两株猪伪狂犬病病毒变异株的分离鉴定及gB、gC和gE基因的分子特征分析

doi:10.16431/j.cnki.1671-7236.2019.03.029

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (3): 881-890.doi: 10.16431/j.cnki.1671-7236.2019.03.029

两株猪伪狂犬病病毒变异株的分离鉴定及gB、gC和gE基因的分子特征分析

李翔¹, 郭振华², 阮海宇³, 乔松林², 张以芳¹, 张改平^2,3

1. 云南农业大学动物医学院, 昆明 650200;
2. 河南省农业科学院动物免疫学重点实验室, 郑州 450002;
3. 河南农业大学牧医工程学院, 郑州 450002

收稿日期:2018-09-25 出版日期:2019-03-20 发布日期:2019-03-20
通讯作者: 张以芳, 张改平 E-mail:zyfkm@qq.com;zhanggaip@126.com
作者简介:李翔(1992-),男,湖北黄冈人,硕士生,研究方向:猪伪狂犬病病毒病原学及致病机制,E-mail:lx.5636125@qq.com郭振华(1984-),男,河南新乡人,博士,助理研究员,研究方向:猪病毒病病原学及分子流行病学,E-mail:gzhuavet@163.com
基金资助:
国家重点研发计划专项"畜禽重要疫病病原学与流行病学研究（2017YFD0500105）"；云南省院士工作站（2014IC034）；河南省农业科学院科研发展专项资金项目（2019CY01）

Isolation and Identification of Two Pseudorabies Virus Variants and Analysis of Molecular Characteristics of gB,gC and gE Genes

LI Xiang¹, GUO Zhenhua², RUAN Haiyu³, QIAO Songlin², ZHANG Yifang¹, ZHANG Gaiping^2,3

1. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650200, China;
2. Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received:2018-09-25 Online:2019-03-20 Published:2019-03-20

摘要/Abstract

摘要：

为探明近年来河南省猪伪狂犬病病毒（PRV）的遗传变异情况，本研究于2017年采集河南漯河和中牟地区疑似伪狂犬病发病养殖场送检的脑组织病料，通过细胞盲传、噬斑纯化、间接免疫荧光试验、Western blotting和透射电镜技术进行病毒分离鉴定。TCID₅₀法测定分离毒株的病毒滴度、生长曲线，通过小鼠感染试验测定分离毒株对小鼠的致死性。对gB、gC和gE基因进行PCR扩增、测序，并与参考毒株序列进行比对分析。结果显示，对PCR鉴定阳性的病料在PK-15细胞盲传后，两份病料在6代内均出现细胞病变，通过间接免疫荧光试验、噬斑纯化和透射电镜技术，成功分离鉴定了两株PRV，分别命名为HeN-LH株及HeN-YM株。分离毒株在PK-15细胞上的生长曲线显示，HeN-LH和HeN-YM株在感染后36 h病毒滴度分别可达10^8.35和10^6.63 TCID₅₀/mL。用不同浓度的病毒接种小鼠，结果显示，HeN-LH和HeN-YM株LD₅₀分别为10^2.13及10^3.25 TCID₅₀。对gB、gC和gE基因全长扩增测序后构建遗传进化树，结果显示，两株PRV毒株与Bartha、Fa和Ea等经典株的亲缘关系相对较远，而与2011年以来国内不同省份分离的PRV变异株亲缘关系较近。氨基酸序列比对分析显示，与其他变异株相似，gB、gC和gE基因均发生了多个氨基酸的变异，且在特定的位点存在特征性的氨基酸插入和缺失。本研究成功分离鉴定了两株PRV变异株，分离株对小鼠均表现出一定的致病性，本试验结果可为河南省伪狂犬病的防控工作和疫苗株的选择提供科学依据。

关键词: 伪狂犬病病毒(PRV); 分离鉴定; 噬斑纯化; 感染试验; 序列分析

Abstract:

To investigate the genetic variation of pseudorabies virus (PRV) in Henan province in recent years,in this study,the virus isolation and identification were performed by cell blind transmission,indirect immunofluorescence,Western blotting,plaque purification and transmission electron microscopy in brain tissue samples from suspected pseudorabies farms in Luohe and Zhongmou in 2017.The viruses titer and growth curve of the isolated strains were determined by 50% tissue culture infective dose (TCID₅₀),and the lethality of the mice was determined by mouse infection test.Futhermore,the gB,gC and gE genes were aligned and analyzed.Two strains of PRV were successfully isolated and identified by indirect immunofluorescence,plaque purification and transmission electron microscopy,named as HeN-LH strain and HeN-YM strain,respectively.The titers of HeN-LH and HeN-YM strains reached 10^8.35 and 10^6.63 TCID₅₀/mL at 36 h post infection on PK-15 cells.The lethality of the mice showed that the median lethal dose (LD₅₀) of HeN-LH and HeN-YM strains was 10^2.13 and 10^3.25 TCID₅₀,respectively.The evolutionary analysis displayed that the two isolates were closer to the PRV variants which were mainly epidemic in China since 2011.Amino acids sequences alignment showed that,similar to other variants,gB,gC and gE genes had multiple amino acid variations,and there were unique amino acid insertions and deletions at specific sites.In short,we successfully isolated and identified two PRV variants,and confirmed the current pseudorabies were mainly caused by the PRV variants,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Henan province.

Key words: pseudorabies virus (PRV); isolation and identification; plaque purification; infection test; sequence analysis

中图分类号:

S852.65

李翔, 郭振华, 阮海宇, 乔松林, 张以芳, 张改平. 两株猪伪狂犬病病毒变异株的分离鉴定及gB、gC和gE基因的分子特征分析[J]. 《中国畜牧兽医》, 2019, 46(3): 881-890.

LI Xiang, GUO Zhenhua, RUAN Haiyu, QIAO Songlin, ZHANG Yifang, ZHANG Gaiping. Isolation and Identification of Two Pseudorabies Virus Variants and Analysis of Molecular Characteristics of gB,gC and gE Genes[J]. , 2019, 46(3): 881-890.

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两株猪伪狂犬病病毒变异株的分离鉴定及gB、gC和gE基因的分子特征分析

Isolation and Identification of Two Pseudorabies Virus Variants and Analysis of Molecular Characteristics of gB,gC and gE Genes

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