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密级：论文编号：中国农业科学院学位论文基于两种表达系统的蓝舌病毒重组亚单位疫苗研制ＤｅｖｅｌｏｐｍｅｎｔｏｆｒｅｃｏｍｂｉｎａｎｔｓｕｂｕｎｉｔｖａｃｃｉｎｅｓａｇａｉｎｓｔｂｌｕｅｔｏｎｕｅｇｖｉｒｕｓｕｓｉｎｇｂａｃｕｌｏｖｉｒｕｓａｎｄＥ．ｃｏｌｉｅｘｐｒｅｓｓｉｏｎｓｙｓｔｅｍｓｒ博士研究生：ＤａｉｅｎＫｈｅｄｅｒＡｌｉＭｏｈａｍｅｄ指导教师：殷宏研究员申请学位类别：农学博士专业：预防兽医学研究方向：．兽医微生物与分子生物学培养单位：兰州兽医研究所研究生院提交日期２０１６年１２月 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Secrecy:No.ChineseAcademyofAgriculturalSciencesDissertationDevelopmentofrecombinantsubunitvaccinesagainstbluetonguevirususingbaculovirusandE.coliexpressionsystemsPh.D.Candidate：DarienKhederAliMohamedSupervisor:ProfessorHongYinMajor：PreventiveVeterinaryMedicineSpecialization:VeterinaryMicrobiologyandMolecularBiologyDecember2016II AbstractBluetongue(BT)isaninsect-bornediseaseoflivestockcausedbybluetonguevirus(BTV),theprototypememberofthegenusOrbiviruswithinthefamilyReoviridae.Thediseaseisclassifiedasanemergingdiseaseinseveralcountriesandcancausemajorlossoflivestockininfectedsheepherds.Infectionofruminantsoccursthroughoutmuchofthetemperateandtropicalregionsoftheworld,coincidentwiththedistributionofspecificspeciesofbitingmidgesbelongingtothespeciesCulicoides.ThespreadofBTVtopreviouslydisease-freeregionswhichprohibittheuseofthecurrentBTVlive-attenuatedvaccinehashighlightedtheneedforanewgenerationofvaccines.Subunitvaccinesareoneoftheattractivealternativestrategies.SubunitvaccinesagainstBTVwouldtargettheoutercapsidproteinVP2,themainneutralization-specificantigen.TheVP2canbeco-expressedwithothercapsidandcoreproteinstoformaparticlethatresemblestheintactBTVviruslikeparticle(VLP).Inthecurrentstudywediscussedindetailstheproductionandevaluationofprotein-basedsubunitvaccinesagainstBTV.Theworkhasbeendissectedintothreeparts.(1)Expressionoffull-lengthBTVproteinsinbaculovirusexpressionsystemInthispartofwork,abaculovirusexpressionsystemwassuccessfullyutilizedtoexpressBTV-16(VP2,VP3,VP7,andVP5),BTV-1(VP2andVP5),andBTV-8(VP2)proteinsforlaterimmunizationofanimalswiththepurifiedproteinsaimingtoproduceaneffectivesubunitvaccineagainstBTV.Baculovirusexpressionsystemoffersacorrectfoldingoftheexpressedproteins.Theexpressionconditions(optimumexpressiontimeandtemperatureforproteinbindingtotheNi-NTAresins,pH,bindingandelutionbuffers)wereoptimizedforeachindividualprotein.Allproteins,exceptforVP5,weresuccessfullyexpressedandtheobtainedyieldsofthepurifiedproteinsweresufficientforlateruseintheproposedsubunitvaccine.(2)Prokaryoticexpressionoffull-lengthBTV-16VP3,VP5,VP7,NS2andtruncatedVP5BTV-16VP3,VP7,NS2andVP5-41aageneswereamplifiedandusedforcloningintopSMK,pET32aorpPROEX-HTbplasmids.TheconfirmedclonesofpSMKBTV16VP3andpSMKBTV16VP7wereusedtotransformE.coliBL21-codonPlus(DE3)-RILcompetentcells,whilepET32aBTV16VP5-41aaandpPROEX-HTbBTV16NS2weretransformedintoBL21(DE3)competentcells,andlaterinducedwithIPTGforexpression.Expressionconditionswereoptimizedinordertogethighexpression.Thelowertemperature(16°C)fortheexpressionoftheHis-sumotaggedproteinswasfoundtoenhancethesolubilityoftheexpressedproteins,whiletheHis-taggedNS2proteinwasexpressedasasolubleproteinat37°C.Incontrast,VP5-41aacouldnotyieldasolubleproteinthereforeithasbeenpurifiedunderthehybridconditionthatassumedtopartiallyenhancesfortherefoldingoftheexpressedprotein.Furthermore,theSUMO-taggedproteinsofVP3andVP7wereassembledintocore-likeparticles(CLP)invitroafterthecleavageoftheHis-sumomoiety.V (3)Immunogenicityevaluationofprotein-basedsubunitvaccinesagainstbluetonguevirusinBALB/cmiceandsheepInthisstudy,themainobjectivewastodevelopasubunitvaccinecandidatetargetingBTV-16asthisstrainhasbeenpreviouslyisolatedfromclinicallyinfectedsheepinChina.Otherobjectiveofthestudywastoexaminethepossibilityofthevaccinecandidatetobeusedforheterologousserotypeprotection.Withthispurpose,BTV-1VP2andBTV-8VP2werealsoexpressedandusedtoreplaceBTV-16VP2.Tocharacterizeovineandmurineimmuneresponsestotheexpressedproteins,bothsheepandmiceweresubcutaneouslyimmunizedtwicewithoneoffiveproteincombinationsinMontanideTMISA201VGadjuvantoraplacebo.(n=5/group).UtilizingBTV-1cELISA,significantlyhigherantibodytitersweredetectedintheimmunizedanimalsthanthecontrols.Thevaccineinducedaproteinspecifichumoralresponseaswellaslymphocyteproliferativeresponsesuponre-stimulationofperipheralbloodmononuclearcells(PBMC)fromimmunizedsheep.SerumneutralizationtestagainstserotypeBTV-1showedapromisingresultsforcrossserotypicprotectionofthevaccineagainstBTV-8andBTV-16.ThedatasuggestthattherecombinantpurifiedproteinscouldrepresentanimportantpartofanovelvaccineagainstBTV.Inconclusion,webeganwitheightimmunologicBTVproteinsthatwereproducedinscalableexpressionsystems.Wefollowedsystematicstepsofproteinidentificationandpurificationmethodoptimizations.ThepurifiedproteinswereutilizedtoultimatelydevelopanexperimentalvaccinecandidatecomposedofthestableandpurifiedrecombinantBTV-16proteinsVP2,VP3,VP7,NS2andVP5-41aa,indifferentcombinationswithMontanideTMISA201VGadjuvant.ThereplacementofBTV-16VP2witheitherBTV-1VP2orBTV-8VP2wasalsodiscussed.Sincetheevaluatednovelvaccineinducedbothhumoralandcell-mediatedimmuneresponsesandallvaccinatedserawereabletoneutralizeBTV-1inpart,indicatingacrossserotyperesponsesinducedbythevaccine,webelievethatthisrationalrecombinantsubunitvaccinecanbeadaptedtoadditionalormultipleBTVserotypesdependingonlocalepidemiologyandthediseaseoccurrenceandwillrepresentadefenselineincaseofBTVoutbreakoremergence.Keywords:Bluetonguevirus,baculovirusexpression,E.coliexpression,subunitvaccine,neutralizingantibodiesVI 摘要蓝舌病是一种由昆虫媒介传播的家畜传染病，其病原蓝舌病毒是呼肠孤病毒科环状病毒属的代表性成员。在一些国家蓝舌病被划分为新发传染病，该病感染绵羊群后可导致巨大损失。蓝舌病经常发生于热带和大部分温带地区，这与其传播媒介库蠓（Culicoides）的地理分布一致。蓝舌病毒在禁用弱毒疫苗无疫区的传播已经提示人们研制新一代疫苗非常必要。研制亚单位疫苗无疑是一种有前景的替代策略。BTV亚单位疫苗的主要蛋白为外层衣壳蛋白VP2，该蛋白是主要的中和抗原，VP2可与其他衣壳蛋白（VP5）和核心蛋白（VP3和VP7）共表达形成一个类似于完整BTV的病毒样颗粒（VLP）。本研究的主要工作集中在BTV亚单位疫苗的研制及免疫效果评价。该研究工作包括三部分：（1）BTV16VP2、BTV16VP3、BTV16VP5、BTV16VP7、BTV1VP2、BTV1VP5和BTV8VP2蛋白在杆状病毒表达系统中的表达本研究中我们首先用杆状病毒表达系统成功地表达了7种蛋白并进行了纯化，包括BTV16VP2、BTV16VP3、BTV16VP5、BTV16VP7、BTV1VP2、BTV1VP5和BTV8VP2，目的是将其免疫动物研制有效的BTV亚单位疫苗。杆状病毒表达系统能对表达的蛋白质提供正确折叠并增强蛋白可溶性。对每种蛋白的表达条件进行了优化，包括表达时间、温度、蛋白与Ni-NTA树脂的结合条件、pH值、结合和洗脱缓冲液等，以上所有蛋白被成功表达，且获得了大量纯化的VP2、VP3和VP7蛋白。（2）BTV-16VP3、VP7、NS2、VP5-41aa蛋白在大肠杆菌中的表达PCR扩增获得BTV-16VP3、VP7、NS2和VP5-41aa基因并分别与pET32a、pPROEX-HTb和pSMK质粒载体连接，构建的重组质粒pSMKBTV16VP3和pSMKBTV16VP7转化BL21-codonPlus(DE3)-RIL感受态细胞，pET32aBTV16VP5-41aa和pPROEX-HTbBTV16NS2质粒转化到BL21（DE3）感受态细胞，用IPTG诱导表达，对每种蛋白的表达条件进行了优化，带有His-sumo标签的VP3和VP7蛋白在低温下(16°C)其可溶性大大增加，然而带有His标签的NS2在37°C条件下为可溶性表达，而VP5-41aa蛋白以不可溶性的包涵体形式表达，所以对其进行复合型纯化以增强蛋白的复性。另外，原核表达的VP3和VP7蛋白在去除His-sumo标签后可在体外可装配成病毒核心样颗粒（CLP）。3、BTV亚单位疫苗在BALB/c小鼠和绵羊上的免疫原性评价VII 中国曾从临床感染的绵羊分离到了BTV-16毒株，因此本研究的主要目的是研制针对BTV-16的亚单位疫苗，同时我们也尝试了异源血清型疫苗保护的可能性。为了评价表达蛋白的免疫原性，五种不同组合的蛋白经MontanideTMISA201佐剂乳化后皮下免疫接种至绵羊和BALB/c小鼠（每组5只动物），结果显示，与空白对照组相比，免疫动物均产生了很高的BTV血清抗体，且该疫苗诱导产生了蛋白特异的体液免疫应答和明显的淋巴细胞增殖反应。血清中和试验表明，针对BTV-1、BTV-8和BTV-16的亚单位疫苗抗体均能有效中和BTV-1病毒。本研究研制的重组蛋白亚单位疫苗有希望成为防控蓝舌病的新型候选疫苗。总之，在本研究中我们首先用两种表达系统表达了8种不同血清型的BTV蛋白，系统地鉴定了表达蛋白的特异性，优化了蛋白纯化方法，评价了表达蛋白的免疫原性，研制了由BTV-16VP2、VP3、VP7、NS2和VP5-41aa五种蛋白组合的候选亚单位疫苗，同时也评价了以BTV1VP2或BTV8VP2替换BTV16VP2构建不同血清型嵌合亚单位疫苗的可能性。本研究研制的重组亚单位疫苗诱导产生了显著的体液免疫和细胞免疫应答，且产生的免疫血清能不同程度地中和BTV-1病毒，提示该疫苗可能对不同血清型的BTV产生交叉保护，因此该重组亚单位疫苗策略可参考当地的BTV流行病学进一步优化，从而应对多个血清型BTV的暴发和流行。关键词：蓝舌病毒、杆状病毒表达、大肠杆菌表达、亚单位疫苗、中和抗体VIII TableofcontentsTitle……………………………………………….………………………………………………..…………IEnglishtitle……………………………………………………………………………………………….…IIDeclarationofOriginality............................................................................................................................IIIAuthorizeduseagreement…………………..............................................................................………….IIISignatureformforrevieweranddefensecommitteeofPh.Dthesis......................................................IVAbstract…......................................................................................................................................................VChineseabstract..................................................................................................................................……VIITableofcontents………………........................................................................................................……IXAbbreviations……...............................................................................................................……….……..XII1CHAPTREILiteraturereview….......................................................................................................…...11.1Introduction…………….…………….…………….…………….…………….…………….………….11.2Bluetonguedisease……………………………………………………………………………………….21.2.1Historyandcurrentglobaldistributionofthedisease………………………………..………….21.2.2Thediseasetransmission…………...……………………………………….…………………...51.2.3Thediseaseepidemiology……………………...………………………………………………..81.2.4Thepathogenesisofbluetonguevirus………….…………………………………………...….101.3Bluetonguevirus………………………………………………………………………………………..121.3.1TheoutermostproteinsVP2andVP5…………………………………………......…………..141.3.2StructuralorganizationofBTVcoreandassociatedproteins……………….……..…….……151.3.3Non-structuralproteins……………………………………..………………….……………….151.3.4ReplicationcycleofBTV……………………………………………….………………………171.4Vaccinedevelopment………..………………………………………………………………….………191.4.1Bluetonguevaccinationstrategies………………………………….…………..…….………..201.4.2TypesofBTVvaccines……………………………………………………………...………….221.4.2.1Conventionalliveandinactivatedvaccines……………….…………………………...221.4.2.2Recombinantviralvectorvaccines………………………….……………….………….231.4.2.3Recombinantsubunitvaccines…………………………………………….………...…241.4.2.4DNAvaccines…………………………………….………..………..…………………..261.4.2.5Reversegenetics-basedvaccines…….....…………………….…………………………271.5Aimsofthecurrentstudy………………………………………………………………………………282CHAPTERII.Expressionoffull-lengthBTVproteinsinbaculovirusexpressionsystem………....302.1Introduction…………………………………………………………………..……………………….302.2Materialsandmethods………………………………………………………………………………….312.2.1Virus,genesandcells………………………………………………………………………….312.2.2PCR,electrophoresisandDNApurification……………………………………………….…...312.2.3CloningintopFastBac™HTbvector………………………………………………………….…332.2.4TranspositionofrecombinantpFastBac™HTbintothebacmidDNA………………………....33IX 2.2.5IsolationofrecombinantplasmidandbacmidDNA……………………..………….…………342.2.6TransfectionofSf9cellswithrecombinantbacmidforproductionoftherecombinantbaculovirus…………………………………………………………………………………………….342.2.7ExpressionandidentificationofrecombinantBTVproteins……..……………………………..362.2.8Purificationandquantificationofrecombinantproteins……………...………………………...362.3Results…………………………………………………….…….……...………………………….……372.3.1ConstructionofrecombinantpFastBac™HTbvectors……………………..…………………...372.3.2Generatingtherecombinantbacmids...…………………………………………………………382.3.3TransfectionofSf9cellsandidentificationoftheexpressedproteins…………………...…....392.3.4Proteinexpressionandpurification……………………….……………………...……………402.4Discussion………………………………………………………………………………………………423CHAPTERIII.Prokaryoticexpressionoffull-lengthBTV-16VP3,VP5,VP7,NS2,andtruncatedversionofVP5…………………………………………………………..…………………………….........453.1Introduction………………………………………………………………………………………….....453.2Materialsandmethods…………………………………………………………………………………463.2.1Virusgenesandcells………………………………………………………..………….…….463.2.2Constructionoftherecombinantexpressionplasmids…………………………………..........463.2.3ExpressionandidentificationofBTV-16proteins……………………………….………..……463.2.4Purificationandquantificationoftheexpressedproteins……………………………………..483.2.5Assemblyofcorelikeparticles..…………………………………………………….…………..493.3Results…………………………………………………………………………………………...……..493.3.1Constructionofrecombinantbacterialexpressionplasmids…………………………………..493.3.2ProkaryoticexpressionandidentificationofBTVproteins…………………………………..493.3.3Proteinpurificationandquantification………………………………………………………..523.3.4AssemblyandcharacterizationofBTVCLPs.…………………………………...……………..523.4Discussion……………………………………………………………………………………………..534CHAPTERIV.Immunogenicityevaluationofprotein-basedsubunitvaccinesagainstbluetonguevirusinBALB/Cmiceandsheep………………………………………………………………………....564.1Introduction……………………………………………………………………………………………564.2Materialsandmethods……………………………………………………………………………….….574.2.1Ethicsstatement……………………………………………………………………..………….574.2.2Celllines,virusesandanimals…………………………………...…………………………….574.2.3Animalimmunization………………………………………………...………………..……….574.2.3.1Immunizationofsheep…………………………………………...………………….574.2.3.2ImmunizationofBALB/c…………………………………………………..…….…….584.2.4Serologicalanalyses……………………………………………………...…………………….584.2.4.1DetectionofBTV-specificantibodiesbycELISA……………………………….…….584.2.4.2Detectionofprotein-specificantibodiesbyindirectELISA………………….………...59X 4.2.4.3Plaquereductionneutralizationassay(PRNA)………………………………...…….....594.2.4.4Antigenspecificlymphocyteproliferationassayinsheep……………..……………….604.3Results…………………………………………………………………………………………...……..604.3.1Immunogenicityoftherecombinantsubunitvaccinesinsheep…………………………...…..604.3.2Immunogenicityoftherecombinantsubunitvaccinesinmice……………………………….614.3.3.Detectionofprotein-specificantibodies……………………………………………….……….664.3.4Plaquereductionneutralizationassay…………………………………………..……………….674.3.5Lymphocyteproliferationresponses………………………………………………………....684.4Discussion……………………………………………………………….……………………………..68Conclusion………………...……………………………………………………………….………………72References……………………………………………………..………………….…………………..…….73Acknowledgements…………………………………………………………………………...……………91CurriculumVitae…………………………………………………………………….…………………….92XI AbbreviationsµgMirogramµlMicroliteraaAminoacidBHKBabyhamasterkidneybpBasepairsbpBasepairBTDBluetonguediseaseBTVBluetongueviruscELISACompetitiveEnzymelinkedimmunosorbentassycDNAComplementaryDeoxyribonucleicCLPCorelikeparticleDNADeoxyribonucleicE.ColiEscherichiacoliELISAEnzymelinkedimmunosorbentassyFBSFetalbovineserumhHour/sHRPHorseradishperoxidaseIFNIntferoneIPTGIsopropylthio-b-galactosideKbKilobasekDaKilodatonLLiterLBLuria-BertaniMMoleM.O.IMultiplicityofinfectionmAMilliampereminMinutesmlMicrolitermMMilimoleNAsNeutrlizingantibodiesNi-NTANickelnitrilotriaceticacidNSNonstructuralproteinODOpticaldensityXII OIEOfficeintaernationaldeepizooticsORFOpenreadingframePAGEpolyacrylamidegelelectrophoresisPBMCPeripheralbloodmononuclarcellsPBSPhosphatebufferedsalinepHpotentialofHydrogenPRNAPlaquereductionneutralizationassayPVDFPolyvinylidenefluorideRNARibonucleicacidrpmRevolutionsperminuteSDSsodium-dodecyl-sulphateSecSecondsSUMOSmallubiquitinmodifierTNFTumornicrosesfactorTrishydroxymethylaminomethaneVVoltVIBsViraliclusionbodiesVLPViruslikeparticleVPViralproteinX-gal5-Bromo-4-chloro-3-indolylβ-D-galactopyranosideXIII 中国农业科学院博士学位论文ChapterICHAPTERILiteraturereview1.1IntroductionBluetongue(BT)isaninsect-borneemergingdiseaseoflivestockcausedbybluetonguevirus(BTV),theprototypememberofthegenusOrbiviruswithinthefamilyReoviridae.BluetonguediseaseisclassifiedasanemergingdiseaseinEuropeandcancausemajorlossesoflivestockininfectedsheepherds(Roy,2001).ThediseaseisnotcontagiousfromanimaltoanimalbutisspreadbybitingmidgesoftheCulicoidesgenusthenaturalbiologicalvectorsoftheBTV(Mellor,etal.,1995;Mellor,etal.,2000a).TheincidenceofbluetonguediseaseisthereforecloselyrelatedtothedistributionofthevariousCulicoidesspecieswhicharenormallyassociatedwithwarmorhotclimaticconditions(Gibbs,etal.,1994;Tabachnick,2004).Itisnowknownthatthemidgescanbeblownforlongdistancesonthewind(Gloster,etal.,2007a;Gloster,etal.,2007b;Gloster,etal.,2008).Worldwide,thereisarealeconomicsignificanceassociatedwithbluetongueinfectionincludingthedeathofanimalsandsubsequentreducedproductionaswellastheeffectofinternationalrestrictionsfortradeofanimalsandanimalproductsfrominfectedareas.Consequently,bluetonguewaslistedasoneofthecompulsorynotifiablediseasesbytheWorldAnimalHealthOrganization(OIE)andstudiedextensivelyasamodelsystemforrelatedviruses.Todate,atleast27serotypesofBTVhavebeendescribedinternationally(Maan,etal.,2007;Maan,etal.,2011a;Schulz,etal.,2016).WhilethevirusesareclassifiedantigenicallyandtaxonomicallyasBTV,eachserotypeisuniqueandmaynotcausethedisease.Thediseaseaffectsmanydomesticandwildruminants.Althoughsheep,goats,cattleandotherruminantscanallbeaffected,themostsevereclinicalsignsofBTaredisplayedinsheep,occasionallyingoats,andrarelyincattle(Roy,2001).Severediseasecanalsooccurinwildruminants,suchaswhitetaileddeer,pronghorn,anddesertbighornsheep.AlthoughBTVinfectionofcattle,goatsandmostwildruminantspeciesistypicallyasymptomaticorsubclinical,specificstrainsofBTVlikethestrainofserotype8currentlycirculatinginEuropecaninduceseverediseaseinotherspeciesincludingcattleandcamelids(Backx,etal.,2007;Darpel,etal.,2007;Henrich,etal.,2007;Toussaint,etal.,2007;Conraths,etal.,2009;Meyer,etal.,2009).Mortalityratescanvarysubstantiallybetweenepizootics,typicallyanywhereupto30%(orevenhigheronrareoccasion)insusceptibleruminants(Verwoerd,etal.,2004).Thisliteraturechapterthereforereviewsfourmainaspects,relevanttothefocusofthisstudy.ThefirstdiscussedtopicisBTdisease.Thisservestoputintoperspectivetheglobaldistribution,epidemiologyandseverityofthediseasethatleadstonegativeimplicationsonanimalsandindustryduetotherapidspreadofthedisease.Thissubstantiatestheneedforaneffectivevaccinestrategy.Intheseconddiscussiontopic,thetransmissionofthediseaseisaddressedwhileinthethirdsectionBTVisdissectedintermsofstructuralandmolecularbiologywithanemphasisplacedontheoutercapsidproteinsthemostimmunogenicviralproteins.Itisnecessarytohaveacompleteunderstandingofthestructuralorganization1 中国农业科学院博士学位论文ChapterIofthevirionaswellasthebiologicalimportanceoftheviralstructuralproteinsinordertorationalizeasubunitvaccine.Thefourthsectiondiscussesvaccinationstrategiesbycriticizingthevariousmethodsforvaccineproduction.Underthissection,thereisafocusontheadvantagesanddisadvantagesofasubunitvaccinationstrategyandtheprevioussubunitvaccinationstrategies.Thisin-turnisweightedagainsttheprosandconsofthecurrentlive-attenuatedandinactivatedBTVvaccinesandlogicallyjustifyingtheaimsofthecurrentstudy.Thefifthdiscussiontopicissummarizationfortheaimsandtheobjectivessuggestedtobeobtainedbytheendofthecurrentstudy.1.2Bluetongue1.2.1HistoryandcurrentglobaldistributionofthediseaseBTwasfirstreportedmorethan125yearsagowiththeintroductionofEuropeanbreedsofsheepintosouthernAfrica(Howell,etal.,1971).AlthoughtheoriginalwrittendescriptionsofBTdiseasewerepublishedinthelate19thandearly20thcenturies,thediseasewaslikelyrecognizedassoonasfine-woolEuropeansheepbreeds,particularlyMerinosheep,wereintroducedtosouthernAfrica(Spreull,1905;Henning,1956).BTisanarthropod-transmittedviraldiseaseofruminants,especiallysheep,thatwasfirstrecognizedinSouthAfrica,andprobablyhasoccurredthereforaslongassheepfarminghasbeenundertaken(Henning,1956).HenningquotedfromtheReportoftheCattleandSheepDiseasesCommissionfor1876that:“Formanyyears,ifnotfromthetimeoftheintroductionoftheMerinosheepintotheColony,therehasbeenprevalentamongsttheflocksofadiseaseknownasfever.Thisdiseaseismostprevalentduringthesummermonths,andisverymuchworseinwetseasons.”Itwasfurthernotedthatthisdiseasewascharacterizedbysorenessofthemouthandfeet,andsharedthesignsoffootandmouthdisease.Hutcheon(Hutcheon,1902)andSepreull(Spreull,1905)wrotethefirstcomprehensivedescriptionsofthediseasedesignatedatthattimeas“malarialcatarrhalfever”or‘‘EpizooticCatarrhofSheep’’.