影响非洲猪瘟病毒对培养细胞感染性因素分析

前言非洲猪瘟病毒概述1.1.1ASFV的病原学非洲猪瘟(Africanswinefever,ASF)是一种具备极高的传染性和致命性的动物疫病。该疾病由非洲猪瘟病毒(Africanswinefevervirus,ASFV)引起，病程短且迅速，一旦感染，发病率与死亡率均显著上升。其主要受害者是家猪和野猪，对养殖业构成了巨大的威胁ADDINZOTERO_ITEMCSL_CITATION{"citationID":"RofCuANe","properties":{"formattedCitation":"(Wang\\uc0\\u31561{},2019)","plainCitation":"(Wang等,2019)","dontUpdate":true,"noteIndex":0},"citationItems":[{"id":658,"uris":["/users/9215574/items/3EFXI629"],"itemData":{"id":658,"type":"article-journal","abstract":"UnveilingAfricanswinefevervirus\n\nAfricanswinefevervirus(ASFV)ishighlycontagiousandoftenlethal.Withnovaccineoreffectivetreatment,infectionsoftenrequirelarge-scalecullingofpigs.Wang\netal.\napplycutting-edgecryo–electronmicroscopytechniquestodeterminethestructureofthisverylargeDNAvirus.An8.8-angstrom-resolutionreconstructionshowsthefivelayersofthevirus,andthefourthcapsidlayercouldbereconstructedat4.8-angstromresolution.ThestructurerevealsepitopesinthemajorcapsidproteinthatdistinguishASFVfromothernucleocytoplasmiclargeDNAvirusesandshowshowtheminorcapsidproteinsstabilizethecapsid.\n\n\nScience\n,thisissuep.\n640\n\n,\nAstructuredeterminedbyelectronmicroscopyprovidesaclose-uplookatavirusthatisoftenlethalinpigs.\n,\nAfricanswinefevervirus(ASFV)isagiantandcomplexDNAvirusthatcausesahighlycontagiousandoftenlethalswinediseaseforwhichnovaccineisavailable.Usinganoptimizedimagereconstructionstrategy,wesolvedtheASFVcapsidstructureupto4.1angstroms,whichisbuiltfrom17,280proteins,includingonemajor(p72)andfourminor(M1249L,p17,p49,andH240R)capsidproteinsorganizedintopentasymmetronsandtrisymmetrons.Theatomicstructureofthep72proteininformsputativeconformationalepitopes,distinguishingASFVfromothernucleocytoplasmiclargeDNAviruses.Theminorcapsidproteinsformacomplicatednetworkbelowtheoutercapsidshell,stabilizingthecapsidbyholdingadjacentcapsomerstogether.Actingascoreorganizers,100-nanometer-longM1249Lproteinsrunalongeachedgeofthetrisymmetronsthatbridgetwoneighboringpentasymmetronsandformextensiveintermolecularnetworkswithothercapsidproteins,drivingtheformationofthecapsidframework.Thesestructuraldetailsunveilthebasisofcapsidstabilityandassembly,openingupnewavenuesforAfricanswinefevervaccinedevelopment.","container-title":"Science","DOI":"10.1126/science.aaz1439","ISSN":"0036-8075,1095-9203","issue":"6465","journalAbbreviation":"Science","language":"en","page":"640-644","source":"DOI.org(Crossref)","title":"ArchitectureofAfricanswinefevervirusandimplicationsforviralassembly","volume":"366","author