2020年~2021年河北省4种猪腹泻病毒流行情况调查及PEDV分离株S1和ORF3基因序列的遗传变异分析  

Epidemic survey of four diarrhea viruses in pig farms and genetic variation analysis of S1 and ORF3 gene sequences of PEDV strains in Hebei Province from 2020 to 2021

作　　者：王挺 李风云 葛生虎 刘慧芳 WANG Ting;LI Feng-yun;GE Sheng-hu;LIU Hui-fang(Hunan Environmental Biology Vocational and Technical College,Hengyang 421005,China;Agriculture and Rural Affairs Bureau of Linxi country,Xingtai 054900,China;Hebei Mingzhu Biotechnology Co.,Ltd.,Xingtai 054000,China)

机构地区：[1]湖南环境生物职业技术学院,湖南衡阳421005 [2]临西县农业农村局,河北邢台054900 [3]河北铭诸生物科技有限公司,河北邢台054000

出　　处：《中国预防兽医学报》2023年第5期473-479,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：2021年度湖南环境生物职业技术学院青年基金项目(ZK2021-01);2022年全国职业院校巩固扶贫成果服务乡村振兴协作联盟研究课题(LM2022035)。

摘　　要：为初步调查河北省近年4种猪腹泻相关病毒的流行情况,本研究于2020年~2021年从河北省部分地区猪场采集244份腹泻猪粪便或肠组织样品,采用荧光定量PCR(qPCR)检测4种猪腹泻相关病毒[(猪流行性腹泻病毒(PEDV)、猪轮状病毒(PoRV)、猪传染性胃肠炎病毒(TGEV)和猪德尔塔冠状病毒(PDCoV)];采用RT-PCR扩增部分PEDV阳性样品S1和ORF3基因并测序,利用DNAStar软件分析这两个基因及其编码氨基酸序列与PEDV相应序列的同源性;采用MEGA 7.0软件构建这两个基因的进化树,分析PEDV的基因型及其遗传进化关系。采用DNAStar比对这两个基因编码的氨基酸序列,分析其氨基酸序列的变异情况。结果显示:病料样品中PEDV、PoRV、PDCoV和TGEV检出率分别为34.02%(83/244)、18.44%(45/244)、10.66%(26/244)和2.87%(7/244)。样品中两种病原混合感染较多,其中PEDV+PoRV和PEDV+PDCoV检出率最高,分别为8.61%(21/244)和2.46%(6/244);仅2份样品为PEDV+PDCoV+PoRV三重感染,占0.81%(2/244)。8株PEDV S1和ORF3基因序列之间的同源性分别为98.2%~99.8%和97.3%~99.6%,氨基酸序列同源性分别为97.4%~100.0%和96.9%~99.2%,且二者均与国内流行变异株(如AJ1102、AH2012和CHGX株)的同源性更高(分别为97.7%~98.9%、97.2%~98.4%和98.3%~99.7%、97.4%~99.3%)。PEDV S1基因的系统进化树结果显示,5株PEDV为GII-a亚型,3株PEDV为GII-b亚型,且均与PEDV变异株亲缘关系较近;PEDV ORF3基因进化树结果显示,8株PEDV均与上述PEDV变异株所属分支亲缘关系较近。以上结果表明,本研究检测的8株PEDV均属于变异株。S1和ORF3氨基酸序列比对结果均显示,部分PEDV与其参考株相比存在独特的变异,其中HeB-QHD、HeB-HD和HeB-TS PEDV的S1存在N^(139)D、I^(289)M和A^(363)T/S变异;HeB-HD和HeB-BD的ORF3存在Q^(202)H等变异。上述结果表明,PEDV和PoRV是导致河北省仔猪腹泻的主要肠道病毒,且该省PEDV流行株主要为GII基因型,且同时存在GII-a和GII-b亚型PEDV的流�In order to investigate the prevalence of four porcine diarrhea related viruses in Hebei Province in recent years,this study collected 244 diarrhea feces or intestinal tissues from pig farms in Hebei Province from 2020 to 2021.Four diarrhea related pathogens(porcine epidemic diarrhea virus(PEDV),porcine rotavirus(PoRV),porcine transmissible gastroenteritis virus(TGEV)and porcine delta coronavirus(PDCoV))were detected by the fluorescent quantitative PCR(qPCR).The S1 and ORF3 genes of 8PEDV positive samples were amplified by RT-PCR and sequenced.The homology of the two genes and their encoded amino acid sequences with their corresponding sequences of PEDV was analyzed by using DNAStar software.The phylogenetic tree of each gene was generated by MEGA 7.0 software and further used to analyze the genotypes and evolutionary relationships.The amino acid sequences encoded by the two genes were compared using DNAStar,and the variation of the amino acid sequences was analyzed by DNAStar software.The results showed that the positive rates of PEDV,PoRV,PDCoV and TGEV in the collected samples were 34.02%(83/244),18.44%(45/244),10.66%(26/244)and 2.87%(7/244),respectively.The mixed infection of two pathogens was common in clinical samples.The detection rates of PEDV+PoRV and PEDV+PDCoV were the highest,which were8.61%(21/244)and 2.46%(6/244),respectively.Two samples were PEDV+PDCoV+PoRV triple infection,accounting for 0.81%(2/244).The nucleotide sequence homologies of the S1 and ORF3 genes of eight PEDV strains were 98.2%-99.8%and 97.3%-99.6%,respectively,and the amino acid sequence homologies were 97.4%-100.0%and 96.9%-99.2%,respectively.Both of the PEDV S1 and ORF3 genes displayed higher sequence homologies with the PEDV variants(AJ1102,AH2012 and CHGX strains)(which were97.7%-98.9%,97.2%-98.4%,and 98.3%-99.7%,97.4%-99.3%,respectively).The analysis of the S1 gene phylogenetic tree showed that 5 PEDV strains were GII-a subtype and 3 strains were GII-b subtype.Both the S1 gene and ORF3 gene phylogenetic tree showed that all 8 P

关 键 词：仔猪腹泻 猪腹泻病毒 流行病学 S1和ORF3基因 遗传进化 

分 类 号：S852.65[农业科学—基础兽医学]