(Henning,1956)andSpreullreportedthatthename‘‘bluetongue’’(bluetongue,blue-tongue)istheEnglishformofthenametwistedfromAfrikaans“Blaauwtong.”Or‘‘bloutong’’,whichwascoinedbyBoerfarmerstodescribethedistinctivecyanotictongueofsomeseverelyaffectedsheep.Afrikaansfarmersalsoreferredtothediseaseas“Bekziekte,”meaning“mouthsickness”(Spreull,1905).Theviraletiologyofthediseasewasfirstdemonstratedin1906bySirArnoldTheilerwhoshowedthatbluetonguewascausedbyafilterablevirus(Henning,1956),andhealsointroducedthefirstwidelyusedbluetonguevaccinethatincorporatedastrainofvirusthatwasattenuatedbyserialpassagesinsheep.Some50milliondosesofthisvaccinewereusedbetween1907and1943,thenitsusewasstoppedwhenitbecameclearthatthismonovalentvaccinedidnotconferimmunityagainstthemanyotherserotypesofBTVthatcirculateinSouthAfrica(Henning,1956).Spreulldescribedtheessentialepidemiologicalfeaturesofbluetongueinhisclassicpublicationin1905,andreportedthat“Hutcheon,inhisearlierwritings,gaveitashisopinionthatsomebitinginsectactedasthecarrierofbluetongue2 中国农业科学院博士学位论文ChapterIinfection”andthatthediseaseoccurred“undertheconditionswhicharerecognizedasmostfavorablefortheproductionofSouthAfricanhorsesickness.”CulicoideswasidentifiedastheinsectvectorofbluetongueandAfricanhorsesickness(DuToit,1944).Priortothe1940s,bluetonguewasthoughttobeconfinedtosouthernAfrica,theMiddleEast(Israel),andMediterraneanislandssuchasCyprusuntilapproximately63yearsagowhenitwasfirstdescribedinCaliforniaandTexas(Hardy,etal.,1952;McKercher,etal.,1953).ThisassumptionwassoonchallengedbythegeographicalrecognitionofthediseaseinwidespreadregionsoutsideAfrica.Thefirstwell-documentedepizooticofBToutsideofAfricaoccurredamongstsheepinCyprusin1943(Gambles,1949),althoughthediseasehadlikelyoccurredtheresinceatleast1924(Polydorou,1985;Rodriguez-Sanchez,etal.,2008).AnextensiveepizooticoccurredontheIberianPeninsulain1956–1957.ThesequentialidentificationofBTVand/orbluetonguethroughoutmuchoftheworldduringthesecondhalfofthe20thcenturywasinterpretedatthetimeastheemergenceofbluetonguefromitspresumedancestralorigininAfrica;thisleadtotheputativeglobalspreadofbluetonguethatjustifieditsinclusionintheformerListAbytheWorldOrganizationforAnimalHealth(OIE)(OfficeInternationaldesEpizooties)(Gibbs,etal.,1994;MacLachlan,etal.,2006).BThasnowexpandedintomanyotherareasoftheglobeincludingIndia,theAmericas,AustraliaandEastAsia.TheincursionsintoCyprusin1943andinPortugalandSpainin1956representedan“earlywarning”thatarbovirusescouldalsocauseproblemsoutsideofthetropicalandsub-tropicalareasoftheplanet.BTwasrecognizedthereafterintheUnitedStates,MiddleEast,AsiaandsouthernEurope(Verwoerd,etal.,2004)..TheincreasedrecognitionofBTinwidelyseparatedregionsoftheworldinthemiddleofthe20thcenturywasthoughttoreflecttheemergenceofBTfromitspresumedancestralorigininAfrica(Gibbs,etal.,1994).Indeed,since1999severalBTVserotypeshaveenteredtheMediterraneanBasinandNorthernEurope,includingBTV-8whichwasresponsibleforoneofthemostdevastatingoutbreaksinthehistoryofbluetongue.OtherarbovirusessuchasWestNilevirus,Chikungunya,SchmallenbergandDenguehavefollowedtheincursionsofBTVintotheEuropeancontinent,furtheremphasizingthepointthatincreasedinternationaltravel,globalization,changesinecosystemsandclimateareexpandingtheterritoryofarboviralvectorsand,inturn,currentlyexoticviruses.ItisnowclearlyapparentthatBTVinfectionoccurswidelythroughouttropical,subtropicalandsometemperateregionsoftheworld.BTVhasbeenisolatedfromruminantsand/orvectorinsectsfromallcontinentsexceptAntarcticaandtheglobaldistributionofBTVcoincideswiththedistributionofthecompetentCulicoidesinsectvectorsinthepresenceofanappropriateclimaticconditions.Furthermore,differentserotypesandstrainsofBTVoccurindifferentregionsoftheworld,frequentlycoincidentwiththepresenceofdistinctspeciesofCulicoidesinsectvectors(Gibbs,etal.,1994;Pritchard,etal.,2004;Tabachnick,2004;MacLachlan,etal.,2006;Balasuriya,etal.,2008).3 中国农业科学院博士学位论文ChapterIAlthoughBTVlikelyhaslongbeenpresentthroughoutmuchoftheworld,theglobaldistributionofBTVinfectionveryrecentlyhasdrasticallyaltered(Purse,etal.,2008;Wilson,etal.,2008b).BTVstrainshavebeenidentifiedinmanytropicalandtemperateareasoftheworldfromlatitudeofapproximately40°Nto35°S.However,duringtherecentnorthernEuropeanepidemic,thevirusspreadfarbeyonditspriorknownuppernorthernlimits(Toussaint,etal.,2007).TheglobaldistributionofindividualBTVserotypesisnotuniformandhasundergonedrasticrecentalteration,perhapsasaconsequenceofclimatechangewhichisinpartresponsibleforthisprofoundchangeintheglobaldistributionofBTV,morelikelybyitsimpactontheCulicoidesinsectpopulationsinpreviouslyvirus-freeregionssuchasmuchoftheMediterraneanBasin(Gerry,etal.,2001;Purse,etal.,2005;Purse,etal.,2008;Wilson,etal.,2008b;Gould,etal.,2009).BTVhasrecentlyspreadthroughoutmostofEuropewhere,untilapproximately1998,onlysporadicandtransientepizooticsofBTVinfectionoccurredpreviously(Gomez-Tejedor,2004;Purse,etal.,2008;Rodriguez-Sanchez,etal.,2008;Saegerman,etal.,2008).Similarly,multiplenovelserotypesofBTVhaverecentlyspreadnorthwardfromtheCaribbeanBasintoinvadethesoutheasternUnitedStates,wheretheypreviouslydidnotidentified(Johnson,etal.,2006)andadditionalserotypesofBTVhaverecentlyoccuredinnorthernAustralia.TheappearanceandsubsequentrapidspreadofBTV-8thatbeganinnorthernEuropein2006(Enserink,2006;Thiry,etal.,2006;Backx,etal.,2007;Toussaint,etal.,2007;Elbers,etal.,2008a;Elbers,etal.,2008b)isespeciallyremarkableforseveralreasons.ThefirstreasonisthatthisvirushasspreadfurthernortherlythanBTVhaseverpreviouslybeenreported.Secondly,thevirusisnotspreadbyCulicoidesimicola,themajorvectorforBTVtransmissioninAfricaandAsia,andPalearcticCulicoidesspeciesareinvolvedinthetransmission(Meiswinkel,etal.,2008).ThestrainofBTV-8thatinvadednorthernEuropeisrecognizedasahighlyvirulentstrain,notonlyforsheepbutalsocattle(Backx,etal.,2007;Darpel,etal.,2007;Elbers,etal.,2008a;Elbers,etal.,2008b;Worwa,etal.,2008;Conraths,etal.,2009),free-rangingandcaptivespeciesofwildruminants(Mauroy,etal.,2008),camelids(Henrich,etal.,2007)andevenzoocarnivoresfedonaborted,virus-infectedfetalruminants(Jauniaux,etal.,2008).Indeed,transplacentaltransmissionofBTVwaspreviouslyconsideredtobelargelyorexclusivelyapropertyoflaboratory-adaptedstrainsofBTV,particularlycertainlive-attenuatedvaccineviruses(MacLachlan,etal.,2000;Kirkland,etal.,2004;Maclachlan,etal.,2008b).However,thisstrainofBTV-8crossestheruminantplacentatocausefetalinfectionswithastonishingfrequency,whichisunusualcomparedwithother‘‘field’’strainsofBTVthatoccurelsewhereintheworld(DeClercq,etal.,2008a;DeClercq,etal.,2008b;Desmecht,etal.,2008;Vercauteren,etal.,2008;Wouda,etal.,2008;Worwa,etal.,2009).In2008,BTV-6emergedinthesamegeneralareainwhichBTV-8hademergedtwoyearsbefore,andBTV-11wasreportedveryrecentlyinBelgiumaswell.TherouteofintroductionofBTVserotypes6,8and11intonorthernEuropeisuncertain,astheseBTVserotypesdidnotfirstinvadetheMediterraneanBasinandthenspreadnorthward,asserotype1hasrecentlydone.4 中国农业科学院博士学位论文ChapterIFurthermore,thestrainsofBTVserotypes6and11apparentlyincludegenesegmentsderivedfromeitherSouthAfricanlaboratoryprototypevirusesorlive-attenuatedvaccinevirusesderivedfromthem(Mertens,2008,2009),whichraisesseriousconcernregardingthepotentialroleofhumaninvolvementintheseoccurrences.Thedisconcertingsignificanceofsuch‘‘translocation’’eventstothegloballivestockindustryisself-evident,includingtheAmericaswhereavarietyofBTVserotypesarealreadyenzootic(Gibbs,etal.,1994;MacLachlan,etal.,2006).TherecentemergenceoftenBTVserotypesinEurope,eightnewserotypesintheUSA,twonewserotypesinAustraliaandisolationofsevendifferentserotypesinIndiainlasttwodecades(Gollapalli,etal.,2012;Rao,etal.,2012),illustratetheincreasedrisksposedbyBTandotheremergingarboviraldiseasesworldwide(Zientara,etal.,2013).AdditionalnovelserotypesofBTVhavealsorecentlyinvadedIsraelandAustralia,countriesinwhichongoingsurveillanceovermanyyearshadconfirmedanapparentlystablecycleofannualBTVinfection.SeveralBTVserotypes(Alexander,1948;Anderson,etal.,1989;Barnard,1997;Bonneau,etal.,2001;Anon,2009)havehistoricallybeenpresentinIsrael,butserotypes8,15,24haveallrecentlyemergedinthecountry(Shimshony,2004).Similarly,eightserotypes(1,3,9,15,16,20,21,and23)ofBTVpreviouslyhavebeenidentifiedinAustralia,ofwhichonlyserotypes1and21annuallyspreadintoeasternAustralia.However,BTVserotypes2and7wereidentifiedin2007/2008forthefirsttimeintheNorthernTerritoryofAustralia(Maclachlan,2010).InterpretationoftheBTVstatusofotherregionsoftheworldiscomplicatedbythelackofadequatesurveillanceinmanyareas,particularlythroughoutmuchofAfrica,AsiaandtheMiddleEast.1.2.2ThediseasetransmissionBitinginsectsofthegenusCulicoidesserveasbiologicalvectorsthattransmitBTVinfectionbetweenruminants(Mellor,2000;Gerry,etal.,2001).OfmorethanonethousandknownspeciesofCulicoidesthatoccurworldwide,aboutthirtyspecieshavebeenincriminatedaspotentialvectorsofBTVinfection(Meiswinkel,etal.,2004;Meiswinkel,etal.,2008).AppropriateclimaticconditionsarealsoimportantintheregionalmaintenanceofBTVinfection,thusthevirusexiststhroughoutmuchoftheworldbetweenlatitudesofapproximately40-50oNorthand35oSouth(Gibbs,etal.,1994;Tabachnick,2004;Verwoerd,etal.,2004).However,thevirusrecentlyhasspreadfarbeyondtheupperlimitsofthistraditionalrangeinportionsofEurope(Saegerman,etal.,2008).ThespeciesofvectorinsectsthattransmitBTVdifferbetweenregionsoftheworld,andareespeciallypoorlycharacterizedinpartsofEuropeandAsia(MacLachlan,etal.,2006).AlthoughvenerealandverticaltransmissionofBTVcanoccurinruminants,withthenotableexceptionofBTV-8inEuropetheseeventsareapparentlyrareandconsideredunimportanttothemaintenanceofBTV(Kirkland,etal.,2004;Walton,2004;Anon,2008).Moreover,otherhostshavebeenimplicatedinthelifecycleofBTVinfection,forexample,serologicalevidenceindicatesthatlargeAfricancarnivoresareinfectedwithBTV,whereassmallerpredatorsthatcohabitwiththemarenot,suggestingthatlargecarnivoresareinfected5 中国农业科学院博士学位论文ChapterIthroughfeedingonBTV-infectedruminants(Alexander,etal.,1994).InadvertentcontaminationofacaninevaccinewithBTVconfirmedthatdogsaresusceptibletoBTVinfection;indeed,pregnantbitchesthatreceivedthiscontaminatedvaccinetypicallyabortedanddied(Akita,etal.,1994;Evermann,etal.,1994;Wilbur,etal.,1994;Brown,etal.,1996;Levings,etal.,1996).Thereisnoevidence,however,thatdogsorothercarnivoresareimportanttothenaturalcycleofBTVinfection.Thenormaltransmissioncycleofavector-bornediseasesuchasBTisshownintheleftpanelofFig(1.1).Infectiousvectorsinjectvirusintohostsviasalivawhilefeeding.Infectedhoststhenpassthroughashortincubationperiodbeforedevelopinganinfectiouslevelofvirus,andvectorsthatbitethehostwhileviruslevelsarehighmaybeinfected.Ingestedvirusreplicatesininfectedvectorsuntilitreachesthesalivaryglands,atwhichpointthevectorbecomesinfectiousandthecycleiscompleted.BTVisolatehastheabilitytheoverwinteringandsurviveduringwinterusingdifferentwaysrepresentedintherightpaneloffigure1.1(Wilson,etal.,2008a).Figure1.1Thetransmissionofbluetonguevirus(Wilson,etal.,2008a).BTVhasalsospreadrapidlythroughoutextensiveportionsofEuropewhereC.imicoladoesnotoccur,utilizingapparentlynewvectorspecies,potentiallyincludingPalaearcticspeciessuchasCulicoidesobsoletussensustrictu,C.pulicaris,C.dewulfi,C.scoticus,andC.chiopterus(Caracappa,etal.,2003;Savini,etal.,2005;Meiswinkel,etal.,2007;Dijkstra,etal.,2008;Wilson,etal.,2008b;Conraths,etal.,2009;Kampen,etal.,2010).TheseinsectswereallresidentinEuropelongbeforetherecentemergenceofmultipleBTVserotypes,suggestingthatenvironmentalchangesmayhavebeenresponsiblefortheirrecentabilitytoserveasefficientvectorsofthevirus(Purse,etal.,2005;Purse,etal.,2008).6 中国农业科学院博士学位论文ChapterIIntheUSA,theprincipalvectorsareCulicoidessonorensisandCulicoidesinsignis,whichlimitthedistributionofBTVtosouthernandwesternregions.BTVserotypes10,11,13and17occurthroughoutmuchofNorthAmericacoincidentwiththedistributionofCulicoidessonorensis,whereasserotype2isrestrictedtothesouth-easternUnitedStatesapparentlybecauseofitsrelianceonCulicoidesinsigniasfortransmission(Gibbs,etal.,1994;Tabachnick,2004).Itislikelythatthe10additionalBTVserotypesthatrecentlyhaveencroachedintothesouth-easternUnitedStatesoriginatedintheadjacenttropicalecosystemtotheimmediatesouth,whereC.insigniasandpossiblyotherspeciessuchasCulicoidespusillus,Culicoidesfurens,CulicoidesfilariferandCulicoidestrilineatusmightactasvectorsofnumerousBTVserotypes.ThebluetonguechapteroftheOIEterrestrialanimalhealthcode(OIE,2003)referstoCulicoidesbygenus,ratherthanbyspecies,thusconveyingtheimpressionthatallspeciesofCulicoidesarecompetentvectorsofBTVserotypes.ThelogicthatfollowsfromthisimpressionisthatallcountriescouldbeconsideredatriskforBTVtransmissionsinceitappearsthatonlyAntarcticacanbeconsideredfreefromCulicoides.Thefactisthatintheabsenceofconfirmedvectorstatus,orcompetenceofindigenousCulicoidesspecies,itisillogicaltoimplythateverycountrywithCulicoidesisatriskifimportsofruminantsarepermittedfromcountriesinwhichlivestockareconsideredinfectedwithBTV.Intheabsenceofacompetentvector,theimportationofinfectedorviraemicruminantsservesasnothreattoanimportingcountry.Restrictionsdo,however,placeapotentialtradingpartneratacompetitivedisadvantagethatisnotbaseduponscience.Similarly,seropositivityofacandidateruminantforproposedimportinnowayconveysanindicationthattheanimalisviraemicanddoesnotjustifylegislationagainsttheimportationofsuchanimals.SeropositivitysimplydocumentsaprevioushistoricexperiencewithaBTVantigenandhasnorelevancetocurrentinfectionorinfectivityofthehost.Furthermore,andperhapsmoreimportantly,theseanimalsareimmunetotheinfectingandcloselyrelatedviruses.Thevirusistraditionallyspreadtoanimalsviabloodfeedingmidges(Culicoidesspp.)whichrepresentthemainvectorandcontributetothegeographicaldistributionofthedisease.Otherbitinginsects,suchasticksmayalsotransferthevirus.Bluetongueisnotcontagiousandisnotspreadbycontactbetweenanimals.However,thevirusmaybespreadbycontaminatedobjects(fomites),suchassurgicalequipmentandneedles.Bluetongueviruscanbetransferredfromthedamduringpregnancytothefetus.Theviruscanbefoundinsemen,butsexualtransmissiondoesnotappeartobeamajorrouteofinfection.ContinuedcyclingofthevirusamongcompetentCulicoidesvectorsandsusceptibleruminantsiscriticaltoviralecology.7 中国农业科学院博士学位论文ChapterI1.2.3ThediseaseepidemiologyThegeographicdistributionofBTViscategorizedintothreeecologiczones:endemic,epidemicandincursion.EndemiczonesaredefinedastypicallytropicalregionswhereBTVtransmissionoccursthroughouttheyearandsubclinicalinfectioniscommon.Inendemiczones,clinicaldiseasegenerallyoccursonlyinintroduced,immunologicallynaïvesusceptiblespecies(Gibbs,etal.,1994;Caporale,etal.,2003).Epidemiczonesincludetemperateareaswhereoutbreaksoccurseasonally,generallyinthelatesummerwhenvectorpopulationspeak.Incursionzonesareareasthatexperienceoutbreaksinfrequentlywhenclimaticconditionsfavordiseasetransmissionbyvectors.BTV,thecausativeagentofbluetongueofruminants,hasnowbeenidentifiedonallcontinentsexceptAntarctica(Gibbs,etal.,1994;Tabachnick,2004;MacLachlan,etal.,2006;Maclachlan,2010).BTVexistsinanextensivebandthatincludestropical,subtropical,andtemperateregionsoftheworld.ExceptionsincluderegionsofAsiaandwesternNorthAmerica,whereBTVinfectionofruminantsoccursasfaras50°North(Clavijo,etal.,2000;Lundervold,etal.,2003;Tabachnick,2004;Shoorijeh,etal.,2010)and,mostrecently,northernEurope.However,thedistributionofspecificinsectvectorsanddifferentBTVserotypesdifferremarkablythroughouttheworld,sospecificvectorsexistwithspecificconstellationsofBTVserotypesandtopotypesinrelativelydistinctglobalecosystems(Gibbs,etal.,1994;Tabachnick,2004;MacLachlan,etal.,2006;Balasuriya,etal.,2008).CompetentvectorinsectsarepersistentlyinfectedwithBTVfortheirentirelifespanafteracquiringinfectionthroughfeedingonaBTV-infectedruminant(Mellor,2000).BTtypicallyoccurswhensusceptiblesheepareintroducedintoareaswhereBTVvirulentstrainscirculate,orextendtheirrangeintopreviouslyunexposedpopulationsofruminants.ThevirusisendemicinmanytropicalandsubtropicalregionsincludingtheUnitedStates,CentralAmerica,partsofSouthAmerica,Africa,SoutheastAsia,andnorthernAustralia,withamorerecentemergenceintoEurope(Stott,etal.,1985;Gorman,1990;Jones,etal.,1997;Zhang,etal.,1999;Kirkland,etal.,2002;Zientara,etal.,2002;Purse,etal.,2005;Ravishankar,etal.,2005;Mehlhorn,etal.,2007;Mertens,etal.,2007)alsopressreleasefromtheWorldOrganisationforAnimalHealth,Paris,France,25August2006,http://www.oie.int/eng/press/en_060823.htm).Since1998multipleoutbreaksofdifferentserotypes(BTV-1,2,4,6,8,9,11,16,and25)havebeenidentifiedinEurope(Mellor,etal.,2008;Carpenter,etal.,2009).AnoutbreakcausedbyBTV-8startedintheNetherlandsduring2006,subsequentlyspreadacrossmostofEurope,causinghighlevelsofmortalityinsheep(15–32%,reaching-50%insomeareas),aswellassignificantclinicalsignsbutlowmortality(<1%)incattle(Maan,etal.,2008;Menzies,etal.,2008).TheseeventshavecausedsubstantialeconomiclossestotheagriculturaleconomiesofEurope.TheBTV-8outbreak,wasnotonlythefirstBTVoutbreaktospreadthroughnorthernEuropebutwasthelargestsingleoutbreakofBTVeverrecordedinEurope.Thisoutbreakwasextensivewith8 中国农业科学院博士学位论文ChapterImorethan2millioninfectedanimals(predominantlysheep)duetotheoverwinteringabilityofthisBTVisolate.BTV-8wasalsovirulentforcattle,whicharegenerallyasymptomatic,with10%ofinfectedcattledisplayingBTresultinginanimalfatality(Elbers,etal.,2008a)Thereareatleast27immunologicallydistinctBTVserotypesvectoredbybitingmidgesofthegenusCulicoidesspp(Maan,etal.,2007;Maan,etal.,2011a;Schulz,etal.,2016),10ofwhich(BTV-1,2,4,6,8,9,11,14,16and25)havebeendetectedinEuropesince1998(Purse,etal.,2005;Purse,etal.,2008;Maclachlan,2010).InChina,multipleBTVstrains(BTV-1-5,7,9,12,15,16and24)havebeenisolatedfromthebloodofsheepandcattlebetween1986and1996whilethefirstisolationofBTVinChinagoesbackto1979inYunnanProvince(Zhang,etal.,1999;Ting,etal.,2005;Lee,etal.,2011;Yang,etal.,2016).Serotypes1and16wereisolatedfromsheepwithobvioussignsofbluetongue,whereasserotypes2,3,4,12and15wereisolatedfromclinicallynormalcattleandthephylogeneticanalysesofthesevirusesindicatethattheyarecloselyrelatedtooneanother,andtoanAustralianstrainofserotype1(Zhang,etal.,1999;Zhang,etal.,2004a).ThefirstreportofthecompletegenomesequenceoftheBTV-16inChinahasbeenreportedin2011(Yang,etal.,2011).UptodatefoureasternBTV-16strains,fromAustralia,India,ChinaandtheSouthAfricanreferencestrainhavebeenfullysequenced(Yang,etal.,2011;Boyle,etal.,2012;Maan,etal.,2012;Minakshi,etal.,2012).PreviousanalysesshowedthattheBTV-16referencestrain(RSArrrr/16),theBTV-16vaccinestrain(RSAvvvv/16)andaChineseBTV-16(strainnumberBN96/16)(Yang,etal.,2011;Maan,etal.,2012),have>99%sequenceidentityinalltengenomesegments,indicatingthattheyarecollectivelyderivedfromarelatedcommonancestor.Figure1.2Epidemiologyofbluetongue9 中国农业科学院博士学位论文ChapterISincethefirstoutbreakofBTinYunnanin1979,BTVshavebeenisolatedfromsheepinYunnan,Hubei,Sichuan,Anhui,ShandongandShanxiProvinces,fromgoatsinXinjianandInnerMongolia,andfromcattleinGansu.ThesevirusstrainswereidentifiedasBTVbyAgargelimmuodifutiontest(AGID),Immunoflurecencetest(IF)andagargelelectrophoresis.EightoftheChinesestrainswereserotypedbymicro-serumneutrlizationtests.TheseshowedthatstrainsS×1fromYunchengCountyinShanxi,S×2fromJiansuCountyinShanxi,Y863P12fromYunnan,×27fromXinjianandYcfromCulicoidestrappedintheanimallaboratoryinKunmingwereallBTVserotype1.BothWP7fromHubeiandSWP7fromSichuanProvincewereBTVserotype16.EarlyresultsindicatethatNMP11fromInnerMongoliamightbeBTV-17butfurtherconfirmatorytestsarerequired(Zhang,etal.,2004b).In1998,110strainsofBTVwereisolatedfromsubclinicallyinfectedcattleinfoursentinelherdsintheYunnanProvince.Usingtheinternationalreferenceantiserum,theBTVstrainswereidentifiedtoserotypesusingthemicroneutralizationtest.Theresultsareasfollows.I)theserotypesisolatedincluded1,2,3,4,12,15and16;II)thepredominantserotypeswere1,4,15and16;III)duringshortperiodsofjustoneortwomonths,twoorthreedifferentBTVserotypeswereisolatedfromthesamecattle.ThissuggeststhatanimalswereeitherinfectedwithmorethanoneBTVserotype,orthattheycanbeinfectedsequentiallywithmultipleserotypes;IV)theseresultshaveprovidedconsiderableinsightintothedistributionandseasonaltransmissionofBTVinChina(Zhang,etal.,1996b;Nianzu,etal.,1998;Zhang,etal.,1999;Kirkland,etal.,2002).1.2.4ThepathogenesisofbluetonguevirusBToccursmostcommonlyinsheepofcertainbreeds,especiallyfine-woolEuropeanbreeds,andinsomespeciesofwildruminantssuchaswhite-taileddeer(Verwoerd,etal.,2004).Bluetonguevirusinfectedsheepcandevelopseriousdiseaseandmayevendiewhilemanyotherinfectedanimalsdonotshowsignsofdisease.ManykeyquestionsonBTpathogenesisremaintobefullyaddressed.BTVinfectioncanbeessentiallyasymptomaticorlethalforthemammalianhost.Thisvariabilityisduetofactorsrelatedtothevirus,thehost,theenvironmentandpossiblytheinsectvector.Thevariablesusceptibilitytobluetonguedisplayedbydifferentspeciesandbreedsofruminantswasnotcompletelyunderstood.DifferentiatingahighlyvirulentBTVstrainfromalessvirulentoneisalsoremainedquestionable.ThisisextremelyimportantbecauseanyBTVincursioninsomegeographicalareascanbetreatedbyregulatoryagenciesinthesameway,potentiallyleadingtosevereeconomicconsequences.BybetterunderstandingthedeterminantsofBTVvirulencewemaybeabletodesigncontrolstrategiesthat“fit”therisksposedbyincursionsofspecificBTVstrains,similartotheapproachtakenforavianinfluenza(Capua,etal.,2009)orNewcastledisease(Alexander,2000).Indeed,itisnoteasytodeterminewhetheroutbreaksofseverecasesofbluetongueareassociatedwith“highlyvirulent”BTVstrains,orwhetherspecificecosystemsandindividualcircumstancesplayamorefundamentalrole.Importantly,differentspeciesofCulicoidesvectors10 中国农业科学院博士学位论文ChapterIcandisseminatedifferentBTVstrainsindifferentecosystems.TheroleofdifferentCulicoidesspeciesinthepathogenesisofbluetongueremainstobedetermined.Thus,itwillbeimportanttoexperimentallyteaseapartthevariablesthatcaninfluencetheclinicaloutcometoBTVinfection.Althoughseveralstudieshavebeguntodissectvirus-hostinteractions,muchremainsunknownastohowthehostcontrolsBTVinfectionand,inturn,howtheviruscontrolsthehost’sresponse.ThesignsofBTinsheepreflectcongestion,oedemaandhaemorrhageasaconsequenceofvirus-mediatedvascularinjury(Erasmus,1975;Pini,1976;Mahrt,etal.,1986;Verwoerd,etal.,2004;MacLachlan,etal.,2008a),andincludefever,excessivesalivation,depression,anddifficultyinbreathing(Figure1.3.A).Thus,infectedsheepusuallyhaveanycombinationoffever,seroustobloodynasaldischargewithcrustingaroundthenares,reddenedandulceratedmuzzle,lips,andears,thelipsandtonguemaybeveryswollen,causingthetonguetostickoutfromthemouth;thetongueisoftenbluishincolor,givingthediseaseitsname(Figure1.3.B),laboredbreathingandprofoundrespiratorydifficultyinanimalswithseverepulmonaryoedema,oralerosionsandulcers,lamenesswithhyperaemiaofthecoronarybandmakinganimalsreluctanttomoveandinsomecasesthehoovesmayactuallysloughoff,andweaknesssecondarytomusclenecrosis.Pregnantewesinfectedduringthefirsttrimestermayhavereproductiveproblems,suchasabortionorbirthof“dummylambs”.Sheepthatdosurvivecanlosesomeoralloftheirwool.Thelesionspresentonpost-mortemexaminationofsheepaffectedwithbluetonguehavebeenwelldescribed,andincludeoralerosionsandulcers,coronitis,weaknessandfacialedema(Spreull,1905;Henning,1956;Moulton,1961;Verwoerd,etal.,2004;MacLachlan,etal.,2008a;Maclachlan,etal.,2009),hyperaemia,haemorrhages,erosionandulcerationofthemucosaoftheuppergastrointestinaltract(oralcavity,oesophagus,forestomachs);oedemaandhaemorrhageoflymphnodes;haemorrhageswithinthesubcutis;subintimalhaemorrhagesinthepulmonaryartery;pulmonaryoedema;pleuraland/orpericardialeffusion;facialandsubmandibularoedema;oedemawithinthefascialplanesofthemusclesoftheabdominalwallandneck,particularlyaroundtheligamentumnuchae;necrosisofskeletalandcardiacmuscle,withthepapillarymuscleoftheleftventriclebeinganespeciallycharacteristicsite(Spreull,1905;Moulton,1961;Kennedy,1968;Erasmus,1975;Verwoerd,etal.,2004;MacLachlan,etal.,2008a).Althoughvascularinjuryclearlyisresponsiblefortheclinicalmanifestationsandlesionsofbluetongue,themechanismbywhichtheviruscausestheseisnotclear.Sheepwithseverebluetonguetypicallydevelopcoagulopathyandexcessivebleeding,however,theydonotinvariablydevelopterminaldisseminatedintravascularcoagulationlikecertainhighlysusceptiblespeciesofwildungulates(MacLachlan,etal.,2008a;Maclachlan,etal.,2009).Furthermore,BTVinfectionofvascularendotheliumisrelativelyrestrictedandtransientininfectedruminants,incontrasttothewidespreadvascularleakagethatcharacterizesfulminantcases(Maclachlan,etal.,2009).11 