single defining property.
So that the recognized hierarchy of viral taxa was prepared and it is as follows.
 Order
 Family
 Subfamily(in some virus families)
 Genus
 Species
The Eighth Report of the ICTV published in 2005 had approved there are the following orders,
families, subfamilies, genera and species of viruses.
 3 orders
 73 families
 9 subfamilies
 287 genera
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  More than 5000 approved species
Nowadays the process of classification and defining nomenclature is an ongoing one because of the
discovery of new viruses and the generation of sequence data on older isolates is necessary. There
are two modern virus classification methods.
 1. Classification of viruses based on genome sequences
 2. Baltimore classification of viruses
1. Classification of viruses based on genome sequences
It is also called classification of viruses based on genome sequences. The modern approach to
virus classification is based on comparisons of genome sequences and organizations. Now those
technologies for sequencing virus genomes and for determining genome organization are readily
available. The degree of similarity between virus genomes can be assessed using computer programs
and can be represented in diagrams known as phylogenetic tree because they show the likely
phylogeny (evolutionary development) of the viruses. Phylogenetic trees may be of various types
but the most commonly used in virus taxonomy are:-
 Rooted phylogenetic tree
 Un rooted phylogenetic tree
In rooted phylogenetic tree the tree begins at a root which is assumed to be the ancestor of the
viruses in the tree; while in un rooted phylogenetic tree; no assumption is made about the ancestor of
the viruses in the tree. The branches of a phylogenetic tree indicate how sequences are related.
The branches may be scaled or un scaled. If they are scaled, their lengths represent genetic distances
between sequences. In many cases analysis of the sequence and organization of virus genomes has
supported earlier classification of viruses. Some of the examples are the genera of the family
Reoviridae and Rhabdoviruses which were originally grouped together because of their bullet-
shaped morphology but it turns out that they are also related genetically.
2. Baltimore classification of viruses
This method of virus classification was made based on:-
 The type of genome and
 The way in which the genome is transcribed and replicated
This approach to virus classi fication was first suggested by David Baltimore, after whom the
scheme is named. Baltimore method of classification of animal viruses groups by genome structure.
This method classifies viruses with regard to the various mechanisms of viral genome replication.
The central theme is that all viruses must generate positive strand mRNAs [(+) RNA) from their
genomes, in order to produce proteins and replicate themselves; so that the precise mechanisms
whereby this is a achieved differ for each virus family. An advantage of the Baltimore classification
is its differentiation between plus-strand RNA viruses and dsDNA viruses. The plus ssRNA viruses
are classified as follows.
 Plus ssRNA viruses in which the RNA serves as mRNA are grouped in class IV
 Plus ssRNA viruses in which the RNA serves as template for DNA synthesis are grouped in
class VI
The dsDNA viruses are classified as follows:-
 The dsDNA viruses that carry out reverse transcription are grouped in class VII
 Those which do not carry out reverse transcription are grouped in class I
The seven classes of animal viruses grouped by type of nucleic acid
Class of viruses Genus of viruses
Papovavirus
I. dsDNA Adenovirus
Herpesvirus
Poxvirus
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 II. ssDNA Parvovirus
III. DsRNA Reovirus
IV. ssRNA that can serve as mRNA Picornavirus
Togavirus
V. ssRNA that is a template for mRNA Rhabdovirus
Paramyxovirus
Orthomyxovirus
VI. ssRNA that is a template for DNA synthesis Retrovirus
VII. dsDNA with RNA intermediate that is a template for Hepadnavirus
DNA synthesis
NB. There are subclasses within each class which differ mainly in capsid structure in the
presence or absence of a membranous envelope.
ds = double stranded ; ss = single - stranded
1.10. 2. Viruses’ nomenclature
In the early time of development of virology, there was no single approach to the naming of viruses
and the names of viruses were derived in a variety of ways. Some of them were:-
 Small icosahedral, single stranded DNA viruses of animals were called parvoviruses (Latin
word “parvus” meaning small).
 By clinical features that they cause; like FMD virus
 Nematode-transmitted icosahedral viruses of plants were called nepoviruses
 Coliphages T2, T4 andT6 were called T- even phages.
In modern nomenclature of viruses somewhat different approaches was taken for viruses of different
host types.
 Bacterial viruses were simply allotted codes, such as T1, T2 and ϕX174
 Viruses of humans and other vertebrates were commonly named after the diseases that they
cause, e.g. measles virus, small poxvirus, foot and mouth disease virus.
 Some were named after the city, town or river where the disease was first reported. e.g.
Newcastle disease virus and Ebolavirus.
As in other areas of biology, many names of virus taxonomic groups are based on Latin words while
some have Greece and Spanish origins. Some of examples are:-
 Filovirus the name originated from “Latin” and “Greece” words “filum” meaning thread as
the viruses have thread like morphology.
 Picornaviruses originated from “Spanish” word “Pico” meaning small as the virus is the
smallest RNA virus.
Name of some viruses’ families and genera derived from places name
Place name Family/genus and species name
Bunyamwera (Uganda) Family Bunyaviridae
Ebola(river in Zaire) Genus Ebolavirus
Hantaan (river in South Korea) Genus Hantavirus
Hendra(Australia)and Nipah(Malaysia) Genus Henipavirus
Norwalk(USA) Genus Norovirus
Newcastle(England) Species Newcastledisease virus
Gumboro(USA) Species Gumborodisease virus
Names of some virus families and genera based on Latin and Greek words
Origin Words Translation Reason for name Family/genus name
Mikros Small Virion size Family Microviridae
Greek Pous Foot Phages with short tails Family Podoviridae

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 Latin Arena Sand Ribosomes in virions Family Arenaviridae
resemble sand grains in
thin section
Baculum Stick Capsid shape Family Baculoviridae

Filum Thread Virion shape Family Filoviridae

Latin Flavus Yellow Yellow fever virus Family Flaviviridae
Parvus Small Virion size Family Parvoviridae
Toga Cloak Virion is enveloped Family Togaviridae
Example of taxonomic group of virus
Taxonomic group Suffix Example
Order -virales Mononegavirales
Family -viridae Paramyxoviridae
Subfamily - virinae Paramyxovirinae
Genus -virus Morbillivirus
Species - Rinderpest virus
For example, it is possible to write like this one can grow canine distemper virus or West Nile virus
in monkey cells without italics but must be capitalized if the name of the virus is a proper noun.

1.11. VIRAL REPLICATION

A fundamental characteristic that separates viruses from other replicating entities is the manner in
which new virus particles are synthesized. Viruses do not use binary fission, budding and
fragmentation as other microbes do. Virus particles are assembled into new virions from the various
structural components synthesized as somewhat independent but synchronized events. The
replication of viruses in susceptible host can be represented by virus replication curve is called one-
step growth curve of viruses. One step virus growth curve shows infectious virus particle
“disappeared” from the infected cultures for a variable period of time, depending on the virus–host-
cell system. The period of time is referred to as the eclipse period of virus replication. Eclipse
period represents the time needed for the various parts of the virus particle to be synthesized and
assembled.
One-step growth curve

Once assembly begins, there is an essentially exponential increase in infectious virus until the host
cell is unable to maintain metabolic integrity. Depending on the type of virus, there may be:-
 Sudden release of virus particles (lysis of the host cell, exemplified by T-even
bacteriophage) or

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  A more slow release (maturation of the virus particle at a cell membrane site, such as with
influenza virus).
You know viruses were defined as obligate intracellular parasites that are unable to direct any bio-
synthetic processes outside the host cell. The genetic complexity of viruses varies greatly between
individual virus families, ranging from those viruses that encode just a few proteins to others that
encode more than 900 proteins. The replication processes used by individual viruses would also be
highly variable. However, all viruses must go through the same general steps for replication to
occur. The one-step virus growth curve can be used to divide the virus replication cycle into its
component parts for discussion of the general replication patterns; however, more specific details
are to be found in the chapters dealing with the individual virus families in systematic virology.

1.11.1. Stages of viral replication

The basic components of the replication cycle of viruses are:-
 1.Attachment to a susceptible host cell
 2.Penetration to the cell
 3.Uncoating
 4.Replication of its own genetic material and associated proteins
 5.Assembly of new virus particles; also called maturation of viruses
 6. Escape from the infected cell(release of virus particles)
So that the eclipse period comprises a period required by different families of viruses for:-
 Un coating
 Replication of component parts
 Assembly of new virus particles called maturation
One-step growth curve of a non-enveloped virus differed from naked viruses. In enveloped viruses;
attachment and penetration are followed by an eclipse period of 2–12 hours during which cell-
associated infectivity cannot be detected. This is followed by a period of several hours during which
virus maturation occurs. The release of enveloped virions occurs concurrently with maturation by
budding from the plasma membrane; however, virions of non-enveloped (naked) viruses are often
released late.
1. Attachment
Attachment is the critical first step in the virus replication cycle; and it is the binding of the virus
particle to a host cell. This binding process may involve a series of interactions that define in part
the host range of the virus and its tissue/organ specificity called tropism of virus. Tissue and organ
specificity tropism largely defines:-

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  The pathogenic potential of the virus and
 The nature of the disease it induces
Virus particles interact with cell surface molecules referred to as attachment factors, entry factors,
receptors, and co-receptors. Frequently, the term “viral receptor” is used to describe the cell-
surface molecules because cells certainly do not maintain receptors specifically for viruses; rather,
viruses have evolved to use host cell-surface molecules critical for cellular processes. Attachment of
a virus particle to the host cell is a temperature-independent process. Among them the most
common virus entry factors are:-
 Ligand-binding receptors (e.g. chemokine receptors)
 Signaling molecules (e.g., CD4)
 Heparan sulfate proteoglycan
 Cell adhesion/signaling receptors [e.g., intercellular cell adhesion molecule-1 (ICAM-1)]
 Enzymes
 Integrins
 Glyco- conjugates with various carbohydrate
 Sialic acid
 Opsonin (Antibody bind to Fc centor of the virus) etc.
The number of specific molecules that play a part in the initial interactions of virus with host cells
will certainly increase as new viruses are identified. However, the existing viruses’ receptors are
better characterized. Different viruses may use the same receptor/entry factor, which simply reflects
the fact that a similar host cell serves as the replication site of the viruses. Furthermore, different
strains of the same virus can utilize different receptors. For example for foot-and-mouth disease
virus, the receptors in the bovine host are integrins but cell-culture passaged virus can use heparan
sulfate. This change in receptor specificity alters the pathogenicity of the virus and clearly indicating
that receptor specificity is a key factor in the disease process. For viruses with a wide host range, the
viruses can use several receptors, accounting for their ability to grow in cells from many hosts.
Virus–receptor interaction is further complicated by those viruses that require several entry factors
to initiate an infection successfully. A prominent example of this phenomenon is human
immunodeficiency virus (HIV).
Examples of a few cellular macromolecules used by viruses as receptors/entry factors
Virus Family Receptor
Human immunodeficiency Retroviridae CCR5, CCR3, CXCR4 (heparan sulfate
virus proteoglycan

Avian leukosis/sarcoma virus Retroviridae Tissue necrosis factor-related protein

Foot-and-mouth disease Picornaviridae Various integrins
virus—wild-type virus
Newcastle disease virus Paramyxoviridae Sialic acid
Rotavirus Reoviridae Various integrins
Blue tongue virus Flaviviridae Heparan sulfate proteoglycan
Rabies virus Rhabdoviridae Acetylcholine, NCAM (neural adhesion
molecule)
Influenza A virus Orthomyxoviridae Sialic acid
Influenza C virus Orthomyxoviridae Acetylsialic acid

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2. Penetration
After binding to receptors animal viruses must cross the plasma membrane to gain entry to the host
cell. Penetration is dependent upon the fluidity of the lipids in the plasma membrane which does
have temperature constraints. Viruses penetrate the plasma membrane either:-
 At the cell surface through pores of cytoplasmic membrane or
 They may cross the membrane by forming endosome
Endosome is a vesicle formed by part of the plasma membrane pinching off into the cytoplasm; and
the process is called endocytosis. You know endocytosis is used by cells for a variety of functions,
including:-
 Nutrient uptake
 Defense against many foreign materials (macrophages and neutrophiles). Endocytosis in
animal cells can occur via several different mechanisms. Among several mechanisms the one
is pinocytic. Pinocytosis is the process of the uptake of fluid, solutes, and small particles by
cells from fluid environment. This includes:-
 Clathrin-mediated pinocytosis
 Macropinocytosis
 Caveolin mediated pinocytosis.
Among them the most common methods used by animal viruses are:-
 Clathrin-mediated endocytosis and
 Caveolin-mediated endocytosis
However, most animal viruses hi - jack one or more of these mechanisms in order to gain access to
their host cells. If a virion binds to a region of the plasma membrane coated with clathrin or caveolin
these protein molecules force the membrane to bend around the virion and are endocytosed at
clathrin coated regions of the plasma membrane. The virions end up in clathrin coated endosomes,
from which the clathrin is soon lost. Some viruses, such as simian virus 40, are endocytosed at
Caveolin-coated regions of the plasma membrane and the virions end up in caveolin-coated
endosomes. Other viruses are taken up by mechanisms that are independent of clathrin and caveolin.
An endosome may fuse with other vesicles such as lysosomes, which has a PH of 4.8– 5.0 results in
lowering the PH within the vesicle. The PH may be further lowered by a process that pumps
hydrogen ions across the membrane; the process involves the hydrolysis of ATP to ADP. This
acidification of the environment of the virion is important for those enveloped viruses that need to
carry out acid-triggered fusion of the envelope with the vesicle membrane. Generally, the
mechanisms of virion penetration (entry) to susceptible host vary with their morphological features.
 1. Entry of naked viruses
 2. Entry of enveloped viruses
1. Entry of naked viruses
Some naked viruses deliver their genomes into their host cells through a pore formed in the plasma
membrane; but for most naked viruses irreversible attachment of the virion to the cell surface leads
to endocytosis. The plasma membrane flows around the virion and more receptors bind, and
eventually the virion is completely enclosed in membrane which pinches off as an endosome. The
endosome contents, however, are part of the external environment and the virus is not yet in the
cytoplasm. The mechanisms by which virions or their genomes are released from endosomes are not
fully understood.
2. Entry of enveloped viruses
Reversible attachment of an enveloped virion may lead to irreversible attachment, as for naked
viruses. There are then two processes where infection of the cell may occur by enveloped viruses.
These are:-
 Fusion of the virion envelope with the plasma membrane or
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  Fusion of the virion envelope with the endosome membrane after endocytosis.
Both processes involve the fusion of the virion envelope with a cell membrane, either at the plasma
membrane or at a vesicle membrane. Lipid bilayer of cells doe not fuse spontaneously, however,
each enveloped virus has a specialized glycoprotein responsible for membrane fusion. Some of the
examples of fusion proteins of enveloped viruses that involve in membrane fusion at cell membrane
are:-
 CD4(receptor for HIV)
 CD155(receptor for poliovirus)
 CAM(Clathrin-mediated endocytosis which is a receptor for most rhinovirus strains)
The fusion sequence normally lies hidden and must become exposed in order for fusion to take
place. Conformational changes to the fusion protein that expose the fusion sequence may come
about either as a result of:-
 The virus binding to a receptor or
 They may be induced by the low PH within an endosome.
If the fusion sequence becomes exposed while the virion is at the cell surface, then infection could
occur either by fusion with the plasma membrane or by endocytosis; but if, however, exposure to
low PH is required to expose the fusion sequence, or then endocytosis is the only option. Therefore;
there are two categories of membrane fusion. These are:-
 PH-independent fusion, e.g. herpesviruses and HIV etc.
 Acid-triggered fusion, e.g. influenza viruses and rhabdoviruses etc.
Once the fusion sequence is exposed it can insert into the target membrane (plasma membrane or
endosome membrane). A further conformational change in the fusion protein pulls the two
membranes together and then mediates their fusion. This process involves a release of energy from
the fusion protein which results in irreversibly changing of shapes of the viruses.
3. Uncoating
Once the nucleocapsid gains entry into the host cell cytoplasm, the process of uncoating occurs.
Uncoating can be defined as the complete or partial removal of the capsid to release the virus
genome (the viral nucleic acid is released from its coat). Uncoating processes are apparently quite
variable and only poorly understood. However, most virologists proposed acidification due to
lysosome fusion in the endosome is known to cause rearrangements in the virus coat proteins. This
probably allows extrusion of the viral core of enveloped viruses into the cytoplasm of infected cells.
Depending on the virus, uncoating takes place at different sites of infected cells; and it can be:-
 At the cell surface(the capsid remaining on the exterior surface of the cell)
 Within the cytoplasm
 At a nuclear pore
 Within the nucleus
It should be noted that successful entry of a virion into a cell is not always followed by virus
replication because the host’s intracellular defenses, such as lysosomal enzymes, may inactivate
infectivity before or after uncoating. In some cases the virus genome may initiate a latent infection
(persistent infection) rather than a complete replication cycle. However, if viruses able perform a
complete replication cycle, the entry of the virus particle, nucleocapsid, or genomic nucleic acid into
the cytoplasm in many cases is not the final step in the initiation of the replication process. In most
cases, the nucleic acid may not be in the correct location for replication to occur which means
intracellular transport is needed. So that again, cellular processes are involved in the transport of the
viral units to the required locations. Destination of viral nucleic acid is reached using one of the
transport systems of infected cell including:-
 Microtubules of cytoplasm of cells
 Nuclear microtubules
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  Nuclear pores
Microtubules are actin - filaments structure found in many eukaryotic cells. Microtubules are hollow
cylinders, 25nm in diameter, and are composed of the proteins tubulin. The ends of each
microtubule are designated as plus or minus. In most animal cells the plus ends are located near the
plasma membrane; while the minus ends are attached to a structure in cytoplasm of infected cells
called centrosome which is located close to the nucleus. Proteins, known as motor proteins, move
themselves and any cargo moves along the microtubules. A number of viruses exploit this transport
system to take their nucleocapsid to nucleus. Some of them are:-
 Herpesviruses
 Adenoviruses
 Parvoviruses
 Retroviruses
The nuclear envelope is composed of two membranes which is a lipid bilayer. Within the nuclear
envelope; there are nuclear pores and their number is between 3000 and 5000 per nuclear
membrane. A few animal viruses replicate on the nuclear pores. Animal viruses in which their
genome replication occurs in nucleus and which are small in size enters to the nucleus through the
pores of nuclear membrane but those which are large enter to the nucleus through nuclear
microtubules. What you must understand is that most RNA viruses of eukaryotes replicate in the
cytoplasm because the majority encode all the enzymes for replication of their genomes and they
have no requirement for the enzymes of the nucleus. However there are some exceptions even if
they are RNA viruses in which their genome needs enzymes that are found in the nucleus of infected
cells. Some of them are:-
 Influenzavirus
 Retroviruses
Influenza viruses with negative sense ssRNA virus that serves as a template for mRNA are
exceptions as they require the cell splicing machinery of the nucleus, so their genomes must be
delivered into the nucleus. Other RNA viruses that replicate their genomes in the nucleus of infected
eukaryotic cells are retroviruses. Retroviruses are reverse – transcribing viruses. They copy their
RNA genomes to DNA in the cytoplasm.
RNA RdDP DNA [RdDP (RNA dependent DNA polymerase of the virus also called reverse
transcriptase)];then most retroviruses DNA and associated proteins must wait in the cytoplasm
until mitosis begins. During mitosis the nuclear envelope is temporarily broken down and the virus
DNA with associated proteins is able to enter the nuclear compartment. In the nuclear compartment
by using the cell nucleus DdRP(DNA dependent RNA polymerase of the nucleus); DNA is
converted into RNA.
DNA DdRP RNA of the virus
These viruses therefore can replicate only in cells nucleus that are dividing; however, some groups
of retroviruses like lentiviruses which include HIV; their DNA can replicate in non-dividing cells.
.4. Replication of viral genome and synthesis of viral proteins
To have a better understanding of viral genome replication and proteins synthesis, let us remember a
few essential molecular biology terms like:-
 Transcription
 Translation
 Transport
Transcription means writing across; while translation means bearing across; and transport means
carrying across.
In viral genome replication; transcription refers to the writing across of genetic information from a
sequence of bases in a nucleic acid to the complementary sequence in messenger RNA (mRNA);
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while translation converts the genetic information from the language of bases in nucleic acids to the
language of amino acids in proteins. Transport is a carrying across of viral proteins and RNAs to
particular locations in infected cells. In viral genome replication and protein synthesis, the processes
that occur in the correct order are:-
 1.Transcription
 2.Translation
 3. Genome replication
1. Transcription and translation
You know in molecular biology in that in 1958 Francis Crick proposed “a central dogma of
molecular biology”. James Watson, Crick’s collaborator in deducing the structure of DNA. The
discovery of the structure of DNA made significant contributions to the formulation of the dogma
which stated that the flow of genetic information is always from DNA to RNA and then to protein
with genetic information transmitted from one generation to the next through copying from DNA to
DNA.However, increasing understanding of how viruses replicate their genomes necessitated some
medications to this dogma in 1970 because many viruses have RNA genomes that are copied to
RNA, and some viruses copy from RNA to DNA, as the result modifications to the central dogma
was known.
Central dogma of molecular biology

Modification of central dogma of molecular biology

3. Viral genome replication

We have seen there are four main categories of virus genome. These are:-
 dsDNA
 ssDNA
 dsRNA
 ssRNA
You know because of distinct modes of transcription within the dsDNA and ssRNA categories of
viruses, a total of seven classes of animal viruses were recognized in Baltimore method of virus
classification. They are :-
 Class I = dsDNA viruses
 Class II = ssDNA viruses

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  Class III = dsRNA viruses
 Class IV = ssRNA viruses in which their RNA that can serve as mRNA and are called
positive sense (polarity) ssRNA viruses.
 Class V= ssRNA viruses inwhich their RNA is template for mRNA and are called negative
sense (polarity) ssRNA viruses.
 Class VI = ssRNA viruses inwhich their RNA is a template for DNA synthesis and are called
reverse transcribing viruses.
 Class VII = dsDNA viruses with RNA intermediate that is a template for DNA synthesis.
Let us see the enzymes of cells and viruses that play in genome replication of viruses that are found
in seven classes of Baltimore replication.
1.11.2. Enzymes used by viruses to replicate their genomes
Many viruses encode a polymerase to replicate their genome but some use a cell enzyme.
1. Virus enzymes
1. DNA DNA-dependent DNA polymerase DNA
2. RNA RNA-dependent RNA polymerase RNA
3. RNA RNA-dependent DNA polymerase(reverse transcriptase) DNA
2. Cell enzymes
1. DNA DNA-dependent DNA polymerase DNA
2. DNA DNA-dependent RNA polymerase(RNA pol II) RNA
Enzymes used by viruses to transcribe their genomes to mRNA.

Genome replication in different classes of Baltimore

Baltimore class Mode of replication
I and II DNA DdDp/DdDp 1 DNA
III, IV and V RNA RdRp RNA
VI RNA RdDp DNA DdRp RNA
VII DNA DdRp RNA RdDp DNA
Virus enzymes : DdDp=DNA-dependent DNA polymerase
RdRp=RNA-dependent RNA polymerase
RdDp=RNA-dependent DNA polymerase (reverse transcriptase)
Cell enzymes: DdDp =DNA-dependent DNA polymerase
DdRp=DNA-dependent RNA polymerase
1
DdDp : Some dsDNA viruses use a cell DdDp, some encode their own.
The genome of the infecting virus is replicated so that viral transcription can be amplified. This
provides copies of the genome for progeny virions. Generally, DNA viruses copy their genomes
directly to DNA and RNA viruses copy their genomes directly to RNA. However, there are:-

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  Some DNA viruses that replicate their genomes via RNA intermediate which are found in
class VII in Baltimore virus classification and are called pararetroviruses.
 Some RNA viruses that replicate their genomes via a DNA intermediate which are found in
class VI in Baltimore virus classification and are called reverse transcribing
viruses(retroviruses).
Single-stranded genomes are designated as plus or minus depending on their relationship to the virus
mRNA. Plus strand genomes have the same sequence as the mRNA (except that in DNA thymine
replaces uracil); while minus-strand genomes have the sequence complementary to the mRNA of the
virus.
Single-stranded DNA is converted to dsDNA prior to copying. There are two classes of viruses with
(+) RNA genomes. These are:-
 Class IV viruses copy their (+) RNA genomes via (−) RNA intermediate.
 While Class VI viruses replicate via a DNA intermediate.
The synthesis of DNA from an RNA template (reverse transcription) is also a characteristic of Class
VII viruses.
1.11.3. Sites of virus genome replication in eukaryotic cells
When viruses infect eukaryotic cells the genomes of some are delivered to the cytoplasm and some
are conveyed to the nucleus. The destination of a virus genome, and hence the location in which it is
replicated, varies with the type of genome. So that the sites of replication of viral genome can be:-
 In cytoplasm only( viruses that have all viral replication enzymes)
 In nucleus
 Both in cytoplasm and nucleus
The genomes of most DNA viruses are replicated in the nucleus (but those of some dsDNA viruses
are replicated in the cytoplasm).
The genomes of most RNA viruses are replicated in the cytoplasm (but those of the minus-strand
RNA viruses with segmented genomes are replicated in the nucleus).
The retroviruses and pararetroviruses are special cases: each replicates RNA to DNA in the
cytoplasm and DNA to RNA in the nucleus.

