2. Sub microscopic entity consisting of a single nucleic acid
surrounded by a protein coat and capable of replication only
within the living cells of bacteria, animals or plants
Viruses have an inner core of nucleic acid surrounded by protein
coat known as an envelope
Most viruses range in sizes from 20 – 250 nm
Viruses are inert (nucleoprotein ) filterable Agents
Viruses are obligate intracellular parasites
They do not have cellular organization.
They contain only one type of nucleic acid either DNA or RNA
but never both.
They lack the enzymes necessary for protein and nucleic acid
synthesis and are dependent for replication on the machinery of
host cells.
They multiply by a complex process and not by binary fission.
They are unaffected by antibacterial antibiotics.
3. Terminology
Virus particle = virion
Protein which coats the genome = capsid
Capsid usually symmetrical
Capsid + genome = nucleocapsid
May have an envelope
Extracellular infectious viral particle is called ‘Virion’
Viruses are much smaller than bacteria. For a time, they were known
as ‘filterable agents’ as they can pass through filters that can hold back
bacteria.
They can not be seen under light microscope hence called as
‘ultramicroscopic’.
The virus particles seen in this manner are known as ‘elementary
bodies’.
Size range: 20-300 nm
Parvovirus: 20 nm (smallest virus) Pox virus: 300 nm (biggest virus
4. Measuring the size of virus
Passing them through collodion membrane
Electron microscopy
Sedimentation in the ultracentrifuge
Comparatative measurements
Shape of the Virus
The overall shape of the virus particle varies in different
groups of viruses.
Most of the animal virus are roughly spherical, irregular
and pleomorphic
Eg. Pox virus are brick shaped, rabies virus are bullet
shaped, tobacco mosaic virus is rod shaped
6. • Varies in size, shape and
symmetry
• Highly impo. for
classification
• 3 types of capsid
symmetry:
– Cubic (icosahedral)
– Helical
– Complex
• 5 basic types of virus
7.
8. • Stable in hostile environment
• Released by lysis of host cells
• Examples:
– Adeno-associated Virus (AAV)
– Adenovirus B19
9. Based Upon
The disease they cause
Polio virus, rabies virus
The type of disease
Murine leukemia virus
Geographic locations
Sendai virus, Coxsackie virus
Their discovers
Epstein-Barr virus
How they were originally thought to be contracted
Dengue virus (“evil spirit”), Influenza virus (the
“influence” of bad air)
Combinations of the above
Rous Sarcoma virus
10. Classification of Viruses
On the Basis of 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, papilloma virus, and
herpes virus
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, polio virus, yellow fever virus, dengue virus, hepatitis C
virus, measles virus, rabies virus, influenza virus and Ebola virus are examples
of RNA virus.
DNA-RNA viruses
11. On the basis of 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
in the wound tumour virus and rice dwarf viruses of plants.
Single-stranded RNA
It is found in most of the RNA viruses eg: tobacco mosaic virus,
influenza virus, poliomyelitis, bacteriophage MS-2, Avian leukemia
12.
13. On the Basis of 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: Herpes viruses, Poxviruses, Hepadna viruses
RNA viruses: Flavivirus, Toga virus, Corona virus, Hepatitis D,
Orthomyxo virus, Paramyxo virus, Rhabdo virus, Bunya virus,
Filo virus
Retroviruses
Non-Enveloped Virus
DNA viruses- parvovirus, adenovirus and papova virus.
RNA viruses- Picorna virus, Hepatitis A virus and Hepatitis E
virus.
14.
15. 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.
On the Basis of Shapes of the Viruses
Most of the animal viruses are roughly spherical with some
exceptions.
Rabies virus: Bullet shaped
Ebola virus: Filamentous shaped
Poxvirus: Brick shaped
16. Classification of Virus on the Basis of
Structure
Cubical virus: They are also known as
icosahedral symmetry virus
Eg. Reo virus, Picorna virus.
Spiral virus: They are also known as helical
symmetry virus
Eg. Paramyxovirus, orthomyxovirus.
Radial symmetry virus: eg. Bacteriophage.
Complex virus: eg. Pox virus.
17. On the Basis of the Type of Host
The virus can be classified on the basis of the type of host. They are:
Animal viruses
Plant viruses
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 and 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
18. Classification of Virus on the Basis of Mode of
Transmission
Virus transmitted through respiratory route:
Eg, Swine flu, Rhino virus
Virus transmitted through faeco-oral route:
Eg. Hepatitis A virus, Polio virus, Rota virus
Virus transmitted through sexual contacts:
Eg. Retro virus
Virus transmitted through blood transfusion:
Eg. Hepatitis B virus, HIV
Zoonotic virus:
Virus transmitted through biting of infected animals;
Eg. Rabies virus, Alpha virus, Flavi virus
19. Classification of Virus on the Basis of Replication
Properties and Site of Replication
Replication and assembly in cytoplasm of host:
All RNA virus replicate and assemble in cytoplasm of host cell
except Influenza virus
Replication in nucleus and assembly in cytoplasm of host:
Influenza virus, Pox virus
Replication and assembly in nucleus of host:
All DNA viruses replicate and assemble in nucleus of host cell
except Pox virus.
