General Properties of Viruses

Kalpesh Zunjarrao

Viruses do not fall under any category of unicellular organisms because:

o Do not possess cellular organisation

o Contain only 1 type of N. A. (DNA / RNA)

o Obligate intracellular parasitesLack enz necessary for protein & n. a. synthesisFor replication depend on synthetic machinery of host cell

o Multiply by complex method

MORPHOLOGY

Size: Extracellular infectious viral particle is called ‘Virion’

Viruses are much smaller than bacteriaFor a time, they were known as ‘filterable agents’

Can not be seen under light microscope

Size range: 20-300 nm

Parvovirus: 20 nm Pox virus: 300 nm (can be seen under light microscope)

Estimation of Size: Earliest method:o Passing through membrane filter of graded pore sizeo Average pore size of finest filter that allows passage of

virion gave an estimate of size

Next method:o Ultracentrifuge: depending on rate of sedimentation,

particle size was calculated

Latest & direct method:o Electron microscope

Structure: Virion consists of nucleic acid core surrounded by protein

coat called ‘capsid’

Capsid: made up of subunits called ‘Capsomers’

Genome + capsid: nucleocapsid

Functions of capsid:o Protection of n.a. core from inactivation by nucleaseso Introduction of viral genome into host by adsorbing on the

host cell surfaceo Antigenic in nature

Types of symmetries:1. Icosahedral: Icosahedron: polygon with 12 vertices or

corners & 20 facetes or sides Each facete has shape of equilateral triangle It’s a rigid structure

2. Helical: N.A. & capsomers are wound together to form

helical tube Tube can be rigid or pliable

Some viruses show complex symmetry

Viruses can be enveloped or non-enveloped Envelope is lipoprotein in natureo Lipid derived from host cello Protein: virus codedo Protein subunits are seen as projecting spikes on surface of

envelope: called ‘Peplomers’

Overall shape of virus varies with different groups of viruses:

o Most animal viruses: spherical, some: irregularo Rabies virus: bullet shapedo Ebola virus: filamentouso Pox virus: brick shapedo TMV: rod shapedo Bacteriophage: complex morphology

Chemical properties: Nucleic acids:o Viruses contain only 1 type of n. a.o Single or double stranded RNA or DNAo N. A. can be extracted by treatment with certain chemicals Proteins:o Capsid & envelopeo Protects n.a. & determines antigenic properties Some viruses contain small amount of carbohydrates Most viruses don’t possess enzymes but some of them

may possess (neuraminidase, reverse transcriptase)

Viral hemmaglutination: Large number of viruses agglutinate erythrocytes of many

species Hemmaglutination by influenza virus is due to presence of

protein spikes ‘Hemagglutinin’ Hemagglutinin has ability to bind glycoprotein receptor

sites on erythrocytes Convenient method of detection of viruses Procedure:

RBCs are added to serial dilutions of viral suspension → highest dilution producing hemmaglutination is ‘titre’

Non agglutinated RBCs settle down at bottom in the form of ‘button’

Agglutinated RBCs spread into shield like pattern

ButtonTitre

Hemagglutination is inhibited by Antibodies to virus.This principle can be used in ‘Hemagglutination inhibition test’.This test is used for detecting antiviral antibodies

Some viruses carry surface enzymes (neuraminidase) which act on receptors on erythrocytes- They are called ‘Receptor destroying enzymes’ (RDE)- Destruction of receptor leads to reversal of hemagglutination. Called as ‘Elution’

MULTIPLICATION

Genetic information required for viral replication is present in viral NA but they lack enzymes