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Locations of viral genome replication in eukaryotic cells
Virus genome Cytoplasm Nucleus
dsDNA Some Some
ssDNA - All
dsRNA All -
(+)RNA All -
(−)RNA(non-segmented genome) All -
(−)RNA(segmented genome) All
Retroviruses[(+)RNA] ssRNA→dsDNA dsDNA→ssRNA
Pararetroviruses[dsDNA]
5. Assembly of new virus particles of eukaryotes
Assembly stage also called stage of virions maturation. Once the synthesis of the various viral
components is complete, the assembly stage begins. Assembly stage comprises:-
 5.1. Nucleocapsid assembly
 5.2. Formation of virion membranes (for only enveloped viruses)
5.1. Nucleocapsid assembly
Once threshold quantities of progeny virus genomes and structural proteins have accumulated in the
infected cell, assembly of virions can commence. These components are assembled into
nucleocapsids. That means the capsomere proteins enclose the nucleic acid to form the viral
nucleocapsid; and this process is called encapsidation. Encapsidation is different in different
viruses with different capsid symmetry. In helical viruses the assembly of virions and nucleocapsids
of ssRNA viruses involves coating of the genome with multiple copies of proteins. In icosahedral
viruses, nucleocapsid assembly involves the construction of an empty protein shell, known as a
procapsid. Then the procapsid is filled with a copy of the virus genome. During or after this process
it may undergo modifications to form the mature capsid. Modifications of the procapsid may result
in a change from a spherical to an icosahedral shape. For some viruses, including adenoviruses and
picornaviruses; modifications of the procapsid involves cleavage of one or more of the structural
proteins. Finally the genome enters the procapsid through a channel located at a site that will
become one of the vertices of the icosahedron. Any enzymes involved in packaging the genome are
located at this site.
Assembly of a helical nucleocapsid

Assembly of an icosahedral nucleocapsid

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5.2. Formation of virion membranes
Enveloped virions acquire their membrane envelopes by one of two mechanisms. These are:-
 1. Budding through the host membrane
 2. De novo synthesis of viral membranes
1. Budding through the host membrane
Most enveloped viruses acquire their envelopes by budding through a membrane of the host cell.For
viruses with eukaryotic hosts this membrane is often the plasma membrane called cytoplasmic
membrane. However, there are a few animal viruses that bud through cell membranes including
nuclear, EPR and Golgi complex. Budding is the mechanism by which the virions modify a host cell
membrane and then nucleocapsids bud through it. The virions of most retroviruses and
rhabdoviruses acquire their envelopes in this way. It is known that budding through plasma
membrane of eukaryotic cells occurs at selected site of infected cells. Regions of membrane through
which budding will occur become modified by the insertion of one or more species of virus protein.
The vast majority of which are glycoproteins. Integral proteins in a lipid bilayer have a degree of
mobility as they are “floating in a sea” of lipid. Virus proteins accumulate in regions of membrane
from which cell proteins, to a large extent, become excluded. This is may be:-
 Either the virus proteins repel the cell proteins and/or
 The virus protein molecules have affinities for each other
However, cell proteins may not be totally excluded from these regions and may become
incorporated into virus envelopes; for example, HIV envelopes contain major histocompatibility
complex class II proteins. Nucleocapsids accumulate adjacent to the regions of membrane
containing virus protein.
Interactions between proteins of enveloped viruses with and without matrix protein

NB. Virus membrane proteins usually glycoproteins become incorporated into regions of a cell
membrane, often the plasma membrane then a nucleocapsid buds through the membrane which
pinches off to form the virion envelope.
Budding of virions involves interaction between the cytoplasmic tail of a virus glycoprotein in the
membrane and another virus protein. In a number of virus groups, including paramyxoviruses and
rhabdoviruses, this protein is the M (membrane matrix) protein. M proteins have an affinity for
membranes, and bind to nucleocapsids as well as to the virus glycoproteins, ‘stitching’ the two
together during budding.

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Acquisition of a virion envelope by budding

However, not all enveloped viruses have a layer of protein between the envelope and the
nucleocapsid. Viruses that bud from the cell do so from particular of the plasma membrane. If the
cell is polarized then budding may take place primarily from one surface. Body surfaces, such as the
respiratory tract, are lined with epithelial cells that are polarized and each cell has:-
 An apical and/or
 A baso – lateral surface
Many viruses bud preferentially from one of these.
Example Influenza A virus buds almost exclusively from the apical surface.
While Vesicularstomatitisvirus buds almost exclusively from the baso – lateral surface.
Viruses that bud from apical surfaces are in position to be shed in respiratory or genital secretions or
intestinal contents but viruses that bud from basal surfaces are in position for systemic spread via
viremia or lymphatics. However, viruses that do not bud usually are released only via cell lysis.
The latest age of budding involves the membrane pinching off and the release of the newly formed
virion. The release of influenza virus particles from cells requires the activity of a vi rion enzyme
called a neuraminidase; and at budding sites cleavage of neuraminic acid, the substrate for the
enzyme, and is essential for the release of virions. That is why some anti influenza virus drugs have
been developed that act by inhibiting the neuraminidase activity.
Sites of budding of various enveloped viruses

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What you must understand is that the envelope of enveloped viruses is not only from plasma
membrane but also from different cell membranes like nuclear membrane, membrane of
endoplasmic reticulum and Golgi complex. Some of the examples are:-
 1. Herpesvirus
 2. Hepadna viruses
In Herpesvirus, the nucleocapsids are constructed in the nucleus and begin their journey to the
cytoplasm by budding through the inner membrane of the nuclear envelope. Then the envelope of
the mature virion is acquired in the cytoplasm by budding into vesicles derived from the Golgi
complex. In Hepadnaviruses bud into a membrane compartment between the endoplasmic reticulum
and the Golgi complex.
2. De novo synthesis of viral membranes
A minority of viruses direct the synthesis of lipid membrane of late in the replication cycle. In some
cases the membrane forms a virion envelope. Example: Poxviruses. In other cases the membrane
forms a layer below the surface of the capsid which is not envelope. Example : Iridoviruses
Summary of virion membrane acquisition of eukaryotic viruses
Site of virion Origin of virion Example
assembly membrane
Virion envelope
membrane Internal virion
Plasma membrane Most retroviruses and
Eukaryotic cell most rhabdoviruses
cytoplasm Post-RER membrane Hepadnaviruses
De novo synthesis
Eukaryotic cell Poxviruses Iridoviruses
nucleus Inner nuclear membrane Nuclear rhabdoviruses
6. Escape of virions from the infected cell
It is also called exit of virions from infected cells. Escape of virions from the infected cell is the
release stage virus replication cycle and is the final event in viral replication which results with the
exit of the mature virions from their host cells. It is known that virus maturation and release occurs
over a considerable period of time. Many viruses do not lyse their host cells; instead, progeny
virions may release from the cells over a period of time; so that the process of releasing of virions
from the cell without cell death called egestion; whereas others are released when cell dies and
disintegrates because virions of many viruses are released from the infected cell when it bursts
(lyses); a process that may be initiated by the virus. Cell lyses by virions may happen because:-
 Many phages produce enzymes (lysins, such as lysozymes) that break bonds in the
peptidoglycan of the host bacterial cell walls.
 Other phages synthesize proteins that inhibit host enzymes with roles in cell wall synthesis.
All these lead to weakening of the cell wall and ultimately to lysis of infected cells. We have already
seen how virions that acquire an envelope from a cell surface membrane leave the cell by budding.
So that in the case of envelope viruses, the nucleocapsid acquire its final envelope from the nuclear
or cell membrane by budding off process called envelopment.Virions that acquire envelopes from
internal membranes of the cell exit the cell in other ways.
 Some are transported to the cell surface in vesicles, which fuse with the plasma membrane to
release the virions.
 Others, such as vaccinia virus, attach via motor proteins to microtubules and use this
transport system to reach the cell surface where they are released.

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Generally, whenever a virus acquires a membrane envelope, it always inserts specific viral proteins
into the envelope; as the result the envelope becomes a unique viral antigen and which will be used
by the virus to gain entry into a new host cell.

Attachment, replication and maturation sites of animal viruses of different families

Family Attachment site Site of Nucleic Site of Maturation
Acid Replication (Budding)
Poxviridae Variable Variable Cytoplasm
Asfaviridae Clathrin-mediated Cytoplasm Plasma membrane
endocytosis
Iridoviridae Variable Nucleus/cytoplasm Cytoplasm
Herpesviridae Variable Nucleus Nuclear membrane
Adenoviridae Clathrin-mediated Nucleus Nucleus
endocytosis
Polyomaviridae Caveolin endocytosis Nucleus Nucleus
Papillomavirida Clathrin/caveolar endocytosis Nucleus Nucleus
e

Parvoviridae Clathrin-mediated Nucleus Nucleus

endocytosis
Hepadnaviridae Clathrin-mediated Nucleus/cytoplasm Endoplasmic
endocytosis reticulum
Retroviridae Plasma membrane fusion or Nucleus Plasma membrane
clathrin-mediated endocytosis
Reoviridae Clathrin-mediated Cytoplasm Cytoplasm
endocytosis
Paramyxoviridae Plasma membrane fusion Cytoplasm Plasma membrane
Rhabdoviridae Plasma membrane fusion Cytoplasm Plasma membrane
Filoviridae Plasma membrane fusion Cytoplasm Plasma membrane
Bornaviridae Clathrin-mediated Nucleus Plasma membrane
endocytosis
Orthomyxovirida Clathrin-mediated Nucleus Plasma membrane
e endocytosis

Bunyaviridae Clathrin-mediated Cytoplasm Golgi membrane

endocytosis
Arenaviridae Clathrin-mediated Cytoplasm Plasma membrane
endocytosis
Coronaviridae Clathrin-mediated Cytoplasm Endoplasmic
endocytosis/plasma reticulum
membrane fusion
Arteriviridae Clathrin-mediated Clathrin-mediated Endoplasmic
endocytosis endocytosis reticulum
Picornaviridae Caveolar endocytosis/plasma Cytoplasm Cytoplasm
membrane insertion
Caliciviridae Caveolar endocytosis/plasma Cytoplasm Cytoplasm
membrane insertion?

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 Astroviridae Caveolin endocytosis/plasma Cytoplasm Cytoplasm
membrane insertion?
Togaviridae Clathrin-mediated Cytoplasm Plasma membrane
endocytosis
Flaviviridae Clathrin-mediated Cytoplasm Endoplasmic
endocytosis reticulum

1.12. VIRAL GENETICS

Viruses are simple entities and having the following characteristics.
 Lacking an energy generating system
 Having a very limited biosynthetic capabilities
 The smallest viruses have only a few genes but the largest viruses have as many as 200 genes
Genetically, however, viruses have many features in common with eukaryotic and prokaryotic cells.
 Viruses are subject to mutation
 The genomes of different viruses can recombine to form novel progeny
 The expression of the viral genome can be regulated and viral gene products can interact.
So that by studying viruses, we can learn more about the mechanisms by which viruses and their
host cells function. Viruses are continuously changing.
1.12.1. Genetic changes in viruses
There are two principal mechanisms by which genetic changes occur in viruses.
 1.Mutation
 2. Recombination
Alterations in the genetic material of a virus may lead to changes in the function of viral proteins.
These changes may result in the creation of new viral serotypes or viruses of altered virulence.
1. Mutation
Mutation is defined as errors in copying the nucleic acid during replication. It is a term used to
differentiate new progeny viruses that are formed because of mutation from original “parental”,
“wild type” or “street viruses”. Mutation in viruses arises by one of three mechanisms.
 1. Through fallibility of the enzymes that replicate the nucleic acid
 2. By the natural behavior of bases that make up the nucleic acid
 3. By different physical and chemical agents which cause mutation called mutagens.
The first two mechanisms act similarly in all viruses. Hence the effect of mutagens and the natural
behavior of nucleotides are relatively constant. Generally, mutation could be:-
 1. Spontaneous mutation
 2. Induced mutations
1. Spontaneous mutation
Spontaneous mutation is a mutation which occurs spontaneously. Viruses on passage in the absence
of any mutagen yield a high proportion of mutants. These spontaneous mutations accumulate in the
genome of viruses and introduce phenotypic variation. RNA viruses exhibit a high rate of mutation
which is 10 -3 to 10 -4 per nucleotide incorporated than DNA viruses which have 10 -8 to
10– 11 per incorporated nucleotide. The difference in mutation rate of RNA and DNA viruses is
because of low fidelity of RNA genome replication that is lack of proof reading activities in RNA
polymerase enzyme.
2. Induced mutations
Mutation of viruses which is induced artificially by human being is called induced mutation.
Substances which can induce mutation are called mutagens. Mutagens may be of physical and
chemical. They may act directly on the DNA, causing direct damage to the DNA, and most often

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result in replication error. Some however may act on the replication mechanism and chromosomal
partition. Many mutagens are not mutagenic by themselves, but can form mutagenic metabolites
through cellular processes. Such mutagens are called promutagens.
A. Physical mutagens
Some of physical mutagens are:-
 Ionizing radiations such as X-rays, gamma rays and alpha particles may cause DNA
breakage and other damages.
 Ultraviolet radiations with wavelength above 260 nm are absorbed strongly by bases,
producing pyrimidines diamers which can cause error in replication.
B. Chemical mutagens
Some of chemical mutagens are:-
 Superoxide, hydroxyl radicals and hydrogen peroxide
 Polycyclic aromatic hydrocarbon (PAH)
 Aromatic amines
 Alkaloid from plants
 Bromine and base analogs which can substitute for DNA bases and cause copying errors.
 Intercalating agents such as ethidium bromide and proflavine are molecules that may insert
between bases in DNA, causing frame shift mutation during replication.
 Many metals, such as arsenic cadmium, chromium and nickel.
The physical and chemical mutagens have helped in study of animal virus genetics. These mutagens
could be:-
 “In vivo” mutagens
 “In vitro” mutagens
Powerful mutagens may result genome instability, causing nucleotide breakages and rearrangement
of the nucleotide because of :-
 Translocation
 Duplication
 Deletion
 Inversion
 Substitution
Such mutagens that cause translocation and / or deletion and /or inversion are called clastogens.
Mutagens may also modify the DNA sequence; the changes in nucleic acid sequences by mutations
include substitution of nucleotide base-pairs and insertions and deletions of one or more nucleotides
in DNA sequences. Many mutations are silent mutations, causing no visible effects at all. This is
because of the following reasons.
 They occur either in non-coding or non-functional sequences or
 They do not change the amino-acid sequence due to the redundancy of codons.
1.12.2. Phenotypic variation of viruses by mutation
It is a mutation that alters the viral phenotype but is not deleterious. Some of the most common
phenotypic variations of viruses which can be induced by mutation are:-
 1. Antibody escape/ resistance mutations
 2. Plaque morphology mutations
 3. Host range mutations
 4. Drug resistance mutations
1. Antibody escape/ resistance mutations
This type of mutation can alter the antigenic determinate site called epitope; as the result it can
create a novel antigenic determinates. For example mutation in the hemagglutinin gene of
influenza A virus can give rise to a hemagglutinin molecule with an altered antigenic
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 site(epitope); as the result, new attachment formation of the new hemagglutinin is intact; so that
the mutant virus can able to initiate an infection in an individual immune to the virus expressing
the previous hemagglutinin antigen. This relatively modest mechanism of antigenic change
through mutation called antigenic drift. This allows influenza A virus to outflank host defenses
and cause disease in previously immune individuals. Antigenic drift in influenza A virus is due
to mutation causing phenotypic (antigenic variation); as mutation of the codon for hydrophobic
amino acid serine which is UCC to the codon UUC results in synthesis of hydrophobic amino
acid phenylalanine. This can change an epitope on viral hemagglutinin protein and thereby alter
its recognition by specific antibody. That is why the mutant virus may then able to infect
previously immune host.
Mutation caused phenotypic (antigenic) variation

2. Plaque morphology mutations

These mutants exhibit altered morphology because of metabolic differences in the mutant and the
wild type. For example large plaque mutants in adenoviruses release virus from the host cell more
rapidly than wild type. Similarly mutants of herpes simplex virus cause neighboring cells to fuse
rather than undergoing typical cytolytic changes. These mutants are helpful in understanding the
egress (release) of virus from the membranes.
3. Host range mutations
Mutation has been a principal tool for virologists in developing live attenuated virus vaccines. It is
developed by culturing in unusual cell cultures. Mutation and selection of mutant viruses that are
growing in unusual cells is used to prepare live attenuated virus vaccines. For example the live
attenuated poliovirus vaccine was developed by culturing the virus in monkey kidney cells; so that
mutation affects the gene that codes for the poliovirus coat proteins in such a way to produce mutant
unable to attach to human neural cells but still able to infect human intestinal cells and as the result
infection of human intestinal cells does not produce paralytic but induce immunity.
4. Drug resistance mutations
Drug resistance has been used to assign functions to specific genes or to specific portions of
proteins. For example mutation conferring resistance to drug amantadine has been mapped in M2
gene of influenza virus. Drug resistant mutants provide useful controls for the specificity of
inhibition.
1.12. 3. Mutation rate and outcomes
DNA viruses have mutation rate similar to those of eukaryotic cells because like eukaryotic cell
DNA polymerases of their replicatory enzymes have a proof reading functions. The mutation rate
for DNA viruses has been calculated to be 10 -8 to 10 – 11 per incorporated nucleotide. Even complex

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DNA virus like poxviruses has lower than this error rate. However, RNA viruses exhibit a high rate
of mutation which is 10 -3 to 10 -4 per nucleotide incorporated. This is because of the lack of proof
reading function in their replicatory enzymes. Even the simplest RNA virus with 7400 nucleotides
per genome generates mutants more frequently and perhaps as once per copy. What you must
understand is not all mutations that occur persist in the virus population but mutations that interfere
with essential functions of attachment, penetration; uncoating, replication, assembly and release do
not permit mis replication and are rapidly lost from the population because of the redundancy of the
genetic code of viruses, so that many mutations in viruses are neutral; this is because of:-
 The absence of change in viral protein or
 Replacement of functional viral amino acids by other functionally similar amino acids.
That is why only viral mutations that do not cripple essential viral functions can persist or become
fixed in virus population.
2. Recombination
Viral recombination occurs when viruses of two different parent strains coinfect the same host cell
because of gen interaction during replication they generate virus progeny that have both genes from
both parents. Recombination generally occurs between members of the same viruses which are
called serotypes. Some of the examples of viruses in which recombination is common during
coinfection (mixed infection) of the same host are:-
 Influenza viruses
 Herpes simplex virus
 Blue tongue virus
There are three mechanisms of virus recombination that have been observed.
 1. Recombination by independent assortment
 2. Recombination by incompletely linked genes
 3. Recombination by copy – choice of incompletely linked genes
1. Recombination by independent assortment of genes
Independent assortment occurs when viruses that have segmented genomes coinfect the same host
cell. Independent gen assortment occurs during replication because the gens are unlinked and assort
at random. Recombination by independent assortment has been reported in many segmented RNA
viruses like:-
 Influenza viruses and other orthomyxoviruses which have 8 segments of single stranded
RNA.
 Retroviruses with 10 segments of double stranded RNA
After infection of a cell with two viruses with two and more genetic segments, reassortment of
replicated segments can occur. Independent assortment results in the generation of progeny viruses
whose genomes contain segments of genome from both types of parental viruses. Frequency of
recombination by independent assortment is 6 to 20% for orthomyxoviruses. There is also
independent assortment between an animal and a human strain of influenza virus because during a
mixed infection they yield an antigenically novel influenza virus strain capable of infecting human
being; so that; even if the novel influenza virus strain carries animal strain hemagglutinin and/ or
neuraminidase surface molecule, it is capable to infect human being. This new recombinant viruses
can infect individuals that are immune to the parent human virus. This mechanism results in an
immediate, a major antigenic change is called antigenic shift. Antigenic shifts in influenza virus
antigens give rise to pandemics (worldwide epidemics) of influenza in domestic animals and
human being. Antigenic shifts have occurred relatively frequently during recent history.

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Recombination by independent assortment of genes during dual infection

Antigen shift resulting from reassortment of genome segments

Year Strain
1890 H2N8
1900 H3N8
1918 H1N1
1957 H2N2(Asian flu)
1968 H3N2(Hong Kong flu)
1977 H1N1
2008 H5N1(bird flu)
2. Recombination by incompletely linked genes
Recombination between genes residing on the same pieces of nucleic acid occurs when two viruses
coinfect the same host cells. Genes that are generally segregate together are called linked genes.
However, if recombination occurs between genes that are residing on the same pieces of nucleic
acid, the linkage is said to be incomplete; so that this recombination is called recombination by
incomplete linked genes. Recombination of incompletely linked genes occurs:-
 In all DNA viruses
 In several RNA viruses
The genetic interaction of DNA viruses can result in break – rejoin recombination, in which the two
DNA molecules of different viruses break and then cross over. Break – rejoin recombination results
in novel progeny viruses with some DNA sequences of both types of parental viruses.
Recombination between incomplete linked genes occurs by means of break and rejoins mechanism
and it occurs in DNA of eukaryotic and prokaryotic cells. The break and re- join mechanism
involves the actual severing of the covalent bonds linking the bases of each of the two strands of
DNA in a DNA molecule. These severed DNA strands are then re – joined to the DNA strands of
different DNA molecules that have been broken in similar sites. Some examples of animal viruses
that form new progeny by recombination with incomplete linked genes are:-
DNA virus: Herpesviruses
Positive sense ssRNA: retroviruses
What is interesting in that recombination in retroviruses occur when the RNA is changed to DNA by
reverse transcriptase in cytoplasm of infected cells. Recombination occurs in the nucleus of

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infected cells by break and re – join mechanism with the DNA of other retroviruses or with DNA of
infected cells. So that these re – joining mechanisms results in:-
 Recombination between two retroviruses gives rise to novel viral progeny with reassorted
genes
 Recombination between retroviruses and host cell give rise to novel viral progeny that carry
non viral genes.
If the host cell genes code for growth factor, growth factor receptors or a number of other specific
proteins, the recombinant retroviruses can be oncogenic.
Recombination by break – rejoin incompletely linked genes during dual infection

3. Recombination by copy – choice of incompletely linked genes

In picornaviruses and coronaviruses, recombination takes place at the level of interaction of the viral
RNA genomes and it is believed on to occur by break - rejoin mechanism. But the mechanism is
currently believed to be a copy – choice mechanism. Copy – choice may occur in these RNA
viruses because the viral RNA polymerase binds to only a few bases of template RNA at any one
time. Such a weak interaction of the polymerase with a template RNA would permit the polymerase,
carrying its RNA strand, to disassociate from the original template nucleic acid strand and then
associate with a new template RNA strand. Frequency of recombination by copy – choice
mechanism is low and found in the range of 0.2 – 0.4%.
Recombination by copy – choice of incompletely linked genes during dual infection

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1.12.4. Phenotypic variation of viruses from recombination
Viral recombination is very important because it can generate novel progeny viruses that express
new antigenic and/ or virulence characteristics. For example the novel progeny viruses may have
new surface proteins that:-
 Permit them to infect previously resistant individuals
 Altered the virulence characteristics
 Make them to be infective to other cells of the original host or a new host.
 Lastly they may carry materials of cellular origin that gives them oncogenic potential.
1.12.5. Practical application of genome recombination
Recombination is being used experimentally by virologists for many veterinary and medical
practices. Among them the most common are:-
 A. For recombinant vaccine production
 B. Gene therapy through recombination
A. Recombinant vaccine production
Vaccinia virus is a DNA virus in the family of Poxviridae which is used to produce recombinant
virus and bacterial vaccines. Live recombinant vaccinia virus with smallpox virus was used to
eradicate small pox in the world. Recombinant vaccinia viruses are being developed that carry
vaccinia virus DNA recombined with DNA of from other sources (exogenous bacterial or viral
DNA). This means vaccinia virus strains carrying DNA coding for bacterial and viral antigens. It is
expected that after vaccination with the recombinant vaccinia virus vaccine, the bacteria or viral
antigen will be produced which is immunogen. The presence of this immunogen will then stimulate
specific antibody production by the host. This results in protection of the host from that immunogen.
Development of recombinant vaccinia virus for immunization against cholera toxin
 Vaccinia virus genomic DNA is cut with an endonuclease.
 Then a specific sequence of DNA (with appropriate regulatory sequences) coding for protein
(e.g., for cholera toxin) to be used as immunogen is ligated into vaccinia virus genome to
make recombinant vaccinia virus.
 The DNA of the bacteria will be transcribed and the immunogen will be produced along with
vaccinia virus proteins in infected cells following vaccination.
 As the result the immunogen will then elicit antibody production by the host which provides
protective immunity.
Development of recombinant vaccinia virus for immunization against cholera toxin

B. Gene therapy through recombination

In a similar manner, recombinant viruses also being developed that carry normal human genes. It is
envisioned that such recombinant viruses with normal genes of human being with special interests

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could be useful for gene therapy. Gene therapy using recombinant viruses are used to treat wide
range of disease which includes:-
 Diabetes
 Cystic fibrosis
 Severe combined immunodeficiency syndrome etc.
Indeed, treatment of cystic fibrosis patients with replication deficient, recombinant adenoviruses
bearing the normal copy of the cystic fibrosis transmembrane regulator gene has already been
approved.

