1VIROLOGYYingchun Hou, M.D., Ph.D., M.D., Ph.D.School of Life SciencesSchool of Life SciencesShaanxi Normal University2Introducing MyselfUniversity Student: Xian Medical CollegeMaster Degree: Kunming Medical CollegeBasic Medical Teacher: FMMUPh.D. Degree: FMMUPhysician (Internal Medicine) in Xi-Jing HospitalResearch Associate: Wayne State UniversityPostdoctoral Scholar: NIHPostdoctoral Research Fellow: Wayne State UniversitySenior Scientist: VA Medical Center, Detroit, MIProfessor: SNNU3Chapter 1: Virus and VirologyVirus:Virus: n nVirusesbelongtomicroorganism,buttheyaretheVirusesbelongtomicroorganism,buttheyarethemostsimplebiologicalorganismintheworldthatmostsimplebiologicalorganismintheworldthatcontainDNAorRNAcoreandproteincoatorcapsidcontainDNAorRNAcoreandproteincoatorcapsidonly(Virion).only(Virion).n nVirusesaresosmallthatyoucanseethemunderaVirusesaresosmallthatyoucanseethemunderapowerfulmicroscopeorelectromicroscopeonly.powerfulmicroscopeorelectromicroscopeonly.n nThelifecycleofvirusesisverysimplethatshowsThelifecycleofvirusesisverysimplethatshowsvirusreplicationonly.virusreplicationonly.n nAllofvirusmustsurviveincells.Allofvirusmustsurviveincells.4n nMostofvirusesareveryharmfultohumanbeing,Mostofvirusesareveryharmfultohumanbeing,animal,orplants.Noanyantibioticissensitivetoanimal,orplants.Noanyantibioticissensitivetovirussofar.So,virusdiseasesaremorethreatenedvirussofar.So,virusdiseasesaremorethreatenedthanothermicro-organismstohumanhealth.thanothermicro-organismstohumanhealth.n nPseudovirus:Some“virus”containsDNAorRNAPseudovirus:Some“virus”containsDNAorRNAinside,buttheDNAorRNAisfromhost.inside,buttheDNAorRNAisfromhost.Pseudoviruscannotbereplicated,anditisnotPseudoviruscannotbereplicated,anditisnotpathogenalso.pathogenalso.n nReversetranscriptvirusorDNAprovirus:SomeReversetranscriptvirusorDNAprovirus:SomeRNAviruscanbereverselytranscribedascDNA,RNAviruscanbereverselytranscribedascDNA,andthecDNAcancombineintothegenomeofandthecDNAcancombineintothegenomeofhost.host.n nViroid:Nocoatorcapsid,so,thenucleicacidisViroid:Nocoatorcapsid,so,thenucleicacidisnude.Viroidisthepathogentoplantsusually.nude.Viroidisthepathogentoplantsusually.n nPhage:Thevirusparasitesinbacteriaonly.Phage:Thevirusparasitesinbacteriaonly.5Virology and Its Importance to UsVirology:Aacademicfieldthatincludestheresearchesonvirusandcontrolthevirustoprotecthumanbeingandplant.Sometimeandforsomevirus,wecanusethevirustomakeresearches.The roles of viruses to the development of life sciences: Virusdiseases,cardiovasculardiseases,andcancersarethemainkillerstomodernhumanbeing.Exceptingthat,virologyisveryimportantforthedevelopmentoflifesciencesasthefollows:6The Role of Animal Viruses in Understanding the Basic Outlines of Eukaryotic Gene Regulation: Thefirsttranscriptionalenhancerelement(actsinanorientation-anddistance-independentfashion)wasdescribedintheSV40genome,aswasadistance-andorientation-dependentpromoterelementobservedwiththesamevirus.Thetranscriptionfactorsthatbindtothepromoter,SP-1,ortotheenhancerelement,suchasAP-1,AP-2,andwhichareessentialtopromotetranscriptionalongwiththebasalfactors,werefirstdescribedwithSV40.AlmosteverythingweknowaboutthestepsofmessengerRNA(mRNA)processingbeganwithobservationsmadewithviruses.Forexamples,RNAsplicingofnewtranscriptswasfirstdescribedwiththeadenoviruses.ThesignalforpolyadenylationinthemRNAwasfirstfoundusingSV40.7Thecapandmethylationofbasesatthe5endofmRNAwasfirstdetectedusingreoviruses.Thediscoveryoftheroleofinterferonininducingasetofgeneproductsthatactontranslationalregulatoryeventsowesitsoriginstovirology.Posttranslationalprocessingofproteinsbyproteases,carbohydrateadditiontoproteinsintheGolgiapparatus,phosphorylationbyawidevarietyofimportantcellularproteinkinases,ortheadditionoffattyacidstomembrane-associatedproteinshaveallbeenprofitablystudiedusingviruses.Indeed,agooddealofourpresent-dayknowledgeincellbiologyofhowproteintraffickingoccursandisregulatedincellscomesfromtheuseofvirus-infectedcellsystems.Clearly,thefieldofgeneregulationhasreliedonvirologyformanyofitscentraltenets.8The Role of Animal Viruses in the Recombinant DNA Revolution:Thediscoveryoftheenzymereversetranscriptaseinretroviruses(5,138)notonlyhelpedtoprovehowretrovirusesreplicatebutalsoprovidedanessentialtooltoproducecomplementaryDNAs(cDNAs).Thefirstrestrictionenzymemapofachromosome,HindII plus III, was with SV40 DNA, and the first DNA to show thespecificityofarestrictionenzymewasSV40DNAwithEcoRI. Some of theearliestDNAcloningexperimentsusedSV40DNAintolambda,orhumanb-hemoglobingenesintoSV40DNA,toconstructthefirstmammalianexpressionvectors.Indeed,adebateabouttheseveryexperimentsledtoatemporarymoratoriumonallsuchrecombinantexperiments.9Fromthebeginning,severalanimalviruseshadbeendevelopedintoexpressionvectorsforforeigngenes,includingSV40,theretroviruses,theadenoviruses,andadeno-associatedvirus,whichhastheremarkablepropertyofsitepreferentialintegration.Moderndaystrategiesofgenetherapywillsurelyrelyonsomeoftheserecombinantviruses.ThefirstcDNAcloningofhemoglobinsequencesutilizedlambdavectorsforthecloningandreplicationofthesemRNAcopies.Inanicetwistofevents,thelong-elusivehepatitisvirusC(non-A,non-B)wasclonedfromserumusingrecombinantDNAtechniques,reversetranscriptase,andlambdaphagevectors. 10The Role of Animal Virology in Oncology:Itisnottoostrongastatementtosaythatweoweagreatproportionofourpresentunderstandingoftheoriginsofhumancancerstotwomajorgroupsofanimalviruses,theretrovirusesandDNAtumorviruses.TheoncogeneswerefirstdiscoveredandproventoexistinavirusandtheninthehostcellgenomeusingRoussarcomavirus.Awidevarietyofretroviruseshavecaptured,altered,anddeliveredoncogenestothevirologists.Theinsertionofretrovirusesintothegenomesofcancerouscellsalsohelpedtolocateadditionaloncogenes.Thesecondgroupofgenesthatcontributetotheoriginsofhumancancers,thetumorsuppressorgenes,hasbeenshowntobeintimatelyassociatedwiththeDNAtumorviruses.11Geneticalterationsatthep53locusarethesinglemostcommonmutationsknowntooccurinhumancancers(60%to65%ofthetime).Thep53proteinwasfirstdiscoveredinassociationwiththeSV40largeT-antigen.SV40,thehumanadenoviruses,andthehumanpapillomavirusesallencodeoncogenesthatproduceproteinsthatinteractwithandinactivatethefunctionsoftwotumorsuppressorgeneproducts,theretinoblastomasusceptibilitygeneproduct(Rb)andp53.Thecellularoncogenesandthetumorsuppressorgenesinhumancancershavebeenstudiedandunderstoodmostprofitablyusingtheseviruses.12Thevirusesthatcausecancershaveprovidedsomeofthemostextraordinaryepisodesinmodernanimalvirology.ThestoryoftheEpstein-Barrvirusanditsroleinseveralcancers,aswellasininfectiousmononucleosis,providesuswiththebestindetectivestoryscience.Thestoryisnotyetcompleteandmanymysteriesremain.Similarly,theidentificationofanewpathologicdisease,adultT-cellleukemia,ledtotheisolationofavirusthatcausesthediseaseandtherealizationthatthisvirushumanT-cellleukemiavirus(HTLV-1)hadbeenfoundpreviously.Althoughthisdiscoveryprovidedthevirus,thereisyettobeasatisfactoryexplanationofhowthisviruscontributestoadultT-cellleukemia.13EquallyinterestingistheroadtothehepatitisBvirusandhepatocellularcarcinomas.By1967,S.KrugmanandhiscolleagueshadgoodevidencedistinguishingbetweenhepatitisAandBviruses,andinthesameyearB.Blumbergetal.detectedtheAustraliaantigen.Throughatortuouspath,iteventuallybecameclearthattheAustraliaantigenwasadiagnosticmarkerforhepatitisB.Althoughthisfreedthebloodsupplyofthisdangerousvirus,HillemanatMerck,SharpandDohmeandtheChironCorporation(whichlaterisolatedthehepatitisCvirus)wentontoproducethefirsthumanvaccinethatpreventshepatitisBinfectionsandverylikelyhepatocellularcarcinomasassociatedwithchronicvirusinfections.Theideaofavaccinethatcanpreventcancercomessome82to85yearsafterthefirstdiscoveriesoftumorviruses.Atpresent,inmanycountries,newborninfantshavebeeninoculatedtopreventhepatitisBinfections.Basedontheepidemiologicpredictions,thisvaccinationprogramshouldresultinsignificantreductionoflivercancercasesin40to50yearsfromnow.14Vaccines:TheSalkandthenSabinpoliovirusvaccineswerethefirstbeneficialproductsofthecellculturerevolution.Intheearly1950sintheUnitedStates,justbeforetheintroductionoftheSalkvaccine,about21,000casesofpoliomyelitiswerereportedannually.Today,thenumberisfewerthan10.Amongthemostremarkableachievementsoflastcenturyisthecompleteeradicationofsmallpox,adiseasewithahistoryofover2,000years.In1966,theWorldHealthOrganizationbeganaprogramtoimmunizeallindividualswhohadcomeintocontactwithaninfectedperson.Thisstrategy,asopposedtotryingtoimmunizeanentirepopulation(whichsimplywasnotpossible),workedand,inOctober1977,AliMaolinofSomaliawasthelastpersonintheworldtohaveanaturallyoccurringcaseofsmallpox.Becausesmallpoxhasnoanimalreservoirandrequiresperson-to-personcontactforitsspread,mostscientistsagreethatwearefreeofthisdisease.Whatmostscientistsdonotagreeoniswhetherweshouldstoresmallpoxvirussamplesasareferenceforthefuture.15Theviralvaccinesusedinthepasthaveincludedliveattenuatedvaccines,killedvirusvaccines,andsubunitvaccines.Boththekilledvirusvaccine(Salk)andtherecombinantsubunitvaccine(hepatitisB,Santigen)werenewtothemoderneraofvirology.Inthefuture,wewillseeonevirus(vacciniavirus)presentingtheantigensofadifferentvirus,theinjectionofDNA-encodingviralantigens,andtheuseofspecificinterleukinsorhormoneswithvaccinestostimulateimmunityatspecificlocationsinthehostandtoelucidatespecificimmunoglobulinclasses.16Chapter 2: Taxology of VirusClassification principles:Classification principles:n n Nucleicacid:DNA,RNA,Doublechain,Singlechain,LinearNucleicacid:DNA,RNA,Doublechain,Singlechain,Linearandloop,RatioofG+C,etc.andloop,RatioofG+C,etc.n nVirusmorphology:Shapes(Band-formorSpherical,etc)Virusmorphology:Shapes(Band-formorSpherical,etc)n nStructuresStructuresn nThesensitivitytofatsolvents:Ether,Chloroform,etc.Thesensitivitytofatsolvents:Ether,Chloroform,etc.n nTherelationbetweentheserumfeaturesandantigen.Therelationbetweentheserumfeaturesandantigen.n nThefeaturesincellculturing.Thefeaturesincellculturing.n nThesensitivitytootherphysicorchemicaleffects:Acid,Hot,Thesensitivitytootherphysicorchemicaleffects:Acid,Hot,andBivalentpositiveionorAmphotericion.andBivalentpositiveionorAmphotericion.n nEpidemicfeatures:Host,Transmissionway,Vectors,ClinicEpidemicfeatures:Host,Transmissionway,Vectors,Clinicfeatures.features.17Classification:Acoherentandworkablesystemofclassification,ataxonomy,isacriticalcomponentofthedisciplineofvirology.However,theuniquenatureofviruseshasdefiedthestrictapplicationofmanyofthetraditionaltoolsoftaxonomyusedinotherdisciplinesofbiology.Thus,scientistswhoconcernthemselveswithglobaltaxonomyoforganismshavetraditionallyleftthevirusesscatteredthroughoutthemajorkingdoms,reasoningthatviruseshavemoreincommonwiththeirindividualhoststhantheydowitheachother.18Usually,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.19TheICTValsosupportsawebsite(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,suchasfamilyHerpesviridae orgenusRotavirus,immediatelyconjuresforthasetofcharacteristicsthatformthebasisforfurtherdiscussionordescription.Virustaxonomyservesanimportantpracticalpurposeaswell,inthattheidentificationofalimitednumberofbiologiccharacteristics,suchasvirionmorphology,genomestructure,orantigenicproperties,quicklyprovidesafocusforidentificationofanunknownagentfortheclinicianorepidemiologistandcansignificantlyimpactfurtherinvestigationintotreatmentorpreventionofavirusdisease.20Virus Properties and Their Use in Taxonomy:Thetaxonomicmethodadoptedforuseinvirologyispolythetic,meaningthatanygivenvirusgroupisdescribedusingacollectionofindividualproperties.Thedescriptionofavirusgroupisnonsystematicinthatthereexistsnofixedlistofpropertiesthatmustbeconsideredforallviruses,andnostrictformulafortheorderedconsiderationofproperties.Instead,asetofpropertiesdescribingagivenvirusissimplycomparedwithothervirusesdescribedinasimilarfashiontoformulaterationalgroupings.Dozensofpropertiescanbelistedfordescriptionofavirus,buttheybreakdowngenerallyintovirionmorphology,includingsize,shape,capsidsymmetry,andpresenceorabsenceofanenvelope,virionphysicalproperties,includinggenomestructure,sensitivitytophysicalorchemicalinsults;specificfeaturesofvirallipids,carbohydrates,andstructuralandnonstructuralproteins;antigenicproperties;andbiologicpropertiesincludingreplicationstrategy,hostrange,modeoftransmission,andpathogenicity.21The Hierarchy:TheICTVhasadoptedauniversalclassificationschemethatemploysthehierarchicallevelsoforder,family,subfamily,genus,andspecies.Becausethepolytheticapproachtoclassificationintroducesvirusesintothemiddleofthehierarchy,andbecausetheICTVhastakenarelativelyconservativeapproachtogroupingtaxa,levelshigherthanorderarenotcurrentlyused.Levelslowerthanspecies,suchasstrainsandvariants,arenotofficiallyconsideredbytheICTVbutarelefttospecialtygroups.22Chapter 3: Structures and Life CycleI. Structures:232425Shapes and Size of Virus:nThe main shapes of virus are as the follows: 1. Spheroid or globoid virus with or without envelope. The number of virus member in this shape is biggest. Usually, they host in human being or animal. 