":[{"family":"Wang","given":"Nan"},{"family":"Zhao","given":"Dongming"},{"family":"Wang","given":"Jialing"},{"family":"Zhang","given":"Yangling"},{"family":"Wang","given":"Ming"},{"family":"Gao","given":"Yan"},{"family":"Li","given":"Fang"},{"family":"Wang","given":"Jingfei"},{"family":"Bu","given":"Zhigao"},{"family":"Rao","given":"Zihe"},{"family":"Wang","given":"Xiangxi"}],"issued":{"date-parts":[["2019",11]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(Wangetal.,2019)。ASFV作为非洲猪瘟病毒家族中的唯一成员，具备独特的传播方式，是目前已知的唯一一种能够通过节肢动物作为媒介进行传播的DNA病毒。ASFV颗粒直径为260-300nm，由包膜、衣壳、内囊膜、核壳和内核组成，具有对称二十面体结构，呈同心圆，基因组结构复杂，包含超过150个开放阅读框(OpenReadingFrame,ORF)，这些阅读框负责编码150至200种病毒蛋白，其中，68种属于结构蛋白，而余下的100余种则为非结构蛋白ADDINZOTERO_ITEMCSL_CITATION{"citationID":"lkzyko67","properties":{"formattedCitation":"\\super[2]\\nosupersub{}","plainCitation":"[2]","dontUpdate":true,"noteIndex":0},"citationItems":[{"id":596,"uris":["/users/9215574/items/6XSVEFBH"],"itemData":{"id":596,"type":"article-journal","abstract":"Africanswinefever(ASF)isanacute,hemorrhagicandhighlycontagiousinfectiousdiseasecausedbyAfricanswinefevervirus(ASFV),whichinfectsdomesticpigsorwildboars.Itischaracterizedbyshortcourseofdisease,highfeverandhemorrhagiclesions,withmortalityofupto100%fromacuteinfection.Uptonow,thelackofcommercialvaccinesandeffectivedrugshasseriouslythreatenedthehealthyeconomicdevelopmentoftheglobalpigindustry.ASFVisadouble-strandedDNAvirusandgenomevariesbetweenabout170-194kb,whichencodes150-200viralproteins,including68structuralproteinsandmorethan100non-structuralproteins.Inrecentyears,althoughtheresearchonstructureandfunctionofASFV-encodedproteinshasbeendeepened,thestructureandinfectionprocessofASFVarestillnotclear.ThisreviewsummarizesthemainprocessofASFVinfection,replicationandfunctionsofrelatedviralproteinstoprovidescientificbasisandtheoreticalbasisforASFVresearchandvaccinedevelopment.","container-title":"FrontiersinImmunology","ISSN":"1664-3224","source":"Frontiers","title":"ResearchprogressontheproteinsinvolvedinAfricanswinefevervirusinfectionandreplication","URL":"/journals/immunology/articles/10.3389/fimmu.2022.947180","volume":"13","author":[{"family":"Duan","given":"Xianghan"},{"family":"Ru","given":"Yi"},{"family":"Yang","given":"Wenping"},{"family":"Ren","given":"Jingjing"},{"family":"Hao","given":"Rongzeng"},{"family":"Qin","given":"Xiaodong"},{"family":"Li","given":"Dan"},{"family":"Zheng","given":"Haixue"}],"accessed":{"date-parts":[["2024",2,23]]},"issued":{"date-parts":[["2022"]]}}}