LECTURE NOTES

ON

VIROLOGY

AND

TISSUE CULTURE
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 COURSE OUTLINES
INTRODUCTION

HISTORICAL ASPECT OF VIROLOGY

GENERAL CHARACTERISTICS OF VIRUSES

STRUCTURE AND MORPHOLOGY OF VIRUSES

CULTIVATION OF VIRUSES

VIRAL REPLICATION

PATHOGENESIS OF VIRAL INFECTION

VIRAL DETECTION AND CYTOPATHIC EFFECT

VIRAL ASSAY AND PURIFICATION

CLASSIFICATION OF VIRUSES

SOME MEDICALLY IMPORTANT VIRUSES

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 INTRODUCTION

Definition

Virology: is the science which deals with study of viruses as causative agents of very important
diseases that occurs in human, animals, plants and other living organisms (insects, bacteria etc).

Viruses:

are the smallest and simplest form of life on earth, which can replicate only in living
susceptible cells.

are sub-cellular, non-living, infectious entities which only become part of a living
system when they have infected host cells, a form of borrowed life

are sub-microscopic infectious agent that can infect all types of life forms, from animals
and plants to microorganisms, including bacteria (bacteriophages) and archaea
(archaeoviruses).

Are small obligate intracellular parasites which contain either a DNA or RNA genome
surrounded by a protective virus-encoded protein coat.

Unlike living organisms, viruses lack cellular structure and cannot carry out metabolic
processes on their own. They rely entirely on the hoist cell’s machinery for replication and
propagation. Viruses consist of: (1) A nucleic acid genome either DNA or RNA. (2) A protein
coat (capsid) that enclosed the genome. (3) In some cases, a lipid membrane (envelope).

HISTORICAL ASPECT OF VIROLOGY

In 1884, Louis Pasteur developed the first rabies (a deadly viral disease) vaccine, although he
never directly saw or identified viruses, his work on rabies suggested that infectious agents
smaller than bacteria existed. He hypothesized that some infectious agents could pass through
filters that trap bacteria, a concept that was later key in virus discovery.

In 1892, Dmitri Ivanovsky, a Russian botanist discovered that a substance smaller than
bacteria could infect plants. He passed the sap of infected tobacco plants through a filter that
removed bacteria, but the filtered fluid still caused disease in healthy plants. This led to the
identification of a new type of infectious agent. Nevertheless, his work was foundational to the
discovery of viruses.

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In 1898, Martinus Beijerinck furthered the work of Ivanovsky by recognizing that the agent
causing tobacco mosaic disease was not a bacterium but a new type of infectious agent called
a “virus”

GENERAL CHARACTERISTICS OF VIRUSES

 Virus particles are very small in size; they are between 20-500 nm (nanometer) in
diameter.

 They are much smaller than bacteria and cannot be seen by ordinary microscope, but
only by Electron microscope (EM).

 Viruses are not made of cells. They are considered acellular particles, meaning they
lack the typical structure of living cells, such as organelles, cytoplasm, or a cell
membrane.

 Viruses are obligate intra-cellular parasites; hence, there is no metabolic activity outside
the cell.

 Multiply inside the cells by replicating their genomes (either DNA or RNA, but not
both).

 Viruses are not affected by antibiotics.

 Most viruses are sensitive to interferon.

 Some viruses cause latent infection e.g. Herpesviruses, HIV, HBV.

 Viruses cannot grow on artificial media (chemically defined media), but only in living
cells (such as specific host, laboratory animals, chicken embryonated eggs & tissue
culture).

 Viruses are often specific to the organisms they infect. For example, bacteriophages
infect bacteria, while plant viruses infect plants, and animal viruses (such as those that
infect humans) target specific tissues or cells in animals.

Atypical Virus-like Agents

Atypical virus-like agents are infectious agents that share some similarities with viruses but
differ in some fundamental ways, particularly in their structure, replication methods or genetic
material.

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1. Viroids: are small, single molecule of circular RNA without a protein coat and the RNA
does not code for any protein. They cause several plant diseases by disrupting gene expression
but are not implicated in human diseases. E.g. Potato spindle tuber viroid (PSTVd).

2. Prions: are infectious, misfolded proteins without nucleic acids. They cause fatal
neurodegenerative diseases in animals and humans. Example: Creutzfeldt-Jakob disease
(CJD), mad cow disease (BSE).

3. Defective Interfering Particles (DIPs): Defective virus-like particles lacking essential

genetic material. Depend on a helper virus for replication and interfere with its replication.
Example: DIPs in influenza virus or vesicular stomatitis virus (VSV).

4. Satellite Viruses and Satellite RNAs: Require a helper virus for replication but have their
own genetic material. Example: Hepatitis D virus (HDV), which depends on hepatitis B virus
(HBV).

5. Pseudovirions: are viral particles that contain host cell genetic material (DNA) instead of
viral genome (DNA) within the capsid. They can infect cells but do no replicate.

6. Plasmids: are circular DNA molecules, typically in bacteria, that replicate independently.
They can transfer genes, including antibiotic resistance. Example: F-plasmid in bacterial
conjugation.

7. Plasmaviruses: Extrachromosomal, virus-like elements found in bacteria which can affect

cellular processes. They behave like viruses but lack a structured capsid and may take the form
of plasmid-like structures.

STRUCTURE AND MORPHOLOGY OF VIRUSES

Viruses exhibit a wide range of structural diversity, and their morphology is directly tied to
their infective mechanisms, replication strategies, and how they interact with the host's immune
system. Despite their diversity, viruses share some fundamental structural features: genetic
material, a protective protein coat (capsid), and, in some cases, an outer lipid envelope. This
breakdown covers all major aspects of viral morphology and structure

Virion: This refers to a complete infective virus particle.

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Genome: is a core of DNA or RNA which may be single-stranded (ss) or double stranded (ds),
circular or linear.

Capsid: is a protein coat that encloses the viral genome, protecting it from the environment
and playing a key role in viral attachment to host cells. It is made up of repeating protein
subunits called capsomeres, which assemble in a highly ordered and symmetrical fashion.
Capsid has the following functions:

• provides structural symmetry,

• participates in attachment to susceptible host,

• facilitates transfer of viral nucleic acid in to host cell,

• protects the viral genome from damage by enzymes (e.g., nucleases in blood stream).

