斜带石斑鱼(Epinephelus coioides)病毒性神经坏死病毒的纯化分析及检测方法建立

根据GenBank中已有的鱼类病毒性神经坏死病毒(NNV)RNA2基因序列,设计引物,从福建厦门具有典型NNV发病症状的斜带石斑鱼中克隆了RNA2基因的全长序列,并将序列提交到GenBank获得登录号为MF510920,命名为XMNNV。系统进化树分析结果表明XMNNV与RGNNV聚类在一起,与SJNNV、BFNNV和TPNNV等其他鱼类神经坏死病毒亲缘关系较远,说明本研究分离得到的XMNNV属于RGNNV基因型。通过超速离心方法对病毒进行提纯,得到了纯化的NNV病毒。电镜观察结果表明,病毒粒子直径20～25 nm,结构为正二十面体,与已经报道的NNV结构一致。通过对XMNNV与其它RGNNV RNA2基因进行序列比对,在保守区设计引物,运用RT-PCR方法建立了RGNNV的PCR检测方法,该方法灵敏度高达67 copies/μL。纯化的病毒对E11(条纹月鳢细胞系)和大黄鱼肌肉细胞进行感染,结果表明该病毒可以感染这两种鱼类细胞。E11细胞被感染病毒后,细胞出现空泡化,并最终导致细胞分解死亡;大黄鱼肌肉细胞感染后,细胞变圆,慢慢从培养皿壁脱落,最终解体死亡。另外,对感染后细胞进行PCR检测,结果显示为阳性,进一步确定了分离的NNV具有感染这两种细胞的能力。本研究通过电镜观察和PCR检测两种方法确定了患病石斑鱼携带NNV,通过对两种鱼类细胞的感染实验,确定了该病毒具有一定的感染能力。综上,本研究为石斑鱼NNV疾病的诊断提供了有效的方法,对石斑鱼NNV疾病的预防具有一定的指导意义。     

石斑鱼神经坏死病毒传播途径阻断的初步研究

本文报告了斜带石斑鱼（Epinephelus coioides）苗种培育中亲鱼、卵、饵料等几个关键环节的病毒携带检测以及几种药物对病毒的消除效果的试验。用RT—PCR法检测斜带石斑鱼亲鱼的粪便、卵、仔鱼、稚鱼,饵料轮虫、桡足类中都有检出神经坏死病毒（nervous necrosis virus,NNV）。聚维酮碘5—10mg／L浸泡卵20min、浸泡轮虫和桡足类30min,盐酸吗啉胍3～5mg／L浸泡卵30min、浸泡轮虫和桡足类40min,聚维酮碘5mg／L＋盐酸吗啉胍3mg／L浸泡卵20min、浸泡轮虫和桡足类30min可有效灭活所携带的神经坏死病毒活性。     

闽南地区斜带石斑鱼(Epinephelus coioides)神经坏死病毒的基因型与分子进化研究

本研究以闽南地区具有典型神经坏死病症状的斜带石斑鱼为材料,采用RT-PCR法对其进行病毒检测,对检测到的阳性序列进行双向测序和构建系统发育树,对病毒颗粒进行分离纯化,并通过分子进化模型分析病毒衣壳蛋白基因所受的选择压力。结果显示,采集到的6个石斑鱼样品均呈NNV阳性,系统树分析发现6个样品的PCR扩增片段均为RGNNV基因型序列,表明闽南地区感染石斑鱼的神经坏死病毒主要为RGNNV基因型病毒;通过PEG法对病毒进行分离提纯,获得直径为25~28 nm、呈二十面立体对称结构的无囊膜病毒颗粒;分子进化分析显示NNV外壳蛋白基因经历了纯化选择,表明病毒在进化过程中没有出现遗传变异并以相对恒定保守的速率进化。     

