猪流行性腹泻病毒CH/SCJY/2022株的分离鉴定及遗传进化分析  

Isolation,identification and genetic evolution analysis of Porcine epidemic diarrhea virus strain CH/SCJY/2022

作　　者：吴学婧 于吉锋[1] 肖璐 毛从剑 叶勇刚[1] 曾凯[1] 潘梦 谢晶[1] 曹冶[1] 李兴玉[1] 林毅[1] 叶健强[1] 张先惠[1] 魏勇[1] 青易 康润敏[1] WU Xuejing;YU Jifeng;XIAO Lu;MAO Congjian;YE Yonggang;ZENG Kai;PAN Meng;XIE Jing;CAO Ye;LI Xingyu;LIN Yi;YE Jianqiang;ZHANG Xianhui;WEI Yong;QING Yi;KANG Runmin(Sichuan Provincial Key Laboratory of Animal Genetics and Breeding,Sichuan Animal Science Academy,Chengdu 610066,China;Chengdu Livestock and Poultry Genetic Resources Protection Center,Chengdu 610081,China)

机构地区：[1]四川省畜牧科学研究院动物遗传育种四川省重点实验室,成都610066 [2]成都市畜禽遗传资源保护中心,成都610081

出　　处：《黑龙江畜牧兽医》2024年第18期64-74,133,共12页Heilongjiang Animal Science And veterinary Medicine

基　　金：四川省省级科研院所基本科研业务费项目(SASA202202);国家现代农业体系四川创新团队建设项目(Sccxtd-2020-08);四川省区域创新合作项目(2022YFQ0024);四川省“十四五”川猪重大科技专项(2021ZDZX0010);四川省重点研发项目(2021YFYZ0030,2020YFN0147);四川省财政运行专项(SASA2022CZYX006);畜禽遗传资源推广经费项目(51010024T000010479592)。

摘　　要：2022年四川省简阳地区某规模化猪场仔猪发生严重腹泻甚至死亡,疑似猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染。为确定病原并了解其遗传进化特征,试验无菌采集病死腹泻仔猪小肠组织样本,提取RNA后采用RT-PCR方法对病原进行检测,并对病毒进行分离和半数组织培养感染剂量(TCID_(50))测定、间接免疫荧光试验、全基因组测序和遗传进化分析。结果表明:腹泻仔猪小肠组织样本为PEDV阳性,小肠组织过滤上清液接种Vero细胞盲传至第3代出现致细胞病变效应(cytopathic effect,CPE),盲传至第9代时出现稳定的CPE,第9代病毒液的TCID_(50)为1×10^(-5.2)/0.1 mL;在以鼠源抗PEDV-S蛋白单克隆抗体为一抗、FITC标记的羊抗鼠IgG为二抗的间接免疫荧光试验中,接种分离毒株的Vero细胞呈现特异性绿色荧光,未接种分离毒株的Vero细胞无特异性绿色荧光。将分离毒株命名为CH/SCJY/2022。经PCR分段扩增、测序、拼接后得到CH/SCJY/2022株全基因组序列,大小为28038 bp。CH/SCJY/2022株S基因、ORF3基因、M基因、N基因与参考毒株(CV777、AJ1102、LW/L、virulent DR13、LZC)核苷酸相似性分别为93.11%~97.71%、95.41%~98.37%、96.62%~98.09%、94.72%~96.38%。CH/SCJY/2022株全基因组序列与30株PEDV参考毒株核苷酸相似性为96.0%~98.9%,其中与强毒变异株YC2014的相似性最高(98.9%),与韩国毒株SM98的相似性最低(96.0%),与疫苗毒株CV777、AJ1102的相似性分别为96.4%和98.3%。基于全基因组序列构建的遗传进化树中可见CH/SCJY/2022株与CH/SCZY103/2017位于同一小分支,亲缘关系最近,属于GⅡb基因亚群;与疫苗毒株LW/L、AJ1102、XJ-DB2、CV777、attenuated DR13的亲缘关系较远。与参考毒株(attenuated DR13、LW/L、CV777、AJ1102、XJ-DB2)相比,CH/SCJY/2022株S蛋白氨基酸序列存在6处特有的氨基酸位点突变,分别为N139D、I289M、R492K、F541L、L1000M、S1080L。说明试验成功从该养猪场腹泻仔In 2022,severe diarrhea and even death occurred in piglets in a large-scale pig farm in Jianyang area,Sichuan Province,which was suspected to be Porcine epidemic diarrhea virus(PEDV)infection.In order to identify the pathogen and understand its genetic evolutionary characteristics,in the test,small intestinal tissue samples were collected aseptically from dead diarrheic piglets;RNA was extracted and detected by RT-PCR;the virus was isolated and half of the tissue culture infectious dose(TCID_(50))was determined,and indirect immunofluorescence assay,whole-genome sequencing,and genetic evolution analysis were performed.The results showed that the small intestinal tissue samples of piglets with diarrhea were positive for PEDV.Small intestine tissue filtration supernatant was inoculated with Vero cells and blindly transmitted to the third passage,resulting in cytopathic effect(CPE).Stable CPE appeared during blind transmission to the 9th generation,and the TCID_(50)of the 9th generation virus solution was 1×10^(-5.2)/0.1 mL.In an indirect immunofluorescence assay using mouse anti-PEDV-S protein monoclonal antibody as primary antibody and FITC-labeled sheep anti-mouse IgG as secondary antibody,Vero cells inoculated with the isolated strain showed specific green fluorescence;Vero cells uninoculated with the isolated strain did not show specific green fluorescence.The isolate was named CH/SCJY/2022;the whole genome sequence of CH/SCJY/2022 was obtained after PCR amplifying,sequencing and splicing,with a size of 28038 bp.The nucleotide similarity between the whole genome sequence of strain CH/SCJY/2022 and the 30 PEDV reference strains ranged from 96.0%to 98.9%;the highest similarity was found with virulent variant strain YC2014(98.9%),the lowest with Korean strain SM98(96.0%),96.4%and 98.3%with the vaccine strain CV777 and AJ1102,respectively.The nucleotide similarities between the S,ORF3,M and N genes of strain CH/SCJY/2022 and the reference strains(CV777,AJ1102,LW/L,virulent DR13 and LZC)ranged from 93.11%to 97.71%,9

关 键 词：猪流行性腹泻病毒 全基因组测序 同源性分析 遗传进化分析 分离 鉴定 

分 类 号：S855.3[农业科学—临床兽医学]