Virology-To Students病毒学.ppt

1 VIROLOGY YingchunHou M D Ph D SchoolofLifeSciencesShaanxiNormalUniversity 2 IntroducingMyself UniversityStudent Xi anMedicalCollegeMasterDegree KunmingMedicalCollegeBasicMedicalTeacher FMMUPh D Degree FMMUPhysician InternalMedicine inXi JingHospitalResearchAssociate WayneStateUniversityPostdoctoralScholar NIHPostdoctoralResearchFellow WayneStateUniversitySeniorScientist VAMedicalCenter Detroit MIProfessor SNNU 3 Chapter1 VirusandVirology Virus Virusesbelongtomicroorganism buttheyarethemostsimplebiologicalorganismintheworldthatcontainDNAorRNAcoreandproteincoatorcapsidonly Virion Virusesaresosmallthatyoucanseethemunderapowerfulmicroscopeorelectromicroscopeonly Thelifecycleofvirusesisverysimplethatshowsvirusreplicationonly Allofvirusmustsurviveincells 4 Mostofvirusesareveryharmfultohumanbeing animal orplants Noanyantibioticissensitivetovirussofar So virusdiseasesaremorethreatenedthanothermicro organismstohumanhealth Pseudovirus Some virus containsDNAorRNAinside buttheDNAorRNAisfromhost Pseudoviruscannotbereplicated anditisnotpathogenalso ReversetranscriptvirusorDNAprovirus SomeRNAviruscanbereverselytranscribedascDNA andthecDNAcancombineintothegenomeofhost Viroid Nocoatorcapsid so thenucleicacidisnude Viroidisthepathogentoplantsusually Phage Thevirusparasitesinbacteriaonly 5 VirologyandItsImportancetoUsVirology Aacademicfieldthatincludestheresearchesonvirusandcontrolthevirustoprotecthumanbeingandplant Sometimeandforsomevirus wecanusethevirustomakeresearches Therolesofvirusestothedevelopmentoflifesciences Virusdiseases cardiovasculardiseases andcancersarethemainkillerstomodernhumanbeing Exceptingthat virologyisveryimportantforthedevelopmentoflifesciencesasthefollows 6 TheRoleofAnimalVirusesinUnderstandingtheBasicOutlinesofEukaryoticGeneRegulation Thefirsttranscriptionalenhancerelement actsinanorientation anddistance independentfashion wasdescribedintheSV40genome aswasadistance andorientation dependentpromoterelementobservedwiththesamevirus Thetranscriptionfactorsthatbindtothepromoter SP 1 ortotheenhancerelement suchasAP 1 AP 2 andwhichareessentialtopromotetranscriptionalongwiththebasalfactors werefirstdescribedwithSV40 AlmosteverythingweknowaboutthestepsofmessengerRNA mRNA processingbeganwithobservationsmadewithviruses Forexamples RNAsplicingofnewtranscriptswasfirstdescribedwiththeadenoviruses ThesignalforpolyadenylationinthemRNAwasfirstfoundusingSV40 7 Thecapandmethylationofbasesatthe5 endofmRNAwasfirstdetectedusingreoviruses Thediscoveryoftheroleofinterferonininducingasetofgeneproductsthatactontranslationalregulatoryeventsowesitsoriginstovirology Posttranslationalprocessingofproteinsbyproteases carbohydrateadditiontoproteinsintheGolgiapparatus phosphorylationbyawidevarietyofimportantcellularproteinkinases ortheadditionoffattyacidstomembrane associatedproteinshaveallbeenprofitablystudiedusingviruses Indeed agooddealofourpresent dayknowledgeincellbiologyofhowproteintraffickingoccursandisregulatedincellscomesfromtheuseofvirus infectedcellsystems Clearly thefieldofgeneregulationhasreliedonvirologyformanyofitscentraltenets 8 TheRoleofAnimalVirusesintheRecombinantDNARevolution Thediscoveryoftheenzymereversetranscriptaseinretroviruses 5 138 notonlyhelpedtoprovehowretrovirusesreplicatebutalsoprovidedanessentialtooltoproducecomplementaryDNAs cDNAs Thefirstrestrictionenzymemapofachromosome HindIIplusIII waswithSV40DNA andthefirstDNAtoshowthespecificityofarestrictionenzymewasSV40DNAwithEcoRI SomeoftheearliestDNAcloningexperimentsusedSV40DNAintolambda orhumanb hemoglobingenesintoSV40DNA toconstructthefirstmammalianexpressionvectors