VIRUS

A virus is a sub microscopic entity consisting of a single nucleic acid surrounded by aprotein
coat and capable of replication only within the living cells of bacteria, animals or plants.
They are obligate intracellular parasite and once inside the living host cell, the virus gets
integrated into the metabolism of its host, making it difficult to control by chemical means. It
is difficult to kill a virus with antibiotics. Drugs that destroy the host’s ability to be used by a
virus for replication, tend to be highly toxic and have a negatively and sometimes deadly
effect on the host cell.
Properties of Viruses.
 They do not have cellular organization.
 They contain only one type of nucleic acid either DNA or RNA but never both.
 They are obligate intracellular parasites.
 They lack the enzymes necessary for protein and nucleic acid synthesis and are
dependent for replication on the machinery of host cells.
 They are easily transmitted from one organism to another, not affected by antibiotics
and can be crystallised.
 They multiply by a complex process and not by binary fission.
 They are unaffected by antibacterial antibiotics.

Before a virus enters a cell, it is a free virus particle called a virion. A virion cannot grow or
carry out any biosynthetic or biochemical functions because it is metabolically inert. Viruses
are not cells. They vary in size from 20 nanometers (polio virus) to 300 nanometers
(smallpox virus) and cannot be seen under a light microscope.
Measuring the size of viruses
 Passing them through collodion membrane
 Electron microscopy
 Sedimentation in the ultracentrifuge
 Comparatative measurements.
Shape of viruses
 The overall shape of the virus particle varies in different groups of viruses.
 Most of the animal virus is roughly spherical, irregular and pleomorphic.
 Eg. Pox virus are brick shaped, rabies virus are bullet shaped, tobacco mosaic virus is
rod shaped

VIRAL STRUCTURE
The major components of a virus are:
• Nucleic acid core. The nucleic acid core can either be DNA or RNA that makes up the
genetic information (genome) of the virus. RNA genomes only occur in viruses. The genome
may be single stranded or double stranded, circular or linear, segmented or nonsegmented.
According to nucleic acid present, viruses can be classified in to DNA viruses and RNA
viruses.
• Capsid. A capsid is the protein coat that encapsulates a virus and protects the nucleic acid
from the environment. Capsid is composed of large number of capsomer which is made up of

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polypeptide molecules. The capsid with the enclosed nucleic acid is known as nucleocapsid.
It also plays a role in how some viruses attach to a host cell. A capsid consists of one or more
proteins that are unique to the virus and determine the shape of the virus.

Functions of capsid
 It protects the viral genome from physical destruction and enzymatic inactivation by
nucleases in biological material.
 It provides the binding site which enable the virus to attach to specific site on the host
cell.
 It facilitates the assembly and packaging of viral genetic informataion.
 It serves as a vehicle of transmission from host to another.
 It is antigenic and specific for each viruses
 It provides the structural symmetry to the virus particle.

Envelope. An envelope is a membrane bilayer that some viruses have outside their capsid. If
a virus does not have an envelope, the virus is called a naked virus. Environmental factors
that can damage the envelope are:
• Increased temperature
• Freezing temperature
• pH below 6 or above 8
• Lipid solvents
• Some chemical disinfectants (chlorine, hydrogen peroxide, and phenol)
Enveloped Virus:
 The envelope or outer covering of virus containing lipid is derived from the plasma
membrane of the host cell during the release by budding from the cell surface.
 The envelop is glycoprotein in nature.
 Enveloped viruses are susceptible to the action of lipid solvent such as ether,
chloroform and detergent.
 Eg. Herpes virus, Hepatitis B virus, HIV virus
A naked virus is more resistant to changes and is less likely to be affected by conditions that
can damage the envelope. Naked viruses are more resistant to changes in temperature and pH.

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Examples of diseases caused by naked viruses are chickenpox, shingles, mononucleosis, and
herpes simplex.
Peplomers:
In mature virus particle, the glycoproteins often appear as projecting spikes on the outer
surface of the envelope which are known as peplomers.
A virus may have more than one type of peplomers. E.g the influenza virus carries two types
of peplomers, the hemagglutinin and neuraminidase.
Functions of peplomers
 It helps for attachment of virus to the host cell receptors to initiate the entrance of the
virion into the cell.
 It attach to receptors on red blood cells, causing these cell to agglutinate.
 It has enzymatic activity like neuraminidase which cleave neuraminic acid from host
cellglycoproteins.
 It has antigenic properties.