],"sch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summarizesthemainprocessofASFVinfection,replicationandfunctionsofrelatedviralproteinstoprovidescientificbasisandtheoreticalbasisforASFVresearchandvaccinedevelopment.","container-title":"FrontiersinImmunology","ISSN":"1664-3224","source":"Frontiers","title":"ResearchprogressontheproteinsinvolvedinAfricanswinefevervirusinfectionandreplication","URL":"/journals/immunology/articles/10.3389/fimmu.2022.947180","volume":"13","author":[{"family":"Duan","given":"Xianghan"},{"family":"Ru","given":"Yi"},{"family":"Yang","given":"Wenping"},{"family":"Ren","given":"Jingjing"},{"family":"Hao","given":"Rongzeng"},{"family":"Qin","given":"Xiaodong"},{"family":"Li","given":"Dan"},{"family":"Zheng","given":"Haixue"}],"accessed":{"date-parts":[["2024",2,23]]},"issued":{"date-parts":[["2022"]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(Duanetal.,2022)。1.1.2ASFV的流行病学ASF于1921年首次在肯尼亚被发现，随后传播到非洲大陆的其他区域，接着众多欧洲国家也暴发了ASF，随后亚洲的部分地区也出现该疾病，是唯一一种已知的通过节肢动物传播的双链DNA病毒ADDINZOTERO_ITEMCSL_CITATION{"citationID":"dQfjeMSS","properties":{"formattedCitation":"(\\uc0\\u24352{}\\uc0\\u31561{},2023)","plainCitation":"(张等,2023)","noteIndex":0},"citationItems":[{"id":661,"uris":["/users/9215574/items/YPGWVT5L"],"itemData":{"id":661,"type":"article-journal","abstract":"<正>非洲猪瘟于1921年在肯尼亚首次发现，由非洲猪瘟病毒科、非洲猪瘟病毒属的非洲猪瘟病毒(ASFV)感染引起的，仅1个血清型，但可分为24个基因型，ASFV基因组为双股线性DNA，大小为170～190kb，该病毒在自然环境中抵抗力较强，室温干燥条件或冰冻数年均能存活，是对全世界家猪和野猪最具毁灭性的疾病病原之一。","container-title":"猪业科学","ISSN":"1673-5358","issue":"12","language":"zh-CN","page":"90-92","source":"CNKI","title":"非洲猪瘟的流行现状及防控措施","volume":"40","author":[{"family":"张","given":"依玲"},{"family":"易","given":"文毅"},{"family":"肖","given":"静"},{"family":"张","given":"淑娟"},{"family":"马","given":"秀云"},{"family":"李","given":"文杰"},{"family":"张","given":"传师"}],"issued":{"date-parts":[["2023"]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(张百东，2023)。2018年，在辽宁省沈阳市首次发现了非洲猪瘟疫情，此后疫情迅速蔓延至国内多个省份，对我国的养猪业造成了严重的经济损失ADDINZOTERO_ITEMCSL_CITATION{"citationID":"RWsB9mHI","properties":{"formattedCitation":"(Li\\uc0\\u31561{},2019)","plainCitation":"(Li等,2019)","dontUpdate":true,"noteIndex":0},"citationItems":[{"id":669,"uris":["/users/9215574/items/4EH337Y7"],"itemData":{"id":669,"type":"article-journal","abstract":"On16November2018,awildboarinfectedwithAfricanswinefeverwasreportedinChina.Thephylogeneticanalysisshowedthatitscausativestrainbelongedtothep72genotypeII,CD2vserogroup8andcontainednoadditionaltandemrepeatsequencesbetweentheI73RandtheI329Lproteingenes,whichwasdifferentfrompreviouslyreportedstrainsinChina.","container-title":"TransboundaryandEmergingDiseases","DOI":"10.1111/tbed.13114","ISSN":"1865-1682","issue":"3","journalAbbreviation":"TransboundEmergDis","language":"eng","note":"PMID:30592384","page":"1395-1398","source":"PubMed","title":"InfectionofAfricanswinefeverinwildboar,China,2018","volume":"66","author":[{"family":"Li","given":"Lin"},{"family":"Ren","given":"Zhaowen"},{"family":"Wang","given":"Qinghua"},{"family":"Ge","given":"Shengqiang"},{"family":"Liu","given":"Yutian"},{"family":"Liu","given":"Chunju"},{"family":"Liu","given":"Fuxiao"},{"family":"Hu","given":"Yongxin"},{"family":"Li","given":"Jinming"},{"family":"Bao","given":"Jingyue"},{"family":"Ren","given":"Weijie"},{"family":"Zhang","given":"Yongqiang"},{"family":"Xu","given":"Tiangang"},{"family":"Sun","given":"Chengyou"},{"family":"Li","given":"Ling"},{"family":"Wang","given":"Shujuan"},{"family":"Fan","given":"Xiaoxu"},{"family":"Wu","given":"Zongbo"},{"family":"Huang","given":"Baoxu"},{"family":"Guo","given":"Huancheng"},{"family":"Wu","given":"Xiaodong"},{"family":"Wang","given":"Zhiliang"}],"issued":{"date-parts":[["2019",5]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(Lietal.,2019)。这一疫情的爆发凸显了ASF对养殖业的潜在威胁，也强调了加强疫情防控和动物健康管理的重要性。目前，ASF仍然在全球范围内中迅速传播，对全球养猪业和饲料业造成了持续性的严重影响。ASFV的主要宿主是野猪和家猪，它们均能无症状地长期携带并传播该病毒，ASFV的传播途径广泛，其中软蜱作为其传播的主要节肢动物媒介，使得ASFV在猪群中的传播更为隐匿和难以控制。此外，受感染动物间的直接接触，如伤口或飞沫传播以及通过感染动物的排泄物或受污染生肉之间的接触传染，也是ASFV传播的重要途径；除此之外，受污染的运输车辆、饲料、衣物、畜舍物品和场地设施设备等污染物也可作为传染源ADDINZOTERO_ITEMCSL_CITATION{"citationID":"xXFTbjQJ","properties":{"formattedCitation":"(\\uc0\\u38886{}\\uc0\\u31561{},2023)","plainCitation":"(韦等,2023)","noteIndex":0},"citationItems":[{"id":665,"uris":["/users/9215574/items/9E96B89B"],"itemData":{"id":665,"type":"article-journal","abstract":"非洲猪瘟是由非洲猪瘟病毒引起的一种烈性传染病性疾病，在家猪和欧亚野猪中具有极高的致死率。在过去的十年里，非洲猪瘟在欧洲、亚洲和美洲等国家中相继出现，目前已广泛分布于全球各地。在此背景下，本文综述了非洲猪瘟病毒的病原学、流行病学、诊断及防控措施等方面的最新研究进展，以期为非洲猪瘟的防控提供理论参考。","container-title":"中国动物保健","ISSN":"1008-4754","issue":"5","language":"zh-CN","page":"117-118+121","source":"CNKI","title":"非洲猪瘟流行病学与防控措施研究进展","volume":"25","author":[{"family":"韦","given":"雪华"},{"family":"刘","given":"存"},{"family":"王","given":"贵升"},{"family":"孙","given":"圣福"},{"family":"厉","given":"磊"},{"family":"兰","given":"邹然"},{"family":"李","given":"云岗"}],"issued":{"date-parts":[["2023"]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(韦雪华等，2023)。