斑点叉尾鮰出血病病原呼肠孤病毒的分离与鉴定

Isolation and Identification of Channel Catfish(Ictalurus punctatus) Hemorrhage Reovirus

采用细胞培养分离病毒技术从患出血病的斑点叉尾鮰体内分离到一株新病毒。经人工感染试验、电镜观察、理化特性试验以及病毒基因组分析,确认为斑点叉尾鮰呼肠孤病毒（Channel Catfish Reovirus,CCRV）。人工感染试验可复制出与自然发病相同的症状;病毒感染斑点叉尾鮰卵巢组织细胞系和肾脏组织细胞系可产生典型细胞病变效应。感染细胞的超薄切片电镜观察结果显示,病毒在细胞质内复制,呈晶格状排列,病毒颗粒具双层衣壳,无囊膜,直径约60-70nm;冻融、56℃1h、氯仿、乙醚等处理对病毒滴度影响不显著,65℃1h处理病毒滴度下降极显著。病毒基因组SDS-PAGE分析和核酸酶敏感试验结果表明,其基因组由11个分段的RNA核酸节段组成,节段大小范围为0.9-4.4kb,可分为三大类,即L1、L2、L3,M1、M2、M3和S1、S2、S3、S4、S5,为典型的水生呼肠孤病毒（Aquareovirus）基因组结构。病毒基因组S4节段的克隆与序列分析结果表明,其与GenBank中登录的草鱼呼肠孤病毒S4节段（AF403396）、美洲金体鳊呼肠孤病毒（GSRV）的S4节段（AF403407）长度均为909bp,其序列同源性分别为99％和90％。

By using cell culture and virus infection methods, a new reovirus had been isolated from channel catfish（Ictalurus punctatus） suffered with severe hemorrhage and had been identified as channel catfish reovirus （CCRV） after artificial infection in fish, electron microscopy observation, physical-chemical tests, genomic SDS-PAGE analysis and sequencing. In artificial infection test, the typical symptoms of channel cat{ish hemorrhage as naturally occurred could be reproduced. The isolated virus could cause typical cytopathic effect in CCO and CCK ceil＇lines. Electron microscopy observation of ultra-thin section samples of CCRV infected CCO and CCK cells revealed that the virus replicated in cytoplasm, arrayed in crystalline,and had a non-enveloped double capsid with a diameter of 60-70nm. Frozen-thawed, 56℃ lh, chloroform and ether had no significant effects on CCRV titer, 65℃ lh could significantly inactivated the viral infectivity. The CCRV genome SDS-PAGE analysis and nuclease sensitivity test showed that the virus genome was the same as that of viruses in classes L1, L2, L3; M1, M2 Cloning and sequencing of the Aquareovirudae and consisted of 11 segments of dsRNA assigned into three , M3 and S1, S2,S3, S4,S5 with a range of size from 0.9 to 4.4kb. The CCRV S4 segment indicated the nucleic acid number of CCRV S4 was 909bp in length, which was exactly the same as that of GCRV S4 （AF403396） and GSRV S4 （AF403407） segments. The BLAST of CCRV S4 sequence in NCBI GenBank showed that it had a 99 % and 90 % similarity in sequence to the GCRV S4 and GSRV S4 segments, respectively.