中国农业科学院博士学位论文ChapterIAlthoughBTVinfectionofruminantsisoftensubclinicalorunapparent,theinfectioncanleadtoseverediseasewithhighmortalityinsusceptibleanimals.Bluetongueprincipallyisadiseaseofsheepandcertainspeciesofon-Africanwildlife(Verwoerd,etal.,2004;Maclachlan,etal.,2009).AsimilaroridenticaldiseasesyndromeoccurssporadicallyinBTV-infectedcattle,SouthAmericancamelids,andcertainotheranimalspecies.Figure1.3Clinicalsignsofthedisease.BTdiseasemanifestationinsheepshowsacharacteristicclearandstringysalivationwithnasaldischarge(A)andcyanosisoftheoralcavity(B).Adaptedfrom“BluetonguevirusinEurope:anupdateaftertenyears.IthasbeenrecentlyshowninvitrothattheincreasedpermeabilitythatoccursinculturedmonolayersofruminantendothelialcellsinfectedwithBTVisexclusivelyaconsequenceofdeathofvirus-infectedcells.Incontrast,BTVinfectionofruminantmacrophagesresultsinproductionofcytokinemediatorssuchastissuenecrosisfactor(TNF)thatincreasethepermeabilityofendothelialmonolayersbycausingredistributionofVascularendothelial(VE)-cadherin,withoutcelldeath(Drew,etal.,2010a;Drew,etal.,2010b).Inthisscenario,virusinfectionofmacrophagesanddendriticcellswouldleadtotheproductionofvasoactivecytokinemediatorsthatincreaseparacellularpermeabilityoftheendotheliumthroughtheireffectonthecellcytoskeletonandadherensjunction,withresultantfluidleakagefromthevasculatureleadingtohypovolemic(circulatory)shock.Itishypovolemicshockthatisresponsibleformortalityinmanyinstancesoffatalhemorrhagicviralfever(Gowen,etal.,2008).Incontrast,directvirus-mediatedinjurytovascularendotheliumislikelytoberesponsiblefortissueischemiaandnecroticsignssuchasthedistinctiveandcharacteristic,ifuncommon,cyanotictonguefromwhichbluetonguederivesitsname.1.3BluetonguevirusBluetonguevirus(BTV)isacomplex,non-enveloped,doublestrandedRNA(dsRNA)virusandistheprototypememberofthegenusOrbivirusofthefamilyReoviridae.ThisfamilycomprisesofsegmenteddsRNAviruses,possessingicosahedralnon-envelopedcapsidswithadoublelayeredarchitecture.This12 中国农业科学院博士学位论文ChapterIlargegroupofdsRNAvirusesrepresentsthecausativeagentsofseveraleconomicallyandmedicallysignificantdiseasesandassuchpresentagreatchallengeinvirologicalunderstanding.Asamodelsystemforrelatedviruses,BTVhasbeenstudiedextensivelyandnotableprogresshasbeenaccomplishedinsequence,structuralandbiochemicalknowledgeofthevirus.BTVprovidedthefirstwholegenomesequenceofadsRNAvirusandthefirstvirus-likeparticle(VLP)nowinwidespreaduseformanyvirusesbothforvaccinedevelopmentandasatoolforbasicresearch.BTVvirionsarearchitecturallycomplexandarecomposedofsevendiscreteproteinsinspecificbutnonequimolarratiosthatareorganizedintotwocapsids(Hewat,etal.,1992a;Hewat,etal.,1992b;Prasad,etal.,1992;Roy,1996;Ruiz,etal.,1997).Thevirionproteinsencapsidatethegenomeof10double-strandedRNA(dsRNA)segments(S1toS10)inadescendingorder,whichencode7structuralproteins(VP1,VP2,VP3toVP7)and4nonstructuralproteins(NS1toNS4).ThestructureofanintegralBTVcoreproteinbecameaparadigminthefieldandthisstructureconstitutedthefirstcomplexviralcapsidtobesolvedatatomicresolution.ThesevenstructuralproteinsformthreeconsecutivelayersofproteinsintheBTVparticlesthatareorganizedintotwocapsids,anoutermostcapsidconsistsoftwoproteins(VP2andVP5)andaresurfaceexposedtoavaryingextent,andaninnericosahedralcapsid(core)comprisesoftwomajorproteinsVP7andVP3arestronglyassociatedandencapsidatethetengenomicdsRNAsegmentsalongwithminorproteincomponentswhichareVP1,VP4,andVP6havinglowrelativeconcentrationsintheparticleinrelationtothemajorcomponentsofVP2,VP3,VP5andVP7,inadditiontotheviralgenome.Theoutercapsid,whichiscomposedoftwomajorstructuralproteins,alarger110kDaprotein,VP2,andasmaller60kDaprotein,VP5,isinvolvedwithcellattachmentandviruspenetrationduringtheinitialstagesofinfection,respectively.Afterentryintocells,thevirusisuncoated(VP2andVP5removed)toyieldatranscriptionallyactivecoreparticlewhichiscomposedoftwomajorproteins(VP7andVP3)andthreeminorproteins(VP1,VP4,andVP6)inadditiontothedsRNAgenome.Theseminorcorecomponentsarenotnecessarytoreconstitutevirusandcore-likeparticles(CLP)byrecombinantbaculovirusexpression,indicatingthattheyarenotcomponentsoftheproteincapsidandserveasencapsidatedcargo.Thereisnoevidencethatanytraceoftheoutercapsidremainsassociatedwiththesecores,ashasbeendescribedforReovirus(Eaton,etal.,1990).Table(1.1)summarizesthesize,locationandfunctionoftheproteinsencodedbyBTVsegments.Usingcryo-electronmicroscopicanalysisofunfixedandunstainedvirionsandVLP,ithasbeenshownthatthetwooutershellproteinshavedistinctiveshapes.Oneoftheseproteinsisglobular,almostspherical,with360moleculessittingneatlyoneachofthesix-memberedringsoftheunderlyingVP7trimers,andtheotherissail-shapedwithprotrudingtriskelionspikes,locatedabove180oftheVP7trimers(Hewat,etal.,1992a;Hewat,etal.,1992b).Together,thetwoproteinsformaroundthecoreacontinuouslayerca.86nmindiameter.VP2,whichisbelievedtobethespike-likeprotein,hasbeenshowntoberesponsibleforelicitingvirusneutralizingantibodies,hemagglutinationactivity,andserotypespecificity13 中国农业科学院博士学位论文ChapterI(Huismans,etal.,1981b;Grubman,etal.,1983;Kahlon,etal.,1983;Huismans,etal.,1987c;Eaton,etal.,1989b;Roy,etal.,1990b).AntibodyneutralizationexperimentssupportedbyrecentstudiesondirectcellbindingwithVP2anditssubsequentinternalizationsuggestthatVP2istheproteinassociatedwiththecellattachmentandentryofvirions(Hassan,etal.,1999).ThesecondoutercapsidproteinVP5mayalsoplayaroleinvirusneutralizationactivityasVP5enhancestheprotectiveneutralizationactivityofVP2insheep(Roy,etal.,1990b).Figure1.4MorphologyofBluetonguevirus.Thebluetonguevirusparticleconsistsofthedouble-strandedRNA(dsRNA)genomesurroundedbythreeconcentricproteinlayers.TheviralgenomeisdividedintotenlineardsRNAmoleculeslocatedatthecentreoftheparticle.Theviralgenomeissurroundedbytheicosahedralinnerproteinlayer,whichconsistsofVP3.Ontheinnersurfaceofthislayer,atthefive-foldsymmetryaxesoftheparticlearetheRNA-dependentRNApolymerase(VP1),cappingenzyme(VP4)andviralhelicase(VP6)..TheVP3layerissurroundedbyVP7,atrimericprotein.Theouterlayer(whichconsistsofVP2andVP5)ofthevirusparticleisresponsibleforattachmentandpenetrationoftargetcells(Roy,etal.,2009).1.3.1TheoutermostproteinsVP2andVP5ImmunologicalstudiestodatehavegivenmanycluesaboutwhatBTVproteinsaremoreimportanttoinduceprotectivehostimmuneresponsesagainstthevirus.TheBTVparticleismadeofsevenstructuralproteins(VP1toVP7)(Attoui,etal.,2011;Belhouchet,etal.,2011a;Ratinier,etal.,2011a).VP2representsaprimarytargetfortheneutralizingantibodies(vanderWalt,1980;Hassan,etal.,14 中国农业科学院博士学位论文ChapterI1999;Mertens,etal.,2005;Attoui,etal.,2011),determinesvirusserotypeandshowsabout22.4%–73%aasequencevariationbetweenBTVserotypes(Maan,etal.,2007).EpitopemappingstudiesrevealedthatthemajorvirusneutralizingepitopesarelocatedintheproteinVP2(Roy,1992).ThisproteinisthemostvariableproteinamongBTVserotypesandisthedeterminantoftheserotype(Mertens,etal.,1989).However27serotypesofBTVhavebeenreportedandlowlevelsofcross-protectionaredescribed,complicatingvaccinationstrategies.Incontrast,theBTVVP5isastructuralproteinwhichisserotypespecificandcouldenhancetheabilityofneutralizingantibodyinductioninsheepandarecapableofprotectingagainsttheinfection.VP5representsthesecondmostvariableBTVproteinandpossessanaaidentityof41%–79%betweenBTVserotypes(Maan,etal.,2011a;Maan,etal.,2011b)enhancesneutralizingantibodyresponsetoVP2proteins(Mertens,etal.,1987;Hassan,etal.,2001;Mertens,etal.,2005;Attoui,etal.,2011),andseemstoexertsomeinfluenceintheconformationofVP2(Cowley,etal.,1989;Mertens,etal.,1989).(Hassan,etal.,2001)suggestedaninvolvementofVP5proteininmembranepenetrationanddemonstratedarolefortheproteininvirus-cellpenetrationconsistentwithitsrevelationintheentryvesiclesubsequenttocellbindingandendocytosis.1.3.2StructuralorganizationofBTVcoreandassociatedproteinsTheVP3proteinisamajorcomponentoftheBTVcoreandformsasubcoretogetherwiththethreeminorstructuralproteins(VP1,VP4andVP6)andthe10double-strandedRNA(dsRNA)segmentsofthegenome.VP3formsascaffoldforthedepositionofVP7protein,thesurfaceproteinofthecore.Co-expressionofVP3andVP7proteinsininsectcellsusingbaculovirusexpressionsystemwasdemonstratedasareliabletechniquefortheassemblyofcore-likeparticles(CLPs)whichstructurallyresemblevirus-derivedcoresexceptfortheabsenceofthedsRNAgenomeandtheminorproteins(French,etal.,1990).VP3formsalayerthatservesasascaffoldforthedepositionoftheproteinVP7formingadoublelayeredcore(VP3+VP7).TheoutercapsidproteinsVP2andVP5isthenarrangedaroundcore(Roy,etal.,2009)(Figure1.5).1.3.3Non-structuralproteinsNSproteinshavebeenidentifiedinBTVinfectedcells.TheseNSproteinsshareahighdegreeofsequenceconservationamongtheBTVserotypes(Roy,etal.,1990a).NS1andNS2,encodedbyS5andS8,respectively,aresynthesizedinabundanceandareassociatedwithviraltubulesandVIBsrespectively.TheviraltubuleswhichareattachedtothefilamentcomponentofthecellcytoskeletonaremadeupofNS1only.Thepresenceofmanysuchtubulesinavirallyinfectedcellisthereforethemultimericformoftheprotein(Urakawa,etal.,1988)andthepresenceofthetubulesinthecytoplasmofBTV-infectedcellsfavorsviralproteinsynthesis(Monastyrskaya,etal.,1995;Owens,etal.,2004;Boyce,etal.,2012a).15 中国农业科学院博士学位论文ChapterIFigure1.5ElectronmicrographoftheconcentricproteinslayersoftheBTVparticle.PanelAshowstheinnermostsubcoreproteinVP3.ThislayerservesasascaffoldforthedepositionoftheproteinVP7formingadoublelayeredcoreshowninPanelB(VP3+VP7).ThespikyappearanceofthecorerepresentativeoftheVP7trimersandfinallyPanelCshowingtheroughenedappearanceofnegativelystainedBTVvirionwithoutercapsidproteinsVP2andVP5arrangedaroundcore(Roy,etal.,2009).Table1.1Summarizationofthesize,locationandfunctionoftheproteinsencodedbyBTVsegmentswithspecialreferenceforBTVserotype10,thetablemodifiedfrom(Roy,1992;Roy,2001;Patel,etal.,2014).Protein/sencodedSegment(sizeinLocation(sizeinkiloProteinfunctionbasepairs)AndnatureDaltons)S1(3944)VP1(150kDa)SubcoreRNAdependentRNApolymeraseS2(2926)VP2(111kDa)OutercapsidReceptorbindingandcellentry,serotypeStructuraldeterminant,S3(2772)VP3(103kDa)SubcoreSub-corestructuralprotein,localizesviralStructuralpolymerasecomplex,scaffoldforVP7,S4(2011)VP4(76kDa)SubcoreCappingenzymeS5(1769)NS1(64kDa)NonstructuralViralproteintranslation,tubules???enhancerS6(1638)VP5(59kDa)OutercapsidMembranepermeabilizationprotein,StructuralS7(1156)VP7(38kDa)CoresurfaceCorestructuralproteinandreceptorbindingStructuralproteinforCulicoidescellsS8(1124)NS2(42kDa)NonstructuralConcentratorofcorecomponents,viralinclusionbodyformationS9(1046)VP6(36kDa)SubcoreHelicaseNS4(17kDa)NonstructuralViralfitnesstointerferonresponseS10(822)NS3(26kDa)NonstructuralAdaptorproteinfacilitatingegressNS3A(25kDa)IncontrasttothehighlyexpressedNS1andNS2ininfectedcells,NS3andNS3AencodedbyS10aresynthesizedinrelativelysmallamounts(Mertens,etal.,1984).RecombinantvacciniavirusexpressingNS3andNS3Arevealedacloserelationshipbetweentheseproteins,intracellularvesiclesandtheplasma16 中国农业科学院博士学位论文ChapterImembraneandithasbeenlaterfoundtoplayacriticalroleinvirusintracellulartraffickingandegress(Beaton,etal.,2002;Celma,etal.,2009).NS3hasalsobeenshowntodownregulatetranscriptionfromthebetainterferon(IFN-)promoterinreporterassays(Chauveau,etal.,2013).Aputativefifthnonstructuralproteinmaybeexpressedfromaconservedsmallopenreadingframe(ORF)insegment10(Stewart,etal.,2015).Theinvolvementofthe41kDaNS2invirusassemblyisalsograduallybeingdelineated.NS2istheonlyBTVproteinthatundergoesphosphorylation(Huismans,etal.,1987b;Devaney,etal.,1988).Itisahighlychargedhydrophilicproteinandexistsasa7Smultimericcomplex(Thomas,etal.,1990;Uitenweerde,etal.,1995;Theron,etal.,1997;Lymperopoulos,etal.,2003;Butan,etal.,2004;Markotter,etal.,2004).BiochemicalevidenceshowedthatNS2couldbeassociatedwiththecoreproteins(VP1,VP3,VP4,VP6andVP7)(Kar,etal.,2005;Modrof,etal.,2005;Kar,etal.,2007)aswellasaspecificbindingactivitytowardsBTV-ssRNAtranscriptsandalsohasanucleotidylphosphataseactivity(Horscroft,etal.,2000;Taraporewala,etal.,2001;Lymperopoulos,etal.,2003;Lymperopoulos,etal.,2006).TheseobservationssuggestthatNS2mayplayacentralroleintheselection,recruitmentand/orretentionofBTVmessengerRNA(mRNA)segmentsforincorporationintotheprogenyvirionsandsubsequentlyforgenomereplicationinsidetheBTVcores.Furthermore,invitroexpressionofNS2resultsinthesynthesisofvirualinclusionbodies(VIB)-likestructuressimilartothatfoundinvirallyinfectedcells(Huismans,etal.,1979),andithasbeenfoundtobethemajorcomponentofviralinclusionbodies(Lymperopoulos,etal.,2006;Kar,etal.,2007;Jerath,etal.,2010)whicharebelievedtobevirusassemblysites.1.3.4ReplicationcycleofBTVBTVandrelatedorbivirusesconstitutetheonlynon-membranousmammalianvirusestobevectoredbyaninsectspecies,whicharethebitingmidgegenusCulicoides(Mellor,2000).VirusisinoculatedintothehostbloodstreambyCulicoidessalviaduringabloodmeal.Uponwhichitdisseminatesleadingtoprimaryinfectioninendothelialcellsandmononuclearphagocytes,namelydendriticcellspresentinthelymphnodedrainingthesiteofinoculation(Barratt-Boyes,etal.,1994)(Figure1.6.A).Theviruslifecycleisclassicalinthesensethatitfollowswellcharacterized,discretestepsofreplicationcomprehensivelydescribedinothervirusesi.e.entry,replication,assemblyandegress.AnoverviewofthisprocessisillustratedinFigure(1.6.B)Infectionofthecelloccursbycelladhesionviabindingtosurfaceglycoproteinsowingtothereportedhemagglutinationactivity(Eaton,etal.,1989a),onceBTVhasattachedtothesurfaceofasusceptiblecell,thevirusisinternalizedbyreceptor-mediatedendocytosis,formingclathrin-coatedvesiclescontainingvirusparticles(Eaton,etal.,1990).Boundvirusisinternalizedbythehostcellendocyticmachinery,throughwhichthevirustranslocatesintothecytoplasm.Followinginternalization,theclathrincoatsofendocytosedvesiclesarerapidlylost,resultinginformationoflargeendocyticvesicleswhereVP2isdegradedandthecoreissubsequentlybecomingtranscriptionallyactive.ThereleaseoftheBTVcorehasbeenshowntobedependentonacidicpHsincetheadditionof17 中国农业科学院博士学位论文ChapterIFigure1.6.ABTVreplicationinwildanddomesticruminants.BTVreplicatesinbothwildanddomesticruminants,includingsomespeciesofdeer,whichmakesmassvaccinationofdomesticlivestockanincompletemeasureforimmunizationofthesusceptiblehostpopulation.BTVreplicatesinboththemammalianhostandtheCulicoidesinsectvector.Transmissiontotheinsectvectoroccurswhenafemaleinsecttakesabloodmealfromaninfectedmammal.Thevirusistransmittedtonaiveruminantswhentheinsectwithinfectedsalivaryglandsfeedsagain(Roy,etal.,2009).Figure1.6.BASchematicdiagramforthereplicationcycleofBTVinsidetheinfectedcells.VirusentryoccursviasialicacidattachmentbyVP2followedbyclathrinmediatedendocytosis.ParticlesarethentraffickedtotheendosomeuponwhichacidicpHmediatesVP5membranepermeabilizationleadingtovirionuncoatingandegressofthecoreparticletothehostcellcytosol.Transcriptionandtranslationofviralproteinsoccursleadingtocellularmorphogenesisbynonstructuralproteins.NS1formstubulesandNS2assemblestheVIBwherecomponentsforcoreassemblyareconcentrated.AssembledcoreparticlesleavetheVIBandaretraffickedonexocytoticvesiclesbyNS3interactionwithcalpactin.DuringthisprocesstheoutercapsidproteinsVP5andVP2areacquiredforacompleteparticle.ParticlesegressthecellviabuddingmediatedbyTumorsusceptibilitygene101,Tsg101interactionwithNS3orviahostcelllysis(Patel,etal.,2014).18 中国农业科学院博士学位论文ChapterIcompoundsthatraisethelysosomalorendosomalpHpreventsendocytosedvirusparticlesfromenteringthecytoplasm(Eaton,etal.,1990).Thus,althoughtheoutercapsidproteinsareclearlyresponsibleforvirusentryandpenetration,theprecisemechanismofmembranetraverseandthestructuralchangesintheoutercapsidproteinsassociatedwithit,remainunclear.ThepositionofVP5inthecapsidwould,however,beconsistentwitharoleinthetranslocationofthecoreintothecytoplasmaftercellentry.Tenviralsingle-strandedRNAs(ssRNA)areproducedbytranscriptionofgenomicsegments(Fukusho,etal.,1989).ThesearethentranslatedbyhostcellribosomesyieldingthesevenviralstructuralproteinsVP1throughtoVP7andfournonstructuralproteins,NS1,NS2,NS3andNS4(Mertens,etal.,2001;Ratinier,etal.,2011a).NS1preferentiallypromotestranslationofBTVssRNAsenhancingviraltiter(Boyce,etal.,2012b).NS2formsVIBswhichrecruitviralssRNAsandviralproteincomponentsrequiredforgenomicpackaging,replicationandcoreassembly(Kar,etal.,2007).UponassemblycoresarereleasedfromVIBsandenterintotheexocytosispathway,facilitatedbyNS3,wherebytheoutercapsidproteinsVP5andVP2areacquiredandthematurevirionparticlesegressfromthehostcelltopropagateinfection(Beaton,etal.,2002;Wirblich,etal.,2006).1.4VaccinedevelopmentTheterm“vaccine”(fromtheLatinterm“vacca,”meaningcow)wasfirstcoinedbyEdwardJennerwhoinitiatedthefirstscientificattemptstocontrolaninfectiousdiseasein1796byinoculatingan8-year-oldboywithcowpox(vaccinia)thatwereabletoprotecttheboyagainstsubsequentchallengewithvirulentsmallpox.Thankstothesuccessfuldevelopmentofvaccines,manymajordiseases,suchasdiphtheria,poliomyelitisandmeasles,arenowadayskeptundercontrol,andinthecaseofsmallpox,thedreamoferadicationhasbeenfulfilled.However,thereisagrowingneedforimprovementsofexistinganimalandhumanvaccinesintermsofincreasedefficacyandimprovedsafety,besidesthedevelopmentofcompletelynewvaccines.Bettertechnologicalpossibilities,combinedwithincreasedknowledgeinrelatedfields,suchasimmunologyandmolecularbiology,allowfornewvaccinationstrategies.Besidestheclassicalwhole-cellvaccines,consistingofkilledorattenuatedpathogens,newvaccinesbasedonthesubunitprinciple,havebeendeveloped(Liljeqvist,etal.,1999).Astherearenobroad-spectrumantiviralpharmaceuticalsavailable,hygienicmeasurestolimitexposureandvaccinationaretheonlymeanstopreventorcontrolviralinfections.Viruses(especiallyRNAviruses)arehighlyvariable,andmanyviralinfectionsareduetoviruseswithmultipleserotypes(e.g.,bluetonguevirus,footandmouthdiseasevirus,andinfluenzaviruses).Asaconsequence,manyoftheexistingviralvaccinesareoftenunabletocopewiththeprevailingstrainsinthefield,andnewoneshavetobegeneratedfromfieldstrainswithnewoutbreaks.Numerousconventionalliveandinactivatedviralvaccineshavebeenproducedbyanimalhealthcompaniesandhavebeenusedformanydecadesinroutinevaccinationprotocolsforbothcompanionandproductionanimals.Increasingly,anumberofrationallydesignedsubunitvaccinesarereachingthemarket(Meeusen,etal.,2007).19 中国农业科学院博士学位论文ChapterIAddressingtherequirementsofanidealvaccine,thedesignofanidealvaccineshouldencompassthefollowingcharacteristics:1)displayhighimmunogenicityinallindividualswiththecapacitytoelicitlong-termprotection,2)haveminimaltonosideeffects,3)shouldbeeffectivewiththefirstorfewadministrations,4)shouldbedirectedagainstmultipleserotypes,5)shouldallowforthedifferentiationbetweeninfectedandvaccinatedanimals(DIVA),6)thevaccinemustbethermallystableandmaintainitspotencyunderdifferentenvironmentalconditionsthusnegatingtherequirementforexpensivestorageanddeliveryinstruments,7)mustbepracticalintermsofdeliverysystemandusagethereforecircumventingtheneedforspecializedtraininginhandlingandadministrationofthevaccine,8)thevaccineshouldbemanufacturedinacostefficientmanne(Dorner,etal.,1999;Savini,etal.,2008).Therefore,manyrecombinanttechniquesarenowdominatinginstriveforanidealvaccine,beingsafeandcheap,heat-stableandeasytoadminister,andcapableofinducingbroadimmuneresponsewithlife-longmemorybothinhumanandinanimals(Liljeqvist,etal.,1999).1.4.1BluetonguevaccinationstrategiesThemajorcontrolmeasuresincluderestrictionofanimalmovement,vectorcontrol,slaughterofinfectedanimalsandvaccination.Thefirsttwostrategiesaredifficulttoattain,andtheslaughterofinfectedanimalsisimpracticableduetosocioeconomicandethicalrestrictions.VaccinationagainstBTisabletominimizedirectlosses,reduceviruscirculationandenablesafemovementofanimalsfromenzooticareas(Savini,etal.,2008).BTvaccinesmaybeusedfordifferentpurposesorstrategies,dependingontheepidemiologicalsituationoftheaffectedareaandstrategydesired.ThemainpurposesofBTvaccinationstrategiesare1)topreventclinicaldisease,2)tolimittheregionalextensionofBTVinfectionthroughreductionofthespreadofthevirus,3)toallowregionalorcountryeradicationofthediseasebasedonthereductionofviruscirculationand,4)toauthorizethesafemovementofsusceptibleanimalsbetweenaffectedandfreezones.SincetheincursionofBTVintopreviouslynon-endemiczones,asinsomeMediterraneancountries,BTVvaccinesareusedasanaidtopreventfurtherextensionoftheinfectiontoborderzones.Thisprovideslocal/regionalreductionofviruscirculationandforsafemovementsofanimals,whichplayanimportantroleinthelivestockindustry.Dependingontheiravailability,multivalentvaccinesorinactivatedserotype-specificvaccinescanbeused.ClimaticandgeographicfactorsaswellasabundanceofsuitableBTVinsectvectorsareprobablyallimportantfortheoutcomeandpersistence(reemergence)ofBTVinfectioninanarea.Itisthereforecommonlyacceptedthatvaccinescanhelplimitthespreadofthedisease.Ideally,forthepurposeoferadication,asuccessfulvaccinationcampaignshouldcoverallsusceptibleruminantspecies,attainahighdegreeofherdimmunityandencompassextensiveareassurroundinganyactiveBToutbreak(McVey,etal.,2015).Avarietyofvaccinestrategiesarepotentiallyavailableforimmunizationofruminantlivestockagainstorbiviraldiseases.Oftheseoptions,onlymultivalentvaccines(MLVs)andinactivatedvaccines20 