猪伪狂犬病病毒GZYY2015变异株的分离鉴定

doi:10.16431/j.cnki.1671-7236.2021.05.035

摘要/Abstract

摘要： 本试验旨在从贵州省贵阳市云岩区某养猪场送检的经检测为猪伪狂犬病（PR）的样品中分离猪伪狂犬病病毒（PRV）。用细胞接毒试验、理化试验和透射电镜观察等方法分离、鉴定病原，测定病原的毒价后进行动物试验和gD、gE基因序列比对分析。结果显示，细胞接毒试验成功分离能致Vero细胞产生典型病变的PRV。理化试验表明，该分离毒株对5-碘脱氧尿核苷（idoxuridine，IUDR）和有机溶剂氯仿敏感，不耐酸和热。在透射电镜下可见近圆形、有囊膜、150~160 nm大小的成熟病毒粒子，在胞核中可见衣壳呈晶格状排列的病毒包涵体，有实心和空心2种形态；动物试验表明，该毒株对兔具有较强的致病性，可导致接种部位奇痒；核酸鉴定结果表明，扩增获得的PRV gD基因开放阅读框（ORF）为1 209 bp，gE基因ORF为1 740 bp。GenBank比对结果显示，gD基因与PRV JS 2012株（登录号：KP257591）、PRV HNB株（登录号：KM189914.3）核苷酸序列相似性均为99.8%，与疫苗株Bartha-K61（登录号：JF797217.1）相似性为98.7%；gE基因与PRV JS2012株（登录号：KP257591）、PRV HNB株（登录号：KM189914.3）和PRV Qihe547（登录号：KU056477）核苷酸序列相似性均为99.9%。基于gD、gE基因推导的氨基酸序列的遗传进化分析发现，gD、gE基因与国内变异株均位于同一进化分支，对gE基因的遗传变异性分析结果表明，该分离毒株符合PRV变异株的变异模式。本试验结果表明，成功分离PRV变异株，可为贵州省PRV的流行病学遗传进化分析及免疫防控等提供参考。

关键词: 伪狂犬病病毒; 变异株; 分离鉴定; gE基因; gD基因

Abstract: The purpose of this experiment was to isolate Pseudorabies virus (PRV) from a sample tested as pseudorabies (PR) sent by a pig farm in Yunyan district,Guiyang city,Guizhou province.Methods such as cytotoxicity experiments,physical and chemical experiments,and transmission electron microscopy observations were used to isolate and identify the pathogen,and then to determine the virus value of the pathogen and then conduct animal experiments and comparative analysis of gD and gE genes sequences.The results showed that the cell inoculation experiment successfully isolated PRV that could cause Vero cells to produce typical lesions.The physicochemical experiment showed that the isolated virus was unstable in the environment of acid and heat,and sensitive to idoxuridine(IUDR) and chloroform.Transmission electron microscopy results showed that the mature virus particles with envelope in nucleus were approximatively round with a diameter of 150-160 nm,and the virus particles in nucleus were hollow or stuffed without envelope,and the capsids were lattice arrangement in mature viral inclusion bodies.Animal experiment showed that the strain had strong pathogenicity to rabbits,which could cause itching at the inoculation site.The results of nucleic acid identification showed that the open reading frame(ORF) of the amplified PRV gD gene was 1 209 bp,and the ORF of gE gene was 1 740 bp.The results of GenBank comparison showed that the gD gene of the isolated strain was 99.8% similarity with the reference strains of PRV JS2012 strain (accession No.:KP257591) and PRV HNB strain (accession No.:KM189914.3),and the similarity with vaccine strain Bartha-K61 (accession No.:JF797217.1) was 98.7%.The gE gene nucleotide sequence was 99.9% similar to PRV JS 2012 strain (accession No.:KP257591),PRV HNB strain (accession No.:KM189914.3) and PRV Qihe547 (accession No.:KU056477).According to the phylogenetic tree of gD and gE amino acid sequences,the isolated strain belonged to the same branch of the domestic mutant strains.These results indicated that the PRV variant strain was successfully isolated,which could provide references on PRV epidemiology,genetic evolution analysis and immune control of PR in Guizhou province.

Key words: Pseudorabies virus; mutant strain; isolation and identification; gE gene; gD gene

中图分类号:

S852.65⁺1

黄二素, 咸文, 张爱琼, 王彬, 曾智勇, 汤德元, 梁海英, 何小莉, 徐玉, 徐松平, 祝羊. 猪伪狂犬病病毒GZYY2015变异株的分离鉴定[J]. 中国畜牧兽医, 2021, 48(5): 1832-1840.

HUANG Ersu, XIAN Wen, ZHANG Aiqiong, WANG Bin, ZENG Zhiyong, TANG Deyuan, LIANG Haiying, HE Xiaoli, XU Yu, XU Songping, ZHU Yang. Isolation and Identification of Porcine Pseudorabies Virus GZYY2015 Mutant Strain[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1832-1840.

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