Nucleocapsid: The protein-nucleic acid complex.

Envelope: is a lipoprotein surrounding the capsid in some viruses which may contain material
of host cell as well as viral origin. Virus-encoded glycoproteins are exposed on the surface of
the envelope. Most human helical viruses are enveloped while icosahedral are either enveloped
or non-enveloped. Viral envelope has the following significance:

• Enveloped viruses are more unstable i.e. are more sensitive to heat, drying, detergents
and alcohols

• Enveloped viruses are often transmitted by direct contact as by blood, sexual contact
like HIV, HBV, HCV, rabies virus, measles, mumps, rubella viruses etc

Non-Enveloped (Naked) Viruses: consist only of the nucleocapsid (capsid + nucleic acid)
and do not have a surrounding lipid envelope. These viruses are often more resistant to
environmental stressors such as heat, acids, and detergents. Examples include Poliovirus,
Adenovirus, Norovirus

Viral Symmetry and Arrangement

The symmetry of viral capsids is important for viral assembly and function. The repetitive
arrangement of capsomeres gives viruses their characteristic shapes.

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Icosahedral Symmetry: provides the most efficient way to enclose genetic material in a
spherical shape. The capsid is made of 20 triangular faces, forming 12 vertices. Examples
include: Adenovirus, Herpesvirus, Poliovirus.

Helical Symmetry (Rod-Like Structure): Found in viruses with helical capsids, the protein
subunits are wound around the viral nucleic acid in a spiral or helical manner. Examples
include: Tobacco Mosaic Virus (TMV), Influenza virus and Rabie virus.

Complex Symmetry: Specialized Features: Bacteriophages and poxviruses have complex

morphologies with features like tails and fibers for attaching to and penetrating host cells.
Example: Bacteriophage and Poxviruses

Viral mutation and attachment

Mutation: is the most important mechanism of genetic modification in viruses. It occurs

spontaneously and readily in viral genomes causing frequent changes in the nucleic acids. This
results in production of new viral strains showing properties different from parental or wild-
type virus. Mutations occur during every viral infection and new variants of strains are
identified by their nucleotide sequence, antigenic differences, or by differences in their
structural or functional properties. Mutations in the essential genes of virus cause inactivation
of the virus. This is known as lethal mutation. However, mutations in the other genes alter
antigenicity and pathogenicity of the virus and induce drug resistance in viruses.

Attachment: Attachment or adsorption is the first event in the infection of the cell by a virus.
The viruses have attachment sites that attach to the complementary receptor sites on the host
cell surface. These receptor proteins in the virus are distributed on surface of the virus. These

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receptor proteins vary from one virus to another. For example, in influenza virus these receptor
proteins are the spikes present on the surface of the envelope, whereas in adenovirus these
receptor proteins are small fibers present at the corner of the icosahedron.

The attachment sites of the virus bind specifically to the complementary receptors on the
surface of the host. These receptor sites on the cell vary depending on the nature of the virus:
■ Rabies virus binds specifically to the acetylcholine receptors found on neural cells.

■ HIV-1 binds specifically to the CD4, a glycoprotein on the surface of mature T lymphocytes.
■ Influenza virus binds specifically to sialic acid residue of glycoprotein receptor sites on the
surface of respiratory epithelium.

CULTIVATION OF VIRUSES

Cultivation of viruses refers to the methods used to grow viruses in a laboratory setting,
enabling scientists to study them, produce vaccines, or conduct research on viral behavior.
Since viruses require a host to replicate, they cannot be cultured in the same way as bacteria or
fungi. Instead, they need living cells to grow and propagate. The most common methods of
virus cultivation are:

1. Cultivation in Animals (animal models)

Some viruses are grown by infecting live animals (such as mice, rabbits, guinea pigs or
monkeys). This method is often used for viruses that require specific tissue or environmental
conditions that are difficult to replicate in other systems. Viruses can also be inoculated in lab
animals by intraperitoneal, subcutaneous, intracerebral and intranasal routes. After inoculation,
virus multiplies in host and develops disease. The animals are observed for symptoms of
disease and death. Then the virus is isolated and purified from the tissue of these animals.
Examples of viruses introduced into animals via different inoculation routes:

(a). Intraperitoneal (IP) Route e.g. Zika virus, Dengue virus, West Nile virus

(b). Subcutaneous (SC) Route e.g. Yellow fever virus, Vaccinia virus

(c). Intracerebral (IC) Route e.g. Rabies virus, Herpes simplex virus (HSV-1), Poliovirus

(d). Intranasal (IN) Route e.g. Influenza virus, SARS-CoV-2

Advantages of animal models:

Diagnosis, pathogenesis and clinical symptoms are determined.

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 Production of antibodies can be identified.

Primary isolation of certain viruses.

Mice provide a reliable model for studying viral replication.

Used for the study of immune responses, epidemiology and oncogenesis.

Disadvantages of animal models

Expensive and difficulties in maintenance and handling of animals.

Difficulty in choosing of animals for particular virus.

Some viruses cannot be grown in animals or can be grown but do not cause disease.

Mice do not provide models for vaccine development.

Issues related to animal welfare systems/ethical issues.

2. Cultivation in Embryonated Eggs

Dr. Ernest William Goodpasture in 1931 first used the embryonated hen’s egg for the
cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the
type of egg which used. Viruses are inoculated into chick embryo of 7-12 days old. The egg
used for cultivation must be sterile and the shell should be intact and healthy. For inoculation,
eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and
penetrated with a small sterile drill. After inoculation, the opening is sealed with gelatin or
paraffin and incubated at 36 °C for 2-3 days. After incubation, the egg is broken and virus is
isolated from tissue of egg. Viral growth and multiplication in the egg embryo is indicated by
the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions
on the egg membranes. Viruses can be cultivated in various parts of egg like chorioallantoic
membrane, allantoic cavity, amniotic sac and yolk sac. This is a standard method used in the
production of some vaccines (e.g., influenza vaccines).

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Inoculation in embryonated eggs are:

(a) Chorioallantoic Membrane (CAM):

Inoculation is mainly in 10-13 day

Inoculation is mainly for growing poxvirus.

After inoculation and incubation, visible lesions called pocks are observed, which is a grey
white area in transparent CAM.

Herpes simplex virus is also grown.