CLASSIFICATION
In (1927), Johanson was the first to attempt for the classification of plant viruses.
Holmes (1948) kept viruses under the order virales and classified them into three
orders on the basis of host attacked.
1. Phaginae - infect bacteria
2. Phytophaginae - infect plants
3. Zoophagineae - infect animals

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Lwoff, Home and Toumier (1962) proposed a system of classification called Lm
system based on :
(i) Type of nucleic acid (DNA/RNA)
(ii) Symmetry (helical/cubical/bilateral)
(iii) Presence or absence of envelope around nucleocapsid.
(iv) Diameter of helical capsid.
(v) Number of capsomeres in cubic types.
(vi) Molecular weight of virus.
(vii) Shape and size of virus.
(viii) Diameter of coiled nucleocapsid/number of capsomers in cuboidal shape.
(ix) Diameter of nucleocapsid in coiling.
(x) Intracellular multiplication.
(xi) Mode of virus transmission.
The LHT system is not a natural classification system and does not show any evolutionary
phylogentic relationship. It classifies virus on the basis of common chemical and structural
features which can be accurately determined.
Classification according to the provisional committee on nomenclature of viruses (PNVC) of
the International Association of Microbiological societies based on the system of Lwoff,
Horne and Tournier (1962) is as follows

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Classification by Gasjens and King (1975)
The major groups of virus are classified according to the type of nucleic acid, symmetry,
presence or absence of an envelope and site of assembly (nuclear or cytoplasmic) of capsid
with genetic material.

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Baltimore classification
David Baltimore, a nobel laureate, proposed a scheme that encon"passed all viruses, based on
their genomes nature and their modes of replication and gene expression. The international
committee on taxonomy of viruses (ICTV) uses these, together with other parameters to place
viruses into families and genera.

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The revised Baltimore scheme is based on the fundamental importance of mRNA in the
replication cycle of viruses. Baltimore classification (first defined in 1971) is a classification
system that places viruses into one of seven groups depending on a combination of their
nucleic acid (DNA or RNA), strandedness (singlestranded or double-stranded), Sense, and
method of replication. Accordingly, viruses are grouped according to their mechanism of
mRNA synthesis and their replication strategy. By convention all mRNA is designated as
positive (+) sense RNA. Strands of viral DNA and RNA that are complementary to the
mRNA are designated as negative (-) sense and those having the same sequence are termed
+ve sense. In this way 7 classes of nature of virus genomes in that class.

Nomenclature: A new method of classification is given by International committee for virus

nomenclature to name viruses. Binomial system of nomenclature is not suitablefor naming
viruses. According to new nomenclature a virus name has two parts - 1st name is the common
name of virus while in the lInd part it contain' the code adopted for describing a virus- is
called Cryptogram. It is based on 4 pairs of symbols.
(a) The 1st pair indicates, type of nucleic acid/number of strands of nucleic acid.
(b) The lInd pair indicates, molecular weight of nucleic acid (in millions)/
percentage of nucleic acid.
(c) The 3rd pair indicates, outline of the particlej outline of nucleocapsid.
(d) The 4th pair indicates, kind of host infected/nature of vector ego Cryptogram of TMV is
R/1 : 2/5: E/E : 5/0. It means it contain RNA - one stranded (55); molecular weight of RNA is
2 millions, it makes 5% of virus particle, the particle and nucelocapsid are elongated with
parallel sides; ends not rounded it infects seed plants and no vector is needed

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 SHAPES OF VIRUSES
A virus can have one of two structures. These are:
• Helical virus. A helical virus is rod- or thread-shaped. The nucleic acid and capsomers are
wound together in the form of helix or spiral. Eg. Influenza virus, parainfluenz virus, rabies
virus

• Icosahedral virus. An icosahedral virus is spherically shaped. An icosahedral (icosa,

meaning 20 in greek) is a polygon with 12 vertices or corners and 20 facets or sides.Each
facet is in the shape of an equilateral triangle. Pentagonal capsomers at the vertices (pentons)
and hexagonal capsomers making up the facets (hexons) Viruses that cause poliomyelitis and
herpes simplex are icosahedral viruses.