1.13. CULTIVATION OF VIRUSES

It is known that viruses cannot replicate in synthetic media; so that they require living cells as hosts
to replicate. For example:-
 Phages are supplied with bacterial cultures
 Plant viruses may be supplied with specially cultivated plants or with cultures of protoplasts
(plant cells from which the cell wall has been removed).
 Animal viruses may be supplied with whole organisms, such as mice, eggs containing chick
embryos and cell culture.
 Insect viruses are supplied insect larvae.
Until the 1931s, the only tools available to grow and study viruses were animals. As new infectious
agents were discovered and tests developed for their detection, the research animals became more
“clean” and the concept of “specific pathogen-free” (SPF) animals were born. It is noteworthy,
however, that animals apparently specific pathogen-free” could be infected with pathogens that
were still undefined or undeclared. The search for suitable culture systems for viruses led to the
discovery, in 1931, that vaccinia virus and herpes simplex virus could be grown on the
chorioallantoic membrane of embryonated chicken eggs, as was also known for fowl-pox virus, a
naturally occurring pathogen of birds. The use of embryonated chicken eggs then became routine for
propagation efforts, not only for avian viruses, but also for viruses infecting mammalian species.
Various “in-vitro” cell culture systems have been utilized since 1950s as artificial medium was
developed to maintain cell viability outside the source species.
1.13.1. Methods of virus cultivation
Nowadays, there are three methods to cultivate animal viruses.
 1. Cell and tissue culture
 2. Embryonated egg
 3. Naturally susceptible and laboratory animals
1. Cultivation of viruses using cell culture and their types
Though cultivation of viruses in tissue/ cell cultures has long been used, the difficulties in supplying
cells with suitable medium and of preventing contamination of bacteria and fungi limited its
use till introduction of antibiotics. This and the use of trypsin for cell dispersion and use of synthetic
media have made cell culture technique within the scope of any virology laboratory. Cell and tissue
culture technique for virus cultivation is more advantageous than using animals and embryonated
eggs because cell cultures are:-
 Easier and more economical to prepare, maintain and manipulate.
 No antibody or other inhibitory factors (if it is embryonated and fetal cells) to interfere with
the growth of the virus
 The effect of viruses infections can be detected in a very short period of time( hours to days)
 In some cases all cultures may be more susceptible to the effect of viruses than animals and
chick embryos.

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  Cell cultures can be produced and preserved at -700Cor – 196 0C. Cell cultures can be radio –
labeled to study the details of virus multiplication.
There are three types of tissue/ cell culture.
 1.Organ culture
 2. Tissue culture
 3. Cell culture
1. Organ culture
Slices of organs carefully handled to maintain their original architecture and functions for days and
weeks. Some organ cultures are:-
 Tracheal ring cultures
 Organ cultures of fetal intestinal epithelium
Organ cultures are not often used in virology these days. But they can be used to study the
histopathogenesis of viruses and propagation of certain respiratory and enteric viruses.
2. Tissue culture
Tissue fragments taken from living or recently killed animals incubates in a nutrient medium in well
aerates flasks but they are not used for virus propagation these days.
3. Cell culture
Cells are disaggregated / dispersed by trypsinization and incubated in test tubes, bottles/ flasks and
Petridishes. The cells adhere to inside of the container and grow to form a sheet of monolayer. Cell
cultures are the method of choice than embryonated eggs and animals because:-
 Cell culture requires less space
 Cell culture is less expensive than the two methods
 Cell culture is more convenient than the use of animals and embryonated eggs.
But the use of embryonated eggs are still the method of choice in large scale propagation of
influenza virus for vaccine production Cell culture allows:-
 Primary isolation of viruses
 To study the infectivity assay of viruses
 To study biochemical changes in cells because of viruses
 Large scale vaccine production
However, the use of cell cultures for virus replication is limited. This is because:-
 The cells sensitivity is different for different viruses which depend on tropism of viruses.
 The physical or biological conditions present that may interfere with virus replication
Cultures of animal cells are usually divided into 3 classes:-
 1. Primary cells(primary cell cultures)
 2. Cell strains ( secondary cell cultures)
 3. Cell lines
The ultimate source of cells for cell culture is the intact animal. The cells may be obtained from
various organs and tissues of embryonic, infants and adult origin; so that the cell obtained may be
normal or cancerous. Regardless of their source, cell cultures obtained directly from the animal’s
organs and tissues are referred to as primary cell cultures.
1. Primary cell cultures
Primary cell cultures are cell cultures that are obtained directly from an animal’s organs and tissues.
Primary cell cultures have a short “life span” in laboratory since the culture dies off after a few
passages (5 – 10) divisions. Primary cell cultures:-
 They usually contain a variety of cell types and support the growth of a variety of viruses.
 Primary cell cultures may be obtained from embryos or adult animals.
Cells obtained from embryos are preferable than cells from adult animals because:-
 Cells from embryos are rapidly dividing cells which grow easily “in vitro”.
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  Cells from embryos are easily disaggregated and undergo mitosis soon after transplant.
 Sterility is not a problem like that of primary cells from adult animals.
Some good examples of primary cell culture cells are:-
 Chick embryo fibroblastic cells
 Foetal lamb kidney monolayer cells
 Calf kidney cell
But cells obtained from adult animals are:-
 More difficult to disaggregate and usually grow more slowly in culture.
 Sterility is often a problem with adult animals since the normal microflora may contaminate
the cell culture.
Generally, to obtain primary cell culture, tissues are cutup into small fragments and enzymatically
digested, usually with trypsin into constituent cells. The cell suspension and the appropriate growth
medium are then added to flat – bottomed vessel and placed in a Co 2 incubator. After a period of
time the cells attach to the bottom of the flask and start dividing until a monolayer (a one – cell thick
continues layer) is formed; then after the formation of a mono – layer primary cells stop to divide
because of contact inhibition between cells(inhibition of growth under crowded conditions).
With a few exceptions such as nerve and muscle cells all cells in culture can be classified in two
general morphological types.
 Fibroblast like cells which are thin and elongated.
 Epithelial like cells which are polygonal in shape and tend to form sheet.
Depending on the requirement of surface for attachment, all primary cells are divided into two.
 1. Anchorage dependent primary cell cultures
 2. Anchorage independent primary cell cultures
1. Anchorage dependent primary cell cultures
Cells that require a surface for attachment are called anchorage dependent cells; so that these cells
must be attached on the surface of flat – bottomed vessel to grow. Most primary cell cultures are
anchorage dependent cells.
2. Anchorage independent primary cell cultures
Those cells which do not require attachment on the surface of flat – bottomed vessel are called
anchorage independent cells; so that anchorage independent cells can grow and cultivate in
suspension cultures. Suspension cell culture is preferred when a larger yield of cells are required.
Cells are grown in suspension without allowing them to attach on the surface of the vessel and this
is achieved by constant stirring of the vessel content. That means after adding the cell into suitable
culture vessel with culture medium, the vessel is placed on a magnetic stirrer so that cells are
maintained continuously in suspension. Nowadays, for suspension culture on a large scale, large
sized fermenter vessels of several hundred liter capacity are employed with electronic controls for
maintaining all the require optimum conditions. Suspension cultures are mainly employed for the
production of viruses on large scale for vaccine production.
Primary cell cultures retain some of the characteristics of the tissue from which they were derived
and usually contain more than one cell types; and that is why primary cell cultures are often
preferred particularly for diagnostic purposes because of its greater virus susceptibility. Most of the
primary culture cells have finite lifespan of 5 – 10 divisions “in vitro”. The presences of different
cell types in primary cell cultures make them to be sensitive to a wide range of viruses. However,
due to their limited lifespan, one cannot use for long term experiment with these cells.
2. Cell strains
These cells are also called secondary cell cultures because they are obtained upon serial transfers
of primary cells. Upon serial transfers of primary cells, a gradual selection from various cells may
occur until a particular cell type becomes predominant. If these cells continue to grow at a constant
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rate over successive passage, the primary cells are refered to as a cell strain. Cell strains have a
finite lifespan of less than 100 divisions “in vitro”. Secondary cells (cell strains) are also called
diploid cell strains to differentiate them from cell lines because they retain their original diploid
chromosome number and are not malignant(do not induce tumors in test animals).Because of their
satisfactory sensitivity and high yield of virus, cell strains are widely used: -
 In diagnostic virology(isolation and identification of viruses)
 In vaccine production
Examples of cell strain are fibroblasts established from human embryo lung W1 – 38 and MRC –
5.
3. Cell lines
Cell lines are cells that are obtained from cell strain that have under gone transformation process.
The transformation process can be spontaneously or induced. The transformation change is in
karyotype morphology or growth properties that make them “immortal”(able to divide
indefinitely). These cells which are “immortal” but are able to divide indefinitely are called cell
lines. It is not clearly known how cell strains (diploid cell strains) become a cell lines, although this
event may be mimicked by infection with oncogenic viruses or by exposure to chemical
carcinogens. Cell lines often have abnormal chromosome numbers and can be tumorigenic when
inoculated into susceptible animals. Cell lines that have been derived from tumors often do not
exhibit contact – inhibition (inhibition of growth under crowded conditions) but rather continue to
pile up. Some of the examples of continuous cell lines are:-
 HeLa - 6 derived from African green monkey kidney
 Hep - 2 and KB derived from human carcinomas
 L - 929 and 3T3derived from mice
 BHK - 21 from baby hamster kidney
 PK - 15 from normal pig kidney cells
 RK - 13 from rabbit kidney cells
 CCI from duck embryo fibroblasts
 Other cell lines derived from variety of animals
With some exceptions, the fully” normal” cell is considered to have:-
 Original number of chromosomes
 A finite lifespan
 Be anchorage – dependent
 Exhibit contact – inhibition
 To be non – malignant and non – transformed
Generally cell cultures have the following applications:-
 Isolation of viruses(diagnostic purpose)
 Specific identification of viruses by virus serum neutralization tests
1.1. Cell growth and cell passage
1.1.1. Normal cell growth
There are four phases of cell growth and multiplication.
 Lag phase
 Exponential phase
 Stationary phase
 Decline or death phase
Upon being seeded in a new culture vessel and attach to the surface, the cells enter a lag phase
during which there is no increase in cell number. In time, this followed by log phase of growth with
an exponential increase in cell number and increased metabolically activity. The length of the log

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phase varies among cell types as different cell types divided at different rates called generation
time.
A stationary phase is reached when nutrients are depleted and waste or toxic products are
accumulated. At stationary phase the cell number remains constant and this often happens when
confluency is reached and at this stage the cell culture is termed confluent cell culture. Confluent
cell cultures are those cell cultures when the cells occupy all of the available growth area. At this
stage the cells stop to proliferate because of contact inhibition due to cell – to – cell contact; as
some cells are sensitive to contact inhibition(primary cell cultures)but other types of cells like cell
lines(tumor cells) are not sensitive to contact inhibition.
Cells become adversely affected and eventually die at decline or death phase because of the
accumulation of waste and toxic products. To overcome this problem cells must be subcultured into
new cell growth medium. Subculturing of cells into a new medium to maintain viability of cells by
supplying optimum cell culture conditions is called passage. The cells that are subcultured are
called passaged cells; so that passage number refers to the number of times the cells have been
subcultured or transferred since the cell culture was obtained.
1.1. 2. Methods of cell passage
Different methods can be used to passage anchorage dependent cells. Generally there are three
methods to passage cell cultures.
 Mechanical
 Enzymatical
 Chemical with/ without enzyme
Cells may be mechanically scraped from the cell culture surface with a wedge of soft rubber
attached to the end of a glass rod. This removes the cells in clumps but some cells may be damaged.
The cell monolayer can also be taken after the treatment of the cell culture with a calcium and
magnesium free enzyme solution. The most common enzymes that are used to disintegrate the cell
layer are:-
 Trypsin( very often used)
 Collagenase
 Pronase
What you must understand is that enzymatic treatment can damage the cell surface; so that it is
important to minimize the time of the cells’ exposure to the enzyme. EthyleneDiamine Tetra Acetic
Acid (EDTA) is a chelating agent and sometimes used alone or in combination with trypsin. But
anchorage independent cells that grow in suspension culture do not need to be trypsinized. The cell
suspension is centrifuged, the medium is discarded then the cells are resuspended into a small
amount of fresh medium. A given quantity of the cells is then transferred into a new culture vessel
with fresh medium. When passaging cell cultures is needed, one must do cell count by using
hemacytometer.
2. Cultivation of viruses using embryonated eggs and routes of inoculation
2.1. Virus replication in embryonated eggs
Prior to 1950s the standard host for the propagation of many viruses was the embryonated eggs.
Now adays, a variety of embryonic cell types and routes of inoculation are available; so that the age
of embryos when most viruses propagate in developing embryos and their routes of inoculation is
known. Although cell cultures have become widely adopted for the cultivation of many viruses,
embryonated eggs are still used for the isolation of different viruses and for vaccine production. It is
used to isolate viruses like:-
 Avian viruses
 Influenza viruses

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Embryonated egg is the first choice for the propagation of a number of viruses such as:-
 Poxvirus
 Orthomyxovirus
 Some paramyxoviruses
The advantages of using embryonated eggs for cultivation of viruses when we compare to cell
cultures and experimental animals are that:-
 Avian embryos are bacteriologically sterile and immunologically incompetent
 They are relatively inexpensive and easy to handle
 Embryonated eggs do not require care, facilities, technical skills as culture cells
 Embryonated eggs require less time necessary for preparation and do not require
maintenance like that of cell culture.
 Embryonated eggs require less space than experimental animals.
However, in some cases using of embryonated eggs for cultivation of viruses has disadvantages
because:-
 The embryonated egg may possess maternal antibodies to some poultry pathogen which may
present in yolk.
 The embryonated egg may be contaminated by some infectious agents like:-
• Bacteria (Salmonella, Mycoplasma and Chlamydia) etc.
• Viruses that may pass through the egg like Newcastle disease virus, avian encephalomyelitis
virus and avian leucosis virus etc.)
2.1.1. Anatomy of embryonated egg and their functions
Embryonated egg has the following parts.
 Shell with shell membrane
 Air sac
 The chorioallantoic sac with its membrane called Chorio Allantoic Membrane(CAM)
 Yolk sac
 The amniotic cavity with its thin membrane called amniotic membrane
 Albumin
 Embryo/ embryos
The shell and the shell membrane function both as a barrier and as an exchange system for gases
and liquid molecules. The air sac is important for the development of embryo because it is
important for respiration and for pressure adjustments. The chorioallantoic sac and its membrane
called Chorio Allantoic Membrane (CAM) enclose the allantoic cavity and its contents remove
waste products produced by the developing embryo; this membrane and its contents increase in size
as the embryo grows. The yolk sac is the source of nourishment for the developing embryo; and as
the embryo develops, the yolk sac decreases in size until it is completely absorbed into the digestive
system of the mature embryo. The amniotic cavity with its is a thin membrane called amnion
encloses the embryo and protects the embryo from physical damage; and it also serves as an
exchange system and is the best seen in the younger embryos. Albumin is the protein part of the egg
and occupied the pointed end of the egg and is transparent during candling. Embryo is located in
the amniotic sac which is mobile and has different size in different days of incubation. Embryonated
eggs are obtained from fertile eggs which are incubated better artificially with appropriate
temperature, humidity and with forced air circulation. Appropriate humidity and forced air
circulation are important to prevent the embryo from desiccation of the shell membrane that serves
as an exchange system for the developing embryo. Once the hen lays the egg, and as long as the egg
is not exposed to extremes of temperature, further development will be suspended until incubation

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occurs( better to store the fertile egg at 10 0C - 15 0C not for more than 5 – 8 days).To cultivate
viruses using embryonated eggs the following general procedure must be applied by virologists.
 1. Selection of eggs
 2. Incubation of eggs
 3. Candling of eggs
 4. Inoculation of viruses
1. Selection of eggs
Eggs for embryonation should be naturally clean, medium sized (45 gm – 70 gm), white and thin
shelled and obtained from fertile egg supplier who can generate 95% fertility. If available, eggs laid
from Specific Pathogen Free (SPF) hens should be preferred and incubated eggs if necessary may
be kept in cold room(10 0C – 15 0C) for not more than 5 – 8 days after being laid. The period of
storage of fertile eggs with range of 10 0C – 15 0C should not be exceeded 5 – 8 days because there
may be significant reduction in viability of fertile eggs as the length of storage time exceeded 5 – 8
days.
2. Incubation of eggs
Incubation of eggs can be done either in commercial egg incubator or in bacteriological incubator.
The temperature of incubation ranges from 38 0C to 38.5 0C and the relative humidity 45 – 70%
which depends with the age of embryos. Eggs that are used to harvest viruses must be incubated by
putting them at 45 0C with air sac up right and turned twice daily. This helps:-
 Symmetric embryonic development
 Prevent adhesion of embryo to the membranes
3. Candling of eggs
Since viruses need living tissues to replicate, candling is an important first step after incubation.
Incubated eggs must be candled before and after inoculation of viruses. Candling of eggs is best
done in dark room with box that have hole and strong beam of light or with candling table and tray
if it is available. Candling of eggs with strong beam of light in dark room is also called ovoscopy.
Candling before inoculation helps:-
 To determine the normal development of eggs
 To locate the embryo and to mark on it
 To check the viability of the embryo
 To locate the position of the air sac and to mark on it
Candling must be also done after inoculation to check the viability of the embryo. When you
candled the egg , you observe different features in the egg.
 Infertile egg and fertile eggs incubated for less than 4 days show a diffuse shadow from the
yolk which is slightly mobile in a clear environment representing the albumin.
 Fertile eggs starting from the fourth day of incubation on ward reveal fringe of blood vessels
centered on a dark spot firmly adherent to the shell and close to the air chamber.
 As the age of the embryo progresses:-
 The vascular system will progress over the circumference of the shell and over the base of
the air chamber.
 But the pointed end will remain free and transparent as it is occupied by albumin.
 On the sixth days on ward:-
 The embryo will be seen as freely moving shadow and may show independent movement
occasional
 The umbilical vein, leading from CAM to the embryo can be distinguished as a floating large
blood vessel.
 But if the embryo die early before the 4 th days of incubation:-
 The dead embryo will be visible as clear yellow with lack of vasculature.
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  A black, brown or green cast is indicative of contamination of these eggs with bacteria or
fungi and they should be properly discarded.
 If the embryos die in older age:-
 The immobile outline of the dead embryo can be distinguished.
 Blood vessels are absent or faintly visible.
What you must understand is that the first ten days of the 21 days incubation period:-
 Proliferation of cells is at its highest rate.
 Tissues of the embryo are not well differentiated.
That is why the embryos at this age of incubation are susceptible to most animal viruses. However,
between day 10 and 15 there is greater differentiation of tissues and susceptibility spectrum of the
tissues of embryos becomes narrow. Finally towards the end of incubation the embryos become
susceptible only to avian viruses only.
3. Cultivation of viruses using animals
Viruses can be cultivated using live animals that can be:-
 Naturally susceptible animals
 Laboratory experimental animals
Cultivation of viruses using animals helps to confirm the infectious nature of the disease; so that the
inoculum should be made bacteriologically and mycological sterile. That is why the material that we
are going to inoculate must be filtered or treated with broad spectrum antibiotics and antifungal
agents. Nowadays, animals are not longer frequently used for propagation of viruses. This is because
chick embryos and cell cultures are simpler to handle and are more versatile. However, laboratory
animals are still used for many kinds of virological works such as:-
 To study the pathogenicity viruses.
 To study the potency of new vaccines.
 To produce diagnostic and prophylactic antisera for many viral diseases.
 In addition few viruses are to be cultivated in experimental animals.
But to use animals for virus cultivation and isolation we must fulfill the following criteria.
 1.Selection of animals
 2.Susceptibility of the animal to given viruses
 3. Route of inoculation
 4. Prepare control and inoculated animals
 5. Control of contamination
1. Selection of animals
Ideally experimental animals are the ones which are:-
 Uniformly and fully susceptible to the virus under study
 Free from intercurrent infection and adventitious lesions
 Inexpensive to purchase and to maintain
 If possible animals are better to use germ free, Specific Pathogen Free (SPF) or colostrum
deprived animals.
2. Susceptibility of animals
Selected animals must be susceptible to the virus under experiment. It is depend on species, strain,
age and presence or absence of maternal antibodies.
3. Route of inoculation
To cultivate viruses in animals, we must also know the correct route of inoculation. It depends on
tropism of viruses. It is selected in such a way that the viruses get direct contact with the organs in
which it is most likely to produce pathological effects.
4. Control and inoculated animals

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Use adequate number of control and inoculated animals under experiment; so that careful
examination of controls and inoculated animals is essential during necropsy.
5. Control of contamination
After inoculation, strict measures should be taken to these animals (control and inoculated).
 To prevent the contamination of the inoculated and control animals by other pathogenic
organism
 To avoid infection resulting from inoculation to spread to other animals including human
being.
So that the control and inoculated animals must be kept in different isolators and the isolator for
inoculated animals must be made of rigid materials and kept under slight negative pressure with
facilities to filter or to incinerate the outgoing air.

