四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析

doi:10.3969/j.issn.1004-1524.2019.09.05

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (9): 1429-1436.DOI: 10.3969/j.issn.1004-1524.2019.09.05

四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析

易可可¹, 阴文奇², 周远成³, 蒋金蓁¹, 张白玉¹, 李中银⁴, 颜其贵^1,*

1.四川农业大学动物医学院,四川成都 611130;
2.四川省畜牧科学研究院,四川成都 610066;
3.四川华神兽用生物制品有限公司,四川成都 610200;
4.四川伊禾动物药品有限公司,四川成都 610066

收稿日期:2019-02-07 出版日期:2019-09-25 发布日期:2019-10-11
通讯作者: *颜其贵,E-mail: yanqigui@126.com
作者简介:易可可(1994—),女,湖南岳阳人,硕士,研究方向为兽医微生物与免疫学。E-mail: kkky1024@163.com

Isolation and identification of 4 strains of porcine pseudorabies virus and analysis of main virulence genes

YI Keke¹, YIN Wenqi², ZHOU Yuancheng³, JIANG Jinzhen¹, ZHANG Baiyu¹, LI Zhongyin⁴, YAN Qigui^1,*

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Sichuan Academy of Animal Sciences, Chengdu 610066, China;
3. Sichuan Huashen Veterinary Biological Products Co., Ltd., Chengdu 610200, China;
4. Sichuan Yihe Animal Pharmaceutical Co., Ltd., Chengdu 610066, China

Received:2019-02-07 Online:2019-09-25 Published:2019-10-11

摘要/Abstract

摘要： 猪伪狂犬病毒(PRV)的流行对中国养猪业造成巨大损失,而2011年后许多已免疫PRV疫苗的猪场频繁出现gE抗体转为阳性现象,感染猪出现PRV的临床症状,并出现所谓的“流产风暴”,学者们怀疑PRV的重新流行与病毒毒力增强和基因变异有关。为了解PRV变异情况,从各地疑似PRV阳性病料中,通过PK-15细胞分离出4个毒株,对毒株传代培养,进行TCID₅₀与LD₅₀测定,对主要毒力基因gB、gC、gE和TK进行扩增测序后分析,确定该4株病毒为PRV株,分别命名为FJ01株、FJ03株、YK株和MS2018株,滴度分别为10^-6.63、10^-7.08、10^-8.10、10^-7.18 TCID_50s·0.1mL^-1,对Balb/c小鼠的LD₅₀分别为10^2.17、10^2.72、10^3.44、10^3.51 TCID_50s,可见FJ01株的毒力最强。对4个毒株的毒力基因与其他PRV毒株进行同源性比对并建立进化树,FJ01株、FJ03株、MS2018株与中国近几年流行的变异毒株如HNX株、HNB株、JS-2012株等在一个大进化分支上,亲缘性较近,而与疫苗株Bartha-K61、SA215等,国际经典毒株Becker、Kaplan等亲缘性较远,变异较大。YK株与国际毒株亲缘性更近,其原因有待进一步探索。

关键词: 猪伪狂犬病毒, 分离鉴定, 毒力基因, 序列分析

Abstract: Pseudorabies virus(PRV)’s prevalence brings huge economic losses to the domestic pig industry, however, many large-scale immunized pig herds broke out pseudorabies, and gE antybodies turned from negative to positive since 2011, causing PRV clinical symptoms infected pigs, and then resulting in “abortion storm”. Most scholars believed that national outbreak of PRV might be related to factors such as viral virulence and genetic variation. In this study, four pseudorabies viruses were successfully isolated from suspected PRV infected pigs by PK-15 cells, moreover, their TCID₅₀ and LD₅₀ were determined. Their main immunogenic genes and virulence-related genes gB, gC, gE and TK were amplified, sequenced and analyzed. The 4 pseudorabies viruses were affirmed, named as FJ01, FJ03, YK and MS2018 strain, respectively, whose titers were 10^-6.63, 10^-7.08, 10^-8.10, 10^-7.18 TCID_50s·0.1mL^-1, respectively, and the results of virulence test in Balb/c mice showed as 10^2.17, 10^2.72, 10^3.44, 10^3.51 TCID_50s, respectively. The virulence of FJ01 strain was the strongest. Their nucleotide and amino acid homologies were analyzed and evolutionary trees were established. The results showed that the genes of FJ01, FJ03 and MS2018 strains had higher homology with the mutants such as HNX, HNB and JS-2012 strains which were isolated in recent years. In evolutionary relationship, they belonged to a large branch, however, they belonged to different evolutionary branches from vaccine Bartha-K61,SA215, and foreign strains Becker and Kaplan with evident variations. YK had the closer affinity with foreign strains in evolutionary relationship, but the reason remained to be further explored.

Key words: pseudorabies virus, isolation and identification, virulence gene, sequence analysis

中图分类号:

S858.28

易可可, 阴文奇, 周远成, 蒋金蓁, 张白玉, 李中银, 颜其贵. 四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析[J]. 浙江农业学报, 2019, 31(9): 1429-1436.

YI Keke, YIN Wenqi, ZHOU Yuancheng, JIANG Jinzhen, ZHANG Baiyu, LI Zhongyin, YAN Qigui. Isolation and identification of 4 strains of porcine pseudorabies virus and analysis of main virulence genes[J]. , 2019, 31(9): 1429-1436.

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四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析

Isolation and identification of 4 strains of porcine pseudorabies virus and analysis of main virulence genes

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