石斑鱼病害防治

本文结合国内外资料介绍了近年来石斑鱼养殖过程中发现的主要疾病及其防治方法,供广大业者参考。 1石斑鱼苗培育过程常见病害 1．1病毒 石斑鱼苗培育过程,极易遭受病毒性病原的侵害,而简称为NNV的病毒性神经坏死症（Nervous Necrosis Virus）,是造成石斑鱼苗培育中经常发生的主要病毒性疾病。神经坏死病毒（NNV）主要感染寸苗前期的多种类石斑鱼苗,感染后的鱼体出现异常泳姿,     

斜带石斑神经坏死病毒外壳蛋白基因克隆与序列分析

从患病毒性神经坏死病的斜带石斑鱼(Epinephelus coioids)的头部提取总RNA，根据已发表的神经坏死病毒外壳蛋白基因设计引物进行RT-PCR扩增，得到预期大小的基因片段。将此基因片段转入pET载体进行序列测定和分析，结果表明：编码斜带石斑神经坏死病毒(Orange-spoued grouper nervous necrosis virus，OGNNV)外壳蛋白基因的阅读框核苷酸数为1017bp，编码338个氨基酸；基因的核苷酸序列与野田村病毒科(Nodaviridae)的几种病毒的外壳蛋白基因序列比较结果显示，该病毒与p野田村病毒属(Betanodavirus)中的赤点石斑神经坏死病毒(red-spotted grouper nervous necrosis virus，RGNNV)的同源性最高(99％)，说明该病毒株是RGNNY血清型的成员。     

引起半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗大规模死亡的神经坏死病毒病

2012和2013年,山东某育苗场15–20日龄的半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗出现暴发性大规模死亡,7 d内死亡率高达90%–100%。本研究调查了疾病的发生情况和临床特征,采集病鱼样品进行了组织病理学检查,并运用RT-PCR方法进行了病原的检测和基因序列分析。结果发现,半滑舌鳎鱼苗一般在7月和8月发病,发病时养殖水温为22–24℃。病鱼游泳行为异常,表现为上下翻游、螺旋性游动、全身大幅度波浪状浮动症状,但病鱼体表无出血和溃疡症状。组织病理检查发现,病鱼脑和视网膜组织出现严重的空泡化及坏死。病鱼样品的RT-PCR检测结果全部呈鱼类神经坏死病毒阳性。对得到的RT-PCR产物测序,进行BLAST比对,发现该病毒与鱼类神经坏死病毒的赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus,RGNNV)基因型的相似性达98%以上,而与鱼类神经坏死病毒的其他3个基因型:黄带拟鲹神经坏死病毒(Striped jack nervous necrosis virus,SJNNV)、红鳍东方鲀神经坏死病毒(Tiger puffer nervous necrosis virus,TPNNV)和条斑星鲽神经坏死病毒(Barfin flounder nervous necrosis virus,BFNNV)的相似性仅为71%–78%。由此可以判定,本研究发现的引起半滑舌鳎鱼苗大规模死亡的神经坏死病毒为RGNNV基因型,半滑舌鳎也是鱼类神经坏死病毒的天然宿主。该发现在半滑舌鳎疾病防治和鱼类神经坏死病毒的流行机制研究方面都具有重要意义。     

引起半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗大规模死亡的神经坏死病毒病

2012和2013年,山东某育苗场15–20日龄的半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗出现暴发性大规模死亡,7 d内死亡率高达90%–100%。本研究调查了疾病的发生情况和临床特征,采集病鱼样品进行了组织病理学检查,并运用RT-PCR方法进行了病原的检测和基因序列分析。结果发现,半滑舌鳎鱼苗一般在7月和8月发病,发病时养殖水温为22–24℃。病鱼游泳行为异常,表现为上下翻游、螺旋性游动、全身大幅度波浪状浮动症状,但病鱼体表无出血和溃疡症状。组织病理检查发现,病鱼脑和视网膜组织出现严重的空泡化及坏死。病鱼样品的RT-PCR检测结果全部呈鱼类神经坏死病毒阳性。对得到的RT-PCR产物测序,进行BLAST比对,发现该病毒与鱼类神经坏死病毒的赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus, RGNNV)基因型的相似性达98%以上,而与鱼类神经坏死病毒的其他3个基因型：黄带拟鲹神经坏死病毒(Striped jack nervous necrosis virus,SJNNV)、红鳍东方鲀神经坏死病毒(Tiger puffer nervous necrosis virus, TPNNV)和条斑星鲽神经坏死病毒(Barfin flounder nervous necrosis virus,BFNNV)的相似性仅为71%–78%。由此可以判定,本研究发现的引起半滑舌鳎鱼苗大规模死亡的神经坏死病毒为RGNNV基因型,半滑舌鳎也是鱼类神经坏死病毒的天然宿主。该发现在半滑舌鳎疾病防治和鱼类神经坏死病毒的流行机制研究方面都具有重要意义。     