Single virus gives single pocks

This method is suitable for plaque studies.

(b) Allantoic cavity:

Inoculation is mainly in 9-11 day

Inoculation is mainly done for production of vaccine of influenza virus, yellow fever, rabies.

Most of avian viruses can be isolated using this method.

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(c) Amniotic sac:

Inoculation is mainly in 10-12 day

Inoculation is mainly done for primary isolation of influenza virus and the mumps virus.

Growth and replication of virus in egg embryo can be detected by haemagglutination assay.

(d) Yolk sac inoculation:

Inoculation is mainly in 7 day

It is also a simplest method for growth and multiplication of virus.

It is inoculated for cultivation of some viruses and some bacteria (Chlamydia, Rickettsiae)

Immune interference mechanism can be detected in most of avian viruses.

Advantages of Inoculation into embryonated egg

Widely used method for the isolation of virus and growth.

Ideal substrate for the viral growth and replication.

Isolation and cultivation of many avian and few mammalian viruses.

Cost effective and maintenance is much easier.

Less labor is needed.

The embryonated eggs are readily available.

Sterile and wide range of tissues and fluids

They are free from contaminating bacteria and many latent viruses.

Specific and non-specific factors of defense are not involved in embryonated eggs.

Widely used method to grow virus for some vaccine production.

Disadvantages of Inoculation into embryonated egg

The site of inoculation varies with different virus (i.e. each virus has different sites for their
growth and replication).

eggs from vaccinated flock may carry antibodies in yolk which can interfere in growth of
specific microorganisms.

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 some microbes like Salmonella, Mycoplasma etc. can pass from infected hen to eggs.

3. Cultivation in Cell Cultures

Cell cultures involve growing cells in a controlled, sterile environment such as petri dishes or
flasks. There are different types of cell cultures:

Primary Cell Cultures: Derived directly from animal tissues, these have a limited lifespan
(short-lived). They are used for experiments that require cells with characteristics close to
normal physiological function such as drug testing, toxicity studies and cancer research.

Diploid Cell Lines: These consist of cells that can divide a number of times and are derived
from human or animal sources. They are commonly used in vaccine production because they
retain diploid characteristics and are safer and more stable for industrial scale production.

Continuous Cell Lines: These are immortalized cells that can divide indefinitely (e.g., HeLa
cells, MCF-7 cells, Vero cells). They often undergo genetic changes that allow them bypass
the normal cellular checkpoints that would lead to cell death. It is used for cancer research,
drug development etc.

Advantages:

Cell culture is the most commonly used method because it provides a controlled environment.

allows for observation of the cytopathic effects (CPE) of the virus on cells.

suitable for many different types of viruses.

Disadvantages:

Not all viruses grow well in cell cultures

contamination or maintenance of the cells can be difficult.

4. Bacterial Cultures for Bacteriophages

Bacteriophages (viruses that infect bacteria) can be grown in bacterial cultures. The bacteria
are cultured in a liquid medium or on solid agar plates. The bacteriophage is introduced to the
culture, where it infects and lyses the bacteria, creating clear zones called plaques.

Advantages: Bacteriophage cultivation is simple and fast, with clear results.

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Disadvantages: This method is specific to bacteriophages and cannot be used for viruses that
infect animal or plant cells.

5. Organ Culture

In some cases, whole organ tissues (e.g., respiratory or intestinal tissues) can be used to grow
viruses. This method maintains the architecture of the organ, providing a more realistic
environment for the virus. It's less commonly used but useful for studying tissue-specific viral
infections.

VIRAL REPLICATION

1. Attachment (adsorption): Surface proteins of the virus interact with specific receptors on
the target cell surface. These may be glycoproteins, phospholipids or glycolipids with limited
distribution or molecules that are more widely distributed on tissues throughout the body. The
presence of a virus-specific receptor is necessary but not sufficient for viruses to infect cells
and complete the replicative cycle.

2. Penetration (entry): The viruses (e.g., HIV, influenza virus) penetrate cells through fusion
of the viral envelope with the host cell membrane. Some viruses (non-enveloped viruses)
penetrate cells by translocation of the virion across the host cell membrane or receptor mediated
endocytosis of the virion in clathrin coated pits with accumulation of viruses in cytoplasmic
vesicles.

3. Uncoating (disassembly): The viral capsid is removed and degraded by viral enzymes or
host enzymes to release the viral genomic nucleic acid. Inside the host cell, the viral lipid
envelope or capsid is shed and the viral nucleic acids are released. This process makes the
nucleic acid available for transcription to permit multiplication of the virus. At this stage, the
virus ceases to be infective and will only regain infectivity after new virions have been formed.

4. Transcription and Translation (replication / biosynthesis): How a virus undergoes

replication relies on the type of genetic material the virus possesses. Based on their genetic
material, viruses will hijack the corresponding cellular machinery for said genetic material.
Viruses that contain double-stranded DNA (dsDNA) share the same kind of genetic material
as all organisms, and can therefore use the replication enzymes in the host cell nucleus to
replicate the viral genome. Many RNA viruses typically replicate in the cytosol, and can

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directly access the host cell's ribosomes to manufacture viral proteins once the RNA is in a
replicative form.

Viruses may undergo two types of life cycles: the lytic cycle and the lysogenic cycle. In the
lytic cycle, the virus introduces its genome into a host cell and initiates replication by hijacking
the host's cellular machinery to make new copies of the virus. In the lysogenic life cycle, the
viral genome is incorporated into the host genome. The host genome will undergo its normal
life cycle, replicating and dividing the viral genome along with its own. The viral genome can
be triggered to begin viral production via chemical and environmental stimulants. Once a
lysogenic virus enters the lytic life cycle, it will continue in the viral production pathways and
proceed with transcription /mRNA production. (e.g. Cold sores, herpes simplex virus (HSV)-
1, lysogenic bacteriophages, etc.)

5. Assembly (maturation): The process of virion assembly involves bringing together newly
formed viral nucleic acid and the structural proteins to form the nucleocapsid of the virus.

6. Release: Virions are released from the cell either by lysis or budding. In lysis, the infected
cell dies and the virions are released. In budding, the virion takes some of the host cell’s
membrane with it as it leaves (this normally does not kill the infected cell).