2. Band or rod form virus. Usually, host in plants or insects. 3. Brick form virus. Brick form viruses are biggest and most complex viruses. Usually, host in human being or animal. 4. Virus with a globoid head and a rod tail. Usually, host in bacteria, and we give them a different name: Phage. 5. Insect viruses inside inclusion body. One inclusion body contain many virus particles inside.26nVirus size is very different between viruses from hundreds nm to ten more nm. So, usually, we use electron microscope to check or make observation on virus particles. nYou have to remember the follows in your mind: 1. The resolving power of an optical microscope = 0.25m (250nm or 2500) 2. The resolving power of an electron microscope = 2.5 3. The diameter of a RBC = 8m, The diameter of a DNA double chain helix = 20, The diameter of an -helix = 10, The diameter of an atom = 2 - 3.27Virus Life Cycle:Viruseshavetoparasiteinanalivecelltosurviveandreplicatebecausetheyaresosimplethattheyhavenoanybasicstructuretosurviveindependently.Replicative Cycle:AdsorptiontocellInvasionintocellRemovetheirenvelopeorcoatSynthesizetheirDNAorRNAwithnucleotidesfromhostAssembleanewvirusparticlewiththeproteinandothersfromhostReleasethemselvesfromthehostedcelltooutAnotheradsorptiontoanewcell28How to culture and amplify virus?nBacterialcultureforphage:E. colistrainsandPlaque.nCellcultureforhumanoranimalvirus:Cellculturetechnology.nProtoplasmcultureforplantvirus:Protoplasm:PlantcellRemoveawaytheshellorwallRemainedpartcanbeculturedandgrowup.29nAdsorption to cell:1.VirusSurfaceAdsorptionProtein:Thepropertyandtheelectrostaticchargecanaffectordecidethecombinationofvirusandcell.But,thecombinationmediatedbyelectrostaticchargeisreversibleusually.Iwanttoyouknowgp120forHIVatleast.2.VirusReceptoronCellSurface:Thereceptorcanberecognizedbyvirusadsorptionprotein.Receptorisveryimportanttothehostandtissuespecificity,andthediseasescausedbyvirus.3.Temperature,Concentrationofion,andpHcanputeffectsontheelectrostaticchargeandthemobilityofcellsandvirus.So,theirchangeswillresultinthevirusadsorptionandinvasion.30nInvasion (Penetration, Insert) into cell:1.Invasionofphages:(1)Injectionwithanalfilament,and(2)infectbacteriawithsexfimbria.(1)(2)312.Invasionofanimalviruses:(1)Endocytosis,(2)Fusion(EnvelopeandMembrane),(3)Translocation.(1)(2)(3)32n nRemove their envelope and coat (Uncoating) or capsid:CellBacterium333.Invasionofplantviruses:Plantvirusescannotinfectplantbythemselvesbecausethehardcellwall.Theyinvadeplantbyothervectors,forexample,grafting,pollengrain.34n nSynthesize their DNA or RNA with nucleotides from hosted cells: A. Combine into genome of host cell as Combine into genome of host cell as provirus provirusThe futures of a virus in cellB.Synthesize new virus particles with Synthesize new virus particles with the material resource from host cell the material resource from host cell35n nAssemble a new virus particle with the protein and others from hosted cells: AfterthesynthesizeofacompletemoleculewasAfterthesynthesizeofacompletemoleculewasfinished,themoleculeisnotacompletevirusyet.Itfinished,themoleculeisnotacompletevirusyet.Ithastobeassembledwithcapsidorcoattoformahastobeassembledwithcapsidorcoattoformavirusthatcaninfectanothercell.virusthatcaninfectanothercell. 1.Phage:Base-plate Caudal tube & sheath Head Base-plate Caudal tube & sheath Head Caudal part Anal filament Hosted cell is broken Caudal part Anal filament Hosted cell is broken Viruses are released out Viruses are released out (For many detailed steps, you can read them but I don (For many detailed steps, you can read them but I don not want to you remember them in mind)not want to you remember them in mind)36 2.AnimalVirus: Capsid(Proteinfromcellnucleimembraneorcellmembrane)CapsidcombinetoDNA/RNAEnvelope(Proteinisfromcellnucleimembrane,plasmaorcellmembrane.Someviruseshavenoenvelope)Virusisreleasedoutbysomespecialtunnelortransportationstructureoncellmembraneorcellbreakage.37 3. Plant virus: Most of plant viruses have no envelope, so, they are more simple than animal viruses. Capsid is formed automatically. Plant viruses do not break cell to release out usually. Plant viruses can transmit to other cells by filament between cells. 38Host CellParasited CellCellReceptorVirusParticleRemovedEnvelopeAssemblingVirusParticleDying CellReplicatingVirusDNA/RNAReleasedCompleteVirusParticle39Chapter 4: Synthesizing of Virus Molecule n nThereplicationofvirusDNAorRNAA virus particle has two futures in a cell: Combine its nucleotides into A virus particle has two futures in a cell: Combine its nucleotides into cell genome and become a provirus; Synthesize its DNA or RNA and cell genome and become a provirus; Synthesize its DNA or RNA and assemble a new virus particle.assemble a new virus particle.But most virus select second way.But most virus select second way. The ways of virus replication:The ways of virus replication: 1. Semi-conservative replication (Most of dsDNA virus) 1. Semi-conservative replication (Most of dsDNA virus) dsDNA ssDNA dsDNA dsDNA ssDNA dsDNA2 (For each dsDNA, one ssDNA is 2 (For each dsDNA, one ssDNA is from original , another one is new synthesized)from original , another one is new synthesized) 2. Conservative replication (Most of dsRNA virus) 2. Conservative replication (Most of dsRNA virus) dsRNA dsRNA2 (For both, one is original, another one is new dsRNA dsRNA2 (For both, one is original, another one is new synthesized)synthesized) 3. Replication of circular DNA 3. Replication of circular DNA Some viruses have their genome like a loop, for examples, some Some viruses have their genome like a loop, for examples, some phages and animal or human virus. Circular DNA viruses are very phages and animal or human virus. Circular DNA viruses are very important to scientific researches because we use them as vectors to important to scientific researches because we use them as vectors to clone a gene and others.clone a gene and others.40 Most of circular DNA viruses take the semi-conservative replication Most of circular DNA viruses take the semi-conservative replication way. There are 3 models for that: way. There are 3 models for that: 1. 1. CainesCaines replication ( replication ( ) ); ; 2. Rolling-circle replication ( 2. Rolling-circle replication ( ); ); 3. Butterfly shaped replication. 3. Butterfly shaped replication. The types of virus replication:The types of virus replication: 1. Type I: Virus genome is double strand normal chain DNA. 1. Type I: Virus genome is double strand normal chain DNA. a. Replication happens in nucleus. a. Replication happens in nucleus. b. Replication happens in cytoplasm. b. Replication happens in cytoplasm. 2. Type II: Virus genome is single strand normal chain DNA. 2. Type II: Virus genome is single strand normal chain DNA. Replication happens in nucleus. A minus chain can be formed by the Replication happens in nucleus. A minus chain can be formed by the replication and it can be the template to the synthesis of the normal replication and it can be the template to the synthesis of the normal chain. chain. 3. Type III: Virus genome is double strand RNA. 3. Type III: Virus genome is double strand RNA. Genome was divided into many fragments and each fragment can Genome was divided into many fragments and each fragment can be be transcriptedtranscripted to an mRNA of single to an mRNA of single cistroncistron. . 41 4. Type IV: Virus genome is single normal chain RNA. 4. Type IV: Virus genome is single normal chain RNA. Virus with multiple cistron mRNA. The mRNA is Virus with multiple cistron mRNA. The mRNA is genomic RNA, and it will be translated into protein.genomic RNA, and it will be translated into protein. 5. Type V: Virus genome is single strand minus chain 5. Type V: Virus genome is single strand minus chain RNA. RNA. Single cistron is from genomic RNA. Single cistron is from genomic RNA. 6. Type VI: Virus genome is single strand normal chain 6. Type VI: Virus genome is single strand normal chain RNA with the DNA midbody:RNA with the DNA midbody: RNA DNA Virus RNA DNA Virus 7. Type VII: Virus genome is double strand DNA with the 7. Type VII: Virus genome is double strand DNA with the RNA midbody:RNA midbody: DNA RNA Virus DNA RNA Virus42Thefeaturesofvirusgenomicreplication:n nPhase of replication: 6-48hrs (Phages: 20-60min).Phase of replication: 6-48hrs (Phages: 20-60min).n nLength: 3kb-hundreds kb, include 3 genes to more than Length: 3kb-hundreds kb, include 3 genes to more than 300 genes.300 genes.n nVirus genomic replication can inhibit the synthesis of the Virus genomic replication can inhibit the synthesis of the DNA, RNA and protein of the hosted cell.DNA, RNA and protein of the hosted cell.43The features of the virus genomic nucleotides: n nOnly one DNA or RNA included.Only one DNA or RNA included.n nExcepting reverse transcription virus, virus genes are Excepting reverse transcription virus, virus genes are always monoploid, that means every gene appears one time always monoploid, that means every gene appears one time only.only.n nGene overlap: Some DNA sequence can encode more than Gene overlap: Some DNA sequence can encode more than 1 proteins. 1 proteins. n nGenomes of phages are continued that have no intron. Genomes of phages are continued that have no intron. n nMost of virus genomes are composed by double strands or Most of virus genomes are composed by double strands or single strand, but some are composed by fragments only, single strand, but some are composed by fragments only, and these fragments are not linked together naturally. and these fragments are not linked together naturally. n nVirus genome include a little bit of non-encoding sequence Virus genome include a little bit of non-encoding sequence only, that means the ratio of intron and regulatory only, that means the ratio of intron and regulatory sequence is very small.sequence is very small.44VirusGenomeTypesTypes Characters of genome Characters of replicating genomeTypes Characters of genome Characters of replicating genome IDoubleStrands(IDoubleStrands()DNA)DNADoubleStrands()DNADoubleStrands()DNAIISingleStrand(+)DNADoubleStrands()DNAIISingleStrand(+)DNADoubleStrands()DNAIIIDoubleStrands()RNADoubleStrands()RNAIIIDoubleStrands()RNADoubleStrands()RNAIVSingleStrand(+)RNASingleStrand(-)RNAIVSingleStrand(+)RNASingleStrand(-)RNAVSingleStrand(-)RNASingleStrand(+)RNAVSingleStrand(-)RNASingleStrand(+)RNAVISingleStrand(+)RNASingleStrand()DNAVISingleStrand(+)RNASingleStrand()DNA45SomeImportantAnimalVirus Names Types of genome Carcinogenesis Names Types of genome Carcinogenesis AdenoVirusdsDNASomeAdenoVirusdsDNASomeHerpesVirusdsDNASomeHerpesVirusdsDNASomePoxVirusdsDNASomePoxVirusdsDNASomeSV40VirusdsDNAYesSV40VirusdsDNAYesReoVirusdsRNANoReoVirusdsRNANoPolioVirusssRNANoPolioVirusssRNANoRousVirusssRNAYesRousVirusssRNAYes(RTVirus)(RTVirus)46n nThetranscriptionandtranslationofvirusgenomeVirusamplificationandinfectionisbasedonthereplicationVirusamplificationandinfectionisbasedonthereplicationofvirusgenome.ofvirusgenome.1. Phages: 1. Phages: (1)(1)NointronNointron(2)(2)PhagemRNAhasno5-Capand3-PolyAPhagemRNAhasno5-Capand3-PolyA(3)(3)Poly-cistronsPoly-cistrons47M13KE482. Animal Virus:2. Animal Virus: (1)(1) (+) RNA Translation Protein (+) RNA Translation Protein (2)(2) ( -) RNA mRNA Translation Protein ( -) RNA mRNA Translation Protein (3)(3) ( ( ) RNA (+) RNA mRNA Translation ) RNA (+) RNA mRNA Translation Protein Protein (4)(4) () DNA (+) DNA mRNA Translation () DNA (+) DNA mRNA Translation Protein Protein (5)(5) ( -) DNA () DNA (+) DNA mRNA ( -) DNA () DNA (+) DNA mRNA Translation Protein Translation Protein (6)(6) (+) RNA Reversely Transcription (-) DNA (+) RNA Reversely Transcription (-) DNA () DNA Combine into host genome () DNA Combine into host genome mRNA Translation Protein mRNA Translation Protein493. Plant Virus:3. Plant Virus:MostofplantvirusesaressRNAandalmostsametootherMostofplantvirusesaressRNAandalmostsametootherRNAvirusfortranscriptionandtranslation.RNAvirusfortranscriptionandtranslation.Replication of genome:Replication of genome:(+)RNA(+)RNA()RNA(-)RNA(+)RNA(Newvirusgenome)RNA(-)RNA(+)RNA(Newvirusgenome)mRNATranslationProteinmRNATranslationProtein50Chapter 5: Mutation of VirusMutation:Mutation: Any change of the genes sequence with that causes any Any change of the genes sequence with that causes any change of virus characteristic. The mutated virus is called change of virus characteristic. The mutated virus is called “mutant”. The non-mutated virus is called “wild-type”. “mutant”. The non-mutated virus is called “wild-type”. Usually, virus mutation is site-mutation. Usually, virus mutation is site-mutation. Self-mutation:Self-mutation: Some mutation without any inducement. RNA viruses Some mutation without any inducement. RNA viruses are much easier to self-mutation than DNA viruses because are much easier to self-mutation than DNA viruses because RNA polymerase can not revise the replication mistakes RNA polymerase can not revise the replication mistakes effectively. effectively. 