【作者】 陈蕾；

【导师】 才学鹏；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 小反刍兽疫（Peste des petitus ruminants，PPR）的流行加剧了欠发达国家的贫困，严重影响了动物健康与食品安全，联合国粮农组织（Food andAgriculture Organization of the UnitedNations, FAO）于2012年提出开启全球范围的多国家多地区全方位合作，从而控制和消灭PPR。然而，小反刍兽疫病毒（Peste des petitus ruminants virus，PPRV）的基础研究还很薄弱，近年来PPRV跨地区跨国界传播以及感染野生动物，给全球消灭计划提出了严峻挑战。基于对同属麻疹病毒（Measles virus, MV）的研究证实，与受体的相互作用，不仅是病毒入侵宿主的第一步，也是导致宿主机体组织病理变化分布、宿主呈现临床症状表现的重要原因。下面几个问题值得关注：PPRV除了同MV一样利用SLAM作为受体开启病毒的入侵，是否也同样利用宿主呼吸道等上皮细胞基底层的Nectin4分子作为受体从而介导子代病毒的排出？PPRV疫苗株不具备向外“排毒”的特点，是否因为其不能与Nectin4结合？PPRV利用病毒囊膜上的血凝素蛋白（Hemaggluntinin, H）特异性识别宿主受体，H能否单独决定PPRV的宿主嗜性？运用计算机模拟对PPRV野毒株H蛋白（Hw）、疫苗株H蛋白（Hv）分别与羊源SLAM/Nectin4的相互作用模式、关键区域、关键氨基酸等做了预测，并设计试验进行了验证，结果如下：1. Hw和Hv的重组真核表达质粒均能与绵羊Nectin4的重组真核表达质粒共定位于CHO、293T细胞的细胞质中；Co-Ip确认Hw和Hv蛋白均能与绵羊Nectin4蛋白相互作用；荧光定量PCR显示Nectin4主要在绵羊的呼吸道、消化道等上皮组织中表达，结合本课题组以往人工感染PPRV的病毒载量、组织嗜性，以及绵羊SLAM在各组织中的分布表达来看，PPRV在唇、口腔黏膜、会厌、扁桃体等呼吸道组织和网胃、瓣胃、瘤胃等消化道组织中的分布与Nectin4的表达量成正相关，而PPRV在各种淋巴结、脾脏等免疫相关组织中的分布与SLAM的表达量成正相关；病毒感染实验表明羊源Nectin4可以提高PPRV Neriga75/1株在Vero细胞中的繁殖效率。综上所述，可以得出结论Nectin4是PPRV野毒株和疫苗株利用的受体。2.羊源Nectin4分子胞外区IgV结构域参与了同PPRV Hw、Hv蛋白的相互作用；3.计算机模拟计算得出PPRV Hw和Hv具备与人源SLAM相互作用的分子基础，无论是Hw还是Hv，发生Ser550Trp、Arg191Tyr、Arg191Phe、Thr545Trp、Ser548Trp、Ser534Trp这六种突变后，与人源SLAM相互作用的能力会加强，存在跨种间传播的风险。本研究成功鉴定了PPRV的受体Nectin4，并通过对PPRV野毒株和疫苗株分别与受体SLAM、Nectin4相互作用的模式、机理进行探究，为PPRV疫苗生产和诊断监测均提出了有益的思路和理论参考。更多还原

【Abstract】 The epidemics of peste des petits ruminants (PPR) increase the poverty of undeveloped countriesand threaten animal health and food security heavily, thus was announced to be controlled anderadicated with global efforts by the Food and Agriculture Organization of the United Nations (FAO) in2012. However, the fundamental research on peste des petits ruminants virus (PPRV) is still backwardand the expansion of its distribution, particularly among wild animals greatly challenged the PPReradication project.The interactions between another morbilivirus-measles virus (MV) and its receptors play animportant role in virus infection. We are keen to figure out whether PPRV also use Nectin4to mediatethe shedding of offspring virions? If so, is it because PPRV vaccine strain cannot interact with Nectin4that it doesn’t ‘shed’ offsprings? Dose the hemaggluntinin (H) protein solely determine host specificity?We predicted the interactions between PPRV H wildtype (Hw)／vaccine (Hv) and sheep SLAM/Nectin4, analyzed their interaction mechanisms, determined the crucial domains and amino acids usingmolecular modeling, and finally acquired the following results by experiments:1. Plasmids containing Hw/Hv and sheep Nectin4locate in the cytoplasm of CHO,293T cells; Co-Ipshows both Hw and Hv have interactions with sheep Nectin4; sheep Nectin4mRNAs were expressedmainly in the epithelial tissues of digestive and respiratory tracts which is consistent with the tissuetropism of PPRV in sheep along with the mRNAs expressions of SLAM; transfection of sheep Nectin4can obviously increase the reproduction of PPRV cultured in Vero cell line. All the results aboveconfirm that Nectin4is the receptor for both PPRV wildtype strain and vaccine strain.2. The IgV domain of sheep Nectin4involves in the interaction with PPRV Hw.3. Both PPRV Hw and Hv possess the molecular basis to bind human SLAM, while the mutations ofSer550Trp，Arg191Tyr，Arg191Phe，Thr545Trp，Ser548Trp，Ser534Trp of Hw/Hv would increase theinteraction energies between Hw/Hv and human SLAM.To sum up, we have successfully identified PPRV’s receptor-Nectin4, and analyzed the interactionmodels and mechanisms between PPRH Hw/Hv and receptor SLAM/Nectin4respectively, whichprovide theoretical significance and valuable reference for future development of PPRV diagnosis andvaccines.更多还原