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General Properties of Viruses

Virology

Virology is the bioscience for study of viral nature,and the relationship


between viruses and hosts

Definition of Virus

Viruses may be defined as acellular organisms whose genomes


consist of nucleic acid, and which obligately replicate inside host cells
using host metabolic machinery and ribosomes to form a pool of
components which assemble into particles called VIRIONS
Viruses are inert (nucleoprotein ) filterable Agents

Viruses are obligate intracellular parasites

Viruses cannot make energy or proteins


independent of a host cell

Viral genome are RNA or DNA but not both

Viruses lack the enzymes necessary for protein and


nucleic acid synthesis

Viruses do not have the genetic capability to


multiply by division

Viruses occupy the twilight zone that separates the


living from the nonliving
Medical importance of viruses

Viraldiseases range from minor ailments such as the common cold


to terrifying diseases such as rabies or AIDS

They may be sporadic like mumps, endemic like infectious hepatitis,


epidemic like dengue fever or pandemic like influenza

They may be localised to circumscribed areas (as some


arbovirus diseases) or worldwide (as Herpes simplex)

Viruses can cause cancer in animals and birds, as well as in


humans
Morphology

Size

Methods of analysis

1. Passing through collodion membrane filters of graded porosity


(gradocol membranes)

2. Ultracentrifuge

3. Electron microscope

4. X-ray crystallography
Size of Viruses

A small virus has a diameter of about 20 nm.


Example: Parvovirus

A large virus have a diameter of up to 400 nm.


Example: Poxviruses
Structure and shape

Virion

The complete infectious unit of virus particle

Capsid

The protein shell, or coat, that encloses the nucleic acid genome.
Functions:
a. Protect the viral nucleic acid.
b. Participate in the viral infection.
c. Antigenic and specific for each virus type
d. Provides structural symmetry to the virus particle

Nucleocapsid

The capsid with the enclosed nucleic acid


envelope

Capsid

Viral core
DNA RNA

Single Stranded + or -

Double Stranded Segmented

Double Stranded Segmented


Circular
Symmetry of Nucleocapsid

1. Cubical/Icosahedral
(Adeno virus, Coxsackie virus, CMV, EBV, Hepatitis virus, HSV,
Polio virus, Rubella virus)

2. Helical
(Influenza, Rubeola, Mumps, Rabies, Hanta, Corona viruses)

3. Complex
(Bacteriophage, Pox viruses)
Cubic or icosahedral symmetry

An icosahedron is a polygon with 12 vertices or corners and


20 facets or sides

Two types of capsomers constitute the icosahedral capsid

1. Petagonal capsomers at the corners (pentons)

2. Hexagonal capsomers making up the sides (hexons)


Helical symmetry

Complex symmetry

T4 bacteriophage Pox virus


Envelope
A lipid-containing membrane that surrounds some viral
particles

It is derived from the plasma membrane of the host cell


during there release by budding from the cell surface

Viruses-encoded glycoproteins are exposed on the surface


of the envelope (peplomers)

A virus may have more than one type of peplomer

Envelopes confer chemical, antigenic and biological


properties on viruses

Not all viruses have the envelope, and viruses can be


divided into 2 kinds: enveloped virus and nonenveloped
(naked) virus
Cubic Helical

Naked
Virus

Enveloped
Virus
Shape of viruses

Overall shape of the virus particle varies in different groups of viruses

Spherical-shape Rod-shape Bullet-shape

Filament-shape Brick-shape Tadpole-shape


Viral Hemagglutination

A large number of viruses agglutinate erythrocytes from different


species

Hemagglutination by the influenza virus is due to the presence of


hemagglutinin spikes on the surface of the virus

In the haemagglutination test RBCs are added to serial dilutions of


viral suspension, the highest dilution that produces haemagglutination
provides the haemagglutination titer (HA units)

The haemagglutination test can be carried out in test tubes or


special plastic trays

The test is a convenient method for detection and assay of the


influenza virus and also serves to titrate killed influenza vaccine
As hemagglutination is specifically inhibited by the antibody to
virus, hemagglutination inhibition assay is useful for determining
antiviral antibody
Replication of viruses

The viral multiplication cycle can be divided into six sequential phases,
though the phases may sometimes be overlapping

1. Adsorption or attachment

2. Penetration

3. Uncoating

4. Biosynthesis

5. Maturation

6. Release
Adsorption
Virions may come into contact with cells by random collision but
adsorption takes place only if there is an affinity between the two

