1

The virus is a Latin word meaning “poison”

“Virus are obligate intracellular parasites which means they replicate only inside a living host.”
“Virus are non-cellular infectious agents consisting of either DNA or RNA, Reproduce only in
living cells and use biosynthetic machinery of the host cell to direct the synthesis of virion,
containing viral genome and transfer them to other cells “.
All viruses consist of two basic components:
1. A core nucleic acid called genome.
2. A surrounding coat of a protein is known as a capsid.

1. GENOME:
The genome contains either DNA or RNA, but not both, and nucleic acid occurs in double-
stranded or single stranded form.
The most common forms of viral genomes found in nature are ssRNA and dsDNA.
Single-stranded viral RNA genomes are further subdivided into those of “positive polarity” (that
is, of messenger RNA sense, which can, therefore, be used as a template for protein synthesis)
and those of “negative polarity” or antisense (that is, complementary to messenger RNA sense,
which cannot, therefore, be used directly as a template for protein synthesis).
Viruses containing these two types of RNA genomes are commonly referred to as positive-strand
and negative-strand RNA viruses, respectively.
Viral Nucleic Acids
The genome of some viruses is DNA, whereas the genome of others is RNA. These DNA and
RNA genomes can be either single-stranded or double-stranded.
Some RNA viruses, such as influenza virus and rotavirus, have a segmented genome, i.e., the
genome is in several pieces.
All viruses have one copy of their genome (haploid) except retroviruses, which have two copies
(diploid).

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 2

2. CAPSID:
The capsid provides a protective covering for the genome because the construction of its amino
acids resists temperature, pH, and other environmental fluctuations.
The protein shell enclosing the genome is, for most virus families, found in either of two
geometric configurations.
 helical (rod-shaped or coiled)
 icosahedral (spherical or symmetric).

Viral Proteins
Viral proteins serve several important functions.
The outer capsid proteins protect the genetic material and facilitate the attachment of the virus to
specific receptors on the host cell surface. This interaction of the viral proteins with the cell
receptor is the major determinant of species and organ specificity.
Outer viral proteins are also important antigens that induce neutralizing antibodies and activate
cytotoxic T cells to kill virus-infected cells. These outer viral proteins not only induce antibodies
but are also the target of antibodies, i.e., antibodies bind to these viral proteins and prevent
("neutralize") the virus from entering the cell and replicating.

3. CAPSOMERS:
Generally, the capsid is composed of smaller protein components referred to as capsomers
4. NUCLEO CAPSID:
The capsid plus genome combination is called a nucleocapsid. It is also known as genocapsid.
5. Envelope:
Many viruses are surrounded by a flexible membrane known as an envelope. The envelope is
composed of lipids and proteins and is similar to the host cell membrane, except that it includes
viral-specific components.
A virus that is not enveloped is referred to as a naked virus.
Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 3

Replication of Virus:
The process of viral replication is one of the most remarkable events in nature
A virion invades a living cell a thousand or more times its size, uses the metabolism of the cell,
and produces copies of itself, often destroying the cell.
Viruses do not replicate in synthetic media.
Replication of virus (bacteriophage) is a five-step process:
1. Adsorption
2. Penetration
3. Transcription
4. Maturation
5. Release

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 4

1) ADSORPTION (attachment):
The 1st step in the replication of a bacteriophage is contact with its host cell. It should be noted
that infection of host bacterial cells cannot occur without adsorption
The tip of the virus tail becomes attached to the cell via specific receptor sites.
Any surface of bacteria can act as a receptor including lipopolysaccharides, flagella, pili
carbohydrates and proteins in the membrane or cell wall.
The actual attachment consists of a weak chemical union between the virus and receptor site.

2) PENETRATION:
The actual penetration of phage into the host is mechanical. However, it is facilitated by
localized digestion of certain cell structures either by phage enzymes (lysozyme) carried on the
tail of the phage or by viral activation of host degradative enzymes.
 The tail fibres of the virus attach to the cell and hold the tail firmly against the cell wall.
 The sheath contracts, driving the tail core into the cell through the cell wall and
membrane, and
 The virus injects its DNA the way a syringe injects a vaccine.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 5

 The protein coat, which forms the phage head and tail structure of viruses remains
outside the cell.