1.14. LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

In earlier times when laboratory diagnostic testing was in its infancy, diagnosis of viral diseases was
related to viral infection was achieved based on:-
 History and signs of viral infections
 Gross pathology and histopathology
But nowadays diagnosis of viral infection is performed by complex method that comprises: -
 Epidemiologically
 History and clinical signs
 Gross pathology and histopathology
 Laboratory
The first three methods of diagnosis are tentative diagnostic methods; whereas laboratory method of
diagnosis is confirmatory diagnostic method. Tests to support or establish a specific diagnosis of a
viral infection are:-
 Those that demonstrate the presence of infectious virus called virus isolation and
identification.
 Those that detect viral antigens
 Those that detect viral nucleic acids
 Those that demonstrate the presence of an agent-specific antibody response called
serological tests.
 Those that directly visualize the virus called electronmicroscopy.
Laboratory diagnosis of viral infection comprises:-
 I. Collection, packaging and transport of specimens
 II. Virus isolation
 III. Virus identification
I. Collection, packaging and transport of specimens
Specimens in broad sense, they are any materials that can be collected from living and/ or dead
organisms (animals) in our case to investigate them in the laboratory to identify the causative agent.
So that specimens from animals for virus isolation and identification can be collected from:-
 Live animals
 Dead animals (animals died because of the disease and/ or animals that are slaughtered for
diagnostic purpose).
Specimens can be:-
 The animal as whole if it is visible
 Organs
 Pieces of tissues

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  Blood during viremia stage
 Exudates and transudate in natural cavities
 Discharges
 Vesicular contents
 Body excretions like faces and urine
 Biopsies etc
Specimens from animals must be collected at the right time from:-
 The most appropriate animals
 At right sites(organs and systems) of animals
Because the chance of detecting of a virus from specimens depends critically on the attention given
to the collection of specimens at right time and from right animals; so that clearly, such specimens
must be taken from the right site (depending on the tropism of virus) from the most appropriate
animal, and at the right time. The right time for specimens collection for virus detection is as soon as
possible after the animal first develops the typical clinical signs of the disease because maximal
amounts (titers) of virus are usually present at the onset of clinical signs; so that the incorrect
collection and storage of specimens, and the sub-mission of inappropriate specimens, will diminish
the likelihood of accurate diagnostic laboratory success. The site from which the specimens can be
collected will be influenced by:-
 The knowledge of epidemiology, clinical signs and gross pathology of the disease
 The knowledge of the pathogenesis of the suspected disease.
Specimens appropriate for laboratory diagnosis of various clinical syndromes from live animal
Syndrome Specimen
Respiratory Nasal or throat swab; nasopharyngeal aspirate, tracheal wash fluid
Enteric Feces
Genital Genital swab
Eye Conjunctival swab
Skin Vesicle swab or scraping; biopsy of solid lesion
Generalized Nasal swab, feces, blood at stage of viremia, serum and urine
Biopsy Relevant organ
Any disease Blood for serology
For example in viral respiratory infection in cattle, the most important diagnostic specimens that
should be collected include:-
 Nasal and throat swabs from live animals
 Bronco -tracheal wash fluid from live animals
 Lung tissue and bronchial lymph nodes from dead animals
But whole-blood samples from this type of case are often useless because the causative viral agents
of bovine respiratory syncytial virus may not produce detectable concentrations of virus in blood
samples. Timing of sample collection is also critical because as the time postponeded after the onset
of typical clinical signs of the disease the titer of the virus is reduced; as there is the formation of
specific antibodies against specific virus in the organism. Specimens for virus isolation,
identification, antigen and nucleic acid detection may be collected from:-
 Live animals
 Dead animals
Samples must be collected aseptically; wherever possible, sampling should include specimens that
allow the use of several diagnostic tests; as no single test will provide an unambiguous diagnosis in
all cases. So that collect separate specimens for:-
 Detection of virus(isolation, antigen detection and nucleic acid detection)

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  Bacteriology
 For histopathology
Specimens for histopathology must be fixed with 10% buffered formalin; while specimens for
bacterial isolation and identification must be collected aseptically and transported in cold condition.
Specimens for detection of virus like for virus isolation and identification; antigen detection and
molecular techniques and for immunohistochemistry staining must be in unfixed form. However, all
specimens for viral detection assay must be moist and cold. So that viruses require virus transport
media and cold chain while you are collecting and transporting specimens. Virus transport media
contain:-
 Protein like gelatin, albumin or fetal bovine serum
 Broad spectrum antimicrobial agent
Protein in viral transport medium protects the virus against inactivation; while antimicrobials
prevent the multiplication of bacteria and fungi. Properly packaging and labeling of specimen for
specimens identification are also crucial in virology laboratory. Another important thing that you
must give attention for safe arrival of the specimens; so that packed and labeled specimens should
be protected from breaking in transit and should be sent refrigerated + 4 0C(but not frozen), with
“cold packs” in ice box with legal body. In general, specimens that are correctly and aseptically
collected will be acceptable for virus isolation and identification; antigen and nucleic acid detection
laboratory tests. However, viability is not necessary for nucleic acid detection like PCR assays.
Specimens should be sent to the diagnostic laboratory with legal letter that has detailed information
about the disease which comprises: -
 The epidemiology of the disease
 Clinical history of the animal and/or herd from which the specimens are derived.
 Gross pathology and histopathology(if possible) of the disease
The above information assist diagnosticians:-
 In selecting the most appropriate tests for the specimens received
 To permit a dialogue with the clinician over additional specimens if needed.
II. Virus isolation
Viruses can be isolated using:-
 1. Cell cultures
 2. Embryonated eggs
 3. Live naturally susceptible or laboratory animals
1. Isolation of viruses using cell cultures
Despite the presence of new techniques like nucleic acid and antigen detections that can identify
viruses from specimens without isolation, virus isolation using different cultures (cell and
embryonated eggs) and live animals (rarely) remains an important procedure because theoretically,
at least a single viable virion present in a specimen can be grown in cultured cells. Thus expanding it
to produce enough virions to permit further detailed characterization of the isolated viruses is
critical. Virus isolation remains the “gold standard” method against which newer methods must be
compared. However, nucleic acid detection tests, particularly quantitative PCR assays, are
challenging that paradigm. Culture is the easiest method of producing a supply of live virus for
further examination by molecular methods (genome sequencing and antigenic variation).Moreover,
large quantities of virus must be grown in cultured cells to produce diagnostic antigens and reagents
such as monoclonal antibodies. Until recently, vaccine development for many viral diseases has also
been reliant on the availability of viruses grown in culture. You know there are three types of cell
cultures.
 Primary cell culture
 Secondary cell cultures(cell strain)
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  Continuous cell lines
The choice of cell culture strategy for the primary isolation of an unknown virus from clinical
specimens is largely empirical. Primary cells derived from fetal tissues of the same species usually
provide the most sensitive cell culture substrates for virus isolation. Continuous cell lines derived
from the homologous species are, in many cases, an acceptable alternative. Most laboratories also
select a cell line that is known to grow many types of viruses, in case an unanticipated agent is
present. Arthropod cell cultures are used frequently as a parallel system for isolating “arboviruses.
Even with the best cell culture systems available, some viruses such as papillomaviruses will not
grow in traditional cell culture conditions; so that special culture systems such as organ cultures and
tissue explants have a great value for isolation of viruses.
2. Isolation of viruses using embryonated eggs
Embryonated eggs from different species of birds in different incubation days (hens, ducks, turkeys
and pheasants) have been used to isolate many viruses mainly avian origin and many animal viruses
like:-
 Paramyxoviruses
 Orthomyxoviruses
 Duck hepatitis virus
 Marble spleen diseases of pheasants
Embryonated hens’ eggs are still used for the isolation of influenza A viruses, even though cell
cultures [Madin–Darby canine kidney (MDCK) cells] are now more commonly used. Embryonated
eggs are still used in many virological laboratories to isolate viruses from specimens because:-
 Many avian viruses replicate much better in eggs than in cell cultures derived from chick
embryo tissues.
 There is a lack of widely available avian cell lines for routine virus isolation from avian.
You know that according to the virus of interest, the diagnostic specimens are inoculated to
embryonated egg into:-
 Amniotic cavity
 Allantoic cavity
 Yolk sac
 ChorioAllantoic Membrane(CAM)
 Rarely into intravenously into the vessels of the shell membrane
 Rarely into embryo(intra - cerebral route)
3. Isolation of viruses using live animals
When standard methods had failed to diagnose what appeared to be an infectious disease,
inoculation of animals was used. This helps virologists:-
 To define the infectious nature of the disease
 Further to isolate and identify the agent
Animals that are commonly used to isolate viruses can be:-
 Naturally susceptible animals for a given virus of interest
 Laboratory animals
Although some specialized laboratories still use animals to isolate different viruses, it has largely
been abandoned, as a result of costs and animal welfare concerns but still most diagnostic
laboratories are continued to inoculate suckling mice, a system that has been valuable for isolating
arboviruses that resist cultivation in cell cultures.
4. Preparation of inoculum from specimens and methods of inoculation
After the appropriate specimens with cold condition have reached in laboratory, the specimens are
macerated in sterile BSS in aseptic condition and are chopped into small pieces. Tissue derbis and
bacteria in the suspension can be purified using bacterial filter which has a size of < 300 nm pores
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that allow only virus but retain most bacteria and derbis of tissues. This suspension of viruses is
inoculated into cell cultures and embryonated eggs. You know the routes and the techniques of
inoculation of embryonated eggs. Inoculate the virus suspension with appropriate dose and routes
and incubate with 37 0C for 24 – 72 hours. However, cell cultures are inoculated by pouring the
suspension with suspected virus on the surface of cell culture with sterile pipette, then allowing the
viruses to be attached on the surface of cell culture for some hours by incubating at 37 0C, then wash
with sterile BSS to remove unattached viruses and incubate for 2 – 3 days. You must have
uninoculated cell culture as negative control for every specimen. Detection of viral growth in cell
culture is dependent upon the detection of a property of the inoculated cells that is not found in
control cultures (uninoculated cell cultures) that are maintained under identical growth conditions.
The changes that may occur in inoculated cell cultures that help us to detect virus growth are:-
 1.Cytopathic effect
 2.Plaques formation
 3. Formation of polykaryocytes
 4. Formation of inclusion bodies
 5. Hemadsorption test
1. Cytopathic effect
Viral infection of cell culture is often detected by the death of infected cells because viruses often
kill the infected cells which have very significant change in cell morphology. This effect of viruses
in infected cell cultures is called CytoPathic Effect of viruses which is usually called by virologists
“CPE”. CPE effect of viruses can be detected by using inverted microscope. To make the CPE
effect of viruses more visible it is possible to stain the cell culture with dyes called trypan blue and
neutral red; as trypan blue stains only dead cell and neutral red stain only live cells; so that dead
cells retain blue colour and live cell retain red colour.
2. Plaques formation
Many viruses can be isolated as a result of their ability to form discrete visible zones (plaques) in
layers of host cells. If a confluent layer of cells is inoculated with virus at a concentration so that
only a small proportion of the cells are infected; then plaques may form where areas of cells are
killed or altered by the virus infection. Each plaque is formed when infection spreads radially from
an infected cell to surrounding cells. Plaques can be also formed by many animal viruses in
monolayers if the cells are over laid with agarose gel to maintain the progeny virus in a discrete
zone. Plaque formation can be detected by looking the inoculated cell culture under inverted
microscope. It is also possible to know the plaque formation by staining the cell culture by trypan
blue and neutral red as CPE.
3. Formation of polykaryocytes
It is other morphological changes that may be manifested in virus-infected cell cultures which is
characterized by fusing infected cells with neighboring uninfected cells to form multinucleated cells
are called polykaryocytes which are also called syncytial cells. They can be detectable under
inverted microscope. But not all viruses form polykaryocytes in cultured cells. The most common
viruses that form syncytial cells in cell cultures are:-
 Avian reoviruses
 Some species of viruses in the family of Paramyxoviridae
4. Formation of inclusion bodies
One of the morphological changes that may occur in virus infected cell by certain viruses can be the
formation of inclusion bodies. Inclusion bodies are bodies that can be formed in some virus infected
cells. Inclusion bodies are composed of either:-
 Aggregates of nucleocapsids or
 Crystalline arrays of mature virus particles
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By their site of formation inclusion bodies can be:-
 Cytoplasmic inclusion bodies
 Nuclear inclusion bodies
Inclusion bodies in infected cells can be seen with a light microscope after fixation of infected cells
with chemical fixation (methanol or acetone) and treatment with cytological stains like
haematoxylin eosin stain. However, all viruses do not produce detectable inclusion bodies in
infected cells. Some of the common viruses which form intranuclear inclusion bodies are:-
 Herpesviruses
 Adenoviruses
 Parvoviruses
Whereas cytoplasmic inclusion bodies are the characteristics of infections with viruses like:-
 Poxviruses
 Orbiviruses
 Paramyxoviruses
 Rabies viruses
It is possible to suspect the type of virus infecting a cell by detecting the type of inclusion bodies
that are formed by some viruses in infected cell cultures. Some of the characteristics of inclusion
bodies that help us to suspect the virus type are:-
 Location of inclusion bodies in infected cells
 Shape of the inclusion bodies in infected cells
 The chemical composition of the inclusion bodies
By their site of location; inclusion bodies can be: -
 Cytoplasmic
 Nuclear
Inclusion bodies have different morphological features and they may have round upto pear shaped
morphology. By their chemical composition, they are different and can be detected by their reaction
with cytological stains. That is why inclusion bodies are classified as:-
 Basophilic
 Eosionphilic
 Neutrophilic
For example the inclusion body which is formed in cytoplasm of infected neurons has eosionphilic
character with almost round shape called Negri bodies. Cytological stains are rarely used to
identify cells infected with specific viruses by detecting inclusion bodies but they are mainly used as
screening test to assess the presence of any virus.
5. Hemadsorption test
Certain enveloped animal viruses may not cause CPE or plaque in infected cells but they acquire the
ability to absorb RBCS. Therefore, to search these unknown viruses with such type of property in
cell cultures, early researchers took an advantage of property of some viruses to bind to red blood
cells. The property of some viruses to adsorb RBCs on the surface of infected cell cultures is called
hemadsorption. Practically it can be done by flooding of RBCs suspension on the layer of infected
cells and incubating it in the incubator for some hours for attachment; then washing out those RBCs
that are not attached to infected cells using sterile BSS. Finally look the cells under inverted
microscope. Some the examples of animal viruses with hemadsorption characteristics are:-
 Bovine parainfluenza virus 3
 Orthomyxoviruses
 Paramyxoviruses
However, viruses can absorb RBCs of different species of animals. For example, cells infected with
bovine parainfluenza virus 3 will show the ability to adsorb chicken red blood cells to the plasma
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membrane; where as in some viruses like orthomyxoviruses and paramyxoviruses in which during
their budding process of virus maturation the viral protein is inserted into the plasma membrane of
infected cells. So that if these proteins bind to receptors on the red blood cells, the infected cells
show adherence of the RBCs cells on their surface of infected cells. Hemadsorption of RBCs to
these viruses is specific for red blood cells of a given animal species. These groups of viruses that
induce hemadsorption also show the ability to agglutinate red blood cells in cell-free medium called
haemagglutination. Haemagglutination is not a serological test but it shows the haemoagglutinating
property of some viruses (the presence of haemoagglutinating antigen). It is possible to conclude
that in these groups of viruses; the same viral proteins are responsible to permit both hemadsorption
and haemagglutination reaction. However, there are a few groups of viruses that can hemagglutinate
red blood cells but do not show hemadsorption in infected cell. Some of the examples are: -
 Adenoviruses
 Alphaviruses
Negative test results with the above mentioned methods like:-
 Absence of cytopathology
 Absence of inducing hemadsorption
 Absence of producing definable cytoplasmic or nuclear inclusions bodies
It may/ may not indicate to reach that the specimen is free from viruses. Cell cultures without CPE,
plaque formation and inclusion bodies) are called blind passages because when a virus is first
isolated it may replicate poorly in cell culture in the laboratory. But after it has gone through a
number of replication cycles it may replicate more efficiently. Each time the virus is’ sub -
cultured’ (to borrow a term from bacteriology) it is said to have been passaged; so that it is possible
to say that the specimen is free from viruses if and only if the viruses are not isolated after at least
three blind passages from a given specimen. In other cases the viruses may not cause cytopathology
but the virus antigen may present in cells. In this situation, the most commonly used tests are
immunologically and molecular biology based assays such as:-
 Immunofluorescence assay
 Immunohistochemistry assay
 Virus nucleic acid hybridization
 Polymerase Chain Reaction(PCR)
The quality of immunological tests depends on the specificity of the antibodies used in the assay; so
that with the development of monoclonal antibodies, this issue has been largely resolved. Another
method that is used to detect viruses in infected cells in the absence cytopathology in cell culture is
the molecular biology techniques as it has enhanced sensitivity.
NB. The above immunological and molecular biology technology are mostly used for identification
of the isolated viruses and you will see each techniques detail in virus identification.
5. Recognition of viral growth in embryonated eggs
There are many methods that help us to know the multiplication of viruses in the embryonated egg
and some of them are:-
 1. Embryo responses to inoculation
 2. Discoloration of the contents of egg
 3. Change in quantity of allantoic/ amniotic fluid
 4. Lesions on CAM
 5. Agglutination test with fluids
 6. Direct electronmicroscopical examination of the fluid by negative staining
1. Common embryo responses to inoculation
Embryos can respond to infection in a number of ways and a few responses which may be noticed
following inoculation of the embryonated egg are:-
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  Death
 Gross embryo abnormalities like stunting, dystrophy of muscle and limbs
 Body distortions
 Lesions of the extracellular membranes like edema or localized lesions as seen in the
formation of pock on CAM during poxviruses.
 Histopathological responses which are visible under bright field microscopy of tissues
stained by haematoxylin eosin.
Evidence of virus multiplication in chick embryos
Indication Virus which cause
1. Death of the embryo NDV(Newcastle disease virus), Fowl
plague(Avian influenza) and paramyxovirus
2. Specific lesions on the embryo
2.1. Pin point hemorrhage at occipital region like
under aspect of the abdomen and inter digital NDV(Newcastle disease virus)
space
2.2. Generalized congestion of the embryo
IBDV(Infectious Bursal Disease Virus) and
duck plague virus
2.3. Necrosis of the liver, enlargement of spleen IBDV,duck plague and IBV(Infectious
Bronchitis Virus)
2.4. Curling and dwarfing of the embryo IBV(Infectious Bronchitis Virus)
2.5. Edematous embryo Duck hepatitis A virus
3. Discoloration of the contents of egg
3.1. Greenish allantoic fluid Duck hepatitis A virus
3.2. Urate precipitate IBV and IBD
4.Change in quantity of allantoic/ amniotic fluid
4.1. Increased in quantity Lentogenic strains of NDV and EDS -76
V(Egg drop syndrome – 76 virus)
5. Lesions on CAM
5.1. Pock lesions on CAM Poxvirus, some herpes viruses and reovirus
5.2. Edematous CAM Duck plague and IBD
5.3. Congested CAM Duck plague and IBD
6. Agglutination test with fluids
6.1. Haemagglutination of allantoic fluid Orthomyxoviruses and paramyxoviruses
6.2. Haemagglutination of amniotic fluid Orthomyxoviruses and paramyxoviruses
7.Direct electronmicroscopical examination of Orthomyxoviruses and paramyxoviruses
the fluid by negative staining
6. Recognition of viral growth in naturally susceptible and laboratory animals
It is possible to recognize the growth of virus in experimental and naturally susceptible animals by
looking:-
 The main clinical signs that may develop after the incubation period.
 The gross pathology and histopathology of affected internal organs.
 By isolating and identification of the virus from specimens that are taken from these animals
by using other alternative cell culture.

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III. Identification of viruses
After a virus has been propagated, it is usually necessary to remove host cell debris and other
contaminants if it is possible, before the virus particles can be used for further laboratory studies
like:-
 Virus identification
 For incorporation into a vaccine
 Antigen preparation for diagnostic purposes
Many virus purification procedures involve centrifugation which is partial virus purification that can
be achieved by differential centrifugation. Differential centrifugation involves alternating cycles of
low-speed centrifugation after which most of the virus is still in the supernatant and high-speed
centrifugation, after which the virus is in the pellet. Differential centrifugation can be done crude
preparation of virus containing host derbis is subjected to low – speed/short- time centrifugation(e.g.
10000 g/ 20 minutes) followed by high – speed/ long time centrifugation(e.g. 100000 g/ 2 hours).
This cycle can be repeated to obtain a higher degree of purity and the final pellet containing partly
virus is resuspended in a small volume of fluid. There are many methods used in virology
laboratory to identify the isolated viruses. Among them the most common methods are:-
 A. Morphological investigations of the isolated virions
 B. Detection of virus antigens
 C. Detection of virus nucleic acids
A. Morphological investigations of the isolated virions
The morphology of isolated virions can be detected by electronmicroscopy. Negative staining
techniques of electronmicroscopy have generated many high quality electron micrographs; so that
the isolated virions can be negatively stained and examined in an electron microscope for the
presence of virions. However, this method has its own imitations and some of them are:-
 Structural distortions resulting from drying during preparation of specimens on the film
 High costs of the equipment
 Limited sensitivity as the method requires the minimum detectable concentration of virions
to be about 106/ml.
NB. The morphological features of some large virus like poxviruses and mimiviruses of
invertebrates are visible under light microscope with special staining technique.
B. Detection of virus antigen
This method of virus identification can be done using virus specific antisera or monoclonal
antibodies. The method is called immunological (serological) method of virus identification. The
antivirus antibody is produced by injecting virus antigen into one animal species; while and the
second antibody called antiimmunoglobulins is produced by injecting immunoglobulin from the
first animal species into a second animal species. The immunological tests that are used to identify
the viral antigens are:-
 1.Primary binding tests
 2.Secondary binding tests
 3. Tertiary binding tests
1. Primary binding tests
The most common primary binding tests which are used to detect the viral antigen are:-
ImmunoFluorescence Assays (IFAs)
ImmunoEnzyme Assays (IEAs)
RadioImmunoAssay (RIA)
In most primary binding techniques positive results are indicated by detecting the presence of a
labeled antibody or antiimmunoglobulins with fluorescence material or enzyme or radioactive
elements; so that if the specific antibody or anti – immunoglobulins are coated with fluorescence
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material, the test is called Fluorescence Antibody Test(FAT). FAT can be direct and indirect. FAT
test can be detected by using fluorescent microscope. But if specific antibody or anti -
immunoglobulin is coated with a known enzyme, the assay we call it Enzyme Linked Immuno
Sorbent Assay (ELISA). ELISA test can be detected by adding a substrate to the given enzyme and
observing the colour change. There are a lot of ELISA techniques but the most common ELISAs
Which are used to detect un known viral antigen are: -
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA( direct and indirect)
 Competitive ELISA
 Blocking ELISA
In RIA the specific antibody or antiimmunoglobulins are coated with radioactive elements like 3H,
14
C and 197I isotopes. The test result can be observed by measuring the radioactive amount which is
attached. Although RIA has high specificity as compared the other primary binding tests, it is not
commonly used in veterinary practice; because:-
 It is expensive
 It is hazardous to health of human beings
2. Secondary binding tests
Secondary binding tests are two – stage process and it is explained as:-
 The first stage is the interaction between antigen and antibody in which a reaction that is
associated with development of covalent bonds.
 The second stage is determined by the physical state of antigen and the results are varies.
If the antibody combines with soluble antigen in solution under appropriate conditions, the complex
precipitate is formed but if the antigen is particulate (bacteria or red blood cells), then they
agglutinate (clump).In other circumstances the combination of antigen and antibody may lead to the
activation of the complement which can be detected and measured.
Secondary binding tests which are commonly used to detect viral antigens are:-
 Precipitation tests example Agar Gel Immuno Diffusion(AGID) test
 Agglutination tests Example HaemAgglutination Inhibition (HAI)test
 Complement Fixation Test(CFT)
3. Tertiary binding tests
Tertiary binding tests are also called assay in living system. That means identification of the virus
using either cell culture or embryonated egg. The assay that is used to identify the virus using living
system is called Virus Neutralization Test (VNT).
B. Detection of viral nucleic acids
This method is called molecular technology method of identification of viruses.The most common
molecular technologies that are used to identify viruses are:-
 Hybridization
 Polymerase Chain Reaction(PCR) and its modifications
In hybridization, virus genomes or virus messenger RNAs (mRNAs) may be detected using
sequence-specific DNA probes carrying appropriate labels. Some of the labels that are used for
antibody detection can be used to label the probes. Hybridization may take place on the surface of a
membrane after Southern blotting (DNA) or northern blotting (RNA). Thin sections of tissue
may be probed for the presence of speci fic nucleic acids, in which case the technique is known as
“in situ” hybridization. Polymerase chain reaction (PCR) is used when a sample is likely to contain
a low number of copies of a virus nucleic acid; so that the probability of detection can be increased
by amplifying virus DNA using a PCR; while RNA can be copied to DNA and amplified using a RT
(reverse transcriptase)-PCR. The procedures require oligonucleotide primers speci fic to viral
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sequences; then an amplified product can be detected by electrophoresis in an agarose gel, followed
by transfer to a nitrocellulose membrane, which is incubated with a labeled probe. There are also
PCR techniques available for determining the number of copies of a specific viral nucleic acid in a
sample; and real-time PCR is commonly used for this purpose.