Indeed adebateabouttheseveryexperimentsledtoatemporarymoratoriumonallsuchrecombinantexperiments 9 Fromthebeginning severalanimalviruseshadbeendevelopedintoexpressionvectorsforforeigngenes includingSV40 theretroviruses theadenoviruses andadeno associatedvirus whichhastheremarkablepropertyofsitepreferentialintegration Moderndaystrategiesofgenetherapywillsurelyrelyonsomeoftheserecombinantviruses ThefirstcDNAcloningofhemoglobinsequencesutilizedlambdavectorsforthecloningandreplicationofthesemRNAcopies Inanicetwistofevents thelong elusivehepatitisvirusC non A non B wasclonedfromserumusingrecombinantDNAtechniques reversetranscriptase andlambdaphagevectors 10 TheRoleofAnimalVirologyinOncology Itisnottoostrongastatementtosaythatweoweagreatproportionofourpresentunderstandingoftheoriginsofhumancancerstotwomajorgroupsofanimalviruses theretrovirusesandDNAtumorviruses TheoncogeneswerefirstdiscoveredandproventoexistinavirusandtheninthehostcellgenomeusingRoussarcomavirus Awidevarietyofretroviruseshavecaptured altered anddeliveredoncogenestothevirologists Theinsertionofretrovirusesintothegenomesofcancerouscellsalsohelpedtolocateadditionaloncogenes Thesecondgroupofgenesthatcontributetotheoriginsofhumancancers thetumorsuppressorgenes hasbeenshowntobeintimatelyassociatedwiththeDNAtumorviruses 11 Geneticalterationsatthep53locusarethesinglemostcommonmutationsknowntooccurinhumancancers 60 to65 ofthetime Thep53proteinwasfirstdiscoveredinassociationwiththeSV40largeT antigen SV40 thehumanadenoviruses andthehumanpapillomavirusesallencodeoncogenesthatproduceproteinsthatinteractwithandinactivatethefunctionsoftwotumorsuppressorgeneproducts theretinoblastomasusceptibilitygeneproduct Rb andp53 Thecellularoncogenesandthetumorsuppressorgenesinhumancancershavebeenstudiedandunderstoodmostprofitablyusingtheseviruses 12 Thevirusesthatcausecancershaveprovidedsomeofthemostextraordinaryepisodesinmodernanimalvirology ThestoryoftheEpstein Barrvirusanditsroleinseveralcancers aswellasininfectiousmononucleosis providesuswiththebestindetectivestoryscience Thestoryisnotyetcompleteandmanymysteriesremain Similarly theidentificationofanewpathologicdisease adultT cellleukemia ledtotheisolationofavirusthatcausesthediseaseandtherealizationthatthisvirus humanT cellleukemiavirus HTLV 1 hadbeenfoundpreviously Althoughthisdiscoveryprovidedthevirus thereisyettobeasatisfactoryexplanationofhowthisviruscontributestoadultT cellleukemia 13 EquallyinterestingistheroadtothehepatitisBvirusandhepatocellularcarcinomas By1967 S KrugmanandhiscolleagueshadgoodevidencedistinguishingbetweenhepatitisAandBviruses andinthesameyearB Blumbergetal detectedtheAustraliaantigen Throughatortuouspath iteventuallybecameclearthattheAustraliaantigenwasadiagnosticmarkerforhepatitisB Althoughthisfreedthebloodsupplyofthisdangerousvirus HillemanatMerck SharpandDohmeandtheChironCorporation whichlaterisolatedthehepatitisCvirus wentontoproducethefirsthumanvaccinethatpreventshepatitisBinfectionsandverylikelyhepatocellularcarcinomasassociatedwithchronicvirusinfections Theideaofavaccinethatcanpreventcancercomessome82to85yearsafterthefirstdiscoveriesoftumorviruses Atpresent inmanycountries newborninfantshavebeeninoculatedtopreventhepatitisBinfections Basedontheepidemiologicpredictions thisvaccinationprogramshouldresultinsignificantreductionoflivercancercasesin40to50yearsfromnow 14 Vaccines TheSalkandthenSabinpoliovirusvaccineswerethefirstbeneficialproductsofthecellculturerevolution Intheearly1950sintheUnitedStates justbeforetheintroductionoftheSalkvaccine about21 000casesofpoliomyelitiswerereportedannually Today thenumberisfewerthan10 