51Induced-mutation:Induced-mutation:Many inducements from physics, chemistry and biology can Many inducements from physics, chemistry and biology can induce virus mutated. Virus mutation is not so good to us. induce virus mutated. Virus mutation is not so good to us. Types of mutation:Types of mutation:Synonymous mutation:Synonymous mutation: Virus gene sequence was changed Virus gene sequence was changed without any change of protein sequence because of the without any change of protein sequence because of the compatibility of amino-acid codons.compatibility of amino-acid codons.Missense mutation:Missense mutation: Virus gene sequence was changed with Virus gene sequence was changed with change of protein sequence. The results of the mutation change of protein sequence. The results of the mutation depend on the mutation location and quantity. depend on the mutation location and quantity. Nonsense mutation:Nonsense mutation: Mutation makes some amino acid codon Mutation makes some amino acid codon became a stop codon. became a stop codon. 52Promoter mutation: Promoter mutation: Mutation happened in the sequence of Mutation happened in the sequence of promoter, that results in promoter up mutation and promoter, that results in promoter up mutation and promoter down mutation. promoter down mutation. Mutants:Mutants:1. 1. Null mutant:Null mutant: A gene of virus was fully knockout or A gene of virus was fully knockout or damaged or became another protein. Null mutant can be damaged or became another protein. Null mutant can be constructed in lab. We can use this method to research a gene constructed in lab. We can use this method to research a gene function. function. 2. 2. Temperature sensitive mutant:Temperature sensitive mutant: Mutation makes some virus Mutation makes some virus sensitive to some temperature.sensitive to some temperature.3. 3. Plaque morphology mutant:Plaque morphology mutant: Mutation makes some animal Mutation makes some animal virus forming a different plaque with wild type virus. virus forming a different plaque with wild type virus. (Animal virus forms plaque on monolayer cell culture)(Animal virus forms plaque on monolayer cell culture)534. 4. Host range mutant:Host range mutant: Mutation makes virus host Mutation makes virus host specification changed. We can use this mutation to specification changed. We can use this mutation to research virus carcinogenesis. research virus carcinogenesis. 5. 5. Medicine resistance mutant:Medicine resistance mutant: Mutation makes virus Mutation makes virus medicine resistance changed. If we culture and passage medicine resistance changed. If we culture and passage virus with some chemicals or antibiotics for a long time, virus with some chemicals or antibiotics for a long time, some medicine resistant mutant can be developed. This some medicine resistant mutant can be developed. This technology can be used to virus genetics research. technology can be used to virus genetics research. 6. 6. Antibody resistant mutant:Antibody resistant mutant: Mutation makes virus antigen Mutation makes virus antigen specification changed, so, this mutation will cause many specification changed, so, this mutation will cause many changes about virus immunological, pathological, changes about virus immunological, pathological, diagnosis, and clinic features diagnosis, and clinic features 7. 7. Revertant:Revertant: Mutant was returned back to wild type by Mutant was returned back to wild type by mutation again. A mutant can return back to wild type by mutation again. A mutant can return back to wild type by the following three ways:the following three ways:54(1)(1)Change the mutated site sequence back to original Change the mutated site sequence back to original sequence.sequence.(2)(2)Make a mutation on another site of the same gene, this Make a mutation on another site of the same gene, this second site mutation may push the mutant return back second site mutation may push the mutant return back to original virus type sometime (The gene is still the to original virus type sometime (The gene is still the mutated gene).mutated gene).(3)(3)Make a mutation on another gene of the same virus, this Make a mutation on another gene of the same virus, this second gene mutation may push the mutant return back second gene mutation may push the mutant return back to original virus type sometime (Actually, virus keeps to original virus type sometime (Actually, virus keeps two mutated genes now). two mutated genes now). Revertant is very useful to research the interaction Revertant is very useful to research the interaction between protein and protein, protein and DNA and between protein and protein, protein and DNA and others. others. 55Recombination of virus: Hosted cellIntramolecular Recombination: ssDNA or ssRNA breakage and exchange.Copy-choice Recombination: ssRNA. RNA polymerase chooses a sequence as template and synthesize RNA.Genome re-assemble: Virus with fragmented genome. Fragments can be exchanged between virus genomesParticles of virus 1Particles of virus 2Or more than 2 virusNew virus with recombined genome56Reactivation: When a multiple infection happens in a cell, by the When a multiple infection happens in a cell, by the recombination as described above, a dead virus particle recombination as described above, a dead virus particle (Some virus particle can not start genome replication) may be (Some virus particle can not start genome replication) may be activated to restart replication. activated to restart replication. 57Interactionaffectingphenotype:1. 1.Phenotypic mixing:Phenotypic mixing: Multiple infection can generate the Multiple infection can generate the filial generation viruses with the coat or capsid from their filial generation viruses with the coat or capsid from their parental viruses. Phenotypic mixing happens in the parental viruses. Phenotypic mixing happens in the replication of the viruses without envelope usually. For replication of the viruses without envelope usually. For phenotypic mixed viruses, their coat or capsid is not phenotypic mixed viruses, their coat or capsid is not encoded by their DNA or RNA. But, after single passages, encoded by their DNA or RNA. But, after single passages, their DNA or RNA will encode their coat or capsid. So, their DNA or RNA will encode their coat or capsid. So, phenotype mixing is temporary only.phenotype mixing is temporary only.2. 2.Homologous interference:Homologous interference: If some virus is passaged If some virus is passaged continually, the quantity of new viruses with infection continually, the quantity of new viruses with infection ability will decrease with the further passages. Happens ability will decrease with the further passages. Happens between the viruses from same resource or the virus with between the viruses from same resource or the virus with close affinity. The mechanism is the deletion of some gene close affinity. The mechanism is the deletion of some gene sequence.sequence.58Chapter 6: Gene and Gene Map of VirusGene map construction: Gene map is a model illustration map for all genes of Gene map is a model illustration map for all genes of genome, and it is very useful to virus gene research and virus genome, and it is very useful to virus gene research and virus vector use. But you do not have to know the detail about gene vector use. But you do not have to know the detail about gene map construction.map construction.1. Recombination mapping:1. Recombination mapping: The frequency is used here as a The frequency is used here as a marker to evaluate two genes are close or not as the follows:marker to evaluate two genes are close or not as the follows:59Your interested sequence data from a virus genomeSequence 1 (Labeled) Sequence 2 (Labeled) Mutant 1Mutant 2Infect same cellRecombinationHigher ratio of recombinantLower ratio of recombinantShorter distance between the two sequences (or genes)Longer distance between the two sequences (or genes)Check the two sequences are from same or different genes, their function and nameRepeat the steps above to other two genesFigure out the virus gene map60 For the virus with the fragment genome, only two recombination results happen: recombined (Yes) and non-recombined (No). “Yes” means the two genes are located on different genome fragment, and “No” means on same fragments.612. Intertype mapping:2. Intertype mapping: By the analysis to the electrophoresis By the analysis to the electrophoresis of virus DNA, RNA or protein, figure out the gene map.of virus DNA, RNA or protein, figure out the gene map.Electrophoresis to virus DNA, RNA genome fragments and protein from different serotype or sero-strain of mutated parental and filial virus particlesAnalyze the phenotype and band poly-morphology Find the fragment on that the mutation locatedCompare the protein bands poly-morphologyRepeat analysis to a lot of mutants of this fragmentFigure out the mutation site and gene location623. Mapping by sequence analysis:3. Mapping by sequence analysis: To get basic data, people To get basic data, people have to sequence virus genome first. have to sequence virus genome first. ORF (One ORF ORF (One ORF may encode more may encode more than one proteins)than one proteins)Compare ORF Compare ORF with some known with some known proteins ORF to proteins ORF to figure its functionfigure its functionFigure out the Figure out the regular or control regular or control elements: Start elements: Start codon, Promoter, codon, Promoter, Enhancer Enhancer AnalysisAnalysisData of full length of sequenceData of full length of sequenceGene Map63The vectors for animal virus expression:The vectors for animal virus expression: 1. SV40:1. SV40:This is the original (Wild type) SV40 map64Modified 165Modified 266Chapter 7: Evolution of Virusn nThe virus original resource: Probably, viruses were originally developed from Probably, viruses were originally developed from some nucleotides molecules that can replicate themselves. some nucleotides molecules that can replicate themselves. For instances, For instances, ViroidViroid and and VirusoidVirusoid (satellite virus): (satellite virus): viroidviroid and and virusoidvirusoid are the smallest molecules (Around 300bp) are the smallest molecules (Around 300bp) that we know so far, they replicate with rolling-circle that we know so far, they replicate with rolling-circle replication way(replication way( ) ); ; Plasmid, insertion fragment, and Plasmid, insertion fragment, and transposontransposon; ; RNA molecules RNA molecules that can replicate that can replicate automatically. automatically. 67n nEvolution ways of viruses:DNAVirusesRNAVirusesMutationRecombinationMutationRecombinationReassortment68 As the described before, RNA viruses are very easy to As the described before, RNA viruses are very easy to mutate because that RNA polymerase has no replication and mutate because that RNA polymerase has no replication and transcription mistake correct function. transcription mistake correct function. Usually, RNA virus Usually, RNA virus mutation rate is one million times faster than DNA virus, it is mutation rate is one million times faster than DNA virus, it is 0.03% - 2.0%/bp/year. So, RNA viruses are more difficult to 0.03% - 2.0%/bp/year. So, RNA viruses are more difficult to be controlled by medicine or vaccine. be controlled by medicine or vaccine. nDivergence rate of RNA viruses:69Chapter 8: Virus Infection and Apoptosisn nApoptosis Apoptosis means the cell dying naturally, and it is Apoptosis means the cell dying naturally, and it is controlled by genes (Apoptosis genes). Some scientists call controlled by genes (Apoptosis genes). Some scientists call apoptosis as “program cell death”. apoptosis as “program cell death”. Apoptosis is very important and a hot research focus Apoptosis is very important and a hot research focus in the modern life science. Apoptosis is involved in aging, in the modern life science. Apoptosis is involved in aging, diseases development, and diseases treatment, for example, diseases development, and diseases treatment, for example, tumor treatment. tumor treatment. Apoptosis is different from necrosis. Apoptosis never Apoptosis is different from necrosis. Apoptosis never form any abscess because apoptosis cells always keep form any abscess because apoptosis cells always keep membrane unbroken, so, there is no inflammation in membrane unbroken, so, there is no inflammation in apoptosis. apoptosis. 