感染唐鱼的草鱼呼肠孤病毒分离与鉴定  

Isolation and Identification of Grass Carp Reovirus from Infected Tanichthys albonubes

作　　者：吴江 赵现锋 陈兵 安微 孙洁 林彦星 王津津 廖立珊 赵永刚[3] 兰文升 Wu Jiang;Zhao Xianfeng;Chen Bing;An Wei;Sun Jie;Lin Yanxing;Wang Jinjin;Liao Lishan;Zhao Yonggang;Lan Wensheng(Animal and Plant Inspection and Quarantine Technology Center,Shenzhen Customs,Shenzhen,Guangdong 518045,China;Technical Center of Chengdu Customs,Chengdu,Sichuan 610041,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China)

机构地区：[1]深圳海关动植物检验检疫技术中心,广东深圳518045 [2]成都海关技术中心,四川成都610041 [3]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《中国动物检疫》2023年第6期31-37,共7页China Animal Health Inspection

基　　金：深圳市科技计划项目(JCYJ20170410103015603);海关总署科技项目(2021HK174,2022HK007)。

摘　　要：广东省某渔场待出境送检唐鱼(Tanichthys albonubes)样品,经病理检查未发现临床症状,取样品的肝、脾和肾等器官组织,用细胞分离培养方法检测病毒性因子,发现该批次唐鱼样品的组织提取物能够造成EPC和CHSE-214细胞发生细胞病变效应(CPE),确定该样品携带病毒性病原。为探究唐鱼携带病原的种类,针对草鱼呼肠孤病毒(GCRV)S9核酸片段设计特异性引物,对产生CPE的细胞培养物进行RT-PCR核酸扩增和序列测定,用EPC细胞扩增该病毒,获得病毒含量为10^(6.25)TCID_(50)/mL的细胞培养物,通过高通量测序技术分析细胞培养物中的病毒类型,并用不同病毒含量的细胞培养物进行唐鱼感染性试验。测序结果经BLAST比对分析发现,扩增序列与GCRV 873株的S9核苷酸序列同源性达98%,鉴定病原为GCRV,将其命名为GCRV SZ0508株。全基因组测序序列注释为GCRV,通过vp2基因遗传进化分析发现,GCRV SZ0508属于I型GCRV。感染性试验中,感染组100%被感染,死亡率为3.3%,表明分离的GCRV能够感染唐鱼,具有造成唐鱼发生草鱼出血病的风险。For the samples of Tanichthys albonubes to be exported by a fish farm in Guangdong Province,no clinical symptoms were found by pathological examination.The samples of liver,spleen,kidney and other organ tissues were collected to detect viral factors by cell isolation and culture.Cytopathic effect(CPE)was produced by both EPC and CHSE-214 cells induced by the sample tissue extracts,indicating viral agents carried by the samples.In order to identify the pathogens carried by Tanichthys albonubes,specific primers were designed based on S9 nucleic acid fragments of grass carp reovirus(GCRV),RT-PCR and sequence determination were conducted for the cell culture producing CPE,the virus was amplified by EPC cells to obtain cell culture with a virus content of 10^(6.25) TCID_(50)/mL.The type of virus in the cell culture was analyzed by high-throughput sequencing technology,then infection test was conducted using the cell culture with different virus contents.BLAST analysis revealed that the amplified sequence was 98%identity to S9 nucleotide sequences of 873 strains of GCRV,the pathogen was identified as GCRV and named as GCRV SZ0508 strain.The sequence was annotated as GCRV by whole genome sequencing.Genetic evolution analysis of vp2 gene revealed that GCRV SZ0508 was categorized into cluster I.In the infection test,the infected group was fully infected,with a mortality rate of 3.3%,indicating that the isolated GCRV could infect Tanichthys albonubes,posing a risk of occurrence of hemorrhage disease to them.

关 键 词：唐鱼 草鱼呼肠孤病毒 Illumina高通量测序技术 

分 类 号：S855.3[农业科学—临床兽医学]