病毒感染的检查方法与防治原则ePPT专业课件.ppt

1,LaboratoryDiagnosis,.,2,CategoryofSample,Blood,Urine,Stool,nasalwashing,nasalswab,throatswab,saliva,sputum,rectalswab,vesiclefluid(scrapingorswab),tissue,brainbiopsy,cerebrospinalfluid,etal.,.,3,LaboratoryDiagnosis,MicroscopyIdentificationVirusisolationandidentificationDetectionofviralproteins(antigensandenzymes)DetectionofviralgeneticmaterialSerologicprocedures,.,4,MicroscopyIdentification,LightmicroscopyFluorescentmicroscopyElectronmicroscopy,.,5,Lightmicroscopy,CharacteristicCPEInclusionBodies,.,6,CelldeathCellroundingDegenerationAggregationLossofattachmentstosubstrateCharacteristichistologicalchanges:inclusionbodiesinthenucleusorcytoplasm,marginationofchromatinSyncytia:multinucleatedgiantcellscausedbyvirus-inducedcell-cellfusion,.,7,Fluorescentmicroscopy,Fluorescent-antibodystaining,.,8,Electronmicroscopy,Directdetection:Humanrotavirus;HAV;HBV;Smallpoxvirus;Herpesvirus.ImmuneElectronmicroscopy:Humanrotavirus;HAV;,.,9,LaboratoryDiagnosis,MicroscopyIdentificationVirusisolationandidentificationDetectionofviralproteins(antigensandenzymes)DetectionofviralgeneticmaterialSerologicprocedures,.,10,ViralisolationandIdentification,ViralGrowthandCellcultureViralDetectionViralIdentificationInterpretationofcultureresults,.,11,SystemsforthePropagationofViruses,PeopleAnimals:cows,chickens,mice,rats,sucklingmiceEmbryonatedeggsOrganandtissuecultureOrganculturePrimarytissuecultureCelllines:diploidTumoror（immortalized）cellline,.,12,Viraldetection,CPEHemadsorptionInterfereMetabolizeofcell,.,13,TCID50(Tissuecultureinfectivedose),TCID50isdefinedasthatdilutionofviruswhichwillcauseCPEin50%ofagivenbatchofcellcultureTCID50=log10ofhighestdilutiongiving100%CPE+1/2(totalnumberoftestunitsshowingCPE)/(numberoftestunitsperdilution),.,14,Viralidentification,Complementfixation：HemagglutinationinhibitionNeutralizationImmunofluorescence(directorindirect)LatexagglutinationInsituEIAELISARIA(radioimmuno,.,15,LaboratoryDiagnosis,MicroscopyIdentificationVirusisolationandidentificationDetectionofviralproteins(antigensandenzymes)DetectionofviralgeneticmaterialSerologicprocedures,.,16,Detectionofviralproteins(antigensandenzymes),Antigendetection(ELISA,RIA,Westernblot)HemagglutinationandhemadsorptionEnzymeactivities(reversetranscriptase)Proteinpatterns(electrophoresis),.,17,LaboratoryDiagnosis,MicroscopyIdentificationVirusisolationandidentificationDetectionofviralproteins(antigensandenzymes)DetectionofviralgeneticmaterialSerologicprocedures,.,18,Detectionofviralgeneticmaterial,PCR(Polymerasechainreaction)RT-PCR(Reversetranscriptasepolymerasechainreaction)Southern（DNA),Northern(RNA),anddotblotsDNAgenomehybridizationinsitu(cytochemistry)ElectrophoreticmobilitiesofRNAforsegmentedRNAviruses(Electrophoresis)Restrictionendonucleasecleavagepatterns,.,19,LaboratoryDiagnosis,MicroscopyIdentificationVirusisolationandidentificationDetectionofviralproteins(antigensandenzymes)DetectionofviralgeneticmaterialSerologicprocedures,.,20,Serologicprocedures,Iftheantibodytiterintheconvalesent-phaseserumsampleisatleast4-foldhigherthanthetiterintheacute-phaseserumsample,thepatientisconsideredtobeinfected.Incertainviraldiseases,thepresenceofIgMantibodyisusedtodiagnosecurrentinfectionOthernonspecificserologictestsareavailable,.,21,Serologicprocedures,Complementfixation：HemagglutinationinhibitionNeutralizationImmunofluorescence(directorindirect)LatexagglutinationInsituEIAELISARIA,.,22,VirusesDiagnosedbySerology,Epstein-BarrvirusRubellavirusHepatitisA,B,C,D,andEvirusesHIVHumanT-cellLeukemiavirusArboviruses(Encephalitisviruses),.,23,Prevention,SuccessesofthePastPossibilitiesfortheFuture,.,24,Activeimmunization,Vaccines,.,25,OverviewofActiveimmunization,Activeimmunization-administrationofantigenresultinginproductionofaspecificimmuneresponsewithimmunologicmemory.Responsemaybecellularorhumoralorboth.Naturalimmunity-todiseasesyouhavecaughtandsuccessfullyfoughtArtificialimmunityVaccination(vaccines),.,26,Attributesofagoodvaccine,AbilitytoelicittheappropriateimmuneresponsefortheparticularpathogenLongtermprotectionideallylife-longSafetyvaccineitselfshouldnotcausediseaseStableretainimmunogenicity,despiteadversestorageconditionspriortoadministrationIn-expensive,.,27,LIVEVACCINES,LiveattenuatedorganismHeterologousvaccinesLiverecombinantvaccinesAttributeslivevaccines,.,28,Liveattenuatedorganism,OrganismswhosevirulencehasbeenartificiallyreducedbyinvitroCultureunderadverseconditions,suchasreducedtemperature.,.,29,Heterologousvaccines,Closelyrelatedorganismoflesservirulence,whichsharesmanyantigenswiththevirulentorganism.Thevaccinestrainreplicationinthehostandinducesanimmuneresponsethatcrossreactswithantigensofthevirulentorganism.Vacciniavirus/cowpoxvirus-Variolavirus,.,30,Liverecombinant,VectorbovinevaccineBCG,.,31,BothcellmediatedimmunityandantibodyresponseActivatesallphasesofimmunesystem.CangethumoralIgGandlocalIgARaisesimmuneresponsetoallprotectiveantigens.Inactivationmayalterantigenicity.Moredurableimmunity;morecross-reactiveImmunityislonglivedSingledose,AdvantagesofAttenuatedVaccines2-1,.,32,AdvantagesofAttenuatedVaccines2-2,LowcostQuickimmunityinmajorityofvaccineesIncaseofpolioandadenovaccines,easyadministrationEasytransportinfieldCanleadtoeliminationofwildtypevirusfromthecommunity,.,33,DisadvantagesofLiveAttenuatedVaccine,Mutation;reversiontovirulence(oftenfrequent)Spreadtocontactsofvaccineewhohavenotconsentedtobevaccinated(couldalsobeanadvantageincommunitieswherevaccinationisnot100%)Spreadvaccinenotstandardized-maybeback-mutatedPoortakeintropicsProbleminimmunodeficiencydisease(mayspreadtothesepatients),.,34,Killedvaccines,Theorganismispropagatedinbulk,invitro,andinactivatedwitheitherbeta-propiolactoneorformaldehyde.Thesevaccinesarenotinfectiousandarethereforerelativelysafe.However,theyareusuallyoflowerimmunogenicityandmultipledosesmaybeneededtoinduceimmunity.Inaddition,theyareusuallyexpensivetoprepare.,.,35,Killedvaccines,Inactivatedorganism:rabiesvirus;epidmictypeBencephalitisvirus.SubunitVaccines:Influenzavirus(HAandNA)Recombinantproteins:HBV,.,36,Advantagesofinactivatedvaccines,GivessufficienthumoralimmunityifboostersgivenNomutationorreversionCanbeusedwithimmuno-deficientpatientsThesevaccinestendtobeabletowithstandmoreadversestorageconditions,Sometimesbetterintropics,.,37,Disadvantagesofinactivatedvaccines,Manyvaccineesdonotraiseimmunitypoor,onlyantibody,nocellimmediatedimmuneresponseresponseisshort-livedandmultipledosesareneededNolocalimmunity(important)Inactivated,thereforecannotreplicateinthehostandcausediseaseFailureininactivationandimmunizationwithvirulentvirusExpense:Expensivetoprepare,.,38,NewMethods,SelectionofattenuatedvirusstrainVaricellaHepatitisAUsemonoclonalantibodiestoselectforviruswithalteredsurfacereceptorRabiesReoUsemutagenandgrowvirusat32degrees.Selectsfortemperature-sensitivevirus.Growsinupperrespiratorytractbutnotlowerflu(newvaccine)respiratorysyncytialvirus,.,39,NewMethods,PassageprogressivelyatcoldtemperaturesTSmutantininternalproteinsCanbere-assortedtosothatcoatisthestrainthatisthisyearsflustrain,.,40,.,41,NewMethods,DeletionmutantsSuppressionunlikely(butcautioninHIV)ViablebutgrowthrestrictionsProblemsOncogenicityinsomecases(adeno,retro),.,42,NewMethods,.,43,Singlegene(subunit)-problems,Surfaceglycoproteinpoorlysoluble-deletion?PoorlyimmunogenicPost-translationalmodificationsPoorCTLresponse,.,44,Singlegene(subunit)inexpressionvector,VaccinatewithlivevirusCanaryPoxInfectshumancellsbutdoesnotreplicateBetterpresentationCTLresponseVacciniaAttenuatedPolioBeingdevelopedforanti-HIVvaccine,.,45,NewMethods,Chemicallysynthesizedpeptidemalariapoorlyimmunogenic,.,46,Newmethods,Anti-idiotypevaccine,epitope,Antibodywithepitopebindingsite,Virus,.,47,Anti-idiotypevaccinecont,Makeantibodyagainstantibodyidiotype,Anti-idiotypeantibodymimicstheepitope,.,48,Anti-idiotypeantibodycont2,Useanti-idiotypeantibodyasinjectablevaccine,Antibodytoanti-idiotypeantibody,Bindsandneutralizesvirus,Useasvaccine,.,49,NewMethods,New“JennerianVaccines”LivevaccinesderivedfromanimalstrainsofsimilarvirusesNaturallyattenuatedforhumansRotavirus:MonkeyRota80%effectiveinsomehumanpopulationsIneffectiveinothersDuetodifferencesincirculatingviralserotypes,.,50,NewMethods,NewJennerianVaccinesBovineparainfluenzaType3Bovinevirusis:InfectioustohumansImmunogenic(61%ofchildrengetgoodresponse)PoorlytransmissablePhenotypicalystable,.,51,NewMethods,.,52,Vaccines,1796Jenner:wildtypeanimal-adaptedvirus1800sPasteur:Attenuatedvirus1996DNAvaccinesThethirdvaccinerevolution,.,53,DNAvaccines,DNAvaccinesareatpresentexperimental,butholdpromiseforfuturetherapysincetheyevokebothhumoralandcell-mediatedimmunity,withoutthedangersassociatedwithlivevirusvaccines,.,54,DNAVaccines,plasmid,Musclecell,Geneforantigen,Musclecellexpressesprotein-antibodymadeCTLresponse,.,55,DNAVaccines,PlasmidsareeasilymanufacturedinlargeamountsDNAisverystableDNAresiststemperatureextremessostorageandtransportarestraightforwardDNAsequencecanbechangedeasilyinthelaboratory.ThismeansthatwecanrespondtochangesintheinfectiousagentByusingtheplasmidinthevaccineetocodeforantigensynthesis,theantigenicprotein(s)thatareproducedareprocessed(post-translationallymodified)inthesamewayastheproteinsofthevirusagainstwhichprotectionistobeproduced.Thismakesafarbetterantigenthanpurifyingthatproteinandusingitasanimmunogen.,.,56,DNAVaccines,Mixturesofplasmidscouldbeusedthatencodemanyproteinfragmentsfromavirus/virusessothatabroadspectrumvaccinecouldbeproducedTheplasmiddoesnotreplicateandencodesonlytheproteinsofinterestNoproteincomponentsotherewillbenoimmuneresponseagainstthevectoritselfBecauseofthewaytheantigenispresented,thereisaCTLresponsethatmaybedirectedagainstanyantigeninthepathogen.ACTLresponsealsooffersprotectionagainstdiseasescausedbycertainobligateintracellularpathogens(e.g.Mycobacteriumtuberculosis),.,57,DNAVaccines,PossibleProblemsPotentialintegrationofplasmidintohostgenomeleadingtoinsertionalmutagenesisInductionofautoimmuneresponses(e.g.pathogenicanti-DNAantibodies)Inductionofimmunologictolerance(e.g.wheretheexpressionoftheantigeninthehostmayleadtospecificnon-responsivenesstothatantigen),.,58,DNAVaccines,DNAvaccinesproduceasituationthatreproducesavirally-infectedcellGives:BroadbasedimmuneresponseLonglastingCTLresponseAdvantageofnewDNAvaccineforflu:CTLresponsecanbeagainstinternalproteinInmiceanucleoproteinDNAvaccineiseffectiveagainstarangeofviruseswithdifferenthemagglutinins,.,59,Adjuvants,Certainsubstances,whenadministeredsimultaneouslywithaspecificantigen,willenhancetheimmuneresponsetothatantigen.,.,60,Adjuvantsincommonuse,AluminiumsaltsLiposomesandimmunostimulatingcomplexesCompletFreundsadjuvantisanemulsionofmycobacteria,oilandwaterIncompleteFreundsadjuvantMuramyldi-peptideCytokines,.,61,Possibleactionmodesofadjuvant,Bytrappingantigeninthetissues,thusallowingmaximalexposuretodendriticcellsandspecificTandBlymphocytesByactivatingantigen-presentingcellstosecretecytokinesthatenhancetherecruitmentofantigen-specificTandBcellstothesiteofinoculation,.,62,Smallpox,.,63,Smallpox,Variolation1%v.25%mortalityLife-longimmunityNodriftorshift,.,64,Smallpox,VaccinationJenner1796:Cowpox/Swinepox1800sCompulsorychildhoodvaccination1930sLastnaturalUKcase1940slastnaturalUScase1958WHOprogramOctober1977:Lastcase(Somalia),.,65,Smallpox,NoanimalreservoirLifelongimmunitySubclinicalcasesrareInfectivitydoesnotprecedeovertsymptomsOneVariolaserotypeEffectivevaccineMajorcommitmentbygovernments,.,66,polio,Killedvirusvaccine(Salk,1954)Liveattenuatedoralpoliovaccine(Sabin,1957)TheinactivatedSalkvaccinesisrecommendedforchildrenwhoareimmunosuppressed.,.,67,PolioVaccine,.,68,Reportedcasesper100000population,100,10,1,0.1,0.001,0.01,1950,1960,1970,1980,1990,Inactivated(Salk)vaccine,Oralvaccine,Casesper100,000populationUnitedStates,.,69,10000,1000,100,10,1,0,Reportedcases,1950,1955,1960,1965,1970,1975,Killed(Salk)vaccine,TotalcasesSwedenandFinland,.,70,.,71,SabinPolioVaccine,AttenuationbypassageinforeignhostMoresuitedtoforeignenvironmentandlesssuitedtooriginalhostGrowslesswellinoriginalhostPolio:MonkeykidneycellsGrowsinepithelialcellsDoesnotgrowinnervesNoparalysisLocalgutimmunity(IgA)Pasteurrabiesvaccinealsoattenuated,.,72,SalkPolioVaccine,Formaldehyde-fixedNoreversion,.,73,PolioVaccine,WhyusetheSabinvaccine?:Localimmunity:VaccinevirusjustlikenaturalinfectionStoppingreplicationinG.I.TractstopsviralreplicationTOTALLYDeadSalkvaccinevirushasnoeffectongutreplicationNoproblemwithselectiveinactivationGreatercrossreactionasvaccinevirusalsohasantigenicdriftLife-longimmunity,.,74,Measles,Liveattenuatedvirusgrowninchickembryofibroblasts,firstintroducedinthe1960s.Etiology:MeaslesvirusIncubation:8to12daysClinicalManifestations:cough,coryza,conjunctivitis,erythematousmaculopapularrashfever,KoplikSpots,complictionsincludeEncephalitis,Pneumonia,andSSPETreatment:Supportive,.,75,Mumps,Liveattenuatedvirusdevelopedinthe1960sMMRvaccineEtiology:MumpsVirusIncubation:16to18daysClinicalManifestations:swellingofthesalivaryglandscomplicationsincludeMeningitis,Orchitis,Encephalitis,andDeafness,.,76,rubella,LiveattenuatedvirusEtiology:RubellaVirusIncubation:14to21daysClinicalManifestations:Congenital,cataractspatentductusarteriosus,deafnessmentalretardation,Postnatalmilddisease,erythematousmaculopapularrash,postauricularlymphadenopathytransientpolyarthralgias,.,77,HepatitisB,Twovaccinesareincurrentuse:AserumderivedvaccineArecombinantvaccineEtiology:HepatitisBVirusIncubation:120days(average)ClinicalManifestations:jaundice;anorexianauseaandvomiting;malaisecomplicationsincludethedevelopmentofachroniccarrierstatewithahighriskforHepatocellularCarcinoma(livercancer),.,78,HepatitisA,Formalin-inactivated,cellcultured-derivedvirus,.,79,Yellowfever,The17Dstrainisaliveattenuatedvaccinedevelopedin1937.Itisahighlyeffectivevaccinewhichisadministeredtoresidentsinthetropicsandtravellerstoendemicareas.,.,80,Rabies,Nosafeattenuatedstrainofrabiesvirushasyetbeendevelopedforhuman.Vaccinesincurrentuseinclude:aTheneurotissuevaccinebhumandiploidcellculture-derivedvaccine,whichismuchsafer.Therearetwosituationwherevaccineisgiven:aPost-exposureprophylaxis,followinfthebiteofarabidanimal,Hyperimmunerabiesglobulinmayalsoadministered.bPro-exposureprophylaxisisusedforprotectionofthoseoccupationputsthematriskofinfectionwithrabies.,.,81,Influenza,Newvaccinesareproducedeveryyear,.,82,Varicella-Zostervirus,Notlicensedvaccines,.,83,.,84,PassiveImmunisation,.,85,Modesofimmunization,Passiveimmunization-administrationofantibody-containingserumtoprovideimmediate,buttemporaryprotection.Doesntactivatealastingspecificimmuneresponse.,.,86,Natural,Providesimmunityfordiphtheria,tetanus,streptococcus,rubeola(redmeasles),rubella(Germanmeasles),mumps,polio,andothers.,.,87,Artificial,Oftenusedasantitoxinsforthingssuchasblackwidowspiderandsnakebites,botulism,andtetanus.Importantforsomeinfectiousdiseasessuchasrabies,sinceitprovidesimmediateprotectionratherthanwaitingthe7-10daysforaprotectiveresponsetodevelopfromactiveimmunization.,.,88,Immunoglobulin,“Normal”ImmuneglobulinHyper-immuneglobulin,.,89,“Normal”Immuneglobulin,LowtitresofantibodytoawiderangeofhumanvirusesHepatitisAvirusinfectionParvovirusinfectionEnterovirusinfections(inneonates)HIV-infectedbabies,.,90,Hyper-immuneglobulin-hightitresofantibodytoparticularviruses,Zosterimmuneglobulin:preve