Introduction to Virology AU0311WE

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Introduction to Virology

DR. MELANIE JANE A. TENDENCIA

August 3, 2011

Pathogenesis of Viral Diseases


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Microorganisms bacteria, protozoans & worms, fungi

- either single cells or composed of many cells

Cells capable of independent replication

- can synthesize their own energy & proteins

- can be seen in the light microscope

Viruses are not cells

- not capable of independent replication

- cannot synthesize own energy & proteins

- too small to be seen in the light microscope


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Capsid protein shell, or coat, that encloses the nucleic acid genome

Capsomeres - morphologic units seen in the electron microscope onthe surface of icosahedral virus particles

- represent clusters of polypeptides

Defective virus a virus particle that is functionally deficient in some aspects

of replication

Envelope

lipid-containing membrane that surrounds some virus particles- acquired during viral maturation by a budding process through a

cellular membrane

Peplomers virus-encoded glycoproteins or projections exposed on the

surface of the envelope

Nucleocapsid the protein-nucleic acid complex representing the packaged

form of the viral genome

Structural units The basic protein building block of the coat

- also called protomer

Subunit a single folded viral polypeptide chain

Virion the complete virus particle; serve to transfer the viral nucleic acid

from one cell to another

VIRAL COMPONENTS


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Characteristics of Viruses

1) Viruses - particles composed of an internal core containing

either DNA or RNA (but not both) covered by a

protein coat. Some viruses have an outer lipoprotein

membrane, called an envelope, external to the coat.

- do not have a nucleus, cytoplasm, mitochondria, or

ribosomes


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Cells both prokaryotic & eukaryotic have both DNA & RNA

= eukaryotic cells have nucleus, cytoplasm,mitochondria & ribosomes

= prokaryotesnot divided into nucleus & cytoplasm- (-) mitochondria

- (+) ribosomes can synthesize own proteins


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2) Viruses must reproduce (replicate) w/in cells, because

they cannot generate energy or synthesize

proteins obligate intracellular parasites

- vs. chlamydiae & rickettsiae cannot synthesize

own energy to replicate independently

3)Viruses replicate in a manner different from that of cells- they do not undergo binary fission or mitosis

- One virus can replicate to produce hundreds ofprogeny viruses, whereas one cell divides toproduce only two daughter cells


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Comparison of Viruses & Cells

Property Viruses Cells

Type of nucleic acid DNA or RNA but not both DNA and RNA

Proteins Few Many

Lipoprotein membrane Envelope present in some Cell membrane in all

Ribosomes Absent Present

Mitochondria Absent Present in eukaryotes

Enzymes None or few Many

Multiplication by binary

fission or mitosis No Yes


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Classification of Viruses

Basis of classification

1.Virion morphology size, shape, type of symmetry, presence or absenceof peplomers, & presence or absence of membranes

2.Virus genome properties type of nucleic acid (DNA or RNA),size of genome in kilobases or kilobase pairs, strandedness

(single or double), whether linear or circular, sense (positive,negative or ambisense), segments (number, size), nucleotidesequence

3.Physicochemical properties of the virion molecular mass,

buoyant density, pH stability, thermal stability &susceptibility to physical & chemical agents, esp. ether & detergents

4. Virus protein properties number, size & functional activitiesof structural & non-structural proteins, amino acid

sequence, modifications, & special functional activities.


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5. Genome organization & replication, including gene order, number &position of open reading frames, strategy of replication &cellular sites (virion assembly & release)

6. Antigenic properties

7. Biologic properties natural host range, mode of transmission,

vectors relationships, pathogenicity, tissue tropisms &pathology

Families - -viridae (virion morphology, genome structure,strategies of replication) , Herpesviridae, Paramyxoviridae

Genera - - virus (physicochemical & serologic differences),Herpesvirus, Paramyxovirus


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DNA- Containing Viruses

Parvoviruses

Polyomaviruses

Papillomaviruses

Adenoviruses

Hepadnaviruses

Herpesviruses

Poxviruses


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Parvoviruses very small, 18-26 nm- cubic symmetry, 32 capsomeres, no envelope- Human parvovirus B 19 replicates in immature erythroid cells

