一株新型云南牛环状病毒的分离鉴定

Identification of a novel Orbivirus strain isolated from sentinel cattle in Yunnan Province

【目的】为加深云南省动物虫媒病毒多样性的认知。【方法】在云南省景洪市设立哨兵牛,定期采血接种细胞进行虫媒病毒的分离;通过RT-PCR、电镜观察与基因组琼脂糖凝胶电泳对分离病毒进行初步鉴定;扩增分离病毒的基因节段2(Seg-2)与基因节段3(Seg-3)全长序列进行分析与系统发生树的构建,明确病毒的分类地位;通过qRT-PCR与血清中和试验对病毒在动物上的感染进行回溯分析。【结果】2019年7月,从哨兵牛上采集的血液中分离到1株可在BHK-21细胞上引起细胞病变的病毒;电镜下观察,病毒粒子呈球形,直径约70 nm;琼脂糖凝胶电泳显示病毒基因组由双链RNA组成,呈现“3-3-3”的带型特征。分离毒株的Seg-2编码环状病毒属病毒保守的T2蛋白,与Mobuck virus具有最近的亲缘关系,核酸与氨基酸序列相似度分别为72.8%与84.0%;分离毒株的Seg-3编码决定环状病毒属病毒血清型的OC1蛋白,与Mobuck virus的核酸与氨基酸序列相似度仅为60.2%与56.5%;在T2与OC1蛋白构建的系统发生树上,分离毒株形成独立于Mobuck virus的进化分支。对哨兵牛血液中的病毒核酸与血清中和抗体回溯分析结果显示,从病毒感染哨兵动物至监测期结束的17周内,动物血液中均可检测到病毒核酸;动物感染病毒1周后,开始产生特异性中和抗体,随后上升至最高水平(抗体效价1:226),至监测期结束,中和抗体仍维持在较高水平(抗体效价1:113)。【结论】本研究首次报道了1种新型Mobuck virus在云南省哨兵牛上的分离与感染特性。研究结果丰富了对我国环状病毒属病毒的认知,为开展病毒的诊断、流行病学与致病性研究奠定了基础。

[Objective]Deepen our understanding about the diversity of epidemic arboviruses of Yunnan Province[Methods]Blood samples regularly collected form sentinel cattle placed in Jinghong city of Yunnan Province,were inoculated on cells for arboviruses isolation.The isolated virus strain was initially identified by RT-PCR,viral genomic agarose gel electrophoresis(AGE)and electron microscope(TM).Full-length segment 2(Seg-2)and segment 3(Seg-3)of the isolated virus were sequenced and the virus classification was conducted by phylogenetic tree construction.The infection kinetics of the isolated virus on the sentinel cattle was retrospectively traced back by qRT-PCR and serum neutralization test.[Results]An unidentified virus strain,causing significant cytopathic effect on BHK-21 cells,was isolated from the blood sample of sentinel cattle.The genome of isolated virus is composed of double stranded RNA showing“3-3-3”migration pattern by AGE analysis.TM observation revealed that the virus particles exhibited typical characteristics of Orbivirus with ring-shaped structure about 70 nm in diameter.Seg-2 of the isolated virus encoding the conserved T2 protein of Orbivirus,showed the closest relationship with Mobuck virus with nucleic acid(nt)and amino acid(aa)sequence identities of 72.8%and 84.0%respectively.Meanwhile,Seeg-3 of the isolated virus encoding Orbivirus serotype decisive OC1 protein,only showed 60.2%(nt)and 56.5%(aa)sequences identity with Mobuck virus.Furthermore,on the phylogenetic tree of T2 and OC1,the isolated virus formed a distinctive branch from Mobuck virus.Retrospective tracing results of the virus infected cattle showed viral RNA lasted in the blood for 17 weeks until supervision terminated.Neutralization antibodies(antibody titer 1:14)were detected one week after virus infection,peaked at the 9^(th) week(antibody titer 1:226)and remained at a high level(antibody titer 1:113)to the end of monitoring.[Conclusion]We reported here,for the first time,the isolation and infection kinetics of a novel Mobuck virus strain isolated from sentinel cattle in Yunnan Province.The results of this study enriched our knowledge about orbiviruses in China,and laid a foundation for the diagnosis,epidemiology and pathogenicity of this novel virus.