猪瘟病毒强毒株和兔化弱毒疫苗株调控干扰素应答差异的分子机制

猪瘟病毒（CSFV）强毒株和猪瘟兔化弱毒疫苗株（C株）在调控干扰素（IFN）应答方面存在明显差异，其分子机制尚不明确。此前我们发现，CSFV强毒和弱毒编码蛋白（如NS3和E2）与天然免疫分子相互作用及其介导IFN的应答存在差异。基于此，本项目利用天然免疫相关分子荧光素酶报告系统高通量筛选和鉴定调控IFN应答差异的CSFV蛋白；通过免疫共沉淀、GST pulldown和荧光素酶活性试验等，确定病毒蛋白与天然免疫分子的相互作用及其对IFN信号通路的调控；通过构建病毒蛋白嵌合突变体和丙氨酸扫描，鉴定影响强毒和弱毒诱导IFN应答差异的关键区域或位点，进一步利用反向遗传操作平台构建突变体病毒，分别在细胞和动物水平比较CSFV突变体病毒、强毒及弱毒诱导IFN应答、病毒复制和致病性的差异。本项目可望揭示CSFV强毒和弱毒调控IFN应答差异的分子机制，并为阐明C株致弱机制提供科学数据。

There exist distinctive differences in the interferon (IFN) response regulated by highly virulent classical swine fever virus (CSFV) and C-strain, a lapinized attenuated vaccine against classical swine fever. However, the underlying molecular mechanisms remain unclear. Previously, we demonstrated that highly virulent CSFV Shimen strain and C-strain show differences in the interactions between major viral proteins (such as NS3 and E2) and innate immunity-related signalling molecules, resulting in differential modulation of the IFN response. This proposal is aimed to: 1) screen and identify viral proteins that are involved in the IFN response using luciferase reporters harboring innate immunity-related molecules; 2) investigate functional aspects of the identified viral proteins interacting with the molecules of innate immunity and the IFN signalling pathway by co-immunoprecipitation, GST pulldown, colocalization and luciferase reporter gene assays; 3) construct a series of chimeric viruses containing the corresponding region(s) of C-strain based on the Shimen strain backbone to map the key region(s) or site(s) responsible for the interactions between the identified viral proteins and innate immunity-related molecules in the IFN signalling pathway; 4) generate a series of chimeric viruses, in which the key region(s) or site(s) was mutated, and evaluate the IFN induction, viral replication, and pathogenicity of the chimeras in macrophages, dendritic cells and pigs. This project is expected to uncover the molecular mechanisms of the differential IFN response regulated by CSFV with different virulence, and provide scientific data for elucidating mechanisms underlying the attenuation of C-strain.