1.15. PATHOGENESIS OF VIRAL INFECTIONS

Pathogenesis is the process by which virus infection leads to disease. Pathogenesis can be also
defines as the mechanism of development of viral infections. Pathogenesis of viral infections
depends on: -
 The virulence of the virus
 The host defense mechanisms( innate and adaptive immunity)
Pathogenic mechanisms of many viruses include:-
 Implantation of the virus at a body site called portal of entry of viruses
 Replication at portal entry sites
 Spread to target organs
 Multiplication of the virus in target organs where disease or shedding of virus into the
environment occurs
It is known that most viral infections are subclinical because, body defenses against many viruses
arrest most infections before disease symptoms become manifest. But the knowledge of most
subclinical viral infections come from serological studies showing that sizeable portions population
have specific antibodies to viruses even though the individuals have no history of the disease. So
that these inapparent infections have great epidemiological importance because:-
 These organisms constitute the major sources for dissemination of virus through the
population.
 They confer immunity to given viral disease.
There are many factors that are affecting the pathogenic mechanism of viruses. Accessibility of
viruses is influenced by physical and physiological barriers of innate immunity of the organisms
which include:-
 Skin
 Mucous membrane with its discharge and cilia
 PH of stomach
 Soluble factors( complement and interferon)
 Inflammatory barriers
 Phagocytic barriers
 Distance traversed within the body
 Natural immunity of the organism
However, if the virus reaches on the target organs, infection occurs only if cells capable of
supporting virus replication are present. That means cellular susceptibility requires:-
 A cell surface attachment site for virions called receptors.
 Intracellular environment that permits viruses to replicate and release.
Even the virus initiates infection in a susceptible organ, replication of sufficient virus to cause
disease may be prevented by host defenses. Other factors that determine whether viral infection or
disease occurs are the many virulence factors of infecting virus. This means to cause the disease, the
infecting virus must be able to overcome the inhibitory effect of physical barriers, distance and host
defense mechanisms. The inhibitory effects are genetically controlled and therefore may vary among
individuals and races. Virulence factors of the virus enable the virus:-
 To initiate infection

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  To spread in the body
 To replicate to large enough numbers (titer) in target organs to impair the functions of target
organs.
These factors include the ability of viruses to replicate under certain circumstance during
inflammation, during the febrile response, in migratory cells and in the presence of natural
body inhibitors and interferon. Viral diseases do not always follow after successful virus
replication in the target organs. So that viral diseases occur if and only if:-
 The virus replicates sufficiently to damage essential cells directly
 Replicated virus cause the release of toxic substances from infected tissues called porins
which cause the formation of pores to cause cells to lyse
 Replicated virus damage to cellular genes or damage organ functions indirectly as the result
of the host immune response to the presence of virus antigens.
In pathogenesis of many viral infections we must understand:-
 1.Portal of entery and implantation of viruses at portal of entery
 2. Local replication and local spread of viruses
 3. Dissemination of viruses from the portal of entery
 4. Multiplication in target organs and tissue tropism of viruses
 5.Cellular pathogenesis
 6. Shedding of virus
 7. Congenital viral infections
1. Portal of entery and implantation of viruses at portal of entery
Viruses are carried into the body of susceptible animals through different routes and viruses use all
possible sites for primary implantation at entery sites. Some of the common routes of virus entery to
susceptible organisms are:-
 Aerosol through respiratory tract
 Together with feeds(ingestion)
 Through wounds
 Vector bites
 Together with contaminated objects(fomites)
 Through placenta from infected mother to fetus
 Through reproductive tract with infected semen and/ or sperm cells
The most common sites for viruses for their primary implantation at the site of entery are all body
surfaces. The frequency of virus implantation is greatest where viruses contact living cells directly.
Some of them are:-
 Respiratory tract
 Alimentary tract
 Genital tract
 Under skin(subcutaneously)
 With some viruses implantation in fetus may occur at the time of fertilization through
infected cells (sperm cell and/or egg cell).
 Later gestation implantation of viruses to the fetus may occur through placenta.
 Viruses may also implant to new born animals from the mother at the time of delivery
However, in the early stage of pathogenesis, even if the virus is implanted to the appropriate site,
there are many factors that determine the outcomes of viral infection. Some of them are:-
 Dose of implanted virus
 Infectivity of implanted virus
 Virulence of implanted virus
 Location of implantation to the site of replication
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  Immunity of infected organism
That is why depending on the above factors, viral infections may have the following forms of
infections.
 Latent( in apparent) infection called subclinical infection
 Mild form of infection
 Severe form of infection
2. Local replication and local spread of viruses
Successful implantation of viruses may be followed by local replication and local spread of viruses.
Viruses that replicate within initially infected cells may spread to adjacent cells through two
mechanisms.
 Extracellularly
 Intracellularly
Extracellular spread of viruses occurs by release of the virions from infected cells into the
extracellular fluid and subsequent infection of the adjacent cells.While intracellular spread of
viruses occurs by:-
 Fusion of infected cells with adjacent cells(uninfected cells) or
 Formation cytoplasmic bridges between infected and uninfected cells.
Intracellular spread of viruses is the reason for the formation of multinucleated cells called
polykaryocytes (syncytial cells) in cell culture. Most animal viruses spread to adjacent cells
extracellularly. However, there are some animal viruses which spread to adjacent cells by both
intracellularly and extracellularly. Some of them are:-
 Herpesviruses
 Paramyxoviruses
 Poxviruses
What you must understand is that intracellular spread of viruses provides them with partial protected
environment than extracellular spread of viruses to adjacent cells. This gives the intracellularly
spreading viruses to be protected from antibodies as the antibodies never cross the cell membrane.
There is also the spread of virions beyond adjacent cells in affected tissues through different
mechanisms. Some of the common mechanisms of spreading of virions beyond adjacent cells in
affected tissues are:-
 Through a liquid in local site called lymph
 By diffusion through surface fluids(secretions from mucous layer of respiratory, digestive
and urogenital systems)
 Through infected migratory cells like lymphocytes and macrophages
After the establishment of infection at the portal of entery, it may be followed by continued virus
multiplication in the tissue. This may lead to divide the viruses into three categories.
 The first category of viruses comprises viruses that cause localized infection with localized
virus shedding.
 The second category of viruses comprises viruses that cause localized infections with the
spread of viruses to infect distant body surfaces and organs.
 The third category of viruses comprises viruses those can cause both localized and
disseminated infections.
In localized infection with localized virus shedding, the local sites of implantation of viruses are
target organs for viruses replication and shedding. Some common viruses which fall into this
category are:-
 Viruses those cause respiratory infections in human beings like influenza viruses, common
cold viruses and parainfluenza virus.
 Viruses that cause alimentary tract infections like rotaviruses and picornaviruses
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  Viruses those cause localized skin infections like papillomaviruses and cowpox virus.
In localized infections with the spread of viruses to infect distant body surfaces and organs are
those viruses that can travel from the sites of primary localized infections to target organs and cause
disseminated form viral infections. Some common viruses which fall into this category are:-
 Viruses those travel to distant target organs from primary sites of infections together with
blood (viremia); example most of viruses that cause generalized form of infection.
 Virus that travel to pharynx and intestine from the eye in the absence of viremia; example
picornavirus.
 Viruses that can spread internally together with feed mass and body secretions to target
organs and site of excretions to cause disseminated infections.
A third category of viruses may cause both local and disseminated viral infections, example
herpes simplex and measles virus in human being.
Pathogenesis of a few selected virus infections (localized viral infections)
Disease Site of Route of Target organ Site of shedding
implantations spread
Influenza Respiratory tract Local Respiratory tract Respiratory tract
Viral Alimentary tract Local Alimentary tract Alimentary tract
gastroenteritis
Warts Skin and mucosa Local Skin and mucosa Skin and mucosa
3. Dissemination of viruses from portal of entery
At portal of entery, multiplying viruses disseminating to the target tissues and organs through:-
 A. Blood and lymph
 B. Nerves
A. Dissemination of viruses in the blood and lymph
They are the principal routes for the wide dissemination of viruses through the body of infected
organisms. Among them the most common route for systemic spread of viruses is blood. However,
in some viruses, the virus progeny may diffuse through the afferent lymphatic vessel to lymphoid
tissues; then through the efferent lymphatic vessels to infect cells which are in close contact with the
blood stream (example endothelial cells, especially those of lymphoreticular organs). The initial
spread of viruses from the site of entery to target tissues through blood cause primary viremia.
Subsequent release of viruses from lymphoreticular organs like spleen directly into bloodstream
induces secondary viremia. This can be lasts for several days and results in putting of viruses in
contact with capillary system of all body tissues; then the virus enters to target organs from
capillaries through different mechanisms.
 By replicating within the capillaries of endothelial cells
 By fixing on macrophages and released on target organ in the side of the capillary
 By diffusing through small gaps in the capillary endothelium
 By penetrating the capillary wall through an infected migrating leucocytes
Then the virus replicates and spreads within the target organ. Disease occurs if the virus replicate in
sufficient numbers in target cells and cause their lyses and death. Finally the virus reaches to the site
of excretion by the same mechanisms of dissemination of the virus from portal of entery to target
organs.
B. Dissemination of viruses in nerves
Dissemination of viruses from portal of entery through nerves is the less common dissemination
method than blood stream dissemination. But it is a means of dissemination in a number of
important animal and human viral diseases. Some of the common viruses those can be disseminated
from portal entery and implantation through nerves are:-

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  Rabies virus
 Herpesvirus
 Poliovirus
Rabies virus implanted by a bite from rabid animals replicates subcutaneously and in muscular
tissues to reach nerve endings. Rabies virus spreads centrally in the axons, dendrites and perineural
cells where the virus is shielded from antibodies. This nerve route leads rabies virus to the central
nervous system where the disease is originated; then rabies virus spreads centrifugally through the
nerves to reach the salivary gland which is the site of shedding. Although there are many additional
factors that determine the length of incubation period of viral infections (dose of virus, virulence,
infectivity and immune status of the organism), distant from portal of entery to target organs also
influence the length of incubation period in many viral infections.
Incubation period of viral infections
During most viral infections, no signs or symptoms of the disease occurs during the stage of virus
dissemination. Thus incubation period of virus infections is defined as the time between exposure to
virus and on set of disease; so that incubation period of viral infections extends from the time of
virus implantation through the phase of virus dissemination, ending when virus replication in target
organs causes disease. Occasionally, mild fever and malaise may occur during viremia but they are
often transient and have low diagnostic value. The incubation period tend to be brief in 1 to 3 days
of infections inwhich the virus travels only short distance to reach target organs; that means
infections inwhich disease is due to virus replication at portal of entery. Conversely, incubation
periods in generalized viral infections are longer because the virus needs stepwise fashion by which
it moves through the body before reaching the target organs. Generalized infections caused by
arboviruses like togaviruses have short incubation period because there is direct injection of rapid
multiplying viruses into vascular system of susceptible animals. The incubation period of some viral
infection can be long (months to years) and this type of viral infection is called persistent viral
infection. In persistently viral infection the infected cells are not often lysed or lysis is delayed; so
that depending on the length of incubation period, viral infections have the following forms.
 Peracute
 Acute
 Subacute
 Chronic
 Latent(persistent infection)
In a few viral infections there can be mutation in the host genetic material resulting in cellular
transformation and cancer.
4. Multiplication of viruses in target organs and tissue tropism of viruses
4.1. Tissue tropism of viruses
Many viruses have an affinity to replicate in specific tissue cells; so that the ability of most viruses
to replicate in specific cells of tissues is called tropism of viruses. Tropism of viruses is named by
the name of tissues / cells where they selectively replicate. Those viruses which are selectively
replicate in epithelial cells of skin and mucous membranes are called eptheliotrophic viruses. Some
of the examples of eptheliotrophic viruses are:-
 Poxvirus
 Papillomavirus
 FMD virus
 Most paramyxoviruses
 Most of orthomyxoviruses etc.
Those viruses which are selectively replicate in nerve cells are called neurotrophic viruses. Some
of the examples of neurotrophic virus are:-
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  Rabies virus
 Polio virus
Those viruses which are selectively replicate in endothelial cells of blood vessel are called
endotheliotrophic viruses. Those viruses which are selectively replicate in lymphocytes are called
lymphocytotrophic viruses. HIV is a good example of lymphocytotrophic virus as it affects pro -
CD4 cells. Those viruses that replicate more than two cells of tissues are called pantrophic viruses.
Tropism of viruses is determined by:-
 The presence or absence of special cell receptor for a given virus.
 The presence or absence of essential molecules for transcription and translation of viral
nucleic acid and protein.
Let us see some examples of tropism of viruses.
 Poliovirus and rabiesvirus selectively infect and destroy certain nerve cells because neurons
have high concentration of surface receptors for poliovirus and rabiesvirus than do poliovirus
and rabiesvirus resistant cells do.
 Rhinoviruses replicate exclusively in the upper respiratory tract because the epithelial cells
of the upper respiratory cells have receptor to these groups of viruses.
4.2. Multiplication of viruses in target organs and cellular pathogenesis
Viruses replication in target organs resembles replication in implantation organs at the portal of
entery except that:-
 The target organ in systemic infections is usually reached late after the stepwise progression
of the virus through the body.
 Clinical diseases are originated from target organs but not from implantation organs
In each step of virus progression through the body, the body local defense mechanisms are activated
which comprise:-
 Interferon
 Local inflammatory reaction
 Local immunity
Thus, when the target organ is infected, the previously infected sites may have reached various
stages of recovery. Circulating interferon and immune responses probably account for termination of
viremia but these responses may be too late to prevent the seeding of viruses into target organs and
into sites of shedding. Nevertheless, these systemic defenses can diffuse in various degrees into
target organs and thereby retard virus replication and disease. Depending on the balance between
virus and host defenses, virus multiplication in the target organs may be sufficient to produce
dysfunctions manifested by disease or death. Clinical features like fever and malaise that is
developed in many viral infections is due to:-
 Diffusion of toxic products produced from infected cells due to cell necrosis.
 Release cytokine called lymphokines and other inflammatory mediators
The release of cytokine like leukotriene C4 during viral respiratory infection may cause
bronchospasm. In addition, impairment of leukocytes and immunosuppression by some viruses may
expose the affected animals to secondary bacterial and fungal diseases. Tissue damage in viral
infections is caused because of the replication of viruses in cells which make up the tissues; so that it
is very important to understand clearly cellular pathogenesis of viral infection at cellular level.
Direct cell damage and death may result from disruption of cellular macromolecular synthesis by the
infecting virus. It is clear that the other reason is also viruses cannot synthesis their genetic and
structural components by their own. An essential aspect of viral pathogenesis at cellular level is the
competition between the synthetic needs of the virus and those of the host cell. Since rely viruses
almost exclusively depend on the host cell for these functions, their parasitic replication therefore
robs the host cell energy and macromolecular components. As viruses must use the cell’s machinery
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to synthesize their own nucleic acid and proteins, they have involved various mechanisms to subvert
the cell’s normal functions to those required for production of viral macromolecules and eventually
viral progeny; as the result it severely impairing the host cell ability to perform these functions for
its own and resulting in cell death and disease.
6. Shedding of virus
The most frequently sites for viruses shedding are respiratory and alimentary tracts. However, blood
and lymph are sites of shedding for the arboviruses since biting insects become infected by this
route. HIV is shed in the blood, semen and milk. Milk is a site of shedding for viruses such as some
RNA tumor viruses like retroviruses and herpes viruses. Urinary tract is the site of shedding for
herpesviruses together with milk. Genital tract is the site of shedding for herpesvirus type 2; and it is
the site through which the virus can be transmitted to female and fetus. Saliva is the primary site of
shedding for rabies virus. Finally, viruses such as tumor viruses that are integrated into the DNA
host cells can be shed through sperm and egg cells.
Pathogenesis of some selected disseminated viral infections (animal and human beings)
Disease Common site of Route of Target Site of shedding
implantation spread organ(organs)
Rabies Subcutaneous and Nerve Brain Salivary gland
muscle(bite)
Chickenpox Respiratory tract Blood and Skin and lung Respiratory tract
nerve and skin
Herpes simplex Respiratory tract Nerve and Brain, liver and Respiratory tract
type 1 leukocytes skin and epithelium
Herpes simplex Genital tract Nerve Genital tract Genital tract
type 2
Poliomyelitis Alimentary tract Blood(nerve) CNS Alimentary tract
Hepatitis A Alimentary tract Blood Liver Alimentary tract
AIDS Injection, trauma and Blood Immune cells Blood and semen
intestine and brain
Arboviruses Subcutaneously(bite) Blood Brain and others Lymph and
infection blood
7. Congenital viral infections
Fetus of animals and human beings can be a target organ for a few viruses. Viruses that multiply in
fetus may cause:-
 Death of the fetus or
 Congenital anomalies
Congenital anomalies caused by replicating viruses in a fetus are called teratogenic effect of
viruses. To cause congenital anomalies, virus must reach to the fetus and multiply on it and cause
maldeveloped organs. Generally, viruses reach the fetus during maternal viremia by passing through
placenta to the fetal circulation and then to fetal target organs. Although the dam may have only a
mild or inapparent infection, virus multiplication in the fetus may lead to congenital anomalies or
fetal death. There are many reasons that make the fetus to be a target organ for some virus
replication.
 The immune and interferon systems of the young fetus are immature.
 The partial placental barrier for transferring of the maternal immunity and interferon from
the dam.
 The undifferentiated state of the fetal cells makes the fetus to be suitable for many viruses to
replicate there.

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Fetuses are highly vulnerable to much virus infection during the first trimester of pregnancy; so
that sufficient virus multiplication may disrupt development of fetal organs, especially during their
rapid development mainly during the first trimester of pregnancy. Although many viruses
occasionally may cause congenital anomalies, the most common offenders are:-
 Herpes simplex virus type 1
 Rubella virus

1.16. IMMUNE RESPONSE TO VIRAL INFECTIONS

The immune responses of the organisms to viral infections are categorized into two major groups.
 I. Innate immunity to viral infections
 II. Adaptive immunity to viral infections
I. Innate immunity to viral infections
Innate immune defenses exhibit neither antigen specificity nor memory but they provide a critical
line of first defense against viral infections because they are constantly present and are operational
immediately after viral infections. Innate immunity is often considered separate from acquired
immune responses but they are inextricably linked and innate responses modulate subsequent
acquired responses in many ways. So that the innate immunity of the organisms to viral infections
comprises:-
 1. Anatomical barrier of skin and mucosa(epithelial barriers)
 2. Soluble factors of serum protein like complement
 3.Phagocytic activity of macrophages, neutrophiles and dendritic cells
 4. Natural killer(NK) cells
 5. Cytokines and chemokines(Interferon(IFN))
 6.Apoptosis
 7.Small RNA molecules which are called RNA interfere (RNAi)
1. Anatomical barrier of skin and mucosa
Viruses that are transmitted horizontally between individuals must first breach the barriers at their
portal of entry before they can cause infection in their respective hosts and some of these barriers
are:-
 The epithelial lining of the skin
 The epithelial lining of the respiratory tract
 The epithelial lining of gastrointestinal tract
 The epithelial lining of urogenital tracts
The epithelial linings provide a mechanical barrier against infection at these common sites of virus
entry. Secretions and other activities at mucosal surfaces provide further non-specific protection
against viral infections. Some of secretions of mucosal surfaces those act as non- specific protection
against viral infections are:-
 Surfactant and the mucociliary apparatus of the respiratory tract
 The mucous barrier, regional PH extremes, sterilizing action of secretions barrier from the
liver and pancreas
 Specific antimicrobial peptides such as defensins of the mucous membrane.
2. Soluble factors of serum proteins
A variety of plasma proteins also exert antimicrobial activity and some of them are:-
 Various complement proteins
 C-reactive protein
 Mannose-binding proteins etc.

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In addition to their antimicrobial activity, they coat the viruses with their surfaces and facilitate to be
phagocytosed by macrophages. So that these proteins make the viruses to be testy for macrophages;
and are called opsonin. The process is called opsonization.
3. Phagocytic activity of macrophages, neutrophiles and dendritic cells
Phagocytic cells that play very crucial role in innate immunity during viral infections are:-
 Macrophages
 Neutrophils
They provide a critical antimicrobial function, as they are attracted to sites of inflammation where
they efficiently ingest and digest foreign materials, including other microorganisms. These cells
possess the intracellular machinery to destroy ingested microbes, particularly bacteria, through:-
 The actions of hydrolytic lysosomal enzymes
 The production of activated oxygen and nitrogen metabolites called superoxide within
phagocytic vacuoles.
Various soluble mediators like chemoattrctants can also attract these cells to sites of inflammation,
and activate them to enhance their antimicrobial activity. There are also cells that play a crucial role
in innate immunity in viral infections. These are Dendritic Cells (DCs). Dendritic Cells (DCs) are
cells which are key players in both innate and adaptive immunity as they play essential role in innate
immunity in viral infections because dendritic cells are important source of type I interferon (I -
IFN) and various other cytokines that inhibit viral replication at portal of entery. That is why
interdigitating dendritic cells are abundant at portals of virus entery; such as respiratory, urogenital,
and GIT tracts and skin. DCs have a capacity to initiate quickly the protective innate immune
responses for invading viruses because they are endowed with pattern recognition receptors for
different viruses during their formation. They participate in adaptive immunity because they are
highly efficient antigen-presenting cells (APCs) for extracellular antigens to CD4+ cells.
4. Natural killer (NK) cells
Natural killer (NK) cells are specialized lymphocytes that are capable of rapid killing of virus-
infected cells. Thus they provide early and non-specific resistance against viral infections.
Specifically, natural killer cells recognize host cells that express altered levels of major
histocompatibility complex (MHC) class I molecules. Natural killer cells mediate death of virus-
infected cells via apoptosis. Natural killer cells also possess surface receptors for the Fc portion of
immunoglobulin molecules and this allows them to bind and lyse antibody-coated target cells
through the process of Antibody Dependent Cell mediated Cytotoxicity(ADCC). Lastly, natural
killer cells synthesize and release a variety of protein molecules like:-
 Cytokines called type II IFN(interferon)
 Several interleukins
These molecules stimulate the proliferation of NK cells and activate their cytolytic activity.
5. Cytokines and chemokines
Cytokines are messengers of the immune system and are responsible for the induction and
regulation of both innate and adaptive immune responses. Chemically cytokines are typically
inducible glycoproteins that are transiently synthesized after appropriate stimulation of the cell that
produces them. Cytokines are soluble mediators that facilitate communication between key cell
populations of the immune system and the cells of the immune system which are communicating
with cytokines are:-
 Various subpopulations of lymphocytes(NK cells, T and B lymphocytes)
 Macrophages
 Dendritic cells
 Neutrophils

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Individual cytokines usually are produced by more than one cell type and perform several,
frequently divergent activities. Different cytokines that are produced by different immune cells may
perform similar activities. This overlapping activity of different cytokines to bring similar effect on
target cells is called redundancy. Cytokines bind to specific receptors on the surface of target cells,
with subsequent transcriptional activation of target cell. These activities of cytokines may act on
different cells. If the effect of cytokine is manifested on cells where they are produced, this effect of
cytokines is called autocrine effect of cytokine. But if the effect of cytokines is manifested on the
adjacent nearby cells of different types, this effect of cytokine is called paracrine effect of cytokine
Lastly, if the effect of cytokine is manifested on many cells types; located distantly, this effect of
cytokine is called endocrine effect of cytokine.
Chemokines are the family of small proteins that are chemotactic for leukocytes; and play crucial
role in innate immunity. Other cytokines which has chemotactic effect for leukocytes is interleukin
– 8(IL – 8). Cytokines which are involved in innate immune responses include:-
 Type I IFNs
 Tissue Necrosis Factor - α (TNF - α)
 IL-6
 IL -8
 Chemokines
Cytokines that are involved in both innate and adaptive immune responses include:-
 IL-12
 Type II IFN
Among all cytokine and chemokine, the interferons (IFNs) are especially critical to antiviral
immunity.
Interferons
In 1957, Isaacs and Lindenmann reported that cells of the chorioallantoic membrane of embryonated
hen’s eggs infected with influenza virus release into the medium a non-viral protein called
“interferon” (IFN). They indicated, this non- viral protein is produced by viral infected cells and
protects uninfected cells against the same or unrelated viruses. It has since been determined that
there are several types and subtypes of interferon (IFN). These proteins are key elements of anti-
viral resistance and are central to both innate and adaptive immune responses to viral infections.
There are three distinct types of IFN, represented as:-
 Types I IFN (type I IFN)
 Type II IFN(type II IFN)
 Type III IFN (type III IFN)
Type I IFN is also called alpha interferon, represented by IFN – α. It is the product of virus infected
cells. IFN – α is important to innate resistance of cells to become resistant to many viral infections
but not with intracellular bacterial pathogens.
Type II interferon is also called gamma interferon, represented by IFN- γ. Gamma interferon is a
product of T cells and NK cells. Thus activated T cells are especially important sources of
production of IFN- γ and are central to the expression of the cell-mediated immune aspects of
adaptive immunity. IFN - γ is particularly important in conferring immunity to intracellular
microorganisms other than viruses.
Type III interferon is also called lambda interferon and is represented by IFN - λ. IFN- λ was
only recently described and appears to represent an ancestral type I IFN. Its function is not well
studied but perhaps the one with regulatory function. It is clear that among cytokines, interferons are
crucial in innate immunity against many viruses. Among interferons, the IFN - α is crucial in viral
infections. However interferons regulate not only the innate immunity but also the adaptive

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immunity; so that in addition to their important antiviral activities, particularly those of type I IFN,
the IFNs also stimulate adaptive immune responses and some of them are:-
 IFNs enhance the cytotoxic adaptive immune responses, including enhancing cytotoxic T-
lymphocyte-mediated cell lysis through increased expression of class I MHC molecule on
virus-infected nucleated cells.
 IFN- γ promotes expression of class II MHC molecules on macrophages and modulates
immunoglobulin synthesis by B lymphocytes.
6. Apoptosis
Apoptosis is an important and common event during many viral infections and is the process of
programmed cell death and this cytocidal activity of NK cells is very important to control viral
infections because the mechanism of cell suicide that the host activates as a last resort to eliminate
viral factories before progeny virus production is complete.
7. Gene silencing (Interfering RNA)
Cells utilize small, interfering, RNA molecules (RNAi) to silence the genes of viruses.
Silencing gene helps virus infected cells:-
 To regulate normal developmental and physiological processes
 To interfere with virus replication.
The RNAi are produced from longer segments of either ssRNA or dsRNA after their cleavage by an
endoribonuclease. Production of RNAi initiates formation of the RNA-silencing complex that
includes an endonuclease that degrades those mRNAs with a sequence that is complementary to that
of the RNAi. Cells can utilize this mechanism to disrupt virus replication through the production of
RNAi that are complementary to specific viral genes.
II. Adaptive immunity to viral infections
Adaptive immunity to virus infection has two major components.
 Humoral component
 Cellular components
Humoral immunity is mediated principally by antibodies released from B – lymphocytes whereas
cellular immunity is mediated by T - lymphocytes. Adaptive immunity is differing from innate
immunity in that:-
 It is antigen specific, mediated by lymphocytes that possess surface receptors that are
specific to each pathogen.
 Adaptive immunity stimulates long-term memory after infection, meaning that protective
immune responses can quickly be reactivated on re-exposure of the organism to the same
pathogen.
 Responses of adaptive immunity take time (several days at least) to develop but the lag
period of secondary immune response is shorter than the primary immune response.
1. Cells and cytokines of adaptive immunity to viral infections
Cells that are playing essential role in adaptive immunity to viral infections are:-
 1. B - lymphocytes
 2. T - lymphocytes
 3. Macrophages
 4. Dendritic cells(DCs)
1. B - lymphocytes
B lymphocytes produce antibodies. Antibodies against specific viruses can neutralize specific
viruses and protect cells from virus infection. B lymphocytes express surface receptors that are
specific to particular antigen. Receptor diversity of B lymphocytes is generated by rearrangement of
the genes encoding portions of individual immunoglobulin molecules. The acquired B cell response

