牛源流行性出血病病毒(EHDV)血清10型毒株在我国的分离鉴定

我国存在多种血清型流行性出血热病毒(Epizootic haemorrhagic disease virus,EHDV)的流行,但尚未有关于EHDV-10型毒株的分离报道。为了解云南省EHDV的流行情况,2012～2015年,本研究在云南省设立江城、师宗、芒市三个监控点,定期采集监控动物血液,接种幼仓鼠肾细胞(Baby hamster kidney cell,BHK-21)进行病毒分离;通过PCR检测、血清中和试验、琼脂糖凝胶电泳和电镜观察等方法对分离病毒进行鉴定;对分离毒株的Seg-2/VP2与Seg-3/VP3基因节段进行克隆、测序与序列分析。2013年在云南省师宗县的哨兵牛上分离出一株EHDV毒株(YNSZ-V277-2013),病毒可引起BHK-21细胞出现圆缩、裂解的细胞病变(Cytopathic effect,CPE);电镜下病毒粒子呈球形,无囊膜,表面有大量纤维突,直径在70～80nm之间;病毒基因组dsRNA的琼脂糖凝胶电泳显示分离毒株与其他血清型EHDV一致,呈现"3-3-3"的电泳带型;序列分析显示YNSZ-V277-2013毒株的Seg-2/VP2与Seg-3/VP3序列与日本EHDV-10型毒株(ON-4/N/98)相似度最高,分别为97.5%/98.5%与98.1%/99.8%,证实分离毒株为EHDV-10型;系统发育分析显示YNSZ-V277-2013毒株的Seg-2与日本EHDV-10型毒株(ON-4/N/98)的亲缘关系最近,Seg-3与分离至日本和澳大利亚的EHDV毒株同属Eastern型。本研究首次报道了EHDV-10型毒株在我国的分离以及分离毒株的Seg-2与Seg-3基因序列特征,为进一步开展中国EHDV-10型的流行病学调查与致病性研究提供了基础。

Isolation and Identification of the Epidemic Hemorrhagic Fever Virus Serotype 10 Strain from Cattle in China

There are many serotypes of the epizootic hemorrhagic disease virus(EHDV) prevalent in China,but there is no report about the isolation of EHDV-10 strains. We wished to investigate the epidemiology of EHDV in Yunnan Province, China. Monitoring points were established at Jiangcheng, Shizong and Mangshi Counties within Yunnan Province from 2012 to 2015. For virus isolation, blood samples were collected regularly from sentinel herds and blind passed on baby hamster kidney cells(BHK-21 cells). The isolates were identified by polymerase chain reaction(PCR) detection, serum neutralization test, agarose-gel electrophoresis and electron microscopy observation. Seg-2/VP2 and Seg-3/VP3 genes of the isolated virus were cloned and sequenced. In 2013, an EHDV strain named YNSZ-V277-2013 was isolated from sentinel cattle at Shizong County. The isolate induced a cytopathic effect in BHK-21 cells that involved "rounding up" and exfoliation. Electronmicroscopic observation showed that the virus particles were spherical, without envelopes, and had fibers distributed on their surface,with a diameter of 70 nm ~80 nm. Agarose-gel electrophoresis of the genomic doublestranded RNA showed that the YNSZ-V277-2013 strain was consistent with other serotypes of EHDV, and showed a "3-3-3" type of migration pattern. Sequencing analyses of Seg-2/VP2 and Seg-3/VP3 showed that the YNSZ-V277-2013 isolate shared the highest nucleotide and amino-acid identity with the Japanese EHDV-10 strain ON-4/N/98(97.5%/98.5% and 98.1%/99.8%, respectively), which indicated that the isolated strain belonged to EHDV serotype 10. Phylogenetic analysis of Seg-2/VP2 revealed that the YNSZ-V277-2013 isolate displayed the greatest similarity with the Japanese EHDV-10 strain ON-4/N/98. Seg-3/VP3 of the isolate belonged to Eastern topotype with EHDV strains isolated from Japan and Australia. In the present study, the isolation of an EHDV-10 strain with sequence characteristics of Seg-2/VP2 and Seg-3/VP3 in China was reported for the first time, which could build a foundation for further epidemiological investigation and pathogenicity research of EHDV-10 in China.