Differences in susceptibility to viral infection are to a large extent


based on the presence or absence of receptors on cells

Penetration
Virus particles may be engulfed by a mechanism resembling
phagocytosis, a process known as viropexis

In the case of enveloped viruses, the viral envelope may fuse with
the plasma membrane of the host cell and release the nucleocapsid
into the cytoplasm
Uncoating
Process of stripping the 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

Biosynthesis
During this phase, viral nucleic acid, capsid protein, enzymes
necessary in the various stages of viral synthesis, assembly
and release will be synthesised

Certain regulatory proteins which serve to shut down the normal


cellular metabolism and direct the sequential production of viral
components are also synthesised

The site of viral synthesis depends on the type of virus


Biosynthesis consists essentially of the following steps

1.Transcription of mRNA from the viral nucleic acid

2. Translation of the mRNA into early proteins. These are enzymes


which initiate and maintain synthesis of virus components

3.Replication of viral nucleic acid

4. Synthesis of late or structural proteins, which are the components


of daughter virion capsids

The critical step in viral biosynthesis is the transcription of mRNA


from the viral nucleic acid

The mechanisms of nucleic acid synthesis differ in the different


type of viruses
Maturation
Assembly of daughter virions follows the synthesis of nucleic acid & proteins

Virion assembly may take place in the host cell nucleus (Herpes and
adenoviruses) or cytoplasm (picorna and poxviruses)

Naked viruses are present intracellularly as fully developed virions but


in the case of enveloped viruses, only the nucleocapsid is complete

Release
Progeny of bacterial viruses release by the lysis of the infected bacterium.
In the case of animal viruses, release usually occurs without cell lysis

Myxoviruses are released by budding from the cell membrane


over a period of time

In the case of some viruses (varicella), transmission occurs directly


from cell to cell and poliovirus causes profound damage to the host cell
and may be released by cell lysis
Stages in the infection of a hosts cell and replication of a virus
Abnormal replicative cycles

Incomplete viruses
Incomplete viruses are seen in large proportions, when cells are
infected with a high dose (MOI) of the influenza virus

This is the result of defective assembly

The virus yield will have a high hemagglutinin titer but low infectivity
and this is known as the von Magnus phenomenon

Abortive infection

In nonpermissive cells, viral components may be synthesised but


maturation or assembly is defective, and either no release occurs
or the progeny is noninfectious
Defective viruses

Some viruses are genetically defective in that when they infect cells,
they are unable to give rise to fully formed progeny

Yield of progeny virions occurs only if the cells are simultaneously


infected with a helper virus, which can supplement the genetic
deficiency

(eg; hepatitis D, adeno-associated satellite viruses, which replicate


only in the presence of their helper viruses hepatitis B and
adenoviruses, respectively)
Cultivation of viruses

Systems for the propagation of viruses

Human volunteers

Animal inoculation

Embryonated eggs

Tissue culture

a) Organ culture
b) Explant culture
c) Cell culture
Animal inoculation

Mice, Infant (suckling) mice, rats, monkeys, chikens, guinea pigs,


rabbits, ferrets are used for inoculation

Growth of the virus in inoculated animals may be indicated


by death, disease or visible lesions

Animal inoculation is also used for the study of pathogenesis,


immune response, epidemiology and oncogenesis

Disadvantages of animal inoculation are that immunity may interfere


with viral growth and that animals often harbour latent viruses
Embryonated eggs

The embryonated egg offers several sites for the cultivation of viruses

Inoculation on the chorioallantoic membrane (CAM) produces visible


lesions (pocks); pock-forming viruses eg: variola or vaccinia

Inoculation into the allantoic cavity provides a rich yield of influenza


and some paramyxo viruses; hence employed for vaccine production

Inoculation into the amniotic sac is employed for the primary isolation
of the influenza virus

Yolk sac inoculation is used for the cultivation of some viruses,


chlamydiae and rickettsiae
Embryonated egg showing different routes of inoculation
Small pox (variola) virus pocks on CAM of embryonated egg
Tissue culture

Organ culture

Small bits of organs can be maintained in vitro for days and weeks,
preserving their original architecture and function

Useful for the isolation of some viruses which appear to be highly


specialised parasites of certain organs (eg: Tracheal ring culture
corona virus, a respiratory pathogen)

Explant culture

Fragments of minced tissue can be grown as explants embedded in


plasma clots or may be cultivated in suspension (eg: adenoid tissue
explant cultures adenoviruses)
Cell culture

