Virology-To Students病毒学.ppt

1,VIROLOGY,YingchunHou,M.D.,Ph.D.SchoolofLifeSciencesShaanxiNormalUniversity,2,IntroducingMyself,UniversityStudent:XianMedicalCollegeMasterDegree: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,Thecapandmethylationofbasesatthe5endofmRNAwasfirstdetectedusingreoviruses.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,ledtotheisolationofavirusthatcausesthediseaseandtherealizationthatthisvirushumanT-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(/ICTV/),whichcontainsallitspublishedinformationinaconvenientlyinteractiveformat,pluslinkstoadditionalsitesofinterest,includingtheuniversalvirusdatabaseoftheICTV(ICTVdB).Ifyouwanttolearnthedetailedinformationaboutthesystem,thefollowingwebsitesareavailableforthat:.au/viruses/welcome.htmhttp:/www.res.bbsrc.ac.uk/mirror/auz/welcome.htm/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=0.25m(250nmor2500)2.Theresolvingpowerofanelectronmicroscope=2.53.ThediameterofaRBC=8m,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-plateCaudaltubeSynthesizeitsDNAorRNAandassembleanewvirusparticle.Butmostvirusselectsecondway.Thewaysofvirusreplication:1.Semi-conservativereplication(MostofdsDNAvirus)dsDNAssDNAdsDNA2(ForeachdsDNA,onessDNAisfromoriginal,anotheroneisnewsynthesized)2.Conservativereplication(MostofdsRNAvirus)dsRNAdsRNA2(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:VirusgenomeisdoublestrandDNAwiththeRNAmidbody:DNARNAVirus,42,Thefeaturesofvirusgenomicreplication:Phaseofreplication:6-48hrs(Phages:20-60min).Length:3kb-hundredskb,include3genestomorethan300genes.VirusgenomicreplicationcaninhibitthesynthesisoftheDNA,RNAandproteinofthehostedcell.,43,Thefeaturesofthevirusgenomicnucleotides:OnlyoneDNAorRNAincluded.Exceptingreversetranscriptionvirus,virusgenesarealwaysmonoploid,thatmeanseverygeneappearsonetimeonly.Geneoverlap:SomeDNAsequencecanencodemorethan1proteins.Genomesofphagesarecontinuedthathavenointron.Mostofvirusgenomesarecomposedbydoublestrandsorsinglestrand,butsomearecomposedbyfragmentsonly,andthesefragmentsarenotlinkedtogethernaturally.Virusgenomeincludealittlebitofnon-encodingsequenceonly,thatmeanstheratioofintronandregulatorysequenceisve