HIV检查 ppt课件.ppt

Retroviruses,Retrovirusesareenveloped,single-strandedplus-senseRNAviruses.TheyencodeanenzymecalledreversetranscriptasethatconvertstheRNAgenomeintoadouble-strandedDNAcopythatsubsequentlybecomesintegratedintothehostcellDNA.,Retrovirusesstructure,Retrovirusesareclassifiedintothreefamilies:Oncoviruses,(onco-,“relatedtoatumor”)Lentiviruses,(lenti-,“relatedtoslow”)Spumaviruses,(humanfoamyvirus)Spumaviruseshasnotyetbeendefinitelyassociatedwithdiseaseinhumans.Representativesoftwomajorgroupsareconsideredinthischapter:theoncovirusesandthelentiviruses.,Membersoftheoncovirusessubgroupofretroviruseshavelongbeenassociatedwithavarietyofcancersinanimals,includingleukemias,lymphomas,andsarcomas,butuntilrecentyearshadnotbeenfoundtoinfecthumans.Thefirsthumanretrovirus,humanT-cellleukemiavirustypeI(HTLV-1),wasdiscoveredinthelate1970s.ItwasshowntocauseadultT-cellleukemia,araremalignancyfoundonlyinJapan,Africa,andtheCaribbean.ArelativeofHTLV-1,HTLV-2,hasbeenassociatedwithafewrarecasesofT-cellmalignancies,includinghairycellleukemia,butitspreciseroleinthesediseasesremainsunclear.,HumanImmunodeficiencyVirus(HIV),ThevirusthatcausesAIDSFirstexposedin1981incentralAfrica,infectionunknownHaskilledmorethan20millionpeopleWith3.1millionpeopledyinglastyearalone.Estimated45millionpeoplethatweknowofarecurrentlylivingwithHIV,Themostimportantdiseaseresultingfromahumanretrovirusinfection,acquiredimmunodeficiencysyndrome(AIDS),iscausedbyeitheroftwolentvirusestermedhumanimmunodeficiencyvirusestypes1and2(HIV-1andHIV-2).Adevastatingdisease,forwhichthereisnopresentcure.,Virusesarealsoetiologicfactorsinthedevelopmentofseveraltypesofhumantumors,includingtwoofgreatsignificanceworldwide-cervicalcancerandlivercancer.Atleast15%ofallhumantumorsworldwidehaveaviralcause.Thesevirusesarecancerassociatedvirusesorhumancancervirusesortumorviruses.,HIVandAIDSaninfectiousagent,1979-5casesofPneumocystiscarinii,Withgiemsastainathighmagnification,thefaintbluishdot-likeintracysticbodiesofPneumocystiscariniiinlungareseeninthiscytologicpreparationfromabronchoalveolarlavage,InLosAngeles1967-1978onlytwocasesofPneumocystiscariniipneumonia,AllHomosexual,HIVandAIDS,Withdisseminationtoextrapulmonarysites,Pneumocystiscariniitendstoproducefociwithprominentcalcification,asseeninthekidneyheregrossly.,HIVandAIDSaninfectiousagentKaposisSarcoma,Early1981MMWR:5casesofKaposissarcomaHitherto:rare(immunocompromization)1981-26casesofKaposissarcomaYoungSanFranciscoandNewYorkAllHomosexuals,HIVisaretrovirus,amemberofthelentivirusgenus,andexhibitsmanyofthephysicochemicalfeaturestypicalofthefamily.TheuniquemorphologiccharacteristicofHIVisacylindricalnucleoidinthematurevirion.Thediagnosticbarshapednucleoidisvisibleinelectronmicrographsinthoseextracellularparticlesthathappentobesectionattheappropriateangle.Thevirionisabout100-120nmindiameter,andbecauseitcontainstwocopiesoftheRNAgenome,itisdiploid.,TheRNAgenomeiscoatedwiththenucleocapsidprotein(NC),andtheRNA-proteincomplexesareenclosedinacapsid(CA)composedofmultiplesubunits.Likeallenvelopedviruses,themembraneisacquiredduringbuddingfromthehostcell,butthesurfaceglycoproteins(SU,alsocalledgp120)andtransmembraneglycoproteins(TM,alsocalledgp41)foundintheenvelopearevirallyencoded.Inadditiontothestructuralproteins,thevirioncorecontainsthreevirusspecificproteinsthatareessentialforviralreplication:reversetranscriptase(RT),protease(PR),andintegrase(IN).,Theviruscontainsthethreestructuregenesrequiredforareplicatingretrovirus-gag,pol,andenv.Uptosixadditionalgenesregulateviralexpressionandareimportantindiseasepathogenesisinvivo.HIV-1contains,inadditiontothegag,pol,andenvgenes,anarrayofothergenes(tat,rev,vef,vif,vpr,andvpu).ExpressionofthesegenesrequiresmRNAsplicing,andallapparentlyencodeproteinsthatserveregulatoryoraccessoryrolesduringtheinfection.Tatistranscriptionalactivatorthatpromotessynthesisoffull-lengthviraltranscripts.Revpromotesexportofunsplicedandpartiallysplicedtranscriptstocytoplasm.Vefdown-regulatesCD4topromotevirusreleaseandalsodown-regulatesMHC-Itointerferewithimmunerecognition.VputargetsCD4destructionandvirionrelease.Vprpromotestransportofsubviralparticlesintonucleusofnondividingcells.VpuandVifincreaseefficiencyofinfectionandyieldofvirus.ActivationofCD4Tlymphocytesvirusincreasesvirusproduction.,Theregionsofgreatestdivergenceamongdifferentisolatesarelocalizedtotheenvgene,whichcodesfortheviralenvelopeproteins.TheSU(gp120)productoftheenvgenecontainsbindingdomainsresponsibleforvirusattachmenttotheCD4moleculeandcoreceptors,determineslymphocyteandmacrophagetropisms,andcarriesthemajorantigenicdeterminantsthatelicitneutralizingantibodies.TheTM(gp41)envproductcontainsbothatransmembranedomainthatanchorstheglycoproteinintheviralenvelopeandafusiondomainthatfacilitatesviralpenetrationintotargetcells.ThedivergenceintheenvelopeofHIVcomplicateseffortstodevelopaneffectivevaccineforAIDS.,MajorretroviralgenesandproteinsofHIV,Surfaceglycoprotein,Transmembraneprotein,matrix,cpsid,nucleocapsid,Reversetranscriptase,Virusreplication,Thevirionsadsorbtocellularmembranereceptorsandenterthecellbydirectfusionwiththeplasmamembrane.ForHIV-1,thevirionattachmentproteinistheSUglycoproteingp120,andthecellularreceptoristheCD4molecule.AllprimatelentivirusesuseasareceptortheCD4molecule,whichisexpressedonmacrophagesandTlymphocytes.AsecondcoreceptorinadditiontoCD4isnecessaryforHIV-1togainentrytocells.Thesecondreceptorisrequiredforfusionoftheviruswiththecellmembrane.,ThevirusfirstbindstoCD4andthentothecoreceptor.Theseinteractionscauseconformationalchangesintheviralenvelope,activatingthegp41fusionpeptideandtriggeringmembranefusion.TheHIV-1transmembraneTMproteingp41isresponsibleforfusionoftheviralandcellmembranes,leadingtoentryofthevirioncorecomplexintothecytoplasmofthecell.,Fusionactivitymayalsoplayanimportantroleinamplificationoftheeffectsofthevirusinfection,particularlyduringthelaterstagesoftheinfection,becauseinfectedcellsexpressingviralglycoproteinsintheirmembranesreadilyfusewithuninfectedCD4+Tlymphocytestoformlargesyncytia.Thisprocessappearstoprovideameansforcell-to-celltransmissionofthevirusthatbypassestheusualextracellularphaseandmaycontributetotheoveralldepletionofCD4lymphocytesinaninfectedindividual.,Soonafterentryoftheviralcoreintothecytoplasmoftheinfectedcell,theRNAiscopiedintodouble-strandedDNAbyreversetranscriptase,thevirion-associatedDNApolymerase.TheoverallprocessesreferredtoasreversetranscriptaseandresultsinalinearDNAmoleculethatentersthenucleusandintegratesmore-or-lessatrandomintoahostcellchromosome.OncetheviralgeneticinformationhasbeenconvertedtoDNAandintegrated,itessentiallybecomespartofthecellulargenome.Theviralgenes,calledtheprovirus,arethereforereplicatedandfaithfullyinheritedaslongastheinfectedcellcontinuestodivide.SpecialsequencescontainedwithintheRNAareduplicatedduringthereversetranscriptionprosesssothattheintegratedproviruscontainsidenticallongterminalrepeats(LTRs)atitsends.,TheLTRsequencescontaintheappropriatepromoter,enhancer,andothersignalsrequiredfortranscriptionoftheviralgenesbythehostRNApolymeraseII.DNApolymeraseactivity,thereversetranscriptasepossessesanRNaseHactivitythatisresponsiblefordegradingtheRNAportionoftheDNA-RNAhybrid(RNA/DNA)producedinthefirstphaseofreversetranscription.Theimmediateproductofreversetranscriptionisalineardouble-strandedDNAmoleculethatisflankedbytheLTRsequences.Theviralintegrase(IN)catalyzestheintegrationofthelinearDNAintohostDNA.,ThechoiceofatargetsiteforintegrationintothecellularDNAappears,however,tobenearlyrandom.ThereplicationprocessiscompletedbytranscriptionoftheproviralDNAbythehostRNApolymeraseII.Virusparticlesmatureandemergefrominfectedhostcellsbybuddingfromcytoplasmicmembranes.TheviralproteasethencleavestheGagandPolproteinsfromtheprecursorpolyprotein,producinganinfectiousvirionpreparedforreversetranscriptionwhenthenextcellisinfected.,Replication,HIVViruseClassification,Lentiviruseshavebeenisolatedfrommanyspeciesincludingatleast26differentAfricannonhumanprimatespecies.TherearetwodistincttypesofhumanAIDSviruses:HIV-1andHIV-2.Thetwotypesaredistinguishedonthebasisofgenomeorganizationandphylogenetic(evolutionary)relation-shipwithotherprimatelentiviruses.,Variation,HIV-1possessesthemosterror-pronereversetranscriptase.TheconsequenceofthishigherrorrateisthateachtimetheviralRNAisreversetranscribed,onetotwonewmutationsareintroducedintotheresultingDNA.BecausetheprocessoftranscriptionoftheintegratedproviralDNAtoproducenewviralgenomesisalsoerrorprone,mutantgenomesaccumulaterapidlyoverthecourseofaninfection.,Theendresultisaquasi-speciesthataccountsforthemanynucleotidedifferencesobservedbetweendifferentisolates(evenfromthesameinfectindividual)andforthevariabilityoftheSUenvelopeproteingp120.Itmayexplain,inpart,thefailureoftheimmunesystemtocontraltheinfectionandalsotheincreasesinviralvirulencethatappearstooccurduringthecourseoftheinfection,Disinfectionandinactivation,HIViscompletelyinactivatedbytreatmentfor10minutesatroomtemperaturewithanyofthefollowing:10%householdbleach,50%ethanol,35%isopropanol,1%NonidetP40,0.5%lysol,0.5%paraformaldehyde,or0.3%hydrogenperoxide.ThevirusisalsoinactivatedbyextremesofpH(pH1.0,pH1.3).However,whenHIVispresentinclottedorunclottedbloodinaneedleorsyringe,exposuretoundilutedbleachforatleast30secondsisnecessaryforinactivation.HIVisreadilyinactivatedinliquidsor10%serumbyheatingat56for10minutes,butdriedproteinaceousmaterialaffordsmarkedprotection.,Pathogenesisandsymptomatology,PathogenesisandpathologyThetypicalcourseofuntreatedHIVinfectionspansaboutadecade.Stagesincludetheprimateinfection,disseminationofvirustolymphoidorgans,clinicallatency,elevatedHIVexpression,clinicaldisease,anddeath.Thedurationbetweenprimaryinfectionandprogressiontoclinicaldiseaseaveragesabout10yesrs.Inuntreatedcases,deathusuallyoccurswithin2yearsaftertheonsetofclinicalsymptoms.Followingprimaryinfection,thereisa4-to11-dayperiodbetweenmucosalinfectionandinitialviremia;theviremiaisdetectableforabout8-12weeks.Vriusiswidelydisseminatedthroughoutthebodyduringthistime,andthelymphoidorgansbecomeseeded.,Anacutemononucleosis-likesyndromedevelopsinmanypatients(50%-75%)3-6weeksafterprimaryinfection.ThereisasignificantdropinnumbersofcirculatingCD4+Tcellsatthisearlytime.AnimmuneresponsetoHIVoccurs1weekto3monthsafterinfection,plasmaviremiadrops,andlevelsofCD4cellsreboundhowever,theimmuneresponseisunabletocleartheinfectioncompletely,andHIV-infectedcellspersistinthelymphnodes.Thisperiodofclinicallatencymaylastforaslongas10years.Duringthistime,thereisahighlevelofongoingviralreplication.Itisestimatedthat10billionHIVparticlesareproducedanddestroyedeachday.Eventually,thepatientwilldevelopconstitutionalsymptomsandclinicallyapparentdisease,suchasopportunisticinfectionsorneoplasms.,BClinicalfeaturesSymptomsofacuteHIVinfectionarenonspecificandincludefatigue,rash,headache,nausea,andnightsweats.AIDSischaracterizedbypronouncesuppressionoftheimmunesystemanddevelopmentofawidevarietyofsevereopportunisticinfectionsorunusualneoplasms.Themoreserioussymptomsinadultsareoftenprecededbyaprodtone(“diarrheaanddwindling”)thatcanincludefatigue,malaise,weightloss,fever,shortnessofbreath,chronicdiarrhea,whitepatchesonthetongue(hairyleukoplakia,oralcandidiasis)diseasesymptomsinthegastrointestinaltractfromtheesophagustothecolonareamajorcauseofdebility.,Thepredominantcausesofmorbidityandmortalityamongpatientswithlate-stageHIVinfectionareopportunisticinfections,i.e.severeinfectionsinducedbyagentsthatrarelycauseseriousdiseaseinimmunecompetentindividuals.OpportunisticinfectionsusuallydonotoccurinHIV-infectedpatientsuntilCD4+Tcellscountshavedroppedfromthenormallevelofabout1000cells/Ltolessthan200cells/L.,AIDSpatientsexhibitamarkedpredispositiontothedevelopmentofcancer,anotherconsequenceofimmunesuppression.AIDS-associatedcancersincludenon-Hodgkin,slymphoma(bothsystemicandcentralnervoussystemtypes),Kaposi,ssarcoma,cervicalcancer,anogenitalcancers,andHodgkin,slymphomaKaposi,ssarcoma,isnow20,000timesmorecommoninuntreatedAIDSpatientsthaninthegeneralpopulation.Kaposi,ssarcoma,associatedherpesvirus,orHHV8,appeartobecausallyrelatedtobecancer.,ThecardinalfeatureofHIVinfectionisthedepletionofThelper-inducerlymphocytes-theresultofHIVreplicationinthispopulationoflymphocytesaswellasthedeathofuninfectedTcellsbyindirectmechanisms.TheTcellsexpresstheCD4phenotypicmarkerontheirsurface.TheCD4moleculeisthemajorreceptorforHIV;ithasahighaffinityfortheviralenvelope.TheHIVcoreceptoronlymphocytesistheCXCR4chemokinereceptor.MonocytesandmacrophagesplayamajorroleinthedisseminationandpathogenesisofHIVinfection.CurtainsubsetsofmonocytesexpresstheCD4surfaceantigenandthereforebindtotheenvelopeofHIV.UnliketheCD4Tlymphocyte,themonocyteisrelativelyrefractorytothecytopathiceffectsofHIV,sothatthevirusnotonlycansurviveinthiscellbutalsocanbetransportedtovariousorgansinthebody(suchasthelungsandbrain).,Infectedmacrophagesmaycontinuetoproducevirusforalongperiodoftime.TheHIVcoreceptoronmonocytesandmacrophagesistheCCR5chemokinereceptor.Inthebrain,themajorcelltypesinfectedwithHIVappeartobethemonocytesandmacrophages,andthismayhaveimportantconsequencesforthedevelopmentofneuropsychiatricmanifestationsassociatedwithHIVinfection.InfectedpulmonaryalveolarmacrophagesmayplayaroleintheinterstitialpneumonitisseenincertainpatientswithAIDS.,C.ImmunityHIV-infectedpersonsdevelopbothhumoralandcellmediatedresponsesagainstHIV-relatedantigens.Antibodiestoanumberofviralantigensdevelopsoonafterinfection.MostinfectedindividualsmakeneutralizingantibodiesagainstHIV,directedagainsttheenvelopeglycoprotein.However,thelevelsofneutralizingactivityarelow;manyanti-envelopeantibodiesarenonneutralizing.Itisbelievedthatthedenseglycosylationmayinhibitbindingofneutralizingantibodytotheenvelopeprotein.Theenvelopeglycoproteinshowsgreatsequencevariability.Thisnaturalvariationmayallowtheevolutionofsuccessiveofresistantvirusthatescaperecognitionbyexistingneutralizingbyneutralizingantibodies.,Laboratorydiagnosis,EvidenceofinfectionbyHIVcanbedetectedinthreeways:a)virusisolation,b)serologicdeterminationofantiviralantibodies,andc)measurementofviralnucleicacidorantigens.,VirusisolationHIVcanbeculturedfromlymphocytesinperipheralblood.Themostsensitivevirusisolationtechniqueistococultivatethetestsamplewithuninfected,mitogen-stimulatedperipheralbloodmononuclearcells.ThevastmajorityofHIV-1antibody-positivepersonswillhavevirusthatcanbeculturedfromtheirbloodcells.However,virusisolationtechniquesaretimeconsumingandlaboriousandarelimitedtoresearchstudies.PCRamplificationtechniquesaremorecommonlyusedfordetectionofvirusinclinicalspecimens.,B.SerologyMostindividualswillhavedetectableantibodieswithin6-12weeksafterinfection,whereasvirtuallyallwillbepositivewithin6months.Testkitsarecommerciallyavailableformeasuringantibodiesbyenzyme-linkedimmunoassay(EIA).Ifproperlyperformed,thesetesthaveasensitivityandspecificityexceeding98%.antibodiestoviralcoreproteinp24orenvelopeglycoproteingp41,gp120,orgp160aremostcommonlydetected.simple,rapidtestsfordetectingHIVantibodiesareavailableforuseinlaboratoriesill-equippedtoperformEIAtest.Thesimpletestsarebasedonprinciplessuchasagglutinationorimmunodotreactions.,C.DetectionofviralnucleicacidorantigensAmplificationassayssuchastheRT-PCR,DNAPCR,ANDbDNAtestsarecommonlyusedtodetectviralRNAinclinicalspecimens.TheHIVRNAlevelsareimportantpredictivemarkersofdiseaseprogressionandvaluabletoolswithwhichtomonitortheeffectivenessofantiviraltherapies.,Preventionandtreatment,TransmissionrouteTheHIVvirusistransmittedbetweenhumansinthreeways:sexually,perinatally,andbyexposuretocontaminatedbloodorbodyfluids.Thevirushasbeendemonstratedinparticularlyhightitersinsemenandcervicalsecretions,andthemajorityofcasesresultfromsexualcontact.Thepresenceofothersexuallytransmitteddiseasessuchassyphilis,gonorrhea,orherpessimplextype2increasestheriskofsexualHIVtransmissionasmuchasahundredfoldbecausetheinflammationandsoresfacilitatethetransferofHIVacrossmucosalbarriers.,Transfusionofinfectiousbloodorbloodproductsisaneffectiverouteforviraltransmission.Forexample,over90%ofhemophiliacrecipientsofcontaminatedclottingfactorconcentratesintheUnitedstates(beforeHIVwasdetected)developedantibodiestoHIV.Injectionusersofillicitdrugsarecommonlyinfectedthroughtheuseofcontaminatedneedles.InjectiondruguseaccountsforasubstantialproportionofnewAIDScases.Theriskofperinataltransmissionfromaninfectedmothertoherchildhasbeenestimatedtorangefrom15%to40%.Infantscanbecomeinfectedinuterus,duringthebirthprocess,ormorecommonly,throughbreastfeeding.,Theroutesoftransmission(blood,sex,andbirth)describedaboveaccountforalmostallHIVinfections.Transmissondoesnotoccurthroughday-to-daynonsexualcontactwithinfectedindividualsorthroughinsectvectors,becauseofthefragilityofthevirusandtheneedfordirectmucosalorbloodcontact.,B.PreventionWithoutcontrolbydrugsorvaccines,theonlywaytoavoidepidemicspreadofHIVistomaintainalifestylethatminimizesoreliminatesthehigh-riskfactorsdiscussedabove.BecauseHIVmaybetransmittedinblood,alldonorbloodshouldbetestedforantibody.Infectedpersonsshouldrefrainfromdonatingblood,plasma,bodyorgans,othertissus,orsperm.,Thereisariskofinfectingothersbysexualintercourse(vaginaloranal),byoral-genitalcontact,orbysharingofneedles.Theconsistentandproperuseofcondomscanreducetransmissionofthevirus,thoughprotectionisnotabsolute.,SeropositivewomenorwomenwithseropositivesexualpartnersarethemselveatincresaedriskofacquiringAIDS.Iftheybecomepregnant,theiroffspringalsoareathighriskofacquiringAIDS.,Devicesthathavepuncturedtheskine.g.hypodermicandacupunctureneedlesshouldbesteamsterilizedbyautoclavingbeforereuseorshouldbesafelydiscarded.Dentalinstrumentsshouldbeheat-sterilizedbetweenpatients.Wheneverpossible,disposableneedlesandequipmentshouldbeused.Toothbrushes,razors,andotherimplementsthatcouldbecomecontaminatedwithbloodshouldnotbeshared.,Thehealtheducationmessagesforthegeneralpublich