中国农业科学院博士学位论文ChapterIhavebeenproducedcommerciallyandusedwidelytopreventBT.Ofthesetwostrategies,inactivatedvaccinesappeartobethesaferoption,andoneortwodosesofinactivatedvaccineprovidesufficientprotectionagainstviremia.WithMLV(live-attenuated)BTVvaccines,viremiacouldbepreventedin>90%ofvaccinatedanimalswithonedoseofvaccine.Thedurationofprotectionispresumablyquitelongbecause>80%ofvaccinatedanimalshasstableneutralizingantibodytiters.However,twoinjectionsofthesevaccinesprovidestableneutralizingantibodytitersandprotectionfromviremia.Studiesofexperimentalefficacy(thatis,byvirulentviruschallengeofimmunity)havebeensupportedbyfieldobservationsthatimmunizedanimalswerenotinfectedbyBTVeventhoughexposedtovector-transmittedcirculatingvirus.InBTendemicregions,vaccineshavebeenusedtopreventclinicaldiseaseanddeathlossesinsheep.Intheseregions,theCulicoidesspp.vectorsmaybepresentaroundtheyearwithcontinuouscirculationofdifferentBTVserotypes.Thishasledtothedesignofmultivalentvaccinescontainingdifferentserotypes,asdoneinSouthAfrica,whereBTVinfectionisendemic.TheSouthAfricanmultivalentvaccinesweredevelopedonlytocontrolclinicalBTdiseaseinsheep,ascattleandotherruminants;althoughsusceptibletoBTVinfection,usuallydonotsufferclinicaldisease.Furthermore,fielduseofattenuatedvaccineshasbeenassociatedwithnearcompleteclinicalprotection,andviruscirculationwasreduced.Furtheranalysisofthesestudiesrevealsthatimmunizationofapproximately80%ofasusceptiblepopulationwillprovideeffectiveherdimmunity.TheinherentshortcomingsandpotentialdisadvantagesofMLVsarewelldocumented(McVey,etal.,2015).AlthoughariskmodelintegratingfactorsdeterminantforBTVpersistence(includingvaccinations)needstobedeveloped,itislikelythateradicationofBTVwithvaccinesmaybeachievableonlyunderconcurrentfavorablegeoclimaticconditions.Theseconditionslimitorpreventvectoractivity,andasaresult,preventBTVcirculation(e.g.coldwinterimpedingoverwintering,geographicalbarriers,lowvectordensities,lowprobabilityofBTVincursionfromneighboringzones),butthisreliesontheassumptionthatverticalBTVtransmissiondoesnotoccurininsects.SuccessfulcontrolalsorequiresrestrictionofanimalmovementsbetweenBT-affectedandBT-freezones.ItistobestressedthatvaccinationismorelikelytobeeffectiveincontrollingBToutbreakswhenonlyasingleBTVserotypeinvadesapreviouslyfreeregion,asoccurredwithBTV-8innorthernEurope.Incontrast,itisclearthatBTVhasremainedendemicincountrieswhereMLVBTVvaccinesare/wereused(e.g.,Israel,Italy,SouthAfrica,UnitedStates)andwheremorethanoneBTVserotypeispresent(Maclachlan,2011).1.4.2TypesofBTVvaccinesDifferentstrategieshavebeenfollowedoverthelastthreedecadestodevelopnovelrecombinantvaccinesforBTV,rangingfromsubunitvaccinestolivevirusvectorvaccines.Todate27serotypesof21 中国农业科学院博士学位论文ChapterIBTVhavebeenreportedandlowlevelsofcross-protectionaredescribed,complicatingvaccinationstrategies.ThespreadofBTVworldwidewas,however,controlledbyvaccination.Comparedtosomeotherarboviraldiseases,vaccinesforbluetonguearerelativelystraightforward:avirusantigenaffordingprotectiveimmunitywasdefined30yearsago(Inumaru,etal.,1987),andinactivatedandattenuatedlivevirusvaccineshavealsobeendeveloped.However,inactivated-virusvaccineswereusedsuccessfully,leadingtotherapideradicationofBTV-8fromEurope.Theseinactivatedvaccines,whichweremadeavailableforserotypes1,2,4and8ofBTV,arethoughttoworkprimarilythroughgenerationofaprotectiveserotype-specificneutralizing-antibodyresponsetargetingtheVP2antigen(vanderWalt,1980;Cowley,etal.,1987;Mertens,etal.,1987;Hassan,etal.,1999;Attoui,etal.,2009;Belhouchet,etal.,2010;Umeshappa,etal.,2010;Attoui,etal.,2011;MohdJaafar,etal.,2014a).ThemultipleoutbreaksofBTVinMediterraneanEuropeinthelasttwodecadesandtheincursionofBTV-8inNorthernEuropein2008havere-stimulatedtheinteresttodevelopimprovedvaccinationstrategiesagainstBTV.Inparticular,safer,cross-reactive,moreefficaciousvaccineswithdifferentialdiagnosticcapabilityhavebeenpursuedbymultipleBTVresearchgroupsandvaccinemanufacturers.Todate,awidevarietyofrecombinantBTVvaccineprototypeshavebeeninvestigated,includingsubunitvaccine,viralvectorvaccine,DNAvaccine,andReversegenetics-basedvaaccine(Calvo-Pinilla,etal.,2014).1.4.2.1ConventionalliveandinactivatedvaccinesAsthefirstvaccineforhumansmallpox,mostliveveterinaryviralvaccinesinducemildinfectionswithliveorganismsderivedfromnontargethostsorattenuatedthroughpassageindifferentcelllinesorchickenembryos(eggs).Attenuatedviralstrainsarealsoobtainedbyinducingrandommutationsandselectingforreducedvirulence.Astheliveorganismcanstillinfecttargetcells,thesevaccinescanreplicateandinducebothcellularandhumoralimmunityandgenerallydonotrequireanadjuvanttobeeffective.Liveproductsalsooffertheadvantageofeaseofadministration,potentiallyindrinkingwater,intranasally,intraocularly,etc.However,theycanposeariskofresidualvirulenceandreversiontopathogenicwildtypesaswellasprovideapotentialsourceofenvironmentalcontamination.Althoughmodernregulatoryprocessesrequiredatatoprovideassuranceontheseissues,problemsinthefieldcanarise.Thiswashighlightedduringaprogramtocontrolporcinerespiratoryandreproductivesyndrome(PRRS)inDenmark.ThisdiseasefirstemergedinNorthAmericainthelate1980sandspreadquicklyinEuropeintheearly1990s(Murtaugh,etal.,1995).ThetwomaintypesofPRRSvirus,EuropeanandNorthAmerican,areonly55to80%identicalatthenucleotidelevelandcausedistinguishableserologicalresponses.Followingvaccinationwiththelive,attenuatedNorthAmericanPRRSvaccineagainsttheEuropeanPRRSvirustypepresentinDenmarkin1996,thevaccinevirusrevertedandspreadwithinvaccinatedherdsaswellasfromvaccinatedtononvaccinatedherds,leavingbothvirustypesintheDanishpigpopulation(Mortensen,etal.,2002).22 中国农业科学院博士学位论文ChapterILive-attenuatedandinactivatedBTVvaccinesarecurrentlycommerciallyavailable,andhavebeenusedovertheyearswithdifferentdegreesofsuccess.Bothtypesofvaccinesareeffective,however,theuseofthesevaccinestocontrolthediseasehashadanumberofunexpectedconsequences,includingthattheincompleteattenuationofvirusvaccineshasbeenlinkedtoteratologicaleffectsinnewbornanimalswhenpregnantsheepandcattlewerevaccinatedandreversionofthevaccinestrainsorre-assortmentbetweenvaccineandfieldstrains,leadingtothegenerationofvirulentstrains(Osburn,1994;Batten,etal.,2008;Roy,etal.,2009;Gonzalez,etal.,2010).Moreover,thereareseveralconcernsoverincompleteattenuationorinactivation,aswellasadversesideeffectsduetovaccinationandthepotentialthreadoftransmissionbybitingmidges(Gethmann,etal.,2009).Theseconcernsareparticularlyrelevantforviruseswithahighdegreeofgeneticexchange,forexample,BTVasoneofthevirusesthathavesegmentedgenomes.InChina,chickenembryo-adaptedattenuatedandinactivatedvaccinesofBTVserotypes1and16weresuccessfullydevelopedandproduced.Theprotectionrateofattenuatedvaccinesandinactivatedvaccinesweregreaterthan90%and75%,respectively,andgaveimmunitythatlastedoneyearandmorethansixmonths,respectively.Atotalof220,000animalsfromYunnan,Sichuan,Hubei,ShanxiandJiangsuprovinceswerevaccinated,whichhascontrolledtheincidenceofclinicalBTdiseaseinChina.Theeffectivevaccinationschemeswereimplementedusingsingleorcombinedvaccinationindifferentepidemicareas(Feng,1996;Li,etal.,1996;Li.Z.,etal.,1996;Shun,etal.,1996;Wang,1996;Zhang,etal.,1996a;Zhang,etal.,1996b)Ontheotherhand,inactivatedvirusvaccinesareverysafeifproperlyproduced.Theirinherentpotentialdisadvantagesincludetheirhighcostsofproduction,asvaccinationrequireslargeamountsofantigen;andtheneedforboosterimmunizations,asinactivatedvaccinesgenerallyinducearelativelytransientimmunity.1.4.2.2RecombinantviralvectorvaccinesWhenconventionalBTVvaccineswereusedinthepast,serotype-specificcontrolofbluetonguewasachievedinresponsetospecificoutbreaks.However,inordertoovercomedisadvantagesofattenuatedandinactivatedvaccines,severaleffectiveexperimentalrecombinantvaccineshavebeendevelopedanddescribedoverthelasttwodecadesandtheyclearlyhavenumerousinherentpotentialbenefits,includingrapidonsetofimmunity,lackoftransmissibilityandevenapolyvalentstrategy.DespitethemajoradvancesintheunderstandingandpreventionagainstBTVintrialswithnewgenerationvaccines,acommercialrecombinantvaccineagainstthisvirusremainselusive.Usuallyanimalhealthpolicyrequirementsforimprovedvaccinesdelaysorprecludestheirimplementationinthefield.Importantadvantagesofnewgenerationvaccinescomparingtoconventionalvaccinesaretheinherentsafetyandthepossibilitytodistinguishserologicallyinfectedfromvaccinatedanimals,whichallowthecontrolofthediseaseandsurveillance.Rightsurveillanceofadiseaseishighlyimportanttoauthorizethesafemovement23 中国农业科学院博士学位论文ChapterIofsusceptibleanimalsbetweenaffectedandfreezones.Moreover,experimentalvaccinesareusefultoolstobetterunderstandtheimmunemechanismsactivatedintheanimaltocounteractthevirus.Forbothreasons,developmentofrecombinantvaccinesagainstBTVisstillessentialnowadays.ArecombinantvacciniavirusthatexpressedbothVP2andVP5ofAustralianBTV-1inducedvariabletitersofneutralizingantibodyinsheepandaffordedprotectionagainsthomologouschallenge(Lobato,etal.,1997),butthisapproachhasnotbeenpursuedfurther.ArecombinantcapripoxvirusexpressingVP7wasshowntoprovidepartialprotectionagainstheterologousBTVchallenge(Wade-Evans,etal.,1996),butliketherecombinantvacciniaBTVvaccine,itsdevelopmentwasnotcontinued.Recently,arecombinantcanarypoxvirus-VP2/VP5vaccinewasdescribedasinducingahighlyeffectiveprotectiveimmunityinsheep(Boone,etal.,2007).ThisvaccinehasamajorinherentadvantageinthattheexistingVP7competitiveELISAassaywoulddistinguishvaccinatedfromnaturallyinfectedanimals(DIVA),anditutilizesanexpressionvectorthatisincorporatedinseveralvaccinesalreadyinuseinseveralcountries,thoughisstillatadevelopmentstage.(Calvo-Pinilla,etal.,2009)showedthatarecombinantmodifiedvacciniavirusAnkara(rMVA)expressingVP2,VP5andVP7proteins,usedasheterologousprimeboostvaccine,wereabletoprotectIFNAR(-/-)miceagainstBTV-4infection.ArecombinantmodifiedvacciniavirusAnkara(rMVA)expressingVP2,VP7andNS1proteinsofBTV-4wasalsousedtoimmunizeIFNAR(-/-)miceandinducedcross-protectionagainstlethaldosesofheterologousBTV-8andBTV-1(Calvo-Pinilla,Navasaetal.2012).1.4.2.3RecombinantsubunitvaccinesProtein-basedvaccinesagainstBTVhavebeendevelopedusingsingleproteinsorbycombiningvariousproteinsinthesamevaccinepreparation.HuismansetalutilizedachemicalmeansusingdivalentsaltstoisolateVP2fromapurifiedBTVandfoundthatvaccinationofsheepwiththisproteinwasabletoinducevirusneutralizingantibodiesandprotectagainstavirulenthomologouschallenge(Huismans,etal.,1987a).Thisprocedurewereefficientforprotection,however,suchastrategycouldnotbedevelopedfurtherduetothelargeamountsofvirusrequiredtoproduceenoughpurifiedproteinforuseasacommercialsubunitvaccine.Followingthesefindings,Royandco-workersdevelopedasubunitvaccinationstrategybasedontheexpressionofBTVproteinsusingrecombinantbaculovirussystems(Roy,etal.,1990b).BTVproteinsderivedindividuallyfrombaculovirusvectorswereadministeredindifferentcombinationsanddosestosheepandprotectionwasevaluatedagainsthomologouschallengewithvirulentBTV-10.Aminimaldoseof100µgofVP2wasneededtoprotectsheepagainstchallenge.However,whenusedincombinationwith20µgofVP5,alowerdoseofVP2(50µg)wassufficienttoelicitequivalentvirusneutralizingantibodytitersandtoprotectimmunizedsheepagainstBTVinfection.VLPsrepresentaspecificclassofsubunitvaccinethatmimicthestructureofauthenticvirusparticles.Theyarerecognizedreadilybytheimmunesystemandpresentviralantigensinamoreauthenticconformationthanothersubunitvaccines.IntactVLPsalsoprovedtheirusefulnessasvaccinesasfarasthe24 中国农业科学院博士学位论文ChapterIepitopesarepresentedinauthenticconformations.TheassemblyofVLPsafterthesynthesisoftheircomponentproteinsisanareawhichpresentsanumberofopportunitiesforfuturestudies.Until1990,onlysingle-shelledVLPsorcoreshavebeenassembledaftertheexpressionofsingleordoubleviralgenes(French,etal.,1990).ThehepatitisBvirus(HBV)vaccinecurrentlygivenroutinelytoinfantsintheUSAisaparticulatevaccinepreparedbyexpressionoftheSgeneofHBVinyeast(McAleer,etal.,1984).Thisvaccinecomprisesstructuresequivalenttothe22nmsub-virionparticlesfoundintheplasmaofindividualsinfectedwithHBV,andisarguablythemosteffectiveVLPpreparationtodatebecauseitisthefirsttobesuccessfullycommercialized.Moreover,theexpressionofthecapsidproteinofHBVinE.coliresultsintheformationofstructuresequivalenttotheHBVcoreparticle.Althoughthisparticledoesnotmimicthematureformofthevirusbecauseitdoesnothaveanenvelope,itdoesformaparticlethatisequivalenttotheHBVnucleocapsid.ThisparticlehasbeendevelopedmoreasavehiclefordisplayepitopesinseveralotherdiseasesratherthanasavaccineforHBV(Stahl,etal.,1989;Kratz,etal.,1999).TheotherexpressionsystemthathasbeenusedeffectivelyforVLPproductionisthemammalianvaccinia-virus-basedexpressionsystem.However,althoughthissystemisthemethodofchoiceforthepreparationofsomeVLPs,itsuseintheproductionofVLPsforvaccinationisnotwidespread.VLPswerefirstsuccessfullydevelopedforBTV-10andsincethenthisapproachhasbeenusedtoproduceVLPsfromavarietyofBTVserotypes(Roy,etal.,1990b).VLPsfromBTV-10orBTV-17orfromfiveserotypesincombination(BTV-1,BTV-2,BTV-10,BTV-13andBTV-17)weretestedinvaccinationstudiesandraisedspecificneutralizingantibodiesandaprotectiveresponseinsheepagainsthomologouschallenge(Roy,etal.,1994).Neutralizingantibodytitersinanimalsthatreceiveddosesof50µgofVLPswerenotsignificantlyhigherthanthosevaccinatedwith10µg.ThisworkalsodemonstratedthatvaccinationwithVLPsfromBTV-10andBTV-17inducedneutralizingantibodiesagainsttherelated(basedontheaminoacidsequenceofVP2)BTV-4,andpartialprotectionwasaffordedagainstthisheterologousserotype.Lateron,anotherstudydescribedthatVLPsfromBTV-8wereabletoaffordcompleteprotectionagainstahomologousviralchallengeinsheep(Stewart,etal.,2013).ThisapproachwasalsoeffectiveincombinationwithVLPsfromBTV-1orBTV-2.AdditionalstudiesdescribedhomologousprotectioninsheepusingVLPsfromBTV-1andinterestinglyinthiscasetheprotectionwasaffordedagainstavirulentvirusfromadifferentgeographicorigin(Stewart,etal.,2012).BacterialandyeastexpressionsystemstoproducerecombinantBTVproteinshavealsobeendevelopedbyseveralauthors(Gould,etal.,1994;Martyn,etal.,1994;Pathak,etal.,2008).Jabbarandco-workersexpressedVP2,VP5andVP7proteinsofBTV-8bybacterialexpressionsystemandweretestedassubunitvaccineinIFNAR(-/-)mice(Jabbar,etal.,2013).TheyutilizedVP2protein,eitherasacompleteproteinorasthreeseparateoverlappingfragments,andexpressedthefull-lengthVP5andVP7tousetheminavaccinationexperiment.WhenIFNAR(-/-)micevaccinatedwithfragmentedVP2togetherwithVP5andVP7,noneutralizingantibodieswereinducedandnoprotectionwasachievedafterinfectionwith100pfuofBTV-8.HoweverwhenmicewerevaccinatedwithcompleteVP2togetherwithVP5andVP7,25 中国农业科学院博士学位论文ChapterIpartialprotectionwasobservedagainstBTV-8challengesinceclinicalscore,mortalityrates(50%)andviremiawerelowerthanintheunvaccinatedcontrolmice.VaccinatedanimalswithcompleteBTVproteinsshowedneutralizingantibodyresponse,albeitatlowlevels.Itwassuggestedthattheinsolubilityoftheproteinsusedintheexperimentmayhavehadaneffectonthelackofcompleteefficacyofthevaccination.1.4.2.4DNAvaccinesAsmentionedbefore,mostofthecurrentviralvaccinesarepreparedusingattenuatedorinactivatedvirus.ControlofBTisparticularlydifficultduetothemultipleserotypesofthevirus.Inaddition,theviralgenomeismadeupof10segmentsallowingexchangingthegenesrandomlybetweendifferentviruses.Thismaycausegenerationofinfectiousviruswithmixedgenes.RecombinantDNAtechnologyhasprovidednovelapproachesfordevelopingintrinsicallysafevaccines,althoughthesevaccinesarenotyetcommerciallyavailable.ImmunizationofanimalswithnakedDNAencodingprotectiveviralantigenswouldinmanywaysbeanidealprocedureforviralvaccines,asitnotonlyovercomesthesafetyconcernsoflivevaccinesandvectorimmunitybutalsopromotestheinductionofcytotoxicTcellsafterintracellularexpressionoftheantigens.Thistechnologyofferssubstantialadvantagesbothintermsofsafetyandthepotentialofdevelopingamarkervaccine.Thelattercouldbeusedasaprophylaxisinareasatrisk,withoutendangeringthe“free”statusoftheregion.Anaccompanyingserologicaltestwouldallowthedifferentiationbetweenvaccinatedandinfectedanimals.DNArecombinanttechnologyinvolvesthesynthesisofimmunogenicproteinsandparticlesthatelicithighlyprotectiveimmuneresponses.Furthermore,DNAvaccinesareverystableanddonotrequireacoldchain.WhileDNAvaccinationoflargeanimalshasnotbeenaseffectiveasinitiallydemonstratedinmice,severalgroupshaveobtainedsignificantimprovementsinimmuneresponsesusinginnovativetechnologiessuchasspecifictargetingofthevaccineantigentoantigen-presentingcells(Kennedy,etal.,2006),priming-boostingwithstimulatingCpGoligodeoxynucleotides(Kennedy,etal.,2006;Liang,etal.,2006;Calvo-Pinilla,etal.,2012;Li,etal.,2015),andinvivoelectroporationofDNA(Scheerlinck,etal.,2004).DNAvaccineshaveadvantagesassafety,easymanufacturing,biologicalstabilityandcosteffectiveness.InthecaseofBTV,thisvaccinationapproachprovidedpartialprotectionwhenplasmidsexpressingVP2,VP7andNS1proteinsfromBTV-4wereusedincombinationtovaccinateIFNAR(-/-)mice(Marín-López,etal.,2014).Micewerevaccinatedtwicewith50µgofeachplasmidtwoweeksapartandchallengedwithBTV-4.Althoughtherewasnoclinicalprotection,viremiawasdelayedinDNA-BTVimmunizedanimalsincomparisonwithnon-immunizedanimals.AlthoughDNAvaccinesoftenhavelowimmunogenicity,theycanbeusefultoprimetheimmunesystemwhenusedinheterologousvaccinationregimesincombinationwithrecombinantvirusesasboostingagents.Thisheterologousvaccinationstrategywassuccessfullyusedinvariousstudies(Calvo-Pinilla,etal.,2009;Calvo-Pinilla,etal.,2012;Jabbar,etal.,2013).Recently,Junpingandco-workerhaveevaluatedDNAvaccinesandrecombinantfowlpoxvirus(rFPV)vaccinesexpressingVP2aloneorVP2incombinationwithVP5orco-expressing26 中国农业科学院博士学位论文ChapterItheVP2andVP5proteinsofBTV-1inbothmiceandsheepandindicatedthatastrategycombiningaDNAvaccineprime(co-expressingVP2andVP5)followedbyanrFPVvaccineboost(co-expressingVP2andVP5)inducedahightiterofneutralizingantibodiesinsheep(Li,etal.,2015)1.4.2.5Reversegenetics-basedvaccinesTheavailabilityofcompleteDNAsequencesandabetterunderstandingofgenefunctionhaveallowedspecificmodificationsordeletionstobeintroducedintotheviralgenome,withtheaimofproducingwell-definedandstablyattenuatedliveorinactivatedviralvaccines.TraditionallivevaccinesforBTVrelyontheattenuationofvirusbypassageineggs.InfectiousBTVisproducedentirelyfromDNAclonesbygeneratingonetranscriptinvitroforeachgenomesegment,andusingthesetranscriptstotransfectpermissivecells(Harper,etal.,2006).ThissystemallowstheintroductionofanymutationintothegenomeofBTV,aslongastheresultingvirusisviable.TheabilitytotestthevirulenceofBTVmutantsintheruminanthostwillallowtheidentificationofthepathogenicitydeterminantsofBTV,andtheseresultscanbeusedtoinformthedesignofvaccinestrainswithmultipleattenuatingmutations.ReversegeneticsdataandtheformationofBTVVLPshaveconfirmedthatoutercapsidproteinsfromphylogeneticallydiverseserotypescanassembleontheconservedcoreproteinstocreateviableBTVstrains(Loudon,etal.,1991;Boyce,etal.,2008).Thisobservationsuggeststhatitwillbepossibletouseadefinedattenuatedgeneticbackgroundandintroducetheantigenicallyimportantoutercapsidproteinsfromtheserotypesofinterest.Reversegeneticsalsoprovidesabasisforthedevelopmentofdisabledinfectioussinglecycle(DISC)vaccinesforBTV(Figure1.7),whichallowsthevirustoinfectthevaccinatedanimal,butstopsitfromcompletingareplicationcycle.Theresultingabortedinfectionallowstheexpressionofviralproteinsatnaturalsitesofinfectionwithouttheproductionofinfectiousvirusordiseaseintheanimal,andcanbeconsideredtobeanextremeformofattenuation.27 中国农业科学院博士学位论文ChapterIFigure1.7ProposedDISCvaccinesforbluetonguevirus.Usingthenewreversegeneticssystemforbluetonguevirus(BTV)itmightbepossibletomakedisabledinfectioussinglecycle(DISC)vaccinesforthevirus.Inthesevaccines,multipleessentialviralgeneswouldbeinactivatedinthevirusandsuppliedduringvaccineproductionusingacomplementingcellline.Inunmodifiedcells,andinthevaccinatedanimal,theviruswouldbeunabletoreplicatebecausethecomplementingproteinswouldbemissing.Toensurethatprogenyvirionsproducedthroughrecombinationwithfieldstrainswerenon-viable,theDISCstraincouldbefurtherattenuatedusingcodonbiasmutationsinalltensegments.(Roy,etal.,2009)ADISCvaccinesstrainwouldexhibitmanyofthesafetyfeaturesofinactivatedvaccines,whilepreservingtheexpressionofviralproteinsatthenaturalsitesofinfection,asobservedwithlivevaccines.DISCvaccinesandcolonbiasvaccinesforBTVrepresentanexcitingfuturepossibility,astheyshouldallowincreasedsafetywithevenbetterimmunogenicity(Roner,etal.,2001;Coleman,etal.,2008).1.5AimsofthecurrentstudySubunitvaccinesprovideanopportunitytodevelopsafeandrationalvaccines,withthepossibilityofdifferentiatingbetweenvaccinatedandinfectedanimals.Inthepresentstudy,weaimedtodeveloptheprotein-basedsubunitvaccinecandidatescomposedofthestableandpurifiedrecombinantBTV-16VP2,VP3,VP7,NS2,andVP5-41aaproteinsemulsifiedwithMONTANIDETMISA201VGadjuvant.Twokindsofexpressionsysytems,baculovirusandE.coli,wereusedtoexpressBTVproteins.TostudytheabilityoftheproposedvaccinetobeadaptedforseveralBTVserotypesdependingonlocalepidemiologyandthediseaseoccurrence,theimmunogenicityofBTV-1VP2orBTV-8VP2serorype-specificproteinscombined28 中国农业科学院博士学位论文ChapterIwithBTV-16VP2,VP5,VP3,VP7,andNS2proteinswillalsobeinvestigated.Therecombinantsubunitvaccinessupposedtoinducebothhumoralandcell-mediatedimmuneresponses,whichwillofferapromisingalternativetothecurrentlyattenuatedandinactivatedvirusvaccines.29 中国农业科学院博士学位论文ChapterIICHAPTERIIExpressionoffull-lengthBTVproteinsinbaculovirusexpressionsystem2.1IntroductionThebaculovirusexpressionsystemisavaluabletoolforhighlevelsynthesisofforeignproteins(Ailor,etal.,1999).Baculovirusexpressionvectorsystem(BEVS)isaneukaryoticexpressionsystemthatusesagroupofDNAviruses,thebaculoviruses,toinfectcertaininsectsandinsectcelllines.BEVShasthemarkedadvantageovertheprokaryoticexpressionsystemduetoitisablilitytomimic,tosomeextend,thepost-translationalprocessingfoundinmammaliancells(Jarvis,etal.,1996;Jarvis,etal.,2001;Kost,etal.,2005).ThemajorityofsubunitvaccinesproducedforBTVhasbeensynthesizedusingthebaculovirusexpressionsystem.Thisinsect-cell-basedproteinproductionsystemhasmanyadvantagesforvaccineproduction.Largeamountsofrecombinantproteinscanbeproducedinhigh-densitycellcultureconditionsineukaryoticcells,resultinginhighrecoveryofcorrectlyfoldedantigen.Astheinsectcellsusedforvaccineproductioncanbeculturedwithoutmammalian-cellderivedsupplements,theriskofculturingopportunisticpathogensisminimized.ThebaculovirususedforrecombinantproteinexpressionhasanarrowhostrangethatincludesonlyafewspeciesofLepidoptera,andthereforerepresentsnothreattovaccinatedindividuals.Moreover,baculovirusiseasilyinactivatedbysimplechemicaltreatment(Rueda,etal.,2000),andislocalizedmainlyinthenucleusandculturemediaofinsectcellpreparations,whereasmostVLPsarepurifiedfromcytoplasmicextracts.Finally,thebaculovirussystemcanbescaledupforlarge-scalevaccineproduction(Maranga,etal.,2002).TheproductivityandflexibilityofbaculovirusexpressionvectorsandtheabilityofthebaculovirusgenometoincorporateandexpresslargeamountsofforeignDNAhavepermittedthissystemtobeusedforbothsingleanddualgeneexpression(Emery,etal.,1987;Vlak,etal.,1988;Weyer,etal.,1991)(Smith,etal.,1983).Bluetongueisoneofthemostimportanteconomicallysignificantvector-borne,noncontagious,viraldiseaseofdomesticandwildruminantsinmanypartsoftheworld.ThediseaseiscausedbyBTV,asegmented,double-stranded(ds)RNAvirus,whichisamemberoftheorbivirusgenuswithintheReoviridaefamily.BTVisanon-envelopedvirusconsistingoftwocapsids(outerandinner)thatenclosethreetranscriptasecomplexproteins(VP1,VP4,andVP6)and10dsRNAsegments(S1-S10)ofthegenome.Theinnercapsid(termedas“core”)hastwoconcentriclayers:thefirstlayerisformedbyVP3,oneofthefourmajorviralproteins,andthesecondbyVP7,anothermajorprotein.BothproteinsarehighlyconservedacrossallBTVserotypes.Thesurfacelayerofthecore,whichisformedbyVP7,actsasascaffoldforthedepositionofothertwomajorcapsidproteins,VP2andVP5thatformtheoutercapsid.VP2isthemostexposedproteinofthe30 中国农业科学院博士学位论文ChapterIIvirus;itisthemostvariableproteinamongthesevenproteinsandisthedeterminantoftheserotypespecificityofBTV.WhileVP2isresponsibleforattachmentofthevirustothecellsurface,VP5isinvolvedinmembranepenetrationandtogethertheyareresponsibleforvirusentryintothehostcells.Previousstudiesexploringrecombinant-expressedBTVstructuralproteinsassubunit-vaccinecandidateshaveevaluatedcrudelysatesofrecombinant-baculovirus-infectedinsectcellsexpressingBTVVP2andVP5(Roy,etal.,1990b;Stewart,etal.,2012;Anderson,etal.,2013a).ImmunisationofsheepwiththeseproteinsprotectedtheanimalsandraisedsignificantNAbtitres.Inthischapter,wediscussindetailsthemethodofconstructionofbaculovirusexpressionvectorsforthefourmajorproteinsofBTV-16(VP2,VP5,VP3,andVP7).TheseproteinsarethemaincomponentsoftheVLPandconsideredthemostimmunogenicproteinsamongallotherstructuralandnon-structuralproteinsofBTV.ThemainobjectiveofthecurrentchapteristoexpressBTVproteinseitherasasubunitvaccineorasanassembledVLPvaccine.2.2Materialsandmethods2.2.1VirusgenesandcellsBTV-16(Isolate7766,SouthAfrica)S2,S3,S6,andS7andBTV-8S2(StrainNET2006/04)geneswereartificiallysynthesizedbyGenScriptcompany,whileBTV-1(GS/11,China)S2andS6geneswereclonedfromalivevirusstrain.EachgenewasutilizedtoconstructtheindividualrecombinantbaculovirusDNA.Spodopterafrugiperdacells(Sf9)(Invitrogen,USA)wereculturedat28°CinSf-900™IISFM(Gibco)supplementedwith1:4Grace'sInsectMedium,Supplemented(Gibco).Themediumweresupplementedwith2%fetalbovineserum(FBS)(Gibco).2.2.2PCR,electrophoresisandDNApurificationThefull-lengthBTV-16S2,S3,S6,andS7,BTV-1S2andS6BTV-8S2geneswereamplifiedfromtherecombinantplasmidtemplatesbyPCRusingappropriateprimerslistedintable2.1inaC1000TMThermalcycler.Theforwardandreverseprimerscontainedeitherstartorstopcodonsinthecorrectreadingframerespectivelyinordertoexpressthetargetproteinwithfusiontag.Therestrictionenzymesitesincludedintheprimerswasusedfortheligationofinsertgenewiththeexpressionvectors,(Fig2.1).ThePCRreactionwascarriedoutinatotalvolumeof50µlandcontained34.5µlDNase-freewater,10µl5xPrimerstarbuffer(includingMgCl2),1µl250mMdNTPsmixture,0.5µMoftheforwardandreverseprimers,50ngtemplateDNAofeachplasmidand1UPrimerstarHSDNAPolymerase;thereactionmixtureswerepreparedonice.ThefollowingPCRprogramwasused:3minat95°Cfortheinitialdenaturationwhichwasfollowedby35cyclesofdenaturationat95°Cfor30seconds,primer31 中国农业科学院博士学位论文ChapterIIannealingat55°Cfor30secondsandelongationat72°Cfor2minandafinalextensionstepperformedat72°Cfor10minutes.DNAwasseparatedona1%(w/v)agarosein1xTris-acetate-EDTA(TAE)(40mMTris-HCI,20mMNa-Acetate,1mMEDTA,pH8.5)containing0.01%Goldview(Solarbio,China).DNAsamplesweremixedwith1:6of6xGelLoadingDye(TaKaRa,China),loadedintothewellsofthegelandseparatedat100-120Vfor30-50minutes.ThesampleswerevisualizedunderUVlightandeachDNAfragmentwasidentifiedbymolecularweightbycomparisontoDNALadder(TaKaRa,China)whichwasrunalongsidethesamples.Table2.1PrimersusedtoamplifyBTV-16S2,BTV-16S3,BTV-16S6,BTV-16S7,BTV-8S2,BTV-1S2,andBTV-1S6genesbyPCR.PrimernamePrimer5’-3’sequenceorientationBTV-16S2-EcoRIForward5’-CCGGAATTCAAATGGAGGAGCTAGTTATAC-3’BTV-16S2-SphIReverse5’-ACATGCATGCTTAAATATTTAGAAGCTTCGT-3’BTV-16S3-SaLIForward5’-CGCGTCGACGATGGCTGCTCAGAACGAGC-3’BTV-16S3-XbaIReverse5’-TGCTCTAGACTACACAGTCGGCGCAGCCAG-3’BTV-16S6-BamHIForward5’-CGCGGATCCATGGGTAAAATCATCAAATCAC-3’BTV-16S6-HindIIIReverse5’-CCCAAGCTTTCACGCGTTTTTTAGGAAG-3’BTV-16S7-EcoRIForward5’-CCGGAATTCAAGACACTATCGCTGCAAG-3’BTV-16S7--HindIIIReverse5’-CCCAAGCTTACTATGCGTAAGCGGCGC-3’BTV-1S2-EcoRIForward5’-CGGAATTCAAATGGATGAACTAGGCATCCCAG3’BTV-1S2-HindIIIReverse5’-CCCAAGCTTTCATACGTTGAGAAGTTTTGTC-3’BTV-1S6-EcoRIForward5’-CCGGAATTCAAATGGGTAAAGTCATACGGTCC3’BTV-1S6-HindIIIReverse5’-CCCAAGCTTTCAAGCATTTCGTAAGAAGAG-3’BTV-8S2-BamHIForward5’-CGCGGATCCATGGAGGAGCTAGCGATTCCG-3’BTV-8S2-HindIIIReverse5’CCCAAGCTTCTATACATTGAGCAGCTTAG3’TheDNApurificationwascarriedoutusingTaKaRaMinibestagarosegelDNAExtractionkitVersion4.0asfollows:ThePCRproductswereseparatedon1%agarosegelelectrophoresisbeforebeingexcisedusingacleansharpscalpelunderminimalUVlightexposure.ThegelslicecontainingtheDNAwasthenmixedwithaDNABindingBuffer(0.5Mpotassiumacetate(pH4.2),4Mguanidinehydrochloride)andexposedtoatheroomtemperaturefor15minutesinordertomelttheagaroseandreleasetheDNA.ThesolutioncontainingtheDNAandmeltedagarosewasthenpassedthroughtheprovidedfiltertubesbydesktopcentrifugation(13,000rpm).TheboundDNAwasthenwashedtwiceintheprovidedwashingbuffercontaining70%ethanoltoremovecontaminatingsaltsandwasthereafterelutedintheprovidedelutionbuffer.32 中国农业科学院博士学位论文ChapterII2.2.3CloningintopFastBac™HTbvectorTheamplifiedPCRproductsandpFastBac™HTbvectorweresubjectedtorestrictionenzymedigestion,andfurtherligatedeachotherbeforebeingtransformedtoE.coliDH5αstrain(Transgen,China).The10µlligationvolumecontained:6µlPCRproducts,1µlpFastBac™HTbvector,1µlT4ligase,and1µlT4ligasebuffer.Thereactionwasincubatedat16°Cfor16h.Approximately100µlofchemicallycompetentcellsweregentlymixedwiththeligationreactionmixtureinapre-chilled1.5mltube.Thecellswereplacedoniceforapproximately30minutesandthereafterheat-shockedat42°Cfor90sec.Thecellswerethencooledonicefor2-4minbeforebeingsupplementedwith800µlSOCmediawithoutantibioticsandincubatedat37°Cfor1hwithshakingat220rpm.Appropriateamountsofthetransformationmixturewereplatedonagarplates(LBbroth+1.5%(w/v)Agar)supplementedwith100µg/mlampicillin.Figure2.1ThecircularmapofpFastBac™HTb(4856bp).RestrictionendonucleasesthatcleavepFastBac™HTbonceareshownontheoutercircle.2.2.4TranspositionofrecombinantpFastBac™HTbintothebacmidDNATransformationofrecombinantpFastBac™HTbintoDH10Bac™wasperformedaspertheinstructionsoftheBac-toBac®BaculovirusExpressionSystemmanual(Invitrogen,USA).Briefly,approximately100ngoftherecombinantpFastBac™HTbwasaddedto100µlchemicallycompetentDH10Bac™inapre-chilled15mleppendorfftubeandplacedonicefor30mintopromoteDNAbindingtothecellmembrane.Thecellswerethenheat-shockedwithoutshakingat42°Cfor60sectoincreasethepermeabilityofthecellsandpromotetheuptakeoftherecombinantvector.Thiswasfollowedby2-33 中国农业科学院博士学位论文ChapterII3minutesincubationonicetoallowthecellstorecover.Thereafter900µlofsterileLBbrothwasaddedtothecells,whichwerethenincubatedat37°Cwithshakingat220rpmfor4h.Theincubationwasnecessarytoallowfortranspositionofthetargetgeneintothebacmidandforcellgrowth.After4hours,a10-foldserialdilutionofthecellsweremadeusingsterileLBbrothand100µlofeachdilutionwasspreadonagarplatescontaining100µg/mlX-gal,40µg/mlIPTG,7µg/mlgentamicin,50µg/mlkanamycinand10µg/mltetracycline.Theplateswereincubatedfor48hoursat37°C.DH10Bac™containingtherecombinantbacmidwasselectedbasedonblue/whitecolonyscreening.Coloniesdisplayingawhitephenotypewereselectedandre-streakedonagarplatescontainingappropriateantibioticsinadditionto100µg/mlX-galand40µg/mlIPTGinordertoverifythephenotype,andPCRwasusedfortheconfirmationofthepositiveconstructs.TheconstructionoftherecombinantpFastBac™HTbwasverifiedbyPCRusingspecificprimerslistedintable2.1whilethetranspositionoftheDNAinsertintothebacmidwasconfirmedusingtheuniversalthepUC/M13forwardandreverseprimers(Table2.2)thathybridizetwositesflankingthemini-attTn7sitewithinthelacZα-complementationregiontofacilitatePCRanalysis(Figure2.2).Vector-specificprimerslistedintable2.2werealsousedforsequencingconfirmation.2.2.5IsolationofrecombinantplasmidandbacmidDNAIsolationofplasmidswascarriedoutusinge.Z.N.A.TMPlasmidMiniKitI(OMEGA)accordingtothemanufacturer’sinstructions.Singlebacterialcoloniescontainingtherecombinantplasmidwereinoculatedinto5mlLBbrothcontainingappropriateantibiotics.Theculturewasmaintainedat37°Cfor16hwithshakingat220rpm.Bacterialcellswerecollectedbycentrifugationat10,000rpmfor1min().Themediumwasdiscardedandthecellsresuspendedin250µlSuspensionbuffer(SolutionI)containing(50mMTris-HCl(pH8.0),10mMEDTA,2.5mgRNaseA)and250µlLysisbuffer(SolutionII)(0.2MNaOH,1%SDS)wereadd.Afterincubationfor5minatroomtemperature,theprecipitationofthebacterialproteinswasperformedupontheadditionof300µlbindingbuffer(SolutionIII)(0.5Mpotassiumacetate(pH4.2),4Mguanidinehydrochloride).Themixturewascentrifugedat14,000rpmfor10min.Thepelletwasdiscardedwhilethesupernatantwastransferredtoaprovidedfiltertubeandcentrifugedat14,000rpmfor2min.Theflow-throughwasdiscardedwhilethefilterwiththeboundDNAwaswashedtwicewith700µlwashbufferI(20mMTris-HCl(pH6.6),5Mguanidinehydrochloride,70%(v/v)ethanol),thencentrifugedat14,000rpmfor1minandfinallyelutedwithelutionbuffer.TheconcentrationoftheisolatedplasmidswasmeasuredwiththeNanodropND2000spectrophotometer.2.2.6TransfectionofSf9cellswithrecombinantbacmidsforproductionofrecombinantbaculovirusesTransfectionofSf9cellswasperformedfollowingthemethoddescribedinBac-to-Bac®BaculovirusExpressionSystemmanualwithslightmodifications.Eachwellof6-wellplatewasseededwith34 中国农业科学院博士学位论文ChapterIIapproximately8x105cellsinthecompleteculturemedium.Thecellswerethenincubatedatroomtemperaturefor1-2htoallowforthecellattachment.Simultaneously,abacmid-lipidcomplexwaspreparedasfellows：1)SolutionA,8μLCellfectin®IIwasdilutedin100μLGrace’sMedium,Unsupplemented(withoutantibioticsandserum),mixedbyvortexingandincubatedatroomtemperaturefor30min,2)SolutionB,1-2μgbacmidDNAwasmixedin100μLGrace’sMedium,Unsupplemented(withoutantibioticsandserum)andmixedgentlybeforebeingaddedtoSolutionA.Thetransfectionmixturewasmixedgentlyandincubatedfor15–25minatroomtemperature.Thevolumewasmadeupto1mlbyadditionof800μlofSf-900™IISFMmedium.Table2.2PrimersusedforverifyingthepresenceofthegeneofinterestintherecombinantbacmidPrimername5’-3’sequenceorientationpUC/M13Forward5′-CCCAGTCACGACGTTGTAAAACG-3′pUC/M13Reverse5′-AGCGGATAACAATTTCACACAGG-3′T7Forward5′-TAATACGACTCACTATAGGG-3′T7Reverse5′-GCTAGTTATTGCTCAGCGG-3′Figure2.2BacmidDNAamplificationusingpUC/M13forwardandreverseprimersSf9cellswerewashedtwicewith2mlSf-900™IISFMmediumandthereafteroverlaidwiththebacmid-lipidcomplex.Thecellswereincubatedat28°Cfor5h.Thetransfectionmixtureswereremovedandthecellswereoverlaidwith2mlofthecompleteculturemedium.Afterincubationfor72to96hat28°C,therecombinantbaculoviruspresentinthesupernatantwasharvestedbycentrifugationat5000rpmfor10min.Forrecombinantbaculovirusamplification,approximately200µlofthesupernatantcontainingtherecombinantbaculoviruswasaddedtoaconfluentSf9cellsoverlaidwith1800µlcompleteculture35 中国农业科学院博士学位论文ChapterIImediumina6-wellplateandincubatedforfurther72hat28°C.TheSf9cellswerecollectedbycentrifugationat5000rpmfor10minandanalyzedwithsodiumdodecylsulphatepolyacrylamidegelelectrophoresis(SDS-PAGE)andwesternblottoconfirmtherecombinantproteinexpression.Therecombinantbaculovirusinmediawasstoredat4°C,protectedfromdirectlight.2.2.7ExpressionandidentificationofrecombinantBTVproteinsEachbaculovirusstockwasamplifieduptopassagethreebeforebeingusedtoinfectSf9cellforexpression.TheSf9cellswereinfectedwithrecombinantbaculovirusataMOIof3andweremaintainedat28°Cfor72hpostinfectionoruntilplaqueformation.Toharvestthebaculovirusinfectedcells,thecultureflaskwereshakenvigorouslytoloosenthecells.Thecellswerethencollectedin50mlFalcontubesbycentrifugationat5000rpmfor5min.Themediumwasdiscardedandthecellpelletwasstoredat-20°Cuntilusedforproteinpurification.InanattempttoassembleofBTV-16VLPs,Sf9cellsweresimultaneouslyinfectedwiththefourrecombinantbaculovirusexpressingVP2,VP5,VP3,andVP7stocksatMOIof3andincubatedat28°Cfor3-4days.Thecellswerethenusedtoperformwesternblotfortheconfirmationofthefourproteinsexpression.Thesampleswerere-suspendedinphosphatebufferedsaline(PBS),mixed1:5in5xSDSloadingbuffer(250mMTrisHClpH6.8,10%SDS,30%Glycerol,5%β-mercapitalethanol,and0.02%bromophenolblue)andboiledat95°Cfor10min.The12.5%separationgeland5%stackinggelwereusedintheseexperiments.Electrophoresiswasperformedatroomtemperatureatmaximum150voltages.ThegelwasstainedwithCoomassie®BrilliantBlueG-250dye.Forwesternblot,SDS-PAGEgelsweretransferredtoPolyvinylidenefluoride(PVDF)membranesintransferbuffer(500mMGlycine,50mMTrisHCl,0.01%SDS,20%methanol)at23Vfor1h.MembraneswerewashedinPBST(8gofNaCl,0.2gofKCl,1.44gofNa2HPO4,0.24gofKH2PO4,5mloftween-20in1litterofddH2O,pHto7.2)andthenblockedwith5%fat-freemilkextractinPBSTfor1h.Membraneswereexposedtoanti-Hismouseprimaryantibody(ZSGB-BIO,China)in5%fat-freemilkextractinPBSTfor2hatroomtemperatureorovernightat4°C,washedthreetimeswithPBSTandincubatedwiththesecondaryhorseanti-mouseperoxidase-conjugatedIgG(ZSGB-BIO,China)in5%fat-freemilkextractinPBSTfor2hatroomtemperatureorovernightat4°C.TheproteinbandswerevisualizedusingBCIP/NBT(Sigma,USA).2.2.8PurificationandquantificationofrecombinantproteinsTopurifytherecombinantproteinsfromtheSf9cells,thecellpelletwasthawedoutatroomtemperature,anyresiduesofPBSormediumwasremovedbyspinningdownat8000rpmfor5minat4°Candthepelletwasre-suspendedin2mlproteinpurificationsolutionA(20mMTris-HCl(pH8.0),0.5MNaCl,5mMDithiothreitol(DTT)and1%TritonX-100,pH=8.5)containingproteaseinhibitorcocktailcompleteMiniEDTAfree(Roche,USA)andincubatedat4°Cfor5min.Thecellswerethenhomogenizedusingultrasonicationfor20minwith3secpulseand3secpausebeforebeingspineddowntwiceat800036 中国农业科学院博士学位论文ChapterIIrpmfor30minat4°C.ThesupernatantwastransferredtoanickelaffinityHistagpurificationresin(Roche,USA)pre-equilibratedwithsolutionA,incubatedfor1-2hatroomtemperaturewithagentleshakingtoallowresintobindwiththetargetprotein.TheresinswerelaterwashedwithsolutionAtowashanyunbindornonspecificproteinsbeforetheelutionofhistaggedproteinsinbufferB(500mMNaCl,20mMTris–HCl,500mMimidazole,2mMDTT,0.1%TritonX-100;pH=8.5).Acolorimetricproteinassay,basedontheBradfordmethod,wasusedforthemeasurementforallthepurifiedproteinsconcentrationinthecurrentstudy.200µlofBradfordreagentwasmixedwith20μlofthesampleinaflatbottommicroplates,incubatedfor30minatroomtemperatureandabsorbancewasdeterminedat595nm.Theproteinassaywasperformedwithbovineserumalbumindilutionsofknownconcentrationsandacalibrationcurvewasestablishedeachtime.Usingthestandardcurve,theconcentrationofeachsamplewasdeterminedaccordingtoitsabsorbancebyinterpolation(Bradford,1976).2.3Results2.3.1ConstructionofrecombinantpFastBac™HTbvectorsTherecombinantplasmidscontainingBTV16S2,BTV16S3,BTV16S6,BTV16S7,BTV1S2,BTV1S6andBTV8S2geneshavebeenutilizedastemplatesforPCRamplificationofthefull-lengthgenesusinggene-specificprimerslistedinTable2.1.ThePCRproductswereanalyzedbyagarosegelelectrophoresis,andthesizeofbandswasapproximately2.9kb,2.8k,1.6k,and1.2kbforVP2,VP3,VP5,andVP7,respectively.ThePCRamplifiedfull-lengthgenesweresuccessfullyclonedintoanentryvectorpFastBac™HTb(Invitrogen,USA).ThepurifiedPCRproductsandpFastBac™HTbvectorweresubjectedtorestrictionenzymesdigestionaccordingtoTable2.1.ThedigestedPCRproductsandvectorwereligatedusingT4ligaseandtransformedintoDH5αcompetentcells.ThetransformantswereselectedonLBagarplatescontainingampicillinafter16hofincubation.Inordertoconfirmthepositivecolonies,colonyPCRwith2-3µloftheculturedbacteriawascarriedoutusingthegenespecificprimers(Table2.1)andthePCRproductswereanalyzedon1%agarosegelelectrophoresis.Thepositivecoloniesshowedtheamplificationof2.9kb,2.8kb,1.6kb,1.2kb,2.9kb,1.6kb,and2.9kbbands,indicatingthepresenceofthepositiveclonesofpFastBac™HTbBTV16VP2,pFastBac™HTbBTV16VP3,pFastBac™HTbBTV16VP5,pFastBac™HTbBTV16VP7,pFastBac™HTbBTV1VP2,pFastBac™HTbBTV1VP5andpFastBac™HTbBTV8VP2,respectively(Figure2.3).TherecombinantplasmidswereisolatedfromthePCRpositivecoloniesusinge.Z.N.A.TMPlasmidMiniKitIandsequenced.TheresultsshowedthatallrecombinantpFastBac™HTbvectorscontainedthegeneofinterestwithcorrectexpressioncassette.(Genbankaccession:No.JX272460.1,No.JX272461.1,No.JX272464.1,No.JX272465.1,No.JX680448.1,No.JN848760.1,andNo.JN848764.1,forBTV-16S2,BTV-16S3,BTV-16S6,BTV-16S7,BTV-8S2,BTV-1S2,andBTV-1S6,respectively).37 中国农业科学院博士学位论文ChapterIIM1234M125000---10,000-3000---7,500--2000---3,500--~2.9kb1500---2000---1000---~1.6kb1000---ABM12M125000---3000---5000---3000---M122000---M12000---1500---1500---1000---1000---CDFigure2.3PCRidentificationoftherecombinantpFastBac™HTbconstructs.Molecularmassmarkers(inbasepairs)arenotedattheleftofeachimage.ArrowheadsindicatethePCRproductsattheexpectedsizes.(laneM):DL-5,000;(A)(Lane1):S2,(Lane2):S3,(Lane3):S6,and(Lane4):S7;(B)(Lane1):pFastBac™HTbBTV16S2,and(Lane2):pFastBac™HTbBTV-16S6;(C)(Lane1and2):pFastBac™HTbBTV16S3;(D)(Lane1and2):pFastBac™HTbBTV16S7.2.3.2GeneratingtherecombinantbacmidsOncetherecombinantpFastBac™HTbconstructswereconstructedandconfirmedbyPCRamplificationandsequencing,thepurifiedplasmidsDNAsweretransformedintotheDH10Bac™competentcellstocreatetherecombinantbacmidscontainingtheindividualgeneofinterest.Blue/whiteselectionmethodwasusedtoidentifycoloniescontainingtherecombinantbacmids.ThepUC/M13forwardandreverseprimerswereusedtoconfirmtherecombinantbacmids.TheresultsshowedthatthePCRproductsbandsofthepositivebacmidswereconsistentwiththeexpectedsize(Figure2.4).Allrecombinantbacmidswerefurtherconfirmedbysequencing.38 中国农业科学院博士学位论文ChapterIITable2.4TheexpectedsizeoftherecombinantbacmidsbyPCR.BacmidS2S3S6S7Theexpectedsize=2430+insert(bp)5,3565,2024,0683,586M5kb12M10kb12M5kb12M5kb12ABCD12345678M10k91011EFigure2.4PCRidentificationofrecombinantbacmidconstructs.Molecularmassmarkers(inbasepairs)arenotedattheleftofeachimage.ArrowheadsindicatethePCRproductsattheexpectedsizes.(laneM):DL(5Kb):5,000bpor(10Kb):10,000;(A)(lane1and2):BTV16S2;(B)(lane1and2):BTV16S3;(C)(lane1and2):BTV16S6;(D)(lane1and2):BTV16S7;(E)(lane1-3):BTV1S2;(lane4-8):BTV1S6;and(lane9-11)BTV8S2:E2.3.3TransfectionofSf9cellsandidentificationoftheexpressedproteinsAftertransfectionofSf9cellswiththeindividualbaculovirusDNAfor3-4days,thevirustiterforeachstockhasbeenestimated.Sf9cellsweretheninfectedatamultiplicityofinfectionMOIof3withrecombinantbaculovirusandharvestedat~72h.TheinfectedSf9cellswerecollectedbycentrifugationfor39 中国农业科学院博士学位论文ChapterIISDS-PAGEanalysis.TheinfectedSf9cellsexpressedspecificproteinbandscorrespondingtotherecombinantbaculoviruses(Figure2.5).AllcellproteinsweretransferredtoaPVDFmembrane(Immobilon,USA)usingaTrans-Blot®Turbotransfersystem(BioRad).Membraneswereincubatedinablockingsolutioncontainingfat-freemilkinPBST.Anti-Hismouseprimaryantibody(ZSGB-BIO,China)dilutedinPBST(1:2,000)wasusedtodetecttheHisfusionproteins.Asecondaryhorseanti-mouseperoxidase-conjugatedIgG(ZSGB-BIO,China)(1:5,000)wasincubatedwiththemembraneandlaterthepositivebandattheexpectedsizewerevisualizedusingBCIP/NBTsolution.Allproteinsweresuccessfullyexpressedwithmolecularweightof110,103,59,38,110,59,and110kDacorrespondingtotheexpressedproteinofBTV16VP2,BTV16VP3,BTV16VP5,BTV16VP7,BTV1VP2,BTV1VP5andBTV8VP2,respectively(Figure2.6).Moreover,theoptimumtimeforeveryexpressedproteininSf9wasdeterminedbyharvestingtheinfectedcellsatseveraltimepoints(48h,72h,96h,and120h)anddetectingwithwesternblot(e.g.,BTV16VP7,representedinFigure2.6.H).Thepositivevirusstockswerethenamplifieduptopassagethreeforlateruseintheprotenexpressionforpurification.M123M123M12170---170----170----130---130----130----100---100---100---70---70---70---55---55---55---40---40---40---35---35---35---ABCFigure2.5SDS-PAGEgelsforSf9cellsinfectedwithrecombinantbaculovirus.Molecularmassmarkers(inkDa)arenotedattheleftofeachimage.ArrowheadsindicaterecombinantBTVproteinsatexpectedmolecularweight.