& causes several adverse consequences aplasticcrisis, fifth disease, & fetal death

Polyyoma viruses small, 45 nnm, nonenveloped, cubic symmetry, 72 caps- JC virus progressive multifocal leukoencephalopathy- BK virus nephropathy in transplant patients

Papillomaviruses 55 nm- wart viruses- certain genotypes cause genital cancers

Adenoviruses medium sized, 70-90 nm, nonenveloped, cubic, 252 caps

- acute respiratory diseases, conjunctivitis, gastroenteritis


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Hepadnaviruses small, 40-48 nm, enveloped- acute & chronic hepatitis; persistent infections associated

w/ a high risk of developing cancer

Herpesviruses 150-200 nm, cubic , 162 capsomeres, lipid-contenvelope

- herpes simplex types 1 and 2 (oral and genital lesions)- varicella-zoster virus (chicken pox & shingles)

- cytomegalovirus, Epstein-Barr virus (inf mononucleosis),human herpesviruses 6 &7 (T lymphotropic) &human herpesvirus 8 (Kaposi sarcoma)

Poxviruses large brick-shaped or ovoid, 220-450 nm (L) x 140-260 nm (W)

x 140-260 nm thick- all poxviruses tend to produce skin lesions- smallpox, vaccinia, molluscum contagiosum


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RNA-Containing Viruses

Picornaviruses

ArenavirusAstroviruses Flaviviruses

Coronaviruses

Caliciviruses Rhabdoviruses

RetrovirusesHepeviruses Paramyxoviruses

Orthomyxoviruses

Reoviruses Filoviruses

Bunyaviruses

Arboviruses TogavirusesBornaviruses

Viroids Prions


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Picornaviruses small, 28-30 nm, cubic- enteroviruses (polioviruses, coxsackieviruses, echoviruses- rhinoviruses common colds- hepatovirus hepatitis A

Reoviruses medium sized, 60-80 nm- rotaviruses gastoenteritis

Arboviruses have a complex cycle involving arthropods as vectors transmit the viruses to vertebrate hosts by their bite

- dengue, yellow fever, encephalitis viruses- not a virus family, ecologic grouping

Coronaviruses- 120-160 nm particles, enveloped

- SARS

Retroviruses spherical, enveloped, 80-110 nm in diameter- the viron contains a reverse transcriptase enzyme that produces

a DNA copy of the RNA genome HIV AIDS


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Structure

Size & Shape

= 20 to 300 nm in diameter

= sphere, rod, bullet, bricks

= complex structures of precise geometric symmetry

= shape of virus particles - determined by the arrangement

of the repeating subunits that form the protein

coat, capsid


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HIV Phage O29 Phage P22

Influenza Smallpox

Filamentous Phage T4 Phage AdenovirusCoronavirus


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Viral Nucleic Acids

= viral nucleic acid (genome) located internally

- can either be single- or double-stranded DNA or

single or double-stranded RNA

- the NA can either be linear or circular

DNA always a single moleculeRNA can exist either as a single molecule or in several pieces

Almost all viruses contain only a single copy of their genome

haploid

Exception: Retrovirus family members have two copies of

their RNA genome diploid


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Capsid & Symmetry

Capsid a protein coat which surrounds the nucleic acid- made up of subunits called capsomers

- each capsomer consist of one or several proteins

E/M spherical particle, w/ a central hole

- the arrangement of capsomers gives the virusstructure its geometric symmetry

2 forms of symmetry in viral capsids:

1. icosahedral (cubic) capsomers are arranged in 20

triangles that form a symmetric figure

(icosahedron) w/ the approx outline of a sphere


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2. helical capsomers are arranged in a hollow coil that

appears rod-shaped

The helix can be either rigid or flexible.

Both the icosahedral & the helical forms can exist

either as a naked nucleocapsid or

with an outer envelope layer


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Viral Proteins

Functions:

1. The outer capsid proteinsprotect the genetic material& mediate the attachment of the virus to specific

receptors on the host cell surface. This interaction of

the viral proteins w/the cell receptor is the major

determinant of species & organ specificity.