Amongthemostremarkableachievementsoflastcenturyisthecompleteeradicationofsmallpox adiseasewithahistoryofover2 000years In1966 theWorldHealthOrganizationbeganaprogramtoimmunizeallindividualswhohadcomeintocontactwithaninfectedperson Thisstrategy asopposedtotryingtoimmunizeanentirepopulation whichsimplywasnotpossible workedand inOctober1977 AliMaolinofSomaliawasthelastpersonintheworldtohaveanaturallyoccurringcaseofsmallpox Becausesmallpoxhasnoanimalreservoirandrequiresperson to personcontactforitsspread mostscientistsagreethatwearefreeofthisdisease Whatmostscientistsdonotagreeoniswhetherweshouldstoresmallpoxvirussamplesasareferenceforthefuture 15 Theviralvaccinesusedinthepasthaveincludedliveattenuatedvaccines killedvirusvaccines andsubunitvaccines Boththekilledvirusvaccine Salk andtherecombinantsubunitvaccine hepatitisB Santigen werenewtothemoderneraofvirology Inthefuture wewillseeonevirus vacciniavirus presentingtheantigensofadifferentvirus theinjectionofDNA encodingviralantigens andtheuseofspecificinterleukinsorhormoneswithvaccinestostimulateimmunityatspecificlocationsinthehostandtoelucidatespecificimmunoglobulinclasses 16 Chapter2 TaxologyofVirus Classificationprinciples Nucleicacid DNA RNA Doublechain Singlechain Linearandloop RatioofG C etc Virusmorphology Shapes Band formorSpherical etc StructuresThesensitivitytofatsolvents Ether Chloroform etc Therelationbetweentheserumfeaturesandantigen Thefeaturesincellculturing Thesensitivitytootherphysicorchemicaleffects Acid Hot andBivalentpositiveionorAmphotericion Epidemicfeatures Host Transmissionway Vectors Clinicfeatures 17 Classification Acoherentandworkablesystemofclassification ataxonomy isacriticalcomponentofthedisciplineofvirology However theuniquenatureofviruseshasdefiedthestrictapplicationofmanyofthetraditionaltoolsoftaxonomyusedinotherdisciplinesofbiology Thus scientistswhoconcernthemselveswithglobaltaxonomyoforganismshavetraditionallyleftthevirusesscatteredthroughoutthemajorkingdoms reasoningthatviruseshavemoreincommonwiththeirindividualhoststhantheydowitheachother 18 Usually basedontheirhosts youcancallsomevirusesasanimalviruses associatedwithhumanhealth plantviruses Importanttoagricultureandnationaleconomy insectviruses avianviruses bacterialviruses phages andothers Also prokaryoticviruses phages andeukaryoticvirusescanbeoftenfoundinsomebooksorpapers Sofar morethan4 000speciesofvirushavebeestablishedintheworld AllvirusesaretaxologicallysortedintoaninternationallyacceptablesystemnamedasInternationalVirusTaxologicalSystem ThissystemishandledbyICTV InternationalCommitteeofTaxonomyofVirus TheICTVisacommitteeoftheVirologyDivisionoftheInternationalUnionofMicrobiologicalSocieties TheobjectivesoftheICTVaretodevelopaninternationallyagreed upontaxonomyandnomenclatureforviruses tomaintainanindexofvirusnames andtocommunicatetheproceedingsofthecommitteetotheinternationalcommunityofvirologists TheICTVpublishesanupdateofthetaxonomyatapproximately3 yearintervals 19 TheICTValsosupportsawebsite http www ncbi nlm nih gov ICTV whichcontainsallitspublishedinformationinaconvenientlyinteractiveformat pluslinkstoadditionalsitesofinterest includingtheuniversalvirusdatabaseoftheICTV ICTVdB Ifyouwanttolearnthedetailedinformationaboutthesystem thefollowingwebsitesareavailableforthat http life anu edu au viruses welcome htmhttp www res bbsrc ac uk mirror auz welcome htmhttp www ncbi nlm nih gov ICTVdB welcome htmMostimportant thevirustaxonomythathasbeendevelopedworkswell Forthetrainedvirologist thementionofavirusfamilyorgenusname suchasfamilyHerpesviridaeorgenusRotavirus