1株库蠓源版纳病毒的分离与全基因组序列分析

Isolation and Whole Genome Sequence Analysis of a Banna Virus Strain from Culicoides

为分析云南省虫媒病毒的种类与遗传特征,在云南省师宗县采集库蠓进行病毒的分离与鉴定;通过全长cDNA扩增与高通量测序技术获取病毒全基因组序列,进行序列比对与系统发生树构建。结果显示,从采集的库蠓样本中分离出1株可在C6/36细胞上引起细胞病变的毒株(YNSZ043),病毒基因组为分节段双链RNA,琼脂糖凝胶电泳呈“2-4-3”的带型特征;电镜观察可见直径为70~80 nm,呈“指环状”,表面具有纤维突起的病毒粒子。全基因组测序结果显示,YNSZ043毒株为版纳病毒(Banna virus,BAV),基因组大小为20683 bp,由Seg-1(3762 bp)至Seg-12(861 bp)12个基因节段组成,与中国BAV毒株各基因节段的核苷酸序列相似性在64.8%~99.6%之间,氨基酸序列相似性在58.8%~100%之间,在系统发生树上YNSZ043毒株与中国分离的BAV聚为一簇,形成独立的中国进化支系。对决定BAV基因型的Seg-12分析结果显示,YNSZ043毒株属于A2基因型,该毒株的Seg-5/VP5与越南分离BAV毒株的核苷酸和氨基酸序列相似性高达97.1%和97.6%,表明该毒株的Seg-5基因节段很可能与越南毒株之间发生了基因重配。研究结果丰富了中国BAV的基因组序列,为开展云南省BAV的流行病学研究提供了参考。

To investigate the variety and genetic characteristics of arboviruses epidemic in Yunnan province,Culicoides were collected in Shizong county and inoculated cells for virus isolation and identification.Whole genome sequence of the isolated virus was obtained by full-length cDNA amplification(FLAC)and next generation sequencing(NGS),then analyzed through sequence alignment and phylogenetic tree.The results showed that a virus strain(YNSZ043)causing cytopathic effect on C6/36 cells was isolated from collected Culicoides specimens.The genome of the isolated virus was composed with double-stranded segmented RNA,showing migration pattern of“2-4-3”by agarose gel electrophoresis.The viral particle of the isolated virus was about 70 to 80 nm in diameter exhibiting morphological feature of“finger ring”,with fibrous protrusions on the surface.The genome size of the YNSZ043 was 20683 bp in length with 12 gene segments ranging from 3762 bp(Seg-1)to 861 bp(Seg-12),with nucleic acid sequence identities ranging from 64.8%to 99.6%and amino acid sequence identities ranging from 58.8%to 100%,compared with other Chinese BAV strains.On the phylogenetic tree,YNSZ043 clustered together with other Chinese BAV strains forming an independent Chinese evolutionary branch.YNSZ043 belonged to A2 genotype by phylogenetic analysis of Seg-12 which decided the genotype of BAV.Maximum nucleic and amino acid sequence identity of Seg-5/VP5 were observed between YNSZ043 and BAV strains isolated from Vietnam,which were 97.1%and 97.6%,respectively,indicated there might be Seg-5 reassortment between Vietnamese strains and YNSZ043 strain.The present study enriched the genome sequence of BAV,provided a foundation for further epidemiological studies of BAV in Yunnan.