猪流行性腹泻病毒变异株感染性克隆构建及致病机制研究

Since the winter of 2010, porcine epidemic diarrhea (PED) with high morbidity and mortality rate, have been prevalent in the pig industry in many provinces of China. The variant strain, PEDV CH1 strain, was confirmed as the mainly agent of the disease which no effectively measurements to control it has been established due to the lack of virulence property and pathogenesis mechanism of the new virus. In this project, the infectious clone of the new PEDV virus (CH1 strain) is constructed and the resultant rescued PEDV with different deletion domains of spike gene and ORF3 are used to infect Vero cell lines in which attachment efficacy, intracellular viremia and titers of the new virus are evaluated, at the same time, the mutated S and ORF3 gene fragments was cloned to pCDNA3.1(+)and transfect Vero cells,respectively， followed by monitoring the intracellular localization, and aims to understanding the function of the deleted domain of the target genes as well as to identifiy potential new pathogenecity-related factors. Meanwhile, by mean of bioinformatics analyses, pathological examination, immunohistochemical test and genechip analyses, using the traditional field virus (CV777) as control strain, PEDV CH1 strain was used to in vivo and in vitro infection experiment focusing on the understanding of molecular pathogenesis and the important characteristics of the new virus isolated by the applicant. Through these researches, the pathogenesis of PEDV CH1 strain is supposed to be clarified and thus provide the way for gene functional analyses and the developing new candidate vaccine, which might greatly be helpful for fighting against PED.

自2010年冬季以来，我国多个省市爆发仔猪腹泻病，发病率和死亡率很高，严重影响养猪业的发展，目前尚无有效控制本病的措施。已证实猪流行性腹泻变异毒株是主要病因，但其毒力特征和致病机理尚未明晰。项目申请者以自行分离的PEDV变异毒株（PEDV CH1株）为研究对象，构建该毒株的感染性克隆，拯救重组突变体，进行细胞感染和动物感染，结合突变基因细胞转染试验，测定病毒增殖能力和细胞病变指标，确定突变的S基因和ORF3与毒株致病性相关的功能域，发现潜在的毒力相关因子；同时，以传统野毒（CV777）为参照，开展PEDV CH1株体内外感染试验，利用生物信息学、病理学、免疫组化技术和基因表达谱芯片等手段，分析该变异毒株的重要生物学特征和宿主体内外感染后的分子病理学特征。通过本项目的研究，不仅阐明PEDV变异株的致病机理，也为进一步研究变异株基因功能和新型弱毒疫苗奠定基础，为该病防控技术的制定提供理论依据。

近年来，多个国家的猪场暴发了仔猪腹泻病，发病率和死亡率极高，给世界养猪业造成巨大经济损失。已证实PEDV变异毒株是主要病因。因此，加强对PEDV的分离、比较基因组学分析、生物学特性和致病性机制的研究显得非常迫切。.本研究首先开展分子病原学研究，分离了临床弱毒株AH-M株和强毒YN1株。以YN1为亲本毒株，经Vero细胞传代致弱,获得致弱毒株YN144。对PEDV经典毒株和变异强毒株及致弱变异毒株进行了比较基因组分析，发现致弱毒株在ORF1a/b和S 蛋白处存在9-26个氨基酸的改变，ORF3在145位氨基酸处提前终止，确定了S蛋白144和145位氨基酸的缺失是PEDV毒株致弱的标志，而ORF3提前终止是病毒适应细胞的标志。.PEDV强弱毒株感染Vero细胞的蛋白质组学分析表明，强毒株显著上调NF-κB通路，诱发更强烈的炎症反应。强毒株通过下调mTOR及其下游靶分子通路活性来抑制细胞蛋白质的合成。强弱毒株感染仔猪后的致病性差异和病理学研究结果表明，强毒株YN13感染后仔猪严重腹泻，而弱毒株YN144感染组未见腹泻症状，免疫组化分析发现PEDV主要分布在胃和小肠，但强毒株的信号明显多于弱毒株。仔猪肠道蛋白质组学分析发现，PEDV感染可以诱导多种干扰素刺激基因的表达，热休克蛋白家族的许多成员在强毒感染组上调表达，而在弱毒感染组下调表达。 .为了研究PEDV S基因的功能，构建了带标签的重组病毒rPEDV-DR13-SCHGDU-Flag-dORF3/GFP，细胞感染试验发现S基因决定了病毒对胰蛋白酶的依赖性。PEDV S基因第888位氨基酸V/R突变，可以改变PEDV对胰蛋白酶的依赖性。发现hTMPRSS2并不能替代胰蛋白酶帮助病毒的释放和感染，PEDV ORF3基因表达可以延长Vero细胞S周期，促进囊泡形成，利于致弱PEDV的增殖。.本研究可以得出如下结论：PEDV的S基因决定了PEDV胰蛋白酶依赖性。S蛋白144和145位氨基酸的缺失是毒力致弱的关键，为研究基因缺失疫苗提供了理论依据。PEDV强毒株通过显著上调NF-κB通路，诱发更强烈的炎症反应，是导致仔猪发生剧烈腹泻的原因。.本研究培养博士生5人，硕士生3人，发表文章11篇，其中SCI文章8篇。课题负责人参加国内外学术会议和技术培训班约15场次，介绍本课题取得成果以及对养猪业对该病防控的策略和措施。

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