云南景洪帕利亚姆亚群达圭勒病毒的分离与鉴定

Isolation and Identification of D’Aguilar Virus from Palyamserogroup Virus in Jinghong,Yunnan Province

为了解牛感染帕利亚姆亚群达圭勒病毒(D’Aguilar virus,DAGV)情况,本研究应用BHK21细胞对2019年云南省景洪市采集的健康黄牛血液样品进行盲传病毒分离,对出现细胞病变的样品进行形态学、基因组带型和分子生物学鉴定,对分离到的病毒进行S2、S3、S7基因序列测定和比对分析。结果显示,有5个血液样品可致BHK21细胞病变,电镜观察到完整病毒颗粒呈球形,直径约50 nm;琼脂糖凝胶电泳结果发现5株新分离病毒基因组为10节段,呈现3-3-4的电泳带型特征,其带型特征与2014年在云南分离到的DAGV V106/YN/2014毒株相似;5株病毒S2、S3、S7基因核苷酸、氨基酸序列相似性均为100%,S3、S7片段核苷酸和氨基酸序列与中国本土分离的帕利亚姆亚群病毒(Palyamserogroup virus,PALV)毒株相似性最高;S2片段氨基酸与核苷酸序列与日本分离的DAGV相似性最高。S2、S3、S7基因遗传进化分析结果显示,5株新分离毒株间基因相似度为100%;5个毒株的S7与S3基因序列均与中国本土分离的已知部分PALV毒株在同一分支,亲缘关系较近,提示这5株毒株为PALV;5株毒株S2片段核苷酸序列与日本分离的部分DAGV毒株位于同一分支上,亲缘关系较近,进一步证实这5株毒株为PALV DAGV。本研究成功分离到5株DAGV毒株,并进行了基因片段遗传进化分析,为进一步开展DAGV流行病学研究提供基础。

In order to understand the bovine infection with D’Aguilar virus(DAGV),BHK21 cells were used to isolate the virus from the blood samples of healthy cattle collected in Jinghong city,Yunnan province in 2019.The samples with cytopathy were identified by morphology,genomic banding and molecular biology.The sequences of S2,S3 and S7 genes were determined and compared.The results showed that five samples of CPE were found.The complete virus particles were observed by electron microscope,it was about 50 nm in diameter.The results of agarose gel electrophoresis showed that the genome of 5 newly isolated viruses was 10 segments,and the banding pattern of the virus genome was characterized by 3-3-4 electrophoresis.The banding pattern of the viruses were similar to that of the DAGV V106/YN/2014 strain isolated in Yunnan province in 2014.The nucleotide and amino acid sequences of S2,S3 and S7 genes of the five strains were 100%,the S3 and S7 genes had the highest homology with PALV isolated in China,and the S2 gene had the highest homology with DAGV isolated from Japan.Genetic analysis of S2,S3,and S7 genes showed that the similarity of the five newly isolated strains were 100%.The S7 and S3 genes were in the same branch with some known PALV strains isolated from China,indicating that these five strains were PALV.The S2 gene of five strains was located in the same branch as some DAGV strains isolated from Japan.It was further confirmed that these five strains were PALV DAGV.In this study,five strains of DAGV were successfully isolated,and the genetic evolution analysis of gene fragments were carried out to provide the basis for further epidemiological study of DAGV.