草鱼呼肠孤病毒分离株HZ08基因组L节段的全长克隆与序列分析  

Cloning and sequence analysis of large segmens from the genome of one grass carp reovirus isolate HZ08

作　　者：张超[1,2,3] 王庆[1] 石存斌[1] 曾伟伟[1] 刘永奎[1,2] 杨淞[1,2] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306 [3]山东省济宁市渔业监测站,山东济宁272073

出　　处：《江苏农业学报》2011年第1期116-123,共8页Jiangsu Journal of Agricultural Sciences

基　　金：农业公益性行业科研专项(200803013);现代农业产业技术体系建设专项基金项目(nycytx-49-14);农业科技跨越计划(2008043)

摘　　要：从浙江省湖州地区采集发病草鱼(Ctenopharyngodon idellus)样品,取症状明显病鱼的肝、脾、肾组织,经过滤除菌处理后接种草鱼肾脏细胞(CIK)进行病毒分离。盲传8代,CIK细胞未出现明显细胞病变,但感染细胞固定后经电镜观察发现,细胞质内有大量病毒聚集,病毒无囊膜,近球形,直径约70nm,形态与已报道的草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)相似。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,病毒基因组由11个dsRNA组成,呈现水生呼肠孤病毒基因组典型特征。为了从分子水平上进一步了解该病毒的特性,应用单引物扩增技术获得了该病毒基因组L节段的全长。序列分析结果表明:完整的L1、L2、L3节段分别由3 927、3 870、3 753个碱基对组成,每个节段正链RNA仅含有1个开放阅读框,通过同源性比较,推测分别编码病毒的结构蛋白VP1、VP2、VP3,其中VP1推测为病毒RNA转录的鸟苷酸转移酶和甲基化酶,VP2为病毒的RNA依赖性RNA聚合酶(RdRp),VP3为病毒NTP酶及解旋酶。3个推测结构蛋白的序列与GCRV 873株的同源性最高,分别为31%、45%、36%,并且具有与其他呼肠孤病毒相同的基序(motif)。各节段都具有相同的5'端和3'端保守序列,即5'-GUAAUUU......UUCAUC-3'。利用RdRp构建系统进化树,HZ08株与水生呼肠孤病毒聚为一簇,但单独列为1支,应为水生呼肠孤病毒属新成员。The tissues of liver,spleen,and kidney from hemorrhagicly diseased grass carps（Ctenopharyngodon idellus） were collected from Huzhou city,Zhejiang Province,to isolate the virus.The CIK ce1ls inoculated with the virus showed nocytopathic effects（CPE） even after passages of eight times.However,plenty of nonenveloped virus particles with subsphaeroidal shape were observed in the cytoplasm under electron microscope（EM）.The particles had an overall diameter of approximately 70 nm,which was highly similar to the reported grass carp reovirus（GCRV）.The SDS-PAGE electropherosis suggested that the virus possessed 11 segments of dsRNA,which is the typical characteristic of GCRV.In order to characterize the virus on the molecular level,the complete nucleotide sequences of large segments L1,L2,and L3 were obtained by the single primer amplification technique.The complete sequences of HZ08 genome segments L1,L2,and L3 were comprised of 3 927 base pairs,3 870 base pairs and 3 753 base pairs,respectively.Each segment contained a single open reading frame（ORF） which presumably encoded structural proteins VP1,VP2 and VP3,respectively.VP1 might represent viral guanylyl transferase and methyl transferase,VP2 is the viral RNA-dependent RNA polymerase（RdRp）,and VP3 is NTPase and helicase.The three structural proteins shared the highest identity of 31%,45% and 36%,respectively,with GCRV strain 873.Conserved terminal sequence 5′-GUAAUUU......UUCAUC-3′ were found at the ends of each segment.Based on the sequence analysis,VP1,VP2 and VP3 had the same motifs with other reovirus.Phylogenetic tree based on the amino acid sequences of RdRp revealed that the HZ08 formed a cluster close to the aquareovirus,but was far from the other isolates,which indicated that HZ08 was likely to be a new member of aquareovirus genus.

关 键 词：草鱼呼肠孤病毒 序列分析 RNA依赖性RNA聚合酶(RdRp) 

分 类 号：S965.112[农业科学—水产养殖]