牙鲆弹状病毒的鉴定和检测方法研究及应用
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摘要
日本和韩国是我国水生动物及其产品的主要进口国,我国非常重视对日韩的水生动物及其产品的出口贸易。2004年,根据国家质检总局与韩国海洋水产部签订的《中韩进出口活水生动物检验检疫协议》,在进出境的养殖和种苗用水生动物中,对于鲈鱼、真鲷、鲤鱼、鲫鱼、大菱鲆、牙鲆等要进行牙鲆弹状病毒(Hirame Rhabdovirus,HRV)、真鲷虹彩病毒(Red sea bream iridovirus,RSIV)、淋巴囊肿病毒(Lymphocystis disease virus,LCDV)和黄尾鰤腹水病毒(Yellow tail ascite virus, YAV)等多种疫病的检测。
2008年,我们从山东荣成某鱼苗场的石鲽(Kareius bisoloratus)中检测到了牙鲆弹状病毒(HRV),并通过EPC和FHM细胞系分离到病原。为了对该病原进行深入的研究,满足《中韩进出口活水生动物检验检疫协议》的要求,有效地防范国外水生动物病原传入传出我国,保护和促进我国对外贸易的发展,我们开展了对牙鲆弹状病毒病原学和基因组学的研究,这对于认识水生动物病毒的基因组结构与功能,研究水生病毒与宿主的相互作用、阐释疫病的发病机理,了解该病毒与其他鱼类弹状病毒的关系等方面有着重要的理论意义;并在此基础上,建立了几种牙鲆弹状病毒(HRV)的快速检测方法。建立常规RT-PCR、实时定量RT-PCR和LAMP等分子生物学方法;制备HRV的单克隆抗体,建立HRV的ELISA等免疫学检测方法,为快速诊断提供技术支持。建立的快速检测方法可应用于检验检疫行业和水产养殖业,做到对病毒的快速诊断,同时为有关部门制定HRV检疫操作规范的行业标准和国家标准提供理论依据。本研究对于促进我国水生动物对外贸易的发展,保护我国水产养殖业的可持续健康发展,具有重要的现实意义。
A strain of rhabdovirus was isolated from cultured stone flounder (Kareius bisoloratus) and its biological and physico-chemical characteristics were studied. Focal areas of cytopathic effect (CPE), such as shrinking, rounding and detaching, was observed in 8 cell lines including FHM and EPC. The optimal replication temperature of the virus was 20℃. The virus was sensitive to chloroform, pH3, pH10 and heat treatment, the virus replication was not inhibited by IUDR. This indicated that the isolate was RNA virus with envelope. Typical bullet-shape particles with the size of (60-80) nm×(160-180) nm were observed in the cytoplasm of infected EPC cells. A 515 bp fragment was amplified from the viral glycoprotein gene by RT-PCR. The fragment was sequenced and it shared more than 99% homology with the Korea reference strain of HRV. Five protein bands were observed by SDS-PAGE with the molecular weights 200 kD, 56 kD, 42 kD, 29 kD and 24 kD respectively. These data indicated that the isolate strain SR080113, was hirame rhabdovirus and was named HRV-Shandong strain.
The complete genome of the strain SR080113 was cloned and sequenced with RT-PCR and RACE. The length of the viral genome was 11,037 nt, similar to the HRV Korea strain (GenBank Acc. No. NC_005093) with the identity of more than 95%. The content of G+C in viral RNA was 50.8%. Six open reading frames (ORFs) was induced and analyzed, coding nucleocapsid protein, Phosphoprotein, matrix protein, glycoprotein, non-virion protein and RNA-dependent RNA polymerase separately. The phylogenetic analysis of nucleotide sequences and amino acid sequences suggested that the isolate had the genomic characters of the genus Novirhabdovirus.
Three molecular detection methods including RT-PCR, real-time RT-PCR, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were set up, with the sensitivity of 103.5TCID50/100μL, 100 copies per test, and 101.5TCID50/100μL, respectively. A fragment of viral RNA, containing target fragment, was transcripted in vitro which could be used as the reference positive control for quantitative analysisin the real-time RT-PCR test.
A sandwich ELISA using monoclonal antibody was set up for the detection of HRV and the sensitivity was 102.5TCID50/100μL. The ELISA kits were prepared and the consistency and stability of the kits were evaluated. The coefficient of variation in one lot was 1.99%-9.51% and was 4.22%-8.70% among different lots.
