【作者】 刘荭；

【导师】 陈焕春； 江育林； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2004， 博士

【摘要】 传染性造血器官坏死毒(IHNV)、鲤春血症病毒(SVCV)、流行性造血器官坏死病毒(EHNV)、病毒性出血性败血症(VHSV)、传染性胰脏坏死(IPNV)、草鱼出血病病毒(GCHV)、真鲷虹彩病毒(RSIV)、病毒性神经坏死病毒(VNNV)、锦鲤疱疹病毒(KHV)、对虾Taura病毒(TSV)、对虾传染性皮下和造血器官坏死病毒(IHHNV)和对虾白斑病毒(WSSV)等十二种水生动物病毒引起的病害是世界范围内对水产动物养殖危害较为严重的病害，它们在世界局部地区造成流行，并随着水产贸易的曰益增加而在逐渐蔓延开来。为此，被世界动物健康组织(OIE)、世界粮农组织亚太水产病害网络(NACA)和各个国家列入水生动物病害名录，在各国对进出境水生动物检疫中，将这些疫病作为检疫的重要对象。本研究建立了十二种水生动物疫病的PCR或RT-PCR检测方法，并将之用于进出境水生动物的检疫和国内水生动物暴发性疫病的检测和诊断中，利用生物信息学手段对扩增的基因片段进行了解析；在上述研究的基础上，设计并制备了各病毒特异性的基因片段，建立基因芯片检测平台，达到用一个基因芯片，对两个样品进行十二种水生动物病毒带毒情况检测的目的。主要研究内容包括： 1．IHNV RT-PCR和nested-PCR检测和序列分析 建立了RT-PCR和Nested-PCR检测IHNV的技术，分别扩增IHNV核蛋白基因786 bp和323 bp的基因片段。在国内养殖、出现暴发性死亡牙鲆和虹鳟鱼中分离并检测出IHNV，扩增产物的基因序列都与IHNV标准株有98.1％的同源性，推导出的氨基酸序列与IHNV标准株分别有97％和99％的同源性。在从美国进口的匙吻鲟鱼卵中检测出IHNV，其扩增产物的基因序列与IHNV标准株有92.5％的同源性，推导出的氨基酸序列与IHNV标准株分别有93％的同源性。 2．SVCV RT-PCR和nested-PER检测和序列分析 建立了RT-PCR和Nested-PCR检测SVCV的技术，分别扩增SVCV糖蛋白基因714和606 bp的基因片段。在国内养殖锦鲤和建鲤中分离并检测出SVCV，扩增产物的基因序列之间有98％的同源性，与GenBank中SVCV其它毒株的基因序列都有88％以上的同源性，系统发育树分析结果表明，与属于Ia基因亚组的毒株亲缘关系最近。 3．EHNV PCR检测和序列分析 建立了PCR检测EHNV的技术，扩增EHNV主要衣壳蛋白基因410 bp的基因片段。从国内患“红脖子病”的养殖甲鱼中分离出病毒，并扩增出该特异性的基因片段，作序列分析和比较发现，该序列与蛙病毒属病毒有高度同源性，因此判定分离出的毒株属于蛙虹彩病毒属。 4．VHSV RT-PCR和nested-PCR检测 建立了用RT-PCR和nested-PR检测VHSV的技术，分别扩增VHSV核蛋白基因1131聚合酶链式反应和基因芯片技术的研究及在主要水生动物病毒检疫和监测中的应用bp和811 bp的基因片段。在国内养殖的水生动物和进出境的水生动物中，未检测到州SV特异性的基因片段。5.工PNV RT一PCR检测和序列分析 建立了RT一PCR方法检测IPNV的技术，扩增IPNV片段B中病毒依赖RNA的RNA聚合酶编码基因304 bp片段。在孵化后出现大量死亡的虹鲜稚鱼中，分离并检测出IPNV特异性的基因片段。序列分析结果表明与IPNv的sP株的基因序列有高达100%的同源性。6.GCHV RT一PCR检测和序列分析 建立了用RT一PCR检测GCHV的技术，扩增GCHV片段510衣壳蛋白编码基因565bP的基因片段。对国内分离出的GCHv的两个毒株进行检测，结果均扩增出565bP的DNA片段。7.RSIV PCR检测和序列分析 建立了用PCR检测RSIV的技术，扩增RSIV DNA聚合酶编码基因698 bp的基因片段。在进境的妒鱼苗中检测到RSIV特异性的基因片段。序列分析结果表明，与国内从绷鱼中分离出的虹彩病毒和RSIV参考株的基因序列有较高程度的同源性。8.vNNv RT一PCR检测和序列分析 建立了用RT一PCR检测VNNV四种不同基因组的技术，分别扩增SJNNV基因组1 147b·、TPNNV基因组523 bp、BFNNV基因组479 bp和RGNNV基因组294 bp的衣壳蛋白基因片断。在进境的海水鱼苗中多次检测到RGNNV基因组中特异性的片段。序列分析结果表明，与RGNNV各毒株基因组的同源性均在94.3%以上。在广东和福建养殖的3批患病石斑鱼中检测出RGNNV基因组特异性的片段。序列分析结果表明，与RGNNV各毒株基因组的同源性均在94.8%以上。所有扩增出的基因片段推导出的氨基酸序列都与玛拉巴石斑鱼神经坏死病毒(MNNV)有100%的同源性。9.KHV PCR和nested一PCR检测和序列分析 建立T PCR和nested一PCR检测KHV的技术，分别扩增KHV 484 bp和412 bp的基因片段。在患病锦鲤中检测到KHV特异性的基因片段，序列分析结果表明，与GenBank中KHV参考株的基因片段有99%的同源性。10.TSv RT一PCR检测和序列分析 建立了用RT一PCR检测TSV的技术，扩增TSV衣壳蛋白基因231bP的片段。在进口的南美白对虾亲虾中检测到TSV特异性的基因片段，序列分析结果表明与TSV墨西哥株和美国株有99.1%的同源性。21.IHHNv PCR检测和序列分析 建立了用PCR检测IHHNV的技术，扩增IHHNV非结构蛋白基因356 bp的基因片段。在国内养殖的南美白对虾亲虾中检测到IHHNV特异性的基因片段，序列分析结果表明，与寄主为对虾的更多还原

