猪萨佩罗病毒受体的筛选验证和互作配体的关键位点鉴定

In 2010, sapelovirus was firstly isolated from pig farms outbreaking with encepholomylities. Following epidemiology investigation showed that sapelovirus infectivity could be as high as 40% in eastern pig farms. Sapelovirus cause severe and mild neurological disorders, reproductive failure, pneumonia, and diarrhea, posing a serious threat to the pig industry. And, the key point of exploring PSV invasion, host cell spectrum and tissue tropism was the molecular model on how sapelovirus virion attaching, binding and entering host cells. It helps finding out sapelovirus replication and pathogenic mechanism. So, we intend to focus on screening and identifying receptors of sapelovirus from host cell surface by Co-IP and Pull-Down, and reconstructing sapelovirus infectious clone with mutants lied on spike ligands. They are usefull in verifing the ligand and receptor interaction model during virus infection, pointing out the key sites of ligands that correlated with receptors, revealing cell tropism and cytopathic effect changes of mutants, and exploring the virulence and antigenicity of mutants. This will provide further information on sapelovirus intruding, infection, immunity and pathogenicity.

2010年我们首次从国内爆发脑脊髓炎的猪群中分离培养出萨佩罗病毒（Porcine Sapelovirus， PSV），随后的流行病学调查显示，华东地区猪群PSV感染率高达40%。PSV可引起猪肠道紊乱、呼吸道疾病、生殖障碍和脑脊髓炎等多系统综合征，对养猪业构成严重威胁。研究病毒粒子吸附感染宿主细胞的受体和配体互作分子模式，探明PSV感染的入侵方式、宿主细胞范围及组织嗜性等，可以深入了解PSV在猪体内的复制和致病机理。本研究拟应用免疫共沉淀、Pull-Down筛选鉴定宿主细胞表面PSV受体，通过改造PSV感染性克隆构建配体氨基酸序列突变体，验证病毒吸附、结合、感染的配体受体互作分子模式，定位与受体作用的配体关键位点，进而揭示PSV克隆突变体细胞嗜性和细胞病变变化，探讨突变体毒力和抗原性变化。本研究对深入阐明PSV入侵与感染、免疫与致病等科学问题具有重要意义。

猪萨佩罗病毒(porcine sapelovirus, PSV)属于小RNA病毒科，萨佩罗病毒属，是单股正链、无囊膜的RNA病毒。PSV感染能引起多种症状，包括急性腹泻、脊髓灰质炎、肺炎和生殖障碍等，给养猪业带来巨大危害。目前PSV的研究主要集中在病毒的基因组特征和流行病学方面，但该病毒是如何入侵其宿主细胞及在宿主细胞内进行复制的等机制还没有被报道。因此，本研究以PSV感染PK15为研究对象，采用多种方法验证PSV入侵细胞依赖小窝蛋白/脂筏介导的内吞途径，而不依赖于网格蛋白和巨胞饮途径，并发现PSV感染PK15细胞能引起细胞微丝骨架重排并依赖Rab7和Rab11。此外，通过蛋白质组学的方法，我们分析了PSV感染PK15细胞前后的差异表达蛋白，共鉴定出181个差异表达蛋白，其中包括59个上调差异表达蛋白和122个下调差异表达蛋白，发现过表达VCL或干扰SDC1能显著降低病毒VP1蛋白表达水平，而干扰VCL或过表达SDC1则能增加VP1蛋白表达水平，表明VCL能抑制PSV在PK15细胞的增殖，而SDC1能促进PSV在PK15细胞的增殖。该研究为揭示和认识PSV与宿主细胞感染与入侵，致病与传播等互作机制提供借鉴和基础依据。.关键词：萨佩罗病毒，入侵，病毒复制