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 PATHOGENESIS OF VIRAL INFECTION

Viruses cause infection of the host first by breaking the natural protective mechanisms of the
body, then evading the immune system of the host, and finally by killing off the host cells and
triggering immune and inflammatory responses. The outcome of viral infection depends upon
response of the host to infection by a virus and on the nature of the host–virus interaction.

Stages of Viral Infections

(A) Entry into the body:

Entry of the viruses into the body is the first step of viral infections. The skin is the best barrier
to infections. In addition to the skin, mucus, ciliated epithelium, gastric acid, bile, tears, etc.

1-Respiratory tract: Many viral infections are caused by entry of virus through the respiratory
tract. The viruses enter the respiratory tract by droplets containing the viruses expelled from
the nose and mouth of infected individuals during the act of coughing, sneezing, or talking.
Some viruses, after entry into body, are confined to the respiratory tract where they multiply
and produce local diseases. These are known as respiratory viruses. Examples of these viruses
are influenza virus. Other viruses after entry, initially multiply at the site of respiratory tract
followed by hematogenous or lymphatic spread to other sites of the body. At these sites, the
viruses replicate in large numbers and cause systemic manifestations of the disease. The
examples of such viruses include measles, mumps, rubella.

2- Skin: Many viruses enter the skin through abrasions or breaks in the skin. Viruses enter the
skin through minor lesions and produce cutaneous lesions at the site of entry for example
cowpox virus. Other viruses, such as papilloma virus, enter the skin through minor injuries on
the surface of skin, arboviruses by bite of insects.

3- Conjunctiva: Some viruses may enter through the conjunctiva and may cause the disease,
e.g. adenovirus.

4- Alimentary tract: The viruses also cause infection by entering through the alimentary tract,
which is another important route of infection by viruses. The viruses, such as rotaviruses,
enteroviruses, reoviruses, hepatitis viruses, and other gastrointestinal viruses cause infections
of the gastrointestinal tract and produce disease. Natural barriers against viral infections include
(a) the acidity of the stomach, (b) the alkalinity of the small intestine, and (c) secretory enzymes
found in the saliva and pancreatic secretions. Intestinal mucus and secretory IgA antibodies are
important and offer partial protection to the intestinal tract. Enveloped viruses usually fail to

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establish infection in the gastrointestinal tract, because these are destroyed by bile secreted in
the gastrointestinal tract.

5- Genital tract: Viruses are also transmitted through sexual contact and enter the body
through the genital tract.

■ HIV, hepatitis B virus, and hepatitis C virus are sexually transmitted and do not produce any
local lesions in the genital tract but produce systemic manifestations.

■ Papilloma viruses and herpes simplex viruses (HSVs) are also transmitted sexually and
produce lesions locally in the genital tract and perineum (area between the anus and
scrotum/vulva).

6-Congenital infections: Few viruses may cause congenital infection in foetus. Rubella and
cytomegaloviruses are the viruses that are more commonly associated with congenital
infections. Depending upon the age of the foetus, these viruses may cause malformations or
even foetal death and abortion.

(B) Initiation of Infection at Primary Site (Infection of the Target Tissue)

The viruses, on entry into the human host, may remain at the primary site of infection, replicate,
and cause infection of the target tissue. The specificity of the virus-attachment proteins and
tissue-specific expression of receptors during replication are two important properties of
viruses to cause infection of target tissues.

(C) Replication of Virus and Spread to Secondary Site

The viruses are spread in the body mainly by the blood stream and the lymphatic system.
Transport of virus in the blood is known as viraemia. After multiplication in the lymph nodes,
the virus enters the blood stream, resulting in primary viraemia. In the blood stream, the virus
may exist either free in the plasma or it may be ingested by the lymphocytes or macrophages.
Viruses enter the brain or central nervous system (a) through the blood stream, (b) through the
infected cerebrospinal fluid or meninges, and/or (c) through the infection of the sensory
neurons.

(D) Manifestations of the Viral Diseases:

The clinical manifestations of viral diseases depend on the complex interaction of virus and
host factors. The outcome of the infection, that is, the disease manifestation depends on the:

■ Age, general health, and immune status of the person

■ Dose of the infective virus

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■ Genetics of the host and the virus

After the host is infected by the virus, the immune status of the host plays an important role
and determines the outcome of viral infection whether it will be an asymptomatic infection, a
benign disease, or a life-threatening disease.

VIRAL DETECTION / VIRAL ASSAY AND CYTOPATHIC EFFECT

These are essential tools in diagnosing viral infections and studying the behaviour of viruses
in host cells as well as design for vaccines.

Viral Detection

Viral detection involves identifying the presence of viruses in clinical samples or research
settings. Various techniques are used, depending on the type of virus and the speed/accuracy
required. Common Viral Detection Methods:

1. Microscopy

• Electron Microscopy (EM): Direct visualization of viruses in clinical samples (e.g.,

blood, tissue) through EM allows identification based on size and shape. However, it
requires specialized equipment and expertise.

• Light Microscopy: Used to observe cytopathic effects (CPE) caused by viral infections
in cultured cells.

2. Immunological Methods

• Enzyme-Linked Immunosorbent Assay (ELISA): Detects and quantifies viral proteins

in infected samples, using antibodies to detect viral antigens. The result can indicate
viral presence and approximate viral load, especially in clinical or diagnostic settings.

• Immunofluorescence assay (IFA): this involves labeling antibodies with fluorescent

dyes to detect viral antigens in infected cells or tissues. It is commonly used for

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 detecting respiratory and herpes viruses in cell cultures. This method is both qualitative
and quantitative.

• Western Blot: Detects specific viral proteins and confirm the presence of antibodies to
a virus. The viral proteins are separated by electrophoresis, transferred to a membrane
and probed with antibodies to reveal specific viral antigens. It is often used to confirm
viral infections such as HIV.

3. Nucleic Acid Detection (Molecular Methods)

• Polymerase Chain Reaction (PCR): Amplifies viral DNA or RNA from clinical
samples, making it one of the most sensitive and specific detection methods. Reverse
transcription PCR (RT-PCR) is used for RNA viruses like SARS-CoV-2.

• Real-Time PCR (qPCR): Provides quantitative data on viral load by measuring the
amplification of genetic material in real-time. It gives result in viral genome copies per
milliliter.

