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 genomesconsist 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

Smallpox was endemic in China by 1000BC. In response, the practice of variolation was developed. Recognizing that survivors of smallpox outbreaks were protected from subsequent infection, variolation involved inhalation of the dried crusts from smallpox lesions like snuff, or in later modifications, inoculation of the pus from a lesion into a scratch on the forearm of a child.

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’

Cellular organisation

Growth on inanimate media

Binary fission

Both DNA and RNA

Ribosome Sensitivity to antibiotics

Sensitivity to Interferons

Bacteria + + + + + + -Mycoplasma + + + + + + -Rickettsiae + - + + + + -Chlamydiae + - + + + + +Viruses - - - - - - +

Properties of bacteria and viruses

Medical importance of viruses

Viral diseases 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

Capsid

Viral core

envelope

DNA RNA

Single Stranded

Double Stranded

Circular

+ or -

Segmented

Double Stranded Segmented

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

Enveloped

Virus

Naked Virus

Cubic Helical

Shape of viruses

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

Filament-shape Brick-shape Tadpole-shape

Spherical-shape Rod-shape Bullet-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

Elution

In myxoviruses, neuraminidase (RDE) acts on the receptor and destroys it and this leads to the reversal of hemagglutination and the release of the virus from the red cell surface

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 host’s 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 forisolation 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 culturesare 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)

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

Some continuous cell lines in common use

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)

(a)Cytoplasmic inclusion body caused by rabies virus in brain tissue (b)Syncytium formed by cell fusion due to infection by measles virus

Normal cell CPE

Metabolic inhibitionWhen viruses grow in cell cultures, cell metabolism is inhibited andthere is no acid production

HemadsorptionWhen hemagglutinating viruses grow in cell culture, their presencecan be indicated by the addition of guinea pig erythrocytes to the cultures

InterferenceThe growth of a non-cytopathogenic virus in cell culture can be testedby the subsequent challenge with a known cytopathogenic virus

TransformationOncogenic 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 assaysOnly 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

Viral Genetics

Two main mechanisms for genetic modification in viruses are mutation and recombination

Mutation

The frequency of mutation in viruses is about 10-4 to 10-8

Mutations may be lethal or sub lethal, may occur spontaneously or induced by mutagens

Mutations affecting virulence, host range, antigenicity and pock plaque morphology are of clinical and laboratory interest

Mutants which are able to grow under certain conditions (permissive conditions) and can not grow or lethal under some specific conditions (restrictive conditions) are called as ‘conditional lethal mutants’

Interaction between viruses (Genetic)

Reassortment

Found in viruses which have segmented genomes (eg: influenza, bunya and arena viruses) In a single cell infected with two related viruses, there is an exchange of segments with production of reassortants It is probably one of the ways by which the pandemic strains of the influenza virus originate in nature

Reactivation

when a cell is infected with an active virus and a different but related inactive virus, recombination may occur and as result of this the progeny possessing one or more genetic traits of inactive virus may be produced. This process is called cross reactivation or marker rescue

Interaction between viral gene products (nongenetic)

1. Phenotypic mixing

When two different viruses infect the same cell, some mix up may take place during assembly, so that progeny genome of one virus may be surrounded by a capsid belonging partially or entirely to other virus

This altered phenotype is not a stable variation

When the genome of one virus is surrounded by the entire capsid of the other virus, it is called ‘transcapsidation’

2. Genotypic mixing

It occurs when more than one complete genomes are accidentally surrounded by a single virus capsid

There is no recombination between the different genomes so that the two kinds of viral progeny are formed on passage

3. Complementation

One virus provides the gene products to the second virus in which the latter is defective, this allows the second virus to replicate

4. Interference

Infection of a cell by one virus inhibits simultaneous or subsequent infection by other virus

The most important mediator of interference is interferon

Interference may also be produced by destruction of cell receptors by an active or inactive virus

Viral interference has been applied in the field in controlling poliomyelitis outbreaks by introducing vaccine strain into the population

5. Enhancement

Mixed infection of cells may sometimes lead to increased virus yield or greater CPE

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

DNA viruses

1. Poxviridae

2. Herpesviridae

3. Adenoviridae

4. Papovaviridae

5. Parvoviridae

6. Hepadnaviridae

RNA viruses

1. Picornaviridae2. Orthomyxoviridae3. Paramyxoviridae4. Togaviridae5. Flaviviridae6. Bunyaviridae7. Arenaviridae8. Rhabdoviridae9. Reoviridae10.Coronaviridae11.Retroviridae12.Caliciviridae13. Filoviridae

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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