Two hundred and twenty-four samples of cultured marine fish were collected in the surveillance program of marine fish diseases in China, in which, 123 samples were detected by isolation of virus, RT-PCR, real-time RT-PCR, LAMP and ELISA. Thirteen samples from Yellow sea and Bohai sea were HRV positive. These fish includs Kareius bicoloratus L. and Scophthalmus maximu.The detection methods were evaluated during the assay and their diagnostic performance characteristics were analyzed.
2008年,我们从山东荣成某鱼苗场的石鲽(Kareius bisoloratus)中检测到了牙鲆弹状病毒(HRV),并通过EPC和FHM细胞系分离到病原。为了对该病原进行深入的研究,满足《中韩进出口活水生动物检验检疫协议》的要求,有效地防范国外水生动物病原传入传出我国,保护和促进我国对外贸易的发展,我们开展了对牙鲆弹状病毒病原学和基因组学的研究,这对于认识水生动物病毒的基因组结构与功能,研究水生病毒与宿主的相互作用、阐释疫病的发病机理,了解该病毒与其他鱼类弹状病毒的关系等方面有着重要的理论意义;并在此基础上,建立了几种牙鲆弹状病毒(HRV)的快速检测方法。建立常规RT-PCR、实时定量RT-PCR和LAMP等分子生物学方法;制备HRV的单克隆抗体,建立HRV的ELISA等免疫学检测方法,为快速诊断提供技术支持。建立的快速检测方法可应用于检验检疫行业和水产养殖业,做到对病毒的快速诊断,同时为有关部门制定HRV检疫操作规范的行业标准和国家标准提供理论依据。本研究对于促进我国水生动物对外贸易的发展,保护我国水产养殖业的可持续健康发展,具有重要的现实意义。
A strain of rhabdovirus was isolated from cultured stone flounder (Kareius bisoloratus) and its biological and physico-chemical characteristics were studied. Focal areas of cytopathic effect (CPE), such as shrinking, rounding and detaching, was observed in 8 cell lines including FHM and EPC. The optimal replication temperature of the virus was 20℃. The virus was sensitive to chloroform, pH3, pH10 and heat treatment, the virus replication was not inhibited by IUDR. This indicated that the isolate was RNA virus with envelope. Typical bullet-shape particles with the size of (60-80) nm×(160-180) nm were observed in the cytoplasm of infected EPC cells. A 515 bp fragment was amplified from the viral glycoprotein gene by RT-PCR. The fragment was sequenced and it shared more than 99% homology with the Korea reference strain of HRV. Five protein bands were observed by SDS-PAGE with the molecular weights 200 kD, 56 kD, 42 kD, 29 kD and 24 kD respectively. These data indicated that the isolate strain SR080113, was hirame rhabdovirus and was named HRV-Shandong strain.
The complete genome of the strain SR080113 was cloned and sequenced with RT-PCR and RACE. The length of the viral genome was 11,037 nt, similar to the HRV Korea strain (GenBank Acc. No. NC_005093) with the identity of more than 95%. The content of G+C in viral RNA was 50.8%. Six open reading frames (ORFs) was induced and analyzed, coding nucleocapsid protein, Phosphoprotein, matrix protein, glycoprotein, non-virion protein and RNA-dependent RNA polymerase separately. The phylogenetic analysis of nucleotide sequences and amino acid sequences suggested that the isolate had the genomic characters of the genus Novirhabdovirus.
Three molecular detection methods including RT-PCR, real-time RT-PCR, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were set up, with the sensitivity of 103.5TCID50/100μL, 100 copies per test, and 101.5TCID50/100μL, respectively. A fragment of viral RNA, containing target fragment, was transcripted in vitro which could be used as the reference positive control for quantitative analysisin the real-time RT-PCR test.
A sandwich ELISA using monoclonal antibody was set up for the detection of HRV and the sensitivity was 102.5TCID50/100μL. The ELISA kits were prepared and the consistency and stability of the kits were evaluated. The coefficient of variation in one lot was 1.99%-9.51% and was 4.22%-8.70% among different lots.
Two hundred and twenty-four samples of cultured marine fish were collected in the surveillance program of marine fish diseases in China, in which, 123 samples were detected by isolation of virus, RT-PCR, real-time RT-PCR, LAMP and ELISA. Thirteen samples from Yellow sea and Bohai sea were HRV positive. These fish includs Kareius bicoloratus L. and Scophthalmus maximu.The detection methods were evaluated during the assay and their diagnostic performance characteristics were analyzed.
引文
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