2. Outer viral proteins are also important antigens that induce

neutralizing antibody & activate cytotoxic T cells

to kill virus-infected cells.= also the target of antibodies (antibodies bind to these

viral proteins & prevent (neutralize) the virus from

entering the cell & replicating.

(after both natural infection & immunization)


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3. internal viral proteins = some are structural

(capsid proteins of enveloped viruses)

= Enzymes (polymerases that synthesize the viral mRNA)

= Superantigens produced by some viruses,

similar in their action to the superantigens of bacteria

- herpesvirus family Epstein-Barr virus & CMV

- retrovirus mouse mammary tumor virus

- activation of CD4+ T cells is required for replication

of these viruses to occur


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Envelope

= a lipoprotein membrane composed of lipid derived from

the host cell membrane & protein that is virus-specific

= glycoproteins - found in the spike-like projections on the

surface, which attach to host cell receptors during the

entry of the virus into the cell

= matrixproteins mediates the interaction between the

capsid proteins & the envelope


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In general, the presence of an envelope confers instability

on the virus

Enveloped virus are more sensitive to heat , detergents, &

& lipid solvents such as alcohol & ether than are

nonenveloped (nucleocapsid) viruses, w/c are

composed only of NA & capsid proteins

The surface proteins of the virus, the capsid proteins or the

envelope glycoproteins the principal antigens against

w/c the host mounts its immune response to viruses.

They are also the determinants of type specificity(serotype)

Ex. Poliovirus types 1, 2, 3 distinguished by the antigenicity

of their capsid proteins


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Atypical viruslike agents

1. Defective NA & proteins but cannot replicate without a

helper virus, w/c provides the missing function2. Pseudovirions contain host cell DNA instead of viral DNA

within the capsid; can infect cells but they dont

replicate

3. Viroids consists solely of a single molecule of circular RNAwithout a protein coat or envelope

4. Prions infectious particles that are composed solely of

proteins; (-) NA

= implicated as the cause of certain slow diseasescalled transmissible spongiform encephalopathies

- Creutzfeldt-Jacob disease humans

- BSE mad cow disease

- scrapie - sheep


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Replication

Viral Growth Curve

= shows the amount of virus produced at different times

after infection

= presented in a typical growth curve the amount ofvirus produced is plotted on a logarithmic scale as a

function of time after infection.

= the time required for the growth cycle varies

it is minutes for some bacterial viruses and hours for

some human viruses


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First event the disappearance of the virus

(solid line dropping to the x axis)

- although the virus particle is no longer present , the viral

NA continues to function & begins to accumulate within

the cell (dotted line)

Eclipse period the time during which no virus is found inside the cell

- ends with the appearance of virus (solid line)

Latent period the time from the onset of infection to the appearance of

the virus extracellularly

- toward the end of this period, there is alteration of cell

morphology & marked derangement of cellular function

Infection begins with one virus particle & ends with several hundred

virus particles having been produced


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Stages of the viral growth cycle

Attachment & penetration by parental virion

Uncoating of the viral genome - viral DNA in the nucleus

Early transcription synthesis of early mRNA

Early translation - synthesis of early proteins

Viral genome replication

Late viral mRNA synthesis

late viral protein synthesis

Progeny virion assembly

Virion release from cell


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Viral Life Cycle


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Overview

- A virus needs to replicate & create progeny

- A virus cannot live on its own- Active only when replicating within a host using a hosts

resources & food- Inside a hosta virus sole purpose is to make as many

copies of itself & infect other host cells

- A viral life cycle is dependent on a host cell- A virus will remain dormant until it is able to infect the

next host, activate & replicate

- Viruses use the most efficient method to locate ahost, create progeny, & spread to other hosts


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Viral infection occurs when a virus enters a host:

* through a physical breach (cut in the skin)

* direct inoculation (mosquito bite)

* direct infection of the surface itself (inhalation ofthe virus onto trachea)

It is only after a virus enters a host that it can gainaccess to possible susceptible cells


