鸭坦布苏病毒XZ-2012株的分离鉴定与基因变异分析

Isolation and gene variation character of duck Tembusu virus strain XZ-2012

[目的]2012年10月徐州地区某鸭场的樱桃谷种鸭突然发生产蛋下降,疑似鸭坦布苏病毒病。为了确诊病因,本试验进行了剖检与病原的分离鉴定。[方法]将病料匀浆处理后尿囊腔接种SPF鸡胚,分离病毒,接种BHK细胞培养,应用RT-PCR扩增病毒全基因,测序并进行基因进化分析。[结果]分离到一株鸭坦布苏病毒,命名为DTMUV XZ-2012株。该病毒株能适应鸡胚,无血凝活性,在BHK-21细胞上第5代的病毒滴度为5×104PFU·m L-1。测序结果表明该病毒株基因组全长为10 990 nt,编码的多聚蛋白含3 426个氨基酸。DTMUV XZ-2012株与BYD株、YY5株和JS_2010株等分离株的全基因同源性为98.5%~99.3%。与最早分离的BYD株等相比,XZ-2012株的基因变异主要分布于E基因、NS1基因、NS2基因和NS5基因,提示该病毒在自然界的流行存在一定的免疫压力。[结论]本试验成功分离鉴定了一株鸭坦布苏病毒,为鸭坦布苏病毒候选疫苗株的筛选及病毒分子流行病学研究提供了参考信息。

[ Objectives ] In October 2012,a duck disease with symptoms of suddenly egg production drop happened on a Cherry Valley duck farm at Xuzhou area,which was suspected to be duck Tembusu virus（DTMUV）disease. In order to diagnose the cause,necropsy and pathogen identification were carried out in the present study. [ Methods] The collected histological materials were homogenized and the filtered supernatant was inoculated into allantoic cavity of SPF chicken embryo. The viral isolates were inoculated into BHK cells and the virus titer was determining. Virus genome was cloned by RT-PCR and then sequencing and phylogenetic analysis were carried out. [ Results] We isolated the causative agent ,a duck Tembusu virus ,from affected ducks and designated XZ-2012 after determined the partial genome sequence. No hemagglutination activity was observed with the isolated virus in allantoic fluid. The DTMUV strain XZ-2012 can adapt to BHK cells and the virus titer of the 5th generation is 5x104 PFU-mL-＇. The genome of DTMUV strain XZ-2012 is 10 990 nucleotides（nt）in length and encodes a putative polyprotein of 3 426 amino acids. It is flanked by a 5＇ and a 3＇ noncoding region of 94 and 618 nt, respectively. The whole genome homology between XZ-2012 and previously reported virus isolates BYD, YY5 and JS_2010 et al. was 98.5% to 99.3%. Nucleotides mutation of DTMUV XZ-2012 strain was mainly located in E,NS1 ,NS2 and NS5 genes, which indicated that corrently transmitted DTMUV may endure immune pressure in nature. [ Conclusions ] We isolated and identified an epidemic strain of duck Tembusu virus, and analysed its gene variation character successfully. Information of the whole sequence of XZ-2012 strain will be useful for vaccine candidate selection and further investigation of the molecular epidemiology of DTMUV in China.