安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

doi:10.3969/j.issn.1004-1524.2020.01.06

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (1): 43-56.DOI: 10.3969/j.issn.1004-1524.2020.01.06

安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

袁献宇¹, 杨龙斌¹, 何赞赞¹, 毛天骄¹, 何长生², 占松鹤², 孙裴¹, 魏建忠¹, 李郁^1,*

1. 安徽农业大学动物科技学院,安徽合肥 230036;
2. 安徽省动物疫病预防与控制中心,安徽合肥 230091

收稿日期:2019-07-25 出版日期:2020-01-25 发布日期:2020-03-11
通讯作者: ^*李郁,E-mail:liyouer@163.com
作者简介:袁献宇(1994—),男,安徽宿州人,硕士研究生,研究方向为动物传染病。E-mail:253017377@qq.com
基金资助:
国家星火计划重点项目(2014GA710002); 安徽省质量工程项目(2013sxzx008); 安徽省生猪产业体系基金(皖农科〔2016〕84号)

Isolation and identification of pseudorabies virus and molecular characterization of its main virulence genes in Anhui

YUAN Xianyu¹, YANG Longbin¹, HE Zanzan¹, MAO Tianjiao¹, HE Changsheng², ZHAN Songhe², SUN Pei¹, WEI Jianzhong¹, LI Yu^1,*

1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;
2. Anhui Animal Epidemic Prevention and Control Center,Hefei 230091, China

Received:2019-07-25 Online:2020-01-25 Published:2020-03-11

摘要/Abstract

摘要： 猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。

关键词: 猪伪狂犬病毒, 分离鉴定, 毒力基因, 序列分析

Abstract: Porcine pseudorabies (PR) is an acute and hot infectious disease caused by pseudorabies virus (PRV). Since the end of 2011, PRV had been mutated. The traditional attenuated PRV vaccine can not provide complete protection for PRV mutants, which brings great challenges to the prevention and control of PRV in China. To understand the molecular characteristics of major virulence genes of PRV clinical epidemic strains in Anhui Province, PCR,cell-inoculation tests, transmission electron microscopy (TEM), indirect immunoinfluscent assay (IFA) and animal test were used to isolate and identify the pathogen. The major virulence genes (gE, gI, TK, gB, gC and gD) of PRV strains were cloned and sequenced by PCR with 6 pairs of specific primers. A total of 15 PRV strains of clinical cases were isolated and identified from Anhui Province during 2016-2018. The sequences of gE, gI, TK, gB, gC and gD genes of PRV were highly homologous with those of PRV mutants which were prevalent in China after 2011. Compared with the sequence of domestic PRV classical strains before 2011, there are multiple substitutions, insertions or deletions of multiple sites in the gE, gB, gC and gD gene sequences of PRV isolates, and some mutations were located in important epitope regions. The above results indicated that 15 PRV strains isolated from Anhui Province from 2016 to 2018 were all variant strains, which have become the main epidemic strains in Anhui Province. The gene sequences of gI and TK of 15 PRV isolates were relatively conservative, but the mutation of amino acids in the epitopes of gE and gC protein might change their virulence and antigenicity. The homology of PRV sequence between some PRV isolates from neighboring areas was 100%, which may be related to the frequent trans-provincial transfer and trans-regional introduction of pigs.

Key words: pseudorabies virus, isolation and identification, virulence gene, molecular characterization

中图分类号:

S855.3

袁献宇, 杨龙斌, 何赞赞, 毛天骄, 何长生, 占松鹤, 孙裴, 魏建忠, 李郁. 安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征[J]. 浙江农业学报, 2020, 32(1): 43-56.

YUAN Xianyu, YANG Longbin, HE Zanzan, MAO Tianjiao, HE Changsheng, ZHAN Songhe, SUN Pei, WEI Jianzhong, LI Yu. Isolation and identification of pseudorabies virus and molecular characterization of its main virulence genes in Anhui[J]. , 2020, 32(1): 43-56.

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安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

Isolation and identification of pseudorabies virus and molecular characterization of its main virulence genes in Anhui

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