4. Serological Tests

• Neutralization Tests: Measure the ability of antibodies in a patient’s serum to neutralize

the virus and prevent infection of cultured cells. It detects neutralizing antibodies that
block viral infection in cell cultures, indicating exposure or immunity to virus. It
provides a qualitative result showing whether neutralizing antibodies are present.

• Hemagglutination Assay: it detects viruses that agglutinate (clump) RBCs such as

influenza. But in Hemagglutination Inhibition test, the patient’s serum is tested for
antibodies against a virus. If the antibodies are present, they will prevent
hemagglutination by binding to the viral particles and blocking their ability to
agglutinate RBCs. This inhibition indirectly confirms the presence of antibodies against
the virus, hence, indicating past infection or vaccination.

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Viral Assay (Quantification of the Virus)

A viral assay aims to quantify the virus or measure its activity, answering the question: how
much of the virus is present or how infectious is it? It assesses viral concentration, infectivity
or biological properties. The result is quantitative which often provide the concentration of
infectious viral particles or virus titres in a sample

• Plaque Assay: is used to quantify the amount of lytic virus present by counting the
number of plaques (clear zones of cell lysis) formed on a cell monolayer. The result is
expressed as plaque-forming units (PFU).

Quantification of bacterial virus by plaque assay using agar overlay technique

• TCID50 (Tissue Culture Infective Dose): is used to determine the dose at which 50%
of cells show infection or cytopathic effects. This is often used to measure viral
infectivity.

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 • Hemagglutination Assay: it used to quantify viruses (like influenza) that can cause
red blood cell to clump together (agglutinate). The extent of agglutination is
proportional to the virus concentration (i.e. it determines the highest dilution of virus
that causes red blood cell to clump together).

Hemagglutination Assay

Cytopathic Effect (CPE)

Cytopathic effect refers to the visible structural changes or damage in host cells caused by viral
infection. CPE is an important indicator of viral activity and can vary significantly between
different viruses. It is typically observed under a microscope when viruses are grown in cell
cultures.

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Types of CPE

1. Cell lysis: The virus causes cells to burst open, releasing viral particles. This is common in
viruses that replicate rapidly, such as bacteriophages or certain animal viruses.

2. Syncytium formation: Some viruses (e.g., herpesviruses, paramyxoviruses) cause infected

cells to fuse, forming large multinucleated cells known as syncytia.

3. Inclusion bodies: Accumulation of viral particles or viral products in infected cells leads to
the formation of visible structures called inclusion bodies. These can be in the nucleus (e.g.,
herpesvirus) or cytoplasm (e.g., rabies virus).

4. Cell rounding: Infected cells lose their shape and round up, often detaching from the culture
plate. This can be caused by disruption of the cytoskeleton. E.g. Adenoviruses

5. Cell shrinkage: Some viruses cause cells to shrink and become dense (pyknosis), eventually
leading to death.

6. Vacuolation: The formation of vacuoles (fluid-filled compartments) within infected cells,

observed in infections with viruses like Simian Virus40 (SV40).

7. Cellular apoptosis: refers to programmed cell death, triggered by viral infection which leads
to the destruction of infected cells without causing a strong inflammatory response. Some
viruses actively induce apoptosis e.g. HSV-1, EBV, while others prevent it to prolong infection
e.g. HPV, SV40 etc.

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 PURIFICATION OF VIRUS

Virus purification is an essential step in virology to obtain a concentrated, biologically active,

and clean preparation of viruses for research or vaccine production. There are several methods
for virus purification: and they include:

1. Centrifugation-Based Methods

 Differential Centrifugation: This is a simple and widely used method that separates
virus particles from contaminants based on size and density. It involves a series of low
and high-speed centrifugations, where larger debris is pelleted first, followed by
pelleting of smaller virus particles.

 Density Gradient Centrifugation: The virus is separated from other materials based
on its buoyant density using a gradient of sucrose, Cesium chloride (CsCl), or iodixanol.
The virus forms a distinct band at its specific density, which can be collected.

2. Filtration

 Membrane Filtration: Filters with defined pore sizes (e.g., 0.2 µm or 0.45 µm) are
used to remove larger particles, while allowing viruses to pass through. This method is
often used in combination with other purification techniques.

 Ultrafiltration: This method concentrates viruses by retaining them on a membrane

while removing smaller molecules like proteins and nucleic acids.

3. Precipitation

 Polyethylene Glycol (PEG) Precipitation: PEG is added to a virus solution, causing

the virus to precipitate out of solution. The precipitate can then be collected by
centrifugation and resuspended in a smaller volume.

4. Chromatography-Based Methods

 Size Exclusion Chromatography (SEC): This separates viruses from smaller

contaminants based on size, where larger viruses elute first, followed by smaller
molecules.

 Ion Exchange Chromatography: Charged resins are used to bind viruses based on
their surface charge, and viruses are eluted with a salt gradient.

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  Affinity Chromatography: Specific ligands or antibodies are immobilized on a
column to selectively capture viruses. After binding, viruses are eluted with a specific
buffer.

5. Ultracentrifugation

 Rate-Zonal Centrifugation: Viruses are separated based on size and shape through a
sucrose gradient by ultracentrifugation. It allows for separation of virus from
contaminating particles of different sizes.

 Equilibrium (Isopycnic) Centrifugation: This technique separates viruses based on

their density in a gradient (e.g., CsCl), leading to highly purified virus preparations.

6. Immobilization Techniques

 Immunoaffinity: Antibodies specific to the viral surface proteins are used to bind
viruses, allowing for their separation from contaminants.

 Lectin Affinity Chromatography: Lectins, which bind to specific glycosylation

patterns on viral envelopes, are used for the purification of glycosylated viruses like
enveloped viruses.

Each method may be used alone or in combination, depending on the type of virus, desired
purity, and downstream applications.

CLASSIFICATION OF VIRUSES

Virus classification is the process of naming viruses and placing them into a taxonomic system.
Viruses are mainly classified by phenotypic characteristics such as morphology, nucleic acid
type, mode of replication, host organisms and the type of disease they cause. Currently, two
main schemes are used for the classification of viruses: The International Committee on
Taxonomy of Viruses (ICTV) system and Baltimore classification system.

