简介：SARSResearchinChina

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简介：AnnotationofthegenomesequenceoftheSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus)isindispensabletounderstanditsevolutionandpathogenesis.WehaveperformedafullannotationoftheSARS-CoVgenomesequencesbyusingannotationprogramspubliclyavailableordevelopedbyourselves.Totally,21openreadingframes(ORFs)ofgenesorputativeuncharacterizedproteins(PUPs)werepredicted.SevenPUPshadnotbeenreportedpreviously,andtwoofthemwerepredictedtocontaintransmembraneregions.EightORFspartiallyoverlappedwithorembeddedintothoseofknowngenes,revealingthattheSARS-CoVgenomeisasmallandcompactonewithoverlappedcodingregions.ThemoststrikingdiscoveryisthatanORFlocatesontheminusstrand.Wehavealsoannotatednon-codingregionsandidentifiedthetranscriptionregulatingsequences(TRS)intheintergenicregions.TheanalysisofTRSsupportstheminusstrandextendingtranscriptionmechanismofcoronavirus.TheSNPanalysisofdifferentisolatesrevealsthatmutationsofthesequencesdonotaffectthepredictionresultsofORFs.

简介：Knowledgeoftheevolutionofpathogensisofgreatmedicalandbiologicalsignificancetotheprevention,diagnosis,andtherapyofinfectiousdiseases.InordertounderstandtheoriginandevolutionoftheSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus),wecollectedcompletegenomesequencesofallvirusesavailableinGenBank,andmadecomparativeanalyseswiththeSARSCoV.GenomicsignatureanalysisdemonstratesthatthecoronavirusesalltaketheTGTTastheirrichesttetranucleotideexcepttheSARS-CoV.Adetailedanalysisoftheforty-twocompleteSARS-CoVgenomesequencesrevealedtheexistenceoftwodistinctgenotypes,andshowedthattheseisolatescouldbeclassifiedintofourgroups.OurmanualanalysisoftheBLASTNresultsdemonstratesthattheHE(hemagglutinin-esterase)geneexistsintheSARS-CoV,andmanymutationsmadeitunfamiliartous.

简介：Adatasetof103SARS-CoVisolates(101humanpatientsand2palmcivets)wasinvestigatedondifferentaspectsofgenomepolymorphismandisolateclassification.Thenumberandthedistributionofsinglenucleotidevariations(SNVs)andinsertionsanddeletions,withrespecttoa"profile",weredeterminedanddiscussed("profile"beingasequencecontainingthemostrepresentedletterperposition).Distributionofsubstitutioncategoriespercodonpositions,aswellassynonymousandnon-synonymoussubstitutionsincodingregionsofannotatedisolates,wasdetermined,alongwithaminoacid(a.a.)propertychanges.Similaranalysiswasperformedforthespike(S)proteininalltheisolates(55ofthembeingpredictedforthefirsttime).TheratioKa/KsconfirmedthattheSgenewassubjectedtotheDarwinianselectionduringvirustransmissionfromanimalstohumans.Isolatesfromthedatasetwereclassifiedaccordingtogenomepolymorphismandgenotypes.Genomepolymorphismyieldstotwogroups,onewithasmallnumberofSNVsandanotherwithalargenumberofSNVs,withuptofoursubgroupswithrespecttoinsertionsanddeletions.Weidentifiedthreebasicnine-locusgenotypes:TTTT/TTCGG,CGCC/TTCAT,andTGCC/TTCGT,withfoursubgenotypes.Bothclassificationsproposedareinaccordancewiththenewinsightsintopossibleepidemiologicalspread,bothinspaceandtime.

简介：SARS-CoV感染动物模型的建立可加深对SARS病原学的了解和发病机制的研究,加速实验室诊断技术的建立、抗病毒药物的筛选和疫苗的开发,同时也有助于给该病一个更精确的定义。可以说SARS动物模型的建立,不但是SARS研究的瓶颈问题,其应用更是贯穿SARS研究的整个过程。到目前为止,已经报道有4种非人灵长类动物(恒河猴、食蟹猴、绒猴、非洲绿猴)和6种啮齿类动物(大鼠、小鼠、豚鼠、田鼠、仓鼠、转基因鼠),以及雪貂、家猫等可以作为SARS动物模型用于实验研究,并已经开始利用动物模型进行疫苗和药物的安全性和有效性评价。本文就已报道的各类SARS动物模型进行综述,并根据动物模型和SARS患者的比对,提出动物模型建立的技术要点。