【Abstract】 The twelve aquatic animal diseases in this study have caused great economic loss in aquaculture throughout the world, which caused by viral pathogens, including Infectious haematopoietic necrosis virus (IHNV), Spring viraemia of carp virus (SVCV), Epizootic haematopoietic necrosis virus (EHNV), Viral Haemorrhagic Septicaemia virus (VHSV), Infectious pancreatic necrosis virus (IPNV), Grass carp haemorrhagic virus (GCHV), Red sea-bream Irodivirus (RSIV), Viral nervous necrosis virus (VNNV), Koi herpesvirus (KHV), Taura syndrome virus (TSV), Infectious hypedermal and haematopoietic necrosis virus (IHHNV) and White spot syndrome virus (WSSV). These viral diseases were prevalent in some countries and spread to other countries along with the commercial activities.These diseased were listed in the important aquatic diseases by World Organisation for Animal Health (OIE), Network of Aquaculture Centres in Asia-Pacific (NACA) and the aquaculture department of each country and are quarantined as in the trades of aquatic animals. Polymerase chain reaction methods (PCR) and Reverse-trancription polymerase chain reaction methods were studied and practised in the quarantine and surveillance of these 12 diseased in aquatic animls cultured, imported or exported. The sequences of amplified products were analyzed with bioinformatical methods. The specific nucleotide fragments were selected, amplified and cloned. They were spotted on glassslides as probes and the gene chip were prepared, with which the 12 viral pathogens were detected from two smples. The main results in this study were described as follow.1. RT-PCR and nested-PCR for IHNV and sequence analysisRT-PCR and nested-PCR for detection of IHNV were developed and the nucleotide fragments of 786 bp and 323 bp coding part of the viral nucleoprotein were amplified. IHNV were detected positively from cultured turbot and rainbow trout with significant clinical signs and high mortalities. The similarity of nucleotide was 98.1% between the amplified products of samples and IHNV reference strain, with the similarity of 97% and 99% separately between the induced peptides. The samples from imported Mississippi paddlefish Polyodon spathala was found to be infected by IHNV and the similarities of nucleotide and peptide were 92.5% and 93% separately compared with reference strain of IHNV.2. RT-PCR and nested-PCR for SVCV and sequence analysisRT-PCR and nested-PCR for detection of SVCV were developed and the nucleotide fragment of 714 bp and 606 bp coding part of the viral nucleoprotein were amplified. Viral strains were isolated from culured koi and common carp and SVCV specific nucleotides were amplified, with the similities of nucleotide 98% between the two samples and 88% with otherSVCV strains. The two strains had closer relationship with the strains of SVCV la sub-genogroup based on phylogenetic analysis.3. PCR for EHNV and sequence analysisPCR for detection of EHNV were developed and the nucleotide fragment of 410 bp coding part of the viral main capsid protein (MCP) was amplified. The specific nucleotide fragment was amplified from the virus isolated from diseased turtle with the clinical sign of red neck. High homology was found beteen the strain and other ranavirus and it was suggested that the strain belonged to ranavirus.4. RT-PCR and nested-PCR for VHSVRT-PCR and nested-PCR for detection of VHSV were developed and the nucleotide fragments of 1131 bp and 811 bp coding part of the viral nucleoprotein were amplified. None of all the samples from cultured fish or imported fish were found to be infected by VHSV.5. RT-PCR for IPNV and sequence analysisRT-PCR for detection of GCHV was developed and the nucleotide fragment of 304 bp coding part of the RNA-dependant RNA polymerase of segment B was amplified. The specific nucleotide was detected from the viral strain isolated from diseased larvae of rainbow trout, with the similarity of nucleotide 100% with the IPNV strain Sp.6. RT-PCR for GCHVRT-PCR for detection of GCHV was developed and the nucleotide frag更多还原