西部散养肉牛病毒性腹泻病毒流行及遗传变异

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)是一种可导致养牛业出现重大经济损失的常见病原体。该文以中国西部散养肉牛为研究对象,拟通过血清学方法和生物信息分析,评估BVDV在中国散养肉牛中的整体流行状况,探究国内流行的BVDV基因多样性,为制定针对BVDV的合理防控措施和疫苗的研发提供理论依据与素材。【方法】2014—2015年分别从云南、新疆、甘肃和内蒙古4省(区)采集散养肉牛的血清样本共1 332份,利用BVDV抗体检测试剂盒进行检测,统计不同地区散养肉牛BVDV抗体阳性率。将抗体阴性的血清样本进行BVDV抗原检测,抗原阳性或可疑血清接种MDBK细胞,连续盲传五代分离病毒。根据BVDV 5'-UTR保守序列设计引物,经RT-PCR扩增5'-UTR序列并测序。利用Sequencher4.2、BLAST、Clustal X、MEGA5.2等软件对序列进行生物信息学分析,绘制流行毒株系统进化树,确定分离毒株的基因型。【结果】4省(区)散养肉牛BVDV整体抗体阳性率为33.93%。血清抗体阳性率从高到低依次为内蒙71.43%,新疆57.69%,甘肃第16.54%,云南10.0%。BVDV抗原整体阳性率为1.58%,新疆抗原阳性率最高。研究共分离13株BVDV流行毒株,分别命名为甘肃120、内蒙1369、伊犁霍清12953、伊犁霍清12960、伊犁霍清12981、博州精河13001、博州精河13023、博州精河13033、新疆奇台13041、新疆奇台13042、新疆奇台13191、新疆奇台13220、新疆奇台13251。进化树分析显示新疆地区散养肉牛中流行的主要是BVDV-1q和BVDV-1f,内蒙地区流行的主要是BVDV-1m,甘肃地区流行的是一种新的基因亚型BVDV-1u。【结论】通过血清学方法检测发现,虽然4个省(市、自治区)散养肉牛整体抗体阳性率低于国内平均水平,但情况却不尽相同。内蒙古和新疆抗体高阳性率表明BVDV在这些地区散养肉牛中已经广泛流行,必须采取适当措施将其危害性降到最低。从基于5'-UTR序列绘制的系统进化树可以看出,不同地区散养肉牛感染的BVDV基因亚型有所不同。总体看来,BVDV在散养肉牛中的分布呈现多样性,跨物种、跨地域传播和高突变性等特点,这些都使疫苗免疫策略面临严峻的挑战。研究通过对各地区散养肉牛的随机大量采样、检测和分析,客观评价BVDV在散养肉牛中的流行情况,为更好的制定预防策略提供参考依据。

Prevalence Study and Phylogenetic Analysis of Bovine Viral Diarrhea Virus in Free-Roaming Beef Cattle in Western China

【Objective】Bovine viral diarrhea virus(BVDV) is commonly recognized as an important pathogen for the cattle and causes significant economic losses in the cattle industry worldwide. To assess the overall prevalence of BVDV in Chinese free- roaming beeves, the free-roaming beeves in the Western China were used in the present study by the methods of serological and bioinformatic analysis. Then, the genetic diversity of BVDV was analyzed. This study will provide a theoretical basis for the development of reasonable prevention and control, and for the vaccine development. 【Method】 A total of 1 332 sera were collected in Yunnan, Xinjiang, Gansu and Inner Mongolia from 2014 to 2015. These samples were analyzed by the commercial antibody (Ab) detection kits, and the Ab positive rate of BVDV in various regions were summarized up. Meanwhile, the positive or suspicious samples, whose S/P were less than 60%, were further detected by the Ag detection kit. Then, to isolate BVDV, the positive samples were inoculated into the MDBK cells and passaged by five generations. In addition, the primer designed based on the 5′-UTR of BVDV which has low frequency variability was used to analyze the genetic diversity of BVDV. Then the nucleotide sequences were analyzed by the bioinformatic softwares, including Sequencher4.2, BLAST, ClustalX and MEGA5.2. Finally, the genotypes and phylogenetic analysis of sequenced BVDV were confirmed based on the primers.【Result】Results showed that the average Ab positive rate of BVDV was 33.93% in the four detected provincial regions. Among them, that in the Inner Mongolia was the highest (71.43%), that in Xinjiang taking the second place (57.69%), that in Gansu was the third (16.54%) and that in Yunnan was the lowest (10.0%). The average Ag positive rate of BVDV was only 1.58%, that in Xinjiang was the highest. In this study, a total of 13 BVDV strains were isolated, they were named as Gansu 120, Mongolia 1369, Yilihuoqing 12953, Yillihuoqing 12960, Yilihuoqing 12981, Bozhoujinghe 13001, Bozhoujinghe 13023, Bozhoujinghe 13033, Xinjiangqitai 13041, Xinjiangqitai 13042, Xinjiangqitai 13191, Xinjiangqitai 13220, and Xinjiangqitai 13251, respectively. The polygenetic analysis indicated that these isolates could be mainly classified into four subtypes: BVDV-1q and BVDV-1f (isolated in Xinjiang), BVDV-1m (isolated in Inner Mongolia) and BVDV-1u which was a new subtype isolated in Gansu.【Conclusion】The serological analysis indicated that, although the free-range beef cattle overall Ab positive rate in the four provinces (autonomous regions) was lower than the national average, their situation was different. The high Ab positive rate of Inner Mongolia and Xinjiang indicated that BVDV is prevalent in the free-roaming beeves, and it is essential to minimize the damage by valuable controlling methods. The phylogenetic tree based on 5′-UTR sequence indicated that various genotypes of BVDV were existed in various regions. Overall, the diversity, cross-species transmission, cross-regional transmission and high mutation rate of BVDV make a great challenge for the immunity of vaccine. The significance of this study is to evaluate the prevalence of BVDV in free-range beef cattle through the method of healthy cattle random sample detection and analysis in various regions, and provide the reference for the prevention strategies.