非洲猪瘟病毒激活巨噬细胞炎症小体的分子机制

非洲猪瘟是由非洲猪瘟病毒（ASFV）引起的一种烈性传染病，严重危害全球养猪业。巨噬细胞是ASFV的靶细胞。ASFV在体内外均可诱导巨噬细胞促炎性细胞因子IL-1β和IL-18的上调表达，这是炎症小体活化的标志。但ASFV感染如何激活炎症小体尚不明确。申请人近期通过单细胞测序意外发现，ASFV感染巨噬细胞后上调炎症小体相关分子IFI16及其下游分子IL-1β的表达。据此推测，ASFV可能通过IFI16通路激活炎症小体。本项目拟利用炎症小体通路阻断、RNA干扰和免疫共沉淀试验明确ASFV激活炎症小体的信号通路；通过炎症小体体外重构系统确定调控炎症小体活化的病毒蛋白；用CRISPR/Cas9技术构建调控其活化的基因缺失突变体病毒，分别在猪巨噬细胞和SPF猪上比较突变体与亲本病毒激活炎症小体、病毒复制和致病性的差异。本项目将揭示ASFV激活炎症小体的分子机制，为快速构建非洲猪瘟减毒疫苗提供新靶标。

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating disease of domestic pigs and wild boar, posing a major threat to the global pig industry. In vitro and in vivo experiments have indicated that ASFV infection induces transcriptional upregulation of proinflammatory cytokines including interleukin (IL)-1β and IL-18 (the biomarkers of inflammasome activation) in macrophages, the primary target cells for ASFV. However, the molecular mechanism underlying the inflammasome activation induced by ASFV infection remains to be elucidated. Interestingly, we have recently demonstrated that the expression of interferon gamma-inducible protein 16 (IFI16), a critical molecule associated with the inflammasome pathways, is increased in ASFV-infected macrophages using single-cell transcriptomic analysis. In addition, we have also shown that IL-1β, a key downstream molecule of the inflammasome pathways, is increased during ASFV infection. Thus, we speculate that IFI16 may be involved in the activation of inflammasome induced by ASFV infection. In this proposal, we will: 1) define the inflammasome pathway induced by ASFV infection using inhibitor blocking, RNA interference and co-immunoprecipitation assays; 2) identify the viral protein(s) that induces the inflammasome activation using in vitro inflammasome reconstruction system; 3) generate a series of ASFV mutants lacking the corresponding region(s) or site(s) responsible for the inflammasome activation and evaluate the inflammasome activation, viral replication and pathogenicity of the mutants in comparison with the parental virus in porcine macrophages and specific-pathogen-free pigs. This project is expected to dissect the molecular mechanism underlying the ASFV-induced inflammasome activation and provide novel targets for ASFV attenuation.