非洲猪瘟在自然情况下，主要有四个独立的流行病学循环：“森林循环”、“软蜱-猪循环”、“家猪-家猪循环”、“野猪-家猪循环”ADDINZOTERO_ITEMCSL_CITATION{"citationID":"bkMlGyNY","properties":{"formattedCitation":"(Brown&Bevins,2018)","plainCitation":"(Brown&Bevins,2018)","noteIndex":0},"citationItems":[{"id":668,"uris":["/users/9215574/items/RS6Q9WRY"],"itemData":{"id":668,"type":"article-journal","abstract":"Africanswinefever(ASF)iscausedbyAfricanswinefevervirus(ASFV),whichcancausesubstantialmorbidityandmortalityeventsinswine.Theviruscanbetransmittedviadirectandindirectcontactswithinfectedswine,theirproducts,orcompetentvectorspecies,especiallyOrnithodorosticks.AfricaandmuchofEasternEuropeareendemicforASF;aviralintroductiontocountriesthatarecurrentlyASFfreecouldhavesevereeconomicconsequencesduetothelossofproductionfrominfectedanimalsandthetraderestrictionsthatwouldlikelybeimposedasaresultofanoutbreak.WeidentifiedvulnerabilitiesthatcouldleadtoASFVintroductionorpersistenceintheUnitedStatesorotherASF-freeregions.Bothlegalandillegalmovementsofliveanimals,aswellastheimportationofanimalproducts,byproducts,andanimalfeed,poseariskofvirusintroduction.Eachrouteisdescribed,andcurrentregulationsdesignedtopreventASFVandotherpathogensfromenteringtheUnitedStatesareoutlined.Furthermore,existingASFVresearchgapsarehighlighted.LaboratoryexperimentstoevaluatemultiplespeciesofOrnithodorosticksthathaveyettobecharacterizedwouldbeusefultounderstandvectorcompetence,hostpreferences,anddistributionofcompetentsofttickvectorsinrelationtohighpigproductionareasaswellasregionswithhighferalswine(wildboarorsimilar)densities.Knowledgerelativetoantigenicviralproteinsthatcontributetohostresponseanddeterminationofimmunemechanismsthatleadtoprotectionarefoundationalinthequestforavaccine.Finally,samplingofillegallyimportedandconfiscatedwildsuidproductsforASFVcouldshedlightonthetypesofproductsbeingimportedandprovideamoreinformedperspectiverelativetotheriskofASFVimportation.","container-title":"FrontiersinVeterinaryScience","DOI":"10.3389/fvets.2018.00011","ISSN":"2297-1769","journalAbbreviation":"Front.Vet.Sci.","language":"en","page":"11","source":"DOI.org(Crossref)","title":"AReviewofAfricanSwineFeverandthePotentialforIntroductionintotheUnitedStatesandthePossibilityofSubsequentEstablishmentinFeralSwineandNativeTicks","volume":"5","author":[{"family":"Brown","given":"ViennaR."},{"family":"Bevins","given":"SarahN."}],"issued":{"date-parts":[["2018",2,6]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(BrownVRetal.,2018)。1.1.3ASFV的临床症状及病理变化ASFV感染宿主后，以内脏器官广泛、严重的出血和发热为主要症状。