immediatelyconjuresforthasetofcharacteristicsthatformthebasisforfurtherdiscussionordescription Virustaxonomyservesanimportantpracticalpurposeaswell inthattheidentificationofalimitednumberofbiologiccharacteristics suchasvirionmorphology genomestructure orantigenicproperties quicklyprovidesafocusforidentificationofanunknownagentfortheclinicianorepidemiologistandcansignificantlyimpactfurtherinvestigationintotreatmentorpreventionofavirusdisease 20 VirusPropertiesandTheirUseinTaxonomy Thetaxonomicmethodadoptedforuseinvirologyispolythetic meaningthatanygivenvirusgroupisdescribedusingacollectionofindividualproperties Thedescriptionofavirusgroupisnonsystematicinthatthereexistsnofixedlistofpropertiesthatmustbeconsideredforallviruses andnostrictformulafortheorderedconsiderationofproperties Instead asetofpropertiesdescribingagivenvirusissimplycomparedwithothervirusesdescribedinasimilarfashiontoformulaterationalgroupings Dozensofpropertiescanbelistedfordescriptionofavirus buttheybreakdowngenerallyintovirionmorphology includingsize shape capsidsymmetry andpresenceorabsenceofanenvelope virionphysicalproperties includinggenomestructure sensitivitytophysicalorchemicalinsults specificfeaturesofvirallipids carbohydrates andstructuralandnonstructuralproteins antigenicproperties andbiologicpropertiesincludingreplicationstrategy hostrange modeoftransmission andpathogenicity 21 TheHierarchy TheICTVhasadoptedauniversalclassificationschemethatemploysthehierarchicallevelsoforder family subfamily genus andspecies Becausethepolytheticapproachtoclassificationintroducesvirusesintothemiddleofthehierarchy andbecausetheICTVhastakenarelativelyconservativeapproachtogroupingtaxa levelshigherthanorderarenotcurrentlyused Levelslowerthanspecies suchasstrainsandvariants arenotofficiallyconsideredbytheICTVbutarelefttospecialtygroups 22 Chapter3 StructuresandLifeCycle I Structures 23 24 25 ShapesandSizeofVirus Themainshapesofvirusareasthefollows 1 Spheroidorgloboidviruswithorwithoutenvelope Thenumberofvirusmemberinthisshapeisbiggest Usually theyhostinhumanbeingoranimal 2 Bandorrodformvirus Usually hostinplantsorinsects 3 Brickformvirus Brickformvirusesarebiggestandmostcomplexviruses Usually hostinhumanbeingoranimal 4 Viruswithagloboidheadandarodtail Usually hostinbacteria andwegivethemadifferentname Phage 5 Insectvirusesinsideinclusionbody Oneinclusionbodycontainmanyvirusparticlesinside 26 Virussizeisverydifferentbetweenvirusesfromhundredsnmtotenmorenm So usually weuseelectronmicroscopetocheckormakeobservationonvirusparticles Youhavetorememberthefollowsinyourmind 1 Theresolvingpowerofanopticalmicroscope 0 25 m 250nmor2500 2 Theresolvingpowerofanelectronmicroscope 2 5 3 ThediameterofaRBC 8 m ThediameterofaDNAdoublechainhelix 20 Thediameterofan helix 10 Thediameterofanatom 2 3 27 VirusLifeCycle Viruseshavetoparasiteinanalivecelltosurviveandreplicatebecausetheyaresosimplethattheyhavenoanybasicstructuretosurviveindependently ReplicativeCycle AdsorptiontocellInvasionintocellRemovetheirenvelopeorcoatSynthesizetheirDNAorRNAwithnucleotidesfromhostAssembleanewvirusparticlewiththeproteinandothersfromhostReleasethemselvesfromthehostedcelltooutAnotheradsorptiontoanewcell 28 Howtocultureandamplifyvirus Bacterialcultureforphage E colistrainsandPlaque Cellcultureforhumanoranimalvirus Cellculturetechnology Protoplasmcultureforplantvirus Protoplasm PlantcellRemoveawaytheshellorwallRemainedpartcanbeculturedandgrowup 29 Adsorptiontocell 1 VirusSurfaceAdsorptionProtein Thepropertyandtheelectrostaticchargecanaffectordecidethecombinationofvirusandcell But thecombinationmediatedbyelectrostaticchargeisreversibleusually Iwanttoyouknowgp120forHIVatleast 