一株鳢科鱼源弹状病毒的分离及鉴定
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中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所,中国水产科学研究院珠江水产研究所

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国家科技支撑计划(2012BAD25B02)


Isolation and characterization of a rhabdovirus from snakehead fish(Ophicephalus striatus)
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Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Key laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong

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    摘要:

    为研究近来鳢科鱼类疫病频发的病因,实验对采集的患病杂交鳢病料分别从细菌学和病毒学两方面进行病原分离和鉴定。排除细菌感染的可能后,通过细胞培养技术、回归感染实验、电镜技术、分子生物学技术等进行病毒学分析,最终分离到一株弹状病毒,命名为HSHRV-C1207(Hybrid Snakehead Rhabdovirus-C1207)。实验结果显示,该病毒在EPC、FHM、GSB、SFC、CIK等 8种细胞中都能复制,并对其中6种细胞产生明显的细胞病变效应(CPE)。用分离的病毒进行回归感染实验,可使感染的杂交鳢复制出自然发病鱼的相同症状,且死亡率达90%。感染病料组织液的EPC细胞固定后经电镜观察,发现细胞质内有大量子弹状病毒聚集,直径约60 nm,长度约为160 nm,形态和排列方式与已报道的鱼类弹状病毒相似。参考OIE中传染性造血器官坏死病毒(IHNV)、病毒性出血性败血症病毒(VHSV)、鲤春病毒血症病毒(SVCV)的检测引物及根据乌鳢弹状病毒(SHRV)、鳜鱼弹状病毒(SCRV)、比目鱼弹状病毒(HIRRV)的G蛋白基因设计特异性引物,分别对自然发病病料组织、接种病料组织的细胞进行RT-PCR扩增,仅针对SCRV的引物能扩增出大小约350 bp 的片段。对HSHRV-C1207株的G基因进行序列测定和分析,结果表明其核苷酸序列同SCRV和SHRV的同源性分别为93.8% 和 20.4%,推导的氨基酸序列同源性分别是93.7%和18.3%。用G基因推导氨基酸序列与其它弹状病毒的G蛋白进行系统进化树分析,表明该毒株与水泡性病毒属聚为一支,其中与SCRV的亲缘关系最近,而与属于诺拉弹状病毒属的SHRV相距甚远,可能为SCRV的变异株或一个新的毒株。

    Abstract:

    The diseased hybrid snakehead(Channa maculate ♀×Channa argus♂)were collected for analysis of the pathogen both in bacteriology and virology to make the pathologic agent clear.None of pathogenic bacteria was obtained using routine bacteria separation method.One rhabdovirus was isolated using cell culture technique,regression tests,electron microscopy,molecular biology techniques and others,which assigned hybrid snakehead rhabdovirus -C1207(HSHRV-C1207).HSHRV-C1207 could replicate and proliferate in EPC,FHM,GSB,SFC,CIK cells,and cause the significant cytopathic effect(CPE).The regression tests in which separated viruses were injected in the abdominal cavity were all presented with symptoms similar to the natural incidence,and the final mortality was 90%.Electron microscopy observation of ultra-thin section samples of specimen-infected EPC cells revealed that the bullet-shaped virus replicated in cytoplasm with regular arrangement like other fish rhabdovirus,and had a non-enveloped structure with a diameter and length of approximately 60 nm and 145 nm respectively.The OIE recommended primers for Infectious haematopoietic necrosis virus(IHNV),spring viraemia of carp virus(SVCV),viral haemorrhagic septicaemia virus(VHSV)and three pairs of specific primers were designed by G gene conserved sequences of snakehead rhabdovirus(SHRV),siniperca chuatsi rhabdovirus(SCRV)and hirame rhabdovirus(HIRRV)respectively,were applied to the RT-PCR amplification for detecting the possible rhabdovirus in diseased fish and virus-infected cells.A specific ladder product about 350 bp only was amplified with primers of SCRV.Sequencing analysis of the G gene of HSHRV-C1207 showed that this gene shares the sequence identities 93.8% and 20.4% with the respective genes of SCRV and SHRV respectively,and the corresponding deduced amino acid sequence identity 93.7% and 18.3% respectively.Phylogenic relationship with other rhabdoviruses using the corresponding amino acid sequences of G protein indicates that HSHRV C1207 is most closely related to the vesiculovirus genus and shares the highest identity with SCRV,but was far from the snakehead rhabdovirus(SHRV)and the other rhabdovirus.The above results indicated that the causes of hybrid snakehead diseases ceaseless breaking out was the rhabdovirus,and this new isolates HSHRV-C1207 belongs to vesiculovirus genus.It may be a novel variant SCRV isolate or a new member.

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曾伟伟,王 庆,王英英,刘 春,谭爱萍,石存斌,吴淑勤.一株鳢科鱼源弹状病毒的分离及鉴定[J].水产学报,2013,37(9):1416~1424

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  • 收稿日期:2013-02-24
  • 最后修改日期:2013-03-30
  • 录用日期:2013-08-10
  • 在线发布日期: 2013-09-13
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