70n nVirus infection and apoptosis 1. Virus infection enhances apoptosis1. Virus infection enhances apoptosis The recent reports show many virus infections can The recent reports show many virus infections can induce hosted cells to apoptosis. induce hosted cells to apoptosis. HCV core protein (capsid) expression Fas expression HCV core protein (capsid) expression Fas expression Fas mediated apoptosis Fas mediated apoptosis HIV gp120 Fas receptor expression apoptosis HIV gp120 Fas receptor expression apoptosis SV40 and HSV are apoptosis inducers also. SV40 and HSV are apoptosis inducers also. 2. Virus infection inhibits apoptosis2. Virus infection inhibits apoptosis To keep their generation and life cycle, viruses must To keep their generation and life cycle, viruses must inhibit apoptosis. inhibit apoptosis. For examples: For examples:71SV40SV40TantigencombinestoICE(InterleukinChangeEnzymefamily)SV40Tantigencombinestop53 (atumorsuppressorgene)Apoptosisbcl-2(aprotooncogene)p35(aapoptosissuppressorgene)Some virusesApoptosisBlock72Chapter 9: Interaction between Virus and Human or Animal Hostn nConditions needed by successful infection 1. Infectivity of virus 1. Infectivity of virus Pathogen Pathogen Virulence Virulence Virus quantity Virus quantity 2. Infection way 2. Infection way 3. Host specificity 3. Host specificity Receptor on the cell Receptor on the cell Immunity status Immunity status Temperature Temperature73n nVirus and HostVirus and Host 1. Changes of cell morphology 1. Changes of cell morphology 2. Cell breakage 2. Cell breakage 3. Fusion of membrane 3. Fusion of membrane 4. Changes of membrane permeability 4. Changes of membrane permeability 5. Inclusion body forming 5. Inclusion body forming 6. Apoptosis 6. Apoptosis 7. Cell transformation: 7. Cell transformation: Cell transformation is associated with tumor Cell transformation is associated with tumor development. development. 74n nInterfering and Interferon Ifusedtwomoretypesofvirustoinfectsamecell,eachviruswillreleaseoutsomeproteintoinhibitthereplicationofotherviruses.WecallthisnegativeeffectasInterferingandthesecretedproteinasinterferon.Interferonisaveryimportantcytokine,anditisknownastheeffectivemedicinetoinhibitvirusreplicationonlysofar.Butonly25%patientscanuseit.75n nIFNIFNIFN-IFN- IFN-IFN- IFN-IFN- n nInducingofIFNInducingofIFN1.Virusinfection(Aliveordeadvirus)canpromoteinterferon1.Virusinfection(Aliveordeadvirus)canpromoteinterferongenetranscription.genetranscription.2.2.CycloheximideCycloheximide, ,dsRNAdsRNA,DMSO,andLPS(frombacteria),DMSO,andLPS(frombacteria)arestrongIFNinducer.arestrongIFNinducer.3.ThegenesofhumanIFN-3.ThegenesofhumanIFN- andIFN-andIFN- arenotsplitgenes.arenotsplitgenes.IFNinducingbyRNAvirusesismuchstrongerthanbyIFNinducingbyRNAvirusesismuchstrongerthanbyDNAviruses.DNAviruses.IFNIIFNII76n nReceptorofIFNReceptorofIFNIFNsIFNs have to bind to their receptor to activate some have to bind to their receptor to activate some proteins else to start the anti-virus function. proteins else to start the anti-virus function. The species specificity of IFN is dependent on IFN The species specificity of IFN is dependent on IFN receptor. receptor. The species specificity of IFN I (IFN- The species specificity of IFN I (IFN- and IFN- and IFN- ) is ) is stronger than that of IFN II (IFN-stronger than that of IFN II (IFN- ). ). n nAbout virus inhibition of IFNAbout virus inhibition of IFN 1. 1. Some products of virus gene, for example, Some products of virus gene, for example, dsRNAdsRNA is is important to inhibit virus by IFN. important to inhibit virus by IFN. 2. 2. Antivirus of IFN can be carried out at many virus Antivirus of IFN can be carried out at many virus life cycle steps, for example, invasion, coat (life cycle steps, for example, invasion, coat (capsidcapsid) or ) or envelope removing, transcription, protein translation, envelope removing, transcription, protein translation, genome replication, assemble and releasing out. genome replication, assemble and releasing out. 77 3. 3. There are two ways to inhibit reverse transcription There are two ways to inhibit reverse transcription virus . virus . a. a. IFN can inhibit DNA synthesis of the reverse IFN can inhibit DNA synthesis of the reverse transcription virus before it combine into host cell genome. transcription virus before it combine into host cell genome. b.b. To inhibit provirus, IFN can inhibit the assemble and To inhibit provirus, IFN can inhibit the assemble and release of the new RT virus particles. But, IFN can not release of the new RT virus particles. But, IFN can not inhibit the provirus in the host cell genome or delete it inhibit the provirus in the host cell genome or delete it away. away. 78n nTheresistanceofvirustoIFNIFN ResistanceIFN ResistanceVirusIFNAnti VirusAnti VirusBalanceEvery virus type has its special way on that the IFN resistance can Every virus type has its special way on that the IFN resistance can be carried out. But all of them are due to secret some special protein or be carried out. But all of them are due to secret some special protein or poly peptides and shoot some special targets on the IFN protein, IFN poly peptides and shoot some special targets on the IFN protein, IFN gene, or IFN function system. gene, or IFN function system. IFN Inducing79Chapter 10: Virus and Tumor This is a very important talking topic. This is a very important talking topic. The development of tumor is very complicated, and a lot The development of tumor is very complicated, and a lot of detail about that keep unknown so far. But we know the of detail about that keep unknown so far. But we know the tumor development is associated with the follows at least: tumor development is associated with the follows at least: 1. Genetics 1. Genetics 2. Environment (Physics, Chemistry and Biology) 2. Environment (Physics, Chemistry and Biology) 3. Endo-Environment of body (Immunity and others) 3. Endo-Environment of body (Immunity and others) 4. Some virus infections 4. Some virus infections80n nThe molecular changes associated with the The molecular changes associated with the development of tumordevelopment of tumor1. 1.Gene mutation Gene mutation 2. 2.Damage of DNA moleculeDamage of DNA molecule3. 3.Change, mutation or damage during the cell cleavage: Change, mutation or damage during the cell cleavage: When a cell is in cleavage its DNA is very easy to be When a cell is in cleavage its DNA is very easy to be damaged or mutated.damaged or mutated.4. 4.Changes of some steps of cell differentiation: These steps Changes of some steps of cell differentiation: These steps can inhibit the gene mutated cells to differentiate.can inhibit the gene mutated cells to differentiate.5. 5.Changes of defense system: For example, glutathione Changes of defense system: For example, glutathione transferase and others can protect DNA from damages, transferase and others can protect DNA from damages, and they are activated by DNA damage.and they are activated by DNA damage.81n nTumorViruses(Oncoviruses)RTvirusesandallofDNAvirusescanalmostcauseRTvirusesandallofDNAvirusescanalmostcausehumanandanimaltumors.ItisnottruethatallDNAviruseshumanandanimaltumors.ItisnottruethatallDNAvirusescausetumorineveryinfection,but,RTvirusinfectionsalwayscausetumorineveryinfection,but,RTvirusinfectionsalwayscausetumor.causetumor.Tumorvirusesexistinhumanbeing,animalandplantTumorvirusesexistinhumanbeing,animalandplantalmosteverywhere.Onetypeofoncoviruscancausemanyalmosteverywhere.Onetypeofoncoviruscancausemanykindsoftumorandmanytypesofoncoviruscancausesamekindsoftumorandmanytypesofoncoviruscancausesametumor.tumor. OncovirusinfectioncanmakethecellsdifferentiationOncovirusinfectioncanmakethecellsdifferentiationchanged,thatis,normalcellscanbechangedtogrowupaschanged,thatis,normalcellscanbechangedtogrowupasbenignormalignanttumorcells.benignormalignanttumorcells.Oncovirusescancausebothbenignandmalignanttumor.Oncovirusescancausebothbenignandmalignanttumor.Whichresultwillhappendependsonthefollows:Whichresultwillhappendependsonthefollows:821. 1.VirulencetocausetumorVirulencetocausetumor2. 2.Natureoftargetcell:Non-blockreceptorandinducerinNatureoftargetcell:Non-blockreceptorandinducerincellcell3. 3.HostimmunityisstrongenoughtoblockthetumorHostimmunityisstrongenoughtoblockthetumordevelopmentornotdevelopmentornot83Tumor Viruses can be sorted to two types: Tumor Viruses can be sorted to two types: DNA oncoviruses and RNA oncoviruses DNA oncoviruses and RNA oncovirusesn nDNAOncovirusesOncovirusescantransferculturingcellstotumorcells,iftheOncovirusescantransferculturingcellstotumorcells,ifthetransferredcellsareinjectedintotheanimalinsamespecies,transferredcellsareinjectedintotheanimalinsamespecies,tumorwilldevelopoutintheinjectedanimal.tumorwilldevelopoutintheinjectedanimal.1. 1. AV (Adenovirus) AV (Adenovirus) AVcanparasiteinhumanbodyandotheranimals.ThereareAVcanparasiteinhumanbodyandotheranimals.Therearemorethan80serumtypeshavebeenfoundsofar.AVcanbemorethan80serumtypeshavebeenfoundsofar.AVcanbedividedinto4groups:A,B,CandD.Allofthemcantransferthedividedinto4groups:A,B,CandD.Allofthemcantransfertheculturingmousecellstotumorcellsinvitro.culturingmousecellstotumorcellsinvitro.AVcanmakenon-permissivecellstransferredwithE1AAVcanmakenon-permissivecellstransferredwithE1AandE1B(primaryproductsofvirusgeneexpression)whentheandE1B(primaryproductsofvirusgeneexpression)whentheinfectionisfailure.infectionisfailure.84 IfusetherecombinantscontainingE1AandE1BtotransfectIfusetherecombinantscontainingE1AandE1Btotransfectculturingcells,youcanobtaintransformedcellsthatcanbeculturingcells,youcanobtaintransformedcellsthatcanbeculturedunlimitedly.culturedunlimitedly.But,ifuseE1AorE1Bonly,youcannotBut,ifuseE1AorE1Bonly,youcannotobtaincompletelytransformedcells.obtaincompletelytransformedcells.n nHSV (Herpes Simplex Virus)HSV (Herpes Simplex Virus) HSVisaspeciesofherpesvirusesthatcanparasiteinmanyHSVisaspeciesofherpesvirusesthatcanparasiteinmanytypesofanimal.HerewearejusttalkingaboutHSVonly.typesofanimal.HerewearejusttalkingaboutHSVonly.TheoncogenicabilityofHSVisconfirmedalreadyanditTheoncogenicabilityofHSVisconfirmedalreadyanditcancausemanykindsoftumor,especiallyforcervicalcarcinoma.cancausemanykindsoftumor,especiallyforcervicalcarcinoma.HSVcanbesortedto2types:HSV-1andHSV-2.HSVcanbesortedto2types:HSV-1andHSV-2.EpidemiologyinvestigationshowsthereisaverycloseEpidemiologyinvestigationshowsthereisaveryclosecorrelationbetweenHSV-2andcervicalcarcinoma.correlationbetweenHSV-2andcervicalcarcinoma.85 HSV-2containsaoncogene,LA-1.LA-1canbeexpressedHSV-2containsaoncogene,LA-1.LA-1canbeexpressedwithahighlevelonhumancervicaltissue.withahighlevelonhumancervicaltissue.n n HPV (HPV (Human Papilloma VirusHuman Papilloma Virus) ) ThereisaclosecorrelationbetweenHPVinfectionandThereisaclosecorrelationbetweenHPVinfectionandhumancarcinomasofurethraandgenital,especiallycervical.humancarcinomasofurethraandgenital,especiallycervical.Sofar,HPVcanberesortedto65serumtypes,and20typesSofar,HPVcanberesortedto65serumtypes,and20typesofthemareassociatedwithhumantumordevelopment.HPV-6ofthemareassociatedwithhumantumordevelopment.HPV-6and11arethelowriskviruses,but,HPV-16,18,31,33,35,39,and11arethelowriskviruses,but,HPV-16,18,31,33,35,39,45,51,52,and56arethehighriskviruses.Usually,HPV-645,51,52,and56arethehighriskviruses.Usually,HPV-6andHPV-11causepointedcondylomaonly.andHPV-11causepointedcondylomaonly.HighriskHPVvirusesexpressE6andE7proteins.E6andHighriskHPVvirusesexpressE6andE7proteins.E6andE7aretheoncogenes.E7aretheoncogenes.E6canenhancep53proteindegradation(p53isaimportantE6canenhancep53proteindegradation(p53isaimportanttumorsuppressorgene),so,thatmayusetoexplainwhyE6cantumorsuppressorgene),so,thatmayusetoexplainwhyE6canpushtumordevelopment.pushtumordevelopment.86n nSV40SV40 SV40 is one of SV40 is one of HPVs(HPVs(Human Polyma VirusesHuman Polyma