一株猪流行性腹泻病毒强毒株的分离与鉴定  

Isolation and Identification of a Highly Pathogenic Strain of Porcine Epidemic Diarrhea Virus

作　　者：刘维哲 罗成刚 袁蓉 廖艺杰 文艺悯 孙莹 俞恩波 曹三杰 黄小波 LIU Weizhe;LUO Chenggang;YUAN Rong;LIAO Yijie;WEN Yimin;SUN Ying;YU Enbo;CAO Sanjie;HUANG Xiaobo(Research Center for Swine Diseases,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Science-observation Experiment Station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture,Chengdu 611130,China;Chengdu Live Stork and Poultry Genetic Resources Protection Center,Chengdu 610081,China)

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130 [2]农业农村部兽用药物与兽医诊断技术四川科学观测实验站,成都611130 [3]成都市畜禽遗传资源保护中心,成都610081

出　　处：《畜牧兽医学报》2024年第7期3049-3063,共15页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省“十四五”川猪重大科技专项(2021ZDZX0010);成华猪保护基地生物危害防控经费项目(ZZSS-2024-193);云南省重大科技专项计划(202102AE090039);四川省产教融合示范项目“饲料工业全产业链转型升级产教融合创新示范”。

摘　　要：为了掌握猪流行性腹泻病毒(PEDV)最新流行毒株的病原特征与致病性,开展了PEDV的分离与鉴定。将PEDV阳性病料样品接种Vero细胞连续盲传,分离PEDV流行毒株,并开展了分离毒株的培养特性、全基因组序列分析和仔猪致病性评价。结果显示:传至第3代(P3)分离出一株病毒,病毒在细胞上形成典型的“合胞体”样病变;电镜下病毒大小60~110 nm;P10到P35代滴度从10^(4.7)TCID_(50)·mL^(-1)升至10^(6.43)TCID_(50)·mL^(-1);CHN-SC2021(P5)基因组全长28014 bp(GenBank收录号:OM505025.1),与中国地区、欧美地区、亚洲其他国家毒株的相似性分别为99.16%~96.32%、99.01%~96.57%、98.94%~96.33%;系统进化树显示CHN-SC2021株与江西CH/JXJA/2017(MF375374.1)、河南CH/HNAY/2015(KT199103.1)、CH/HNKF-16(KY649107.1)毒株亲缘关系较近,属于GⅡa型毒株。S基因序列与参考毒株相似度在98.7%~93.4%。将CHN-SC2021病毒液以每头3 mL 10~5 TCID_(50)·mL^(-1)的剂量口服感染6日龄哺乳仔猪,12 h开始出现食欲不振,20 h时呕吐,24 h出现剧烈呕吐和黄色水样腹泻;剖检病变主要是胃肠臌气、空肠和回肠透明;组织病变显示各肠段均有不同程度的肠绒毛病变、脱落、结缔组织疏松、黏膜肌层与肌层间距增宽等;免疫组化结果显示小肠各段均有病毒,回肠中病毒最多。本研究分离获得一株GⅡa型的PEDV强毒株,有助于丰富我国PEDV的流行毒株信息及为临床防控提供参考。To understand the pathogenic characteristics and pathogenicity of the latest epidemic strain of porcine epidemic diarrhea virus(PEDV),isolation and identification of PEDV were conducted this study.PEDV positive samples were inoculated into Vero cells for continuous transmission for isolating PEDV epidemic strains.Then the culture characteristics,whole genome sequence of the isolated strains were analyzed,and their pathogenicity in piglets were evaluated.Results showed that a PEDV strain(named CHN-SC2021)was isolated from the 3rd passage(P3)Vero cells of continuous inoculation of PEDV positive sample.Under the electron microscope,the diameter of the virus was about 60-110 nm,forming classic"syncytium"on the cell;The virus titer from P10 to P35 generations increased from 10^(4.7) TCID_(50)·mL^(-1) to 10^(6.43) TCID_(50)·mL^(-1);The whole genome sequence of CHN-SC2021(P5)(GenBank accession number:OM505025.1)is 28014 bp,and its nucleotide sequence homology about 99.16%-96.32%,99.01%-96.57%and 98.94%-96.33%compared with the reference strains from China,Europe and America,and other Asian countries,respectively;The phylogenetic analysis showed that the CHN-SC2021 strain is closely related to Jiangxi CH/JXJA/2017(MF375374.1),Henan CH/HNAY/2015(KT199103.1),and CH/HNKF-16(KY649107.1)strains,belonging to the GⅡa genotype type.The similarity between the S gene sequence and other strains ranges from 98.7%to 93.4%.Oral infection of 6-day-old suckling piglets with 3 mL of 10^(5) TCID_(50)·mL^(-1) per pig of CHN-SC2021 virus solution resulted in loss of appetite at 12 hours,vomiting at 20 hours,and severe vomiting and yellow watery diarrhea at 24 hours;The main pathological changes during dissection are gastrointestinal distension,transparent jejunum and ileum;Histopathological changes showed that each segment of the intestine had varying degrees of intestinal villus lesions,shedding,loose connective tissue,and widened space between the muscularis mucosae and the muscle layer;Immunohistochemistry showed that viruses were pr

关 键 词：猪流行性腹泻病毒 分离 鉴定 序列分析 哺乳仔猪 致病性 

分 类 号：S852.659.6[农业科学—基础兽医学]