猪伪狂犬病病毒变异毒株HeNZK-2014的分离鉴定及gE基因序列分析

发布时间：2018-01-24 07:42

本文关键词： 猪伪狂犬病病毒变异毒株分离鉴定 gE基因遗传变异分析　出处：《中国畜牧兽医》2017年07期 　论文类型：期刊论文

【摘要】：为了解猪伪狂犬病病毒(PRV)变异毒株的特点,本研究采集临床疑似PRV感染发病猪的淋巴结等组织样品进行PCR鉴定,选取仅PRV阳性的组织样品经研磨除菌后取上清接种于PK-15细胞进行病毒分离培养、蚀斑纯化、PCR和间接免疫荧光法(IFA)鉴定,采用Reed-Muench法测定PRV的TCID50,接种小鼠并观察临床症状,对纯化的PRV和死亡小鼠脑组织样品进行gE基因PCR扩增及测序分析。结果显示,PRV阳性病料接种于PK-15细胞24h后出现典型细胞病变(CPE),经3轮蚀斑纯化后PCR和IFA鉴定结果均为阳性,分离株命名为HeNZK-2014,其TCID50为10-9.77/0.1mL;以1×108个TCID50病毒悬液接种小鼠22h后可引起小鼠出现奇痒、撕咬、死亡等典型猪伪狂犬病症状,死亡小鼠脑组织样品PRV PCR检测结果为阳性;纯化病毒和死亡小鼠脑组织样品gE基因核苷酸序列同源性为100.0%,与GenBank中2011年以前登录的经典毒株位于不同分支,与2011年之后中国流行毒株位于同一分支,在第48和496位各有1个天冬氨酸(D)的插入,具有变异毒株的典型特征。本研究成功分离了1株PRV变异毒株,为进一步开展针对PRV变异毒株的疫苗及其防控研究奠定了基础。
[Abstract]:In order to understand the characteristics of porcine pseudorabies virus (PRV) variant strains, PCR samples were collected from clinical samples of swine lymph nodes suspected to be infected with pseudorabies virus (PRV). Only PRV positive tissue samples were harvested and inoculated into PK-15 cells after grinding and inoculated with the supernatant for virus isolation and culture. PK-15 was identified by plaque purification and indirect immunofluorescence assay (IFA). The TCID50 of PRV was determined by Reed-Muench method. The mice were inoculated and the clinical symptoms were observed. The purified PRV and brain tissue samples of dead mice were amplified by PCR of GE gene and sequenced. PRV positive venereal material inoculated with PK-15 cells for 24 hours showed typical cytopathic changes. After three rounds of plaque purification, both PCR and IFA were positive. The isolate was named HeNZK-2014 and its TCID50 was 10-9.77 / 0.1 mL; Mice inoculated with 1 脳 108 TCID50 virus suspension for 22 hours could cause typical pig pseudorabies symptoms such as itching, biting and death. PRV PCR was positive in brain tissue of dead mice. The homology of nucleotide sequence of GE gene between purified virus and brain tissue of dead mice was 100.0%, which was located in different branches with the classical virus strains which were registered before 2011 in GenBank. It is located in the same branch as the Chinese epidemic strain after 2011, and there is one aspartic acid D insertion at the 48th and 496th positions, respectively. One PRV variant strain was successfully isolated, which laid a foundation for further research on vaccine and control of PRV variant strain.
【作者单位】：河南省动物疫病预防与控制中心;广东永顺生物制药股份有限公司;
【基金】：河南省科技创新人才计划项目(174200510003)
【分类号】：S852.651
【正文快照】： 伪狂犬病(pseudorabies,PR)是由伪狂犬病病毒(pseudorabies virus,PRV)感染引起的可致多种动物发病的疫病,其中仔猪受到的危害最为严重。感染PRV后怀孕母猪会出现流产,产死胎、木乃伊胎;新生仔猪会出现腹泻、衰竭及严重的精神症状,死亡率可达100%[1-2]。PR给世界养殖业造成的

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