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Viral entry

Virus must enter cells of the host organism in order for it to

reproduce & establish infectionuse the cells materials

Proteins found on the surface of the virus interact withproteins of the cell

- Attachment or adsorption occurs between the viral particle& the host cell membrane

A hole forms in the cell membrane

then the virus particle or its genetic contents are released intothe host cell

in the host cell viral reproduction may commence


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Viral replication

A virus must take control of the hosts replication

mechanisms

A distinction between susceptibility & permissibilityof a host cell is made

Permissibility determines the outcome of the infection

After control is established & the environment is setfor the virus to begin making copies of itself,replication occurs quickly


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Viral Shedding

-After a virus has made many copies of itselfexhaust the cell of its resources

-Cell is no longer useful to the virus must find new

host

Shedding the process by which virus progeny are

released to find new hosts

- final stage in the viral life cycle


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Viral latency

- Virus hide within another cell

- evade the host cell defense or immune system

- it is not the best interest of the virus to continually

replicate

-hiding latency

= virus will not produce any progeny

= will remain inactive until external stimuli (light or

stress) prompts it into activation


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Viral Entry

- the earliest stage of the infection in the viral life cycle

- virus comes into contact with the host cell &

introduces viral material into the cell

Major steps:

1. Attachment or adsorption

2. Membrane fusion or hemifusion state

3. Entry pore formation

4. Viral Penetration


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Attachment or adsorption receptors on the viral envelopebecome connected to complementary receptors on thecell membrane

- this attachment causes the two membranes to remain inmutual proximity, favoring further interactions betweensurface points

- this is also the first requisite that must be satisfied before

a cell can become infected makes the cell susceptible

- enveloped viruses exhibiting this: HIV, Herpes simplexvirus, influenza virus

- non-enveloped viruses: bacteriophages or phages virusthat infect bacteria

- they have long tails on which to attach to receptors on thebacterial surface


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Membrane fusion or hemifusion state the cell membrane ispunctured & made to further connect with the

unfolding viral envelope

Entry pore formation an opening is established for the

stabilization of an opening for which viral particlescan enter

Viral penetration viral capsid or genome is injected into thehost cells cytoplasm


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Entry via Membrane Fusion- viral receptors attach to the receptors on the surface

of the cell & secondary receptors may bepresent to initiate the puncture of the cellmembrane or fusion with the host cell

followed by the unfolding of the viral envelope

- the virus envelope blends with the cell membrane releasing its contents

- can be done only with viruses that contain an envelope- HIV, Herpes simplex, influenza virus


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Entry via Endocytosis

- the virus tricks the cell into thinking that the virusknocking at the door is nothing more thannutrition or harmless goods

- a cell takes in resources from the environment

attach goods into surface receptors

engulf them into the cell inside a vacuole

- inside the cell the virus breaks out of the vacuole

to gain access to the cytoplasm

- Examples: poliovirus, Hepatitis C virus, Foot-and-mouth disease virus


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Entry via Genetic Injection

- virus simply attaches to the surface of the cell viareceptors on the cell, & inject only its gene intothe cell, leaving the rest of the virus on thesurface

- restricted to viruses in which only the gene isrequired for infection of a cell (most all

positive-sense, single-stranded RNA viruses

- Example: phages


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- Once a virus is in a cell will activate formation of proteinsto gain full control of the host cell

- Control mechanisms include:

- suppression of intrinsic cell defenses

- suppression of cell signalling

- suppression of host cellular transcription & translation

cytotoxic effects lead to the death & decline of a

cell infected by a virus

After the introduction of the viral particle unpacking of theviral proteins & the viral genome via some form ofnucleic acid occurs as preparation for viral replication


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Viral Replication

- Formation of biological viruses during the infection processin the target host cells

-Purpose: to allow production & survival of its own kind

= by generating abundant copies of its genome &packaging these copies into viruses to beable to continue infecting new hosts

- Replication between viruses is greatly varied & depends on

the type of genes involved


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Once a virus has entered a target cell it must replicate its

genome & proteins

Replication strategies used by singlestranded RNA-containing

viruses depend on whether the genome can be used as

messenger RNA (mRNA)

