虎源伪狂犬病毒的鉴定及gB基因分析

Identification of Pseudorabies virus(PrV)and analysis of the gB gene from tigers in China

目的对不明原因死亡虎进行病毒感染检测,并对病毒部分保守基因序列进行分析。方法采集不明原因病死虎肺组织,研磨后提取总DNA,使用特异性引物对伪狂犬病毒gB基因、gD基因进行PCR扩增、测序并构建进化树;经负染法染色后用电子显微镜观察病毒形态结构;取研磨组织液接种家兔,观察病毒的致病力。结果 PCR扩增组织样品为gB基因、gD基因阳性,基因序列经blast比对伪狂犬病毒。将gB基因通过序列测定分析并与GenBank中近4年公布的9株伪狂犬病毒gB基因比对,同源性为99.5%-100%。进化树分析该病毒与2012年北京家猪分离株属同一进化分支。组织研磨液置电镜观察,见病毒呈圆形,直径100nm左右,有囊膜,为具有典型特征的疱疹病毒样病毒粒子。家兔接种组织研磨液后出现啃咬接种部位,抽搐,嘶叫等伪狂犬病症状。结论本研究在病死虎体内检测到伪狂犬病毒,表明濒危野生动物虎已受到伪狂犬病毒病的威胁。

Objective This study sought to detect viral infections in tigers that died of unknown causes and sequence some of the conserved genes of those viruses. Methods Total DNA was extracted from homogenate tissue collected from dead tigers and amplified with PCR using special primers for the gB and gD genes of the Pseudorabies virus.Phylogenetic analysis of these two genes was performed using Mega 5.0software.The virus was also identified with electron microscopy and used to infect rabbits in order to evaluate its virulence. Results The PCR product tested positive for the gB and gD genes and BLAST indicated that the virus was the Pseudorabies virus based on the sequences of those genes.The gB gene was from 99.5% to 100% similar to nine Pseudorabies virus strains in GenBank from the last four years.Phylogenetic analysis indicated that the virus was in the same clade as a strain isolated from pigs in Beijing in 2012.In terms of morphology,the virus had an envelope and was round with a diameter of 100 nm according to electron microscopy;these features are typical characteristics of the Pseudorabies virus.Rabbits that were administered the homogenate tissue displayed symptoms of pseudorabies such as biting at the administration site,seizures,and screaming. Conclusion The Pseudorabies virus was detected in dead tigers,indicating that the Pseudorabies virus is a threat to endangered wild tigers.