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Dept. Microbiology, Medical FacultyDept. Microbiology, Medical FacultyPadjadjaran UniversityPadjadjaran University

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a set of genesa set of genescomposed of either DNA or RNAcomposed of either DNA or RNApackages in a protein containing coatpackages in a protein containing coat

The resulting particle The resulting particle VirionVirion

Virus reproduction : virus particle infect a cell & Virus reproduction : virus particle infect a cell & program the cellular machinery to synthesize program the cellular machinery to synthesize the constituents required for the assembly of the constituents required for the assembly of new virions new virions considered an considered an intracellular intracellular parasiteparasite

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IntroductionIntroductionIntroductionIntroduction

Virus :Virus :

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Different viruses can have very different Different viruses can have very different genetics structures genetics structures reflected in their reflected in their replicative strategiesreplicative strategies

Because of their small size, viruses have Because of their small size, viruses have achieved a very high degree of genetic achieved a very high degree of genetic

economyeconomy

Viruses depend to great extent on host cell Viruses depend to great extent on host cell functions functions difficult to combat medically difficult to combat medically They do exhibit unique steps in their They do exhibit unique steps in their

replicative cycles that are potential targets replicative cycles that are potential targets for antiviral therapyfor antiviral therapy

Different viruses can have very different Different viruses can have very different genetics structures genetics structures reflected in their reflected in their replicative strategiesreplicative strategies

Because of their small size, viruses have Because of their small size, viruses have achieved a very high degree of genetic achieved a very high degree of genetic

economyeconomy

Viruses depend to great extent on host cell Viruses depend to great extent on host cell functions functions difficult to combat medically difficult to combat medically They do exhibit unique steps in their They do exhibit unique steps in their

replicative cycles that are potential targets replicative cycles that are potential targets for antiviral therapyfor antiviral therapy

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CHARACTERISTICCHARACTERISTICSS

CHARACTERISTICCHARACTERISTICSS

Small, ranging from about 20 – 300 nm diameter

Totally depend upon a living cell, either eukaryotic or prokaryotic, for replication and existence

Some viruses possess complex enzyme of their own : RNA or DNA polymerases but they cannot amplify & reproduce the information in their own genomes without assistance

Have a component a receptor-binding protein for attaching to cells

Small, ranging from about 20 – 300 nm diameter

Totally depend upon a living cell, either eukaryotic or prokaryotic, for replication and existence

Some viruses possess complex enzyme of their own : RNA or DNA polymerases but they cannot amplify & reproduce the information in their own genomes without assistance

Have a component a receptor-binding protein for attaching to cells

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Viral SizeViral Size

Length 20 - 14,000 nm

Much smaller than bacteria -

therefore filterable (infectious agent that could pass through porcelain filter; many originated in animals)

Length 20 - 14,000 nm

Much smaller than bacteria -

therefore filterable (infectious agent that could pass through porcelain filter; many originated in animals)

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CHARACTERISTICCHARACTERISTICSS

CHARACTERISTICCHARACTERISTICSS

To see the virus electron microscope

Growth need living cells/ tissues Can not growth saprophytic Only have certain enzyme for metabolism

and energy Easy mutated changes antigenic

property Multiplication different from bacteria

To see the virus electron microscope

Growth need living cells/ tissues Can not growth saprophytic Only have certain enzyme for metabolism

and energy Easy mutated changes antigenic

property Multiplication different from bacteria

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Virus structure & Virus structure & MorphologyMorphology

Virus structure & Virus structure & MorphologyMorphology

The basic design of all viruses places the The basic design of all viruses places the nucleic nucleic

acid genome on the inside of a protein shellacid genome on the inside of a protein shell capsid capsid

Two basic types of virions :Two basic types of virions :1. Enveloped viruses1. Enveloped viruses

have a nucleocapsid of nucleic acidhave a nucleocapsid of nucleic acid complexed to proteincomplexed to protein

2. Naked capsid viruses2. Naked capsid viruses have a nucleic acid genome within ahave a nucleic acid genome within a

protein shellprotein shell

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Virus structure & Virus structure & MorphologyMorphology

Virus structure & Virus structure & MorphologyMorphology

Schematic drawing of two basic type of virions

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Two basic shapes of virions :Two basic shapes of virions : 1. Cylindrical1. Cylindrical 2. Spherical2. Spherical

Some bacteriophages combine those 2 basic Some bacteriophages combine those 2 basic shapesshapes

