牛血液中一株新型环状病毒的分离与全基因组序列分析

Isolation and Genomic Characterization of a Novel Orbivirus Strain from the Blood of Cattle

我们从广西壮族自治区哨兵动物牛上采集的血液样本中分离到一株病毒,暂将其命名为广西环状病毒（毒株号：V172/GX/2015）。病毒接种C6/36细胞后可产生明显的细胞病变,表现为细胞聚集、皱缩与脱落。高分辨率琼脂糖凝胶电泳显示,病毒基因组由10节段的双链RNA组成,在凝胶上呈现＂3-4-3＂的带型特征。通过全长cDNA扩增与高通量测序方法获取V172/GX/2015毒株的全基因组序列。病毒基因组大小为19 889bp,基因节段的大小在3 996bp（Seg-1）至829bp（Seg-10）之间,可编码VP1至VP7等7种结构蛋白以及NS1至NS3等3种非结构蛋白。对环状病毒属保守的VP1、VP3（T2）与VP7（T13）蛋白氨基酸序列分析与系统发育树分析显示,V172/GX/2015与蚊传播环状病毒Yunnan orbivirus、Peruvian horse sickness virus以及Mobuck virus具有较近的亲缘关系,氨基酸序列相似度在31.4%至69.9%之间;V172/GX/2015在系统发育树上形成了一个独立于其它环状病毒的进化分支。本研究首次报道了一种新型环状病毒在牛上的分离与全基因组序列,研究结果将进一步丰富我们对环状病毒属病毒的认知,为开展新型环状病毒的流行病学调查与致病性研究提供基础。

In 2015,a virus strain was isolated from the blood samples collected from the sentinel cattle in Guangxi Autonomous Region.The isolated virus was temporarily designated as Guangxi Orbivirus（strain V172/GX/2015）.After inoculation,the virus caused significant cytopathic effect in C6/36 cells,including aggregation,shrinkage and shedding.High resolution agarose gel electrophoresis showed that the viral genome consisted of 10 segments of double-stranded RNA and showed ＂3-4-3＂ migration pattern on the gel.Whole-genome sequence of the isolated virus was obtained by full-length cDNA amplification and highthroughput sequencing.The genome size of V172/GX/2015 strain was 19,889 bp with ten segments ranged from 3,996 bp（Seg-1）to 829 bp（Seg-10）,encoding seven structural proteins designated as VP1 to VP7 and three nonstructural proteins designated as NS1 to NS3.Sequence and phylogenetic analysis of the highly conserved VP1,T2 and T13 proteins of orbivirus species showed that V172/GX/2015 is most closed to Yunnan orbivirus,Peruvian horse sickness virus and Mobuck virus,with amino acid sequence similarity ranged from 31.4% to 69.9% .Furthermore,phylogenetic analysis placed the V172/GX/2015 in a branch distinct from other Orbivirus.This study first reported the genome sequence of a novel species of Orbivirus isolated from cattle.The results will enrich our understanding of Orbivirus and provide basis for the study of epidemiological investigation and pathogenicity of the novel Orbivirus.