Complex Symmetrical virus

Viruses which don not show either icoshedral or helical symmetry due to complexity of their
structure are referred to have complex symmetry. Eg. Pox viruses

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Viral Multiplication/ reproduction
The genomic information necessary for viral replication is contained in the viral nucleic acid
but lacking biosynthetic enzymes. The virus depends on the synthetic machinery of the host
cell for replication. The viral multiplication follows following steps.
I. Adsorption or Attachment
II. Penetration
III. Uncoating
IV. Biosynthesis
V. Maturation
VI. Release

Adsorption or Attachment
Adsorption or attachment is specific and is mediated by the binding of virion surface
structure, known as ligands, to receptors of cell surface. Because of the exact fit required,
viruses have a limited host range.
Eg. In case of Influenza virus, the surface glycoprotein hemagglutinin binds specifically to
siliac acide residue of glycoprotein receptor sites of respiratory tract.
In case of HIV surface glycoprotein gp120 acts as a ligand and binds to CD4 cells of T
lymphocytes.
Penetration
After binding the virus particle is taken up inside the cell. It is occur by system receptor
mediated endocytosis (viropexis). Most non-enveloped viruses enter by receptor mediated
endocytosis. Enveloped viruses fuse their membranes with cellular membranes to deliver the
nucleocapsid or genome directly into the cytoplasm.
Uncoating
This is the process of stripping the virus of its outer layers and capsid so that the nucleic acid
is released into the cell. With most viruses uncoating is effected by the action of lysosomal
enzymes of the host cell. The genome of DNA viruses except poxviruses must be delivered
into the nucleus whereas most RNA viruses remain in the cytoplasm.

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Biosynthesis
This phase includes synthesis not merely of the viral nucleic acid and capsid protein but also
of enzymes necessary in the various stages of viral synthesis, assembly and release. In
addition regulator proteins are alsosynthesised which serve to shut down the normal cellular
metabolism and direct the sequential production of viral components.
Steps of biosynthesis
 Transcription of messenger RNA (mRNA) from the viral nucleic acid.
 Translation of the mRNA into early proteins which initiate and maintain synthesis of
virus component.
 Replication of viral nucleic acid.
 Synthesis of Late or Structural proteins which are the components of daughter virion
capsid.
Maturation
Assembly of the various viral components into virions occurs shortly after the replication of
the viral nucleic acid. It may take place in either the nucleus or cytoplasm. In case of
enveloped viruses the envelop are derived from the host cell nuclear membrane and plasma
membrane.
Release
Viruses can be released from cells after lysis of the cell, by exocytosis, or by budding from
the plasma membrane. Viruses that exist as naked nucleocapsid may be released by the lysis
of the host cell or reverse phagocytosis. Release of enveloped viruses occur after budding
from the plasma membrane without killing the host cell. Anywhere from 3,000 to 100,000
virions may be released, depending on the virus. Entire length of cycle- anywhere from 8 to
36 hours

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The easiest way to understand how viruses replicate is to study the life cycles of viruses
called bacteriophages. Bacteriophages replicate by either a lytic cycle or a lysogenic cycle.
The difference in these two cycles is that the cell dies at the end of the lytic cycle and remains
alive in the lysogenic cycle.