Virus replication through ds DNA intermediate:
All DNA virus, Retro virus and some tumor causing RNA
virus replicates through ds DNA as intermediates.
Virus replication through ss RNA intermediate:
All RNA virus except Reo virus and tumor causing RNA
viruses.
20. • The Baltimore classification system Based on:
– Genetic contents
– Replication strategies of viruses
• Seven classes:
1. dsDNA viruses
2. ssDNA viruses
3. dsRNA viruses
4. (+) sense ssRNA viruses (codes directly for protein)
5. (-) sense ssRNA viruses (does not encode mRNA)
6. RNA reverse transcribing viruses
7. DNA reverse transcribing viruses
21.
22. Baltimore Classification
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.
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
23. 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.
24.
25.
26.
27. Introduction of virus
• Viruses are extremely small infectious agents that
invade cells of all types.
• 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.
• Viruses have been the culprits in many human diseases,
including smallpox, flu, AIDS, and the ever-present
common cold as well as in plant, bacteria and archaea
also.
28. The primary purpose of virus cultivation is:
To isolate and identify viruses in clinical samples.
To do research on viral structure, replication, genetics and
effects on host cell.
To prepare viruses for vaccine production.
Isolation of virus is always considered as a gold standard for
establishing viral etiology of a disease
Techniques in cultivating and identifying animal
viruses
• viruses require living cells as their “medium”.
•In vivo – laboratory-bred animals and embryonic bred tissues.
•In vitro - cell or tissue culture methods.
29.
30. 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
viruses, 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.
31.
32. Advantages of Animal Inoculation
Diagnosis, Pathogenesis and clinical symptoms are
determined.
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 Inoculation
Expensive and difficulties in maintenance of animals.
Difficulty in choosing of animals for particular virus
Some human viruses cannot be grown in animals or can be
grown but do not cause disease.
Mice do not provide models for vaccine development.
It will lead to generation of escape mutants
Issues related to animal welfare systems.
33. 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
34.
35. Chorioallantoic Membrane (CAM):
Inoculation is mainly for growing poxvirus.
After inoculation 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.
Allantoic cavity:
Inoculation is mainly done for production of vaccine
of influenza virus, yellow fever, rabies.
Most of avian viruses can be isolated using this
method.
36. 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.
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.
37. 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
38. 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 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.
39.
40. 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.
Monkey kidney cell culture, human embryonic kidney cell culture,
and chick embryo cell culture are the common examples of
primary cell culture.
Primary monkey kidney cell cultures are highly useful for the
primary isolation of myxovirus, paramyxovirus, many
enteroviruses, and some adenoviruses.
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
41. They are used for the isolation of some fastidious viruses and
production of viral vaccines.
Examples: Human embryonic lung strain, Rhesus embryo cell
strain
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).
42. Advantages of cell culture
Relative ease, broad spectrum, cheaper and sensitivity
Disadvantage of cell culture
The process requires trained technicians with experience in
working on a full time basis.
State health laboratories and hospital laboratories do not isolate
and identify viruses in clinical work.
Tissue or serum for analysis is sent to central laboratories to
identify virus.
43. 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.
44. Virus Replication
Viral replication is the formation of
biological Viruses during the infection process in the
target host cells.
Viruses must first enter the cell before viral replication
can occur.
Through the generation of abundant copies of its
genome and the packaging of these copies, the virus
continues to infect new hosts.
The replication between viruses is very varied and
depends on the type of genes involved in them.
Most DNA viruses assemble in the nucleus, while most
RNA viruses replicate only in the cytoplasm.
45. 1. Virus attachment and
entry
2. Uncoating of virion
3. Migration of
genomenucleic acid to
nucleus
4. Transcription
5. Genome replication
6. Translation of
virus mRNAs
7. Virion assembly
8. Release of new virus
particles
1
2
3
4
5
6
7
8
46. Steps of replication in viruses
The virus does not have its own metabolic system. The infected
host cell has to provide the energy, metabolic machinery and
precursor molecules for the synthesis of viral proteins and nucleic
acids.
Replication in viruses occurs in six steps which are named below.
1. Adsorption/ attachment of the virus to host cell
2. Penetration of Viral components in the host cell
3. Uncoating
4. Synthesis of viral components by mRNA production /
Transcription
5. Virion assembly
6. Release of virus (liberation stage)
47.
48. 1- Adsorption/ attachment of the virus to host cell
It is the first step of viral replication.
The Virus binds to the cell membrane of the host cell by a specific
receptor site on the host cell membrane through binding proteins in
the capsid or by glycoproteins embedded in the viral envelope.
The specificity of this interaction determines the host (and the cells
within the host) that can be infected by a particular virus.