3) TRANSCRIPTION:
The next step is Transcription (biosynthesis). Inside the host cell the viral gene codes for
disruption of the host chromosome.
The viral DNA then uses the bacterial nucleotides and cell enzymes to synthesize multiple copies
of itself.
Messenger RNA molecules transcribed from viral DNA appear in the cytoplasm, and
biosynthesis of viral enzymes and capsid proteins begins. Bacterial ribosomes and amino acids
are all enlisted for biosynthesis.
As viral capsids are repeating units of capsomers, a relatively simple genetic code can be used
over and over.

4) MATURATION/ASSEMBLY:
In this phase, the replicated viral DNA and capsids are reassembled into complete virions.
The enzymes encoded by viral genes guide the assembly in step-by-step fashion.
In one area, phage heads and tails are assembled from protein subunits, in another the heads are
packaged with DNA, and in the third area, the tails are attached to heads.

5) RELEASE:
The final phase of viral replication is the release phase.
For bacteriophage, it is also called the lysis stage, because the cell lyses or breaks open.
For some phages, the important enzyme in this process is lysozyme, encoded by the
bacteriophage genes, late in the sequence of events,
The time that passes from phage attachment to the release of new viruses is commonly referred
to as the latent period or burst time.
The yield of phage per bacterium is called the burst size.
For bacteriophages, the average burst time ranges from 20 to 40 min.
At the end of the process, 50 to 200 new phages emerge from the host cell. This number is
commonly called the burst size

INACTIVATION OF VIRUSES:
Viruses may be inactivated by many of the physical and chemical agents.
A few methods for inactivating viruses are described below:
a) Formaldehyde reacts with free amino groups on the viral genome modifies it and
prevents its replication.
b) Heat alters the structure of viral proteins and nucleic acids, causing them to unfold and
denature.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 6

c) Ultraviolet light inactivates viruses by stimulating adjacent thymine or cytosine bases on

DNA molecules to bind together and form pairs called dimers. The dimers twist the
molecule out of shape, and the distorted viral genome cannot replicate.
d) X-rays cause breaks in the sugar-phosphate backbone of nucleic acid.
e) Lipids solvents such as ether, chloroform and detergents, all of which dissolve the lipids
in the envelope of viruses and inactivate them.
f) Heavy metal compounds such as mercury and silver derivatives inactivate viruses.

Cultivation of viruses:
Viruses are obligate intracellular parasites, so they depend on the host for their survival.
They cannot be grown in non-living culture media or on agar plates alone, they must require
living cells to support their replication.
The primary purpose of virus cultivation is:
1. To isolate and identify viruses in clinical samples.
2. To research viral structure, replication, genetics and effects on host cells.
3. To prepare viruses for vaccine production.
The following techniques are commonly used for Cultivation of viruses:
1. Animal Inoculation
2. Inoculation into embryonated egg
3. Cell Culture

1. Animal Inoculation:
Viruses which are not cultivated in embryonated egg and tissue culture are cultivated in
laboratory animals such as mice, guinea pigs, hamsters, rabbits, and primates.
The selected animals should be healthy and free from any communicable diseases.
Suckling mice (less than 48 hours old) are mostly used.
Suckling mice are susceptible to the toga virus and coxsackie viruses, which are inoculated by
intracerebral and intranasal routes.
Viruses can also be inoculated by intraperitoneal and subcutaneous routes.
After inoculation, the virus multiplies in the 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.
Live inoculation was first used on human volunteers for the study of yellow fever virus.

Advantages of Animal Inoculation

1. Diagnosis, Pathogenesis, and clinical symptoms are determined.
2. Production of antibodies can be identified.
3. Primary isolation of certain viruses.
4. Mice provide a reliable model for studying viral replication.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 7

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

Disadvantages of Animal Inoculation
1. Expensive and difficult to maintain animals.
2. Difficulty in choosing animals for a particular virus.
3. Some human viruses cannot be grown in animals or can be grown but do not cause
disease.
4. Mice do not provide models for vaccine development.
5. It will lead to the generation of escape mutants.
6. Issues related to animal welfare systems.