其感染潜伏期一般为3～15天，临床症状上可表现为多种方式，从无症状死亡（超急性，死亡率约100%）到无症状感染，其中急性感染的患猪表现为精神沉郁、高热、厌食、嗜睡、腹泻或便秘、腹痛、皮肤上有广泛出血点、脾脏、淋巴结等免疫器官水肿，7至10天内死亡；亚急性感染的患猪表现为发热、走路困难，一般在3至4周内发生死亡或恢复；慢性感染的患猪的特征是间歇性或低热，食欲不振，生长迟缓、咳嗽、呼吸困难。持续感染的动物，如亚临床或慢性感染的猪，可能导致流行地区的疾病持续性ADDINZOTERO_ITEMCSL_CITATION{"citationID":"tVwmk8uS","properties":{"formattedCitation":"(\\uc0\\u20309{},2021)","plainCitation":"(何,2021)","noteIndex":0},"citationItems":[{"id":679,"uris":["/users/9215574/items/GKS8N3BR"],"itemData":{"id":679,"type":"article-journal","abstract":"非洲猪瘟(Africanswinefever,ASF)是由非洲猪瘟病毒(Africanswinefevervirus,ASFV)引起的一种急性动物传染病,且有高度接触传染性、急性和出血性等特征,病情传播不受时间和种类的限制,其病死率高达100%。目前针对非洲猪瘟还未研究出有效的治疗方法及疫苗防控,对我国养猪业造成巨大破坏,影响经济效益。非洲猪瘟病毒是非洲猪瘟病毒科非洲猪瘟病毒属的唯一种类,是已知存在的首个虫媒病毒。非洲猪瘟病毒粒子为260～330nm,是具有囊膜结构的二十面体双链线性DNA大分子病毒,基因组长度为170～193kb,包含有150～167个开放阅读框,猪瘟病毒能够编码68种结构蛋白和100多种非结构蛋白。其中已知功能的病毒编码蛋白约有50个,仍有一半以上的ASFV编码蛋白功能尚未探究清楚。非洲猪瘟病毒主要寄宿于家猪和野猪体内,主要在巨噬细胞胞质中进行复制,能够调控IFN产生、炎症反应、细胞调亡、自噬及宿主蛋白合成等生物学过程,影响宿主细胞正常的生长周期及细胞分泌物的生成,进一步抑制宿主细胞体内的天然免疫应答系统。综述非洲猪瘟病毒编码蛋白种类及致病机理研究现状,为非洲猪瘟病毒结构研究及防治方法提供参考。","container-title":"畜禽业","DOI":"10.19567/ki.1008-0414.2021.08.017","ISSN":"1008-0414","issue":"8","language":"zh-CN","page":"29-32","source":"CNKI","title":"非洲猪瘟病毒特征及致病机理研究","volume":"32","author":[{"family":"何","given":"顺宇"}],"issued":{"date-parts":[["2021"]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(何顺宇，2021)。ASFV侵入宿主后首先感染血液中的单核细胞和巨噬细胞，然后扩散到淋巴器官、脾脏、肝脏、肾脏和其他器官。ASFV的主要攻击目标为单核巨噬细胞系统的细胞，这些细胞类型多样，涵盖血液中的单核细胞、巨噬细胞以及树突状细胞，同时还包括内皮细胞等ADDINZOTERO_ITEMCSL_CITATION{"citationID":"thnLMp6Y","properties":{"formattedCitation":"(Cui\\uc0\\u31561{},2014)","plainCitation":"(Cui等,2014)","dontUpdate":true,"noteIndex":0},"citationItems":[{"id":477,"uris":["/users/9215574/items/8HA5EQGK"],"itemData":{"id":477,"type":"article-journal","container-title":"HumanVaccines&Immunotherapeutics","DOI":"10.4161/21645515.2014.979640","ISSN":"2164-5515,2164-554X","issue":"11","journalAbbreviation":"HumanVaccines&Immunotherapeutics","language":"en","page":"3270-3285","source":"DOI.org(Crossref)","title":"Mechanismsandpathwaysofinnateimmuneactivationandregulationinhealthandcancer","volume":"10","author":[{"family":"Cui","given":"Jun"},{"family":"Chen","given":"Yongjun"},{"family":"Wang","given":"HelenY"},{"family":"Wang","given":"Rong-Fu"}],"issued":{"date-parts":[["2014",11,2]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(Cuietal.,2014)。ASFV感染宿主后会引起宿主明显的生理机能上的受损以及代谢紊乱，表现为凝血能力下降从而引起机体严重的出血、肺水肿、肾脏损伤等。对感染ASFV的猪进行解剖，可观察到内脏器官表面有广泛的出血点和出血斑，以脾脏呈紫黑色，边缘梗死，高倍充血肿大和淋巴结充血肿大为主要症状。