Functions of capsid or envelope of viruses :Functions of capsid or envelope of viruses : 1. To protect the NA genome from damage 1. To protect the NA genome from damage during during extra-celullar passage of extra-celullar passage of the virus from the virus from one cell to anotherone cell to another 2. To aid in the process of entry into the cell2. To aid in the process of entry into the cell 3. To package enzymes essential for the early 3. To package enzymes essential for the early steps steps of the infection processof the infection process

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Basic viral Basic viral formsforms

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The structure The structure and relative and relative

sizes of a sizes of a number of number of

DNADNA

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The structure The structure and relative and relative

sizes of a sizes of a number of number of

RNARNA

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DNA VIRUSESDNA VIRUSES

ENVELOPEDENVELOPED NAKED NAKED

Double – strandedDouble – stranded Double – stranded Single – Double – stranded Single – strandedstranded

Icosahedral Complex Icosahedral Icosahedral

HERPES POX PAPOVA PARVO

HEPADNA ADENO

ENVELOPEDENVELOPED NAKED NAKED

Double – strandedDouble – stranded Double – stranded Single – Double – stranded Single – strandedstranded

Icosahedral Complex Icosahedral Icosahedral

HERPES POX PAPOVA PARVO

HEPADNA ADENO

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RNA VIRUSESRNA VIRUSESRNA VIRUSESRNA VIRUSES Single – stranded Double – stranded

Positive – stranded (+) Negative – stranded (-)

Naked Enveloped Enveloped Naked

*PICORNA *TOGA BUNYA *REO*CALICI *FLAVI ORTHOMYXO

CORONA PARAMYXO RETRO RHABDO

ARENA FILO

* Icosahedral; all of the rest have helical symmetry

Single – stranded Double – stranded

Positive – stranded (+) Negative – stranded (-)

Naked Enveloped Enveloped Naked

*PICORNA *TOGA BUNYA *REO*CALICI *FLAVI ORTHOMYXO

CORONA PARAMYXO RETRO RHABDO

ARENA FILO

* Icosahedral; all of the rest have helical symmetry

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The main groups of human viruses

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VIRAL REPLICATIONVIRAL REPLICATIONVIRAL REPLICATIONVIRAL REPLICATION

Virus multiplication cycle consist of 6 phases :

1. Attachment/ adsorption to the host cell2. Penetration or entry

3. Uncoating to release the genome4. Virion component production/ synthesis

5. Assembly/ maturation6. Release from the cells

Viral infections maybe productive/lytic response or nonproductive response (lysogeny)

Lysogeny may be associated with oncogenic transformation by animal viruses

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The outcome of the infection depend on :

the particular virus-host combination other factors : - the extracelullar environment - multiplicity of the infection - physiology & developmental state of the cell

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ADSORPTIONADSORPTIONADSORPTIONADSORPTIONAdsorption is the first step in every viral infection.

Adsorption involves :- virion attachment proteins- cell surface receptor proteins

Examples of viral receptors

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For some viruses co-receptors are involved in For some viruses co-receptors are involved in adsorption adsorption

HIV-1 : CD4 & chemokine receptorsHIV-1 : CD4 & chemokine receptors

Viral spikes & phage tails carry attachment proteins Viral spikes & phage tails carry attachment proteins

In some case, a region of the capsid protein serveIn some case, a region of the capsid protein serve the function of attachment the function of attachment

Adsorption is enhanced by presence of multiple Adsorption is enhanced by presence of multiple attachment & receptor proteins.attachment & receptor proteins.

A particular kind of virus is capable to infecting onlyA particular kind of virus is capable to infecting only a limited spectrum of cell types a limited spectrum of cell types its host rangeits host range Differences in host range & tissue tropism due Differences in host range & tissue tropism due

toto presence or absence of the receptors presence or absence of the receptors

For some viruses co-receptors are involved in For some viruses co-receptors are involved in adsorption adsorption

HIV-1 : CD4 & chemokine receptorsHIV-1 : CD4 & chemokine receptors

Viral spikes & phage tails carry attachment proteins Viral spikes & phage tails carry attachment proteins

In some case, a region of the capsid protein serveIn some case, a region of the capsid protein serve the function of attachment the function of attachment

Adsorption is enhanced by presence of multiple Adsorption is enhanced by presence of multiple attachment & receptor proteins.attachment & receptor proteins.

A particular kind of virus is capable to infecting onlyA particular kind of virus is capable to infecting only a limited spectrum of cell types a limited spectrum of cell types its host rangeits host range Differences in host range & tissue tropism due Differences in host range & tissue tropism due

toto presence or absence of the receptors presence or absence of the receptors

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Entry & UncoatingEntry & UncoatingEntry & UncoatingEntry & Uncoating

Enveloped Animal VirusesEnveloped Animal Viruses Some enveloped viruses enter cells by direct direct

fusionfusion of plasma membrane & envelope, release the nucleocapsid directly into the cytoplasm.

