中国帕利亚姆血清群中山病毒的分离及全基因组序列分析  

Isolation and complete genome characterization of Chuzan virus from Palyam serogroup in China

作　　者：寇美玲 孟锦昕 苗海生[1] 李乐[1] 谢佳芮 高林[1] 廖德芳[1] 李华春[1] 宋建领[1] KOU Meiling;MENG Jinxin;MIAO Haisheng;LI Le;XIE Jiarui;GAO Lin;LIAO Defang;LI Huachun;SONG Jianling(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《畜牧与兽医》2024年第7期65-71,共7页Animal Husbandry & Veterinary Medicine

基　　金：云南省热带亚热带动物病毒病重点实验室自主研究课题(2022RW004);公益性行业(农业)科研专项(201303035)。

摘　　要：旨在获取中国帕利亚姆血清群中山病毒(Chuzan virus, CHUV)毒株的全基因组序列并分析其遗传特征。应用BHK-21、Vero、MDBK细胞对云南省江城县采集的152份健康黄牛血液样品盲传分离病毒,针对出现细胞病变样品,采用CHUV VP7蛋白基因片段进行RT-PCR扩增和测序,并对阳性样品进行全长扩增及高通量测序,获得的全基因组序列进行遗传进化分析。结果:1份血液样品可致BHK 21、Vero、MDBK细胞病变;病毒基因组为10节段的“3-3-3-1”带型;病毒基因全长18 914 bp,各基因节段长度在728 bp(Seg-10)~3 930 bp(Seg-1)之间,编码6 072个氨基酸残基,编码蛋白在211(NS3蛋白)~1 295(VP1蛋白)个氨基酸残基之间;通过对保守基因Seg-1,Seg-3、Seg-7的核苷酸系统发育分析,显示与中国本土CHUV亲缘关系最近,为98.17%~99.65%,形成一簇;变异基因Seg-2的核苷酸序列同样与中国本土CHUV毒株相似度最近,为98.76%~99.57%。本研究表明中国CHUV毒株的Seg-1、Seg-3和Seg-7形成了独特的地域型,为深入揭示我国CHUV的致病性和病原学特征提供了基础数据。The aims of the present study were to obtain the complete genome sequence of a Chuzan virus(CHUV)strain isolated from Palyam serogroup and to analyze its genetic characteristics.BHK21,Vero,MDBK cells were used to isolate virus from the blood samples of healthy cattle collected in Jiangcheng county,Yunnan Province.Then,RT-PCR amplification and sequencing of CHUV VP7 protein gene fragments were performed on the samples with cellular lesions.Next,full-length amplification and Next Generation Sequencing(NGS)were performed on the positive samples,and the whole genome sequence obtained was analyzed for genetic evolution.Finally,a full-length complementary-DNA copy of the genome was amplified and sequenced.The results showed that one sample of CPE were found in BHK 21,Vero and MDBK cells.Agarose-gel electrophoresis indicated a genome consisting of 10 segments of double-stranded RNA molecules that demonstrated a 3-3-3-1 pattern.The size of the virus genome was 18914 bp and consisted of 10 genes from 3930 bp(Seg-1)to 728 bp(Seg-10)and encoded 6072 amino acid residues,with a protein size ranging of Seg-1,Seg-3 and from 211(NS3 protein)to 1295(VP1 protein)aminoacid residues.Phylogenetic analysis of Seg-7 showed that they were most closely related to CHUV in China(98.17%-99.65%),and formed a cluster of its own.The phylogenetic analysis of Seg-2 showed that the similarity of the Seg-2 strain to the CHUV strain in China was 98.76%-99.57%.The Seg-1,Seg-3 and Seg-7 of CHUV strains in China had formed unique geographic types.These results suggested that the CHUV strain was special in evolution.This study provided basic data for further research on the pathogenicity of CHUV in our country and the technology for detection of its pathogens.

关 键 词：中山病毒 分离 全基因组测序 序列分析 帕利亚姆血清群病毒 

分 类 号：S855.3[农业科学—临床兽医学]