猪流行性腹泻病毒HeN21株的分离鉴定及致病性研究  

Isolation,identification and pathogenicity of porcine epidemic diarrhea virus HeN21 strain

作　　者：冷超粮[1] 贾楠 翟洪月 马秀秀 田想 宋佳静 史鸿飞[1] 李娜 姚伦广[1] 阚云超[1] 张瑞 田志军[3] LENG Chao-liang;JIA Nan;ZHAI Hong-yue;MA Xiu-xiu;TIAN Xiang;SONG Jia-jing;SHI Hong-fei;LI Na;YAO Lun-guang;KAN Yun-chao;ZHANG Rui;TIAN Zhi-jun(Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Henan Provincial Engineering Laboratory of Insects Bio-reactor,Nanyang Normal University,Nanyang 473061,China;Agricultural and Rural Bureau of Fengqiu County,Henan Province,Fengqiu 453300,China;State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]南阳师范学院河南省动物疫病诊断与综合防控工程技术研究中心/昆虫生物反应器河南省工程实验室,河南南阳473061 [2]河南省封丘县农业农村局,河南封丘453300 [3]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第11期1108-1115,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省高等学校重点科研项目(22A230016)。

摘　　要：为了解河南地区猪流行性腹泻病毒(PEDV)的流行及变异情况,本研究从该地区某疑似感染PEDV的发病猪场采集粪便样品,经RT-PCR检测并测序,结果显示该猪场为PEDV感染。将阳性样品接种Vero细胞进行病毒分离,并通过RT-PCR扩增、测序及间接免疫荧光试验(IFA)鉴定,结果显示分离到一株PEDV,命名为HeN21株。经PCR分段扩增病毒全基因组,测序、拼接后的结果显示,该病毒基因组全长28036 bp。为进一步分析HeN21株的遗传演化特征,利用MegAlign分析HeN21株全基因组与GenBank中5株PEDV参考株全基因组序列的相似性;采用MEGA 11软件中NJ法构建HeN21株与GenBank中31株PEDV参考株S基因的系统发育树;采用MegAlign分析HeN21株S蛋白氨基酸序列的变异特征;进一步评价HeN21株对哺乳仔猪的致病性。相似性分析结果显示,HeN21株与PEDV参考株的相似性为96.6%~98.9%,其中与经典GIa亚型代表株CV777的相似性最低为96.6%,与GIIa亚型代表株AH2012和GIIb亚型代表株AJ1102的相似性均为98.5%。进化树结果显示,HeN21株与GIIa亚型PEDV聚为一支。S蛋白氨基酸序列分析结果显示,与经典株CV777相比,HeN21株在中和抗原表位类胶原酶消化区(COE)存在11个氨基酸突变,其中K635T是其特有的突变。致病性试验结果显示,3日龄健康仔猪最早于接种HeN21株后12 h出现腹泻和呕吐,随后症状加重并死亡。感染组仔猪的直肠拭子经RT-PCR检测和PCR产物测序,全部为PEDV阳性,且小肠肠壁薄而透明,完全丧失消化吸收功能。上述结果表明,PEDV在我国持续变异,且分离株具有较强的致病性,应高度重视。本研究报道的PEDV分离株为深入了解河南地区PED的流行及防控具有重要意义,并进一步丰富了PEDV病毒库,为开发设计新型疫苗奠定了基础。In order to study the prevalence and variation of porcine epidemic diarrhea virus(PEDV)in Henan Province,fecal samples collected from a suspected PEDV-infected pig farm were detected by RT-PCR.The amplicons were sequenced,which showed the PEDV infection in this pig farm.The positive samples were inoculated into Vero cells for virus isolation,followed by RT-PCR,sequencing,and indirect immunofluorescence assay(IFA).The results showed that a PEDV strain was successfully isolated,which was designated HeN21.The full-length genome of the virus was 28036 nucleotides.To further analyze the genetic evolution characteristics of the HeN21 strain,MegAlign software was used to analyze the whole genome nucleotide similarity among the HeN21 strain and five representative PEDV strains from GenBank.MegAlign was also used to analyze the S protein amino acid sequence variation characteristics of the HeN21 strain.A phylogenetic tree by the NJ method using MEGA 11 software was constructed based on the S gene among the HeN21 strain and 31 representative PEDV strains in GenBank.The pathogenicity of the HeN21 strain to suckling piglets was further evaluated.The results showed that the nucleotide similarities among the whole genome and five representative PRRSV strains were 96.6%-98.9%,among which the similarity between the HeN21 strain and CV777 strain of GIa subtype was 96.6%,and that between the HeN21 strain and AH2012 of GIIa subtype or AJ1102 of GIIb subtype was 98.5%.Phylogenetic tree results showed that the HeN21 strain and GIIa subtype PEDV were clustered into one branch.The amino acid sequence analysis of the S protein showed that compared with the classical strain CV777,the HeN21 strain had 11 amino acid mutations in the collagenase-digested fragment equivalent(COE)region of neutralizing epitope,of which K635T was a unique mutation.The results of the pathogenicity test showed that 3-day-old healthy piglets began to develop diarrhea and vomiting at the earliest 12 hours after inoculation with the HeN21 strain,followed by aggrava

关 键 词：猪流行性腹泻病毒 序列分析 遗传变异 致病性试验 

分 类 号：S852.65[农业科学—基础兽医学]