草鱼呼肠孤病毒湖州分离株的分离及鉴定

Isolation and identification of a strain of Grass carp reovirus in Huzhou

中文关键词: 草鱼  草鱼呼肠孤病毒  草鱼肾细胞  分离  鉴定

英文关键词: Grass carp  Grass carp reovirus  Ctenopharyngodon idellus kidney cell  Isolation  Identification

中文摘要:

      2008年6月从湖州某患病草鱼池塘采集草鱼出血病疑似病样，将除菌过滤后的患病鱼肝、脾、肾组织滤液，注射健康的8～10 cm的草鱼鱼种。5d后草鱼开始发病，且症状与原发病症状一样，死亡率为57%，对照组未有死亡。将无菌处理的病样滤液接种草鱼肾细胞（CIK），连续接5代均出现明显的细胞病变（CPE），并与草鱼呼肠孤病毒参考株所产生的CPE一致。该株病毒的TCID50为10-8/0.1ml。内脏组织经超薄切片，电子显微镜观察，发现组织内有大量的病毒颗粒，大小均一，近似球形，直径约70～75 nm。理化鉴定表明，氯仿、乙醚处理组病毒的感染力和对照组相比并没有多大变化，说明该株病毒对氯仿和乙醚有一定的抗性。经草鱼呼肠孤病毒特异性的RT-PCR检测，获得阳性的目的片段，测序的结果与草鱼呼肠孤病毒相应序列的同源性达99%以上。上述鉴定结果表明所分离的病毒为草鱼呼肠孤病毒, 将其命名为HZ2008。

英文摘要:

      Aquareoviruses, which belong to a newly identified genus of the family Reoviridae, are agents infecting all kinds of aquatic animals. Grass carp reovirus (GCRV) is a kind of pathogenic virus which causes disease outbreak of Grass carp fingerling in freshwater region of China. In June 2008, a strain virus had been isolated from the samples of visceral organs of the diseased Grass carp with Hemorrhage symptoms from a fish pond at Huzhou City in Zhejiang Province. Sample of diseased Grass carp visceral organs was filtered and was injected into healthy Grass carp fingerlings. Clinical signs of Hemorrhage symptoms of Grass Carp were replicated after five days. Mortality of the sample was 57%. By contrast, the control group fishes were still alive. The sample was continually inoculated onto CIK cells and induced typical cytopathic effect (CPE) was observed. The typical CPE was the same with that induced by the GCRV873 strain. The TCID50 of the isolated virus strain was 108/0.1ml. The visceral organs examined by electron microscopy showed that there were a number of viral particles approximately 71~75 nm in diameter with a sphericity shape. Physicochemically, the isolated strain was not sensitive to chloroform and ether. 436bp nucleotide segment was amplified from the isolated virus strain by reverse transcription polymerase chain reaction (RT-PCR). The nucleic acid sequence of the amplicon had been sequenced. It showed 99% identity with those of GCRV strains in GenBank. All of the evidences indicated that the isolated virus stain was GCRV and was named as HZ2008 isolate.