大多数的病猪伴有肾脏病变，表面可见淤血点和肾盂出血；有的还存在严重肺水肿、肺炎；肝脏严重出血，全身淋巴结严重出血肿大，呈“大理石”样；有的可观察到心包积液、腹水和多灶性水肿。还可能导致怀孕母猪流产、死胎、不孕ADDINZOTERO_ITEMCSL_CITATION{"citationID":"thnLMp6Y","properties":{"formattedCitation":"(Cui\\uc0\\u31561{},2014)","plainCitation":"(Cui等,2014)","dontUpdate":true,"noteIndex":0},"citationItems":[{"id":477,"uris":["/users/9215574/items/8HA5EQGK"],"itemData":{"id":477,"type":"article-journal","container-title":"HumanVaccines&Immunotherapeutics","DOI":"10.4161/21645515.2014.979640","ISSN":"2164-5515,2164-554X","issue":"11","journalAbbreviation":"HumanVaccines&Immunotherapeutics","language":"en","page":"3270-3285","source":"DOI.org(Crossref)","title":"Mechanismsandpathwaysofinnateimmuneactivationandregulationinhealthandcancer","volume":"10","author":[{"family":"Cui","given":"Jun"},{"family":"Chen","given":"Yongjun"},{"family":"Wang","given":"HelenY"},{"family":"Wang","given":"Rong-Fu"}],"issued":{"date-parts":[["2014",11,2]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}(LiZetal.,2014)。1.1.4ASFV的防控措施由于病毒本身庞大的基因组及其调节宿主免疫反应的复杂机制，仍然没有开发出有效的药物和疫苗。目前主要以生物安全建设及疾病净化作为有效防控手段，包括病毒的监测和报告、病猪的隔离和扑杀、定期清洁和消毒猪舍和设备、防止野猪和其他野生动物的入侵，避免使用可能受污染的饲料和水、控制猪场的人员和物资流动、猪肉的检疫和运输控制以及加强ASF的监测和报告。1.2研究目的与意义ASF于1921年被发现，距今已有百年历史，对人类健康和经济发展造成严重威胁，2018年我国首次暴发ASF，给全国养猪业造成重大损失。由于ASFV的高度变异性和多样性以及研发疫苗的各种技术难题，能够应用于临床的有效商业化疫苗仍在研制中。由于原代细胞的制备具有难度较大，成本较高，批次差异大的问题，缺乏高效稳定的细胞系是阻碍有效疫苗研发的重要原因之一，因此探索新的可用细胞系是必要的。同时探究影响ASFV对培养细胞感染性因素是理解其致病机理、评估其传播风险、开发可用疫苗的有效手段。2材料与方法2.1主要实验材料与主要仪器2.1.1主要材料表1实验材料名称提供商ASFVGZ/2018毒株3D4/21细胞系PK15细胞系G1/S期抑制剂Nocodazole华南农业大学兽医学院传染病教研室华南农业大学兽医学院传染病教研室华南农业大学兽医学院传染病教研室Sigma公司G2/M期抑制剂PanobinostatVincristineSulfate谷氨酰胺抑制剂BPTES乙酰辅酶A羧化酶-α的变构抑制剂脂肪酸合成酶抑制剂C75糖酵解抑制剂2-Deoxy-D-glucose澳洲级别胎牛血清（FBS）RPMI1640培养基高糖DMEM培养基Nunclon™Sphera™细胞培养皿CellCountingKit-8Sigma公司Sigma公司MCE公司MCE公司MCE公司MCE公司ThermoFisherScientific公司ThermoFisherScientific公司ThermoFisherScientific公司ThermoFisherScientific公司新赛美公司2.1.2主要仪器表2主要仪器名称公司二氧化碳细胞培养箱Thermo生物工程有限公司细胞培养板细胞培养瓶DMIL倒置荧光显微镜T3000ThermocyclerPCR仪普通离心机紫外分光光度计PowerPacTM基础电泳仪Corning公司Corning公司Leica公司WhatmanBiometra公司eppendorf公司Thermo公司Bio-Rad公司2.2方法2.2.1细胞制备与培养将4周龄仔猪肺泡经多次灌洗后得到PAMs，经1640培养基洗涤后离心后接种到T25细胞培养瓶内，放入含有CO2的37°C培养箱；将含1mL3D4/21和PK15细胞悬液冻存管置于37°C水浴上迅速晃动融化后，将其移至5mL的离心管中，充分均匀混合。随后，以1000rpm的转速进行离心3分钟。离心完成后，移除上清液，接着使用含有10%FBS的新鲜培养基对细胞进行重悬处理，以确保细胞在培养基中均匀分布。接着，将这些细胞接种至培养瓶中，随后置于37°C恒温培养箱中进行培养。次日，利用显微镜对细胞的密度与生长情况进行仔细观察，以判断细胞的生长状态。2.2.2病毒感染（1）将PAMs、3D4/21以及PK15细胞接种至细胞培养皿中，随后将培养皿置于37℃、含有5%CO2的恒温培养箱中培养一天。（2）对细胞进行观察，当细胞达到适当的生长密度后，将其1MOI的ASFV加入到细胞培养皿中，并轻轻摇动以确保其在培养基中均匀分布后，将培养皿放入37℃培养箱中培养2h。