International Committee on Taxonomy of Viruses (ICTV Classification)

The International Committee on Taxonomy of Viruses began to devise and implement rules
for the naming and classification of viruses early in the 1970s. ICTV is the only body charged
by the International Union of Microbiological Societies with the task of developing, refining,
and maintaining a universal virus taxonomy.

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Viral classification starts at the level of realm and continues as follows, with the taxon suffixes
given in italics:

Realm (-viria): The highest taxonomic rank. Viruses are grouped into realms based on their
mode of replication, the presence of specific genes, and their evolutionary relationships.
Example: Riboviria.

Kingdom (-virae): Below the realm level, viruses may be classified into kingdoms, though
not all realms contain kingdoms. Example: Orthornavirae

Phylum (-viricota): Viruses are further grouped into phyla based on their genetic
characteristics and replication strategies. Example: Negarnaviricota

Class (-viricetes): Clasification is based on more specific genetic or evolutionary traits.

Example: Monjiviricetes.

Order (-virales): Orders categorize viruses based on genome structure, replication methods,
and other molecular features. Example: Mononegavirales

Family (-viridae): A more specific grouping of viruses with similar morphology, genome
organization, and replication strategies. Example: Coronaviridae (the family of coronaviruses).

Subfamily (-virinae): Orthocoronavirinae

Genus (-virus): Genera group viruses with closely related genetic sequences and structural
features. Example: Betacoronavirus

Species: The most specific classification, defining distinct virus types that cause similar
infections or replicate in similar hosts. Example: Severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) (the species that causes COVID-19).

The species names generally take the form of Disease virus.

Baltimore Classification

The most commonly and currently used system of virus classification was first developed by
Nobel Prize-winning biologist David Baltimore in the early 1970s. In addition to the
differences in morphology and genetics, the Baltimore classification scheme groups viruses
according to how the mRNA is produced during the replicative cycle of the virus.

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◦ Group I viruses: contain double-stranded DNA (dsDNA) as their genome. Their mRNA is
produced by transcription in much the same way as with cellular DNA, using the enzymes of
the host cell. (e.g. Adenoviruses, Herpesviruses, Poxviruses)

◦ Group II viruses: have single-stranded DNA (ssDNA) as their genome. They convert their
single-stranded genomes into a dsDNA intermediate before transcription to mRNA can occur.
(+ strand or "sense") DNA (e.g. Parvoviruses)

Group III viruses: use dsRNA as their genome. The strands separate, and one of them is used
as a template for the generation of mRNA using the RNA-dependent RNA polymerase encoded
by the virus. (e.g. Reoviruses)

◦ Group IV viruses: have ssRNA as their genome with a positive polarity, which means that
the genomic RNA can serve directly as mRNA. Intermediates of dsRNA, called replicative
intermediates, are made in the process of copying the genomic RNA. Multiple, full-length RNA
strands of negative polarity (complementary to the positive-stranded genomic RNA) are
formed from these intermediates, which may then serve as templates for the production of RNA
with positive polarity, including both full-length genomic RNA and shorter viral mRNAs. (+
strand or sense) RNA (e.g. Picornaviruses, Togaviruses)

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◦ Group V viruses: contain ssRNA genomes with a negative polarity, meaning that their
sequence is complementary to the mRNA. As with Group IV viruses, dsRNA intermediates
are used to make copies of the genome and produce mRNA. In this case, the negative-stranded
genome can be converted directly to mRNA. Additionally, full-length positive RNA strands
are made to serve as templates for the production of the negative-stranded genome. (e.g.
Orthomyxoviruses, Rhabdoviruses)

◦ Group VI viruses: have diploid (two copies) ssRNA genomes that must be converted, using
the enzyme reverse transcriptase, to dsDNA; the dsDNA is then transported to the nucleus of
the host cell and inserted into the host genome. Then, mRNA can be produced by transcription
of the viral DNA that was integrated into the host genome. RNA with DNA intermediate in
life-cycle (e.g. Retroviruses)

◦ Group VII viruses: have partial dsDNA genomes and make ssRNA intermediates that act as
mRNA, but are also converted back into dsDNA genomes by reverse transcriptase, necessary
for genome replication. (e.g. Hepadnaviruses)

SOME MEDICALLY IMPORTANT VIRUSES

HERPESVIRIDAE

The herpesvirus family contains seven important human pathogens:

1. Herpes simplex virus types 1 and 2

2. Varicella-zoster virus

3. Cytomegalovirus

4. Epstein–Barr virus

5. Human herpes virus 6 (HHV6)

6. Human herpes virus 7 (HHV7)

7. Human herpesvirus 8 (also known as Kaposi’s sarcoma–associated herpesvirus)

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General characteristics of herpesviruses (all are structurally similar and each has an):

❑ Icosahedral core surrounded by a lipoprotein envelope

❑ The genome is linear double-stranded DNA.

❑ The virion does not contain a polymerase.

❑ They are large (120–200 nm in diameter), second in size only to poxviruses.

❑ Herpesviruses replicate in the nucleus.

❑ The virions of herpesviruses possess a tegument located between the nucleocapsid and
the envelope.

❑ This structure contains regulatory proteins, such as transcription and translation factors,
which play a role in viral replication.

Ability to cause life-long latent infections:

➢ In these infections, the acute disease is followed by an asymptomatic period during

which the virus remains in a quiescent (latent) state.

➢ When the patient is exposed to an inciting agent or immunosuppression occurs,

reactivation of virus replication and disease can occur.

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 1. HERPES SIMPLEX VIRUSES (HSV)

HSV type 1 (HSV-1) and type 2 (HSV-2) are distinguished by two main criteria: antigenicity
and location of lesions. Lesions caused by HSV-1 are, in general, above the waist, whereas
those caused by HSV-2 are below the waist.

Diseases

 HSV-1 causes acute gingivostomatitis (inflammation of the gum and oral mucosa),
recurrent herpes labialis (cold sores), keratoconjunctivitis (inflammation of the cornea
and conjuctiva), and encephalitis (inflammation of the brain tissue), primarily in adults.

 HSV-2 causes herpes genitalis /genital herpes (penis, urethra, cervix, vulva, vagina),
neonatal encephalitis and other forms of neonatal herpes, and aseptic meningitis.
Infection by HSV-1 or HSV-2 is a common cause of erythema multiforme.

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Transmission & Epidemiology

 HSV-1 is transmitted primarily in saliva, whereas HSV-2 is transmitted by sexual

contact.

