猪流行性腹泻病毒变异株对仔猪致病性减弱的分子机制

Recently, novel PEDV variants constantly appear in wordwide and have brought new challenges to prevention and control of PED. Our previous and related studies both showed that novel PEDV variants are obviously distinguished from previous epidemic virulent strains, such as large deletion in S gene and attenuated pathogenicity in piglets. That suggested that the critical molecular basis for attenuated pathogenicity of PEDV variants in piglets could be the deletion mutation of S gene. However, to date, none of any study report could conclusively prove it. Therefore, in this study, the S gene deletion and insertion mutants of PEDV will be constructed and rescued using virulent and variant strains as parents by reverse genetics based on in vitro ligation. Through deletion and insertion of nucleotides in virulent and variant strains, the effect of partial deletion of S gene on the pathogenicity of PEDV will be studied by two-way. Meanwhile, the critical regions and positions that affect pathogenicity of PEDV will be comfirmed and the molecular mechanisms for attenuated pathogenicity of PEDV variant in piglets will be clarified. The results of this study will contribute to clarify pathogenicity of PEDV and provides the basis for the development of new and efficient vaccine.

近年来，新型猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)变异株在世界范围内不断出现，给PED的防控带来了新的挑战。前期研究及相关报道都显示，新型PEDV变异株明显区别于以往流行的强毒力株，其S基因存在较大程度的缺失突变，而且对仔猪的致病性明显减弱，提示S基因的缺失突变可能是导致新型PEDV变异株对仔猪致病性减弱的关键分子基础，但迄今为止尚未有任何的研究报道证明此推论。因此，本项目将以PEDV强毒力株和变异株为亲本病毒，利用基于体外连接的反向遗传操作技术，构建并拯救PEDV S基因缺失和插入突变株，通过强毒力株的碱基缺失和变异株的碱基插入，双向探讨S基因部分序列缺失对PEDV致病性的影响，解析影响PEDV致病性的关键基因区域或位点，阐明PEDV变异毒株对仔猪致病性减弱的分子机制，为揭示PEDV致病的分子机制和研制新型、高效的疫苗奠定基础。

PEDV变异株区别于以往流行的强毒力株，其S基因存在较大程度的缺失突变，而且对仔猪的致病性明显减弱。本项目通过Vero细胞体外连续传代培养，分离获得了当前流行的两株PEDV变异株：CH/HBXT/2018（基因型GⅡa）和CH/HNPJ/2017（基因型GⅡb），并且对两株病毒的生物学特点和在仔猪中的致病性进行了详细的比较分析。通过比较基因组序列差异，确定了PEDV变异株S基因N端缺失突变的确切区域位点，并进一步通过细胞和仔猪感染试验比较了PEDV变异株的致病性差异。进一步地，以上述PEDV变异毒株为亲本毒株，利用基于体外连接的反向遗传操作技术，成功的获得了PEDV S基因缺失突变株基因组全长cDNA，并通过体外转录的方式获得了PEDV S基因缺失突变株的基因组全长感染性克隆。该感染性克隆的鉴定和仔猪致病性试验工作正在进行。综上所述，本研究成功的构建了PEDV基因组全长cDNA，为PEDV反向遗传操作平台的建立和PEDV致病分子机制的深入研究奠定了坚实的工作基础。项目资助发表SCI论文5篇，待发表SCI论文2篇，获得国家发明专利和国际专利各1项，培养研究生3名，其中2人已获得硕士学位，1名在读。项目投入经费19万元，已全部支出，各项支出与预算相符。