Although embryonated eggs and laboratory animals are very useful for
isolation viruses, cell culture is the sole system for virus isolation in
most laboratories

Based on their origin, chromosomal characters and the number of


generations through which they can be maintained, cell cultures
are classified into three types

1. Primary cell cultures

2. Diploid cell cultures (semi-continuous)

3. Continuous cell lines


Primary cell cultures

Prepared directly from animal or human tissues and can be


subcultured only once or twice (eg: monkey kidney, human
embryonic kidney cell cultures)

Useful for the isolation of viruses and their cultivation for vaccine
production

Diploid cell cultures


Derived from human fetal tissue and can be subcultured 20-50 times
(human diploid fibroblasts MRC-5, WI-38 (derived from normal
embryonic lung tissue)

They are useful for the isolation of some fastidious pathogens

They are also useful for the production of viral vaccines


(eg: poliomyelitis, rubella, rabies, CMV, VZV)
Continuous cell lines

These are cells of a single type, usually derived from cancer cells
and capable of continuous serial cultivation indefinitely
(eg: HeLa, HEp-2, Vero, KB cell lines)

Some continuous cell lines in common use


HeLa Human carcinoma of cervix cell line

HEp-2 Human epithelioma of larynx cell line

Vero Vervet monkey kidney cell line

KB Human carcinoma of nasopharynx cell line

McCoy Human synovial carcinoma cell line

BHK-21 Baby hamster kidney cell line


Detection of virus growth in cell cultures

Cytopathic effect
Many viruses cause morphological changes in cultured cells, these
changes can be readily observed by microscopic examination and
these changes are known as cytopathic effects (CPE)

Cytopathic effects may be cytocidal (cell death) or non-cytocidal

Non-cytocidal effects include acidophilic or basophilic inclusion bodies


in the nucleus or cytoplasm, or both; cell fusion, and transformation

Cytopathic effects can be so characteristic of individual viruses that


they can often be used to identify viruses
(Measles syncytium formation; adenovirus large granular clumps)
Normal cell CPE

(a)Cytoplasmic inclusion body caused by rabies virus in brain tissue


(b)Syncytium formed by cell fusion due to infection by measles virus
Metabolic inhibition
When viruses grow in cell cultures, cell metabolism is inhibited and
there is no acid production

Hemadsorption
When hemagglutinating viruses grow in cell culture, their presence
can be indicated by the addition of guinea pig erythrocytes to the
cultures

Interference
The growth of a non-cytopathogenic virus in cell culture can be tested
by the subsequent challenge with a known cytopathogenic virus

Transformation
Oncogenic viruses induce cell transformation and loss of contact
inhibition, so that growth appears in a piled-up fashion producing
microtumors

Immunofluorescence
Viral assay
The virus content of a specimen can be assayed in two ways

1. Total virus particles: Electron microscopy and haemagglutination

2. Assay of infectivity (with reference to the infectious virions only)

a)Quantal assays
Only indicate the presence or absence of infectious viruses but using
serial dilutions of virus suspensions and with the aid of statistical
methods, reasonably accurate estimates of infectivity can be obtained

End points used for infectivity titration are the death of the animal,
production of hemagglutinin in allantoic fluid or the appearance of
CPE in cell cultures

The titers are expressed as the 50 percent infectious dose (ID50)


or LD50
b) Quantitative assays
Similar to the estimation of bacterial viable counts by colony
counting

Two methods are available;

1.Plaque assay
A viral suspension is added to a monolayer of cultured cells in a bottle
or petri dish, and after adsorption, the medium is removed and
replaced with a solid agar gel

Each infectious viral particle gives rise to a localised focus of infected


cells that can be seen with the naked eye. Such foci are known
as plaques

2. Pock assay
Plaque assay Pock assay
Classification and nomenclature of viruses

Viruses are classified on the basis of biological, physical and


chemical properties

Viruses are broadly classified into DNA and RNA viruses and then
further divided into families, subfamilies, genera and species
Viroids

Viroids are single-stranded circular RNA molecules that lacks a


protein coat and they are mainly plant pathogens

Prions

Prions are infectious proteins without any detectable nucleic acid

They are highly resistant to physical and chemical agents

They produce slow infections with very long incubation period

Diseases caused by prions in animals include, scrapie of sheep and


goats,mink encephalopathy, bovine spongiform encephalopathy

Responsible for Kuru, Creutzfeldt-Jakob disease and some other


chronic neurological degenerative diseases of humans

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