Viruses depend on synthetic machinery of host cell Viral multiplication cycle is divided into 6 sequential

phases:o Adsorptiono Penetrationo Uncoatingo Biosynthesiso Maturationo Release

Adsorption (Attachment): Contact between virion & host cell: by random collision Adsorption takes place only if there is affinity between

them Cell surface contains some receptors to which viruses can

attach In case of influenza viruses: hemagglutinin on virus

surface attaches to glycoprotein receptors sites on respiratory epithelium

Destruction of receptors by RDE prevents viral adsorption In HIV virus: attachment between CD4 receptors on host

cell & viral surface glycoprotein ‘gp120’ Susceptibility to viral infection depends on presence or

absence of receptors on cells

Penetration: Bacterial cells possess rigid cell wall. Thus, viruses can

not penetrate into the cell. Only nucleic acid is introduced Animal cells → no cell wall → whole virus can enter into

the cell Virus particle may be engulfed by process resembling

phagocytosis, called ‘Viropexis’ In case of enveloped viruses: viral envelop fuse with

plasma membrane of host cell → nucleocapsid released into the cytoplasm

Uncoating: Stripping the virus of its outer layer & capsid In most cases, uncoating is effected by action of lysosomal

enzymes In pox virus: Uncoating is 2 step process.

1st step in phagosome: outer coat removed by lysosomal enz2nd step in cytoplasm: viral uncoating enz removes protein covering

Biosynthesis: Synthesis of viral nucleic acid, protein capsid & various

enzymes required for synthesis, assembly & release Certain ‘regulator proteins’ are also synthesized Regulator proteins: shut down normal cellular metabolism &

stimulates production of viral components Site of viral synthesis depends on type of virus Most DNA viruses: synthesize n. a. in host cell nucleus

(exception: poxvirus which synthesizes all components in host cytoplasm)

Most RNA viruses: synthesize all components in cytoplasm(Exceptions: Orthomyxoviruses, some paramyxoviruses synthesized partly in nucleus)

Proteins: always synthesized in cytoplasm

Biosynthesis consists of following steps:1. Transcription of mRNA from viral nucleic acid2. Translation of mRNA into ‘early proteins’

Early/non-structural proteins are enzymes which initiate & maintain synthesis of virus componentsThey may also shut down production of host proteins

3. Replication of viral nucleic acid4. Synthesis of ‘late / structural proteins’ required for viral

capsid

Critical step in biosynthesis: transcription of mRNA from viral nucleic acid

Once this is achieved, host cell resources can be used for translating mRNA into viral components

Maturation (Assembly): Assembly of daughter virions follows the synthesis of

viral nucleic acid & proteins Assembly may take place in cytoplasm or nucleus Herpes & Adenoviruses are assembled in nucleus Picorna & Poxviruses are assembled in cytoplasm At this stage, non-enveloped viruses are present

intracellularly as fully developed virions but in case of enveloped viruses, only nucleocapsid is complete

Envelops are derived from host cell membrane during process of budding

Host cell membrane that becomes envelope is modified by addition of virus-specific antigens

Release: In case of bacteriophages, release takes place by lysis of

bacterium In animal viruses, release usually occurs without cell lysis Certain viruses are released by process of budding from the

cell membrane over period of time. Host cell is unaffected & may even divide, daughter cells continuing to release virions

Progeny virions released into surrounding medium may infect other cells

In case of some viruses, transmission occurs directly from cell to cell, very little free viruses being demonstrable extracellularly in the medium

Poliovirus causes profound damage to host cell & may be released by cell lysis

From the stage of penetration till appearance of mature daughter virions, virus can not be demonstrated inside host cell

This period during which virus seems to disappear or go ‘underground’ is called as ‘eclipse phase’

Single life cycle of replication takes 15-30 mins in bacteriophages & about 15-30 hours for animal viruses

Single infected cell releases large number of progeny viruses. This can be demonstrated in bacteriophages but difficult in case of animal viruses which are released over a prolonged period

CULTIVATION

Viruses: Obligate intracellular parasites

Can not be grown on inanimate culture medium

3 methods employed for cultivation of viruses:o Inoculation into animalso Embryonated eggso Tissue culture

