东北虎和华南虎源传染性鼻气管炎病毒的PCR检测和序列分析

Diagnosis of Rhinotracheitis Virus from Siberian Tiger(Panthera tigris altaica) and South China Tiger(Panthera tigris amoyensis) by PCR and Sequence Analysis

为了鉴定东北虎和华南虎疑似传染性鼻气管炎病料中病毒的种类及进行流行病学分析,我们设计并合成了4对引物Fhv1、Fhv2、Fhv3、Fhv4用于扩增大小分别为193 bp,288 bp,312 bp和1088 bp的基因片段,同时对PCR扩增产物进行纯化、克隆至PMD18-T Simple载体、转化、酶切鉴定、测序及序列分析。结果显示设计并合成的4对引物Fhv1、Fhv2、Fhv3、Fhv4均扩增出与目的片段大小相符的明亮条带,分别约为200 bp、290 bp、310 bp、1100 bp;对Fhv3进行单酶切鉴定,出现大小分别约为160 bp、150 bp的2条条带;对Fhv4进行测序及序列分析,与GenBank^(TM)中已公布的猫传染性鼻气管炎病毒毒株(序列号:FJ478159.2)序列相似性为100%,与其他基因序列相似性均较低。本研究首次发现东北虎和华南虎源猫传染性鼻气管炎病毒,可为虎类的饲养管理及疾病的防控提供参考,为虎传染性鼻气管炎病毒的相关研究奠定基础。

We detected and identified the type of virus isolated from the mucosal tissues of Siberian Tiger（Panthera tigris altaica） and South China Tiger（Panthera tigris amoyensis） with clinical manifestations of upper respiratory disease.Four pairs of conserved primers Fhvl,Fhv2,Fhv3 and Fhv4 were prepared for PCR amplification of 193bp,288bp,312bp and 1088bp nucleotides.Then the purified PCR products were cloned into PMD18-T Simple vector,and transformed into DH5α.Finally,the combinant plasmids were extracted and identified by PCR and restriction endonucleases,and sequence analysis based on the Fhv4 sequence was performed using DNAStar software.AD primers had amplified clear strips in agarose gel with expected sizes.The product of Fhv3 was digested into approximately 160bp and 150bp fragments.The sequence similarity of Fhv4 was 100%compared with Feline Herpes virus（FJ478159.2） available in GenBank.This first report of tiger infection with Feline Herpesvirus,which might contribute to prevention,treatment and epidemiological survey of the disease,and lay a foundation for further related studies.