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Lytic Cycle

The most studied bacteriophage is the T-even bacteriophage. The virions of T even
bacteriophages are big, complex, and do not contain envelopes. The T-even bacteriophages
are composed of a head-and-tail structure and contain genomes of double-stranded DNA. The
lytic cycle of replication begins with the collision of the bacteriophage and bacteria, called
attachment. The tail of the bacteriophage attaches to a receptor site on the bacterial cell wall.
After attachment, the bacteriophage uses its tail like a hypodermic needle to inject its DNA
(nucleic acid) into the bacterium. This is called penetration. The bacteriophage uses an
enzyme called lysozyme in its tail to break down the bacterial cell wall, enabling it to inject
its DNA into the cell. The head or capsid of the bacteriophage remains on the outside of the
cell wall. After the DNA is injected into the host’s bacterial cell’s cytoplasm, biosynthesis
occurs. Here the T-even bacteriophage uses the host bacterium’s nucleotides and some
enzymes to make copies of the bacteriophage’s DNA. This DNA is transcribed to mRNA,
which directs the synthesis of viral enzymes and capsid proteins. Several of these viral
enzymes catalyse reactions that make copies of bacteriophage DNA. The bacteriophage DNA
will then direct the synthesis of viral components by the host cell.
Next maturation occurs. Here the T-even bacteriophage DNA and capsids are put together in
order to make virions. The last stage is the release of virions from the host bacterium cell.
The bacteriophage enzyme lysozyme breaks apart the bacterial cell wall (lysis) and the new
virus escapes. The escape of this new bacteriophage virus will then infect neighboring cells
and the cycle will continue in these cells.
The Lysogenic Cycle
Some viruses do not cause lysis and ultimate destruction of their host cells which they infect.
These viruses are called lysogenic phages or temporate phages. These bacteriophages
establish a stable, long-term relationship with their host called lysogeny. The bacterial cells
infected by these phages are called lysogenic cells. The most studied bacteriophage, which
multiplies using the lysogenic cycle, is the bacteriophage Lambda. This bacteriophage infects
the bacterium E. coli.
When the bacteriophage Lambda penetrates an E. coli bacterium, the bacteriophage DNA
forms a circle. The circle recombines with the circular DNA of the bacteria. This
bacteriophage DNA is called a prophage. Every time the bacteria host cell replicates
normally, so does the prophase DNA. On occasion, however, the bacteriophage DNA can
break out of the prophage and initiate the lytic cycle.

Cultivation of viruses

Viruses are obligate intracellular parasites so they depend on 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.

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 2. To do research on viral structure, replication, genetics and effects on host cell.
3. To prepare viruses for vaccine production.

Cultivation of viruses can be discussed under following headings:

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 pig, hamster, rabbits and primates are
used.
 The selected animals should be healthy and free from any communicable diseases.
 Suckling mice(less than 48 hours old) are most commonly used.
 Suckling mice are susceptible to togavirus and coxsackie virues, which are inoculated
by intracerebral and intranasal route.
 Viruses can also be inoculated by intraperitoneal and subcutaneous route.
 After inoculation, virus multiply 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.
 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.
5. Used for the study of immune responses, epidemiology and oncogenesis.

Disadvantages of Animal Inoculation

1. Expensive and difficulties in maintenance of animals.

2. Difficulty in choosing of animals for 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 generation of escape mutants
6. Issues related to animal welfare systems.

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2. Inoculation into embryonated egg

 Good Pasture 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 is used.
 Viruses are inoculated into chick embryo of 7-12 days old.
 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.

1. Chorioallantoic Membrane (CAM):

 Inoculation is mainly for growing poxvirus.

 After incubation and incubation, visible lesions called pocks are observed, which is
grey white area in transparent CAM.
 Herpes simplex virus is also grown.
 Single virus gives single pocks
 This method is suitable for plaque studies.

2. Allantoic cavity:

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  Inoculation is mainly done for production of vaccine of influenza virus, yellow fever,
rabies.
 Most of avian viruses can be isolated using this method.

3. Amniotic sac:

 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.

4. Yolk sac inoculation:

 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

1. Widely used method for the isolation of virus and growth.

2. Ideal substrate for the viral growth and replication.
3. Isolation and cultivation of many avian and few mammalian viruses.
4. Cost effective and maintenance is much easier.
5. Less labor 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. Specific and non specific factors of defense are not involved in embryonated eggs.
10. Widely used method to grow virus for some vaccine production.