Animal Inoculation: Earliest method for cultivation: human volunteers → high

risk involved → used only when virus is relatively harmless

Monkeys were used for isolation of poliovirus → limited application due to cost

Use of white mice: most widely employed in virology Guinea pigs, rabbits: used in some situations Growth of virus in animal can be indicated by death,

disease or visible lesion Disadvantage: immunity may interfere with viral growth

Embryonated eggs: Embryonated hen’s egg was 1st used for cultivation by

Goodpasture (1931) & method was further developed by Burnet

Embryonated eggs offer several sites for cultivation of viruses

Inoculation on chorioallantoic membrane (CAM)o produces visible lesions (pocks)o Different viruses: different pock morphologyo Each infectious viral particle can form 1 pock. Thus, pock

counting can be used for assay of pock-forming viruses like variola & vaccinia

Inoculation into allantoic cavity:o provides rich yield of influenza & paramyxoviruseso Used for growing influenza virus for vaccine production Inoculation into amniotic sac: used for primary isolation of

influenza virus Yolk sac inoculation:

for cultivation of some viruses, Chlamydiae & Rickettsiae

Tissue culture: Tissue & organ culture: used for study of morphogenesis

& wound healing 1st application of tissue culture in virology: for

maintaining vaccinia virus in fragments of rabbit cornea Major obstacle in using tissue culture: bacterial

contamination Antibiotics: prevention of contamination Every human virus can be grown in tissue culture

Types of tissue culture: Organ culture:o Small bits of organs can be maintained in vitro preserving

their architecture & functiono Useful for isolation of viruses which appear to be

specialised parasites of certain organso Tracheal ring organ culture: for coronavirus isolation

Explant culture:o Fragments of tissues can be grown as explants embedded

in plasma clots or in suspensiono Originally known as tissue cultureo Adenoid tissue explant culture: for Adenovirus isolation

Cell culture:o Routinely employed for growing viruseso Tissues dissociated into cells by proteolytic enzymes →

cells washed → counted → suspended in growth mediumo Cell culture medium components:

Amino acids, vitamins, salt, glucose, buffer (HCO3-), fetal

calf serum, antibiotics & phenol redo Cell suspension is dispensed in bottles/ petri plates →

incubated → cells adhere to glass surface → form monolayer of cells

o Bottles incubated at stationary condition or in roller drums for aeration

Cell cultures are classified into 3 types:1. Primary cell culture:o Normal cells freshly taken from bodyo Capable of only limited growth in cultureo Eg: monkey kidney, human embryonic kidney, Human

amnion

2. Diploid cell culture:o Cells of single cell type that retain original diploid

chromosome number & karyotypeo Can be subcultured for limited number of times (due to

senescence)o Eg: Human fibroblasts

3. Continuous cell culture:o Cells of single cell type derived from cancer cellso capable of continuous serial cultivationo Eg: HeLa, HEp-2, Vero cell lines

Detection of virus growth in cell cultures:

Cytopathic effects (CPE):o Morphological changes in cultured cells → can be

observed by microscopic examinationo Viruses causing CPE are called ‘cytopathogenic viruses’o Help in presumptive identification of viruseso Eg:

- Enteroviruses produce rapid CPE by crenation of cells- Measles virus produces syncytium- Adenovirus produces large granular clumps

Metabolic inhibition:o In normal cell cultures, medium becomes acidic due to

metabolismo When viruses grow → metabolism inhibited → no acid

Hemadsorption:o Hemagglutinating viruses can be identified by addition of

guinea pig erythrocyteso If viruses are multiplying, erythrocytes adsorb onto cell

surface

Interference:o Growth of non-cytopathogenic virus in cell culture can be

tested by subsequent challenge with known cytopathogenic virus

o Growth of first inhibits infection by second virus

Transformation:o Tumor forming viruses induce cell transformation → loss

of contact inhibition → piled-up growth ‘Microtumors’

Immunofluorescence:o Cells from virus infected cultures → stained by

fluorescent conjugated antiserum → examined under UV microscope

CLASSIFICATION&

NOMENCLATURE

Till 1950, little was known about viruses They were named haphazardly, based on the disease they

caused or site of isolation They were grouped according to tropism or affinity to

different organs Were classified as Dermotropic, Neurotropic,

pneumotropic & viscerotropic Bawden suggested that nomenclature & classification

should be based on properties of viruses & not the responses

Classification & nomenclature are now official responsibility of International committee on Taxonomy of Viruses (ICTV)