(laneM):prestainedproteinmarker;(A)(lane1-3):BTV16VP2;(B)(lane1-3):BTV16VP3;and(C)(lane1and2):BTV-16VP7.2.3.4ProteinexpressionandpurificationBeforeinfectingthecellsforproteinexpression,theoptimaltimeandvirustiterforeachvirusweretestedin6-wellplates(Figure2.6.H).Theoptimumtimefortheexpressionofallproteinswasfoundat~72h.MonolayersofSf9cellsin75cm2corningflaskswereinfectedusingvirusesatpassagethree(virustiterof~2x107pfu/mL)at0.1MOI.Thecellswerethenincubatedat28°Cfor3days,harvestedandstored40 中国农业科学院博士学位论文ChapterIIat-70°C.ProteinpurificationwasdonebybindingtheHis-taggedproteinstocomplete™His-TagPurificationResins(LifeScience-Roche)insolutionA(20mMTrisHClpH8.0,500mMNaCl,5mMDithiothreitol(DTT),and20mMImidazole)andlaterelutedinsolutionB(20mMTrisHClpH8.0,500mMNaCl,3mMDTT,and300mMImidazole).ThepurityoftheproteinswasinspectedinastainedSDS-PAGEandtheproteinconcentrationwasmeasuredusingBradfordmethod.M123M123M12M123170---130---100---70---55---40---35---ABCD170-----M12M1M1M12345130------100-----70---55---40---35---EFGHFigure2.6WesternblotforSf9cellsinfectedwithrecombinantbaculoviruses.Molecularmassmarkers(inkDa)arenotedattheleft.ArrowheadsindicaterecombinantBTVproteinsatexpectedmolecularweight.(laneM):prestainedproteinmarker;(A)(lane1-3):BTV16VP2;(B)(lane1-3):BTV16VP3;(C)(lane1and2):BTV16VP5;(D)(lane1-2):BTV16VP7;(E)(lane1and2):BTV1VP2;(F)(lane1):BTV8VP2;(G)(lane1and2):BTV1VP5;and(H)(lane1-5):TimeoptimizationofBTV16VP7expressioninSf9infectedcells,controlpositive,48h,72h,96h,and120h,respectively.41 中国农业科学院博士学位论文ChapterIIWithanattempttoproduceVLPs,Sf9cellswereco-infectedwiththerecombinantbaculovirusesexpressing,BTV-16VP2,VP3,VP5,andVP7proteins.Theresultsshowedthatafterharvestingtheco-infectedcells,thefourstructuralproteinbandswithexpectedsizeweredetectedbywesternblot(Figure2.7).M12VP2~110kDaVP3~103kDaVP5~59kDaVP7~38kDaFigure2.7WesternblotforSf9cellsco-infectedwiththerecombinantbaculoviruses.(LaneM):prestainedproteinmarker;(lane1and2):Sf9cellsinfectedwiththerecombinantbaculovirusesexpressingBTV-16VP2,VP3,VP5andVP7proteins.ArrowheadsindicaterecombinantBTVproteinsatexpectedsize.2.4DiscussionExpressionofproteinsbyheterologousexpressionsystems,suchasthebaculovirussystem,canprovidesufficientproteinforexaminationoftheimmunogenicityaffordedbyvaccinationofanimalswithindividualoracombinationofviralproteins.Inthecurrentstudy,wehaveusedthisapproachtoexpresstheBTVproteinswith6×histagintheN-terminal.ThestructuralproteinswereselectedtotargetBTV-16becauseofpreviousoutbreaksinChinathatcausedclinicalsignsinsheepandgoats(Zhang,etal.,1999;Zhang,etal.,2004a).Therefore,BTV-16proteinswereinitiallyselectedtobeusedforthedevelopmentofBTVvaccine(Huismans,etal.,1981a).ThemainpurposeofthestudyistoproduceaneffectivevaccineagainstBTV-16,meanwhile,theabilityofthethisvaccinecandidateforlaterbeadaptedtootherormultipleBTVserotypesbyexchangingorincludingVP2originatingBTV-1andBTV-8serotypeshasalsobeenstudied.Withthisaim,wehavealsoexpressedBTV-1VP2andBTV-8VP2proteins.ThemainreasonbehindtheselectionofofBTV-1wasitspreviousisolationinChina,andBTV-8hasgainedaninternationalimportanceowingtoitsrecentemergenceinEurope.Manyvaccinesaimtoelicithumoralimmuneresponsesagainstspecificepitopespresentonthesurfaceproteinsofpathogensthatcansubsequentlyprotectthehostuponfutureexposuretothepathogen.ThemostexposedBTVprotein,VP2,isknowntobethehighlyvariableproteinamongBTVserotypesandisthedeterminantofthevirusserotypes(Hassan,etal.,1999).WhenthepurifiedVP2wasaddedtoBTV42 中国农业科学院博士学位论文ChapterIIsusceptiblecellsitreadilyattachedtothecellsurfaceviaasialoglycoproteinandwasrapidlyinternalizedbyendocytosis.Moreover,antibodiesraisedagainstVP2neutralisevirusinfectivity,supportingthefactthatVP2isthecellularreceptorbindingproteinofthevirus(Huismans,etal.,1981a).MembranedestabilizationactivityofVP5wassuggestedbythecytotoxicityoftheproteinwhenitwassynthesizedintheabsenceofanyotherviralprotein.DirectevidencewasalsoobtaineddemonstratingthatVP5hasintrinsicmembrane‘pore-formation’activity.Further,thecytotoxicityofVP5wasmappedtotheN-terminalsequencesthatpotentiallyformtheamphipathichelices(Hassan,etal.,2001;Forzan,etal.,2004).SomefindingsrevealedthatVP2alone,whileabletoinduceaneutralizingantibodyresponse,cannotefficientlyprotectagainstBTVinfection.Thedegreeofprotection,however,wasmuchhigherwhenVP2wasusedtogetherwiththeminoroutercapsidproteinVP5,nomatterimmunizedwithrecombinantproteinsorrecombinantviruses(Roy,etal.,1990b;Lobato,etal.,1997;Ma,etal.,2012).Inordertoproducetheindividualrecombinantbaculovirusforeachstructuralprotein,therecombinantplasmidshasbeenconstructedforeachandlatertransposedtothebacmidDNAincludedinDH10BacTMcompetentcells.AftertransfectionofSf9insectcellswiththebacmids,thefusionproteinswereexpressed.TheVP2,VP3andVP7proteinswerelaterpurifiedsuccessfully.However,thepurificationconditionsforVP5couldnotbeoptimized;thereforeithasbeenomittedfromthefurtherstudy.ThisresultisinagreementwithpreviousstudiesthatVP5hasshowntobecytotoxictoSf9cellsandeasilydegradedfollowingexpression(Vlak,etal.,1988;Marshall,etal.,1990).Furthermore,Andersonetal.havealsoreportedasimilarissuewithVP5purification(Anderson,etal.,2014).ThebaculovirusexpressionsystemhasbeenusedinpreviousstudiesinanefforttoproduceaBTVsubunitvaccine.ThesestudieshavebeenfocusedontheuseofVP2ofBTVeitherbyitselforinVLPs(Huismans,etal.,1987a;Roy,etal.,1990b;Roy,etal.,1992;Roy,etal.,1994).TheVLPsareaneffectivevaccinestrategysincetheviralparticleslacknucleicacidsandarepresentedinamorenativeconformation;however,itisdifficulttoexpressmorethanoneproteinsimultaneouslyinthesameexpressionsystem.Theseproteinshavetobeexpressedatamore-or-lessequalquantity.In1987,Huismansetal.demonstratedBTVneutralizationinsheepusingisolatedVP2fromintactviralparticles,andlaterthevaccinepotentialofbaculovirusexpressedBTV-10VP2wasfurtherconfirmedinsheep(Huismans,etal.,1987a;Roy,etal.,1990b;Roy,etal.,1992;Roy,etal.,1994).Immunizationwiththeviralcapsid-proteins(VP2,VP3,VP5andVP7)haslongbeenstudiedfortheirabilitytoproduceNabsagainstthedisease.Previousstudieshaveevaluatedcrudelysatesofbaculovirus-infectedinsectcellsexpressingBTVVP2andVP5(Zientara,etal.,2010;Stewart,etal.,2012;Anderson,etal.,2013b).ImmunizationofsheepwiththeseproteinsprotectedtheanimalsandraisedsignificantNAbstitreswithtransientorundetectableviraemiaafterasubsequenthomologousBTVchallenge(Roy,etal.,1990b).Recently,itwasshownthatbaculovirus-expressedandpurifiedVP2inducedneutralizingantibodiesandisstableat+4°Caswellas−80°Cforalmost2years(Anderson,etal.,2013a;Anderson,etal.,2014).Inthecurrentstudy,baculovirusexpressionsystemwassuccessfullyutilizedto43 中国农业科学院博士学位论文ChapterIIexpressBTVVP2,VP3,VP7andVP5immunogenicproteinsforlaterimmunizationofanimalswiththepurifiedproteinsaimingtoproduceaneffectivesubunitvaccineagainstBTV.Theexpressionconditions(optimumexpressiontimeandtemperatureforproteinbindingtotheNi-NTAresins,pH,bindingandelutionbuffers)wereoptimizedforeachindividualprotein.AlthoughBTV-16VP5andBTV-1VP5weresuccessfullyexpressedininsectcells,thepurificationtrialsforbothproteinswerenotsuccessfulperhapsduetotheinstabilityoftheexpressedproteins.Allotherexpressedproteinsweresuccessfullypurifiedandtheobtainedyieldsweresufficientforlateruseintheproposedsubunitvaccine.CommonlyreferredtoasVLPsorCLPs,theseparticlesresemblethenativevirus,withoutthegeneticmakeupofthevirus.Inthecaseofthebaculovirusexpressionsystem,theseproteinscaneitherbeexpressedfrommultiplebaculoviruseseachcarryingasingleforeigngene(monocistronic),orfromasinglebaculoviruscarryingmultipleforeigngenes(polycistronic).VLPsproducedusingco-infectioninthebaculovirusexpressionsystemareabletoinduceimmuneresponsesinanimalmodels,andinsomecaseshaveresultedinstrongerimmuneresponsesthanthoseachievedbysimilarstrategiesindifferentsystems(Garcea,etal.,2004).Furthermore,immunizationwithVLPswasfoundtoprotectsheepandraisedNAbsgeneratingsterileimmunitypost-homologous-challenge(Stewart,etal.,2012).Inthisstudy,withanattempttoassembletheVLPs,Sf9cellswereco-infectedwiththeindividualfourBTVproteins.Afterco-infection,thesimultaneousexpressionofthefourstructuralproteinswasconfirmedbywesternblot.TheVLPswilllaterbevisualizedusingelectronmicroscopyandevaluatedasavaccineagainstBTVinanimals.44 中国农业科学院博士学位论文ChapterIIICHAPTERIIIProkaryoticexpressionoffull-lengthBTV-16VP3,VP5,VP7,NS2,andtruncatedVP53.1IntroductionAmongstthenumerousdiseasesofruminants,BThasrecentlygainedaconsiderableimportanceinthetropicalandsubtropicalcountriesoftheworld(Mellor,etal.,2000b;Purse,etal.,2005;Mellor,etal.,2008;Wilson,etal.,2008b).ThegenomeofBTVconsistsoftenlineardouble-strandedRNAgenomesegments(Seg-1toSeg-10)encodingstructuralproteinsVP1toVP7,non-structuralproteins,NS1,NS2andNS3/NS3a,andtherecentlydiscoverednon-structuralproteinNS4ofBTV(Roy,2005;2008;Belhouchet,etal.,2011b;Ratinier,etal.,2011b).Thevirusparticlecomposesofthreeshellsofproteins.TheinnershellconsistsofVP3encodedbySeg-3,themiddleshellconsistsofVP7encodedbySeg-7,andtheoutershellisformedbyVP2(Seg-2)andVP5(Seg-6).TheorbivirusparticlefurthercontainsenzymaticproteinsVP1(Seg-1),VP4(Seg-4)andVP6(Seg-9),andonecopyofeachofthetengenomesegmentsintheinnershell.Thenon-structuralproteinsNS1(Seg-5),NS2(Seg-8),NS3/NS3a(Seg-10),andNS4(Seg-9)arenotpartofthevirusparticle.VP5hasrecentlyshowntobeamembranepenetrationproteinthatmediatesthereleaseofviralparticlesfromendosomalcompartmentsintothecytoplasm.VP5undergoespH-dependentconformationalchangesthatallowmembranefusionandsyncytiumformation(Forzan,etal.,2004).VP3andtoalesserextentVP7areconservedproteins,hydrophobicinnatureandareformingmajorcoreprotein(Schwartz-Cornil,etal.,2008).Theyplayanimportantroleinthestructuralintegrityoftheviruscore.Theyexpressgroup-specificantigenicdeterminantsdefiningseveraldistinctphylogeneticgroups(Anthony,etal.,2007).TheVP3/VP7complexprotectstheviraldsRNAgenomefromintracellularsurveillance,thuspreventingactivationoftypeIinterferon(IFN)productionviacytoplasmicsensorssuchascytosolichelicases,orinteractionswithdicerandRNAsilencingmechanisms(Schwartz-Cornil,etal.,2008).ThenonstructuralNS2isthemajorconstituentoftheviralinclusionbodies(VIB)seenininfectedcellsmainlyinthevicinityofthenucleus.ItbindstoviralssRNAandhydrolysesnucleotidetriphosphatestonucleotidemonophosphates(Schwartz-Cornil,etal.,2008).ThesetwopropertiesimplythatNS2mightbeinvolvedinsomewayinselectionandcondensationoftheBTVssRNAsegmentspriortogenomeencapsidation.NS2expressionincellsissufficientforformationofinclusionbodiesanditrecruitsVP3,suggestingthatNS2isakeyplayerinvirusreplicationandcoreassembly(Karetal.,2007).Inthisstudy,selectedstructural-proteinsofBTV-16,includingthefull-lengthVP3,VP7,NS2andatruncatedversionofVP5(VP5-41aa)wereproducedinaprokaryoticexpressionsystemandweresuccessfully45 中国农业科学院博士学位论文ChapterIIIpurified.VP3,VP7,andNS2wereexpressedassolublefusion-proteinswhileVP5-41aawasexpressedasaninsolubleprotein.Theproducedproteinsweretaggedwithapolyhistidinetag(His)tofacilitatetheirpurification.3.2Materialsandmethods3.2.1Virus,genesandcellsBTV-16(Isolate7766,SouthAfrica)S3,S6,S7andS8genesencodingforVP3,VP5,VP7andNS2wereartificiallysynthesizedbyGenScriptcompanyandutilizedtoconstructtheindividualrecombinantvectorsforbacterialexpression.E.coliBL21-codonPlus(DE3)-RILcompetentcells(Stratagene)andBL21(DE3)competentcells(Transgen)wereusedforbacterialexpression.3.2.2ConstructionoftherecombinantexpressionplasmidsPrimesweredesignedforfull-lengthamplificationofBTV16S3,S7andS8inadditiontothetruncatedversionofBTV16S6witha1–123nucleotidedeletion(encodesfor1-41aa)intheN-terminal(Table3.1).Theforwardandreverseprimerscontainedeitherstartorstopcodonsinthecorrectreadingframe.Therestrictionenzymesitesincludedintheprimerswasforinsertverificationandorientationdetermination.EachcDNAwasamplifiedbyPCRinaC1000TMThermalcycler.Theampliconswereanalyzedby1%agarosegelelectrophoresisandlatersubjectedtoenzymedigestionbeforebeingclonedintopET-32a(+)orpPROEX-HTbvectorsthatcarriesanampicillinresistantgene(AmpR)orpSMKvectorwithSUMOmoietythatenhancesthesolubilityoftheexpressedproteinsandcarriesakanamycinresistantgene(KanR).Allproteinsweretaggedwith6×HisresiduesfusedintheN-terminalofeachgenetopurifyproteinsusingnickelaffinitychromatography.3.2.3ExpressionandidentificationofBTV-16proteinsTheconstructedplasmidspSMK-BTV16S3andpSMK-BTV16S7wereusedtotransformE.coliBL21-codonPlus(DE3)-RILcompetentcells(Stratagene),whilepET32a-BTV16S6-41aaandpPROEX-HTb-BTV16S8weretransformedintoBL21(DE3)competentcells(Transgen).Asinglecolonyofeachtransformantwasgrownin5mlLBbrothcontainingkanamycin(50μg/ml)andchloramphenicol(34μg/ml)(forVP3andVP7)orin100μg/mlampicillin(forVP5andNS2)at37°C.Bacterialculturesweregrownuntilopticaldensity(OD600reached~0.8,theninducedbyadditionof0.5mMIPTGandfurtherincubatedat37°Cfor4-6h(VP5andNS2)orat16°Cfor6h(VP3andVP7).46 中国农业科学院博士学位论文ChapterIIIABCFigure3.1Mapsofbacterialexpressionvectorsinthisstudy.(A):pET-32a(+);(B):pPROEX-HTb;(C):pSMKplasmidsofVP3andVP7.47 中国农业科学院博士学位论文ChapterIIITable3.1PrimersusedtoamplifyBTV16S3,BTV16S7,BTV16S8andtruncatedBTV16S6genesbyPCRPrimernamePrimer5’-3’sequenceorientationBTV16S3-BbsIForward5’–GTTGCTGAAGACTTAGGTATGGCTGCTCAGAACGAG-3’BTV16S3-XhoIReverse5’-GATTAACTCGAGCTACACAGTCGGCGCAGC-3’BTV16S6-41aa-BamHIForward5’-CGCGGATCCGAGATTGGGTCCGCGGCAATTG-3’BTV16S6-HindIIIReverse5’-CCCAAGCTTTCACGCGTTTTTTAGGAAG-3’BTV16S7-BsmBIForward5’-GCATAGCGTCTCAAGGTATGGACACTATCGCTGCA-3’BTV16S7-BamHIReverse5’–GTTATAGGATCCCTATGCGTAAGCGGCGCGT-3’BTV16S8-BamHIForward5’-GTGGGATCCATGGAGCAAAAGCAACGTAAAT-3’BTV16S8-XhoIReverse5’–CGACTCGAGCTAAACGCCGACCGGCAATATG-3’Thebacteriawerethenpelletedbycentrifugationat10,000rpmfor5minandusedforSDS-PAGE.Thesampleswerere-suspendedinPBS,mixedin1:5of5xSDSloadingbufferandboiledat95°Cfor10min.SDS-PAGEgelswerestainedwithCoomassie®BrilliantBlueG-250dye.Forwesternblot,SDS-PAGEgelsweretransferredtoPVDFmembranesinwesternblottransferbufferat23Vfor30min.Thefollowingstepsweredescribedindetailsinsection2.3.3.3.2.4PurificationandquantificationoftheexpressedproteinsBacterialculturesweregrownin1LofLBbrothcontainingtheappropriateantibiotic/sat37°CuntiltheOD600reached~0.8.IPTGwasaddedwithafinalconcentrationof0.5mM,andthecultureswereincubatedat16°Cfor16h(VP3andVP7)orfor6hat37°C(VP5andNS2).VP3,VP7andNS2bacterialcultureswerethenharvestedbycentrifugationat10,000rpmat4°Cfor30minandre-suspendedinsolutionA(500mMNaCl,20mMTris–HCl,20mMimidazole,2mMDTT,0.05%TritonX-100;pH=8.0).Thecellswerethenhomogenizedusingultrasonicationfor20minwith3secpulseand3secpausebeforebeingspineddowntwiceat8,000rpmfor30minat4°C.ThesupernatantswerecollectedandtransferredtoanickelaffinityHis-tagpurificationresin(Roche,USA)pre-equilibratedwithsolutionA,incubatedfor1-2hatroomtemperaturewithagentleshakingtoallowresinstobindwiththetargetprotein.TheresinswerewashedwithsolutionAtoremoveanyunboundornonspecificproteinsbeforetheelutionofHis-taggedproteinsinsolutionB(500mMNaCl,20mMTris–HCl,500mMimidazole,2mMDTT,0.1%TritonX-100;pH=8.5).ThepurifiedsampleswereanalyzedbySDS-PAGEand/orWesternblot.pET-32aVP5-41aaHis-taggedproteinwaspurifiedfrominclusionbodies(insolublefraction).Briefly,theproteinwasfirstdissolvedin6MGuanidiniumLysisBuffer,pH7.8,hencedenaturingtheprotein,andthen48 中国农业科学院博士学位论文ChapterIIIpurifiedunderhybridconditionfollowingtheProBondTMPurificationSystemmanual(Invitrogen).Thehybridpurificationprotocolrestorestheproteinactivityfollowingcelllysisandbindingunderdenaturingconditions.AllpurifiedproteinswerequantifiedusingaBradfordassayatwavelength595nm.Usingthestandardcurve,theconcentrationofeachsamplewasdeterminedaccordingtoitsabsorbancebyinterpolation(Bradford,1976).3.2.5AssemblyofcorelikeparticlesInordertoassembleBTVCLP,thepurifiedHis-sumo-VP3andVP7proteinswereplacedinadialysisbagtogetherwithSumoprotease(ULP1)ataratioof100:1(vol/vol).Dialysiswasconductedagainstbuffer(40mMTris-HClbuffer,500mMNaCland1mMCaCl,pH8.0).His-sumo-proteinswerecleavedat4°Cfor15-16hwithcontinuouslystirring.TheformationofCLPwasfurtherexaminedbytransmissionelectronmicroscopy(TEM).3.3Results3.3.1ConstructionofrecombinantbacterialexpressionplasmidsTheresultingbacterialplasmidsarepET-32aBTV16S6-41aa,pSMK-BTV16S3,pSMK-BTV16S7,andpPROEX-HTbBTV16S8.TherecombinantplasmidswereverifiedbycoloniesPCRusingthespecificprimerslistedintable3.1.TheresultsshowedthattherecombinantbacterialexpressionplasmidscontainedthegenesofinterestwiththeexpectedsizeforBTV16S3,BTV16S7,BTV16S8,andBTV16S6-41aagenes(2.8kb,1.2kb,1.1kband1.5kbinlength,,respectively)(Figure3.2).Thesequencingresultswerealsoconfirmedbycomparisontoparentalvirussequences.3.3.2ProkaryoticexpressionandidentificationofBTVproteinsTherecombinantexpressionplasmidsweretransformedintocompetentcellsandtheninducedwithIPTGtoexpresstheproteins.TheresultsshowedthattheexpressedproteinbandscouldbeseenwiththeexpectedsizeofeachproteininSDS-PAGEgel(Figure3.3.).Theproteinsweresubjectedtowesternblotanalysis.Theproteinsweredetectedbyaprimarymouseanti-His-antibodyasdescribedabove.Asshown(Figure3.4),BTV16VP5-41aa,BTV16VP3,BTV16VP7,andBTV16NS2proteinsweresuccessfullyexpressedinE.coliwiththeexpectedmolecularweightof65,115,55and43kDa,respectively.SolubilityanalysisoftheexpressedproteinsshowedthatBTV16VP3,BTV16VP7,andBTV16NS2wereexpressedassolubleproteins,whileBTV16VP5-41aawasexpressedasinclusionbodies(insolublefraction).49 中国农业科学院博士学位论文ChapterIIIM123M1235000---5000---3000---3000---2000---2000---1500---1500---1000---1000---ABM1M1235000---2000---3000---1000---2000---1500---1000---CDFigure3.2PCRidentificationoftherecombinantexpressionplasmids.Molecularmassmarkers(inbasepairs)arenotedattheleftofeachimage.ArrowheadsindicatethePCRproductsattheexpectedsizes.(LaneM):DNAMarker;(A)(Lane1-3):pSMKBTV16S3;(B)(Lane1-3):pSMKBTV16S7;(C)(Lane1):pPROEX-HTbBTV16S8;and(D)(Lane1-3):pET-32aBTV16S6.50 中国农业科学院博士学位论文ChapterIIIM12M123120---100---120---70---100---50---70---40---50---30---40---30---ABM12M12170----170----130----130----100---100---70---70---55---55---40---40---35---35---CDFigure3.3SDS-PAGEgelsforprokaryoticexpressionofVP3,VP7,NS2andVP5-41aa.Molecularmassmarkers(inkDa)arenotedattheleftofeachimage.ArrowheadsindicaterecombinantBTVproteinsatexpectedmolecularweight.(LaneM):prestainedproteinmarker;(A)(lane1):negativecontrol;(lane2):pSMKBTV16VP3;(B)(lane1-3):pSMKBTV16VP7;(C)(lane1and2):pPROEX-HTbBTV16NS2;(D)(lane1and2):pET-32aBTV16VP5-41aa.51 中国农业科学院博士学位论文ChapterIII1M1M1M1170----170---170---120---130----130---130---100---100---100---100---70---70---70---70---55---55---40---55---40---50---35---40---35---40---35---30---ABCDFigure3.4WesternblotsforexpressedVP3,VP7,NS2,andVP5-41aaproteins.Molecularweightmarkers(inkDa)arenotedattheleftofeachimage.ArrowheadsindicaterecombinantBTVproteinsatexpectedmolecularmasses.(LaneM):prestainedproteinmarker;(A)(Lane1):pSMKBTV16VP3;(B)(Lane1):pSMKBTV16VP7;(C)(Lane1):pPROEX-HTbBTV16NS2;and(D)(Lane1):pET-32aBTV16VP5-41aa.3.3.3ProteinpurificationandquantificationRecombinantVP3,NS2,VP7,andVP5-41aaproteinswereexpressedinE.coliBL21andthenconfirmedwithSDS-PAGEandwesternblot.His-sumo-taggedrecombinantVP3andVP7andHis-taggedNS2proteinswerewater-soluble;hence,theseproteinswereusedforfurtherpurificationbyNi-NTAaffinitychromatographyundernativeconditions.However,BTV16VP5-41aawasexpressedasinsolubleprotein(inclusionbodies).Thisproteinwaspurifiedunderhybridconditionasindicatedbefore.Althoughsomeunspecificbandswithlowermolecularweightweredetectedafterelution,specificbandscorrespondingtotheexpectedsizeofVP3,VP7,NS2andVP5-41aawereobservedonSDS-PAGEafterpurification(Figure3.5).Acolorimetricproteinassay,basedontheBradfordmethod,wasusedforthemeasurementforallthepurifiedproteinsconcentrationinthecurrentstudyandasufficientamountofeachproteinwasobtained(VP2,VP3VP5,andVP7yielded~2.5mg,4mg,≥10mg,and≥10mg,respectively).3.3.4AssemblyandcharacterizationofBTVCLPsExtractscontainingSUMOfusionproteinswerepurifiedandthentreatedwithSUMOproteasetoremovetheHis-Sumomoieties.ThecleavageproductscontainedVP3andVP7solubleproteins(with52 中国农业科学院博士学位论文ChapterIIImolecularweightsofapproximately103kDaand38kDa,respectively)andHis-Sumotag(Figure3.6.A),wasusedtoassembletheCLPs.TodeterminetheefficiencyofCLPassemblyunderdifferentconditions,differentpHwasusedtoadjusttheconditioninthedialysisbuffer.Asshowninfigure3.6.B,thesizeofCLPsunderpH7.0isapproximately50-60nm.M12345M123M1170---170---170---130---130---130---100---100---70---70---100---55---55---70---40---40---55---35---35---40---35---ABCFigure3.5SDS-PAGEgelsforthepurifiedVP7,NS2andVP5-41aaproteins.Molecularweightmarkers(inkDa)arenotedattheleftofeachimage.ArrowheadsindicatethepurifiedBTVproteinsatexpectedmolecularweight.