Viruses).).IthasbeenreportedthatSV40isassociatedwithmanykindsIthasbeenreportedthatSV40isassociatedwithmanykindsoftumordevelopmentinmanyanimals.Peoplehavefoundtheoftumordevelopmentinmanyanimals.PeoplehavefoundtheSV40likedsequencesinmanytypesofhumantumortissue,thatSV40likedsequencesinmanytypesofhumantumortissue,thatshowsaclosecorrelationbetweenSV40andhumantomorshowsaclosecorrelationbetweenSV40andhumantomordevelopment.development.SV40containstwoprimaryantigengenes:TantigenandtSV40containstwoprimaryantigengenes:Tantigenandtantigen.Bothofthemareassociatedwithtumordevelopmentantigen.Bothofthemareassociatedwithtumordevelopmentclosely.closely.Tantigen(96kDa)cancausechromosomestructurechanged,Tantigen(96kDa)cancausechromosomestructurechanged,site-mutationandothers.tantigen(17-21kDa)inhibitsthesite-mutationandothers.tantigen(17-21kDa)inhibitsthephosphorylationofsometumorsuppressorproteins.phosphorylationofsometumorsuppressorproteins.87n nHBV (Hepatitis B Virus)HBVisaveryimportanthumanvirusbecauseitcausesaHBVisaveryimportanthumanvirusbecauseitcausesaverypopularhumandisease,hepatitisB.verypopularhumandisease,hepatitisB.HBVisspecifictoinfecthumanlivercellsonly,anditexistsHBVisspecifictoinfecthumanlivercellsonly,anditexistsinhumanbodyattwotypes:(1)ItsDNAfragmentcombinedintoinhumanbodyattwotypes:(1)ItsDNAfragmentcombinedintohumanlivercellgenomewhenthehostedpersonbecomeaHBVhumanlivercellgenomewhenthehostedpersonbecomeaHBVcarrierorchronichepatitispatient;(2)ReleasedHBVDNAcarrierorchronichepatitispatient;(2)ReleasedHBVDNAprobedinbloodofacuteinfectionorhepatitispatient.probedinbloodofacuteinfectionorhepatitispatient.TheratioofHBVinfectionisabout7%atleastforthetotalTheratioofHBVinfectionisabout7%atleastforthetotalpopulationinChina.MostofthemwillbecomenormalcarrierofpopulationinChina.MostofthemwillbecomenormalcarrierofHBV,andsomeofthemwillgetliverdamagedandbecomeHBV,andsomeofthemwillgetliverdamagedandbecomehepatitispatients.25%ofpatientsmaysufferfromprimaryhepatitispatients.25%ofpatientsmaysufferfromprimarycellularhepatocarcinoma.cellularhepatocarcinoma.88nRNAOncovirusesRNARNAoncovirusesoncovirusesaretheretroviruses(Reversetranscriptionaretheretroviruses(Reversetranscriptionvirus,RTvirus).virus,RTvirus).MostofRNAMostofRNAoncovirusesoncovirusesarefromanimalsandcausearefromanimalsandcauseanimaltumors,theyincludeanimaltumors,theyincludeViper type C Viper type C oncovirusoncovirus, ,AviaAvia reticuloendotheliosisreticuloendotheliosis virus virus, ,Baboon type C Baboon type C oncovirusoncovirus, ,Bovine Bovine leukosisleukosis virus virusForhuman,Forhuman,Hepatitis C virusHepatitis C virus(HCV)ismostimportant(HCV)ismostimportantbecauseHCVinfectionisalsopopularandeasiertogetchronicbecauseHCVinfectionisalsopopularandeasiertogetchronicormalignantthanHBV.ormalignantthanHBV.HCVisHCVisssRNAssRNA+ +virus.virus.89MostofRNAoncovirusesarefromRetroviridae,MostofRNAoncovirusesarefromRetroviridae,theyarecharacterizedbythefollows:theyarecharacterizedbythefollows:1.About100nmsize.1.About100nmsize.2.Withenvelopeandcapsid.2.Withenvelopeandcapsid.3.ssRNAgenome.3.ssRNAgenome.4.Moleculeweight:about74.Moleculeweight:about710106 6Da.Da.5.Theycanencodereversetranscriptase.5.Theycanencodereversetranscriptase.6.ssRNADNA(Proviruscanbecombined6.ssRNADNA(Proviruscanbecombinedintohostgenome)ssRNA(Transcriptedfromintohostgenome)ssRNA(Transcriptedfromprovirus).provirus).90 RNAoncovirusesInfection(Speciesspecificity)RNAoncovirusesInfection(Speciesspecificity)MoveintocellnucleusandmixwithcellDNAMoveintocellnucleusandmixwithcellDNAProvirusRecombinationtohostgenomessRNAProvirusRecombinationtohostgenomessRNARNAvirusparticlesRNAvirusparticlesRNAoncovirusescanbetransmittedby:RNAoncovirusescanbetransmittedby: Bodytobody(Horizontaltransmission)Bodytobody(Horizontaltransmission) Parentalgenerationtofilialgeneration(VerticalParentalgenerationtofilialgeneration(Verticaltransmission)transmission)91n nProto-oncogeneProto-oncogenesaresomegenesequencesthatexistinProto-oncogenesaresomegenesequencesthatexistinnormalgenomesequence,buttheycanbeactivatedbysomenormalgenomesequence,buttheycanbeactivatedbysomespecialcondition(Mutation,overexpression)andcausetumor.specialcondition(Mutation,overexpression)andcausetumor.Proto-oncogeneincludesProto-oncogeneincludesrasrasfamily,family,c-erbc-erbBfamily,Bfamily,myc myc family,family,bclbcl-2,-2,c-srcc-src, ,c-rafc-raf, ,c-fosc-fos, ,c-junc-jun, ,c-mybc-mybrasras family: family: rasrasfamilyincludesH-familyincludesH-ras,ras,N-N-ras and ras and K-K-rasras. .rasras familyencodessomeproteinsassociatedwithGTPmetabolismfamilyencodessomeproteinsassociatedwithGTPmetabolismandparticipatesincellsignaltransduction.Ifthenumber12,13,andparticipatesincellsignaltransduction.Ifthenumber12,13,and61codonsofand61codonsofras ras familyaremutated,itwillexpressP21.P21familyaremutated,itwillexpressP21.P21canpresentthesignaltocellforthecontinuingmitochysis.Overcanpresentthesignaltocellforthecontinuingmitochysis.Overmitochysiswillenhancecellstoovergrowthwithoutregulation,mitochysiswillenhancecellstoovergrowthwithoutregulation,andtumorwillbedeveloped.andtumorwillbedeveloped.92 c-erbc-erb B family: B family: c-erbc-erbBfamilyincludesBfamilyincludesc-erbc-erbB-1andB-1andc-erbc-erb B-2.B-2.c-erbc-erbB-1encodesEGFR(Epidermgrowthfactorreceptor).B-1encodesEGFR(Epidermgrowthfactorreceptor).c-erbc-erbB-2encodesP185.EGFRandP185areassociatedwithB-2encodesP185.EGFRandP185areassociatedwithtumordevelopment.tumordevelopment.myc myc family:family: myc myc familyincludesC-familyincludesC-myc, myc, N-N-myc, myc, L-L-mycmyc. . myc myc familyencodesfamilyencodes theproteinsthatcanspecificallycombinetotheproteinsthatcanspecificallycombinetoDNA,andassociatewithcellgrowthregulation.DNA,andassociatewithcellgrowthregulation.bclbcl-2:-2: bclbcl-2isaapoptosissuppressorgene.Butifitis-2isaapoptosissuppressorgene.Butifitisshiftedonchromosome14to18andlinkedtothesiteofthegeneshiftedonchromosome14to18andlinkedtothesiteofthegeneofimmunoglobulinheavychain,ofimmunoglobulinheavychain,bclbcl-2willbeexpressed-2willbeexpressedexcessivelyandenhancethelifeofcells,whichcausestumorexcessivelyandenhancethelifeofcells,whichcausestumordevelopmenteasy.developmenteasy.c-srcc-src: : c-src c-src isaproto-oncogene,andisaproto-oncogene,and src src isaRTvirusisaRTviruscancergene.cancergene.c-src c-src regulatesregulates mitochysisanddifferentiatescells.mitochysisanddifferentiatescells.93n nTumorsuppressorgenesTumorsuppressorgenesincludeTumorsuppressorgenesincludeRb, p16, p15, p53, DCCRb, p16, p15, p53, DCC andothers.andothers.Rb:Rb: Rb Rbistheproteingeneofretinoblastoma,anditistheistheproteingeneofretinoblastoma,anditisthefirsttumorsuppressorgenethatwasisolatedbyscientist.firsttumorsuppressorgenethatwasisolatedbyscientist.RbRbisislocatedonhumanchromosome13q14.locatedonhumanchromosome13q14.RbRbencodesaDNAencodesaDNAbindingproteinthatisassociatedwithcelldifferentiationandbindingproteinthatisassociatedwithcelldifferentiationandinhibitscelltransformation.Incancertissue,inhibitscelltransformation.Incancertissue,RbRbisdeletedorisdeletedormutated.mutated.Non-phosphorylatedNon-phosphorylatedRbRbregulatescellcycle,anditinhibitsregulatescellcycle,anditinhibitsSphasefollowingG1phase.SphasefollowingG1phase.p16:p16: p16p16isasuppressorgenetotheproteinofCDKisasuppressorgenetotheproteinofCDK(Familyofcyclindependentkinase).Productencodedby(Familyofcyclindependentkinase).Productencodedbyp16p16 inhibitsCDK4andCDK6.CDKisanenhancertocellcycle.So,inhibitsCDK4andCDK6.CDKisanenhancertocellcycle.So,ififp16 p16 ismutatedorlost,cellswillgrowupunlimitedly.ismutatedorlost,cellswillgrowupunlimitedly.94 p16p16andandRbRb,theirgrowthanddeclinewasobservedinmany,theirgrowthanddeclinewasobservedinmanytumortissues.tumortissues. p15p15: : p15p15islocatedon9p21,anditsMWis14.7kDa.Thereislocatedon9p21,anditsMWis14.7kDa.Thereare2readingframeson9p21,oneforare2readingframeson9p21,oneforp16 p16 andanotheroneforandanotheroneforp15p15.Thesequencesofp16andp15are97%homologous.The.Thesequencesofp16andp15are97%homologous.Theexpressionofp15canbeenhancedobviouslybyTGFexpressionofp15canbeenhancedobviouslybyTGF ,thatis,thatiswhywhyTGFTGF caninhibitcellgrowth.caninhibitcellgrowth.p15p15iscloselyassociatedwithcellcycle,andbockscelliscloselyassociatedwithcellcycle,andbockscellgrowthatG1phase.DeletionorovergrowthatG1phase.Deletionorovermethylationmethylationofofp15p15waswasfrequentlydetectedinhumanmalignanttumors.frequentlydetectedinhumanmalignanttumors.p53p53: :WhenacellDNAwasdamagedWhenacellDNAwasdamagedp53p53canblockitatcanblockitatG1/Sphase,then,aDNArepairingsystemisstartedimmediately.G1/Sphase,then,aDNArepairingsystemisstartedimmediately.IfthereparationfailedIfthereparationfailedp53p53willinducethecellturnedtowillinducethecellturnedtoapoptosis.Inresult,thedeviation(Tumordevelopment)isapoptosis.Inresult,thedeviation(Tumordevelopment)issuppressed.Thedeletionormutationofsuppressed.Thedeletionormutationofp53p53willcausethatcellswillcausethatcellsgrowunlimitedly.growunlimitedly.95SomeexperimentaldatashowsthatSomeexperimentaldatashowsthatp21p21isaimportantisaimportantproteintothecellcycleinhibitionbyproteintothecellcycleinhibitionby p53 p53becausebecausep53p53caninducecaninducep21p21expression.expression.p53 p53 mutationwasmostoftendetectedinhumantumors,andmutationwasmostoftendetectedinhumantumors,anditisthemostimportantnegativeregulationfactortocellcycle.itisthemostimportantnegativeregulationfactortocellcycle.ThemostfrequentlymutatedsitesforThemostfrequentlymutatedsitesfor p53 p53are175,248and273are175,248and273aminoacids.aminoacids.SV40,AV,HPV(Humanpapillomavirus)andothervirusesSV40,AV,HPV(Humanpapillomavirus)andothervirusesinducetumorspecificproteins,suchasSV40Tantigen,E1A,inducetumorspecificproteins,suchasSV40Tantigen,E1A,E1B,E6,E7andothers.AllofthesetumorproteinscanbindtoE1B,E6,E7andothers.Allofthesetumorproteinscanbindtop53 p53 andinactivateforthefasttumorgrowth.andinactivateforthefasttumorgrowth.96Chapter 11: The purification, detection and diagnosis of virusThe purification of virusTheisolation,amplificationandpurificationareveryimportanttovirusidentification,virusvaccineresearchandselectionofthemedicinesagainstvirus.1.AmplificationThevirusquantityfromnaturalinfectionisnotenoughtoresearchuse.So,youhavetoamplifyvirusforyourresearch.Usually,weinfecttheculturedhostcellwiththetargetvirusforthevirusamplification.972.PrimarypurificationwithbiologicalmethodTheisolatedvirussolutionformnaturallyinfectedbodyortissueisnotpurifiedforonetypevirus.Fortheconvenientproceduresinlatterresearch,theisolatedvirussolutionshouldbepurifiedbyabiologicalmethod.So,youhavetopurifythesolutionasabiologicalpurificationlevelbeforeyougetthesolutionpurifiedasachemicalpurificationlevel.Viruscanformtheplaquesorvirusdotsonmonolayerculturedcells,andsingleplaqueorvirusdotisformbyonevirusparticle.Infectthemonolayerculturedcells(bacteriaoranimalcells)selectplaqueorvirusdot(clone)infectagainselectagainpurifiedvirus.Fortheamplificationandprimarypurificationasthedescribedlikeabove,youhavetousetheextractionstepineachroundtogetvirusextractionsolution.983.ThereleaseandextractionofvirusThebreakageofcell:repeatedlyfreezecells;ultrasonicbreakageandothers.Youhavetobecarefultothevirusisoelectricpoint(pI)becausemostofviruseshavetheirpI at4.0.InsuchpHenvironment,viruseswillformtheirreversibleprecipitationwiththebrokencellpartstogether.ThecellbreakageiseasytoformalowpHenvironment.So,togetahighquantityharvestofvirus,youshouldkeepthecellbreakageinpH7.0environmentusually.Virusparticlesareeasytobindtogethertoformalargemassthatiseasytobeprecipitatedawayfollowingthecentrifuge.So,youhavetoaddsomedetergent(suchasTween-80)toblockthebinding.994.Fractionalpurification(1)Isolatetheorganelles:Usecentrifugemethod.Bufferandspeedselectionareimportanthere.(2)Isolateproteins:Useproteinprecipitationreagents,suchasammoniumsulfate,PEG(polyethyleneglycol).(3)Centrifugation:Usecentrifugetoisolatevirusparticlesfromthemixture.Eachvirushasitssedimentationcoefficient(ornamedsedimentationconstant),so,viruscanbeprecipitatedwithadifferentsedimentationspeed(s20)withothervirusesorpartsfromcells.a.Differentialcentrifugation;b.Densitygradientcentrifugation.1005.ElectrophoresisIftheenvironmentpHisnotthevirusisoelectricpoint,eachtypeofviruswillchargedwithdifferentelectricpower.So,wecanuseelectrophoresismethodtoisolatevirus.101The detection of virus and virus infectionThedetectionofvirusorvirusinfectionincludequantitativeassayandqualitativeassay.1.TestofinfectivityUsetitrationmethod.Thesmallestquantityofvirustoinfecthostandcausesomespecificresponseiscalledinfectionunit.Todescribetheconcentrationofvirusparticles,weusepfu(plaqueformingunits).2.ViruscultureUsecellculturemethodandharvestvirusparticlesfromtheculture.3.Immunologicalmethods4.Assayofvirusnuclearacidsa.HybridizationtechnologyUselabeledprobe,suchashybridizationinsitu.102b.PCRc.RT-PCRd.RealtimePCRe.Microarray103Chapter 12: Important Viruses Viruses DiseasesHepatitisvirusesHBVHepatitisBHCVHepatitisCHumanimmunodeficiencyvirus(HIV)AIDSHerpesvirusesHSV1Herpesonmouth,eyes,facesandotherHSV2HerpesongenitalsurfacesVaricellozostervirus(VZV)HerpesZosterEBLymphoma,pharynxcancerCytomegalovirus(CMV)Thediseasesofliver,kidney,whitebloodcells,genitalsystem,cancersHumanpapillomavirus(HPV)Pointedcondyloma AvianInfluenza(BirdFlu)A(H5N1)VirusAvianandhumanflu104HBV(HepatitisBVirus)105n n About HBVAbout HBV HBVisdsDNAviruswithenvelopeandspecifictoHBVisdsDNAviruswithenvelopeandspecifictoparasiteinhumanlivercells.parasiteinhumanlivercells.HBVisasphericalparticlewithadiameterof42nmHBVisasphericalparticlewithadiameterof42nm(1nm=0.000000001meter)andiscomposedasfollows.