Viruses are classified into 2 main divisions:Riboviruses & Deoxyriboviruses

Further classification is based on properties such as strandedness of n.a., symmetry of nucleocapsid, presence of envelop, size & shape of virion & number of capsomers

DNA viruses: Poxviridae family:o Large, brick-shaped or ovoid (300 X 240 X 100 nm)o Complex structure, having lipid containing outer coat & core

carrying single linear ds-DNAo Multiplication & maturation: in cytoplasmo Several genera

Herpesviridae family:o Medium sized containing linear ds-DNAo Icosahedral nucleocapsid has 162 capsomers surrounded by

lipid containing envelopeo Multiplication: in nucleuso Maturation: by budding through nuclear membraneo Only 1 genus: Herpesvirus

Adenoviridae family:o Medium sized (70-90 nm) non-enveloped, icosahedral

viruses with 252 capsomerso 2 genera: Mastadenovirus & Aviadenovirus

Papovaviridae family:o Small (40-55 nm), non-enveloped, ds-DNA viruses with

72 capsomerso 2 genera: Papillomavirus & Polyomavirus

Parvoviridae family:o Very small (18-26 nm) non-enveloped, ss-DNA viruses

with 31 capsomerso 3 genera: Parvovirus, Adenosatellovirus, Densovirus

Hepadnaviridae family:o Spherical (42 nm) virus with core surrounded by envelope

having specific antigenso Human Hepatitis type B virus & related viruses of animals

RNA viruses: Picornaviridae family:o Small (20-30 nm), non-enveloped, icosahedral, ss-RNA

viruseso 3 genera: Enterovirus, Rhinovirus, Hepatovirus (HAV)

Orthomyxoviridae family:o Medium sized (80-120 nm), spherical or elongated,

enveloped viruses with hemagglutinin & neuraminidase peplomers

o Genome consists of ss-RNA in several pieceso 1 genus: Influenzavirus

Paramyxoviridae family:o Pleomorphic virions with lipid envelope having surface

projectionso Genome: un-segmented, linear ss-RNAo 3 genera: Paramyxovirus, Morbillivirus, Pneumovirus

Togaviridae family:o Spherical viruses (40-70 nm) with lipoprotein envelope &

ss-RNAo Multiply in arthropods & vertebrateso 3 genera: Alphavirus (Group A arboviruses), Rubivirus,

Pestivirus

Flaviviridae family:o Formerly grouped as group B arboviruses under togaviridae

Bunyaviridae family:o Spherical, enveloped virions (90-100 nm)o Arthropod-borne viruseso 5 genera: Bunyavirus, Hantavirus, Nairovirus, Phlebovirus,

Ukuvirus

Arenaviridae family:o Spherical or pleomorphic viruses (50-300 nm) with number of

electron dense particles giving sandy appearanceo Rodent parasites but can infect humans rarelyo 1 genus: Arenavirus

Rhabdoviridae family:o Bullet shaped viruses (130-300 nm long & 70 nm wide) with

lipoprotein envelope carrying peplomerso 2 genera: Vesiculovirus, Lyssavirus

Reoviridae family:o Icosahedral, non-enveloped viruses (60-80 nm) with double

layered capsido Genome: ds-RNA in 10-12 pieceso 3 genera: Reovirus, Orbivirus, Rotavirus

Coronaviridae familyo Pleomorphic, enveloped viruses (100 nm) with club-shaped

peplomers. Only 1 genus: Coronavirus

Retroviridae family:o Icosahedral viruses (100 nm) with lipoprotein envelopeo They have RNA dependent DNA polymerase (Reverse

transcriptase)o 3 subfamilies: Oncovirinae, Splumivirinae, Lentivirinae

Calciviridae family:o Naked spherical particles (35-39 nm) with 32 cup shaped

depressions arranged in symmetry

Filoviridae family:o Long, filamentous, enveloped viruses (80 nm in diameter &

14,000 nm long) with helical nucleocapsid & ss-RNA

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