简介：在感染人类SARS病毒株中尚未发现具有抗体依赖的病毒入侵增强作用的抗体。该抗体作用因能影响疫苗抗病毒能力甚至会加重疾病，而一直备受疫苗研制者们的重视。迄今为止，抗体依赖性增强作用并未在感染人SARS-CoV病毒株系中发现，这也许能减轻人们对疫苗这种抗体依赖的增强作用产生的忧虑，但在该类疫苗投入使用前必须做好相关的动物模型试验。

简介：Thenucleocapsidprotein(Nprotein)hasbeenfoundtobeanantigenicproteininanumberofcoronaviruses.WhethertheNproteininsevereacuterespiratorysyndrome-associatedcoronavirus(SARS-CoV)isantigenicremainstobeelucidated.UsingWesternblotandEnzyme-linkedImmunosorbentAssay(ELISA),therecombinantNproteinsandthesynthesizedpeptidesderivedfromtheNproteinwerescreenedinserafromSARSpatients.AllpatientserainthisstudydisplayedstrongpositiveimmunoreactivitiesagainsttherecombinantNproteins,whereasnormalseragavenegativeimmunoresponsestotheseproteins,indicatingthattheNproteinofSARS-CoVisanantigenicprotein.Furthermore,theepitopesitesintheNproteinweredeterminedbycompetitionexperiments,inwhichtherecombinantproteinsorthesynthesizedpeptidescompetedagainsttheSARS-CoVproteinstobindtotheantibodiesraisedinSARSsera.OneepitopesitelocatedattheC-terminuswasconfirmedasthemostantigenicregioninthisprotein.AdetailedscreeningofpeptidewithELISAdemonstratedthattheaminosequencefromCodons371to407wastheepitopesiteattheC-terminusoftheNprotein.UnderstandingoftheepitopesitescouldbeverysignificantfordevelopinganeffectivediagnosticapproachtoSARS.

简介：RobertsA等人为了评价疫苗和抗SARS病毒药物的有效性，建立了金黄地鼠小动物模型。他们用滴鼻的方法用SARS病毒感染了5周龄金黄地鼠。根据检测结果，SARS病毒在肺脏和鼻甲骨中以高滴度复制。下呼吸道的病毒滴度在攻毒第2天达到高峰，第7天后消失。在攻毒后第14天，在部分地鼠鼻甲骨检测出有低滴度病毒复制。病毒在攻毒早期就在上呼吸道复制，病理检查发现呼吸道上皮细胞坏死，随后引起炎症反应，局部硬化，病毒被清除，肺组织最终被修复。

简介：TheE(envelope)proteinisthesmalleststructuralproteininallcoronavirusesandistheonlyviralstructuralproteininwhichnovariationhasbeendetected.WeconductedgenomesequencingandphylogeneticanalysesofSARS-CoV.Basedongenomesequencing,wepredictedtheEproteinisatransmembrane(TM)pro-teincharacterizedbyaTMregionwithstronghydrophobicityandα-helixcon-formation.Weidentifiedasegment(NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH)inthecarboxyl-terminalregionoftheEproteinthatappearstoformthreedisulfidebondswithanothersegmentofcorrespondingcysteinesinthecarboxyl-terminusoftheS(spike)protein.ThesebondspointtoapossiblestructuralassociationbetweentheEandSproteins.OurphylogeneticanalysesoftheEproteinsequencesinallpub-lishedcoronavirusesplaceSARS-CoVinanindependentgroupinCoronaviridaeandsuggestanon-humananimalorigin.

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简介：Thecorona-likespikesorpeplomersonthesurfaceofthevirionunderelectronicmicroscopearethemoststrikingfeaturesofcoronaviruses.TheS(spike)proteinisthelargeststructuralprotein,with1,255aminoacids,intheviralgenome.Itsstructurecanbedividedintothreeregions:alongN-terminalregionintheexte-rior,acharacteristictransmembrane(TM)region,andashortC-terminusintheinteriorofavirion.WedetectedfifteensubstitutionsofnucleotidesbycomparisonswiththeseventeenpublishedSARS-CoVgenomesequences,eight(53.3%)ofwhicharenon-synonymousmutationsleadingtoaminoacidalternationswithpredictedphysiochemicalchanges.ThepossibleantigenicdeterminantsoftheSproteinarepredicted,andtheresultisconfirmedbyELISA(enzyme-linkedimmunosorbentassay)withsynthesizedpeptides.AnotherprofoundfindingisthatthreedisulfidebondsaredefinedattheC-terminuswiththeN-terminusoftheE(envelope)pro-tein,basedonthetypicalsequenceandpositions,thusestablishingthestructuralconnectionwiththesetwoimportantstructuralproteins,ifconfirmed.Phyloge-neticanalysisrevealsseveralconservedregionsthatmightbepotentdrugtargets.