（3）为了去除未吸附的病毒，我们使用PBS进行了三次洗涤操作。随后，将PAMs细胞置于含有10%FBS的RPMI1640培养基中。同时，3D4/21和PK15细胞则分别放置于含有10%FBS的DMEM培养基中，以提供适宜的生长环境。2.2.3感染细胞DNA样品的获取使用FastPureViralDNA/RNAMiniKit试剂盒提取ASFV感染PAMs、3D4/21、PK15细胞的DNA/RNA样品，用于后续实验。大致步骤如下：（1）首先往离心管中加入200μL的样本，随后沿管壁缓慢加入500μL的BufferVL溶液，并轻柔地上下颠倒，使两者充分混合。（2）接着，将这一混合液体小心地转移至试剂盒内附带的2mL收集管中。随后，以12,000rpm的转速进行1分钟的离心操作，弃去滤液。（3）向离心管中加入600μLBufferRW，12,000rpm(13,400g)离心30sec，随后弃去滤液。（4）重复上述步骤3的操作。（5）接着，不加入其他溶液空柱12,000rpm(13,400×g)离心2min。（6）小心地取出FastPureRNAColumns，将其转移至试剂盒里提供的新的1.5mL管中，悬空加入30μL试剂盒提供的ddH2O至膜中央，室温环境下静置1min，随后在12,000rpm的条件下离心1min。（7）吸取下层液体用于后续实验，或在-30℃~-15℃条件下短期保存或者在-85℃~-65℃条件下长期保存。2.2.4ASFV全基因转录谱分析（1）通过步骤2.2.3获得感染ASFV的PAMs、PK15和3D4/21总RNA，用逆转录酶将其反转录为cDNA，再通过PCR获得其全基因组。（2）我们将获得的数据与NCBI上的参考序列GCF_000003025.6_Sscrofa11.1（NCBI）进行比对分析。（3）通过比较各组的差异表达基因（DEGs）的差异表达分析。p≤0.05且Foldchange>2的单基因可认定是显著的差异表达。（4）为进一步研究，我们利用BGI公司的Dr.Tom多组学数据挖掘工具，成功地分析了聚类热图和KEGG通路，并呈现了差异性基因的研究成果。（5）最后通过GO、京都基因和KEGG数据库对差异表达基因进行功能注释以及通路分析。2.2.5悬浮培养将含有3D4/21和PK15的细胞悬液轻柔地吹打混匀，然后全部加入含培养基的3D培养板中，将其置于培养箱中进行培养。通过显微镜持续观察细胞生长状况，待细胞生长至合适密度时进行传代处理，从培养板中吸取适量细胞悬液，在1000r/min条件下离心5min，随后去除上清液并加入新鲜且清洁的悬浮培养液，加入培养板中混合均匀，放回至培养箱中继续培养。生长良好状态下以MOI=1将ASFV接种至细胞中，观察细胞状态并在一定时间内取样进行后续实验。2.2.6细胞周期/代谢的调节与感染为了研究3种细胞周期药物和4种代谢药物对3D4/21和PK15细胞的毒性，我们通过CellCountingKit-8(CCK8)试剂盒将不同浓度的药物接种于细胞。首先将细胞铺在96孔板中（100μL/孔），然后将孔板放入37°C，5%CO2的培养箱中培养一段时间，再更换为含有不同浓度的化合物的培养基。接着，将培养板置于培养箱中，进行48小时的孵育。孵育完成后，我们向每个孔中加入10μL的CCK8试剂，用手拍匀。之后，再次将培养板放入培养箱中，进行1至4小时的孵育。最后，通过酶标仪测量了各孔在450nm下的吸光度。若无法即时测定OD值，需在每孔中加入10μL浓度为0.1M的HCL溶液或1%w/v的SDS溶液，随后，将培养板盖好，在室温下避光存放，24小时以内其吸光度值不会发生改变。2.2.7用于检测ASFV基因组拷贝数的实时定量PCR（RealtimefluorescencequantitativePCR,qPCR）（1）设计并合成特异性引物和探针：表3引物序列引物序列正向引物5’-ATAGAGATACAGCTCTTCCAG-3’反向引物探针5’-GTATGTAAGAGCTGCAGAAC-3’5’-FAM-TATCGATAAGATTGAT-MGB-3’（2）准备反应混合液，每个反应总体积为20μL，包括以下组分：表4反应组分t组分体积2×AceQqPCRSYBRGreenMasterMix10.0µLForwardPrimer(10μM)0.4µLReversePrimer(10μM)0.4µL50×ROXReferenceDye10.4µLTemplateDNA/cDNAddH2O2.0μL7.2μL（3）按下列条件进行qPCR反应：表5反应程序步骤温度时间循环次数预变性95℃30sec1变性95℃10sec40退火60℃30sec40溶解曲线95℃/15s，60℃/60s，95℃/15s2.2.8病毒滴度测定使用HAD50法测定ASFV的滴度，步骤如下：（1）铺细胞：在96孔板中接种消化重悬后的悬浮培养的3D4/21和PK15细胞培养至单层细胞。（2）病毒的稀释：首先将待检测的ASFV病毒溶液加入到不含血清的DMEM培养基中，接着按照10倍的浓度梯度进行稀释处理。（3）感染细胞：首先弃去细胞原有的培养基，然后以每孔100µL的量，将稀释后的病毒液分别加入96孔板中，对每个稀释度均设置8个重复孔，作为阴性对照，再选取8个孔，仅加入不含血清的DMEM培养基。（4）随后，每个孔中加入10µL10%的新鲜猪红细胞。（5）观察培养：把96孔板放置于细胞培养箱中培养，通过倒置显微镜密切观察红细胞吸附现象，并详细记录观察结果。（6）计算病毒滴度：ASFV的HAD50通过Reed-Muench法得出。2.2.9目的蛋白Western-blotWB实验的步骤如下：（1）收集细胞：将感染ASFV的悬浮培养的3D4/21和PK15细胞分别吸取并转移到1.5mL的离心管中，用PBS溶液洗涤两次，然后在1000g的条件下离心5min，并将上清液弃去。（2）细胞的裂解：首先，向每个