Paramyxoviruses (eg. measles) retroviruses (eg. HIV-1) & herpesviruses

Other enveloped & naked viruses are taken in by receptor-mediated endocytosisreceptor-mediated endocytosis (viropexis).

orthomycovirus (eg. influenza viruses), togaviruses (eg. rubella viruses), rhabdoviruses

(eg. rabies) & coronaviruses

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The two basic modes of entry of an enveloped animal virus

into the host cell

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Entry by Entry by

direct fusiondirect fusion

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ViropexisViropexis

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Naked Capsid Animal VirusesNaked Capsid Animal Viruses

Naked capsid viruses (eg. poliovirus, reovirus,Naked capsid viruses (eg. poliovirus, reovirus, adenovirus) also appear to enter by adenovirus) also appear to enter by viropexis, butviropexis, but in this case, virus can’t escape the in this case, virus can’t escape the endosomalendosomal vesicle by membrane fusion vesicle by membrane fusion

For poliovirus : acidified endosome exposeFor poliovirus : acidified endosome expose hydrophobic domains hydrophobic domains result in the binding result in the binding ofof the virions to membrane & releases the virions to membrane & releases nucleocapsidnucleocapsid to cytoplasm to cytoplasm

For reovirus : the content of the endosome areFor reovirus : the content of the endosome are transferred to a lysosome transferred to a lysosome the lysosomal the lysosomal

proteases strip away part of the capsid proteases strip away part of the capsid protein & protein & active virionsactive virions

SUNARYATI 28Schematic of receptor-mediated endocytosis ulitilized by poliovirus for entry into host cell

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Insertion of Insertion of

glycoprotein glycoprotein

into the cell’s into the cell’s

membrane structures membrane structures

and formation of and formation of

the viral envelopethe viral envelope

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Viral release by Budding

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VIRAL LIFE

CYCLE

VIRAL LIFE

CYCLE

ATTACHMENT

PENETRATIONPENETRATION HOSTFUNCTIONS

ASSEMBLY(MATURATION)

Transcription

REPLICATION

RELEASE

UNCOATINGUNCOATING

Translation

MULTIPLICATION

Click after each step to view process

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Mechanisms of genetic changeMechanisms of genetic changeMechanisms of genetic changeMechanisms of genetic change

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MutationMutation

Many DNA viruses use the host DNA synthesis Many DNA viruses use the host DNA synthesis machinerymachinery

for replicating their genomes for replicating their genomes

However the largest animal viruses code for their However the largest animal viruses code for their own own DNA polymerases, & these enzymes are not DNA polymerases, & these enzymes are not as as effective at proofreading as the cellular effective at proofreading as the cellular polymerases polymerases

The resulting higher error rates in DNA The resulting higher error rates in DNA replication replication endow the viruses with the potential endow the viruses with the potential for a high rate for a high rate of evolution, but they are of evolution, but they are partially responsible for partially responsible for the high frequency of the high frequency of defective viral particles defective viral particles

High mutation rates permit adaptation to High mutation rates permit adaptation to changed changed

conditionsconditions

MutationMutation

Many DNA viruses use the host DNA synthesis Many DNA viruses use the host DNA synthesis machinerymachinery

for replicating their genomes for replicating their genomes

However the largest animal viruses code for their However the largest animal viruses code for their own own DNA polymerases, & these enzymes are not DNA polymerases, & these enzymes are not as as effective at proofreading as the cellular effective at proofreading as the cellular polymerases polymerases

The resulting higher error rates in DNA The resulting higher error rates in DNA replication replication endow the viruses with the potential endow the viruses with the potential for a high rate for a high rate of evolution, but they are of evolution, but they are partially responsible for partially responsible for the high frequency of the high frequency of defective viral particles defective viral particles

High mutation rates permit adaptation to High mutation rates permit adaptation to changed changed

conditionsconditions

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RecombinationRecombinationHomologous recombination is common in DNA Homologous recombination is common in DNA virusesviruses

There are 2 mechanisms : 1. Which is unique to the viruses with segmented genomes, involves reassortment of the segments during a mixed infections involving 2 different viral strains. 2. Exemplified by the genetic recombination between different forms of poliovirus Recombination occurs during replication by a “copy choice” type of mechanism Poliovirus replicase switches templates to generate recombinants 33

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COMMON METHODS OF INACTIVATING COMMON METHODS OF INACTIVATING VIRUSES FOR VARIOUS PURPOSEVIRUSES FOR VARIOUS PURPOSE