This can be imagined by thinking of multiple keys with multiple
locks where each key will fit a single specific lock.
Viral proteins on the capsid or phospholipid envelope interact with
specific receptors on the host cellular surface. This specificity
determines the host range (tropism) of a virus.
49. 2- Penetration of Viral components in the host cell
Then the virus injects its DNA or RNA into the host to start the
infection.
Bacteriophage nucleic acid enters the host cell naked, leaving the
capsid outside the cell.
Plant and animal viruses can enter through endocytosis, in which
the cell membrane surrounds and engulfs the entire virus.
In the plant, the cell membrane of the host cell invaginates the virus
particle, enclosing it in a pinocytotic vacuole.
Some enveloped viruses enter the cell when the viral envelope fuses
directly with the cell membrane of the host cell.
The process of attachment to a specific receptor can induce
conformational changes in viral capsid proteins, or the lipid envelope,
that results in the fusion of viral and cellular membranes.
Some DNA viruses can also enter the host cell through receptor-
50. 3- Uncoating
Once inside the cell, the viral capsid is broken down by the
cellular enzymes (from lysosomes) of the host and the viral
nucleic acid is released, which is then available for replication
and transcription.
The viral capsid is removed and degraded by viral enzymes or
host enzymes releasing the viral genomic nucleic acid.
51. 4- Synthesis of viral components by mRNA production /
Transcription
The virus uses cellular structures of the host cell to replicate. The
replication mechanism depends on the viral genome.
DNA viruses generally use proteins and enzymes from the host cell to
produce additional DNA that is transcribed into messenger RNA
(mRNA), which is then used to direct protein synthesis.
RNA viruses generally use their RNA core as a template for the
synthesis of viral genomic RNA (to be incorporated in the structure of
new virus) and mRNA. Viral mRNA directs the host cell to synthesize
two types of proteins.
a) Structural: The proteins that make up the viral particle are
manufactured and assembled.
b) Non-structural: it is not found in viral particles. It is composed of
enzymes for the replication of the virus genome.
If a host cell does not provide the enzymes necessary for viral
replication, the viral genes provide the information to direct the
synthesis of the missing proteins.
52. Replication:
After the viral genome has been uncoated, transcription or
translation of the viral genome is initiated.
It is this stage of viral replication that differs greatly between
DNA and RNA viruses and viruses with opposite nucleic acid
polarity.
The viral mRNA directs the host cell to synthesize viral enzymes
and capsid proteins, and to assemble new virions.
Retroviruses, like HIV, have an RNA genome that
must be reverse transcribed into DNA, which is then
incorporated into the host cell genome.
To convert RNA to DNA, retroviruses must contain genes that
encode the enzyme reverse transcriptase for the virus-specific
enzyme, which transcribes an RNA template into DNA.
.
53. To convert RNA into DNA, retroviruses must contain genes that encode the
virus-specific enzyme reverse transcriptase, which transcribes an RNA
template to DNA.
Reverse transcription never occurs in uninfected host cells; the needed
enzyme, reverse transcriptase, is only derived from the expression of viral
genes within the infected host cells.
The fact that HIV produces some of its own enzymes not found in the host
has allowed researchers to develop drugs that inhibit these enzymes.
These drugs, including the reverse transcriptase inhibitor AZT, inhibit HIV
replication by reducing the activity of the enzyme without affecting the
host’s metabolism.
This approach has led to the development of a variety of drugs used to treat
HIV and has been effective at reducing the number of infectious virions
54. 5- Virion assembly
A virion is simply an intact or active virus particle. At this stage, the
newly synthesized genome (nucleic acid) and proteins assemble to form
new virus particles.
This can take place in the cell nucleus, the cytoplasm, or in the plasma
membrane of most developed viruses.
6- Release of virus (liberation stage)
It is the last stage of viral replication in which the viruses, which are now
mature, are released in the host organism.
They can then infect adjacent cells and repeat the replication cycle.
Viruses are released by sudden cell disruption or by gradual extrusion
(budding) of viruses enveloped through the cell membrane.
New viruses can invade or attack other cells, or remain dormant in the cell.
In the case of bacterial viruses, the virions are released from the progeny by
lysis of the infected bacteria. However, in the case of animal viruses, release
generally occurs without cell lysis.
55. Virion release:
There are two methods of viral release: lysis or budding.
Lysis results in the death of an infected host cell, these types of
viruses are referred to as cytolytic.
An example is variola major also known as smallpox. Enveloped
viruses, such as influenza A virus, are typically released from the
host cell by budding.
It is this process that results in the acquisition of the viral
phospholipid envelope. These types of virus do not usually kill
the infected cell and are termed cytopathic viruses.
After virion release some viral proteins remain within the host’s
cell membrane, which acts as potential targets for circulating
antibodies.
Residual viral proteins that remain within the cytoplasm of the
host cell can be processed and presented at the cell surface on
MHC class-I molecules, where they are recognised by T cells.