简介：TheCoronaviridaefamilyischaracterizedbyanucleocapsidthatiscomposedofthegenomeRNAmoleculeincombinationwiththenucleoprotein(Nprotein)withinavirion.ThemoststrikingphysiochemicalfeatureoftheNproteinofSARS-CoVisthatitisatypicalbasicproteinwithahighpredictedpIandhighhydrophilicity,whichisconsistentwithitsfunctionofbindingtotheribophosphatebackboneoftheRNAmolecule.ThepredictedhighextentofphosphorylationoftheNproteinonmultiplecandidatephosphorylationsitesdemonstratesthatitwouldberelatedtoimportantfunctions,suchasRNA-bindingandlocalizationtothenucleolusofhostcells.SubsequentstudyshowsthatthereisanSR-richregionintheNproteinandthisregionmightbeinvolvedintheprotein-proteininteraction.TheabundantantigenicsitespredictedintheNprotein,aswellasexperimentalevidencewithsynthesizedpolypeptides,indicatethattheNproteinisoneofthemajorantigensoftheSARS-CoV.Comparedwithotherviralstructuralproteins,thelowvariationrateoftheNproteinwithregardstoitssizesuggestsitsimportancetothesurvivalofthevirus.

简介：WestudiedstructuralandimmunologicalpropertiesoftheSARS-CoVM(mem-brane)protein,basedoncomparativeanalysesofsequencefeatures,phylogeneticinvestigation,andexperimentalresults.TheMproteinispredictedtocontainatriple-spanningtransmembrane(TM)region,asingleN-glycosylationsitenearitsN-terminusthatisintheexteriorofthevirion,andalongC-terminalregionintheinterior.TheMproteinharborsahighersubstitutionrate(0.6%correlatedtoitssize)amongviralopenreadingframes(ORFs)frompublisheddata.ThefoursubstitutionsdetectedintheMprotein,whichcausenon-synonymouschanges,canbeclassifiedintothreetypes.OneofthemresultsinchangesofpI(isoelectricpoint)andcharge,affectingantigenicity.ThesecondchangeshydrophobicityoftheTMregion,andthethirdonerelatestohydrophilicityoftheinteriorstructure.PhylogenetictreebuildingbasedonthevariationsoftheMproteinappearstosupportthenon-humanoriginofSARS-CoV.Toinvestigateitsimmunogenicity,wesynthesizedeightoligopeptidescovering69.2%oftheentireORFandscreenedthembyusingELISA(enzyme-linkedimmunosorbentassay)withserafromSARSpatients.Theresultsconfirmedourpredictionsonantigenicsites.

简介：Inthefaceoftheworldwidethreatofsevereacuterespiratorysyndrome(SARS)tohumanlife,someofthemosturgentchallengesaretodevelopfastandaccurateanalyticalmethodsforearlydiagnosisofthisdiseaseaswellastocreateasafeanti-viralvaccineforprevention.Totheseends,weinvestigatedtheantigenicityofthespikeprotein(Sprotein),amajorstructuralproteinintheSARS-coronavirus(SARS-CoV).BaseduponthetheoreticalanalysisforhydrophobicityoftheSprotein,18peptidesweresynthesized.UsingEnzyme-LinkedImmunosorbentAssay(ELISA),thesepeptideswerescreenedintheserafromSARSpatients.Accordingtotheseresults,twofragmentsoftheSgenewereamplifiedbyPCRandclonedintopET-32a.BothSfragmentswereexpressedintheBL-21strainandfurtherpurifiedwithanaffinitychromatography.TheserecombinantSfragmentswereconfirmedtohavepositivecross-reactionswithSARSsera,eitherbyWesternblotorbyELISA.OurresultsdemonstratedthatthepotentialepitoperegionswerelocatedatCodons469-882intheSprotein,andoneepitopesitewaslocatedatCodons599-620.IdentificationofantigenicregionsintheSARS-CoVSproteinmaybeimportantforthefunctionalstudiesofthisvirusorthedevelopmentofclinicaldiagnosis.

简介：InordertodevelopclinicaldiagnostictoolsforrapiddetectionofSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus)andtoidentifycandidateproteinsforvaccinedevelopment,theC-terminalportionofthenucleocapsid(NC)genewasamplifiedusingRT-PCRfromtheSARS-CoVgenome,clonedintoayeastexpressionvector(pEGH),andexpressedasaglutathioneS-transferase(GST)andHisx6double-taggedfusionproteinunderthecontrolofaninduciblepromoter.WesternanalysisonthepurifiedproteinconfirmedtheexpressionandpurificationoftheNCfusionproteinsfromyeast.Todetermineitsantigenicity,thefusionproteinwaschallengedwithserumsamplesfromSARSpatientsandnormalcontrols.TheNCfusionproteindemonstratedhighantigenicitywithhighspecificity,andtherefore,itshouldhavegreatpotentialindesigningclinicaldiagnostictoolsandprovideusefulinformationforvaccinedevelopment.

简介：Themultiplexpolymerasechainreaction(PCR)techniquewasappliedtodetecttheSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus)specifictargetcDNAfragmentsinthepresentstudy.ThetargetcDNAfragmentsofSARS-CoVweresynthesizedartificiallyaccordingtothegenomesequenceofSARS-CoVinGenBanksubmittedbyTheChineseUniversityofHongKong,andwereusedassimulatedpositivesamples.FiveprimersrecommendedbyWorldHealthOrganization(WHO)wereusedtoamplifythefragmentsbysinglePCRandmultiplexPCR.ThreetargetcDNAfragments(121,182and302bp),aswellasthethreedifferentcombinationsofanytwoofthesefragments,wereamplifiedbysinglePCR.ThecombinationofthesethreefragmentswasamplifiedbymultiplexPCR.TheresultsindicatedthatthemultiplexPCRtechniquecouldbeappliedtodetecttheSARS-CoVspecifictargetcDNAfragmentssuccessfully.

简介：Thesuddenoutbreakofsevereacuterespiratorysyndrome(SARS)in2002promptedtheestablishmentofaglobalscientificnetworksubsumingmostofthetraditionalrivalriesinthecompetitivefieldofvirology.WithinmonthsoftheSARSoutbreak,collaborativeworkrevealedtheidentityofthedisastrouspathogenasSARS-associatedcoronavirus(SARS-CoV).However,althoughtherapididentificationoftheagentrepresentedanimportantbreakthrough,ourunderstandingofthedeadlyvirusremainslimited.Detailedbiologicalknowledgeiscrucialforthedevelopmentofeffectivecountermeasures,diagnostictests,vaccinesandantiviraldrugsagainsttheSARS-CoV.ThisarticlereviewsthepresentstateofmolecularknowledgeaboutSARS-CoV,fromtheaspectsofcomparativegenomics,molecularbiologyofviralgenes,evolution,andepidemiology,anddescribesthediagnostictestsandtheanti-viraldrugsderivedsofarbasedontheavailablemolecularinformation.

简介：BeijinghasbeenoneoftheepicentersattackedmostseverelybytheSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus)sincethefirstpatientwasdiagnosedinoneofthecity'shospitals.WenowreportcompletegenomesequencesoftheBJGroup,includingfourisolates(IsolatesBJ01,BJ02,BJ03,andBJ04)oftheSARS-CoV.ItisremarkablethatallmembersoftheBJGroupshareacommonhaplotype,consistingofsevenlocithatdifferentiatethegroupfromotherisolatespublishedtodate.Among42substitutionsuniquelyidentifledfromtheBJgroup,32arenon-synonymouschangesattheaminoacidlevel.Rootedphylogenetictrees,proposedonthebasisofhaplotypesandothersequencevariationsofSARS-CoVisolatesfromCanada,USA,Singapore,andChina,gaverisetodifferentparadigmsbutpositionedtheBJGroup,togetherwiththenewlydiscoveredGD01(GD-Ins29)inthesameclade,followedbytheH-UGroup(fromHongKongtoUSA)andtheH-TGroup(fromHongKongtoToronto),leavingtheSPGroup(Singapore)moredistant.ThisresultappearstosuggestapossibletransmissionpathfromGuangdongtoBeijing/HongKong,thentoothercountriesandregions.

简介：目的：研究SARS冠状病毒（SARS-CoV）N蛋白与CYP4F3的相互作用。方法：应用免疫共沉淀、GST—pulldown、BIACORE实验验证SARS—CoVN蛋白与CYP4F3的相互作用。结果：SARS—CoVN蛋白与CYP4F3能够相互作用，BIA-CORE实验证实CYP4F3与SARS—CoVN蛋白的亲和常数为Ko=4．3×10^-11。结论：SARS—CoVN蛋白与CYP4F3在细胞内、外均能形成复合物，这为进一步探讨SARS—CoVN蛋白在SARS致病机理中的作用奠定了基础。