COMMON METHODS OF INACTIVATING COMMON METHODS OF INACTIVATING VIRUSES FOR VARIOUS PURPOSEVIRUSES FOR VARIOUS PURPOSE

Virus may be inactivated for : Sterilize laboratory supplies

Sterilization : steam, dry heat, ethylene oxide,

-irradiation Disinfect surfaces or skin

Surface disinfection : sodium hypochlorite, glutaraldehyde.

formaldehyde, peracetic acidSkin disinfection : chlorhexidine, 70% ethanol,

iodophores Vaccine production :

formaldehyde, -propiolactone, psoralen + uv irradiation, detergents (subunit vaccines)

Virus may be inactivated for : Sterilize laboratory supplies

Sterilization : steam, dry heat, ethylene oxide,

-irradiation Disinfect surfaces or skin

Surface disinfection : sodium hypochlorite, glutaraldehyde.

formaldehyde, peracetic acidSkin disinfection : chlorhexidine, 70% ethanol,

iodophores Vaccine production :

formaldehyde, -propiolactone, psoralen + uv irradiation, detergents (subunit vaccines)

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Cultivation in embryonated eggCultivation in embryonated egg

Virus injected into appropriate region

Method widely used for production of vaccines

Virus injected into appropriate region

Method widely used for production of vaccines

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Cultivation in cell cultures

Cultivation in cell cultures

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Cultivation in cell cultureCultivation in cell culture

Cytopathic effect - normal monolayer structure is disrupted by viral

infection Cell lines developed from embryonic

tissue Continuous cell lines (immortal) - HeLa Maintenance of cell culture lines is

technically difficult; must be kept free of microbial contamination.

Cytopathic effect - normal monolayer structure is disrupted by viral

infection Cell lines developed from embryonic

tissue Continuous cell lines (immortal) - HeLa Maintenance of cell culture lines is

technically difficult; must be kept free of microbial contamination.

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TRANSMISSION OF VIRAL INFECTIONTRANSMISSION OF VIRAL INFECTIONTRANSMISSION OF VIRAL INFECTIONTRANSMISSION OF VIRAL INFECTION

DIRECT CONTACTDIRECT CONTACTdroplet/aerosol

influenza virusmorbilli virussmallpox virus

GASTROINTESTINAL TRACT (ORALLY)GASTROINTESTINAL TRACT (ORALLY)enterovirusHepatitis A, E viruspoliomyelitis virus

ANIMAL BITEANIMAL BITE : rabies virus VECTORVECTOR : dengue virus PARENTERALPARENTERAL : Hepatitis B, C virus, HIV

DIRECT CONTACTDIRECT CONTACTdroplet/aerosol

influenza virusmorbilli virussmallpox virus

GASTROINTESTINAL TRACT (ORALLY)GASTROINTESTINAL TRACT (ORALLY)enterovirusHepatitis A, E viruspoliomyelitis virus

ANIMAL BITEANIMAL BITE : rabies virus VECTORVECTOR : dengue virus PARENTERALPARENTERAL : Hepatitis B, C virus, HIV

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PATHOGENESIS OF VIRAL DISEASESPATHOGENESIS OF VIRAL DISEASESPATHOGENESIS OF VIRAL DISEASESPATHOGENESIS OF VIRAL DISEASES

Viral pathogenesis :interaction of viral and host factors

leads to disease production

Virus pathogenic if : can infect and cause signs of disease of the host

Virus virulent : produce more severe disease Steps in viral pathogenesis :

Viral entry & primary replicationViral spread and cell tropismCell injury & clinical illnessRecovery from infectionVirus shedding

Viral pathogenesis :interaction of viral and host factors

leads to disease production

Virus pathogenic if : can infect and cause signs of disease of the host

Virus virulent : produce more severe disease Steps in viral pathogenesis :

Viral entry & primary replicationViral spread and cell tropismCell injury & clinical illnessRecovery from infectionVirus shedding

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DIAGNOSTIC METHODS OF VIRAL DIAGNOSTIC METHODS OF VIRAL INFECTIONINFECTION

DIAGNOSTIC METHODS OF VIRAL DIAGNOSTIC METHODS OF VIRAL INFECTIONINFECTION

1. Clinical symptoms2. Laboratory diagnosis

Typical clinical symptoms Polyomyelitis Chicken Pox Measles Mumps

1. Clinical symptoms2. Laboratory diagnosis

Typical clinical symptoms Polyomyelitis Chicken Pox Measles Mumps

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Laboratory Laboratory diagnosisdiagnosis

