伪狂犬病毒诱导宿主炎症小体激活及IL-1β分泌的机制研究

Recently an emergent pseudorabies (PR) has been widespread in China. However, study on pathogenesis of pseudorabies virus (PRV) and mechanism of immune response induced by PRV is very lacking. Inflammasomes play important roles in inducing host innate immune responses and antiviral effects. We have previously found that the PRV epidemic strain could induce Caspase-1-dependent IL-1β secretion, but gE/gI gene-deleted vaccine strain could not induce the corresponding immune response. This project aims to study the mechanism of PRV-induced inflammasome activation, Caspase-1 activation and IL-1β maturation and secretion. The activation and antiviral effects of inflammasome during PRV infection will be studied, and the activation of inflammasome receptor molecule, adaptor protein and Caspase-1 and also the secretion of IL-1β will be analysed, by using C57BL/6 wild-type and inflammasome-associated genes knockout mice and macrophages, in vivo and in vitro, respectively. Furthermore, the gE/gI gene-deleted and -revertant PRV epidemic strains constructed by CRISPR/Cas9 technology will be used to identify the role of gE/gI proteins for PRV-induced inflammasome activation. This study will lay a theoretical foundation for research on immune prevention and control of the emergent PR, and also provide new ideas for study on inflammation-related mechanisms of other herpesviruses.

近年新发伪狂犬病在我国广泛肆虐，但伪狂犬病毒（PRV）致病和免疫应答的机制研究较为缺乏。炎症小体在宿主天然免疫应答和抗病毒效应中起重要作用。我们前期发现PRV流行株能诱导半胱天冬酶-1（Caspase-1）依赖的IL-1β分泌，但gE/gI基因缺失的疫苗株不能诱导相应免疫应答。本项目拟探究PRV诱导宿主炎症小体激活、Caspase-1活化及IL-1β成熟与分泌的机制。用PRV感染C57BL/6野生型和炎症小体相关基因敲除小鼠的体内外模型，研究在PRV感染中炎症小体的激活及其抗病毒作用，并分析炎症小体受体分子、接头蛋白及Caspase-1活化和IL-1β分泌；进一步通过CRISPR/Cas9技术构建PRV流行株gE/gI基因的敲除和回复病毒，验证gE/gI蛋白在PRV诱导炎症小体激活中的作用。本研究将为新发伪狂犬病的免疫防控研究奠定理论基础，也为其他疱疹病毒的炎症相关机制研究提供新的思路。

伪狂犬病毒（PRV）是一种α疱疹病毒，其感染猪可导致仔猪神经炎和急性死亡，怀孕母猪生殖障碍，成年猪出现呼吸道症状，是威胁生猪产业的重要病原。近年，新发伪狂犬病在我国广泛肆虐，给养猪业造成重大经济损失，但伪狂犬病毒（PRV）感染致病和诱导宿主免疫应答的相关分子机制研究尚较为缺乏。本项目选取PRV感染与宿主炎症小体激活之间关系这一角度开展研究，从PRV诱导宿主促炎细胞因子IL-1β表达和分泌、PRV诱导宿主炎症小体激活以及促进IL-1β分泌机制、炎症小体激活在宿主抵御PRV感染中的作用这三个方面探讨PRV诱导宿主炎症小体激活及IL-1β分泌的分子机制。ELISA结果表明，PRV感染小鼠巨噬细胞能诱导IL-1β等多种促炎细胞因子产生。进一步，通过Western-blot实验研究发现，PRV感染诱导IL-1β表达依赖于NF-κB典型通路的激活。此外，ATP能够促进PRV感染巨噬细胞炎症小体激活以及IL-1β的显著分泌，而且研究结果表明NLRP3炎症小体参与调控PRV诱导IL-1β的分泌。小鼠感染实验结果显示，ATP促进的炎症小体激活会显著增强PRV对小鼠的致病性，表明炎症小体激活等相关宿主炎症反应对PRV感染致病具有重要影响。今后可以聚焦PRV引起宿主炎症反应的分子机制这一研究方向进行深入研究，并针对病毒致炎的分子路径开展特异性靶向治疗研究，为伪狂犬病免疫治疗药物开发提供新思路。