鱼类病毒性神经坏死病毒衣壳蛋白在昆虫细胞中的表达及其特性

利用RT-PCR技术获得病毒性神经坏死病毒0603株的衣壳蛋白基因,将其插入到杆状病毒Bac-To-Bac表达系统的pFastBacI质粒中,构建了pFastBac-cp质粒.转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-cp,脂质体介导将其转染Sf9细胞产生有感染性的重组杆状病毒AcNPV-cp.利用AcNPV-cp感染Sf9细胞后,SDS-PAGE分析可见大小约为37 ku的特异性蛋白带,Western-blotting分析发现,其可以与病毒性神经坏死病毒阳性血清反应出现特异性的杂交带.试验结果表明,AcNPV-cp在Sf9细胞中成功地表达了病毒性神经坏死病毒的衣壳蛋白,其具有良好的免疫学活性.负染电镜观察发现,CP蛋白可自行装配成病毒样颗粒,其大小形态类似于病毒性神经坏死病毒.制备超薄切片后电镜观察发现,CP蛋白自行装配成的病毒样颗粒呈晶格状排列在细胞质中.为研制有效防控鱼类病毒性神经坏死病的新型颗粒性疫苗奠定了基础.     

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).     

石斑鱼神经坏死病毒研究进展

简述了石斑鱼神经坏死病毒的分类方式和结构,以及侵染石斑鱼引发的免疫反应机制;介绍了神经坏死病毒的检测方法、预防措施和治疗方法。指出,目前神经坏死病毒侵染石斑鱼的具体致病机制仍未完全阐明,感染后的治疗手段也尚未成熟。提出,接种疫苗等其他预先建立免疫屏障的防治措施,比感染后再治疗的效果更好,利用碳酸盐缓冲液进行辅助治疗及复合益生菌疗法是防治神经坏死病毒的新思路,在一定程度上解决了抗生素耐药性及水产养殖的抗生素的残留问题。     

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,...     

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。     

鱼类神经坏死病病毒衣壳蛋白在昆虫细胞中的表达及其特性

鱼类病毒性神经坏死病毒能引起多种海水鱼类中枢神经组织病变,给各国海水养殖业造成了巨大的损失。本研究利用RT-PCR技术获得病毒性神经坏死病毒0603株的衣壳蛋白基因,将其插入到杆状病毒Bac-To-Bac表达系统的pFastBacⅠ质粒中,构建了pFastBac-cp质粒。转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-cp,脂质体介导将其转染Sf9细胞产生有感染性的重组杆状病毒AcNPV-cp。利用AcNPV-cp感染Sf9细胞后,SDS-PAGE分析可见大小约为37kD的特异性蛋白带, Western-blotting分析发现,其可以与病毒性神经坏死病毒阳性血清反应出现特异性的杂交带,试验结果表明AcNPV-cp在Sf9细胞中成功地表达了病毒性神经坏死病毒的衣壳蛋白,其具有良好的免疫学活性。负染电镜观察发现,CP蛋白可自行装配成病毒样颗粒,其大小形态类似于病毒性神经坏死病毒。制备超薄切片后电镜观察发现,CP蛋白自行装配成的病毒样颗粒呈晶格状排列在细胞质中。 本研究为研制有效防控鱼类病毒性神经坏死病的新型颗粒性疫苗奠定了基础。     

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。     

斜带石斑鱼神经坏死病毒主衣壳蛋白抗体的制备

将含有斜带石斑鱼（Epinephelus coioides）神经坏死病毒（orange-spotted grouper nervous necrosis virus，OGNNV）的主衣壳蛋白（main capsid protein，MCP）基因的重组质粒pET32a-MCP转入大肠杆菌BL21后，用异丙基硫代-β-半乳糖苷（IPTG）诱导表达，用柱层析纯化表达的融合蛋白作为抗原免疫新西兰大白兔，制备抗MCP融合蛋白的血清。用ELISA方法检测抗血清的效价，用Western—blot检测抗血清的特异性。结果显示，获得的抗血清稀释1：22000倍时仍呈阳性，并能有效中和OGNNV，实验组的相对存活率达54．50％。这说明，本研究用纯化的MCP融合蛋白制备兔抗OGNNVMCP抗体是成功的，并证实了OGNNV主衣壳蛋白的免疫原性。本研究旨为重组表达主衣壳蛋白基因制备抗OGNNV疫苗提供科学依据，并为进一步研究提供重要的实验材料。     