 HSV-1 infections occur mainly on the face, whereas HSV-2 lesions occur in the genital
area.

 Although transmission occurs most often when active lesions are present,
asymptomatic shedding of both HSV-1 and HSV-2 does occur and plays an important
role in transmission.

Pathogenesis & Immunity

 1) The virus replicates in the skin or mucous membrane at the initial site of infection.

 2) Migrates up the neuron by retrograde axonal flow and becomes latent in the sensory
ganglion cells.

 3)HSV-1 becomes latent in the trigeminal ganglia, whereas HSV-2 becomes latent in
the lumbar and sacral ganglia.

 4)The virus can be reactivated from the latent state by a variety of inducers (e.g.,
sunlight, hormonal changes, trauma, stress, and fever), at which time it migrates down
the neuron and replicates in the skin, causing lesions.

Laboratory Diagnosis

 Isolation of the virus from the lesion by growth in cell culture.

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  ELISAs Detecting virus-specific glycoproteins in enzyme-linked immunosorbent
assays.

 A rapid diagnosis from skin lesions can be made by using the Tzanck smear, in which
cells from the base of the vesicle are stained with Giemsa stain.

 Serologic tests

Treatment

Acyclovir, famciclovir, valacyclovir

2. VARICELLA-ZOSTER VIRUS (VZV)

Disease:

 Varicella (chickenpox) is the primary disease; zoster (shingles) is the recurrent form.

Important Properties:

 VZV is structurally and morphologically similar to other herpesviruses but is

antigenically different.

 It has a single serotype. The same virus causes both varicella and zoster. Humans are
the natural hosts.

Transmission & Epidemiology

The virus is transmitted by respiratory droplets and by direct contact with the lesions. Varicella
is a highly contagious disease of childhood; more than 90% Varicella occurs worldwide. but
the widespread use of the vaccine has significantly reduced the number of cases.

❑ Immunity following varicella is lifelong: A person gets varicella only once, but zoster
can occur despite this immunity to varicella.

❑ Zoster usually occurs only once. The frequency of zoster increases with advancing age,
perhaps as a consequence of waning immunity.

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Clinical Findings

 Varicella: After an incubation period of 14 to 21 days, brief prodromal (early/before)

symptoms of fever and malaise occur. A papulovesicular rash then appears in crops on
the trunk and spreads to the head and extremities.

 Zoster: The occurrence of painful vesicles along the course of a sensory nerve of the
head or trunk is the usual picture.

Treatment:

Acyclovir and vidarabine are effective in the treatment of severe varicella and zoster

3. CYTOMEGALOVIRUS (CMV)

CMV has the largest genome of all the herpes viruses and appears only to replicate in human
cells. It can form multi-nucleated cells (syncytia) with characteristically staining inclusions.
Transmission
The virus may be transmitted from person to person through saliva, respiratory mucus, milk,
urine, semen, cervical secretions, blood transfusion and faeces. It can be sexually transmitted.
Virulent form of disease:
Perinatal (foetuses) CMV infection is mostly asymptomatic, or pneumonitis, & a mon-
onucleosis-like syndrome.

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 New-borns may exhibit enlarged liver & spleen, jaundice, capillary bleeding micro-
cephaly, & ocular inflammation and it may be fatal
Babies who survive develop neurological sequelae; hearing, visual disturbances &
mental retardation
Immunodeficient adults such as in AIDS patients – (CMV mononucleosis, dissemi-
nated CMV, retinitis) and Transplant patients – (pneumonitis, hepatitis, myocarditis,
meningoencephalitis).
Laboratory diagnosis
The disease is commonly latent in various tissues and most infections are asymptomatic and
therefore go undiagnosed. There are fluorescent antibody and ELIZA tests. Multi-nucleated
(cytomegalinic) cells with characteristic inclusions can be seen in biopsies of many tissues.

Treatment:

For the treatment of severe infection, ganciclovir, foscarnet can be used. Acyclovir is not
effective.

4. EPSTEIN-BARR VIRUS (EBV)

It is an enveloped virus with icosahedral nucleocapsid. The viral genome is ds-DNA and it
replicate in the nucleus. It remains latent in B- cells even after the patient has recovered.

Clinical syndromes associated with EBV infection

I. Infectious mononucleosis.

It is a contagious viral infection caused by EBV which is most common among teenagers and
young adults, though it can affect people of any age. The virus spreads through saliva, and
hence, it is called ‘kissing disease’ also known as glandular fever, but it can also be transmitted
through coughing, sneezing or sharing drinks and utensils.

Signs and symptoms

Children infected with EBV develops no symptoms. When infection occurs in adolescence, it
causes infectious mononucleosis.

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Other symptoms include hepatosplenomegaly, malaise, skin rash, loss of appetite etc.

Prognosis and duration

It is a self-limiting disease, but the virus remains latent in B-cells even after recovery. The
disease seldom lasts for more than 4-months.

Lab diagnosis

Recent infection: by detection of Ig-M antibody to the viral capsid antigen (VCA). Immunity:
When Ig-G antibodies to both viral capsid antigen (VCA) and nuclear antigen (EBNA) are
present.

Treatment

There is no specific anti-viral drug therapy, treatment is supportive.

II. Chronic EBV infection.

It is a rare condition characterized by persistent or recurrent symptoms. If the disease lasts for
more than 6- months, it is called chronic EBV infection.

III. Burkitt lymphoma (BL).

There are three main types of Burkitt lymphoma: endemic, sporadic and immunodeficiency-
related.

 The endemic form of the disease is very common in Africa, where EBV and malaria
are endemic. The African form of BL most often present with swelling of the affected
jaw or other facial bones, loosing teeth, swelling of the lymph nodes, which are tender
and rapidly growing in the neck below the jaw.

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  The sporadic form occurs throughout the world. Patients with the sporadic form of BL
most commonly present with abdominal tumors, causing swelling and pain in the
affected area. It may affect the CNS, kidneys, ovaries and other organs.

 The immunodeficiency is most often seen in AIDS patients.

BL is an aggressive B-cell non-Hodgkin lymphoma, it may affect the jaw, CNS, kidneys and
ovaries in adults.

Burkitt lymphoma

1V. Nasopharyngeal carcinoma.