(1nm=0.000000001meter)andiscomposedasfollows.Thereisanoutershell(orenvelope)composedofseveralThereisanoutershell(orenvelope)composedofseveralproteinsknowncollectivelyasHBsorsurfaceproteins.proteinsknowncollectivelyasHBsorsurfaceproteins.Thisoutershellisfrequentlyreferredtoastheenvelope.Thisoutershellisfrequentlyreferredtoastheenvelope.Theenvelopesurroundsaninnerproteinshell,composedofTheenvelopesurroundsaninnerproteinshell,composedofHBcprotein.ThisinnershellisreferredtoasthecoreHBcprotein.Thisinnershellisreferredtoasthecoreparticleorcapsid.Finallythecoreparticlesurroundstheparticleorcapsid.FinallythecoreparticlesurroundstheviralDNAandanenzymeDNAPolymerase.Coreof27nmviralDNAandanenzymeDNAPolymerase.Coreof27nmindiameter,surroundedbyanoutercoatapproximatelyindiameter,surroundedbyanoutercoatapproximately4nmthick.4nmthick.106A model fig of HBV107108109Neighbour-joiningphylogenetictreesofcoreandsurfacegenesequences.Analignmentof262completesequenceswasperformedwithclustalwintheprogramDNA-star.Thealignmentwasfurtheranalysedbyboot-strappingusingtheneighbour-joiningmethodcontainedinmegaversion2.1110Schematic representation of the genomic organization of elements regulating the transcription of HBV. Relative positions of S, C, Pol and X open-reading frames (ORFs) are shown. S1, S2, CP and XP represent the promoters of HBV. The enhancers are indicated as ENI and ENII. Positions of the EcoRI cleavage site and of the direct repeats DR1 and DR2 are also shown. 111n n ORFs and Mutation of HBV:ORFs and Mutation of HBV: S(S,Pro-S1andPro-S2):S(S,Pro-S1andPro-S2):Encodingenvelope.HBsAg(226aa)Encodingenvelope.HBsAg(226aa)isencodedbyS.S1encodesTcellidentifyingsiteandtheliverisencodedbyS.S1encodesTcellidentifyingsiteandthelivercellreceptorbindingsiteontheenvelope.S2encodessomecellreceptorbindingsiteontheenvelope.S2encodessomepeptideofenvelopeassociatedwithHBVinvasion,andTcellpeptideofenvelopeassociatedwithHBVinvasion,andTcellandBcellidentifyingsites.andBcellidentifyingsites. C(CandPro-C):C(CandPro-C):Encodingcoreprotein,HBeAg.CandPro-CEncodingcoreprotein,HBeAg.CandPro-ChavetheirownATGoneachNterminal,but,theyhaveoneTAGhavetheirownATGoneachNterminal,but,theyhaveoneTAGattheirsameCterminal.attheirsameCterminal. X:X:EncodingXantigen,HBxAg.HBxAgisnotsoimportantforEncodingXantigen,HBxAg.HBxAgisnotsoimportantforclinicdiagnosis.XisthesmallestORFofHBV.clinicdiagnosis.XisthesmallestORFofHBV. P:P:EncodingDNApolymerase.PisthelongestORFofHBVEncodingDNApolymerase.PisthelongestORFofHBVandoverlapswithotherORFs.andoverlapswithotherORFs.ENIandENIIareessentialforpromoteractivity.ENIandENIIareessentialforpromoteractivity.112Virusmutationmakeshugedifficultiestocontrolvirusinfection.HBVmutationVirusmutationmakeshugedifficultiestocontrolvirusinfection.HBVmutationrateis2/10kb/yearthatisslower100-1000timesthanRNAvirusbutfaster100timesrateis2/10kb/yearthatisslower100-1000timesthanRNAvirusbutfaster100timesthanotherDNAvirus.HBVmutationcanbeformedinanygeneregion.thanotherDNAvirus.HBVmutationcanbeformedinanygeneregion.S mutation:S mutation:SmutationcausethechangesofHBsAgsubtypes,deletionofHBsAgSmutationcausethechangesofHBsAgsubtypes,deletionofHBsAgexpression,bothHBsAgandAnti-HBsexistinbloodtogether,andbypassingtheattackexpression,bothHBsAgandAnti-HBsexistinbloodtogether,andbypassingtheattackfromhostimmunity.fromhostimmunity.Pro-S (S1 and S2) mutation:Pro-S (S1 and S2) mutation:Pro-SmutationcausestheirproductscannotbePro-Smutationcausestheirproductscannotbedetected,TandBcellscannotidentifyvirus,butHBVinvasionkeepsnochange.detected,TandBcellscannotidentifyvirus,butHBVinvasionkeepsnochange.ManypapersreporttherearethePro-SmutationsinchronichepatitisBpatientsastheManypapersreporttherearethePro-SmutationsinchronichepatitisBpatientsasthefollows:Insertion,Deletion,andRecombination.So,Pro-Smutationmaybeassociatedfollows:Insertion,Deletion,andRecombination.So,Pro-Smutationmaybeassociatedwithviruscarrier,chronichepatitis,hardliver,andprimarycellularhepatocarcinoma.withviruscarrier,chronichepatitis,hardliver,andprimarycellularhepatocarcinoma.C and Pro-C mutation:C and Pro-C mutation:ThemutationrateofCandPro-CisdifferentbetweenThemutationrateofCandPro-Cisdifferentbetweencountries,areasandbodies.CandPro-Cmutationsareassociatedwithcountries,areasandbodies.CandPro-Cmutationsareassociatedwithchronic active chronic active hepatitis Bhepatitis B.PositiveHBeAgcheckingresultmeansthepatientsvirusisreplicatingand.PositiveHBeAgcheckingresultmeansthepatientsvirusisreplicatingandcaninfectotherbodies.PapersreportedthatPro-Cmutationisalsoassociatedwiththecaninfectotherbodies.PapersreportedthatPro-CmutationisalsoassociatedwiththedevelopmentofseverehepatitisB.Pro-CmutationplusCmutationcausethechangeofdevelopmentofseverehepatitisB.Pro-CmutationplusCmutationcausethechangeofHBeAg.Pro-CmutationmayaffecttheIFNtherapeuticefficiency.HBeAg.Pro-CmutationmayaffecttheIFNtherapeuticefficiency.HBVissohighmutationDNAvirusthatyoucannotfindanycoupleHBVstrainsHBVissohighmutationDNAvirusthatyoucannotfindanycoupleHBVstrainswithcompletelysamesequencesintheworld.withcompletelysamesequencesintheworld.113HBVinvasionandhost114n nPathologyandClinicFeaturesYellowingskinoreyes(Jaundice)Yellowingskinoreyes(Jaundice)NotfeelinghungryNotfeelinghungryFeelingtiredFeelingtiredMuscle,joint,orstomachpainMuscle,joint,orstomachpainStomachupset,diarrhea,orvomitingStomachupset,diarrhea,orvomitingHardliverandascitesHardliverandascitesHepaticcomaHepaticcomaPrimarycellularhepatocarcinomaPrimarycellularhepatocarcinoma115Disorderofendocrinebalanceofhepatitispatient116Skinandhandofhepatitispatient117Hardliver118Surfacelocalofhardliver119Cutofhardliver120Sectionofhardliver121Sectionoffattyliver122Handsofhepatitispatient123Ascitesofhepatitispatients124SectionofnormalliverSectionoflivercancer125Primarycellularhepatocarcinoma126Primarycellularhepatocarcinoma127n nTransmissionandInfection1. 1.Oralroute(Mainroute)Oralroute(Mainroute) 2. 2.Bloodroute(Mainroute):injection,bloodtransfusion,Bloodroute(Mainroute):injection,bloodtransfusion,acupuncture,wounds,insects(?)acupuncture,wounds,insects(?) 3. 3.MothertoinfantMothertoinfant 4. 4.Closetouch:Kiss,crossuseofprivatelifeusingstuffs(?),Closetouch:Kiss,crossuseofprivatelifeusingstuffs(?),pool(?),sex(?)pool(?),sex(?)a. a.Spontaneouscureafteranapparentinfectionorsub-Spontaneouscureafteranapparentinfectionorsub-clinicalinfectionclinicalinfectionHBVHBVb.b.Normalcarrier(Majorityofinfections)Normalcarrier(Majorityofinfections)a. a.HepatitisHepatitisc. c.CHBCHBb.b.Hardliver,Hardliver,ascitesascitesandhepaticcomaandhepaticcomac. c.PrimarycellularPrimarycellularhepatocarcinomahepatocarcinoma 128n nDiagnosisinLaboratory1. Serology1. Serology HBsAg:HBsAg:NormalcarrierandhepatitisBNormalcarrierandhepatitisBHBsAb:HBsAb:ImmunizedorcuredbodiesImmunizedorcuredbodiesHBeAg:HBeAg:NormalcarrierandhepatitisBwhenHBVisNormalcarrierandhepatitisBwhenHBVisreplicatingandeasytoinfecttheothers.replicatingandeasytoinfecttheothers.HBeAb:HBeAb:NormalcarrierandhepatitisBwhenHBVisNormalcarrierandhepatitisBwhenHBVisreplicatingandeasytoinfecttheothers.replicatingandeasytoinfecttheothers.HBcAg:Itisnoteasytobecheckedoutfromblood.HBcAg:Itisnoteasytobecheckedoutfromblood.HBcAb:HBcAb:NormalcarrierandhepatitisBwhenHBVisNormalcarrierandhepatitisBwhenHBVisreplicatingandeasytoinfecttheothers.replicatingandeasytoinfecttheothers.129 2. Function checking for liver:2. Function checking for liver:ThemainparameterfortheliverfunctioncheckingisALTThemainparameterfortheliverfunctioncheckingisALTlevel.IftheHBsAgispositivefromthebloodofsomebody,thatlevel.IftheHBsAgispositivefromthebloodofsomebody,thatmeansthisbodyisnormalcarrierorhepatitisBpatient.FormermeansthisbodyisnormalcarrierorhepatitisBpatient.Formerorlater?Youhavetoidentifythembythefollows:orlater?Youhavetoidentifythembythefollows: HBsAgorothersarepositive,but,HBsAbisnegativeHBsAgorothersarepositive,but,HBsAbisnegative HighALTlevel.ASTmaybehighalso.HighALTlevel.ASTmaybehighalso. ClinicalsignsorsyndromesofhepatitisBClinicalsignsorsyndromesofhepatitisB3. PCR3. PCRQualitativePCR(HBVPCRkitfromRoche)QualitativePCR(HBVPCRkitfromRoche)QuantitativePCR(HBVPCRkitfromRoche)QuantitativePCR(HBVPCRkitfromRoche)130n nPreventionandTherapyPrevention:Prevention:HBVvaccine,becarefultotakebloodHBVvaccine,becarefultotakebloodtransfusion,andchangesomefoodandlifehabitsthatmaketransfusion,andchangesomefoodandlifehabitsthatmakeinfectioneasy.infectioneasy.Therapy:Therapy:IFNIFN isknownastheeffectivemedicinetoinhibitisknownastheeffectivemedicinetoinhibitHBVonlysofar,anditiseffectiveto25%patientsbecausetheHBVonlysofar,anditiseffectiveto25%patientsbecausetheIFNantibodyandpro-Cmutation.OthermeasurestotreatIFNantibodyandpro-Cmutation.OthermeasurestotreathepatitisBpatientsareasthefollows:Completerestinbed,takehepatitisBpatientsareasthefollows:Completerestinbed,takethefoodthatiseasytobedigested,mindtherapy,andvitaminsthefoodthatiseasytobedigested,mindtherapy,andvitaminsandsomemedicinestoprotectormaintainlivercells.andsomemedicinestoprotectormaintainlivercells.131Hepatitis C Virus (HCV)132EverythingaboutHCVisalmostsametoHBV.But,HCVisaRNAvirus.ItisdifferentfromHBVbythefollows:1.VeryeasytobecomeCHC.2.TherateofcancerishigherthanHBV3.SextransmissionismoreimportantforHCV4.EasiertomutatethanHBV5.Moredifficulttotreatment,control,anddiagnosis6.Diagnosis:Anti-HCV,qualitativeandquantitativePCR133Herpes Viruses134Herpes viruses:Thereare8speciesofherpesvirusforhuman,buttheimportantandpopularspeciesareasthefollows:Herpessimplevirustype1(HSV1)Herpesonmouth,eyes,facesandother,pregnantfailureHerpessimplevirustype2(HSV2)Herpesongenitalsurfaces,pregnantfailureVaricellozostervirus(VZV)HerpesZosterEBLymphoma,pharynxcancerCytomegalovirus(CMV)Thediseasesofliver,kidney,whitebloodcells,genitalsystem,cancersIwillintroduceHSVandVZVofthemforyou.135 HSVisdsDNAvirusthatparasiteinhumanneuronsandcausestheinflammationofthemucosaofeyes,oralcavity,andtheherpesofskinandgenitals.HSVisanuclearreplicating,icosahedral,envelopedDNAvirus.TheHSVenvelopecontainsatleast8glycoproteins.Thematrixortegumentwhichcontactsboththeenvelopeandthecapsidcontainsatleast15-20proteins.HSVandHSVStructureThebasicbiologyforHSVandVZVisalmostsame,sowejusttalkabouttheirbiologywithHSVasexampleasthefollows:136137138139HSV140Herpessimplexvirusgenomemustenterthecellfortheinitiationofinfection.TheinitialassociationisbetweenproteoglycansofthecellsurfaceandgC.ThisisfollowedbyaspecificinteractionwithoneofseveralcellularreceptorscollectivelytermedHVEMforherpesvirusentrymediators.Thesearerelatedtoreceptorsfornervegrowthfactorsandtumornecrosisfactor.TheassociationrequiresthespecificinteractionwiththeglycoproteingD.Fusionwiththecellularmembranefollows.ThisrequirestheactionofanumberofviralglycoproteinsincludinggB,gH,gI,andgL.Theviralcapsidwithsometegumentproteinsthenmigratetonuclearporesalongcellularmicrotubulesutilizingcellulartransportmachinery.ThisdockingisthoughttoresultintheviralDNAbeinginjectedthroughtheporewhilethecapsidremainsinthecytoplasm.Sometegumentproteins,suchasa-TIF,alsoenterthenucleuswiththeviralgenome.ReceptorBinding141142143144145ThetranscriptionoftheHSVgenomeduringThetranscriptionoftheHSVgenomeduringproductiveinfectionoccurswithcellulartranscriptionalproductiveinfectionoccurswithcellulartranscriptionalmachineryandviralpromotersutilizingcellularmachineryandviralpromotersutilizingcellulartranscriptionfactorbindingsites.Mostviraltranscriptstranscriptionfactorbindingsites.Mostviraltranscriptsarenotspliced.arenotspliced.Therearetwomainphasesoftranscription-early,Therearetwomainphasesoftranscription-early,whichtakesplacepriortogenomereplication,andlate,whichtakesplacepriortogenomereplication,andlate,whichtakesplaceuponreplicatedgenomesinviruswhichtakesplaceuponreplicatedgenomesinvirusreplicationcompartmentsformedintheinfectedcellreplicationcompartmentsformedintheinfectedcellnucleus.nucleus.RNATranscriptionDuringProductiveInfection