应用表达性克隆发现新的猪流行性腹泻病毒（PEDV）细胞受体

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus causing an enteric disease characterized by acute vomiting and watery diarrhea with high mortality in newborn piglets. The disease, porcine epidemic diarrhea (PED), was first reported in 1971 in the United Kingdom and was soon identified in many European and Asian countries. Emergences of variant porcine epidemic diarrhea virus (PEDV) fatal to young pigs in late 2010 in China and Southeast Asia, and later in 2013 in the United States, have been posing a serious threat to the pork industry. . So far, PEDV is propagated only in Vero cells in the presence of trypsin. However, most of the PEDV field strains are difficult to be adapted in Vero cells even if the trypsin is added. This growth property of PEDV is a big challenge for PEDV fundamental study and vaccine development. Based on updated PEDV molecular epidemiological analysis and comparative genomic alignment with other coronaviruses, we hypothesize that there exists genetic determinants on the S gene of a genotype-G1b PEDV mutant that make this mutant growing in the Vero cells without trypsin (designated “trypsin-independent” hereafter). We also hypothesize that there exists an alternative cellular receptor or co-receptor for PEDV on Vero cells, which is different from the known porcine aminopeptidase N (APN/CD13), due to the strictly species-specific interaction between alphacoronavirus and its receptor APN (Vero cell is derived from Africa green monkey whereas PEDV is a porcine virus).. In order to test these two hypotheses, we first plan to construct a reverse genetics system based on a PEDV cell-adapted and trypsin-independent strain pZJU-2. Using this system, mutagenesis will be perform to change the S gene between G1 and G2 PEDV, to examine if the property of trypsin-independent can be reverted to trypsin-dependent. A recombinant PEDV expressing GFP, named ZJU2-GFP, will be also generated by replacement of the ORF3 gene with a GFP fusion gene. We next plan to identify the alternative PEDV receptor using ZJU2-GFP as a reporter virus by expression cloning approach.. The results from this study will provide much needed information on PEDV entry into the cell and molecular basis on PEDV cell adaption. Furthermore, the results will aid in developing a common trypsin-independent PEDV cell culture system, in studying virus-host interaction, and in devising effective vaccination strategies against PEDV.

猪流行性腹泻病毒(PEDV)属于冠状病毒科，是目前危害亚洲和北美洲养猪业最严重的几种病毒之一。制约PEDV基础研究和疫苗研发的一个难点在于：病毒的细胞培养需要加入胰酶才能使PEDV感染并增殖；即使加入胰酶，并非所有的野毒株都能在非洲绿猴肾上皮Vero细胞上分离并适应生长。本研究拟通过病毒反向遗传学方法，构建PEDV 基因1型/2型S基因相互置换的嵌合感染性克隆，验证S基因上存在“不依赖胰酶”生长的遗传决定因子这一猜想。同时构建可以表达GFP的重组PEDV作为报告病毒,以此为实验工具，采用表达性克隆的策略分离与鉴定Vero细胞上不同于已知的PEDV受体（氨肽酶N）的第二种功能性受体或共受体。本研究将为阐明PEDV入侵细胞的机制、揭示以PEDV为代表的某些冠状病毒难以适应细胞培养的原因提供理论依据。

第1部分：非洲绿猴Vero细胞系（ATCC CCL-81）常用于体外培养猪流行性腹泻病毒（PEDV）。然而，猪氨肽酶N（APN）是否PEDV在Vero细胞上的受体它仍然是有争议的。本研究发现，内源性表达Vero APN的mRNA和蛋白的检测不到，而广谱APN抗体的处理没有减少PEDV感染细胞。我们克隆了部分vero APN基因（3340 bp），其含有外显子1到9，并进一步通过CRISPR/Cas9系统生成了两个APN敲除细胞系。。PEDV感染APN基因敲除细胞具有与野生型细胞相同的效率。研究结果表明，Vero细胞存在不依赖于APN的另一个受体介导PEDV进入。.第2部分：本研究（1）首先研制了一种可以表达绿色荧光蛋白GFP的重组PEDV作为reporter virus，建立了实时、直观测量PEDV感染细胞的病毒增殖量化系统。（2）通过预测分析PEDV Spike蛋白的S2亚基中存在两个高度保守的七肽重复区HR1和HR（图），设计了六个衍生多肽并在大肠杆菌中融合表达和纯化。（3）通过PEDV-GFP感染系统进行融合多肽的体外竞争性抑制病毒试验，筛选出三个具有抗病毒作用的融合多肽（HR2M，HR2L，HR2P），其中HR2P的抑制作用最强，并且这些多肽的单独作用对细胞没有毒性。（4）加入这三种多肽可以在病毒感染细胞时、感染细胞前及感染细胞后发挥不同程度的抗病毒作用。（5）三种多肽可以抑制两种不同基因型的PEDV感染，具有广谱性。（6）HR2P多肽免疫小鼠产生的抗血清在体外有效中和PEDV的感染。.第3部分：研究首次从病原分离、血清学、病毒形态、全基因组克隆、进化分析、S蛋白结构及病毒回归感染试验符合“科赫法则”等各方面确证了SeACoV是一种全新的猪肠道冠状病毒，也是迄今所发现的第五种猪冠状病毒。由于SeACoV不与目前猪场流行的三种冠状病毒PEDV，TGEV及PDCoV的抗体产生交叉反应，提示现有的猪冠状病毒疫苗对SeACoV感染可能无效，这对我国新生仔猪腹泻病原的检测与防控提出新的挑战，同时也开辟了新的研究方向，其结果具有重要意义。