It is a rare tumor of the nasopharynx, but more common in Africa, where EBV infection and
malaria are endemic.

Signs and symptoms:

cervical lymphadenopathy (enlargement of lymph node in the neck region), bloody discharge
from the nose, nasal congestion, hearing loss in one ear, frequent ear infections, double vision,
headache and pain in the face and neck.

5. HUMAN HERPES VIRUS 6 (HHV6)

They specifically target and infect T-cells and can affect immune function and sometimes leads
to death (T-lymphotropic virus). They are transmitted by close contact and they are very com-
mon, about 95% prevalent. It causes roseola, an acute febrile disease in babies 2 – 12 months
begins with fever, followed by a faint maculopapular rash usually self-limiting. Adults may get
mono-like symptoms, lymphadenopathy, hepatitis. It can cause encephalitis, cancer, etc.
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 6. HUMAN HERPES VIRUS 7 (HHV 7)
It is closely related to HHV6 and causes similar diseases.

7. KAPOSI’S SARCOMA ASSOCIATED VIRUS (HHV 8)

It is linked with common tumour of AIDS patients, also may be involved in multiple mye-
loma.

PAPILLOMAVIRIDAE

Human papillomavirus (HPV) is a common virus that can affect different part of the body.
There are over 100 types of HPV, including strains that cause warts on the hands, feet and face.
More than 30 different strains of the HPV can affect the genitals. HPV that affects the genitals
is sexually transmitted infection. HPV is divided into high risk HPV and low risk HPV. The
low-risk types cause warts and the high-risk types can cause lesions or cancer. Incubation is
from 2 weeks to more than a year.

Transmission of HPV

HPV is transmitted through intimate skin to skin contact; having vaginal, anal or oral sex with
someone who has the virus. It is most commonly spread during vaginal or anal sex. It is
common that nearly all men and women get it at some point in their lives. It can be transmitted
even when an infected person has no signs or symptoms. Symptoms can be developed years
after being infected, making it difficult to know when one first became infected.

In most cases, HPV goes away on its own and does not cause any health problems. But when
it does not go away, it can cause health problems like genital warts and cancer.

Symptoms of HPV

HPV may not cause symptoms at once but can appear years later. Some types can lead to warts
while others can cause cancer.

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 Genital wart

Warts

Genital warts: this may appear as a small bump, cluster of bumps or stem-like protrusions.
They commonly affect the vulva, cervix, penis, scrotum, around the anus, rectum and in the
groin.

Other warts: common warts (found in hands, fingers and elbows); plantar warts (commonly
found on the heels or balls of the feet); flat wart (found on the face, neck or areas that have
been scratched).

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Cancer

These cancers include cancer of the cervix, vulva, vagina, penis, anus and oropharynx or the
base of the tongue and tonsils. It may take years or decades to develop. Individuals infected
with both HPV and HIV have an increased risk of developing cervical or anal cancer.

As in many cancers, there may be no signs or symptoms of cervical cancer until it has
progressed to a dangerous stage. However, if symptoms do occur, they may include:

Abnormal vaginal bleeding, pain in very advanced stages, any bleeding from the vagina other
than during menstruation, abnormal vaginal discharge, pain and discomfort during sex

Cervical cancer

Diagnosis

The traditional methods of viral diagnosis such as electron microscopy, cell culture and certain
immunological methods are not suitable for HPV detection. HPV cannot be cultured in cell
cultures. But the important methods to diagnose HPV infection include:

o Colposcopy: a procedure that allows illuminated stereoscopic and magnified viewing

of the cervix

o Acetic acid test: a vinegar solution applied to HPV-infected genital areas turns them
white. This may help to identify difficult-to-see flat lesions

o Pap smear test: a screening to test for premalignant and malignant changes, viral
infections like HPV and Herpes can also be detected.

Treatment

There is currently no specific treatments for HPV infection. However, warts can be treated.

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  Salicylic acid: over-the-counter treatments that contain salicylic acid works by
removing layers of a wart s little at a time.

 Imiquimod (Aldara, zyclara): this prescription cream might enhance your immune
system’s ability to fight HPV. Common side effects include redness and swelling at the
application site.

 Podofilox (Condylox): is a topical prescription that destroys genital wart tissue. It may
cause pain and itching where it is applied.

 Trichloroacetic acid: a chemical treatment that burns off warts on the palm, soles and
genitals. It may cause local irritation.

Surgical and other procedures

▪ Cryotherapy: freezing warts or destroying abnormal cells with liquid nitrogen.

▪ Electrocautery: burning with an electrical current.

▪ Loop electrosurgical excision procedure (LEEP): using a special wire loop to remove
warts or abnormal cells on the cervix

Prevention

• Vaccination

• Abstain from all forms all sexual intercourse including; oral, anal and vaginal sex. This
is because the virus is most infectious through sexual organs, the mouth and throat.

• Condom does not completely eliminate the risk of contracting the virus as HPV is
spread through skin to skin contact.

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 PRACTICE QUESTIONS
1. What are viruses?

2. Outline the general characteristics of viruses.

3. Write short notes on the historical aspect of virology

4. Explain the following atypical virus-like agents: (a) viroids (b) prions (c) defective interfering
particles (d) satellite viruses and satellite RNAs (e) pseudovirions (f) plasmids (g) plasma-
viruses

5. Write short notes on the following: (a) virion (b) genome (c) capsid (d) nucleocapsid (e) viral
envelope

6. Write short notes on viral symmetry and arrangement

7. Explain viral mutation and attachment

8. Succinctly discuss the viral cultivation in animal model

9. Outline the different sites in which viruses can be cultivated in embryonated eggs, giving ex-
amples in each case, enumerating the merits and demerits

10. Briefly but concise, discuss the process of viral replication.

11. Discuss cytopathic effect

12. Discuss CMV under the following heading: (a) transmission (b) virulent form of the disease
(c) laboratory diagnosis (d) treatment

13. Discuss HSV 1 and 2 under the following headings: (a) diseases (b) transmission and epidemi-
ology (c) laboratory diagnosis (d) treatment

14. Discuss VZV under the following headings: (a) disease (b) transmission and epidemiology (c)
clinical findings (d) treatment

15. Discuss HPV under the following headings: (a) transmission (b) symptoms (c) diagnosis (d)
treatment (e) prevention

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