2 VirusReceptoronCellSurface Thereceptorcanberecognizedbyvirusadsorptionprotein Receptorisveryimportanttothehostandtissuespecificity andthediseasescausedbyvirus 3 Temperature Concentrationofion andpHcanputeffectsontheelectrostaticchargeandthemobilityofcellsandvirus So theirchangeswillresultinthevirusadsorptionandinvasion 30 Invasion Penetration Insert intocell 1 Invasionofphages 1 Injectionwithanalfilament and 2 infectbacteriawithsexfimbria 1 2 31 2 Invasionofanimalviruses 1 Endocytosis 2 Fusion EnvelopeandMembrane 3 Translocation 1 2 3 32 Removetheirenvelopeandcoat Uncoating orcapsid Cell Bacterium 33 3 Invasionofplantviruses Plantvirusescannotinfectplantbythemselvesbecausethehardcellwall Theyinvadeplantbyothervectors forexample grafting pollengrain 34 SynthesizetheirDNAorRNAwithnucleotidesfromhostedcells A CombineintogenomeofhostcellasprovirusThefuturesofavirusincellB Synthesizenewvirusparticleswiththematerialresourcefromhostcell 35 Assembleanewvirusparticlewiththeproteinandothersfromhostedcells Afterthesynthesizeofacompletemoleculewasfinished themoleculeisnotacompletevirusyet Ithastobeassembledwithcapsidorcoattoformavirusthatcaninfectanothercell 1 Phage Base plateCaudaltube sheathHeadCaudalpartAnalfilamentHostedcellisbrokenVirusesarereleasedout Formanydetailedsteps youcanreadthembutIdonnotwanttoyouremembertheminmind 36 2 AnimalVirus Capsid Proteinfromcellnucleimembraneorcellmembrane CapsidcombinetoDNA RNAEnvelope Proteinisfromcellnucleimembrane plasmaorcellmembrane Someviruseshavenoenvelope Virusisreleasedoutbysomespecialtunnelortransportationstructureoncellmembraneorcellbreakage 37 3 Plantvirus Mostofplantviruseshavenoenvelope so theyaremoresimplethananimalviruses Capsidisformedautomatically Plantvirusesdonotbreakcelltoreleaseoutusually Plantvirusescantransmittoothercellsbyfilamentbetweencells 38 HostCell ParasitedCell CellReceptor VirusParticle RemovedEnvelope AssemblingVirusParticle DyingCell ReplicatingVirusDNA RNA ReleasedCompleteVirusParticle 39 Chapter4 SynthesizingofVirusMolecule ThereplicationofvirusDNAorRNAAvirusparticlehastwofuturesinacell Combineitsnucleotidesintocellgenomeandbecomeaprovirus SynthesizeitsDNAorRNAandassembleanewvirusparticle Butmostvirusselectsecondway Thewaysofvirusreplication 1 Semi conservativereplication MostofdsDNAvirus dsDNAssDNAdsDNA 2 ForeachdsDNA onessDNAisfromoriginal anotheroneisnewsynthesized 2 Conservativereplication MostofdsRNAvirus dsRNAdsRNA 2 Forboth oneisoriginal anotheroneisnewsynthesized 3 ReplicationofcircularDNASomeviruseshavetheirgenomelikealoop forexamples somephagesandanimalorhumanvirus CircularDNAvirusesareveryimportanttoscientificresearchesbecauseweusethemasvectorstocloneageneandothers 40 MostofcircularDNAvirusestakethesemi conservativereplicationway Thereare3modelsforthat 1 Cainesreplication 2 Rolling circlereplication 3 Butterflyshapedreplication Thetypesofvirusreplication 1 TypeI VirusgenomeisdoublestrandnormalchainDNA a Replicationhappensinnucleus b Replicationhappensincytoplasm 2 TypeII VirusgenomeissinglestrandnormalchainDNA Replicationhappensinnucleus Aminuschaincanbeformedbythereplicationanditcanbethetemplatetothesynthesisofthenormalchain 3 TypeIII VirusgenomeisdoublestrandRNA GenomewasdividedintomanyfragmentsandeachfragmentcanbetranscriptedtoanmRNAofsinglecistron 41 4 TypeIV VirusgenomeissinglenormalchainRNA ViruswithmultiplecistronmRNA ThemRNAisgenomicRNA anditwillbetranslatedintoprotein 5 TypeV VirusgenomeissinglestrandminuschainRNA SinglecistronisfromgenomicRNA 6 TypeVI VirusgenomeissinglestrandnormalchainRNAwiththeDNAmidbody RNADNAVirus7 TypeVII Virusgeno