2. Embryonic inoculation:
Good Pasture in 1931 first used the embryonated hen’s egg for the cultivation of the virus.
The process of cultivation of viruses in embryonated eggs depends on the type of egg which is
used.
Viruses are inoculated into chick embryos of 7-12 days old.
For inoculation, eggs are first prepared for cultivation, and 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 the virus is isolated from the tissue of the egg.
Viral growth and multiplication in the egg embryo are 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 the egg like chorioallantoic membrane, allantoic
cavity, amniotic sac and yolk sac.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 8

Advantages of Inoculation into embryonated egg

1. Widely used method for the isolation of virus and growth.
2. Ideal substrate for viral growth and replication.
3. Isolation and cultivation of many avian and mammalian viruses.
4. Cost-effective and maintenance is much easier.
5. Less labour is needed.
6. The embryonated eggs are readily available.
7. Sterile and wide range of tissues and fluids
8. They are free from contaminating bacteria and many latent viruses.
9. Widely used method to grow virus for some vaccine production.

Disadvantages of Inoculation into embryonated egg

The site of inoculation varies with different viruses. That is, each virus has different sites for its
growth and replication.

3. Cell Culture (Tissue Culture):

There are three types of tissue culture: organ culture, explant culture and cell culture.
Organ cultures are mainly done for highly specialized parasites of certain organs e.g., tracheal
ring culture is done for isolation of coronavirus.
Explant culture is rarely done.
Cell culture is mostly used for the identification and cultivation of viruses.
Cell culture is the process by which cells are grown under controlled conditions.
Cells are grown in-vitro on glass or a treated plastic surface in a suitable growth medium.
At first growth medium, is usually a balanced salt solution containing all essential nutrients. On
incubation, the cell divides and spreads out on the glass surface to form a confluent monolayer.

Advantages of cell culture

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 9

The tissue culture technique is the most widely used method for the isolation and propagation of
viruses from clinical material.
Relative ease, broad-spectrum, cheaper and sensitivity.
Tissue culture techniques have an advantage over embryo culture vaccines in minimizing the
possibility of patients developing hypersensitivity to egg albumin.

Unique Characteristics:
1. They are energy less.
2. They are basic life forms, composed of a protein coat, called a capsid, that surrounds
genetic material, viruses are further enclosed by a lipid bilayer membrane that surrounds
the capsid.
3. Genetic material is either DNA or RNA never both.
4. Replication of the genetic material occurs when the virus takes control of the host cell’s
synthetic machinery.
5. A plasma cell is another name for a virus.
Typical virus-like agents:
 DEFECTIVE VIRUSES – composed of nucleic acid & proteins but cannot replicate
without a helper virus.
 PSEUDOVIRION – contains host cell DNA instead of viral DNA within the capsid.
 VIROIDS – consists solely of a single molecule of circular RNA without a protein coat
or envelope.
 PRIONS – infectious protein particles composed solely of proteins.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 10

Classification of Viruses

Based on the Genetic Material Present

 Viruses are small, nonliving parasites, which cannot replicate outside of a host cell.
 A virus consists of genetic information — either DNA or RNA — coated by a protein.
 Accordingly, they are classified as DNA viruses and RNA viruses.
 The nucleic acid may be single or double-stranded, circular or linear, segmented or
unsegmented.
DNA viruses
 As their name implies, DNA viruses use DNA as their genetic material.
 Some common examples of DNA viruses are parvovirus, papillomavirus, and
herpesvirus.
 DNA viruses can affect both humans and animals and can range from causing benign
symptoms to posing very serious health.
RNA viruses
 The virus that possesses RNA as genetic material are called RNA viruses.
 Rotavirus, poliovirus, yellow fever virus, dengue virus, hepatitis C
virus, measles virus, rabies virus, influenza virus and Ebola virus are examples of
RNA viruses.
DNA-RNA viruses
 The RNA tumour viruses called Leukoviruses and Rous’s viruses unusually contain both
DNA and RNA as genetic material.
Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 11

Based on the presence of a number of strands

 Double-stranded DNA
It is found in pox viruses, the bacteriophages T2, T4, T6, T3, T7 and Lamda, herpes viruses,
adenoviruses etc.
 Single-stranded DNA
It is found in bacteriophagesφ, X, 74 bacteriophages.
 Double-stranded RNA
It has been found within viral capsid in the reoviruses of animals and the wound tumour virus
and rice dwarf viruses of plants.
 Single-stranded RNA
It is found in most RNA viruses eg: tobacco mosaic viruses, influenza viruses, poliomyelitis,
bacteriophage MS-2, and Avian leukaemia virus.

