猪伪狂犬病病毒流行毒株JZ-45的分离鉴定与变异分析  

Isolationand identification of pseudorabies virus strain JZ-45 and variability analysis

作　　者：苗信永 孔令昊 陈雨 赵世杰 崔志莹 李闻 徐朋丽 陈静[1] 张宜娜[1] 李新生[1] 夏平安[1] 吴斌[2] MIAO Xinyong;KONG Linghao;CHEN Yu;ZHAO Shijie;CUI Zhiying;LI Wen;XU Pengli;CHEN Jing;ZHANG Yina;LI Xinsheng;XIA Pingan;WU Bin(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450002 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2022年第8期1541-1547,共7页Chinese Journal of Veterinary Science

基　　金：国家“十三五”重点研发计划资助项目(2018YFD0500800);河南省高校科技创新团队支持计划资助项目(19IRTSTHN007);中原千人计划科技创新领军人才资助项目(204200510015)。

摘　　要：利用ST细胞从猪伪狂犬病(PR)阳性样品中分离到1株猪伪狂犬病病毒(PRV),并对其生物学特性进行鉴定,扩增该毒株gC、gE全基因,进行测序和遗传进化分析。结果显示:该毒株在ST细胞盲传2~3代后出现稳定且典型的细胞病变(CPE),经6轮空斑纯化后,测其TCID_(50)为10^(-6.58)/0.1 mL;接毒培养12 h后,间接免疫荧光方法检测,可在荧光显微镜下观察到特异性免疫荧光。将纯化后的病毒液接种小鼠,测其LD_(50)为10^(-2.68)/0.1 mL,组织病理切片分析显示死亡小鼠的心脏、肝脏、脾脏等各组织具有明显的病理损伤,将100个LD_(50)病毒液接种家兔,2 d后接种兔子出现PR典型症状;同源性对比结果显示gC基因与国内经典、变异毒株同源性分别为99.2%~99.7%和99.9%~100.0%,与国外经典株同源性为96.0%~96.2%;氨基酸序列与国外经典株相比,在63位氨基酸位点处有连续7个氨基酸插入(AAASTPA);gE基因与国内经典、变异毒株同源性分别为96.7%%~99.4%和99.7%~99.8%,与国外经典株同源性为95.3%~97.8%;氨基酸序列与国外经典株相比,在492位氨基酸位点处有2个氨基酸插入(DG);遗传进化分析显示,该毒株的gC、gE基因与国内变异株位于同一进化分支,将该毒株命名为JZ-45。本试验研究结果为河南省PRV的流行病学遗传进化分析以及防控提供参考。In this experiment,a porcine PRV strain isolated from porcine pseudorabies positive samples using ST cells was biologically characterized,and the gC and gE genes of this strain were amplified,sequenced and analyzed for genetic evolution.The results showed that the virulent strain showed stable typical cytopathic effect(CPE)in ST cells,and its TCID_(50) was measured as 10^(-6.58)/0.1 mL after 6 rounds of empty spot purification;after 12 h of inoculation and incubation,the specific immunofluorescence could be observed under fluorescence microscope by indirect immunofluorescence method.The purified viral solution was inoculated into mice and its LD_(50) was measured as 10^(-2.68)/0.1 mL.Histopathological section analysis showed that various tissues of dead mice,such as heart,liver and spleen,had obvious pathological damage.100 LD_(50) viral solutions were inoculated into rabbits,and typical symptoms of PR appeared in the inoculated rabbits after 2 d.The results of homology comparison showed that the gC gene was 99.2%-99.7%and 99.9%-100.0%homologous with domestic classical and variant strains,and 96.0%-96.2%homologous with foreign classical strains;the amino acid sequence had 7consecutive amino acid insertions(AAASTPA)at amino acid site 63compared with foreign classical strains;the gE gene was 96.7%%-99.4%and 99.7%-99.8%homologous with domestic classical and variant strains.The genetic evolutionary analysis showed that the gC and gE genes of this strain were located in the same evolutionary branch as the domestic variant,and the strain was named JZ-45.The results of this provide a reference for epidemiological genetic analysis and prevention and control of PRV in Henan Province.

关 键 词：猪伪狂犬病病毒 分离鉴定 GC基因 GE基因 生物学特性 

分 类 号：S852.651[农业科学—基础兽医学]