146147148149150151152153154LatentinfectionandreactivationbyHSVtakesLatentinfectionandreactivationbyHSVtakesplaceinsensoryneurons,primarilyinthetrigeminalplaceinsensoryneurons,primarilyinthetrigeminalgangliaforHSV-1.TheprocessofestablishmentgangliaforHSV-1.Theprocessofestablishmentinvolvesvirusenteringneuronsattheperiphery,andtheinvolvesvirusenteringneuronsattheperiphery,andtheviralgenometravelinguptheaxonandenteringtheviralgenometravelinguptheaxonandenteringthenucleus.Whilesomeneuronsaredestroyedasvirusnucleus.Whilesomeneuronsaredestroyedasvirusreplicates,mostneuronsarerefractorytovirusreplicates,mostneuronsarerefractorytovirusreplicationandtheviralgenomesbecomeassociatedreplicationandtheviralgenomesbecomeassociatedwithhosthistonesandpersistasmini-chromosomes.withhosthistonesandpersistasmini-chromosomes.TheexpressionofmostviralgenesisabsentduringTheexpressionofmostviralgenesisabsentduringlatentinfection,butanumberoflatentlyinfectedlatentinfection,butanumberoflatentlyinfectedneuronsexpressasingletranscript-thelatencyneuronsexpressasingletranscript-thelatencyassociatedtranscriptorLAT-whichisencodedintheassociatedtranscriptorLAT-whichisencodedintherepeatregionsofthegenome.repeatregionsofthegenome.LatentInfections155156157158159HSVinitiatesroundsofDNAreplicationatoneorHSVinitiatesroundsofDNAreplicationatoneorallofthethreeoriginsofreplication(Ori1,Ori2,andallofthethreeoriginsofreplication(Ori1,Ori2,andOri3).TheinitialstepofHSVDNAreplicationisOri3).TheinitialstepofHSVDNAreplicationisdenaturationoftheDNAatthereplicationoriginwithdenaturationoftheDNAatthereplicationoriginwithoriginbindingprotein(UL9).Thehelicase/primaseoriginbindingprotein(UL9).Thehelicase/primase(UL5/UL8/UL52)andsinglestrandedDNAbinding(UL5/UL8/UL52)andsinglestrandedDNAbindingproteins(UL29)associatetoallowtheDNAproteins(UL29)associatetoallowtheDNApolymerase/UL42complextobeginDNAsynthesis.polymerase/UL42complextobeginDNAsynthesis.Oncenewstrandgrowthprogresses,thecircularOncenewstrandgrowthprogresses,thecircularreplicationstructureisnickedtoformarollingcirclereplicationstructureisnickedtoformarollingcircleintermediate.Longconcatemericstrandsofprogenyintermediate.LongconcatemericstrandsofprogenyDNAareencapsidatedbytheinteractionofDNAareencapsidatedbytheinteractionofcleavage/packagingproteinswiththespecificpackagingcleavage/packagingproteinswiththespecificpackagingsignals(asequences)attheendoftheviralgenomes.signals(asequences)attheendoftheviralgenomes. HSVDNAReplication160161162163164Theprocapsidproteins(UL18,UL19andUL38)Theprocapsidproteins(UL18,UL19andUL38)assemblearoundscaffoldingproteins(UL26andassemblearoundscaffoldingproteins(UL26andUL26.5)thatarethendigestedaway.TheemptycapsidUL26.5)thatarethendigestedaway.TheemptycapsidincorporatesDNAbymeansoftheactionofincorporatesDNAbymeansoftheactionofcleavage/packagingproteins(seetheDNAreplicationcleavage/packagingproteins(seetheDNAreplicationanimation).Thecapsidmigratestothenuclearanimation).Thecapsidmigratestothenuclearmembraneandbudsintothelumenbetweentheinnermembraneandbudsintothelumenbetweentheinnerandouternuclearmembrane.Thisenvelopedvirionandouternuclearmembrane.Thisenvelopedvirionthenentersthecytoplasmthroughfusionwiththeouterthenentersthecytoplasmthroughfusionwiththeouternuclearmembrane.nuclearmembrane.EncapsidationandNuclearEgress165166167168169170171172173174ViralglycoproteinsaretranslatedfromHSVViralglycoproteinsaretranslatedfromHSVRNAontheroughendoplasmicreticulumthenRNAontheroughendoplasmicreticulumthentransportedtothegolgibodyinvesiclestocontinuethetransportedtothegolgibodyinvesiclestocontinuetheglycosylationprocess.Theglycoproteinsarethenglycosylationprocess.Theglycoproteinsarethentransportedinvesiclestothenuclearorplasmatransportedinvesiclestothenuclearorplasmamembrane.membrane.TheHSVcapsidassociateswithtegumentTheHSVcapsidassociateswithtegumentproteinsthenacquiresamatureenvelopebybuddingproteinsthenacquiresamatureenvelopebybuddingintoanexocytoticvesicle.Theenvelopedinfectiousintoanexocytoticvesicle.Theenvelopedinfectiousvirionmigratestothevirusmodifiedmembraneandisvirionmigratestothevirusmodifiedmembraneandisreleasedoutsideofthecell.releasedoutsideofthecell. EnvelopmentandRelease175176177178179180181182PathogenesisofHSVPathogenesisofHSVHumanbodyisthehostforHSVonly.HumaninfectionrateHumanbodyisthehostforHSVonly.Humaninfectionrateisabout80-90%.10%ofthemarethesub-clinicalinfection(Noisabout80-90%.10%ofthemarethesub-clinicalinfection(Nosignsorsyndrome).signsorsyndrome).ThetegumentofHSViscomposedbylipidandprotein,so,ThetegumentofHSViscomposedbylipidandprotein,so,itissensitivetoetherandfatsolvent.HSVkeepsinfectiveitissensitivetoetherandfatsolvent.HSVkeepsinfectiveabilityformonthsatcoldandwillbeinactivatedwithin30abilityformonthsatcoldandwillbeinactivatedwithin30minutesatmoisthot(50minutesatmoisthot(50C)C)ordryhot(90C).ordryhot(90C).183HSVType IType IITransmitted by close air and close touch Transmitted by sexInfect the lumbar region and over, such as oral mucosa and eyesInfect the genitals, such as inner and outer vagina, cervical mucosa, penis 184Herpesonmouth185HerpesonhandHerpesongenitalorgan186Congenitalinfection187 Latent period of the herpes of genital organs and others is about 2-20 days. The person whose immunity is inefficient can get severe infection, recurrence, or attack to die. Within 1 3 weeks after infection the antibody against HSV will appear in blood. But, the survival virus particles will stay in the trigeminal ganglion (Type I virus) or sacral ganglia (Type II virus) forever. When the immunity of the host goes down HSV will make a severe or fatal re-attack. 188Pathogenesis of VZVVZVparticles189Chicken Pox190Shingles191Clinicalfeaturesplusmicrobiologicalandimmunologicaldetections.Diagnosis of HSV and VZVPrevention of HSV and VZVPayyourattentiontobecarefultotouchpatientsandanystuffthatpatientstouched!Noinactivevaccineisavailablebecauseofitscancerinducingrole!Butrecombinantorpeptidevaccinesareavailable!Treatment of HSV and VZVACV(无环鸟苷)isspecificallyeffectivetoHSVandVZV.192Human papilloma virus (HPV)193HPVisthepathogenforPointedCondyloma,averypopularsextransmitteddisease.HPVgenomeincludes10importantORF.Theirfunctionsareshownasthetablebelow:HPVcanbeeasilytransmittedbyanytouchingways,includingsex,kissingandothers.Basic biology of HPV: 194GenomicmapofHPV-16.Thegenomeisadouble-strandedcircularDNAmoleculeof7904basepairs.Transcriptionoccursinaclockwisemanner;theonlytranscriptionalpromoterpresentlymappedforHPV-16isdesignatedP97.TheopenreadingframesdeducedfromtheDNAsequencearedesignedE1toE7,L1,andL2andareindicatedoutsideofthecirculargenome.AEandALrepresenttheearlyandlatepolyadenylationsites.Thevirallongcontrolregion(LCR)containstranscriptionalandreplicationregulatoryelements.195Infection and clinical features of HPV:HumancanbeeasilyinfectedbyHPVwithanydirectandindirectclosetouchwaysincludingsexandothers.Theprimaryinfectionpresentsnoanyclinicfeaturesorsyndromebecauseofthelatency.Thelatencystageistheverydangeroustransmissiontimetootherbodies.TheHPVinfectionclinicfeaturesarealmostlocatedonskin,oralcavity,especiallygenitalsurfaces!IjustshowyouthephotosabouttheHPVclinicasthefollows:196197198199200Prevention and therapy of PC:NoefficientmethodtotreatPCsofar.So,preventionisimportanttoHPVinfection.BecarefultohavesexandclosetouchwithdangerouspeopleismostimportantforthepreventionofPC.The therapy of PC includes:1.VirusantibioticsandIFN.2.Keepthegenitalsurfacecleananddry.3.Someantibioticscreamorsolutioncanbeusedontheskinsurfaces.4.Physicmethods:Laser,freezingandothers.5.Surgeryoperation.201Avian Influenza (Bird Flu) A (H5N1) Virus 202HAProtein(Bindingtoreceptorforinvasion)NProtein(Makevirusestotransmitinsidehostbody)EnvelopeTegumentCapsidGenome(ssRNA8)H5N1203Avian influenza in birdsGenomeofGenomeofAvian influenza A virusAvian influenza A virus(H5N1)is(H5N1)isssRNAssRNA- -. .AvianinfluenzaisaninfectioncausedbyavianAvianinfluenzaisaninfectioncausedbyavian(bird)influenza(flu)viruses.Theseinfluenzaviruses(bird)influenza(flu)viruses.Theseinfluenzavirusesoccurnaturallyamongbirds.Wildbirdsworldwideoccurnaturallyamongbirds.Wildbirdsworldwidecarrythevirusesintheirintestines,butusuallydonotcarrythevirusesintheirintestines,butusuallydonotgetsickfromthem.However,avianinfluenzaisverygetsickfromthem.However,avianinfluenzaisverycontagiousamongbirdsandcanmakesomecontagiousamongbirdsandcanmakesomedomesticatedbirds,includingchickens,ducks,anddomesticatedbirds,includingchickens,ducks,andturkeys,verysickandkillthem.turkeys,verysickandkillthem.204Infectedbirdsshedinfluenzavirusintheirsaliva,Infectedbirdsshedinfluenzavirusintheirsaliva,nasalsecretions,andfeces.Susceptiblebirdsbecomenasalsecretions,andfeces.Susceptiblebirdsbecomeinfectedwhentheyhavecontactwithcontaminatedinfectedwhentheyhavecontactwithcontaminatedsecretionsorexcretionsorwithsurfacesthataresecretionsorexcretionsorwithsurfacesthatarecontaminatedwithsecretionsorexcretionsfromcontaminatedwithsecretionsorexcretionsfrominfectedbirds.Domesticatedbirdsmaybecomeinfectedinfectedbirds.Domesticatedbirdsmaybecomeinfectedwithavianinfluenzavirusthroughdirectcontactwithwithavianinfluenzavirusthroughdirectcontactwithinfectedwaterfowlorotherinfectedpoultry,orthroughinfectedwaterfowlorotherinfectedpoultry,orthroughcontactwithsurfaces(suchasdirtorcages)ormaterialscontactwithsurfaces(suchasdirtorcages)ormaterials(suchaswaterorfeed)thathavebeencontaminated(suchaswaterorfeed)thathavebeencontaminatedwiththevirus.withthevirus.205Human infection with avian influenza virusesTherearemanydifferentsubtypesoftypeATherearemanydifferentsubtypesoftypeAinfluenzaviruses.Thesesubtypesdifferbecauseofinfluenzaviruses.ThesesubtypesdifferbecauseofchangesincertainproteinsonthesurfaceofthechangesincertainproteinsonthesurfaceoftheinfluenzaAvirus(hemagglutininHAandinfluenzaAvirus(hemagglutininHAandneuraminidaseNAproteins).Thereare16knownHAneuraminidaseNAproteins).Thereare16knownHAsubtypesand9knownNAsubtypesofinfluenzaAsubtypesand9knownNAsubtypesofinfluenzaAviruses.ManydifferentcombinationsofHAandNAviruses.ManydifferentcombinationsofHAandNAproteinsarepossible.Eachcombinationrepresentsaproteinsarepossible.Eachcombinationrepresentsadifferentsubtype.AllknownsubtypesofinfluenzaAdifferentsubtype.AllknownsubtypesofinfluenzaAvirusescanbefoundinbirds.virusescanbefoundinbirds.206Usually,“avianinfluenzavirus”referstoUsually,“avianinfluenzavirus”referstoinfluenzaAvirusesfoundchieflyinbirds,butinfectionsinfluenzaAvirusesfoundchieflyinbirds,butinfectionswiththesevirusescanoccurinhumans.Theriskfromwiththesevirusescanoccurinhumans.Theriskfromavianinfluenzaisgenerallylowtomostpeople,becauseavianinfluenzaisgenerallylowtomostpeople,becausethevirusesdonotusuallyinfecthumans.However,thevirusesdonotusuallyinfecthumans.However,confirmedcasesofhumaninfectionfromseveralconfirmedcasesofhumaninfectionfromseveralsubtypesofavianinfluenzainfectionhavebeenreportedsubtypesofavianinfluenzainfectionhavebeenreportedsince1997.Mostcasesofavianinfluenzainfectioninsince1997.Mostcasesofavianinfluenzainfectioninhumanshaveresultedfromcontactwithinfectedpoultryhumanshaveresultedfromcontactwithinfectedpoultryorsurfacescontaminatedwithsecretion/excretionsfromorsurfacescontaminatedwithsecretion/excretionsfrominfectedbirds.Thespreadofavianinfluenzavirusesinfectedbirds.Thespreadofavianinfluenzavirusesfromoneillpersontoanotherhasbeenreportedveryfromoneillpersontoanotherhasbeenreportedveryrarely,andtransmissionhasnotbeenobservedtorarely,andtransmissionhasnotbeenobservedtocontinuebeyondoneperson.continuebeyondoneperson.207“ “Humaninfluenzavirus”usuallyreferstothoseHumaninfluenzavirus”usuallyreferstothosesubtypesthatspreadwidelyamonghumans.Therearesubtypesthatspreadwidelyamonghumans.ThereareonlythreeknownAsubtypesofinfluenzavirusesonlythreeknownAsubtypesofinfluenzaviruses(H1N1,H1N2,andH3N2)currentlycirculatingamong(H1N1,H1N2,andH3N2)currentlycirculatingamonghumans.Itislikelythatsomegeneticpartsofcurrenthumans.ItislikelythatsomegeneticpartsofcurrenthumaninfluenzaAvirusescamefrombirdsoriginally.humaninfluenzaAvirusescamefrombirdsoriginally.Duringanoutbreakofavianinfluenzaamongpoultry,Duringanoutbreakofavianinfluenzaamongpoultry,thereisapossiblerisktopeoplewhohavecontactwiththereisapossiblerisktopeoplewhohavecontactwithinfectedbirdsorsurfacesthathavebeencontaminatedinfectedbirdsorsurfacesthathavebeencontaminatedwithsecretionsorexcretionsfrominfectedbirds.withsecretionsorexcretionsfrominfectedbirds.208CartoonofpH-mediatedviralentryintotargetcells.Interactionsoftheviruswithcellularattachmentfactorsleadtoendocytosisoftheviralparticle.ThesubsequentpHdropwithintheendocyticparticlestheninducesconformationalchangesintheviralglycoproteinsthatleadtofusionofviralandcellularmembranes.209Human