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to infection involves immunoglobulin class switching and progressive specificity, known as affinity
maturation; so that after exposure to antigen such as a viral infection, B cells differentiate into:-
 Effector B cells called plasma cells which secrete antibodies with the same antigen
specificity as that of the surface receptors of the B cells from which they were derived.
 Memory cells which will differentiate into effector plasma cells and memory cells during re-
exposure of the organism to the same antigen in the second time.
What you must understand is that nowadays it is known that there is a subclass of B lymphocytes,
designated B1 cells which secrete broadly reactive immunoglobulin without specific antigen
stimulation. This immunoglobulin that is secreted from B1 cells known as natural antibodies.
Natural antibodies are not antigen specific but they provide linkage between the innate and acquired
humoral responses because B1 cells can present the virus peptides to CD4+ by MHC class II
molecule. So that it is possible to say antibodies produced by B1 cells provide a first line of humoral
defense and B1 cells are Antigen Presenting Cells (APCs).
2. T - lymphocytes
T lymphocytes also possess antigen-specific surface receptors. Like B-cell antigen recognition,
diversity and receptor specificity is generated by somatic rearrangement of the genes that encode the
T cell receptor. According to the way of recognition of antigens, T lymphocytes are classified into
two major groups.
 T – lymphocytes that recognize viral antigens which are either presented by MHC molecules
or without MHC molecules.
 T – lymphocytes that recognize viral antigens if and only if the antigens are presented by
MHC molecules of nucleated virus infected cells and MHC molecule of APCs.
T – lymphocytes that recognize viral antigens which are either presented by MHC molecules or
without MHC molecules have receptor composed of a heterodimer of γ and σ chains are called (γ/ σ
T cells). This means (γ/ σ T cells) recognize small viral peptides expressed on the cell surface by
MHC molecule, they do so without the requirement of MHC molecule. So that γ/ σ T cells are called
lack MHC restriction T – cells. The lack of MHC restriction, coupled with the fact that γ/ σ T cells
are especially abundant at mucosal surfaces that serve as portals of virus entry (like the mucosal
lining of the gastrointestinal tract). It is possible to suggest that these cells serve as sentinels to
remove virus-infected cells. T – lymphocytes that recognize viral antigens if and only if the antigens
are presented by MHC molecules of nucleated virus infected cells and MHC molecule of APCs.
These T- lymphocytes have receptors that consist of a heterodimer of α and β chains so-called α/β T
cells. The α/β T cells recognize small peptides expressed on the surface of cells in association with
MHC antigens and these T – cells are called MHC restricted T - cells. MHC restricted cells play
essential role in cell mediated and antibody mediated (humoral immunity) of adaptive immunity
during viral infections. So that those MHC restricted T – cells which recognize virus peptides that
are presented by Antigen Processing and Presenting Cells (APCs) are called CD4 +. APCs present
the virus peptide by MHC molecule which is encoded in their loci of gen and is synthesized in ER
(endoplasmic reticulum) is called MHC – class II molecule; as the result CD4+ cells attach to virus
peptides that are presented by MHC class II molecule of APCs and will be activated and produce
cytokines. The cytokines that are produced by CD4+ cells carry messages to the B- lymphocytes; as
the result the B – lymphocytes will be activated and start to proliferate and to differentiate into:-
 Effector B – cells called plasma cells
 Memory B- cells
As the result effector B – cells (plasma cells) will secrete specific antibodies against specific viral
antigen; while memory B – cells will have a memory to specific viral antigen to be changed into
effector B – cells and memory cells during re – exposure of the organism to the same antigen in the
second time. So that CD4+ cells are T helper lymphocyte cells that play an essential role in humoral
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immunity of viral infections. Other MHC restricted T – cells which recognize the virus peptides that
are presented by MHC class I molecule of nucleated cells are called CD8+. Lymphocytes that
mediate cell lysis are also called cytotoxic T lymphocytes (CTLs) that typically express (CD8) on
their cell surface (CD8+ CTLs). CTLs lyse cells that express viral antigen in the context of
appropriate class I MHC molecules. Portions of immunogenic viral proteins produced in the cytosol
of infected cell are transported to the endoplasmic reticulum, where they associate with class I MHC
molecules. This complex is directed to the cell surface, where the viral peptides can be recognized
by antigen-specific cytotoxic T cells. CD8+ cells kill virus infected nucleated cells through the
process is called apoptosis. So that CD8+ T cells are responsible for cell mediated immunity also
called cellular immunity for virus infections. This is because when the virus peptides are
presented by MHC class I molecule, the CD8+ cells will be differentiated into:-
 Effector CD8+
 Memory CD8+ cells
The effector CD8+ cells lyse all virus infected cells; while memory CD8 + will have a memory to
the specific virus antigen. So that when there is re – exposure of the organism to the same antigen in
the second time, memory CD8 + will be differentiated to effector and memory CD8+ cells again.
3. Macrophages
Cellular immunity (cell-mediated immunity) of many viral infections is mediated not only by
effector T - lymphocytes but also by macrophage. Macrophages are important bone marrow derived
cells that are responsible for microbial phagocytosis and killing. Un matured macrophages in the
blood vessels is called monocytes. After maturation, they migrate in different tissues and have
different names. They can be activated by cytokines into effector cells that mediate cellular
immunity through their enhanced antimicrobial capacity. Macrophage activation is mediated by
IFN-γ that is released by antigen specific T cells and by NK cells.
4. Dendritic cells (DCs)
Dendritic cells are also key players in adaptive and innate immunity of viral infections. They derive
their names from their abundant fine cytoplasmic processes called “dendrites”. There are two major
types of dendritic cells:-
 1. Interdigitating dendritic cells
 2. Follicular dendritic cells
1. Interdigitating dendritic cells
Interdigitating dendritic cells are critical antigen presenting cells that are located at portals of virus
entry such as:-
 Skin
 Within/beneath the mucosal epithelial surfaces lining the gastrointestinal, respiratory, and
urogenital tracts.
 Within the interstitial of virtually all tissues
These dendritic cells express surface pattern recognition receptors that can quickly and generically
respond to the presence of viral triggers (PAMPs) by the production and release of antiviral
cytokines such as IFN.
2. Follicular dendritic cells
Follicular dendritic cells occur within germinal centers of lymphoid tissues such as lymph node and
spleen. These cells efficiently capture (phagocytose) circulating antigens then they present to B
lymphocytes that express the relevant surface receptor specificity, leading to B cell activation and
development of humoral (antibody mediated) immunity.
Cytokines of adaptive immunity in viral infections
Cytokines play essential role not only in innate immunity but also in adaptive immunity. In adaptive
immunity, the major cytokines are produced by CD4+after their stimulation by specific antigen
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which is presented by APCs (Macrophages, B1- lymphocytes and DCs) through MHC class II
molecule. Important cytokines of adaptive immunity which are produced by CD4+ are interleukins.
Interleukins of the adaptive immunity comprise:-
 IL-2
 IL-4
 IL-5
 IL-17
Interleukins that are produced by CD4+ cells promote the activation, proliferation and
differentiation of B – lymphocytes as the result the B – lymphocytes become activated, start to
proliferate and are differentiated into:-
 Effector B- cells called plasma cells
 Memory cells
III. Avoidance and escaping mechanisms of viruses from host defense
Viruses have developed diverse and complex mechanisms of avoiding protective host innate and
adaptive immune responses. Some of the mechanisms that are developed by viruses to escape out
from the host innate and adaptive immunity include:-
 1. Shutdown of host macromolecular synthesis
 2. Avoidance of CTL-mediated killing of virus infected cells
 3. Interference with apoptosis
 4. Counter defenses against cytokines
 5. Evasion of the antiviral state
 6. Virus specific gene silencing pathways
1. Shutdown of host macromolecular synthesis
Many viruses, soon after infection, inhibit normal transcription and/or translation of cellular
proteins. They rapidly divert the machinery of the infected cell for production of progeny virions.
This rapid shutdown of the host cell macromolecule synthesis quickly impairs the innate immune
response to the infecting virus. Some of the shutdown cellular mechanisms are:-
 Production of critical proteins such as class I MHC molecule
 Antiviral cytokines such as type I IFN(IFN – α)
The result is that, without effective innate immune responses, the infecting virus can quickly
replicate and disseminate before the host can develop an adaptive immune response. This strategy is
widely used by RNA viruses, many of which have very rapid replication cycles.
2. Avoidance of CTL-mediated killing of virus infected cells
Cytotoxic T lymphocyte (CTL) also called CD8 + mediated killing of virus infected nucleated cells
requires the presentation of viral antigens on the surface of the infected cell. You know that the viral
peptide is presented on the surface of virus infected nucleated cells by MHC class I molecule. But
viruses have developed different strategies to suppress the normal expression of class I MHC
molecule so as to inhibit CTL(CD8+)mediated cell lysis. Some of the strategies used by viruses to
suppress the synthesis of MHC class I molecules are:-
 Suppression of cellular production of class I MHC molecules by shutdown of host protein
synthesis.
 Production of virus encoded proteins that disrupt normal production of class I MHC proteins,
or their transport from the endoplasmic reticulum to the Golgi apparatus or to the cell
surface.
 Production of virus encoded proteins that disrupt the function or viability of class I MHC
molecules.
 Production of virus encoded homologs of class I MHC molecules that can bind with viral
peptides to be presented on the cell surface but not recognized by CD 8+
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All these lead to the virus antigen no to be recognized by CD 8+ (CTLs) so that CTLs mediated
cellular immunity will be unfunctional.
3. Interference with apoptosis
In addition to apoptosis induced by NK-cell or CTL mediated cell lysis, viral infection alone can
initiate apoptosis. Apoptosis is especially deleterious to the relatively slow growing DNA viruses,
including:-
 Poxviruses
 Herpesviruses
 Adenoviruses
Because apoptosis can result in death of cells infected with these viruses before maximal levels of
virus replication have been completed. Thus these DNA viruses in particular have developed a
remarkable variety of strategies to optimize their replication by inhibiting the various pathways that
normally lead to virus infected cell apoptosis; so that the need for these viruses to prevent apoptosis
to promote their own survival used different strategies to prevent cell apoptosis. The common
strategies used by the above viruses are:-
1. Inhibition of the activity of caspases of the cell called protease inhibitor
As caspases of the virus infected cells is very important:-
 To break down the cytoskeleton of infected cell
 For activation of endonuclease of virus infected cells which causes fragmentation of DNA of
the cell in the nucleus.
So that inhibition of caspases of virus infected cell means inhibition of apoptosis of these cells.
2. Production of virus encoded homologs like apoptotic protein but it is anti - apoptotic protein of
virus
3. Production of proteins that sequester pro-apoptotic molecules that accumulates in cells infected
with certain viruses
4. Counter defenses against cytokines
Now you know that cytokines are the central messengers to both innate and adaptive immune
responses of animals to viral infections. However, viruses also have developed effective strategies to
combat the activities of these important mediators of antiviral immunity. The common strategies
that are used by viruses to stand counter defenses against cytokines are:-
 Modifying cellular genes that create viral genes that encode proteins that are homologs of
cellular cytokines.
By modifying cellular genes they create viral genes that encode proteins that are homologs of
cytokines’ receptors. This homologs cytokines created by viral genes are called virokines.
Virokines and homologs cytokines’ receptors inhibit the function of cellular cytokines by:-
• Mimic the biological effect of cellular cytokines
• Making them non – functional
They make them non – functional by:-
o Simply binding and blocking the specific cytokine receptor to neutralize their activities.
o Simply binding and blocking the specific cytokine receptor to neutralize the relevant
cytokines.
5. Evasion of the antiviral state
Viruses also have developed elaborated strategies to disturb the path ways by which virus infected
cells induced IFN – α which is very important to protect virus free adjacent cells. Viruses perform
this by using:-
 Production of virus encoded proteins
 RNA interfere (RNAi)

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These molecules bind to critical enzymes or genes encoding them which are involved in the IFN – α
production path ways of virus infected cells as the result the enzymes become inactive or non –
functional enzyme homologs are produced and the production IFN - α is disturbed.
6. Virus-specific gene silencing pathways
Viruses have also developed counter defenses to cellular anti-viral RNA interference pathways.
Viruses perform this by:-
 Production of virus-encoded proteins or
 Production of small interfering mRNA (si mRNA) molecules that inhibit key steps of the
cellular RNA interfering pathway
 Other viruses themselves produce RNAi molecules to silence key cellular genes involved in
antiviral immunity.

1.17. TREATMENT, CONTROL AND PREVENTION OF VIRAL

INFECTIONS
1.17.1. TREATMENT OF VIRAL INFECTIONS
Therapy of viral infections is based on:-
 1. The use of drugs that kills or inhibits viruses replication called viral chemotheraptic
agents
 2.The use of immuno – modulators
1. Viral chemotherapy
Viral chemotheraptic agents can have the following actions in viruses. Those viral chemotheraptic
agents which kill the virus are called virocidal chemotheraptic agents while viral chemotheraptic
agents which inhibit the replication of viruses are called virus inhibitors. Generally antiviral
chemotherapeutic agents are not in common use in veterinary practice because of their very high
cost but some of the antiviral drugs used in human medicine have already also been utilized in
veterinary medicine. Most antiviral drugs were prepared and are preparing based on the knowledge
of the biochemistry of virus replication. Several steps in the virus replication cycle represent
potential targets for selective antiviral drug attack. Theoretically, all virus-encoded enzymes are
vulnerable, as are all processes enzymatic or non-enzymatic that are more essential to the replication
of the virus than to the survival of the cell. Thus most antiviral drugs were/ are prepared to act in
most vulnerable steps in virus replication. A logical approach to the development of new antiviral
drugs is to isolate or synthesize substances that might be predicted to serve as inhibitors of a known
virus-encoded enzyme such as:-
 Transcriptase
 Replicase
 Protease
So that analogs of the prototype drugs are then synthesized with a view to enhancing activity and/or
selectivity. Some of the common analogs that are synthesized to use as prototype drug is nucleoside
analog which is an inhibitor DNA polymerase. The common antiviral chemotheraptic agent which is
nucleoside analog and inhibitor of DNA polymerase is acycloguanosine (aciclovir). So that virus
activities that are potential drug targets are:-
 Attachment
 Penetration with membrane fusion
 Uncoating
 Primary transcription of viral genome(DNA and RNA replication)
 Processing of RNA transcripts
 Translation of viral RNA into protein
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  Post-translational cleavage of proteins
 Release of enveloped viruses
This can be achieved using drugs that are:-
 Receptor analogs
 Uncoating inhibitors
 Nucleoside analogues
 Transcriptase inhibitors
 Protease inhibitors
 Replicase inhibitors
 Fusion inhibitors
 Neuraminidase inhibitors
Possible targets for antiviral chemotherapy in veterinary medicine
Target Prototype drug
Attachment of virion to cell receptor Receptor analogs
Uncoating Rimantadine
Primary transcription from viral genome Transcriptase inhibitors
Reverse transcription Zidovudine
Processing of RNA transcripts Ribavirina
Translation of viral RNA into protein Interferons
Post-translational cleavage of proteins Protease inhibitors
Replication of viral DNA genome Acycloguanosine
Replication of viral RNA genome Replicase inhibitors
1.1. Some common examples of antiviral drugs and their mechanism of actions
Antiviral drugs are not widely used in veterinary medicine practice because they are expensive.
However, they are used in many developed countries for high value domestic and pet animals. So
that it is important to know and to understand their mechanism of actions as it helps us to improve
our knowledge. By their mechanism of actions some common antiviral drugs which are used in
human medicine and in veterinary medicine practices are classified into:-
 1. Nucleoside analogues that inhibits DNA synthesis
 2. Nucleoside analogues that inhibit reverse transcription
 3. Protease inhibitors
 4. Fusion inhibitors
 5. Neuraminidase inhibitors
1. Nucleoside analogues that inhibits DNA synthesis
The number of antiviral drugs is synthetic compounds which are structurally similar to nucleosides
such as:-
 Guanosine
 2-deoxyguanosine
 2-deoxythymidine
 2-deoxycytidine
They act by interfering with the synthesis of virus nucleic acids because after they are being taken
into a cell a nucleoside analogue, like a nucleoside is phosphorylated and become a nucleotide
analogue; so that during nucleic acid synthesis if one of these nucleotide analogues is incorporated
into a growing strand, the nucleic acid synthesis is terminated. As in the structure of the nucleotide
analogue prevents it from accepting the next nucleotide. Some of antiviral drugs which are
nucleoside analogues that inhibit DNA synthesis are:-
 A. Ribavirin

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  B. Aciclovir
 C. Ganciclovir
A. Ribavirin
Ribavirin is an analogue of guanosine and is used for the treatment of infection with several RNA
viruses. It is effective especially in persistent infections with hepatitis C virus. Combined treatment
with ribavirin and α-interferon has eradicated HCV infection in many patients. Ribavirin has also
been recommended for treatment of young children infected with respiratory syncytial virus.
B. Aciclovir
Aciclovir is also an analogue of guanosine. Aciclovir strongly inhibits virus DNA synthesis but has
very little effect on cell DNA synthesis. It is widely used for the treatment and prevention of
diseases caused by two of the herpesviruses (herpes simplex viruses 1and2 (HSV-1and HSV-2)
C. Ganciclovir
Ganciclovir, like a ciclovir is an analogue of guanosine. Since it has side effect, it is not widely
used.
2. Nucleoside analogues that inhibit reverse transcription
This group of antiviral drugs inhibits reverse transcription but it can also interfere with cell DNA
synthesis. It inhibits reverse transcriptase and is used usually in combination with other therapies to
treat diseases caused by retroviruses like HIV and Hepatitis B Virus (HBV). The drug does not
eliminate the infections but it inhibits viral replication. Antiviral drugs that inhibit reverse
transcription are:-
 Azidothymidine (Zidovudine)
 Lamivudine
 Dideoxycytidine
 Dideoxyinosine
3. Protease inhibitors
Maturation of retrovirus virions involves the cleavage of virion proteins during assembly. This
activity is performed by a virus protease. Inhibition of the activity of protease means cleavage of
virion protein is disturbed because peptide mimics of the cleavage site in the protein have been
developed and these compounds canfit into the active site of the virus protease. The result is that
fewer virions bud from virus infected cells and those virions that do bud are non-infectious. It is
mostly used in human medicine to treat HIV patients. An example of an HIV protease inhibitor is
ritonavir.
4. Fusion inhibitors
You know in some enveloped viruses after certain virions have bound to a cell, the transmembrane
glycoprotein must fuse the envelope with the membrane of the cell if the virion contents are to be
delivered into the cytoplasm of the cell. In order for this to take place glycoprotein of the virions
must undergo a conformational change. The conformational change in glycoprotein of virions is
initiated by interaction between different regions of the molecule. Number of drugs have been
developed that can inhibit fusion between the membranes of enveloped virion and a potential host
cell. They do this by binding to glycoprotein and inhibiting the conformational change. A good
example of antiviral drug that inhibits the fusion of the glycoprotein of the virion and the membrane
of cell is enfuvirtide. Therefore, enfuvirtide prevents the penetration of the virions into a cell after
attachment. Enfuvirtide is used also to treat HIV patients. However, it is not widely used because
enfuvirtide has to be given by subcutaneous injection where as the other anti-HIV drugs can be
given orally.
5. Neuraminidase inhibitors
One of the surface proteins of most orthomyxoviruses for example in influenza A and B viruses is a
neuraminidase. This enzyme plays a vital role in thefinal stages of virion budding from infected
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cells. So that if the enzyme is inhibited virions is not released. It is known that each neuraminidase
spike on the virion surface is made up of four monomers, each consisting of a ‘balloon’ on a‘stick’.
In 1983 an influenza virus neuraminidase was crystallized and its structure was determined. It was
found that the monomer has a deep cleft and that this forms the active site of the enzyme.
Compounds were designed to bind in the cleft and several compounds were found to be active to
inhibit neuraminidase activity. One such compound which is active to inhibit neuraminidase is
oseltamivir. Nowadays, oseltamivir is approved to use as an anti - influenza virus drug in human
medicine.
1.2. Limitations of antiviral drugs
Clinical use of most currently available antiviral drugs is limited by their toxic effects to the host
cells. There are also some general limitations inherent in antiviral chemotherapy. Some of them are:-
 1. The narrow spectrum of their antiviral activity.
 2. Since antiviral drugs target steps in viral replication, the latent infection is the
characteristics of some viral infections and are not amenable to chemotherapy.
Thus eradication of viral latent infections is not visible currently. Some of the common viruses
which have latent infection are:-
• Herpesviruses
• Retroviruses
 3. Antiviral drug treatment should be started before irreversible tissue damage occurs.
Such timely treatment is not possible without early and accurate diagnosis which is difficult in many
viral infections (such as viral infections of respiratory tract).
 4. The emergency of drug resistance to virus strains
It is dramatically demonstrated in AIDS patients from who drug – resistance human
immunodeficiency virus strains have been isolated against virtually all drugs that has been tried so
far.
1.3. Resistance of viruses to antiviral drugs
Soon after the introduction of anti-viral drugs into clinical practice, drug-resistant strains of the
target viruses began to emerge. This should not have been surprising, as antibiotic-resistant bacteria,
insecticide-resistant insects and rodenticide-resistant rats had emerged as a result of natural
selection. The fact that viruses especially RNA viruses can mutate at high frequencies. They evolve
rapidly means that genotypes encoding drug resistance can arise rapidly in RNA viruses than DNA
viruses. A virus strain is considered to be ‘resistant’ to a drug if it is able to replicate in the body in
the presence of a concentration of the drug that inhibits replication of‘sensitive’strains called
Inhibitory Concentration (IC 50 ). Drug resistant virus isolates are found to have one or more
mutations in the genes encoding the proteins that are the drug targets. For example, the vast majority
of a ciclovir-resistant HSV (Herpes Simplex Virus) isolates have a mutated tk gene. While a small
While a small number of isolates have mutations in the DNA pol gene. Mutations in the HIV-1 RT
gene that confer resistance to a nucleoside analogue (e.g. AZT) are in different codons.
Some examples of mutated genes in drug-resistant virus strains of HIV and Herpes Simplex
Virus (HSV)
Drug Virus Mutated gene
Aciclovir HSV-1 tk
AZT HIV-1 RT
Nevirapine HIV-1 RT
Ritonavir HIV-1 PR
Enfuvirtide HIV-1 env(gp41)
NB. tk : thymidine kinase