(LaneM):pestainedproteinmarker;(A)(Lane1-5):pSMKBTV16VP7;(B)(Lane1-3):pET-32aBTV16VP5-41aa;(C)(Lane1):pPROEX-HTbBTV16NS2.3.4DiscussionE.coliexpressionsystemcontinuestodominatethebacterialexpressionsystemsandremaintobethefirstchoiceforlaboratoryinvestigationsandinitialdevelopmentincommercialactivitiesorasausefulbenchmarkforcomparisonamongvariousexpressionplatforms.E.coliexpressionsystemisalsothebasisforeffortsinproteinengineeringandhigh-throughputstructuralanalysis(Koehn,etal.,2009;Gordon,etal.,2011).Inthischapter,thefull-lengthaasequencesofBTV-16S3,S7,S8,andtruncatedS6(1-123bpdeletion)geneswereamplifiedbyPCRandclonedintotheexpressionvectors.Withtheaimofproducingimmunogenicproteins,threestructuralandonenon-structuralproteinsofBTVserotype16wereexpressedusingdifferentbacterialexpressionvectors.Expressionconditionswereoptimizedforeachindividualproteininordertogetthehighestexpression.ThelowertemperaturefortheexpressionoftheHis-sumotaggedproteinswasfoundtoenhancethesolubilityoftheexpressedproteins,whiletheHis-taggedNS2proteinwasexpressedasasolubleproteinat37°C.Incontrast,thetruncatedversionofVP5couldnotyieldasolubleprotein,thereforeithas53 中国农业科学院博士学位论文ChapterIIIbeenpurifiedunderthehybridconditionthatassumedtopartiallyenhancesfortherefoldingoftheexpressedprotein.M12120---100---VP370---50---40---VP730---ABFigure3.6BTVCLPsassemblyusingaprokaryoticexpressionsystem.Molecularweightmarkers(inkDa)arenotedattheleftofeachimage.ArrowheadsindicatethepurifiedBTVproteinsatexpectedmolecularweight.(LaneM):prestainedproteinmarker;(A)(Lane1-2):His-sumo-VP3andVP7proteinsdigestedbyenzyme.ThemolecularweightofthecleavedVP3andVP7proteinsareapproximately103and38kDa,respectively;(B):TEMimageoftheassembledBTVCLPs.Recently,VP5wasexpressedwitha1-100aadeletionandevaluatedasanimmunogeninmice,whichdemonstratedtheuseofthisversionoftheproteininacombinationwithtwodomainsofVP2aspotentialforabacterial-expressedBTV-subunitvaccine,basedprincipallyonVP2andVP5(MohdJaafar,etal.,2014b).Inapreviousstudy,aseriesofVP5deletionmutants(aa1to20andaa22to41)weregeneratedbasedonthepredictedstructuralfeaturesofVP5andbysubsequentassessmentoftheireffectonmembranepermeablisation.Bothhelices,exhibitedcytotoxicitywiththemost-amino-terminalhelix(aa1to20)showingahigheractivitythantheadjacentpeptide(aa22to41)(Hassan,etal.,2001).HerewehaveusedthissystemtoexpressatruncatedversionofVP5lackingaa1-41attheN-terminalinordertoovercomethecytotoxicity.NS2belongstoaclassofhighlyconservedproteinsfoundinOrbivirusesoftheReoviridaefamily.NS2formslargemultimericcomplexesandlocalizestocytoplasmicinclusionsininfectedcells.Duetoitsabilitytobindsingle-strandedRNA(ssRNA),ithasbeensuggestedthattheproteinparticipatesintheselectionandsequestrationofthe10differentBTV-ssRNAsegments,priortotheirencapsidationandconversionintotheBTVdouble-strandedRNA(dsRNA)genome.ItisthemajorcomponentofVIBs;therefore,antibodiesraisedagainstsuchaproteinwouldinterferewiththevirusassemblyandprovideacrossserotypesprotection.NS2waspreviouslyexpressedinmammalian,baculovirusandE.cloiexpressionsystems(Taraporewala,etal.,2001;Modrof,etal.,2005;Anderson,etal.,2014).Toexaminetheabilityoftheproteintoenhancethecross54 中国农业科学院博士学位论文ChapterIIIreactivityofourproposedsubunitvaccine,wehaveexpressedthesolubleNS2proteinusingE.coliexpressionsystem.Theproteinwasexpressedandyieldedasufficientquantityofthepurifiedprotein.Althoughmanystrategieshavebeenproposedtopromotethesolubilityofheterologousproteins,heterologousproteinexpressionbytagfusionisapotentialdevelopment.Amongthesefusiontags,smallubiquitin-likemodifier(SUMO)tagprovidesadvantagescomparedwithotherfusiontagtechnologies,suchascorrectfusionproteinfolding,targetproteinprotectionfromdegradation,simplepurification,andfusionproteindetection.SUMOproteinhasaspecificstructurecomprisingahydrophilicgroupintheouterlayerandahydrophobicgroupintheinnerlayer;thisstructureallowsSUMOproteintofunctionasemulsifierstointerferewithinclusionbodyformationeffectively,therebyimprovingsolubility(Malakhov,etal.,2004).Inaddition,SUMOproteasecanrecognizethetertiarystructureoftheSUMO-fusiontag,whichcanproduceaccurateN-terminalaminoacidsoffusionproteinsandavoidproducingextraneousresiduesattheNterminusofatargetprotein(Butt,etal.,2005).Withthepurposeofproducingsolubleproteins,severalBTV-pSMKproteinshavebeenexpressedduringthestudyperiodwithtwomainaims.ThefirstistoincludetheexpressedproteinsaproposedsubunitvaccineagainstBTV.ThesecondistousethepurifiedproteinsforinvitroassemblyofBTVCLPsandVLPs.Theexpressionofallproteinwassuccessful.TheoptimizationofthepurificationconditionsofpSMKVP3andpSMKVP7proteinswassuccessfullywithasuperiorexpressionandahigheryieldofVP7proteinthatitcanrepresentagoodsourceofVP7asapartofasubunitvaccine,fortheCLPsandVLPsassemblyandmoreoveritcouldbeusedasabindingantigeninELISAkitforthediagnosisofBTV.AlthoughpSMKVP2andpSMKVP5proteinsweresuccessfullyexpressed,theoptimizationoftheirpurificationconditionsisstillinprogress.Furthermore,utilizationofSUMOtaggedproteinshasallowedforselfassemblyofseveralvirusVLPs.Forinstance,VLPscomposedentirelyoffootandmouthdiseasevirus(FMDV)(Asia1/Jiangsu/China/2005)capsidproteins(VP0,VP1andVP3)weresimultaneouslyproducedasSUMOfusionproteinsbyanimprovedSUMOfusionproteinsysteminE.coli.TheproteolyticremovaloftheSUMOmoietyfromthefusionproteinsresultedintheassemblyofVLPwithsizeandshaperesemblingtheauthenticFMDV(Guo,etal.,2013).SUMOfusionmotifwaspreviouslyutilizedtoexpressawhole,naturalVP2proteinofCanineparvovirus(CPV).Afterthecleavageofthefusionmotif,theCPVVP2proteinhasself-assembledintoVLPs.TheVLPswasassembledwithasizeandshapethatresembledtheauthenticviruscapsid(Xu,etal.,2014).Inthiswork,wehaveexpressedtheCLPproteinsofBTVinaframewiththeSUMOtag.TheCLPswasachievedwiththeexpectedsize(50~60nm).TheabilityofthecoreproteinstoassembleintoCLPsuggeststhattheSUMOexpressiontagcanrepresentagoodbasefortheproductionofcommerciallyVLPvaccinesagainstBTV.55 中国农业科学院博士学位论文ChapterIVCHAPTERIVImmunogenicityevaluationofprotein-basedsubunitvaccinesagainstbluetonguevirusinBALB/cmiceandsheep4.1IntroductionRecently,bluetongueisclassifiedasanemergingdiseaseinEuropeandcancausemajorlossoflivestockininfectedsheepherds.DuetothepotentialrapidspreadofBTVandthethreattotheinternationaltradeoflivestockandlivestockproducts,bluetonguewaslistedasoneofthecompulsorynotifiablediseasesbytheWorldAnimalHealthOrganization(OIE).BTVhasanonenvelopedicosahedralvirionconsistsoffourmainstructuralproteins(VP3,VP7,VP5andVP2)arrangedinconcentricshellsaroundthesegmenteddouble-strandedRNAgenomeinadditiontothreeminorstructural(VP1,VP4andVP6)andfournonstructuralproteins(NS1-NS4)involvedinvirusreplicationBTVcanbeassignedto27distinctserotypes,whichweredeterminedprimarilybytheouterproteinVP2(Maan,etal.,2007;Maan,etal.,2011a;Schulz,etal.,2016).InChina,multipleBTVstrains(BTV-1-5,7,9,12,15,16and24)havebeenisolatedfromthebloodofsheepandcattlebetween1986and1996,whilethefirstisolationofBTVinChinagoesbackto1979inYunnanProvince(Zhang,etal.,1999;Ting,etal.,2005;Lee,etal.,2011;Yang,etal.,2016).Serotypes1and16wereisolatedfromsheepwithobvioussignsofbluetongue,whereasserotypes2,3,4,12and15wereisolatedfromclinicallynormalcattleandthephylogeneticanalysesofthesevirusesindicatedthattheywerecloselyrelatedtooneanother,andtoanAustralianstrainofserotype1(Zhang,etal.,1999;Zhang,etal.,2004a).ThefirstreportofthecompletegenomesequenceoftheBTV-16inChinahasbeenreportedin2011by(Yang,etal.,2011).UptodatefoureasternBTV-16strains,fromAustralia,India,ChinaandtheSouthAfricanreferencestrainhavebeenfullysequenced(Yang,etal.,2011;Boyle,etal.,2012;Maan,etal.,2012;Minakshi,etal.,2012).PreviousanalysesshowedthattheBTV-16referencestrain(RSArrrr/16),theBTV-16vaccinestrain(RSAvvvv/16)andaChineseBTV-16(strainnumberBN96/16)(Yang,etal.,2011;Maan,etal.,2012),have>99%sequenceidentityinalltengenomesegments,indicatingthattheyarecollectivelyderivedfromarelatedcommonancestor.Asotherviralvector-bornediseases,rigorousvaccinationprogramsaretheonlyeffectivecontrolstrategythatcanbeadoptedagainstbluetongue.Todate,theavailablecommercialvaccinesarebasedonbothinactivatedvirusesandliveattenuatedstrains.ThereisevidencethattheuseofcertainmodifiedlivevirusvaccinescancausesufficientviremiainvaccinatedanimalstoallowtransmissionofthevaccinestraintounprotectedanimalsbycompetentCulicoidesmidgesortoallowreassortmentbetweenfieldandvaccineBT56 中国农业科学院博士学位论文ChapterIVstrains(Ferrari,etal.,2005;Purse,etal.,2005;Veronesi,etal.,2005;Savini,etal.,2008;Stewart,etal.,2012).Thereareseveralconcernsoverincompleteattenuationorinactivation,aswellasadversesideeffectsduetovaccination(Gethmann,etal.,2009)andthepotentiallythreadoftransmissionbybitingmidges,therefore,thereisacriticalneedfornewnonreplicativevaccinesthosearemoreefficientthanorasefficaciousastraditionalvaccinesandcanovercometheirdrawbacks.ThemainobjectiveofthecurrentstudyistogenerateaneffectivesubunitvaccinethatcaninduceastrongandspecificimmunityagainstBTV.Forthispurpose,theexpressedproteinswereutilizedindifferentcombinationswithMontanideTMISA201VGadjuvanttoimmunizeBALB/cmiceandsheep,andtheimmuneresponseswereevaluated.4.2.Materialsandmethods4.2.1EthicsstatementAllanimalexperimentswereperformedaccordingtotheprotocolsapprovedbytheAnimalCareandUseCommitteeoftheLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences(CAAS),China.4.2.2Celllines,virusesandanimalsBHK-21cellsweregrowninminimumessentialmedium(MEM)(Hyclone,UnitedKingdom)supplementedwith5%FBSat37°Cin5%CO2.TheBTV-1strain(GS/11)wasisolatedinwesternChinaandpropagatedinBHK-21cells(ATCC-CCL-10)obtainedfromtheAmericanTypeCultureCollection.Theviraltiter,determinedusingaplaqueformationassayonamonolayerofBHK-21cells,wasestimatedasplaqueformationunitspermilliliter(pfu/mL).HealthySmall-TailedHanSheep,conventionallyreared,BTVfreeandnotbeenvaccinatedagainstBTV(5-6monthsage)wereboughtatLanzhou.Formurinemodel,sixtoeightweeksoldfemaleBALB/CmicewereobtainedfromLanzhouveterinaryresearchinstitute.Bothanimalswererandomlydividedintosixgroupsoffiveanimalseachandimmunizedwithrecombinantsubunitvaccine.4.2.3Animalimmunization4.2.3.1ImmunizationofsheepThirty(5-6monthsold)BTV-freesheepwereusedinthisstudy.Sixgroupsoffivesheepeachwereinjectedsubcutaneouslyatdays0and29with50µgofeachrecombinantproteininMontanideTMISA201VG57 中国农业科学院博士学位论文ChapterIVemulsionoraplacebo(Table4.1).Bloodsamplesfromallgroupswereobtainedatweeks1,2,3,4,5,6,7,8,12,and15afterthefirstimmunization.Forvaccinepreparation,thedifferentpurifiedproteinsweremixedwiththeequalvolumeofMontanideTMISA201VGandstirredat~300rpmat30°Cfor5mintoformawater-in-oil-in-waterblend.Theamountsofantigenswerepre-dilutedfromtheoriginalproteinsbeforemixedwiththeadjuvanttomaintainthesameamountofantigenperdoseofvaccine.AnimalsinGroup1and2receivedbaculovirusexpressedVP3andVP7proteins,whileallothervaccinatedanimalswerevaccinatedwiththebacterialexpressedVP3andVP7.4.2.3.2ImmunizationofBALB/cmice6-8weeksoldfemaleBALB/cmiceweredividedintosixgroupsoffivemiceeachandinjectedsubcutaneouslyatdays0and13with10µgofeachprotein,asindicatedinTable4.1,inMontanideTMISA201VGemulsion.AgroupoffivecontrolBALB/cmicethatwereonlyinjectedwithadjuvantwasincluded.Bloodsampleswereobtainedfromthetailatweeks1,2(booster),3,4,5,6,8and12afterthefirstimmunization.ThedesignsoftheanimalexperimentsarepresentedinTable4.1.Table4.1DesignoftheexperimentalsubunitvaccinesVP2VP5VP3VP7NS2G1BTV16BTV16BTV16BTV16BTV16G2BTV16BTV16BTV16BTV16-G3--BTV16BTV16BTV16G4BTV1BTV16BTV16BTV16BTV16G5BTV8BTV16BTV16BTV16BTV16G6-----4.2.4Serologicalanalyses4.2.4.1DetectionofBTV-specificantibodiesbycELISAThebloodsampleswerecollectedfromallsheepbyjugularpunctureatweeks1,2,3,4(booster),5,6,7,8,12and15.Serawerecollected,aliquotedandstoredat−80°Cuntiluse.AllserumsamplesweretestedwithcELISAmethod.Briefly,ELISAplates(Corning,USA)werecoatedwiththepurifiedBTV-1ata58 中国农业科学院博士学位论文ChapterIVconcentrationof0.5µg/mlincarbonate-bicarbonatebuffer(SIGMA,USA)andincubatedovernightat4°C.AfterwashingwithPBSTthreetimesandblockingin2%fat-freemilkinPBSTfor1hat37°C,theserumwereaddedandserialdilutionsstartingfrom1:8werepreparedforeachsample,negativecontrol,andpositivecontrolseraaswell.Rabbitanti-BTVcompetitiveantibodywasaddedtoeachwell,mixedgentlywiththedilutedserumandincubatedat37°Cfor30min.Asecondwashingstepwasperformedbeforetheadditionofgoatanti-rabbitIgGantibodyconjugatedtoHorseRadishPeroxidase(HRP)(Abcam,USA)(1:10,000).Incubationfor30minat37°Cfollowedbyafinalwashstepwascarriedoutbeforetheadditionof100µl3,3′,5,5′-Tetramethylbenzidine(TMB).Theenzymaticreactionwasstoppedbyadding100μLof2Msulfuricacid(2MH2SO4),andtheopticaldensity(OD)wasmeasuredat450nm.TheresultsofcELISAareexpressedasaninhibitionpercentage(IP)asfollow:Percentageofinhibition=ODsample/ODnegativereference×100.Lowpercentageindicatestheseropositivesera,andserawithhighpercentagewereregardedasnegative.SerawithanIPinferiorto35%wereconsideredaspositive.4.2.4.2Detectionofprotein-specificantibodiesbyindirectELISAAntibodiesspecifictoBTV-16VP5andNS2weredetectedusingindirectELISA.Briefly,ELISAplateswerecoatedwitheachpurifiedproteindilutedincarbonate-bicarbonatebufferataconcentrationof0.5µg/mlandincubatedovernightat4°C.ELISAplateswerewashedthreetimeswithPBST,saturatedwithblockingbuffer(2%fat-freemilkinPBST)for1hat37°C.TheplateswerethenwashedthreetimeswithPBSTand100μLofaserialdilutionofserumstartingfrom1:8ofsamples,negativecontrol,andpositivecontrolserawasadded.Theplateswerefurtherincubatedat37°Cfor2handwashedthreetimeswithPBST,andthena1:10,000dilutionofrabbitanti-sheepIgGantibodyconjugatedtoHRP(Abcam,USA)wasaddedandincubatedat37°Cfor1h.Afterafinalwashstep,100µlTMBwasaddedtoenablethecolorimetricanalysis.Thereactionwasstoppedbyadditionof100μLof2MH2SO4.TheODweremeasuredat450nmandthecorrectedODvalueswerecalculated(COD=ODsample-ODbackground).4.2.4.3Plaquereductionneutralizationassay(PRNA)Sheepseraobtainedat3weeksafterthesecondimmunizationwereanalyzedforanti-BTV-1neutralizingantibodies(Nabs)usingaplaquereductionneutralizationassay(PRNA).Sheepserawereinactivatedat56°Cfor30min,andthena2-folddilutionsfromeachserawereprepared(therangeofdilutionswas1/4to1/32)inminimalessentialmedium(Hyclone,UnitedKingdom)andmixedwith2.4x104PFUofBTV-1.Followingincubationfor1hat37°C,theserawereaddedontoconfluentmonolayersofBHK-21cells.After2hoursincubation,thesupernatantwasreplacedwith1.5%lowmeltingpointagarose(Sigma,USA)inMEMsupplementedwith2%FBS.Theplatesweresubsequentlyincubatedfor3to4daysandthenthecellswere59 中国农业科学院博士学位论文ChapterIVfixedby10%formaldehyde.Thefixedcellmonolayerswerestainedwithcrystalvioletsolution,andthenumberofplaqueformationunits(PFU)wasdetermined.4.2.4.4AntigenspecificlymphocyteproliferationassayinsheepPeripheralbloodmononuclearcells(PBMCs)wereisolatedfromEDTA-treatedvenousbloodatweeks3and11afterboosterdose.SheepPBMCswasisolatedandlymphoproliferationassayswereperformed.Briefly,bloodwasdiluted1:3inPBSandcentrifugedatroomtemperatureat3000rpmfor30minusinglymphocyteseparatingkit(TianjinHaoYangBiotechnology,China).ThePBMCswerethenre-suspendedinRPMI1640mediumwithL-Glutamine(Hyclone,UnitedKingdom)supplementedwith10%FBS,penicillin(100µg/ml)andstreptomycin(100µg/ml)andseededinround-bottommicrotiterplates.Isolatedcellswerere-stimulated,inquadruplicatewellsofa96-wellplate,withindividualproteinsat0.06µgofprotein(BTV16VP5-41aaorVP7)perwell.After5daysofincubationat37°Cin5%CO2,cellproliferationwasquantifiedusingalamarBlue®reagent(Invitrogen,USA),whichwasaddedat20µl/well(10%)for18-24h.Absorbancewasmeasuredat570nmand600nm,andtheODvalueswerecalculatedaccordingtotheformulaformeasuringreductionofalamarBlue®(equation1)(alamarBlue®protocol,BIO-RAD).Theresultingpercentageindicatestheamountofcellgrowthinhibition(averageofquadruplicates)inthetestwellcomparedtothatinthecontrolwell.(O2xA1)-(O1xA2)PercentagereductionofalamarBlue®(equation1)=x100(R1xN2)-(R2xN1)Equation1:O1=molarextinctioncoefficient(E)ofoxidizedalamarBlue®(Blue)at570nm*,O2=EofoxidizedalamarBlue®at600nm*R1=EofreducedalamarBlue®(Red)at570nm,R2=EofreducedalamarBlue®at600nm,A1=absorbanceoftestwellsat570nm,A2=absorbanceoftestwellsat600nm,N1=absorbanceofnegativecontrolwell(mediaplusalamarBlue®butnocells)at570nm,N2=absorbanceofnegativecontrolwell(mediaplusalamarBlue®butnocells)at600nm,*Onlyoneappropriatesubstitutewavelengthmaybeused.4.3Results4.3.1ImmunogenicityoftherecombinantsubunitvaccinesinsheepAsshowninTable4.1,theexpressedBTVproteinsVP2,VP3,VP7,NS2,andVP5-41aafrombaculovirusandE.coliexpressionsystemwerecombinedandemulsifiedwithaMontanideTMISA201VGadjuvant.Sixgroupsoffivesheepeachwerevaccinatedsubcutaneouslyatdays0and29.Individual,meanandstandarddeviationofinhibitionpercentage(IP)valuesofBTV-1cELISAforsheeparepresentedinTable4.2,andagraphicalsummaryoftheIPresultsobtainedperweekaregivenforeachgroupinfigure4.2.A.AllsheepwereseronegativebeforevaccinationandtherewasnodetectableBTVgroup-specificantibodyinthecontrolsheepserainallserologicalassaysthroughouttheexperiment.Followingfirstvaccination,inhibition60 中国农业科学院博士学位论文ChapterIVpercentagedramaticallyreducedinallvaccinatedanimalsexceptonesheepingroupG1thatdidnotdevelopanyantibodyresponseduringthestudyperiod;therefore,ithasbeenexcludedfromthefurtherexperiment.Theantibodieslevelsremainedstableandat3weeksafterthefirstvaccinationallvaccinatedanimalsshowedamarkedboostinantibodylevelsandbecameELISApositive(IP<35).AllvaccinatedsheepseroconvertedtoBTVpositiveat4weeksaftervaccination(meanofinhibitionpercentage12.4±9.1in(groupG1),12.5±5.5(groupG2),10.6±5.2(groupG3),23.6±18.1(groupG4),25.7±18.0(groupG5),and94.3±3.0incontrols(groupG6).Allvaccinatedanimalswerestronglypositive(IP≤10)betweenweeks3to15aftersecondvaccination.ComparedtothecontrolgroupG6,allgroupshavehadasignificantdifferenceatweek2afterthefirstimmunizationandthroughoutthestudyperiod(P<0.00001).However,therewerenosignificantdifferencesbetweengroupsG1,G2,G4,andG5obtainedresults(P≥0.05),whilegroupG3atweek3afterthesecondimmunizationstartedtogiveasignificantpvalue(P<0.05)againstgroupallvaccinatedgorupsandremainedstableuptoweek15.4.3.2ImmunogenicityoftherecombinantsubunitvaccineinmiceMiceserumsamplesfromweeks1,2(booster),3,4,5,6,8and12afterthefirstvaccinationweretestedusingBTV-1cELISA.Mostvaccinatedmicewerepositiveatweek4afterthesecondvaccination,andoneweeklaterconvertedtoseropositive(IP<30),(meanofinhibitionpercentage16.9±3.7in(groupA),21.2±12.4(groupGB),12.3±2.8(groupGC),14.9±6.1(groupGD),17.8±4.2(groupGE),and104.6±1.9incontrols(groupGF),withoneexceptioningroupGB,whereonemouse(No.3)hadanIPof41.8,(Table4.2.B).AnillustrationoftheavailabledataisshowninFigure4.2.B.Theantibodyresponseremainedfairlystable,andoverthepositivethresholduntiltheendoftheexperiment.Whencomparedtothecontrolgroup,nosignificancedifferenceswereobservedinthethreevaccinatedgroupsuntilfirstweekafterthesecondvaccination.61 中国农业科学院博士学位论文ChapterIVTable4.2.AIndividual,meanandstandarddeviationofinhibitionpercentage(IP)valuesofBTV-1cELISAforvaccinatedsheep(weeks2-15).Thestatisticaldifferenceforeachgroupagainstthecontrolgroupisindicated.