鱼类神经坏死病毒的检测与细胞培养技术研究进展

鱼类病毒性神经坏死病(Viral nervous necrosis,VNN)又称病毒性脑病和视网膜病(Viral encephlopathy and retinopathy,VER),在全世界多种鱼类中流行,对仔鱼和幼鱼危害很大,死亡率可达100%,对成鱼也有很高的致死率.由于其极高的传染性和危害性,被国际兽疫组织(OIE)列为重要的鱼类病害.该传染病的病原为神经坏死病毒(Nervous necrosis virus,NNV),是罗达病毒科(Nodaviridae)的一种小RNA病毒.罗达病毒科包括α-罗达病毒属(Alphanodavirus)和β-罗达病毒属(Betanodavirus).     

海水鱼神经坏死病毒致病机理研究进展

神经坏死病毒(nervous necrosis virus, NNV)主要分布在地中海和太平洋西海岸,已在120多种鱼中被检测到,能引起鱼类病毒性神经坏死病(viral nervous necrosis, VNN),是对海水鱼类危害重大的病原之一。目前,随着神经坏死病毒的基因序列、类型、诊断和预防技术等方面研究的不断深入,其致病机理的报道也越来越多。本文概述了病毒附着、致病基因、细胞凋亡以及免疫逃避等几个方面的研究现状。     

鱼病毒性神经坏死病病毒（VNNV）不同基因型鉴别方法的建立及在VNN检疫和监测中的应用

从GenBank中查找出乙型野田村病毒组中海水鱼类各病毒的序列,并用Sequencher多重序列比较软件将其分到条纹踢神经坏死病毒(striped jack nervous necrosis virus,SJNNV)组、条纹星鲽神经坏死病毒(barfin flounder nervous necrosis virus,BFNNV)组、红点石斑鱼神经坏死病毒(redspotted grouper nervous necrosis virus,RGNNV)组和虎斑东方纯神经坏死病毒(tiger puffer nervous necrosis virus,TPNNV)组4个基因型的组别中。用DNAsis序列比较软件比较同一基因型各基因序列之间的同源性,均在815％以上;不同基因型之间序列的同源性,均在66％以下。结合Premier引物设计软件和Sequencher序列多重比较软件,设计了4对引物,采用逆转录聚合酶链式反应(RT—PCR)来鉴别这4个不同的基因型。对从深圳口岸进境的产地为台湾的海水鱼苗和广东、福建两省养殖的主要海水鱼类进行检疫和监测,结果在进境的海水鱼苗中检出有RGNNV基因型的VNNV,在福建和广东省养殖的石斑鱼成鱼和鱼苗的病鱼体内均检测到RGNNV基因型的VNNV,对上述扩增产物基因序列进行比较,相似性均在96．5％以上,推导出的氨基酸序列与玛拉巴石斑鱼神经坏死病毒(MNNV)序列相似性均为100％。     