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 RT: Reverse transcriptase
PR: Protease
Env(gp): Envolped glycoprotein
Some mutations confer cross-resistance to other drugs of the same type. Most mutations to drug
resistance of most viruses are:-
 Amino acid substitutions
 Deletions
 Insertions
Clinical problems arise:-
 When drug-resistant virus strains emerge in patients undergoing treatment.
 When resistant strains are transmitted to other individuals.
So that when such problems arise patients may be treated with alternative drugs. For example;
infections with aciclovir-resistant HSV strains can be treated with:-
 Foscarnet or
 Cidofovir
This is because neither of the above drugs depends on the HSVTK gen.
1.4. Methods to combat antiviral drug resistance of viruses
The best method to combat the problem of antiviral drug resistance is identifying new effective
antiviral therapeutics. Some approaches are under investigation that may lead to future approved
therapies are:-
 1. Combination therapy
 2. Discovery of a new antiviral drugs
 3. Evaluating available drugs for a new indications
1. Combination therapy
The use of multiple drugs with different mechanisms of action is being studied as a method of
improving clinical effectiveness of antiviral drugs. Such combinations may offer advantageous over
mono - drug therapy because:-
 It improves antiviral activity(synergetic effect)
 It prevents or delays the development of antiviral drug resistance.
 It helps to use lower dose and less toxic drugs.
In addition of the above methods used to combat antiviral viral drug resistance, it is also possible to
use a combination of cytokines with one or more antiviral agents.
2. Discovery of new antiviral drugs
New drugs with novel mechanisms of actions are being sought and developed. Some of them have
displayed considerable antiviral activities. For example protease inhibiter like saquinevir, ritonavir
and indinavir are new antiretroviral drugs for HIV – 1 in humans.
3. Evaluating available drugs for a new indications
Nowadays, a cytokine called interleukin – 2 currently approved to treat renal cell carcinoma and
have shown considerable effect to treat HIV – patients.
2. Treatment of viral infections using immuno – modulators
Nowadays it is possible to use many immuno – modulators to treat animals and human beings from
many viral infections. These chemicals which are produced in the body of virus infected cell are
called cytokines. Among cytokines; interferons are immuno - modulator which are effective to treat
many viral infections. You know among interferons (IFN –α, IFN – β and IFN – γ), IFN – α is
produced by virus infected cells and effective to treat many viral infections. Early in the
development of virology it was difficult to produce interferon – α to perform many clinical trials.
However, nowadays it is possible to produce IFN - α using:-
 Cell cultures
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  Recombinant DNA technology
1.17.2. Control and prophylaxis of viral infections
To control and prophylaxis many viral diseases, we must consider the triads of epidemiological
process that include:-
 Host
 Agent
 Environment
There are enormous variations in viral infections not only in agent (virus), in their epidemiology
(host and environment) but also in pathogenesis of many viral infections. So that there is no single
magic – bullet approach to control and prophylaxis many viral infections of animals, as each virus
presents its own set of problems. There are many methods that are developed to control many viral
infections in animals. The most common approaches are:-
 1. Using of different antiviral chemotherapeutic agents and immunomodulators.
 2. Using of immunoprophylaxis
 3. Sanitation and vector control
1. Control of viral infections using antiviral chemotheraptic agents and
immunomodulators
You know that there are number of chemotherapeutic antiviral agents and immunomodulators that
are used to treat some of very serious viral infectious diseases but they are not widely used in
veterinary practice because they are expensive.
2. Immunoprophylaxis
Immunoprophylaxis against many viral infections include:-
 The use of vaccines
 Antibody containing preparates
The two methods of immunoprophylaxis against many viral infections used to provide susceptible
individuals with immunologic protection against specific disease; so that immunization against
many viral infections can be:-
 2. 1. Active immunization
 2.2. Passive immunization
2.1. Active immunization
Active immunization involves administrating antigen to an animal; so that it responds by mounting
an immune response. Reimmunization or exposure to infection in the same animal will result in a
secondary immune response and greatly enhanced immunity. However, it has disadvantage in that
active immunization is with all acquired immune response, protection is not confirmed immediately
but once the acquired active immunity is established the immunity is long lasting and capable of re -
stimulation. Some of the examples of biological agents that are used for active immunization of
animal in many viral infections are viral vaccines.
2. 2. Passive immunization
Passive immunization requires that antibodies be produced in donor animals by active immunization
then injection to another animal. Passive immunization produces temporary immunity by
transferring antibodies that is taken from immunized animals to susceptible animals that these
antibodies (Igs) be given to susceptible animals to confer immediate protection. Serum contain these
antibodies is called antisera. These passively transferred antibodies give immediate protection.
However, since the Igs are gradually catabolized within 2 – 3 weeks; so that the immunized animals
eventually become susceptible to infection again after 2 – 3 weeks. Some of good examples of
biological preparates that are used for artificial passive immunization of animals to many viral
infections are:-
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 Hyper immune serum
 γ - globulins
The level of serum antibodies and the degree of protection conferred by active and passive
immunization

2.3. Types of viral vaccines

Viral vaccines are biological preparates that are given to animals to prevent and control them from
many viral diseases. Ideal viral vaccines that are used for active immunization for animals should:-
 Give prolonged strong immunity
 Be free from adverse reaction
 Be cheap, stable and adaptable to mass vaccination
In addition to the requirements listed above effective vaccines must have the following critical
properties which include:-
 Antigen must be delivered efficiently so that Antigen Processing and Presenting Cells(APCs)
can process and present the antigen to CD4+ cells by MHC class II molecules and activate
CD4+ and able to release appropriately cytokines to activate B lymphocytes (humoral
immunity).
 Antigen must be delivered efficiently to the surface of virus infected nucleated cells by MHC
class I molecules to CD8+ (CTLs)(cellular immunity).
 The antigen must have a capacity to stimulate both T and B cells so that they generate large
number of effector and memory cells in both humoral and cellular components.
Unfortunately, two of the prerequisites of an ideal vaccine are:-
 Having high antigenicity
 Absence of adverse side effect
Generally, there are three types of vaccines that are produced from the whole viral particle and
purified proteins and nucleic acid of viruses.
 I. Live attenuated viral vaccine
 II. Killed( inactivated) viral vaccine
 III. Viral vaccines produced by modern technology
I. Live attenuated viral vaccine
In some cases, viruses can be attenuated so that they lose their ability to cause significant disease
(pathogenicity) but retain their capacity to replicate within an inoculated host. For live attenuated
viral vaccines to be successful, the vaccine virus must replicate in the recipient, there by eliciting a
lasting immune response while causing little or no disease. In effect, a live-attenuated virus vaccine
mimics a subclinical infection.

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Methods used to prepare live attenuated viral vaccines
The individual virus strain incorporated in a live-attenuated virus vaccine can be derived from many
methods. Among them, some of the common methods used in veterinary practice to prepare live
attenuated viral vaccines are:-
 1. Vaccines produced from naturally occurring attenuated viruses
 2. Vaccines produced by attenuation of viruses by serial passage in cultured cells
 3. Vaccines produced by attenuation of viruses using serial passage in heterologous hosts
 4. Vaccines produced by attenuation of viruses by selection of cold-adapted mutants and
reassortants
1. Vaccines produced from naturally occurring attenuated viruses
The original vaccine (Vacca meaning cow) was introduced by Jenner in 1798 for the control of
human smallpox. He utilized cowpox virus, a natural pathogen of the cow to vaccinate human
beings against small pox. Cow pox virus produced only a mild infection and lesions in humans.
Since it is antigenically related to smallpox virus, it conferred protection against small pox. The
same principle has been applied to other diseases like:-
 For the protection of chickens against Marek’s disease using a vaccine derived from a related
herpesvirus of turkeys
 For protection of piglets against porcine rotavirus infection using a vaccine derived from a
bovine rotavirus.
2. Vaccines produced by attenuation of viruses by serial passage in cultured cells
Most of live-attenuated virus vaccines in common use today were derived empirically by serial
passage of virulent field” virus (syn. “wild-type” virus) using cultured cells. The cells may be of
homologous or more commonly heterologous host origin. Typically, adaptation of virus to more
vigorous growth in cultured cells is accompanied by progressive loss of virulence for the natural
host. Loss of virulence may be demonstrated initially in a convenient laboratory model such as a
mouse before being confirmed by clinical trials in the species of interest.
3. Vaccines produced by attenuation of viruses by serial passage in heterologous hosts
Serial passage in a heterologous host was a historically important means of empirically attenuating
viruses for use as vaccines. Some of the examples of live attenuated viral vaccines those were
prepared by serial passage in heterologous hosts are:-
 Rinderpest and classical swine fever (hog cholera) viruses which were adapted to grow in
rabbits and after serial passage, became sufficiently attenuated to be used as vaccines.
 Bluetongue virus vaccine which was prepared by serial passage in embryonated hens’ eggs
in similar fashion.
Although some such passaged viruses acquired novel but have very undesirable properties. For
example, live-attenuated bluetongue virus vaccines propagated in embryonated eggs can cross the
placenta of ruminants that are vaccinated during pregnancy; as the result there is fetal infection
which results in mass abortion or in developmental defects in fetuses called anomaly.
4. Vaccines produced by attenuation of viruses by selection of cold-adapted mutants and
reassortants
There were two attempts to prepare live attenuated virus vaccines by culturing them below and
above their optimal replication temperature (37 0C) to prepare temperature sensitive virus mutants.
Cultivation of viruses above the normal body temperature of mammals makes unable to replicate
satisfactorily; so that they generally display reduced virulence suggested that they might make
satisfactory live-attenuated vaccines but this method is not widely used as some viruses with
temperature sensitive mutations have displayed undesired characteristics like reversion toward
virulence during replication in vaccinated animals. That is why temperature adapted mutants is not
used to prepare live attenuated virus vaccines. So attention accordingly moved to cold-adapted
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mutants, derived by adaptation of virus to grow at suboptimal temperatures. The vaccines were safer
and do not revert toward virulence. Live attenuated viral vaccines that are prepared by attenuation of
viruses by selection of cold – adapted mutants are safer vaccines for intranasal administration
because viruses would replicate well at the lower temperature of nasal cavity (about 330C) in most
mammalian species but not in lower respiratory tract. Some of the examples of live attenuated virus
vaccines produced from cold adapted virus mutants are:-
 Cold-adapted influenza vaccines( for human being) that contain mutations in most viral
genes which do not revert to virulence
 Vaccines against equine influenza
II. Killed (inactivated) viral vaccines
They are vaccines produced from inactivated whole virions and are also called non-replicating
virus vaccines. Inactivated (syn. killed) virus vaccines are usually made from virulent strain viruses.
When prepared properly, such vaccines are remarkably safe but they need to contain relatively large
amounts of antigen to elicit an antibody response commensurate with that induced by a much
smaller dose of live-attenuated virus vaccine. Normally, the primary vaccination course comprises
two or three injections and further (“booster”) doses may be required at regular intervals thereafter
to maintain immunity. Killed vaccines usually must be formulated with chemical adjuvants to
enhance the immune response but these also can result in more adverse reactions to vaccination like
anaphylaxis.
Methods used to prepare inactivated (killed) viral vaccines
Viruses are killed for use in vaccines must remain as antigenically similar to the living organisms as
possible. Therefore crude methods of killing that cause extensive changes in antigen structure as a
result of protein denaturation are usually unsatisfactory; so that chemical or physical agents are used
to destroy infectivity while maintaining immunogenicity of viruses. The most common physical
methods which are used to prepare killed (inactivated) viral vaccines are:-
 Heat
 γ – radiation
Inactivation by heat is not commonly used method as it causes denaturation of the antigens; so that
the most common method which is used to prepare inactivated virus vaccines is gamma radiation.
However, if chemicals are used, they must not alter the antigens responsible for stimulating
protective immunity. The most common chemical that are used to inactivate viruses are:-
 Formaldehyde
 Alkylating agent
Formaldehyde cross - links proteins and nucleic acids and confers structural rigidity. Alkylating
agents that cross - link nucleic acid chains are also suitable for preparation of killed inactivated viral
vaccines because they leave the surface protein of viruses unchanged as the result they do not
interfere with antigenicity. Some examples of alkylating agents that are used for inactivation of
viruses are:-
 Ethylene oxide
 Ethyleneimine
 Acetylethyleneimine
 β – propiolactone
One of the advantages of β - propiolactone which is used in the manufacture of rabies vaccines and
ethylenimine which is used in the manufacture of foot-and-mouth disease vaccines, is that they are
completely hydrolyzed within hours to non- toxic products. However, during inactivation the virions
may aggregate in the center and can be shielded from inactivation; so that it is important that
aggregates be broken up before inactivation. In the past, failure to do this occasionally resulted in

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vaccine-associated disease outbreaks in many countries of the world. For example, several post
vaccination foot-and-mouth disease outbreaks in many countries have been traced to this problem.
Merits and demerits of live attenuated and inactivated vaccines
Attenuated vaccines have some advantageous and some dis advantageous. The advantages of
attenuated live viral vaccines are:-
 It forms prolonged immunity as the result these vaccines often require only a single dose of
immunization and they eliminate the need of repeated boostering.
 The immunity is formed with short lag periods after 5– 7 days.
 The ability of many attenuated vaccines to replicate within host cells makes them
particularly suitable for inducing a cell mediated immune response.
Major disadvantages of live attenuated viral vaccines are:-
 The possibility of their reversion to a virulent form
 They need cold chain for storage and transportation
 The presence of other viruses as contaminates
That is why post vaccination complications may render a potential of live attenuated viral vaccine to
be unacceptable.
Comparison of attenuated (live) and inactivated (killed) vaccines
Characteristics Attenuated vaccine Inactivated vaccine
Production Selection for avirulent organisms: Virulent pathogen is
virulent pathogen is grown under inactivated by heat, chemicals
adverse culture conditions or or irradiations(γ – ray)
prolonged passage of a virulent
human pathogen through different
hosts.
Booster requirement Generally requires only a single Requires multiple boosters
dose
Relative stability Less stable and require cold chain Stable on storage
Necessity of Adjuvants No need of Adjuvants Mostly require adjuvants
Types of immunity Produces both humoral and CMI Produce only humoral
induced immunity
Tendency to spread the Yes No
agent
Route of administration Can be given through natural routes Cannot be given through
natural routes
Cost Cheap Relatively Expensive
Reversion tendency May revert to virulent form Cannot revert to virulent form
Hypersensitivity Less chance to cause May induce hypersensitivity
hypersensitivity reaction
Application in Not safe Safe
immunodefficient patients
III. Viral vaccines produced by modern technology
Although both killed and live attenuated viral vaccines have been successful in controlling many
infectious diseases, there is always a need to make them more effective, cheaper and safe; so that
some of the risks associated with the attenuated or killed whole organism vaccines can be avoided
by:-
 1.Vaccines that consists of specific purified macromolecules derived from the virus
 2. Vaccines produced by using modern molecular techniques

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1. Vaccines produced from purified native viral proteins
Lipid solvents such as sodium deoxycholate (bile salt) are used in the case of enveloped viruses, to
solubilize the virion and release the components like glycoprotein spikes of the viral envelope.
Then differential centrifugation is used to semi-purify these glycoproteins. Glycoproteins are then
formulated for use as vaccine to control and prevent many viral infections. These types of vaccines
which are formulated from purified native viral proteins are called split vaccines. Some of the
examples of split viral vaccines are:-
 Vaccine of herpesviruses
 Vaccine of influenza viruses
 Vaccine of coronaviruses
2. Viral vaccine production using modern molecular techniques
The use of modern molecular techniques can produce new and improved vaccines. The modern
technologies that are used for vaccine production can be classified into 5 categories.
 A. Antigens generated by gene cloning
 B. Genetically attenuated organisms
 C. Live recombinant organisms
 D. DNA vaccines (polynucleotide vaccines)
 E. Synthetic peptides
A. Antigens generated by gene cloning
Gene cloning can be used to produce large quantities of purified antigen. In this process, DNA
coding for an antigen of interest is cloned into a bacterium, yeast or other cells and the recombinant
antigen is expressed. The first successful use of gene cloning to prepare an antigen in this way
involved in FMDV. Gene cloning in FMDV was done in this way:-
 The protective antigen (VP1) of FMDV is well recognized.
 Then the genes that code for this protein have been mapped.
 The RNA genome of FMDV was isolated and transcribed into DNA by the enzyme reverse
transcriptase.
 The DNA was then carefully cut by restriction endonuclease so that it contained only the
gene for VP1.
 This DNA was then inserted into a plasmid of bacteria.
 The plasmid inserted into E. coli and allowed the bacteria to grow.
 Finally the recombinant bacteria synthesized large quantities of VP1 that was harvested,
purified and incorporated into a vaccine.
Nowadays, rather than cloning the gene of interest in a microorganism is possible to clone antigen
genes in plants. This method of cloning is successfully achieved for viruses such as transmissible
gastroenteritis and for Newcastle disease. The plants employed include tobacco, potato and corn.
In some cases these plants contain very high concentration of antigen and so can simply be fed to
animals.
Production of a recombinant viral protein for use in vaccine

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B. Genetically attenuated organisms
Attenuation by prolonged tissues culture can be considered as primitive form of genetic engineering.
The desired result is the development of a strain of organism that cannot cause disease. Attenuation
by prolonged tissue culture may be difficult and reversion to virulence is an ever present risk.
Molecular genetics techniques however make it possible to modify the genes of an organism so that
it becomes avirulent and irreversibly attenuated. This has been done with a herpesvirus vaccine for
pigs in which thymidine kinase gene was removed. Since thymidine kinase is required for the virus
to grow in certain types of cells (e.g. neurons); so that removal this gene rendered the virus
incapable of causing disease.

Production of an attenuated virus by removal of a gene required for virulence

C. Live recombinant organisms

It is also called recombinant vector vaccines. It is known that, it is possible to introduce genes that
encode major antigens of virulent pathogens into attenuated viruses. The attenuated organisms serve
as a vector, replicating within the host and expressing the gene product of the pathogen. Numbers of
attenuated viruses have been used for vector vaccines. Among them the most common are:-
 Vaccinia virus
 The canary pox virus
 Attenuated poliovirus
 Adenovirus
Vaccinia virus, the attenuated vaccine used to eradicate smallpox has been widely used as a vector
vaccine. This large complex virus, with a genome of about 200 genes can be engineered to carry
several dozen foreign genes without impairing its capacity to infect host cells and replicate there.
Genetically engineered vaccinia express high levels of the inserted gene products which can then
serve as a potent immunogen in an inoculated host. Like the small pox vaccine, genetically
engineered vaccinia vector vaccines can be administered simply by scratching the skin and this
causing a localized infection in host cells. If the foreign gene product expressed by the vaccinia is a
viral envelope protein, it is inserted into the membrane of the infected host cell. All this result in the
development of cell – mediated immunity (CMI) as well as antibody – mediated immunity.
Production of a vaccinia recombinant vaccine

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D. DNA vaccines (polynucleotide vaccines)
DNA vaccine, unlike viral vectors cannot replicate in mammalian cells. Experience has shown that
plasmid incorporation is enhanced by the use of some adjuvants. These may include the following
adjuvants.
 Lipid complexes
 Microcapsules
 Nonionic copolymers
 Aluminum phosphate
Aluminum phosphate seems especially effective in improving vaccine efficiency; as the result
transinfected host cells express the vaccine protein in association with MHC class I molecules as do
other endogenous antigens. This can lead to the development of not only neutralizing antibodies but
also cytotoxic T cells (CMI). DNA vaccines offer advantages over many of existing vaccines. This
is because:-
 The encoded protein is expressed in the host in its natural form so that there is no
denaturation or modification of the antigen; that is why the immune response will be directed
to the antigen exactly as it is expressed by the pathogen.
 DNA vaccine induces both humoral and cellular immunity as live attenuated vaccine
 DNA vaccines cause prolonged expression of the antigen results to have long immunological
memory.
 The practical aspects of DNA vaccines are also promising because no need for refrigeration
for handling and storage.
However, it has its own drawn back because DNA vaccines are expensive and have complexity of
delivery.
Structure of typical DNA plasmid used for vaccination purpose

Mechanism by which DNA vaccines can work in which the DNA that enters a cell is functional
and can code for endogenous antigen

E. Synthetic peptides
Nowadays complete viral genomes are available. It is possible to identify potential vaccine antigens
by computer analysis. The procedure involved a complete sequencing of the antigens of interest

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followed by identification of their important epitopes. These epitopes may be predicated by the use
of computer models of the protein or by the use of monoclonal antibodies to identify critical
protective components. Once the epitope is identified, it can be chemically synthesized. Some of the
synthetic vaccines those were developed include:-
 Virus hepatitis B
 FMDV
 Canine parvovirus
 Influenza A virus
3. Sanitation and vector control
Sanitation of the farm and the environment are also very important to control many viral diseases in
animals. Sanitation includes:-
 Regular mechanical removing of wastes
 Regular washing of animal houses
 Disinfection of the animal houses
 Deratization of the farm
Disinfection and deratization are methods that are used to control infectious agents and rodents
respectively. Disinfection is the killing of pathogenic viruses on inanimate objects using appropriate
disinfectants. It is used to prevent and control many infectious viral diseases of animals.
Disinfection can be:-
 Prophylaxis disinfection
 Control disinfection
Prophylaxis disinfection is regular disinfection of the farm without disease occurrence with
appropriate disinfectant. Control disinfection is disinfection of the farm during disease occurrence
with appropriate disinfectant. The effectiveness of disinfectants is depending on types,
concentration, the presence and absence of organic matter and time of exposure; so that it is
important to use effective disinfectants with appropriate concentration and time of exposure after
removing of organic matter. Controlling of rodents is also very important to prevent and to control
many viral infectious diseases of animals because they can be carriers for many viral diseases of
animals. Rodents can be controlled by three methods.
 Physical method by using mouse traps
 Chemical method by using different chemicals like Zink phosphide.
 Biological method by using rodents predators like cat.
Controlling of insect vectors is crucial to control many viral diseases of animas because there are
many viral diseases which are vector born (arboviruses). Vector control can be achieved using the
following methods.
 By draining of swamps as it is a place where insects multiply
 By using insecticides and insect repellent; and accaricides to control insects and ticks
respectively.
LABORATORY PRACTICAL SECTION
1. Preparation of cell cultures
In virology, the following materials must be available in laboratory to prepare primary cell cultures.
 Appropriate organs to obtain cells
 Proteolytic enzymes
 Different chemicals
 Bi(triple) – distilled water
 Sterile glass wares
 Different sterile laboratory equipments

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  Sterile cell culture media
 Incubator etc.
A number of different techniques have been developed to obtain cell suspensions from various
organs by disaggregation of cells from different organs. These disaggregation methods can be
broadly classified into three general categories.
 1. Mechanical methods
 2. Chemical methods
 3. Enzymatic digestion of tissues
Most of the techniques that are used in virology to prepare the primary cell cultures involve a
combination methods from two or even all three of the above three general categories.
1. Mechanical methods
Mechanical method includes activities like cutting, mincing, shearing and sieving of tissues. This
method may cause extensive damage to the cells and are normally used only in the initial stages of
the disaggregation procedures to increase the surface area available to enzymatic digestion.
2. Chemical methods
This method usually involves the removal of divalent cations with the presence of a chelating agent
such as EthyleneDiamineTetraAcetic Acid (EDTA).
3. Enzymatic digestion of tissues
It is performed with enzymes such as trypsin, collagenase or pronase. The enzymatic treatment
breaks the connective tissues that are found between cells.
Laboratory precautions
The water which is used in virology laboratory must be bi (triple) - distilled water. All metallic
instruments like scissors and forceps are sterilized in hot air oven at 160 0C for two hours and must
be sterilized by flaming after each step. All glasswares that are used in preparation of cell and tissue
cultures are sterilized as follows.
 Autoclave all glassware before washing at 121 0C for 20 minutes at 15 Pascal pressure.
 Clean the outside surfaces of glasswares with Vim and inside with only Teepol using
glassware brush
 Wash with tap water and leave in 5% Teepol solution over night
 Next morning wash in tap water at least 20 times and leave in 10% HCl over night
 Next day wash in tap water and rinse in 2 changes of bi- distilled water at least 10 times in
each, leave in third bucket of bi – distilled water overnight.
 Take out next day, dry it in hot air oven, pack and sterilize in hot air oven at 160 oC for 2
hours.
Packing and sterilization
Pipettes: Wrap the pipettes with brown paper and sterilize in hot air oven at 160 0C for two hours.
Petridishes: wrap the Petridishes with brown paper and tie with twine and sterilize by hot air oven
at 160 0C for two hours.
Culture and media storage bottles, measuring cylinders and beakers: Cover the mouth with
aluminum foil, over with brown paper and tie with twine at the neck and sterilize in hot air oven at
160 0C for two hours.
Plastic measuring cylinders and beakers are autoclaved at 121 0C, 15 Pascal for 30 minutes.
Plastic centrifuge tubes, Eppendorf tubes, screw cap vials and tubes: Arrange them neatly in
plastic or glass beakers and cover the mouth with aluminum foil and over that wrap brown paper
tied with twine at the neck. Sterilize by autoclaving at 1210C, 15 Pascal for 30 minutes.
Filter apparatus: Wrap filter apparatus first with aluminum foil and then with brown paper, tie it
with twine tightly. Sterilize by autoclaving at 121 0C at 15 Pascal for 30 minutes.
2. Cell culture media
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Cells have complex nutritional requirements that must be met to permit their propagation “in vitro”.
Different types of cells have different growth requirements and a number of chemically defined
formulations have developed that support the growth of a variety of established cell lines. Generally
media that are used to culture cells “in vitro” are classified into two.
 Growth media
 Maintenance media
Growth media are those media that permit cellular proliferation and contain 5 – 20% of
complement free sterile serum.
Maintenance media are those media that permit cells to metabolize at satisfactory level but do not
support cellular proliferation and contain 1 – 5% complement free sterile serum.
So that by frequent replacement of maintenance medium cell sheets may be kept for several weeks.
There are so many media that help to culture cells “in vitro”. The most commonly cell culture media
that are used in virology are:-
 Medium 199
 Eagle’s - Minimum Essential Medium(E - MEM)
 Alpha Minimum Essential Medium(Alpha MEM)
 DMEM
 McCoys 5A etc.
But the most commonly used medium is E - MEM. Although some serum – free media are available
and some cell lines have been adapted to grow in such medium, most cell lines require the addition
of 5 – 20% serum as a supplement to promote cellular multiplication. The best serum is Fetal
Bovine Serum (FBS) but it is possible to use:-
 Calf serum(CS)
 Horse serum
Serum which must be added into cell culture media is used: -
 To be a source of protein
 Provides unknown growth factors
 Concerned in adhesion of cells to the surface of the glass
Serum must be inactivated in water bath at 560C for 30 minutes to make free from complement
and sterilized by filtration prior to its addition to the cell culture medium. A PH indicator such as
phenol red may be included in the original formulation to permit direct observation of the PH of the
medium. The PH of the medium is adjusted by the drop wise addition of a sterile sodium
bicarbonate solution that is used both as a nutrient and a buffering agent. Antibiotics such as
penicillin and streptomycin are added to resist accidental bacterial contamination of the culture.
Once prepared, the cell culture medium has to be properly stored. For long – term storage, it should
be frozen but on a short – term basis the medium should be kept at +4 0C and warmed upto 37 0C
only for the time necessary to perform a given experiment. Among cell culture media available
nowadays, Eagle’s - Minimum Essential Medium (E – MEM) is the most common medium used
in virology to grow many different types of cells because it is relatively simple and easy to prepare.
E- MEM is commercially available in a powder form that is dissolved in glass with triple distilled
water; then the final solution has to be filter sterilized and dispensed into sterile containers. E –
MEM may be obtained in a liquid sterilized form that does not require any filtration.
Some selected suitable medium and cells that can be cultured
Cells or cell lines Medium used Types of serum added
Chicken embryo Eagle’s MEM CS
fibroblasts
Continuous cell lines Eagle’s MEM, DMEM CS
Endothelium DMEM, M199, MEM CS

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 HeLa cells Eagle’s MEM CS
BHK 21 MEM,GMEM,DMEM CS
Melanocytes M199 FBS
Melanoma MEM,DMEM/F12 FBS
Neurons DMEM FBS
Cells or cell lines Medium used Types of serum added
Skeletal muscle DMEM,F12 FBS,HoS
Hematopoietic cells RPMI1640, Fischer’s, FBS
αMEM
3. Media preparation for cell culture (dehydrated tissue culture media)
 Take the necessary amount of dehydrated medium
 Dissolve into necessary amount of triple – distilled water and stir until dissolved
 Add NaHCO 3 and adjust the PH between 7.1 – 7.4 using 1N HCl or 1NNaoH as the
dehydrated medium has phenol indicator
 Add inactivated sterile Fetal Bovine Serum(FBS) or Calf Serum(CS)
 Sterilize the media by filtering through sterile filter of 0.22 micron or less porosity
 Finally, incorporate into the medium with antibiotic mixture usually penicillin and
streptomycin
NB. Strict observation of asepsis is needed during work.

