AIM2/NLRP3炎症小体在非洲猪瘟病毒诱导并调控宿主炎症反应中的功能研究

African swine fever(ASF), a fatal animal infectious disease caused by African swine fever virus(ASFV), is currently posing a severe threat to Chinese pig industry ever since the first report in August 2018. Since all the traditional strateges in ASF vaccine development have been proven to be failed, the elucidation of the infection and pahogenic mechanisms becomes therefore urgent to provide new power source for ASF prevention and control. During the past two decades, inflammasome mediated inflammation triggered by the host, and the evasion of this defense mechanism developed by the pathogens have been shown to play vital roles in virus pathogenesis. Due to the fact of increased IL-1β secretion detected in ASFV-infected pigs, it is very likely that the host recognizes the ASFV via AIM2&NLRP3 inflammasomes which are proven to present the ability to sense cytosolic viral genomic DNA, and further triggers the IL-1β secretion and pyroptosis, inducing the comprehensive inflammation. On the other hand, as the ASFV targets the monocyte-macrophage system that is the major source for inflammation initiation, it should have developed strateges to antagonise, espetially in the early phase of infection, the inflammation signaling cascade to ensure its own survival. In the proposed research, we designed several experiments to certify the hypothesis raised above by various levels. Firstly, using the SY-18 strain of ASFV that we isolated from the materials collected from the ASF outbreak we firstly reported in 2018 as a model strain, we propose to investigate the dynamic characteristics of AIM2/NLRP3 inflammasome mediated IL-1β secretion and pyroptosis both in vitro and in vivo, providing evidence for the rise of inflammasome associated inflammation in host after ASFV infection. In addition, we also propose to screen the potential of the 166 putative ASFV genes to suppress the inflammasome activation and signaling in an activation model constructed by treatment of the primary macrophage with specific stimuli, and further to construct recombinant ASFV lacking the genes screened to confirm their potentials of inflammasome inhibition and to clarify the precise mechanisms how they work. The prospective results derived from the proposed research could largely add new knowledge, in a special perspective of inflammation, on understanding the host-virus interaction and viral pathogenesis, which may provide insights in novel tactics for ASF vaccine development.

非洲猪瘟是一种由非洲猪瘟病毒（ASFV）引起的烈性动物疫病，严重威胁我国养猪业。炎症小体介导的炎症反应是宿主抵抗病毒感染的重要机制。家猪很可能通过单核巨噬细胞中AIM2/NLRP3炎症小体识别ASFV产生炎症反应，而ASFV为了逃逸宿主的炎症反应很可能在感染的不同时期通过自身蛋白调控了宿主炎症小体的激活。为论证这一假说，申请人拟开展以下研究：以本实验室分离到的SY-18株为模式毒株，从体内体外分析AIM2/NLRP3炎症小体介导的IL-1β分泌及细胞焦亡随感染过程的动态变化特征；建立AIM2/NLRP3炎症小体激活模型，筛选ASFV全部166个蛋白对炎症小体的抑制能力，并进一步通过构建相应蛋白缺失或敲低毒株，验证分析ASFV调控炎症小体介导的炎症反应的分子机制。本研究将从炎症反应角度为明确宿主与ASFV相互对抗的分子致病机制提供新的知识，并为构建基因缺失弱毒疫苗的靶基因选择提供理论支撑。

非洲猪瘟(ASF)严重威胁我国养猪业，病毒的致病机制尚不清晰。非洲猪瘟病毒（ASFV）可能通过炎症小体诱导IL-1b分泌，引发炎症反应，参与ASFV的致病过程。我们通过动物实验分析了体内感染SY18强毒株后，病毒引发的炎症反应状态动态变化过程，结果发现ASFV可迅速诱导家猪产生以IL-1b、TNF-a、IL-6、IFN-a为代表的促炎细胞因子显著上升，而抑制抑炎因子的表达，从而诱发细胞因子风暴，导致家猪的直接死亡。我们进一步在巨噬细胞上构建了体外炎症小体活化平台，通过共表达ASFV结构蛋白筛选ASFV对NLRP3炎症小体具有抑制能力的基因，结果未发现单独可以抑制炎症小体通路的ASFV结构蛋白。我们通过敲除基因簇的方式，发现ASFV L7-L11结构基因串敲除后，可以反转ASFV诱导的炎症因子风暴，诱发强烈的IL-1ra分泌，从而降低ASFV的毒力，进一步的攻毒保护试验发现该基因缺失毒株可以提供对亲本毒株的100%保护。我们通过口服感染模型评价了干扰素Ⅰ/Ⅲ型复合制剂对ASFV的预防作用，结果发现干扰素Ⅰ/Ⅲ型复合制剂可以延缓ASFV的感染过程减轻炎症损伤程度，但并不能逆转ASFV诱导的炎症因子风暴。本项目从炎症反应角度首次证明了ASFV通过炎症细胞因子风暴造成家猪系统性组织病例损伤乃至死亡。发现L7-L11基因串的缺失可显著降低病毒的诱导的炎症反应，可以作为基因确实疫苗开发的候选基因。发现干扰素Ⅰ/Ⅲ型复合制剂可以延缓ASF疾病进程。这些研究结果为非洲猪瘟的疫苗开发和临床防控提供了新的选择。