猪伪狂犬病病毒流行株HLJ-01的分离鉴定及致病性分析

Isolation and pathogenicity of a pseudorabies virus variant HLJ-01

【背景】自2011年以来,猪伪狂犬病毒(pseudorabies virus,PRV)发生变异,经典的疫苗株已不能完全抵抗PRV变异株的感染,国内多个猪场出现伪狂犬病的暴发,PRV变异毒株开始在我国大规模流行。【目的】通过分离PRV流行变异毒株,并对其进行遗传进化和致病性分析,为PRV流行病学调查及疫苗研制提供实验数据。【方法】采集黑龙江某猪场感染PRV的脑组织病料,根据GenBank PRV gE和gB保守序列设计引物,进行PCR鉴定。通过对gE和gC基因进行序列测定和遗传进化分析。利用BHK-21细胞分离病毒,采用噬斑纯化方法对病毒进行纯化。通过电镜、间接免疫荧光对病毒进行鉴定,测定病毒生长曲线并进行致病性研究。【结果】经PCR和测序鉴定分离株为PRV流行株,将其命名为HLJ-01。遗传进化分析结果显示,该分离毒株与我国近几年分离的流行变异株位于同一分支;氨基酸序列分析结果显示,gE和gC存在国内流行变异株的特征序列,表明该分离毒株为流行变异株。生长曲线显示,分离株HLJ-01在感染48 h时滴度最高(108.5 TCID50/mL)。电镜观察结果显示,病毒颗粒直径约150 nm,呈球形,有囊膜,囊膜外有放射状纤突,呈现典型PRV病毒特征。动物感染实验结果显示,107.0 TCID50剂量感染组死亡率为100%;106.0 TCID50剂量感染组死亡率为80%;105.0 TCID50剂量感染组死亡率为60%。仔猪在接种病毒后均出现PRV感染的典型症状和病理变化,证实分离毒株对仔猪有较强致病力。【结论】分离获得一株猪伪狂犬病毒,经鉴定该分离株为流行变异株,而且具有较强的致病力,这为PRV流行病学分析及疫苗候选株的筛选奠定了基础。

[Background]Pseudorabies virus(PRV)has been mutating since 2011 and the classical vaccine strain can no longer completely resist the infections of PRV variants.There have been outbreaks of pseudorabies in many domestic pig farms and the PRV variants have become prevalent in China.[Objective]To isolate a PRV variant,analyze its genetic evolution and pathogenicity,and provide experimental data for the epidemiological investigation and vaccine development of PRV.[Methods]The brain tissue samples infected with PRV were collected from a pig farm in Heilongjiang province.The primers were designed according to gE and gB conserved sequences of PRV in GenBank for PCR identification.The gE and gC genes were sequenced for phylogenetic analysis.The virus was isolated from BHK-21 cells by plaque purification method and further identified by electron microscopy and indirect immunofluorescence.The growth curve was established and the pathogenicity was studied.[Results]The isolate was identified as a PRV epidemic strain by PCR and sequencing and named HLJ-01.The phylogenetic tree showed that the isolate was in the same clade with the epidemic variants isolated in recent years in China.The results of amino acid sequence analysis showed that gE and gC of the isolate had the characteristic sequences of domestic epidemic variants,indicating that the isolate was an epidemic variant.The growth curve showed that the titer of HLJ-01 was the highest(108.5 TCID50/mL)48 h after infection.The virus particle of HLJ-01 had the diameter of about 150 nm,and it was a sphere with envelope and radial spikes outside,presenting typical characteristics of PRV.The animal infection experiments showed that the mortality rates of 107.0,106.0,and 105.0 TCID50 infection groups were 100%,80%,and 60%,respectively.After inoculation of HLJ-01,all the piglets showed typical symptoms and pathological changes of PRV infection,which confirmed that HLJ-01 had strong pathogenicity to piglets.[Conclusion]An epidemic variant of PRV with strong pathogenicity was isolated and identified,which laid a foundation for the epidemiological analysis of PRV and the screening of candidate vaccine strains.