Translation-competent genomes (alphaviruses, flavivruses, &

picornaviruses)

are termed plus sense or (+) sense

aretranslated by cellular ribosomes immediately after the

entry of the genome into the cytoplasm

Genome replication of (+) sense RNA-containing viruses requires

the synthesis of a minus sense or (-) sense , RNAintermediat w/c acts as template for the production of

(+) sense genomic RNA


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Retroviruses are RNA-containing viruses that replicate using a DNA

intermediate

= viral genomic RNA is (+) sense & single-stranded

-however does not serve as m-RNA

= The retrovirus RNA genome is the template for the synthesis of

a double- stranded DNA copy, termed as the provirus

- synthesis of the provirus is mediated by a

virus- encoded RNA-dependent DNA polymerase or

reverse transcriptase so named because of the

reversal of genetic information from RNA to DNA

- provirus is translocated to the nucleus & is integrated

to the host chromosomal DNA

Cl 1 D bl t d d DNA i


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Class 1: Double stranded DNA viruses

- must enter the host nucleus before it its able to replicate- requires host cell polymerases to replicate its genome highly

dependent on the cell cycle- Proper infection & production of progeny requires that the cell be

in replication as that is when the cells polymerases are

active- virus may induce the cell to forcefully undergo cell division

chronically this may lead to transformation of the cell cancer

- Example: Adenoviridae

- Poxvirus family class I virus which does not replicate within

the nucleus. (e.g. smallpox virus)


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Class 2: Single stranded DNA viruses

- replicate within the nucleus & form a double stranded DNA

intermediate during replication- Circoviridae, Parvoviridae

Class 3: Double stranded RNA viruses

- replicates in the cytoplasm as w/ most RNA viruses

- do not use the host replication polymerases to as much degree asDNA viruses

- Reoviridae, Birnaviridae

- replication is monocistronic & includes individual, segmented

genomes each of the genes code for only one protein

Class 4 & 5: Single stranded RNA viruses


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Class 4 & 5: Single stranded RNA viruses

- two types = replication is primarily in the cytoplasm

= replication is not as dependent on the cell cycle as

other DNA virusesClass 4: Single stranded RNA viruses positive (+) sense

- can be directly accessed by host polymerases to immediatelyform proteins two groups:

> viruses where the genome RNA forms the mRNA & is translatedinto a polyprotein product that is subsequently cleaved toform the mature proteins.

> viruses with complex transcription, for which subgenomic

mRNAs, ribosomal frameshifting & proteolytic processingof polyproteins may be used

- Examples: Coronaviridae, Flaviviridae, Picornaviridae

Class 5: Single stranded RNA viruses Negative (-) sense


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Class 5: Single stranded RNA viruses Negative ( ) sense

- cannot be directly accessed by host polymerases to immediatelyform proteins. Instead they must be transcripted by viralpolymerases into a readable form, which is the positive

sense reciprocal two groups:

> viruses containing non segmented genomes for which the first stepin replication is transcription from the (-) stranded genomeby the viral RNA-dependent RNA polymerase to yieldmonocistronic mRNAs that code for the various viralproteins. A (+) sense genome copy is then produced thatserves as template for the production of the (-) strandgenome. Replication is within the cytoplasm

> viruses with segmented genomes for which replication occurs in

the nucleus & for which the viral RNA- dependent RNApolymerase produces monocistronic m RNAs from eachgenome segment.

- Examples: Orthomyxoviridae, Paramyxoviridae, Bunyaviridae,

Filoviridae & Rhabdoviridae

C


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Class 6: Positive (+) sense single stranded RNA viruses that replicatethrough a DNA intermediate

- use reverse transcriptase to convert the positive sense RNA into

DNA- use DNA to create templates of proteins (instead of using RNA),

which is spliced into the host genome using integrasereplication can then commence with the help of the host cells

polymerases

= Example: HIV

Class 7: Double-stranded DNA viruses that replicate through a singlestranded RNA intermediate

