一株猪流行性腹泻病毒的分离鉴定及其ORF3和S基因序列分析  

Isolation and identification of a PEDV stain and sequence analysis of its ORF3 and S genes

作　　者：沈海燕 黄俊 张琬玓 聂晶晶 瞿云芝 张建峰 李智丽 张春红 SHEN Haiyan;HUANG Jun;ZHANG Wandi;NIE Jingjing;QU Yunzhi;ZHANG Jianfeng;LI Zhili;ZHANG Chunhong(Guangdong Academy of Agricultural Sciences,Institute of Animal Health/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province/Guangdong Provincial Key Laboratory of Livestock Disease Prevention,Guangzhou 510640,China;Guangdong Guangken Animal Husbandry Group Co.,Ltd.,Guangzhou 510612,China;College of Life Science and Engineering,Foshan University,Foshan 528231,China)

机构地区：[1]广东省农业科学院动物卫生研究所/农业农村部兽用药物与诊断技术广东科学观测实验站/广东省畜禽疫病防治研究重点实验室,广州510640 [2]广东广垦畜牧集团股份有限公司,广州510612 [3]佛山科学技术学院生命科学与工程学院,广东佛山528231

出　　处：《黑龙江畜牧兽医》2024年第2期79-87,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省重点领域研发计划项目(2020B0202080004);广州市重点研发计划项目(202103000096);广东省基础与应用基础研究基金项目(2020A1515010950;2021A1515011125);广州市基础研究计划项目(202002030456)。

摘　　要：为了探明辽宁省沈阳市某猪场仔猪发生腹泻的病原及其遗传进化特征,试验首先对该猪场发生腹泻的仔猪腹腔进行剖检,记录小肠组织的病理变化;收集病理变化较明显的组织进行切片和H.E.染色,观察并记录结果;取病料后提取病毒核酸,用猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus,TGEV)、猪丁型冠状病毒(Porcine delta coronavirus,PDCoV)、猪轮状病毒(Porcine rotavirus,PRoV)和猪急性腹泻综合征冠状病毒(Swine acute diarrhea syndrome coronavirus,SADS-CoV)检测引物进行RT-PCR检测;然后进行病毒的分离、致细胞病变效应(CPE)观察、传代、间接免疫荧光试验与效价的测定;最后对病毒的关键基因进行PCR扩增及遗传进化分析。结果表明:腹泻仔猪小肠肠壁变薄、胀满、充满水样内容物,肠系膜呈树枝状充血,肠系膜淋巴结水肿;十二指肠肠绒毛破碎、细胞坏死,固有层细胞充血;空肠肠绒毛萎缩变短,上皮细胞空泡样变性,少量淋巴细胞浸润,与PEDV感染猪只的肠组织病理变化相同。腹泻仔猪病料组织PEDV核酸检测结果为阳性,其他4种病原检测结果均为阴性。将盲传6代的病毒培养液接种于Vero细胞,培养48 h后出现明显的CPE,以PEDV N特异性单克隆抗体为一抗的间接免疫荧光试验出现特异性的绿色荧光,经测定病毒效价为1×10~(5.83)TCID_(50)/mL。将分离毒株命名为CH/XF1.C.3/2020,其ORF3基因和S基因与国内外PEDV参考株的相似性分别为89.9%~99.6%和92.7%~98.4%。该毒株与近年来分离的国内外的PEDV变异株亲缘关系较近,与经典毒株CV777(GenBank登录号为KT323979.1)的亲缘关系较远,属于GⅡ/GⅡa型PEDV。说明本试验成功分离到一株GⅡ/GⅡa型PEDV,该毒株为该猪场仔猪发生腹泻的病原。In order to investigate the etiology and genetic evolution of diarrhea in piglets from a pig farm in Shenyang City,Liaoning Province,abdominal dissection was performed on piglets with diarrhea and pathological changes of small intestine were recorded.The tissues with obvious pathological changes were collected for section and H.E.staining,and the results were observed and recorded.Virus nucleic acid was extracted from the samples,and RT-PCR was performed by PEDV,TCEV,PDCoV,PRoV and SADS-CoV detection primers.Then the virus was isolated,cytopathic effect(CPE)observation,passage,indirect immunofluorescence assay and titer determination were performed.Finally the key genes of the virus were amplified by PCR and genetic evolution analysis.The results showed that the intestinal wall was thin,swollen and filled with watery contents;the mesentery was dendritic hyperemia,and the mesentery lymph node edema was observed.Duodenal and intestinal villi were broken,with cell necrosis,and lamina propria cell hyperemia;jejunal villi atrophied and shortened;epithelial cells vacuolated and a small amount of lymphocytes infiltrated,which were the same as the pathological changes of PEDV-infected pigs.The nucleic acid test results of PEDV were positive,and the test results of other 4 pathogens were negative.The 6-generation blind virus culture medium was inoculated into Vero cells,and obvious CPE appeared after 48 h of culture.The indirect immunofluorescence test using PEDV N-specific monoclonal antibody as the primary antibody showed specific geen fluorescence,and the virus titer was measured to be 1x105.83 TCIDso/mL.The isolated strain was named CH/XF1.C.3/2020.The genetic similarities of PEDV isolates CH/XF1.C.3/2020 ORF3 gene and S gene with domestic and foreign PEDV reference strains were 89.9%-99.6%and 92.7%-98.4%,respectively.This strain was closely related to PEDV variants isolated at home and abroad in recent years,but far related to the classic strain CV777(GenBank accession number is KT323979.1),and belonged to the G Ⅱ/

关 键 词：猪流行性腹泻病毒(PEDV) 流行病学 病理变化 组织切片 遗传进化分析 分离 鉴定 

分 类 号：S855.3[农业科学—临床兽医学]