-Day6Day13Day22Day29AnimalGroup1week2weeks3weeks4weeksBoosterBTV16VP2,VP5,VP3,VP7andNS2431G193.240.534.19.5432G186.613.315.923.9433G187.815.121.77.7468G1116.27.015.18.4Mean102.319.021.712.4SD18.512.87.69.1Significant.differencep>0.05P<0.05P<0.05P<0.05NodifferSignificantExtr.significantExtr.significantBTV16VP2,VP5,VP3andVP7425G263.113.121.318.1426G289.47.813.39.2427G299.019.624.118.6428G296.440.720.010.4463G294.220.312.26.4Mean88.420.318.212.5SD14.612.55.25.5Significant.differencep>0.05P<0.05P<0.05P<0.05NodifferSignificantExtr.significantExtr.significantBTV16VP3,VP7andNS2421G384.58.213.78.4422G386.411.415.015.0423G377.112.122.810.4424G392.121.417.515.9465G343.35.88.43.2Mean76.711.815.510.6SD19.45.95.35.2Significant.differencep>0.05P<0.05P<0.05P<0.05NodifferSignificantExtr.significantExtr.significantBTV1VP2,BTV16VP5,VP3,VP7andNS2435G484.015.726.033.2437G474.228.535.824.5438G474.213.616.513.2436G4NANANANA460G4NANANANAMean77.219.226.123.6SD5.85.76.818.1p>0.05P<0.05P<0.05P<0.05Significant.differenceNodifferSignificantExtr.significantExtr.significantBTV8VP2,BTV16VP5,VP3,VP7andNS2429G588.117.916.211.0467G573.629.637.312.4466G582.712.118.631.8469G580.013.724.019.2430NANANANANAMean77.619.924.025.7SD9.57.78.218.0p>0.05P<0.05P<0.05P<0.05Significant.differenceNodifferSignificantExtr.significantExtr.significantPBS440G679.190.9100.991.7439G682.090.297.599.1461G678.484.395.191.9462G676.492.097.894.9476G687.294.293.194.1Mean80.690.396.994.3SD4.23.73.03.062 中国农业科学院博士学位论文ChapterIVTable4.2.A.Continued.Day37Day44Day51Day58Day80Day1185weeks6weeks7weeks8weeks12weeks15weeksBTV16VP2,VP5,VP3,VP7andNS28.98.67.97.72.41.78.210.97.58.66.83.98.211.59.08.24.62.415.88.98.011.917.59.110.310.08.19.17.84.33.21.24.11.65.82.9P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05Extr.SignificantExtr.significantExtr.significantExtr.significantExtr.significantExtr.significantBTV16VP2,VP5,VP3andVP77.27.98.48.67.07.213.216.29.318.223.17.27.29.27.27.32.61.713.812.810.015.49.78.48.210.28.111.27.05.39.911.38.612.19.96.03.33.31.14.67.82.6P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05Extr.significantExtr.significantExtr.significantExtr.significantExtr.significantExtr.significantBTV16VP3,VP7andNS27.08.11.57.14.83.38.76.81.67.34.73.010.18.01.87.33.92.08.76.51.97.84.93.08.15.91.47.13.61.58.57.11.67.34.42.61.10.90.20.30.60.8P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05Extr.significantExtr.significantExtr.significantExtr.significantExtr.significantExtr.significantBTV1VP2,BTV16VP5,VP3,VP7andNS29.07.81.69.90.210.327.017.04.78.50.217.811.26.01.510.00.13.4NANANANANANANANANANANANA15.810.33.610.416.910.56.94.22.71.715.85.1P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05Extr.significantExtr.significantExtr.significantExtr.significantExtr.significantExtr.significantBTV8VP2,BTV16VP5,VP3,VP7andNS219.812.19.914.818.68.711.68.77.412.413.95.810.89.76.211.215.65.112.311.27.214.216.79.1NANANANANANA13.610.78.813.116.27.23.61.03.01.51.71.7P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05Extr.significantExtr.significantExtr.significantExtr.significantExtr.significantExtr.significantPBS110.4112.188.5128.8107.097.1125.2110.994.6148.7111.895.088.8107.7110.8143.4107.299.6118.2110.9101.0134.8110.398.193.995.190.6121.9104.397.6107.3107.397.1135.5108.197.515.67.19.010.83.01.763 中国农业科学院博士学位论文ChapterIVTable4.2.B.Individual,meanandstandarddeviationofinhibitionpercentage(IP)valuesofBTV-1cELISAforvaccinatedmice(weeks2-12).Thestatisticaldifferenceforeachgroupagainstthecontrolgroupisindicated.Day7Day13Day23Day29Day34Day41Day55Day81AnimalGroup12weeks3weeks4weeks5weeks6weeks8weeks12weeksweekBoosterBTV16VP2,VP5,VP3,VP7andNS21A99.498.866.623.620.013.219.115.52A99.397.079.021.314.74.118.613.83A99.696.039.623.113.14.915.013.04A97.796.862.120.515.08.413.413.95A100.197.336.313.121.64.314.6NAMean99.297.256.720.316.97.016.214.0SD0.91.118.34.23.73.92.60.9Significant.differencep>0.05p>0.05P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05NodifferNodifferSignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantBTV16VP2,VP5,VP3andVP71B99.295.135.935.79.04.010.615.62B99.099.050.531.521.510.517.315.33B93.490.559.949.441.85.343.327.34B99.097.665.522.117.27.113.512.25B99.098.861.538.816.66.613.015.1Mean97.996.254.735.521.26.719.517.1SD2.53.611.810.012.42.513.55.9Significant.differencep>0.05p>0.05P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05NodifferNodifferSignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantBTV16VP3,VP7andNS21C94.492.733.214.010.93.9NANA2C92.792.264.8NANA8.5NANA3C95.296.459.928.215.811.5NANA4C97.698.525.521.313.93.7NANA5C93.994.260.99.98.45.318.518.1Mean94.894.848.818.412.36.618.518.1SD1.82.618.17.02.83.300Significant.differencep>0.05p>0.05P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05NodifferNodifferSignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantBTV1VP2,BTV16VP2,VP5,VP3,VP7andNS21D89.291.045.929.724.74.521.025.92D84.792.073.021.112.39.79.814.33D91.497.924.631.216.93.610.28.84D94.697.021.812.311.03.511.012.75D94.852.4106.410.59.77.49.512.7Mean90.986.154.321.014.95.812.314.9SD4.219.135.69.66.12.74.96.5Significant.differencep>0.05p>0.05P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05NodifferNodifferSignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantBTV8VP2,BTV16VP5,VP3,VP7andNS21E98.9115.855.728.817.111.418.915.52E99.7122.930.917.119.25.010.619.73E96.6128.253.219.418.76.912.519.74E94.9115.833.4NA11.24.310.0NA5E102.7122.644.013.022.717.314.622.9Mean98.5121.143.419.617.89.013.319.4SD3.05.311.25.84.25.43.62.6Significant.differencep>0.05p>0.05P<0.05P<0.05P<0.05P<0.05P<0.05P<0.05NodifferNodifferSignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantExtr.SignificantPBS1F102.4123.995.8NANA97.4105.1107.72F101.9128.097.193.8102.895.2125.6112.33F97.4126.3101.895.3104.593.0117.0113.44F101.2127.9106.295.8106.998.0131.4110.15F101.1123.3107.385.1101.9100.6119.5116.2Mean100.8125.9101.692.5104.096.8119.7111.9SD2.02.25.24.31.92.99.92.364 中国农业科学院博士学位论文ChapterIV(A)(B)Figure4.1Evolutionofmeaninhibitionpercentages.Individual,meanandstandarddeviationofinhibitionpercentage(IP)valuesforeachgroup(A)(G1-G6):sheep;and(B)(GA-GF):mice]testedwithcELISA.Vaccinationswereperformedondays0and29,insheep,days0and13inmice.65 中国农业科学院博士学位论文ChapterIV4.3.3Detectionofprotein-specificantibodiesBTV16VP5-41aaandNS2specificantibodiesweredetectedbyindirectELISAsinallsheep(Figure4.3.A).AntibodyresponsesagainstNS2weretestedatweeks4,5,7and15andtheantibodytiterswerehigherthanthecontrolsuntilweek15.Thecontrolgroupremainedseronegativeforthewholestudyperiod.AlthoughtheNS2-specificantibodylevelsinducedbythesubunitvaccineswerehigherthanthoseinthecontrolgroupatweek4,theselevelscontinuedtoberisenatweek7(3weeksafterthesecondvaccination),andpeakedatweek15(11weeksafterthesecondvaccination).WhentestedbyindirectELISAagainstBTV16VP5-41aa(Figure4.3.B),ahigherCODvalueswereobservedingroupG1,G2,G4,andG5vaccinatedsheepseracomparedtogroup6(control).ABFigure4.3Protein-specificserumantibodiesdirectedagainstNS2andVP5-41aaofBTV16invaccinatedsheep.(A):IndirectELISAforBTV16NS2-Specificantibodies(G1,3,4,5,and6);and(B):IndirectELISAforBTV16VP5-41aaspecificantibodies(G1,2,4,5,and6).Correctedopticaldensity(COD)valueswerecalculatedas(COD=ODSample–ODbackground).66 中国农业科学院博士学位论文ChapterIV4.3.4PlaquereductionneutralizationassayBytheendofthesecondweek,notitersofneutralizingantibodiesdirectedagainstBTV-1weredetectedinsheepserafromallimmunizedanimalsneitherinthecontrolgroup.Threeweeksaftersecondvaccination,alloftheimmunized25sheepshowedhighdetectableneutralizingantibody(NA)levelsmeasuredbyplaquereductionneutralizationtest.Thenumberofplaqueformationunits(PFU)wasdeterminedusingthesameapproach,whileomittingtheuseoftestedseraandtheplaqueformationinhibitionpercentageateachsampledilutionwerecalculatedagainstthecontrolwells.Noinhibitionpercentagescouldbedetectedinthecontrolanimalsandthedifferencesinvirustitersbetweenvaccinatedandcontrolanimalswerestatisticallysignificantforallthetestedserumdilutions.DifferencesbetweenresultsobtainedforgroupsG1andG2onneutralizingantibodytiters,werenotstatisticallysignificantathigherserumdilutionswhileithadastatisticallysignificantvalue(p≤0.01)inthelowerdilutions.SameresultswereobtainedbycomparinggroupG2andG3(Pvalue=0.04and0.02for1/4and1/8dilutions,respectively).GroupG4andG5howeverhadthehigherpotentialtoneutralizethevirusatallthetestedserumdilutionsandshowedstatisticallyhigherinhibitionpercentagesthanallothergroupsatthelowerserumdilutions(1/8-1/32).Figure4.3VirusplaquesreductionpercentageoftheneutralizingantibodiesdirectedagainstBTV-1followingvaccinationwithanexperimentalsubunitvaccines.Seraobtained3weeksafterthesecondvaccination(givenwitha4-weekinterval).Neutralizingantibodywerecalculatedandexpressedasapercentageofvirusinhibitionforserialserumdilutions.Eachlinerepresentstheaverageofduplicatedeterminations.67 中国农业科学院博士学位论文ChapterIV4.3.5LymphocyteproliferationresponsesInordertoevaluateofthecellularimmuneresponseagainstthesubunitvaccines,re-stimulationofisolatedlymphocytesfromsheepbloodsampleswithpurifiedVP7andVP5-41aaproteinswereperformed.SignificantdifferencesinVP7andVP5-41aa-specificlymphocyteproliferationamongimmunizedsheepwereobservedwhencomparedtothecontrolgroup.AsshowninFigure4.4,bothproteinsstimulatedthelymphocyteswithlowerpercentagesinG1andG4thanothergroups,indicatingabetterimmunogenicityinthatgroup.ComparedtoVP7proteins,VP5-41aainducedhigherlymphocyteresponses.4.4DiscussionHistorically,inactivatedorattenuatedvaccineshavebeenusedduetotheirlowcostandeasyproduction,despitetheriskassociatedwiththeirproduction.ThespreadofBTVtopreviouslydisease-freeregionswhichprohibittheuseofthecurrentBTVlive-attenuatedvaccinehashighlightedtheneedfornewgenerationofvaccine(Ferrari,etal.,2005;Veronesi,etal.,2005).Subunitvaccinesareoneoftheattractivealternativestrategies.SubunitvaccinesagainstBTVwouldtargettheoutercapsidproteinVP2,themainneutralization-specificantigen.AsubunitvaccinebasedontheuseofBTV-VP2maybeachievedbyeitherusingVP2byitselforbymeansofvirus-likeparticles(VLPs)onwhichVP2proteinsareexposed.InVLPs,theVP2isco-expressedwithothercapsidandcoreproteinstoformaparticlethatresemblestheintactBTV.TheBTV-VLPvaccinestrategyisadvantageoussinceitpresentstheneutralizingepitopesofmorethanoneviralproteininamoreauthenticmannerasfoundonthevirusitselfHowevertherearedifficultiesassociatedwithlargescaleproductionandadecreaseinthestabilityoftheparticlesovertime(Berg,etal.,2005;Wang,etal.,2006).Recently,Jabbarandco-workersexpressedVP2,VP5andVP7proteinsofBTVbybacterialexpressionsystemandtestedthemassubunitvaccineinIFNAR(-/-)mice(Jabbar,etal.,2013),whenthecompleteVP2togetherwithVP5andVP7usedformicevaccination,partialprotectionwasobservedagainstBTV-8withneutralizingantibodyresponse,albeitatlowlevels.Additionalstudiesusingbaculovirusexpressedantigensshowedthatco-expressionofVP3andVP7proteinsresultedinthegenerationofCLP,however,theydidnotinducecompleteimmunityagainstBTV(Stewart,etal.,2012;Thuenemann,etal.,2013),suggestingthatVP2andpossiblyVP5areessentialcomponentsofapotentialsubunitBTVvaccine.ImmuniaationwithVLPcontainingcapsid-proteins(VP3,VP7,VP2andVP5)alsoprotectedsheepandraisedNAbsgeneratingsterileimmunitypost-homologous-challenge(Stewart,etal.,2012).Moreover,therewasnointerferenceonthedevelopmentofastrongprotectiveimmuneresponseduetothecombinationofdifferentphylogeneticallyunrelatedBTVserotypesinthevaccinatedanimals(Stewart,etal.,2013).However,VLPsarethoughttoberelativelyunstableandhavealimitedshelflife.68 中国农业科学院博士学位论文ChapterIVABFigure4.4LymphocyteproliferationsinresponsetoBTV16VP5-41aaandVP7-specificre-stimulation.Bloodwascollectedatweek3.PBMCswereisolatedfromsheepandre-stimulatedwith(A):VP7;and(B)VP5.Proliferationisexpressedaspercentageofreductionofinhibitionofthecellsgrowthafter5daysofstimulation.69 中国农业科学院博士学位论文ChapterIVThevaccinewasdesignedtoinduceserotype-specificneutralizingantibodiesaswellasapotentiallycross-protectivecell-mediatedimmuneresponse.Subsequently,proteinswereinitiallyselectedtoinduceprotectionagainstBTV-16throughserotype-specificneutralizingantibodyresponsestotheoutercapsidproteins(VP2andVP5),thoseareresponsibleforvirusattachmentandpenetrationofhostcells,respectively.Further,theVP2fromBTV-1andBTV-8wereusedinplaceofBTV-16andtheimmuneresponsewasevaluated.In1987,HuismansandcoworkerfirstdemonstratedthatVP2wasabletoinduceNAsandprotectionagainsthomologouschallengeinsheepandotherreportsinwhichVP2wasexpressedusingdifferentexpressionsystemshaveconfirmeditsroleinprotection.Furthermore,epitopesmappingstudiesrevealedthatthemajorvirusneutralizingepitopesarelocatedintheproteinVP2(Roy,1992).BTVVP5representsthesecondmostvariableBTVproteinandpossessanaaidentityof41–79%betweenBTVserotypes(Maan,etal.,2011a;Maan,etal.,2011b),enhancesneutralizingantibodyresponsetoVP2proteins(Mertens,etal.,1987;Hassan,etal.,2001),andseemstoexertsomeinfluenceintheconformationofVP2(Cowley,etal.,1989;Mertens,etal.,1989).TheCLP(VP3andVP7)waspreviouslyusedasavaccineandfoundtoalleviatetheseverityofdiseasemanifestationandviremiainvaccinatedsheepcomparedtocontrols(Stewart,etal.,2012),andBTVNS2werefoundtoinduceacytotoxicT-cellresponseinmicewhichaffordspartialprotection(Jones,etal.,1997),togethertheseinformationsuggeststhatamulticomponentvaccineincorporatingthemajorcytotoxicTlymphocytesCTLepitopesfromtheseproteins,VP3,VP7,andNS2,couldpotentiallyconfereffectivecross-protectionbetweenBTVserotypes.Weobservedthattheantibodiesproducedbythecombinationofthesethreeproteinswerestatisticallysignificantincomparisontothecontrolgroupandlastsformorethan15weeksinsheep.Previously,thestructuralproteinVP7havebeenfoundtobeconservedacrossBTVserotypes(Mecham,etal.,1986),consideredtoinduceaserologicalimmuneresponsemorethanacell-mediatedimmuneresponse(Hamblin,2004;Vandenbussche,etal.,2008).VaccinationwithBTV-VP7hasbeenshowntoinduceapartially-protectivecytotoxicT-cellresponsethatmayreduceviremia(Jeggo,etal.,1985),whiletheinclusionofnonstructuralproteinNS2wasbasedonevidenceoftheprotein’santigeniccross-reactivitybetweendifferentstrainsandserotypesofBTV(Maan,etal.,2008).IthasbeenspeculatedthattheinductionofTcellsmaycontributetomulti-serotypeprotectionagainstBTV,particularlywheninducedbythenonstructuralproteins(Calvo-Pinilla,etal.,2012).Toevaluatetheimmuneresponse,differentexperimentalformulationsforsheepandmiceimmunizationsweresuggestedtoallowforcomparisonsofprotein-specificimmuneresponses.Outoftheevaluatedproteins,theinductionofhumoraland/orcell-mediatedimmunogenicitywasdetected.Protein-specifichumoralanalysesofNS2andVP5-41aadetectedspecificserumantibodiestobothproteinsinimmunizedsheep.Additionally,andinagreementwithJeggoandWardleywhohaveshownthatvaccinationwithBTV-VP7inducedapartially-protectivecytotoxicT-cellresponsethatmayreduce70 中国农业科学院博士学位论文ChapterIVviremia(Jeggo,etal.,1985),weobservedsignificantdifferencesinVP7andVP5-41aa-specificlymphocyteproliferationamongimmunizedsheepwhencomparedtothecontrols.Insummary,themainobjectivewastodevelopasubunitvaccinecandidatetargetingBTV-16asthisstrainhasbeenpreviouslyisolatedfromclinicallyinfectedsheepinChina.Withthisaim,fivepolyHistidine-taggedrecombinantproteins,BTV16VP2,VP3,VP7,VP5-41aaandNS2,wereexpressedinbaculovirusandorE.coliexpressionsystemsforfurthercharacterizationoftheaffordedprotection.Otherobjectiveofthestudywastoexaminethepossibilityofthevaccinecandidatetobeusedformoreserotypes.Withthispurpose,BTV-1VP2andBTV-8VP2werealsoexpressedandusedtoreplaceBTV-16VP2.Tocharacterizeovineandmurineimmuneresponsestotheexpressedproteins,bothsheepandmiceweresubcutaneouslyimmunizedtwicewithoneoffiveproteincombinationsinMontanideTMISA201VGadjuvantoraplacebo.(n=5/group).UtilizingBTV-1cELISA,significantlyhigherantibodytitersweredetectedintheimmunizedanimalsthanthecontrols.TheexpressedVP5-41aaandNS2proteinsinducedaproteinspecifichumoralresponse,whileVP7andVP5werefoundtoinducespecificlymphocyteproliferationresponsesuponre-stimulationofperipheralbloodmononuclearcells(PBMC)fromimmunizedsheep.SerumneutralizationtestagainstBTV-1serotypeshowedapromisingresultforcrossserotypicprotectionofthevaccineagainstBTV-8andBTV-16.ThedatasuggestthattherecombinantsubunitvaccinecouldrepresentanimportantpartofanovelvaccineagainstBTV.71 中国农业科学院博士学位论文ConclusionConclusion(1)Thefull-lengthBTV16VP2,BTV16VP3,BTV16VP7,BTV16VP5,BTV-1VP2,BTV1VP5,andBTV8VP2proteinsweresuccessfullyexpressedassolublefractionsutilizingabaculovirusexpressionsystem.ExceptforBTV16VP5andBTV1VP5,otherexpressedproteinswerepurifiedandtheobtainedyieldsweresufficientforlateruseintheproposedsubunitvaccine.FourstructuralproteinbandsweredetectedwhenSf9cellswereco-infectedwiththerecombinantbaculovirusesexpressingBTV-16VP2,VP3,VP5,andVP7proteins,theassemblyBTVVLPwillbefurtherstudied.(2)E.coliexpressionsystemwasusedtoexpressthesolubleproteinsofBTV-16VP3,VP7,andNS2andthethreeproteinswerepurifiedundernativeconditions.AtruncatedVP5lackedtheinitial41aminoacidshasalsobeenexpressedinE.coliasaninsolubleprotein,andpurifiedunderhybridpurificationconditions.TheBTV-16CLPwasassembledwithHis-SUMOtaggedVP3andVP7afterthecleavageoftheSUMOmoiety.(3)TheexpressedBTVproteinsVP2,VP3,VP7,NS2,andVP5-41aafrombaculovirusandE.coliexpressionsystemwerecombinedandemulsifiedwithaMontanideTMISA201VGadjuvant.Sheepandmiceweresubcutaneouslyimmunizedtwicewithoneoffiveproteincombinationstoevaluatetheimmunogenicityofdifferentsubunitvaccines.Significantlyhigherantibodytitersweredetectedintheimmunizedanimalsthanthecontrols.Thevaccineinducedaproteinspecifichumoralresponseaswellaslymphocyteproliferativeresponsesuponre-stimulationofPBMC.SerumneutralizationtestagainstBTV-1serotypeshowedapromisingresultsforcrossserotypicprotectionofthevaccineagainstBTV-1,BTV-8andBTV-16.ThedatasuggestedthattheexpressedproteinsinthisstudycouldbecomeapromisingsubunitvaccinecandidateagainstBTV-1,8,and16forruminantsincaseofdiseaseoccurrence.72 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中国农业科学院博士学位论文AcknowledgementsAcknowledgementsFirstofall,IwouldliketoexpressmysincerethankstoAlmightyAllahwhohasgivenmestrengthandgoodhealthtocompletemyresearchatGSCAAS(LVRI).AllandeverykindofrespecttotheHolyProphetMuhammadforhisunique,comprehensiveandeverlastingsourceofguidanceandknowledgeforhumanity.IamgratefultoChinaScholarshipCouncil(CSC)andGSCAASforfullscholarshipsupportforPh.D.MymostsinceregratitudegoestomysupervisorProfessorHongYinandtoDr.JunzhengDufortheirinstinctedhelpthroughconstructivecritismandvaluableguidanceandwithouttheirinsistenceandpersistencethismanuscriptwouldnothaveseenlightofday.ReallyfortunateIam,togetanopportunitytoworkundertheirguidance.IamalsogratefultoallmembersinProf.HongYin’slaboratory,especiallyProf.JianxunLuo,Prof.GuangyuanLiu,Prof.GuiquanGuan,Dr.ZhijieLiu,Dr.ZhanchengTian,andMr.LinZhangfortheirvaluablesuggestionsandhelpfulcriticismuntiltheendofthisstudy.SincerethankstoDr.ShandianGaoforadministrativeassistanceandsupport.SpecialthankstoDr.ShiqiSunforhishelpandguidanceduringthestudy,andforhisgroupspeciallyDr.HuichenGuo,HuDong,YanyanChang.Tocolleaguesinmyresearchgroup,DexuanHuang,GuoruiZhang,RongshengDu,BiaoKang,andZhiLiIwanttothankyouguysformakingthisafriendlyworkenvironmentwiththeaddedhelpandsupport.ThankstoMr.GuangqinZhou,forhishelpinperformingcELISA.Tothestaffofthescientificdivisionespecially,Mrs.XiaopingLithanksforyouradministrativehelpduringthestudyperiod.IalsowouldliketoexpressmyrecognitiontothetechnicalstaffDaliXu.Iexpressmyhumbleappreciationtomycountry(Sudan)speciallytheministryofHigherEducationandScientificresearch,andtotheUniversityofKhartoum,fortheirhelptofacilitatemynominationandagreeandtheirsupportstojointhestudyinChinaandcompletemyPh.D.degreehere.Iexpressmyheartiestthankstoallwhohelpeddirectlyandindirectlyforbuilding-upmycareer.Lastbutnotleasttomylovingfamily,especiallymyMather(Elham),withoutyourunconditionallove,kindness,respectandsupport,thisPh.D.wouldnothavebeenpossible.IamsorryforthoselonghoursIspentawayfromyou.ThisstudywasperformedattheStateKeyLaboratoryofVeterinaryEtiologicalBiology.91 中国农业科学院博士学位论文CurriculumVitaeCurriculumVitaeName:DarienKhederAliMohamed.Gender:Female.Nationality:Sudanese.Placeofbirth:Libya.Religion:Islam.Address:E-mail:Phone:Qualifications:-2013–2016:Ph.D.inMicrobiologyandMolecularBiology,PreventiveVeterinaryMedicine,LanzhouveterinaryresearchInstitute,ChineseAcademyofAgriculturalSciences,Beijing,P.R.China-2007–2009:Masterdegreeintropicalanimalhealth,DepartmentofVeterinaryPreventiveMedicineandpublichealth,FacultyofVeterinaryMedicine,UniversityofKhartoum,Sudan.-2000–2005:BachelordegreeinVeterinaryMedicine,CollegeofVeterinaryMedicine,UniversityofKhartoum,Sudan.Academicstatus:-TeachingAssistant.DepartmentofVeterinaryPreventiveMedicineandPublichealth.FacultyofVeterinaryMedicine.UniversityofKhartoum,2006-2009.-Lecturer.DepartmentofVeterinaryPreventiveMedicineandPublichealth.FacultyofVeterinaryMedicine.UniversityofKhartoum,2009-now.Trainingcourses:-TrainingcourseinEmpiricalpopulationgenetics,FacultyofVeterinaryMedicine.UniversityofKhartoum,incollaborationwithCRDandCIRDES15-18October2012.-Trainingcourseinveterinarylaboratorydiagnosis,FacultyofVeterinaryMedicine.UniversityofKhartoum,7-11October2012.-Datahandlingandstatisticalanalysis(shortcourse),UniversityofYangzhou,China(December2011).-FundamentalsofBiostatistics(withimplementationofSPSS),shortcourseatfacultyofMathematicalscience,UniversityofKhartoum(February2012)-Teachingmethodsandsyllabusdesign,IncludesEducationaltechnologies,educationalpsychologyanduseofeducationalinstruments,organizedbytheAcademicDevelopmentCentre,FacultyofEducation,UniversityofKhartoum(2006).92 中国农业科学院博士学位论文CurriculumVitaeWorkshops-Introductiontomolecularbiologyandbioinformaticsworkshop,InternationalLivestockResearchInstitute(ILRI),Nairobi,Kenya(May2012).-AworkshoponclimatechangeinSudanatTheUniversityofKhartoumFacultyofForestry(2009).Publications:Thesis:-Darien,Kh.Ali.(2009),Post-mortemcausesofcondemnationsofmeatinslaughterhousesinKhartoumstate-Sudan.(M.Sc.Thesis)Depart.OfVet.Prev.Med.andpublichealth,FacultyofVeterinaryMedicine,UofK.-Darien,Kh.Ali,HayfaM.I,Elamin,A.A.(2012),StatusofmilkandmeathygieneinSudan,review,UofK.J.Vet.Med.&Anim.Prod.Vol.3,No1,2012(107-124)Paperinaconference:Oralpresentation-Darien,Kh.Ali,HayfaM.I,Elamin,A.A.(2012),StatusoffoodsafetyresearchinSudanwithspecialreferencetomilkandmeathygiene,Agriculturalsymposium,China-Sudancollaboration,Yangzhouuniversity,China(6thNovember).Jobtakenexperiences:-Lectureroffoodsafety(meathygiene&inspection)forthefinalyearstudents(since2009)atTheUniversityofKhartoumFacultyofVeterinaryMedicine.-SecretaryofthedepartmentalboardfromOctober2009-Septermber2013.-TrainingoffinalyearstudentsinthemeathygieneandinspectioninseveralslaughterhousesinKhartoum.-Participatingintrainingofstudentsindifferentmethodsofdiagnosisandcontrolofpoultrydiseases.-Trainingofstudentsinvariousmethodsofdiagnosisandcontrollingofzoonoticdiseases.-Trainingofstudentstoproducedifferenttypesofveterinaryextensionmethods.Paperfromthecurrentwork(1)DarienKhederAliMohd,JunzhengDu,ShandianGao,ZhanchengTian,GuoruiZhang,DexuanHuang,RongshenDu,BiaoKang,GuangyuanLiu,JianxunLuo,HongYin.Evaluationofimmunogenicityaffordedbyaprotein-basedsubunitvaccineagainstbluetonguevirusinBALB/Cmiceandsheep.Vaccine.2016(submitted)(2)DarienKhederAliMohd,JunzhengDu,HuDong,DexuanHuang,GuoruiZhang,ShandianGao,YanyanChang,ZhanchengTian,ShiqiSun,GuangyuanLiu,HuichenGuo,JianxunLuo,HongYin.Evaluationofhumoralresponseofchimericsubunitvaccinesagainstbluetonguevirusserotypes1,8and16.AppliedMicrobiologyandBiotechnology.2017.(Inpreparation)93
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