高致病性猪繁殖与呼吸综合征病毒的分子致病机制

Under the support of NSFC, we constructed a differentially expressed gene library of porcine alveolar macrophages (PAM) by SSH method, and obtained the differentially expressed gene spectrum of PAM caused by highly pathogenic PRRSV infection. Using 2D-DIGE technology, 13 differential proteins of PAM which were caused by infection of highly pathogenic PRRSV or classical PRRSV were obtained, and the differential proteins were validated in vivo. To our knowledge, this is the first time to report that PRRSV infection could increase the expression of Annexin A2. Functional analysis suggests that Annexin A2, proteasome and cathepsin B may be involved into the pathogenicity of highly pathogenic PRRSV. . By now, we have trained 1 PhD candidate, 2 graduate students. And 1 person was awarded to the Young Scholar by Chinese Immunology Society. Four SCI papers were published; the cumulative impact factor is 16.25. . Future research will focus on: the enhancement of Annexin A2 in the process of highly pathogenic PRRSV infection; the enhancement of fiber protein dissolving enzyme in the process of replication; the relationship of inflammatory cytokines expression level and proteases cathepsin B. In the area of PRRSV, we will construct chimeric viruses of high pathogenic PRRSV/ classical PRRSV, and detect differential expressed protein levels in infected PAM cells, and detect the pathogenicity of chimeric viruses in piglets, to determine the PRRSV protein (or domain) which was involved in the enhanced pathogenicity. Those findings will be preliminary clarified the molecular pathogenic mechanism of highly pathogenic PRRSV.

在青年基金资助下，构建了高致病性PRRSV感染肺泡巨噬细胞(PAM)的消减杂交文库，获得了差异表达基因谱。利用2D-DIGE技术获得了13条高致病性/经典PRRSV感染PAM的差异蛋白，验证了差异蛋白在仔猪体内的表达。首次发现PRRSV感染后Annexin A2表达上升。功能分析发现Annexin A2、组织蛋白酶B和蛋白酶体等可能参与PRRSV的致病过程。已毕业博士生1人、硕士生2人，获中国免疫学青年学者奖1人。已发表SCI论文4篇，累计影响因子16.25。今后将研究高致病性PRRSV感染复制过程中Annexin A2、纤维蛋白溶酶的促进作用，组织蛋白酶B和蛋白酶体对自身过激免疫的促进作用。构建高致病性/经典PRRSV的嵌合病毒，感染PAM细胞，检测差异蛋白的表达水平，对嵌合病毒进行猪体试验，确定PRRSV中参与致病性增强的蛋白（或结构域），揭示高致病性PRRSV致病性增加分子机制。

高致病性PRRSV 在我国及东南亚国家大面积流行，给养猪业造成了巨大的经济损失，但其致病分子机制尚不清楚。此前，我们发现Annexin A2 (膜联蛋白A2，ANXA2) 在高致病性PRRSV感染后表达升高。本研究开展了高致病性PRRSV与宿主蛋白ANXA2等的相互作用研究、基于PRRSV反向遗传操作的致病性研究。发现了ANXA2与PRRSV的Vimentin受体蛋白结合。单独的ANXA2或Vimentin均不与PRRSV的蛋白结合，只有膜联蛋白A2与 Vimentin蛋白结合后，Vimentin才能和PRRSV蛋白结合。在结合PRRSV蛋白时，仅结合PRRSV的N蛋白，不与PRRSV的其他结构蛋白结合。ANXA2-Vimentin互作在PRRSV的复制过程中发挥重要作用。我们发现PRRSV致病性的高低，与PRRSV在猪体内的复制水平有关。在猪体内复制水平高的PRRSV，其致病性较高，反之亦然。本研究发现，与经典PRRSV相比，高致病性PRRSV在RdRp复制酶中存在规律性的、一致性的2处氨基酸变异位点。在PRRSV反向遗传操作系统上，将经典PRRSV、高致病性PRRSV中的ORF互换，将突变氨基酸进行突变或缺失，并拯救了嵌合/突变病毒。体内、体外试验表明突变病毒的复制效率、致病性均较高致病性PRRSV明显下降、较经典PRRSV有所升高。从而证实了PRRSV RdRp复制酶中规律性氨基酸突变对PRRSV致病性的影响。研究发现高致病性PRRSV 感染后，诱发机体产生大量的炎性因子和趋化因子，引起剧烈的炎性反应，加剧PRRSV的致病性。高致病PRRSV诱发的过激免疫应答可能是重要的致病因素之一。本研究首次报道了高致病性PRRSV与由北美传入的L1分支PRRSV发生重组，重组病毒具有一定的致病性。本研究发现了PRRSV复制过程中的重要环节和参与蛋白，发现了PRRSV致病性的高低与PRRSV在猪体内的复制水平有关，发现了复制酶中规律性氨基酸变异位点对PRRSV复制效率、致病性的影响，发现了高致病性PRRSV感染后诱发产生过激天然免疫反应，对于揭示PRRSV的分子致病机制具有重要的科学意义。受该基金资助，发表了8篇文章，其中SCI文章4篇，中文核心文章4篇。