3.1. Media sterility test

All solutions used for growth and maintenance of cells or tissues cultures must be carefully
presented for sterility. As a routine, two tubes of fluid thioglycolate, two tubes of Trypticase Soya
Agar slants(TSA) and two Petridishes of Sabround Dextrose Agar(SDA) from them one of the
tubes and SDA are inoculated with one ml of the medium for each and incubated at 37 0C(bacteria)
and 25 0C(fungi) for 7 days; and leave the rest of the two tubes and one of SDA uninoculated for
negative control and use the cell culture medium if there is no growth of bacteria and fungi in
inoculated test tubes and Petridishes.
3.2. Preparation of monolayer cell cultures
The most common monolayer primary cell cultures that are prepared in virology laboratory are:-
 Chicken embryo fibroblast monolayer cell culture
 Foetal lamb kidney monolayers
 Calf kidney cells culture
Let us see the procedure that must be under taken during the preparation of the first two cell culture
media.
Materials and reagents required
A) Materials required
1. Dehydrated cell culture medium (Eagle’s Minimum Essential Medium)
2. Triple distilled water
Dissolve the necessary amount of cell culture into triple distilled water and sterilize by filtration.
3. Organs removed aseptically from freshly killed animals or embryonated egg.
4. Sterile dissection instruments (forceps, scissors, blades etc)
5. Sterile tubes, centrifuge tubes, pipettes, beakers, flasks, Petridishes and trypsinization flasks etc.
6. Sterile Teflon coated magnetic bar and stirrer outfit
7. Sterile gauze
8. Sterile cell culture tubes, bottles and Petridishes etc.
B) Reagents required
1. Hanks balanced salt solution
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2. Stock antibiotic solution
 Prepare sterile solution containing 100000 IU of penicillin and 100000µg streptomycin/ ml
using sterile powders in sterile distilled water/ Hanks BSS and store at – 20 0C.
 Add 0.1 ml concentrate to each 100 ml of tissue culture fluid.
3. Sodium bicarbonate
 Using analytical grade NaHCO 3 and prepare 2.8% and 7.5% solution sterilze by filtration
 Store at 4 0C
4. Hydrochloric acid solution
 Prepare 0.1N HCl solution from concentrated HCl
 Sterilize by autoclaving at 1210C for 15 minutes
 NaHCO 3 and HCl are used for adjusting PH of tissues culture fluids.
5. Trypsin (1:250) – (Difco)
 Trypsin is used for dispersing and harvesting tissue cells from organs.
 Prepare 0.25% solution in Hanks BSS phenol red indicator.
 Adjust the PH to 7.4 to 7.6 and sterilze by filtration.
 Store at – 20 0C
6. Serum
Use fetal bovine serum or calf serum and inactivate the complement in water bath at 56 0C for 30
minutes and sterilize by filtration.
Add 10% and 1 – 2% sterile fetal bovine serum (calf serum) if you want to prepare growth and
maintenance Eagle’s minimum essential medium respectively.
3.2.1. Embryo fibroblast cell culture preparation
 Select 10 - 12 days embryonated eggs (only well developed active embryos with good blood
supply need to be selected; and it can be selected by candling called ovo - scopy.
 Apply tincture of iodine on the entire surface of the egg and keep the egg upright by making
the air sack up.
 With the help of a sterile forceps, break the shell at the air sack
 Remove the shell membrane and Chorionic Allantoic Membrane (CAM).
 Take out the embryo using a curved forceps by catching on the neck and place it in a
petridish containing BSS. About 5 embryos can be placed in a petridish.
 Wash the embryos several times with BSS so that it becomes clean.
 Using pair of forceps and scissors remove the head and limbs then remove the internal
organs by tearing the abdominal wall with scissors and discard.
 Transfer the embryo into another petridish containing BSS and cut into small pieces and
transfer into a beaker containing cold BSS.
 Wash several times to remove the blood and other debris by shaking the beaker well.
 Allow the tissue fragments to settle and decant the supernatant.
 Wash with pre warmed trypsin and transfer the tissue pieces into a trypsinization flask
containing teflon coated magnetic bar and pour sufficient amount of trypsin(for chicken
fibroblast the percentage of trypsin should be reduced from 0.25% to 0.1%).
 Then trypsinize by stirring the suspension over a magnetic stirrer at room temperature for
five minutes.
 Allow the tissue pieces to settle and then discard the supernatant.
 Add fresh trypsin and trypsinate for 20 – 30 minutes.
 Filter the resulting cell suspension through a double layered cheese cloth into sterile flask
 Then transfer into sterile centrifuge tubes and centrifuge at 4 0C for 10 minutes at 800 RPM.
 Resuspend the cell pack in growth medium and give two more washing

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  Finally resuspend the packed cells in growth medium count the number of cells and adjust
the cell concentration with growth medium so as to contain 105 cell/ ml
 Distribute the cells in culture tubes/ bottle and petridishes etc.
 Incubate at 37 0C in the incubator and confluent monolayer will be formed in 48 – 72 hrs of
incubation
Method of cell counting
Cells in suspension are counting by using hemacytometer with microscope. The hematocytometer
consists of two chambers that are covered by cover slip. Each chamber is divided into a grid with
nine large squares and each large square is 1mm × 1mm (area of 1 mm2) and the depth under cover
slip is 0.1 mm; so that the volume of each square is 1 mm × 1 mm × 0.1 mm = 0.1 mm3 or 10 – 4
cm3. Since a cm 3 is equivalent to a ml; so that the number of cells per ml(cells/ ml of suspension)
can be calculated by multiplying the average count per large square by 10 4 and the reciprocal of
dilution factor. A total cell count does not distinguish between living and dead cells. However, the
number of viable cells can be determined by staining the cell population with certain dyes such as
trypan blue or erythrosine B. Living cells are able to exclude trypan blue and appear clear;
whereas dead cells cannot and therefore appear blue. It is important to count the cells rapidly upon
exposure to this dye under certain conditions viable cells will begin to take up trypan blue over
period of time.
4. Preservation and thawing of cell culture
4. 1. Preservation of cell culture
When cells are maintained at reduced temperatures, their metabolic rate is decreased and they
require less frequent passaging. At extremely cold temperature, destructive processes as well as
normal metabolic processes are inhibited. Cells can be stored frozen at – 70 0C in the presence of
glycerol but over a period of months to years there is a gradual loss of viability. This method of
preservation of cells at low temperature is called cryopreservation. So that storage of mammalian
cells in liquid nitrogen (- 196 0C) is presently the accepted procedure that results in indefinitely
prolonged preservation of viability. In the absence of a cryoprotective agent such as glycerol or
Dimethylsulfoxide (DMSO), the freezing process is lethal to most mammalian cells. The damage
that occurs to cells is caused by the mechanical injury due to ice crystal formation. Procedures that
have evolved to offset or minimize these damaging conditions are as follows:-
 Glycerol or DMSO is added to the cells to lower the freezing point and to protect the
membrane damage by ice crystal.
 A slowing cooling rate allows water to move out of the cells before freezing.
 Storage of cells below – 130 0C retards ice crystal formation.
Cryopreservation of cell culture protocol
 Observe the cells under inverted microscope; and cells should be healthy and not over
confluent.
Note: when setting up a laboratory cell stock, the culture medium is changed 24 hours prior to
freezing to enhance the cells’ metabolic activity.
 Decant the medium and add 5 ml of E – MEM which has 5% FBS with 10% glycerol
(alternatively, the cells could be trypsinized and then resuspended in freezing medium).
 Scrape the cell monolayer with wedge of soft rubber connected with pipette.
 Pipet the cells up and down several times to break up the clumps and to get an even
suspension.
 Withdraw 0.6 ml of the cell suspension; and dispense 0. 5 ml into Tryptcase Soya Agar,
thioglycolate and SDA for sterility test and the rest 0.1 into test tube to check the viability of
cells. Add 0.4 trypan blue into test tube with 0. 1 ml of cells (1: 5 dilution); and load the

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 hemacytometer and count the blue (non – viable) and the clear (viable) cells. Calculate the
percent viability and determine the number of viable cells per ml.
 Adjust the original cell suspension to obtain 4 – 5 ml of 2 ×106 viable cells/ ml in E-MEM
with 5%FBS and 10 % glycerol.
 Dispense 2 ml of the cell suspension into each of sterile ampules
 Flame seal the ampules and properly label them
 Place the ampules in a beaker containing ethanol and transfer to a refrigerator for 1 hour. The
ethanol is used as a bath to distribute the heat exchange from different parts of the ampules.
Ethanol has a lower freezing point than water and liquid at – 70 0C.
 Transfer the beaker containing the ampules to a – 20 0C freezer for an hour.
 Transfer the beaker containing the ampules from – 20 0C to the – 70 0C freezer for 1 hour.
 After an hour remove the beaker and place the ampules in appropriately labeled freezer box
at – 70 0C and the ampules should have a label with the following information:-
• Cell type
• Passage number
• Date of passage
• The first letter of the name of the person who performed passage
NB. In virology laboratory cell stocks should be frozen with glycerol or Dimethylsulfoxide (DMSO)
in liquid nitrogen at – 1960C for long term cell stock maintenance.
4.2. Thawing of frozen cell
Cells are thawed rapidly when they are taken out of the frozen state in order to limit the cell damage
and the correct thawing of frozen cell culture protocol is as follows.
 Remove the ampule to be thawed from the – 700C or – 196 0C freezers and immediately
place in a 37 0C water bath.
 Open the ampule aseptically and transfer the cell suspension into sterile centrifuge tube.
 Centrifuge at 600 RPM for 10 minutes and discard the supernatant and resuspend the
sediment of cells in 10 ml of E – MEM with 5% FBS.
 Transfer 0.1 ml of the cell suspension to small test tube and add 0.1 ml of trypan blue
solution(1:2) and determine the total and viable cell count and calculate the % of viability.
Compare this figure to the percent viability of the cell culture before freezing.
 Dispense 5 ml of the cell suspension into 2 – 25 cm3 cell culture flask with E – MEM which
has 5 – 10% FBS (CS) and incubate.
 Change the medium after 24 hours of incubation and observe the cells growth after 2 – 3
days under inverted microscope.
5. Inoculation of viruses
5.1. Procedure of embryonated egg inoculation
The embryonated chicken egg is a living organism and highly susceptible to bacterial and mycotic
infections. Hence inoculations and all surgical interventions must be done with sterile precautions.
To avoid contamination, all instruments (scissors, forceps and file) must be sterilized by dipping in
95% of alcohol and igniting in surgical try. You must keep the ignited instruments horizontal in
instrumental try with 95% alcohol and igniting them and beware of flaming alcohol, the flame is
badly visible. Before puncturing or drilling the egg shell, swab the site with iodine or alcohol but
never flame the iodine or alcohol on the egg shell and delineated the air sac with a lead pencil and
mark several dots.
 One dot on the center of the air sac
 4 – 5 dots on the circumference of the air sac

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With an egg punch/ dental drill, puncture the cell at the chosen sites; so the shell should be cracked
and the hole should be just large enough to let the inoculating needle to enter. The suspension is
injected into the appropriate sites of embryonated eggs of 5 – 14 days old. Finally sealing of the
hole/ holes on the egg shell with melted sterile paraffin or collodion or cellotape; as sealing of the
hole is necessary:-
 To prevent dehydration
 To maintain the sterility of inoculated egg
Lastly, incubate the inoculated embryonated egg in the incubator with appropriate temperature and
humidity in different positions. It should be noted that inoculation usually contains pathogenic
viruses and should be handled with care. If in a case, it spilled, wipe it away with alcohol swab and
remove the air bubble in the syringe by injecting into a pad of cotton moistened with alcohol.
Drops that remain on the needle tip after inoculation must be removed by touching to the inside of
the container of inoculum or onto an alcohol swab.
5.2. Common routes of inoculation of embryonated eggs
The choice of the routes of inoculation into embryonated eggs depends on:-
 The age of the embryo
 The viruses type that will be propagated
In virology there are many routes of inoculation of embryonated eggs.
 1.Yolk sac route of inoculation
 2.Allantoic cavity route of inoculation
 3.Amniotic cavity route of inoculation
 4. ChorioAllantoic Membrane (CAM) route of inoculation
 5. Intracerebral route of inoculation
 6. Intravenous route of inoculation
1. Yolk sac route of inoculation
Embryos of 5 -7 days of incubation are used. The technique of inoculation is done as follows.
 Take 5 -7 days old of incubated embryonated egg.
 Candle the egg and mark the edge of the air sac.
 Sterilize the air sac with iodine or alcohol.
 Drill a simple hole in the shell over lying at the center of air space.
 Puncture the under lying shell membrane with sterile needle or with other sharp instrument.
 Inoculate 0.1ml to 0.25 ml of the required suspension of specimen directly into yolk sac
using 11/2” 29 gauge needles.
 This can be best done by holding the egg upright and inserting the needle vertically for
almost its entire length.
 Finally seal the hole and label the egg before placing it over the tray to be returned to the
incubator.
This route of inoculation is used to cultivate:-
 Chlamydia
 Rickettsia
 Rabies virus
 Herpes simplex virus
 Avian encephalomyelitis virus
 Marek’s disease virus
2. Allantoic cavity route of inoculation
This route of inoculation is employed with embryonated egg of 9 – 10 days of incubation. The
technique of inoculation is done as follows.
 Candle the eggs, delineated the air sac with lid pencil.
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  Mark a spot 0.5 cm above the base of the air sac
 Locate the embryo and mark 0.5 cm above it.
 Disinfect the shell with iodine or alcohol
 Puncture the shell at the spot located 0.5 cm above the base of air sac.
 With 24 gauge 5/ 8 “ needle attached to a tuberculin syringe draw up the inoculum
 Then insert the full length of the needle slightly slanting against the position of the embryo
through the hole in the shell and inject 0.2 ml of the suspension.
 Finally seal the puncture site with collodion, paraffin or cellophane tape and label the before
placing it over the tray to be turned to the incubator.
Some of the viruses that can grow in the cells lining this cavity are:-
 Fowl plague(AIV)
 Newcastle disease virus
 Infectious bronchitis virus
 Influenza virus
 Equine encephalomyelitis virus
Allantoic cavity route of inoculation has an advantage over other routes of inoculation because:-
 Its simplicity of inoculation
 It is used to cultivate large quantity of viruses which is used in vaccine production and in
preparation of antigens for serological tests.

3. Amniotic cavity route of inoculation

This method of inoculation is commonly employed with 10 – 12 days of embryonated egg. The
technique of inoculation is done as follows.
 Candle the egg and mark the edge of the air sac
 Disinfect the shell of the air sac
 Cut the shell round the entire surface of the air sac by keeping 1 – 2 mm above the edge of
the base by taking care not to damage the underlying shell membrane.
 Carefully detach and discard the shell cap by keeping the egg upright.
 Paint the entire shell membrane exposed at the base of the air sac with cotton swab soaked
with sterile paraffin (makes the membrane transparent and enables the embryo and its
associated structures situated underneath clearly seen).
 Carefully tear the shell membrane and adjacent CAM with pair of spatulate forceps and
grasp the amniotic sac enveloping the embryo.
 With extreme care, because of its fragility, lift the amnion until it protrudes slightly above
the surface of the allantoic fluid.
 Then inoculate 0.05 ml to 0.1 ml of the inoculum directly into the amniotic cavity using a 26
gauge, 2.5 cm needle
 Finally seal the egg with cover slip and wax and incubate.
This route of inoculation is commonly used for primary isolation of influenza virus.
4. Chorioallantoic membrane (CAM) route of inoculation
This route of inoculation used 10 – 12 days of embryonated egg and preparation and technique of
inoculation of the embryonated egg is done as follows.
 Candle the eggs and mark the edge of the air sac.
 Mark also a point on the side of the egg which is not adjacent to any major underlying blood
vessel.
 Sterilze the shell surface in the region of both marked areas.
 Drill a simple hole in the shell overlying the center of the air sac and puncture the underlying
shell membrane with a sterile needle or other sharp instrument without damaging the CAM.
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  Cut a triangular window with approximately 5mm sides in the marked side of the egg by
taking care not to damage the underlying shell membrane.
 Remove this triangular portion of the shell carefully using sterile leasing needle without
injuring the CAM which adheres to it.
 Then using a rubber bulb suck the air from the air sac through the small hole that is created.
 This will force the CAM to drop from the shell membrane at the window forming an
artificial air cell.
 Deposit 0.2 ml of the inoculum onto the CAM using a 1ml tuberculin syringe fitted with 22
gauges, 2 – 5 cm needle.
 After inoculation, the window is closed with melted paraffin or cellotape.
 The hole over the air space also must be closed as above.
 Finally rotate the inoculated eggs gently to spread the inoculum on the CAM.
 Keep the eggs in the incubator in such a way that the inoculation site is facing upwards.
CAM route of inoculation is particular effective for primary isolation and cultivation of viruses
like:-
 Vaccinia virus
 Variola virus
 Fowlpox virus
 Larygotrachitis virus of chicken
 Pseudorabies virus

These viruses produce easily visible foci on CAM which is called pocks and the lesion called pock
lesion. The CAM is suitable site to study:-
 The development of pathologic alteration and inclusion bodies
 Titration of viruses and antisera by the “pock” counting techniques
 Chemotheraptic activity of some antiviral drugs.
5.3. Post inoculation care
Post inoculation care includes:-
 Sealing
 Incubation temperature and humidity
 Position of the embryos during incubation
The eggs must be sealed immediately after inoculation to prevent dehydration and to maintain
sterility. Common sealing agents that are used in virology laboratory are:-
 Melted paraffin
 Collodion
 Cellotape
 Sterile cover slips with wax
The temperature of incubation usually ranges between 33 0C – 37 0C and the same relative humidity
as pre – inoculation period is used. Position of the embryos during incubation depends on the
location of the largest opening on the shell. The largest opening on the shell must remain upper
most, so that there is least risk of eggs contents being lost; so that eggs inoculated through allantoic
route, yolk sac route and amniotic route must be incubated vertically; while eggs inoculated through
CAM route must be incubated in horizontal position. In all cases the inoculated eggs must not be
turned at any time in the incubator.

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Common routes of inoculation of embryonated eggs and viruses that can be cultivated or
isolated
Routes of inoculation Age of the embryo Viruses can be cultivated or isolated
Yolk sac 5 – 7 days Rabies virus, Herpes simplex virus, avian
encephalomyelitis virus and Marek’s disease
virus
Allantoic cavity 9 – 10 days Avian influenza virus, Newcastle disease virus,
duck hepatitis virus, Avian adenovirus and
hepatitis virus
Amniotic cavity 10 - 12 days Influenza virus type C and mumps virus
CAM 10 – 12 days Poxvirus, some herpes virus, IBD(Infectious
Bursal Disease) and duck plague virus
Intracerebral 13 – 15 days Rabies virus
Intravenous 15 days Encephalomyelitis virus and blue tongue virus
6. Methods to harvest viruses from inoculated embryonated eggs
After incubation of the inoculated embryonated eggs with appropriate temperature, humidity and
position, the eggs must be opened sterile to collect the virus for its further identification.
1. Procedure to harvest virus from allantoic fluid
 Flame the forceps with alcohol and make an opening on the center of the air sac.
 Re – disinfect the forceps then peel back the shell membrane to expose the underlying CAM.
 Examine the CAM for the presence of edematous swelling, necrotic foci, cloudy infiltrations
or hemorrhages.
 Then tear open the CAM without damaging the yolk sac.
 Smell the content of the egg.
Do the eggs smell stringly?
If so, it is most likely contaminated by bacteria.
 Examine and note transparency and colour of the allantoic fluid.
Cloudiness may be from urate precipitate, bacterial contamination or a broken yolk sac.
 Set sterile test tubes and mount rubber bulb to Pasteur pipettes.
 Disinfect the forceps and gently depress the embryo yolk sac and membrane with forceps.
 Finally aspirate the allantoic fluid with Pasteur pipette (upto 10 ml of the fluid can be
collected from each embryo).
 Label the test tubes and store at – 20 0Cfor further study.
2. Procedure to harvest viruses from CAM
 After the collection of allantoic fluid as described above, invert the egg and deposit the
embryo, yolk sac and membranes into Petridish.
 If the CAM adheres to the shell membrane in the shell, remove the albumin with forceps
CAM from the shell membrane.
 Clean the CAM of extraneous materials by holding it with one pair of forceps and stripping
it with another pair of forceps.
 Then spread the membrane in a petridish with normal saline and look for various types of
abnormalities.
 Finally, transfer the membrane into a container, label and store at – 20 0C for further study.
Inoculation of the virus on CAM helps to quantify the viruses by titration of viruses and counting of
pock lesions on CAM.
3. Harvesting of the virus from amniotic fluid

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  After collection of allantoic fluid as described earlier, open the amniotic membrane
containing embryo with sterile fine pointed forceps.
 Then collect the amniotic fluid after puncturing the amniotic membrane.
 The usual yield is between 0.2 ml to 1 ml.
 Place the fluid in sterile tube and label and store at – 20 0C for further study.
4. Harvesting of viruses from yolk sac
Since most of the viruses which are cultivated in the yolk sac are highly pathogenic viruses, the egg
should be opened in a sterile cabinet; and follow the following technique.
 Disinfect the shell over the air sac with 70% alcohol.
 With sterile scissors or curved forceps cut the shell along the marked air space and expose
underlying membrane.
 Carefully tear the shell membrane and CAM and gently pour the content of egg into a
Petridish.
 Remove the yolk sac with sterile forceps by grasping the tissues with forceps and lifting
away as membrane usually rupture while releasing the yolk.
 Empty the yolk and wash the membrane in sterile saline to get rid of the yolk completely.
 Take the yolk sample in sterile test tubes, label and store at – 20 0C for further identification.
What you must understand is that in primary isolation of viruses using embryonated eggs
particularly in diagnostic virology, negative samples should be blindly passage for a minimum of
three times before it is conformed as negative. Depending upon the virus that you want to propagate,
it is possible to use other embryonated eggs in special cases like:-
 Duck embryonated eggs to propagate duck plague virus and EDS – 76 virus
 Turkey embryonated egg to propagate haemorrhagic enteritis virus of turkeys
 Pheasant’s eggs to propagate marble spleen disease virus.

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