- viruses have a double-stranded, gapped genome that is filled in to

form a covalently closed circle (ccc DNA) that serves a atemplate for production of viral mRNAs & a subgenomicRNA. The pregenomic RNA serves as template for theviral reverse transcriptase & for production of DNA genome

= Example: Hepatitis B virus

Viral Shedding


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Viral Shedding

- refers to the successful production of virus progeny &that the progeny is leaving the cell to infect otherhost cells

- shedding from a single cell, from one part of the body to

another part, and shedding from bodies into theenvironment where the viruses may infect other bodies

= via budding

= via apoptosis

= via reverse endocytosis


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Via budding

- budding through the cell envelope

- use the cells membrane for the virus itself most effective forviruses that need an envelope

- Prior to budding, the virus may put its own receptor on to thesurface of the cell in preparation for the virus to bud

through forming an envelope with the viral receptorsalready on it

- This process will slowly use up the cell membrane & eventuallylead to the demise of the cell

- this is also how antiviral responses are able to detect virusinfected cells

= Examples: HSV, SARS, smallpox

Vi t i


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Via apoptosis

- cell suicide release of progeny into the extracellular space

- usually controlledresults in the cells genome being chopped

up, before apoptotic bodies of dead cell materialclump off the cell to be absorbed bymacrophages.

- way for a virus to get into macrophages either to infect them or

simply travel to other tissues in the body- primarily used by non-enveloped viruses but enveloped viruses

may also use this

= HIV enveloped virus that exhibits this process for the infection

of macrophages


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Via reverse endocytosis

- viral progeny are synthesized within the cell & the host cellstransport system is used to enclose vacuoles of virus

progeny for release into the extracellular space

- used primarily by non-enveloped viruses, although enveloped

viruses may do this too

= Example: use of recycling viral particles in the enveloped

Varicella-Zoster virus

Vi l l t


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Viral latency

- the ability of a pathogenic virus to lie dormant within acell, denoted as the lysogenic part of the viral life cycle

- a phase in certain viruses life cycles in which afterinitial infection, virus production ceases, however the

the viral genome is nor fully eradicated

- the virus can reactivate & begin producing largeamounts of viral progeny without the host beinginfected by new outside virus stays within the host

indefinitely

- Mechanisms: episomal, proviral

Episomal latenc


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Episomal latency

- refers to the use of genetic episomes during latency

- viral genes are floating in the cytoplasm or nucleus as distinct

objects

- more vulnerable to marauding ribozymes or host foreign genedegradation than provirus latency

- Herpesviridae Herpes simplex virus undergoes episomal

latency in neuron cells & leaves genetic material floatingin the cytoplasm

- Advantages: > the virus does not need to enter the nucleus &hence may avoid ND10 domains from activating interferonvia that pathway

> far easier to maintain & reactivate than provirallatency

- Disadvantages: more exposure to cellular defenses leading topossible degadation of viral gene via cellular enzymes

Proviral latency


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Proviral latency

- begins when the virus genome integrates into the host genome

becomes a provirus

- requires that the viral gene get into the nucleus & insert itselfinto the host genome

- Example: RetrovirusesHIV the nucleus inserts its gene between Long Terminal

Repeats using integrase & remains within the host owngene

- Advantages: automatic host cell division results in replication of the

viruses gene

- Disadvantages: include the need to enter the nucleus & increaseddifficulty in maintaining the latency


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Antiviral Agents

1.Nucleoside Analogs inhibit NA replication by inhibition of

polymerases for NA replication- acyclovir, lamivudine, zidovudine

2.Nucleotide attached phosphate group- cidofovir

3. Nonnucleoside Reverse transcriptase inhibitor

- binds directly to reverse transcriptase, disrupt catalysis- nevirapine

4. Protease inhibitors- saquinavir

5. Fusion inhibitor - blocks the virus & cellular membrane fusion step

6. Others Amantadine & rimantadine- foscarnet

INTERFERON host-coded proteins that are members of the largecytokine family which inhibit viral replication


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Viral Vaccines

Killed-Virus VaccinesAttenuated Live-virus vaccines

Table 30-9

Introduction to Virology AU0311WE

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