Immunodeficiency Virus (HIV)210The“new”syndromediscovered25yearsagohasbecomeoneofthedeadliestThe“new”syndromediscovered25yearsagohasbecomeoneofthedeadliestepidemicsinhumanhistory,killingmorethan25millionpeoplearoundtheworld,epidemicsinhumanhistory,killingmorethan25millionpeoplearoundtheworld,includingmorethan500,000Americans.Inthelastdecade,majoradvancesinincludingmorethan500,000Americans.Inthelastdecade,majoradvancesinpreventionandtreatmentforHIV/AIDShaveprolongedandimprovedthelivesofpreventionandtreatmentforHIV/AIDShaveprolongedandimprovedthelivesofmany,butdespiteextremelybeneficialadvances,theepidemicisfarfromover.Anmany,butdespiteextremelybeneficialadvances,theepidemicisfarfromover.Anestimated40,000AmericansstillbecomeinfectedwithHIVeveryyear,andmanyofestimated40,000AmericansstillbecomeinfectedwithHIVeveryyear,andmanyoftheseareyoungpersonsundertheageof25.AfricanAmericanmenandwomenaretheseareyoungpersonsundertheageof25.AfricanAmericanmenandwomenareamongthehardesthitpopulationsintheU.S.In2004,theyaccountedforhalfofallamongthehardesthitpopulationsintheU.S.In2004,theyaccountedforhalfofallnewHIVdiagnosesinthiscountryandmorethanathirdofAIDSdeathstodate.newHIVdiagnosesinthiscountryandmorethanathirdofAIDSdeathstodate.AfricanAmericanmenwhohavesexwithmen(MSM)areespeciallyhardhit.AfricanAmericanmenwhohavesexwithmen(MSM)areespeciallyhardhit.RecentdatashowsignificantdeclinesinHIVdiagnosesinnearlyeverygroupofRecentdatashowsignificantdeclinesinHIVdiagnosesinnearlyeverygroupofAfricanAmericansexceptblackMSM.WomenalsoremainaparticularlyvulnerableAfricanAmericansexceptblackMSM.Womenalsoremainaparticularlyvulnerablepopulation.population.Nearly25yearsafterthefirstreportofahandfulofNearly25yearsafterthefirstreportofahandfulofcasesofanamelessdeadlydiseaseamonggaymencasesofanamelessdeadlydiseaseamonggaymeninNewYorkandLosAngeles,therearestillover1inNewYorkandLosAngeles,therearestillover1millionpersonslivingwithHIVintheUnitedmillionpersonslivingwithHIVintheUnitedStates.Aboutone-fourthofthosewithHIVhaveStates.Aboutone-fourthofthosewithHIVhavenotyetbeendiagnosedandareunawareoftheirnotyetbeendiagnosedandareunawareoftheirinfection.infection.211212213214215216217218AIDS in China219220221222TheAIDSvillageinHenanChina223224225226AIDSpatientinChinesejail227228229230231232233234235236237HIVparasitedCD4+cellunderelectronmicroscope238AIDSvirusHIVisRTviruswithHIVisRTviruswithssRNAssRNAgenome.Itssizeisabout90-140nmgenome.Itssizeisabout90-140nmindiameter.Thelengthofgenomeis9.7kb.HIVstructuralindiameter.Thelengthofgenomeis9.7kb.HIVstructuralproteins(genes)includep24,p15,p32,p66(Reverseproteins(genes)includep24,p15,p32,p66(Reversetranscriptase),gp120andgp41.gp120ismostimportantforthetranscriptase),gp120andgp41.gp120ismostimportantfortheresearchprojectsbetweenvirusandimmunesystem.researchprojectsbetweenvirusandimmunesystem.TypeI(HIV-1):Transmittedaroundtheworld(includingTypeI(HIV-1):Transmittedaroundtheworld(includingHIVAfrica)HIVAfrica)TypeII(HIV-2):PopularlytransmittedinAfricaTypeII(HIV-2):PopularlytransmittedinAfricaThefollowingmethodscaninactivatevirus:56Thefollowingmethodscaninactivatevirus:56Cfor30minCfor30mindamagestheenzymesystem.damagestheenzymesystem.60Cfor3hor80Cfor30min60Cfor3hor80Cfor30mininactivatevirus.70%alcohol,5-8%formaldehyde,2%inactivatevirus.70%alcohol,5-8%formaldehyde,2%lysollysol,5%,5%phenol,bleachandotherscankillthevirus.ButHIVisnotphenol,bleachandotherscankillthevirus.ButHIVisnotsensitivetoUV.sensitivetoUV.239TransmissionHIV patient and carrierNormal peopleSexBloodOtherroutesMothertoinfant240The people facing high risk:The people facing high risk:1. 1.People with uranism (MSM)/homosexuality: Lesbians, People with uranism (MSM)/homosexuality: Lesbians, GaysGays2. 2.People who have many sex partners: BisexualsPeople who have many sex partners: Bisexuals3. 3.Drug usersDrug users4. 4.Hemophilia patientsHemophilia patients5. 5.The infants of the people infected by HIVThe infants of the people infected by HIVThe features of infection:The features of infection:1. 1.Almost 10,000 persons are infected by HIV each day in Almost 10,000 persons are infected by HIV each day in the worldthe world2. 2.The infection rate of Asian people is going upThe infection rate of Asian people is going up3. 3.In China, Yunnan and Guangdong are the high infection In China, Yunnan and Guangdong are the high infection areasareas4. 4.In China, the infection rate by sex is going upIn China, the infection rate by sex is going up241PathologyCD4+gp120CD4RemovedenvelopeCD4+Expressedgp120CD4+cellsweredamagedImmunecellslostfunctionImmunitylostInfections+TumorsPatientdiedCD4isthereceptorforgp120HIV Carrier/PatientHIV242DiagnosisAfterpeopleareinfectedbyHIV,theycanliveasHIVcarrierswithoutanyclinicsignorsyndromefor210years.ButthespecificantibodyagainstHIVcanbecheckedoutfromtheirblood.Usually,ifHIVcarriersgetAIDSdeveloped,over50%willdiewithinoneyear,and100%willdiewithin45years.2431. Reaction tests for cellular immunity are weaker than normal2. Total immunoglobulin level is higher than normal (but no function to bind to HIV)3. Anti-HIV (Anti-HIV1/Anti-HIV2): Usually, ELISA is used for this test. Now, there are many commercial products available for this check, and they are very easy to use in home or anywhere, and can be finished in 2min.Positive result means HIV infection and the threat to other people. Negative result means no infection until two months ago at least. So, negative result can not be used to make sure some body has no HIV infection to date. 4. Antigen of HIV: Usually, ELISA is used for this test too. In comparison of anti-HIV antibody, antigen is not so easy to check out. Positive result means HIV infection and threat to others. Negative result means no infection until to now, if you can make sure your checking and performance protocol is fine with no any problem.5. Qualitative RT-PCR/Quantitative RT-PCR: Positive result means same to the described above. Negative result means there is no HIV infection probably.6. Isolation of HIV particles: This method is complex and difficult to perform, so, it is not used to diagnose HIV usually.7. Clinic signs and syndrome. 8. Sex history and other life and medical history.244Therapy So far, there is no any completely effective medicine or method to AIDS therapy or clean HIV from body, in other words, no any medicine or method to protect AIDS patients from death. So, for AIDS, prevention is more important than treatment absolutely! But, we have some medicines to prolong the life of AIDS patients limitedly. They are as the follows:I. Anti-virus Reverse transcriptase inhibitors of HIV: AZT, ddI, d4T, 3TC. Protease inhibitors of HIV: Crixivan, R031-8959, A-80987. The medicines to bind to gp120 or gp41: sCD4. II. Chinese medicine and herbs.III. The medicines to enhance immunity of AIDS patients.IV. Anti-infections and tumors.245Chapter 13: Phage Display TechnologyPhage display technology was developed about in the end of 80s and the beginning of 90s of last century. It is a very important technology that is still using in many research fields. Phage display technology mimics molecules that have or can be used as ligand, for examples, antibody, antigen, receptor and peptide So, it plays a important role in the researches of microbiology, immunology, oncology, biochemistry and others. 246Phage DisplayUsed to construct a repertoire of antibodies displayed on phages and mimic antigensUsed to construct a peptides library displayed on phages and mimic some ligands, marker, or target domain247VectorsM13KE248249250Inserts: ScFv, Peptide (7mers, 12mers)ScFv is used to construct the repertoire of antibodies displayed on phages. After screening to the repertoire, the specific phage with displayed ScFv can be obtained. To get a huge repertoire that includes total antibodies matching to most of antigens in the world, the universal ScFv primers and linker have to be designed and used. I give you a design example as the follows:ScFv251Example for Primer Design: AGATACATCTGGAGACTTTCGGAAAGCCTTGCTCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGCCGCGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAAGGCCATGGTTGTGTATCTCTAAGAATCTAAGAA Upper A2 BamH I:5-AAAAAAGGATCCAGATACATCTGGAGACTTTCGGAAAGCCTTGCT-3(Length: 45bp, TM: 75C, GC: 42.2%)Lower A2 Kpn I:5-CCCACCGGTACCTTCTTAGATTCTTAGAGATACACAACCATGGCC-3(Length: 45bp, TM: 748C, GC: 48.9%)252The sequences of the primers and linker:Hu Vh back (23bp)5-CAGGTGCAGYTRNDGSAGTCDGS-3Hu Jh forward (24bp)5-TGAGGAGACGGTGACCRKKGTBCC-3Hu Vh back Sfi I & Nco I (56bp)5-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGYTRNDGSAGTCDGS-3Hu Jh forward Not I (48bp)5-GAGTCATTCTCGACTTGCGGCCGCTGAGGAGACGGTGACCRKKGTBCC-3Hu Vk back (23bp)5-GAMATYSWGMTSACBCAGTCTCC-3253Hu V back (23bp)5-YMBKYTRTRYTGACKCARBMNBC-3Hu Jk forward Not I (48bp)5-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATYTCCASYYKKGTCCC-3Hu J forward Not I (48bp)5-GAGTCATTCTCGACTTGCGGCCGCACCTARRACGGTSASSTKGGTCCC-3Linker (45bp)5-GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG-3Reverse Jh for ScFv linker (40bp)5-GVACMMYGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGG-3Reverse Vk for ScFv linker (42bp)5-GGAGACTGVGTSAKCWSRATKTCCGATCCGCCACCGCCAGAG-3Reverse V for ScFv linker (42bp)5-GVNKVYTGMGTCARYAYARMVKRCGATCCGCCACCGCCAGAG-3254FcVHLCDRFRAmodelofIgGmolecule255A linker will link HV and LV togetherA.RecombinedScFvgeneinsertB.RecombinedScFvantibodyPrimerVHLinkerVLAB256Peptide (12mers)NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNRandomlysynthesizedpeptidegeneinsertTerminalslinkedtotheMCSofphagePeptide gene insert257BuildingofthelibrarybyoverlapPCR.Tobuildthelibrary,fourPCRwereperformed,twoofthemincorporatingmutationsintheCDRH3andL3.PurifiedproductswerethenlinkedusingoverlapPCRtoobtainfull-sizemutatedgenes.Fromlefttoright,theprimersusedare5PelB,RPMH3,PMH3,LINKINF,LINKSUP,RPML3,PML3,and3CMYC.Example258Example259Example for peptide 260DisplayM13KEgenome+Insert(RecombinedM13KE)DisplayedgeneDisplayedproteinM13KE+Insert(RecombinedM13KE)261Bio-panning Bio-panning is a protocol to screen out the specific ligand that mimicked by phage display technology. It requires careful performed procedures. I present a good bio-panning protocol that was modified by me when I worked in American NIH. The bio-panning protocol is as the follows:262263Ex-phageareobtainedbyinfectingthemutantphageintoTG1suppressingE.colistrain,andusedforpackagingofsingle-chainantibodydisplayphagemidpIGT3inJS5non-suppressingE.colistrain.Theresultingrecombinantphagecarriesseveralcopiesoftheantibodymolecules.ThetrypsinandEKcleavagesiteslinkingthescFvandpIIIcanbeusedforproteaseelutionafterpanning,andrestorationofwild-typepIIIenablestherecombinantphagetoreinfectE.coli.Example264An improved bio-panning protocol for peptides mimickingThisprotocolwasdesignedandmodifiedbymewhenIwasapostdoctoralvisitorinNIH,USA.Becauseofintellectualpropertyrights,Idonotwanttoshowsomedetailedstepinformationhere(Totally,thisprotocolshouldtake62 basic steps at least).But,ifyouwanttouseitinfuture,youwillbewelcometoaskmedetailaboutitatanytime.Ifyouholdyourexperimentsaccordingtothelibrarykitinstructionbook,youwillwastelongtimeandalotmoneytodoitwithoutresultingdataprobably.(1)Coatwells(2wellsof6-wellsplate)forlibrarybinding,4C,O/N(2)Block,librarybinding,nonspecificbinding,wash(3)Elutetheboundphageswiththeligandofthetarget.Collecttheeluateintoatube,thisistheprimary eluate.Storeitat4C(4)Titertheprimaryeluate(5)AmplifytheprimaryeluateinE.coli.(6)Getthespeciallytreatedsupernatantfromtheamplifiedprimaryeluate.Then,precipitateittwotimeswithPEGtogetprimary sub-library.265(7)Titertheprimarysub-library.(8)Take1-21011pfufromprimarysub-library,makesecondscreeningandenrichingroundlikeabove.(9)Getsecondsub-eluate.(10)Getsecondsub-libraryafteramplifying,precipitatinglikeabove.(11)Titerthesecondsub-library.(12)Makethirdscreeningandenrichingroundtogetthirdsub-eluate.(13)Monoclonizethethirdsub-eluatewithX-galplate(Ifyouwanttomakemoreroundstoscreenandenrichthephages,youneedtocontinuetheamplifying,precipitatingcyclelikeabove).(14)SelecttheplaquesfromtheX-galplatesandmakeamplifiedmonoclonalphagestocks.(15)SelectpositiveclonesfromtheamplifiedmonoclonalphagestocksusingELISA.(16)Sequencethepositiveclones.Analyzethesequencedatatogetmotifsequences.The motif sequences are the sequences that mimic the target you want to mimic.(Thisimprovedprotocoltakes62basicstepsatleast)266The motif sequences can be used to many important research projects, for example, DNA vaccine with pVAX1 vector.267267The presentation of virology endsI hope you every one will get a very good test grade for virology!You will have an exam test next time