Based on the Presence of Envelope

 The envelope is a lipid-containing membrane that surrounds some virus particles. It is
acquired during viral maturation by a budding process through a cellular membrane.
 Virus-encoded glycoproteins are exposed on the surface of the envelope. These
projections are called peplomers.
Enveloped Virus
 DNA viruses: Herpesviruses, Poxviruses, Hepadnaviruses
 RNA viruses: Flavivirus, Toga virus, Coronavirus, Hepatitis D, Orthomyxovirus,
Paramyxovirus, Rhabdovirus, Bunyavirus, Filovirus
 Retroviruses
Non-Enveloped Virus
 DNA viruses- parvovirus, adenovirus and papovavirus.
 RNA viruses- Picornavirus, Hepatitis A virus and Hepatitis E virus.

Virus Classification by Capsid Structure

 Naked icosahedral: Hepatitis A virus, polioviruses
 Enveloped icosahedral: Epstein-Barr virus, herpes simplex virus, rubella virus, yellow
fever virus, HIV-1
 Enveloped helical: Influenza viruses, mumps virus, measles virus, rabies virus.
 Naked helical: Tobacco mosaic virus
 Complex with many proteins: some have combinations of icosahedral and helical
capsid structures. Herpesviruses, smallpox virus, hepatitis B virus, T4 bacteriophage.

Based on Shapes of the Viruses

 Most of the animal viruses are roughly spherical with some exceptions.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 12

 Rabies virus: Bullet-shaped

 Ebola virus: Filamentous shaped
 Poxvirus: Brick shaped
 Adenovirus: Space vehicle shaped

Classification of Virus based on Structure.

1. Cubical virus: They are also known as icosahedral symmetry virus.
Eg. Reo virus, Picorna virus.
2. Spiral virus: They are also known as helical symmetry virus.
Eg. Paramyxovirus, orthomyxovirus.
3. Radial symmetry virus: eg. Bacteriophage.
4. Complex virus: eg. Pox virus.

Based on the Type of Host

The virus can be classified based on the type of host. They are:
1. Animal viruses
2. Plant viruses
3. Bacteriophage
Animal Viruses
The viruses which infect and live inside the animal cell including man are called animal viruses.
Eg; influenza virus, rabies virus, mumps virus, poliovirus etc. Their genetic material is RNA or
DNA.

Plant Viruses
The viruses that infect plants are called plant viruses. Their genetic material is RNA which
remains enclosed in the protein coat. Some plant viruses are tobacco mosaic virus, potato virus,
beet yellow virus, turnip yellow virus etc.

Bacteriophages
Viruses which infect bacterial cells are known as bacteriophage or bacteria eaters. They contain
DNA as genetic material. There are many varieties of bacteriophages. Usually, each kind of
bacteriophage will attack only one species or only one strain of bacteria.

Classification of Virus based on Mode of Transmission

1. Virus transmitted through the respiratory route:
Eg, Swine flu, Rhinovirus
2. Virus transmitted through faecal-oral route:

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 13

Eg. Hepatitis A virus, Poliovirus, Rotavirus

3. Virus transmitted through sexual contacts:
Eg. Retrovirus
4. Virus transmitted through blood transfusion:
Eg. Hepatitis B virus, HIV
5. Zoonotic virus:
Virus transmitted through biting of infected animals.
Eg. Rabies virus, Alphavirus, Flavivirus

Classification of Viruses based on Replication Properties and Site of Replication

1. Replication and assembly in the cytoplasm of the host:
 All RNA viruses replicate and assemble in the cytoplasm of the host cell except Influenza
virus.
2. Replication in the nucleus and assembly in the cytoplasm of the host:
 Influenza virus, Pox virus
3. Replication and assembly in the nucleus of the host:
 All DNA viruses replicate and assemble in the nucleus of the host cell except the Pox
virus.
4. Virus replication through ds DNA intermediate:
 All DNA virus, Retro viruses and some tumour-causing RNA viruses replicate through
dsDNA as intermediates.
5. Virus replication through ss RNA intermediate:
 All RNA viruses except Reovirus and tumour-causing RNA viruses.

Baltimore Classification of Viruses

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 14

The Baltimore Classification of Viruses

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

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics
 15

 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.
 Group IV viruses have ssRNA as their genome with a positive polarity. Positive
polarity 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.
 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.
 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.
 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.

Composed by:
Dr. Muhammad Rehan Malik
M.Phil Pharmaceutics