猪流行性腹泻病毒流行毒株的分离与鉴定  

Isolation and identification of a variant porcine epidemic diarrhea virus

作　　者：周书亭 谢洋洋[2] 丁思嘉[2] 郑煜名 蔡雨豪 罗依然[2] 袁聪俐[2] 杨志彪[2] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海交通大学农业与生物学院,上海市兽医生物技术重点实验室,上海200240

出　　处：《中国兽医科学》2017年第8期957-963,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31472211);国家重点研发计划项目(2016YFD0500100);上海市自然基金项目(14ZR1421400)

摘　　要：2016年3月江苏某猪场发生了哺乳仔猪腹泻疫情,导致大部分哺乳仔猪严重腹泻和死亡。为确定此次腹泻的病原,从该猪场采集了6份仔猪腹泻样品,利用RT-PCR方法对可引起仔猪腹泻的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)三种主要腹泻病毒进行了检测。结果显示,6份样品均呈现PEDV阳性,而TGEV和PoRV为阴性。无菌处理6份阳性样品后分别接种于Vero细胞,进行病毒分离培养;结果,6份样品中仅JSqd3/2016接种后可产生明显细胞病变,且连续传至第5代时细胞病变仍稳定存在。对JSqd3/2016分离株利用针对PEDV S蛋白的特异单抗进行间接免疫荧光鉴定结果显示,PEDV S蛋白单抗能够特异性识别JSqd3/2016分离株感染的Vero细胞。对JSqd3/2016-F5分离株S基因和ORF3基因进行克隆测序和序列比较分析结果显示,分离获得的JSqd3/2016毒株与目前的流行毒株亲缘关系较近,与经典毒株相比S基因存在较大变异,氨基酸同源性为93.0%~93.3%。本研究结果证实,PEDV变异毒株感染是该猪场仔猪腹泻疫情的主要病因。A diarrhea outbreak characterized by watery diarrhea and high mortality among neonates occurred in a pig farm in Jiangsu province in March 2016.To identify the pathogen that caused this diarrheal disease,six feces samples were collected and subjected to RT-PCR analysis.The results showed six samples were positive for PEDV,while all of them were negative for TGEV and Po RV.Then all the positive samples were processed and inoculated into the monolayers of Vero cells for virus isolation.Only one sample designated JSqd3/2016 could induce visible cytopathogenic effect（CPE）and the virus isolated from this sample was serially passaged to the 5th generation（JSqd3/2016-F5）.After that,the Vero cells inoculated with JSqd3/2016 were tested by indirect immunofluorescence assay（IFA）and the result showed it could be specifically recognized by the monoclonal antibody（MAb）against the PEDV spike（S）protein.Also,the S gene and ORF3 gene of JSqd3/2016-F5 were amplified,cloned,sequenced and used for sequence analysis.The result showed JSqd3/2016 was more closely related to the currently circulatingvariant PEDV strains;its S gene had pronounced mutations compared with the classical strains and the deduced amino acid sequence similarity between S protein of JSqd3/2016-F5 and the circulating variant PEDV strains ranged from 93.0%to 93.7%.These results suggested JSqd3/2016 was a variant PEDV strain and it was the causative agent of this diarrheal outbreak.

关 键 词：猪流行性腹泻病毒 JSqd3/2016 分离 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]