Disadvantages of Inoculation into embryonated egg

1. The site of inoculation for varies with different virus. That is, each virus have
different sites for their growth and replication.
2. Cell Culture (Tissue Culture)

In recent years use of animal cell culture in vitro as the growth medium for the viruses has
replaced the embryonated egg. Cells are cultured artificially in laboratory. The cultured
animal cells act as bacterial cells. To start the cell culture, small slice of animal tissue is
treated with enzymes which separate each cell. The cells are suspended in a solution of
proper osmotic pressure, nutrients and growth hormones that are required for the cell
growth.The cells get attached to the surface of glass and form a layer which is covered with
viral inoculation and waited to settle the virions on the cell surfaces.Cells are covered with a
thin layer of agar to prevent the spread of virions and facilitates cell infection. Viruses

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inoculated in the cell suspension destroy the monolayer.The phenomenon is called cytopathic
effect which is detected as the plaques formed by the bacteriophages.Plaques are formed on
the infected cells which are dtetected by staining with specific dyes. Eg. Tryphan blue or
neutral red.which differentiate the dead cells fro the living cells.

The grown cells obtained from the sliced tissue are known as the primary cell lines. The
primary cell lines starts degenerating after few generations.. Therefore the diploid cell lines
can be established from human embryos. The diploid cell lines can be maintained for about
100 generations.

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 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, usually balanced salt solution containing 13 amino acids,
sugar, proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the
host tissue or cell is inoculated.
 On incubation the cell divide and spread out on the glass surface to form a confluent
monolayer.

Types of cell culture

1. Primary cell culture:

 These are normal cells derived from animal or human cells.

 They are able to grow only for limited time and cannot be maintained in serial culture.
 They are used for the primary isolation of viruses and production of vaccine.
 Examples: Monkey kidney cell culture, Human amnion cell culture

2. Diploid cell culture (Semi-continuous cell lines):

 They are diploid and contain the same number of chromosomes as the parent cells.
 They can be sub-cultured up to 50 times by serial transfer following senescence and
the cell strain is lost.
 They are used for the isolation of some fastidious viruses and production of viral
vaccines.
 Examples: Human embryonic lung strain, Rhesus embryo cell strain

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3. Heteroploid cultures (Continuous cell lines):

 They are derived from cancer cells.

 They can be serially cultured indefinitely so named as continuous cell lines
 They can be maintained either by serial subculture or by storing in deep freeze at -
70°c.
 Due to derivation from cancer cells they are not useful for vaccine production.
 Examples: HeLa (Human Carcinoma of cervix cell line), HEP-2 (Humman
Epithelioma of larynx cell line), Vero (Vervet monkey) kidney cell lines, BHK-21
(Baby Hamster Kidney cell line).

Susceptible Cell Lines

1. Herpes Simplex Vero Hep-2, human diploid (HEK and HEL),human amnion
2. VZV human diploid (HEL, HEK)
3. CMV human diploid fibroblasts
4. Adenovirus Hep2, HEK,
5. Poliovirus MK, BGM, LLC-MK2, human diploid, Vero, Hep-2,Rhadomyosarcoma
6. Coxsackie B MK, BGM, LLC-MK2, vero, hep-2
7. Echo MK, BGM, LLC-MK2, human diploid, Rd
8. Influenza A MK, LLC-MK2, MDCK
9. Influenza B MK, LLC-MK2, MDCK
10. Parainfluenza MK, LLC-MK2
11. Mumps MK, LLC-MK2, HEK, Vero
12. RSV Hep-2, Vero
13. Rhinovirus human diploid (HEK, HEL)
14. Measles MK, HEK
15. Rubella Vero, RK13

Advantages of cell culture

1. Relative ease, broad spectrum, cheaper and sensitivity

Disadvantage of cell culture

1. The process requires trained technicians with experience in working on a full time
basis.
2. State health laboratories and hospital laboratories do not isolate and identify viruses in
clinical work.
3. Tissue or serum for analysis is sent to central laboratories to identify virus.

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Cultivation of plant viruses and bacteriophages

Cultivation of plant viruses

There are some methods of Cultivation of plant viruses such as plant tissue cultures, cultures
of separated cells, or cultures of protoplasts, etc. viruses can be grown in whole plants.

Leaves are mechanically inoculated by rubbing with a mixture of viruses and an abrasive.
When the cell wall is broken by the abrasive, the viruses directly contact the plasma
membrane and infect the exposed host cells. A localized necrotic lesion often develops due to
the rapid death of cells in the infected area. Some plant viruses can be transmitted only if a
diseased part is grafted onto a healthy plant.

Cultivation of bacteriophages

Bacteriophages are cultivated in either broth or agar cultures of young, actively growing
bacterial cells.

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