鱼类神经坏死病毒研究进展与发展趋势

神经坏死病毒(nervous necrosis virus,NNV)是一种世界范围内流行、严重危害多种海水和淡水鱼类的传染性病原。NNV为单一正链、2节段RNA病毒,基因组由RNA1(3.1 kb)和RNA2(1.4 kb)组成。在病毒复制过程中,会合成亚基因组RNA3。RNA1编码RNA聚合酶。RNA2编码衣壳蛋白,为病毒的唯一结构蛋白。RNA3编码B1和B2两种非结构蛋白。根据病毒衣壳蛋白的基因序列,神经坏死病毒可以分成4种基因型,分别为拟鲹、红鳍东方鲀、条斑星鲽和赤点石斑神经坏死病毒基因型。但是,目前只发现A、B、C三种病毒血清型,A对应拟鲹神经坏死病毒基因型,B对应红鳍东方鲀神经坏死病毒基因型、C对应条斑星鲽神经坏死病毒和赤点石斑神经坏死病毒基因型。病毒存在垂直和水平两种传播途径,而且广泛分布于养殖和野生鱼类中。阻断病毒在野生与养殖鱼类之间的传播和开展新型鱼类疫苗研发是将来研究趋势。     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

Field tests of Poly(I:C) immunization with nervous necrosis virus (NNV) in sevenband grouper, Epinephelus septemfasciatus (Thunberg)

It was recently reported that Poly(I:C) immunization with live nervous necrosis virus (NNV) confers protection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), from NNV infection. In the present study, we conducted field tests with sevenband grouper for the evaluation of Poly(I:C) immunization efficacy. In the first experiment, sevenband grouper were immunized with NNV followed by Poly(I:C) administration 7 weeks before natural occurrence of viral nervous necrosis (VNN). Survival rate of the naïve fish was 71.0%, whereas that of the immunized fish was 99.8%. In the second experiment, sevenband grouper were immunized 10 months before VNN occurrence and survival rate of the non‐treated and vaccinated fish was 79.5% and 97.5%, respectively. In the third experiment, we administered Poly(I:C) to sevenband grouper at 20 days after natural occurrence of VNN. The survival rate of the non‐treated fish was 9.8%, whereas that of fish administered Poly(I:C) was 93.7%. Based on these results, it was concluded that Poly(I:C) immunization conferred protection in fish against NNV infection in field tests and the protection lasted more than 10 months. Furthermore, even after occurrence of VNN, fish mortality could be reduced by Poly(I:C) administration and there was an unexpected curative effect on VNN‐affected fish.     

Infections of nervous necrosis virus in wild and cage‐reared marine fish from South China Sea with unexpected wide host ranges

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage‐reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage‐reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage‐reared fish, respectively. However, by nested RT‐PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage‐reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage‐reared fish by seminested PCR assay, respectively. However, by nested RT‐PCR, the positive signal was observed in 65.2% and 100% species of wild and cage‐reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red‐spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty‐five species of the marine fish were the new hosts of NNV.     

Age dependency of nervous necrosis virus infection in barramundi Lates calcarifer (Bloch)

Age‐dependent susceptibility to nervous necrosis virus (NNV) was demonstrated for barramundi (Lates calcarifer). The experiment used juvenile barramundi produced from a single spawning that were challenged consecutively by immersion with a redspotted grouper nervous necrosis virus (RGNNV) isolate. The dose and environmental conditions (35 ppt salinity and 30 °C) were constant. Fish and water were sampled longitudinally for histopathology and RT‐qPCR analysis to examine the evolution of the disease, virus replication, immune response and release of virus into water. Viral nervous necrosis (VNN) disease occurred in barramundi challenged at 3 and 4 weeks of age while fish challenged at 5, 7 and 9 weeks of age developed subclinical infection. Replication of NNV occurred faster and the concentration of virus reached higher concentrations in the younger fish with clinical disease. Virus isolation and qPCR tests indicated that infectious NNV was released from carcasses into water when fish were affected with clinical disease but not when NNV infection was subclinical. Based on these observations, we consider that carcasses from clinically infected fish have a potentially important role in the horizontal transmission of NNV, and barramundi juveniles should be protected from exposure to NNV until they are 5 weeks of age and reach the disease resistance threshold.     

Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.     

Viral encephalopathy and retinopathy is endemic in wild groupers (genus Epinephelus spp.) of the Algerian coast

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population.