Laboratory Laboratory diagnosisdiagnosis

1. Electron microscope morphology2. Light microscope special staining

~ type of virus Variola virus inclusion bodies

Gispen staining : PASCHEN BODIES Rabies virus specimen : brain

Sellers staining : inclusion bodies in nerve cells NEGRI BODIES

Molluscum contagiosum virus skin nodule

Lugol staining : inclusion bodies in cytoplasm of epithel cell

MOLLUSCUM BODIES

1. Electron microscope morphology2. Light microscope special staining

~ type of virus Variola virus inclusion bodies

Gispen staining : PASCHEN BODIES Rabies virus specimen : brain

Sellers staining : inclusion bodies in nerve cells NEGRI BODIES

Molluscum contagiosum virus skin nodule

Lugol staining : inclusion bodies in cytoplasm of epithel cell

MOLLUSCUM BODIES

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Laboratory diagnosisLaboratory diagnosis Laboratory diagnosisLaboratory diagnosis

3. Culture specimen : depend on the diseasein vitro, in ovo, or in vivo

4. Serology Raise of antibody titer Antigen detection from the specimen Viral type identification :

agglutination, precipitation, complement fixation test, neutralization, inhibition haemagglutination, FAT, ELISA, RIA, LIA

3. Culture specimen : depend on the diseasein vitro, in ovo, or in vivo

4. Serology Raise of antibody titer Antigen detection from the specimen Viral type identification :

agglutination, precipitation, complement fixation test, neutralization, inhibition haemagglutination, FAT, ELISA, RIA, LIA

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PREVENTION AND TREATMENT OF VIRAL PREVENTION AND TREATMENT OF VIRAL INFECTIONSINFECTIONS

PREVENTION AND TREATMENT OF VIRAL PREVENTION AND TREATMENT OF VIRAL INFECTIONSINFECTIONS

1. VIRAL VACCINES Killed-virus vaccines Attenuated live-virus vaccines Future prospect : - attenuation of viruses by

genetic mapping - avirulent viral vectors - purified proteins produced using

cloned genes - synthetic peptides - subunit vaccines

- DNA vaccines

1. VIRAL VACCINES Killed-virus vaccines Attenuated live-virus vaccines Future prospect : - attenuation of viruses by

genetic mapping - avirulent viral vectors - purified proteins produced using

cloned genes - synthetic peptides - subunit vaccines

- DNA vaccines

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2. INTERFERONS2. INTERFERONS2. INTERFERONS2. INTERFERONS

IFNs : host-coded proteins of large

cytokine family inhibit viral replication produced by intact animal or

cell culture in response to viral infection or other inducers

first line of defense against viral infection

IFNs : host-coded proteins of large

cytokine family inhibit viral replication produced by intact animal or

cell culture in response to viral infection or other inducers

first line of defense against viral infection

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3. ANTIVIRAL CHEMOTHERAPY3. ANTIVIRAL CHEMOTHERAPY3. ANTIVIRAL CHEMOTHERAPY3. ANTIVIRAL CHEMOTHERAPY

A.A. Nucleoside Nucleoside analogsanalogs

Acyclovir & valacyclovir

Didanosine Gancyclovir Idoxuridine Lamivudin (3TC)

A.A. Nucleoside Nucleoside analogsanalogs

Acyclovir & valacyclovir

Didanosine Gancyclovir Idoxuridine Lamivudin (3TC)

Ribavirin Stavudine (d4T) Trifluridine Vidarabine Zalzitabine (ddC) Zidovudine (AZT)

Ribavirin Stavudine (d4T) Trifluridine Vidarabine Zalzitabine (ddC) Zidovudine (AZT)

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B. Nucleotide analogsB. Nucleotide analogs

Cidofovir : active against CMV & HSV inhibit s viral DNA

polymeraseC. Nonnucleoside reverse transcriptase C. Nonnucleoside reverse transcriptase

inhibitorinhibitorNevirapine : inhibit reverse transcriptase of Nevirapine : inhibit reverse transcriptase of HIV HIV

D. Protease inhibitorsRitonavir, Saquinavir HIV

E. Other types Amantadine & rimantadineFoscarnetMethiasone

Cidofovir : active against CMV & HSV inhibit s viral DNA

polymeraseC. Nonnucleoside reverse transcriptase C. Nonnucleoside reverse transcriptase

inhibitorinhibitorNevirapine : inhibit reverse transcriptase of Nevirapine : inhibit reverse transcriptase of HIV HIV

D. Protease inhibitorsRitonavir, Saquinavir HIV

E. Other types Amantadine & rimantadineFoscarnetMethiasone

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Questions ?Questions ?Questions ?Questions ?