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％。     

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。     

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。     

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).     

卵形鲳鲹一例疑似神经性病毒病的初步分析

对疑患病毒性神经坏死症、身体失去平衡、漂浮水面螺旋状游泳、体色发黑、大规模死亡的海水网箱养殖卵形鲳鲹幼鱼,采用组织病理切片观察和分子生物学技术对其病原进行初步分析和跟踪调查。组织病理学结果显示,病鱼脑和眼均呈现神经坏死病毒典型的空泡化现象;利用神经坏死病毒特异性引物nest-PCR检测发现,病鱼一扩阳性率100%,摄食游泳正常鱼一扩和二扩阳性率均为46.67%,显示港区苗种多自身携带病毒株;序列比对显示该病毒株属于赤点石斑神经坏死病毒基因型;对神经坏死病毒跟踪发现,成鱼一扩均为阴性,二扩阳性率则高达86.7%。初步认为,外界因子可能作为诱导因子,致使本身携带病毒鱼苗免疫降低致使此次流行病爆发,且神经坏死病毒潜伏于鱼体的时间较长。     

逆转录聚合酶链式反应(RT-PCR)检测5种养殖石斑鱼的神经坏死病毒

神经坏死病毒(Nervous necrosis virus)是导致多种海水鱼类神经性病害的致病原.发病及死亡的石斑鱼除了表现神经异常症状外,无明显的临床病症,体表及内脏组织也未发现明显病变及寄生虫感染.2003年4～8月,应用逆转录聚合酶链式反应(RT-PCR)技术从福建南部人工养殖的5种石斑鱼即紫石斑鱼(Epinephelus lanceolatus)、马拉巴石斑鱼(E. malabaricus)、青石斑鱼(E. awoara)、赤点石斑鱼(E. akaara)和云纹石斑鱼(E. moara)中检出5个神经坏死病毒分离株.检测了76份石斑鱼样品,这些石斑鱼NNV病毒的平均感染率约为90%.对这些病毒的RT-PCR产物421 bp核酸进行了测序和序列分析,其相同的序列超过99%.将这些序列与GenBank的石斑鱼(Epinephelus spp.)神经坏死病毒相关基因序列作比较,同源性在97%以上.对神经坏死病毒在石斑鱼体内的分布也进行了分析,在脑和眼组织的检出率最高,部分病鱼的肝、脾和肾组织也能检出病毒.结合流行病学特征,可确认神经坏死病毒为该传染病的主要致病原.RT-PCR方法是检测NNV等病原的一种理想的诊断方法.     

传染性造血器官坏死病防控试验研究

正传染性造血器官坏死病(Infectious hematopoietic necrosis,IHN)是鲑科鱼类常见的急性病毒性传染病,病原是传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)。在我国,IHN病毒于1985年首次随进口鱼带入国内,并在辽宁本溪暴发IHN,死亡率近100%。随后扩散到东北和华北一带,呈散在性流行,近几年来流行有逐渐扩大和日趋严重的倾     

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.     

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass,Dicentrarchus labrax (L.)

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red‐spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV‐type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.     

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.     

Experimental infection of <Emphasis Type="Italic">Betanodavirus</Emphasis> in freshwater fish <Emphasis Type="Italic">Gambusia affinis</Emphasis> (Baird and Girard, 1853)—a potential infection model for viral encephalopathy and retinopathy

Betanodaviruses are the causative agents of the disease known as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in a variety of marine and freshwater fish species. The aim of this study was to demonstrate experimental infection of an isolate of betanodavirus (RGNNV genotype) in freshwater fish, Gambusia affinis, for elucidation of transmission mechanism and potential use as a laboratory model. Morbidity and mortality rate was significantly higher by injection route of infection as compared to immersion by bath and resembled the natural infection of juvenile marine fish. The fish in disease affected group showed severe neurological disorders accompanied by extensive vacuolar degeneration and mild to moderate neuronal necrosis of the brain in comparison to control. Amplification of ~?427 bp product in the variable region of the coat protein gene of betanodavirus was achieved by RT-PCR with 100% sequence homology to RGNNV genotype.     

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.