赤点石斑鱼神经坏死病毒主衣壳重组蛋白多克隆抗体的制备

把赤点石斑鱼（Epinephelus akaara）神经坏死病毒（RGNNV）主衣壳蛋白（MCP）基因的重组表达质粒载体pRSETA-MCP转化至大肠杆菌（Estherichia coli）BL21（DE3）,经IPTG诱导表达,SDS-PAGE显示表达的重组蛋白主要以不可溶的包涵体形式存在,分子量约44.5kD。通过Ni-NTA-Agarose亲和层析柱纯化,经分析纯度达90%,之后免疫新西兰兔制备抗血清,ELISA效价达1：12800以上。Western-blot分析结果显示,该血清与表达的重组蛋白有较强反应,说明通过原核表达的重组蛋白具有良好的免疫原性。     

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％。     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red‐spotted grouper nervous necrosis virus genotype in shellfish

Using two serially executed PCRs, the discriminative multiplex two‐step RT‐PCR (DMT‐2 RT‐PCR) following the detection seminested two‐step RT‐PCR (DSN‐2 RT‐PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable in vitro. These results are the first report of high level prevalence of in vitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.     

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.     

神经坏死症病毒对长缟鲹仔鱼的感染及在体内的传播

以长缟鲹(Pseudocaranx dentex)孵化仔鱼为材料，通过神经坏死病毒(Nervous Necrosis Virus，NNV)浸浴感染实验，明确病毒浓度越高，供试鱼被病毒感染的时间越早，病毒检出率越高；运用细胞组织病理学和酶联免疫(Enzyme-1inked immunosorbent assays，ELISA)的原理和方法，了解了神经坏死病毒的侵入、感染和在神经组织中的扩散方式；利用ELISA技术，结合电子显微镜对NNV感染细胞进行超显微观察，确认育苗水环境中神经坏死病毒可水平感染供试鱼皮肤上皮的基底细胞；病毒感染细胞发生细胞质内质网膨胀、细胞核变性、细胞小器官数减少等病理变化；病毒感染细胞质中病毒粒子有散布、类结晶和二者兼有3种存在形式。本研究成果可为了解海水鱼类人工育苗的病毒性神经坏死症感染途径、传播和预防病毒性神经坏死症的发生提供科学依据。     

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.     

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.     

Modelling the effect of vaccination on transmission dynamics of nervous necrosis virus in grouper larvae Epinephelus coioides

Nervous necrosis virus (NNV) infection in susceptible grouper larvae has been reported to cause high mortalities, leading to great economic losses in aquaculture industry. Although the effects of NNV vaccines on grouper have been broadly investigated, vaccination strategies have not been fully established. To this end, we introduced the parsimonious epidemiological models that explored the assessment of key epidemiological parameters and how they changed when vaccinations showed the effects. We showed that the models capture the published cumulative mortality data accurately. We estimated a basic reproduction number R₀ = 2.44 for NNV transmission in grouper larvae without vaccination. To effectively control NNV transmission by vaccination, a model for disease control was also generalized to attain the goals of controlled reproduction number less than 1. Our results indicated that at least 60% of grouper population needed to be immunized for ~75 min. Our data-driven modelling approach that links the transmission dynamics of NNV and vaccination strategies for grouper has the potential to support evidence-based planning and adaptation of integrated control measures. We encourage that the epidemiology-based framework introduced here can be further implemented for establishing effective vaccination and mitigation actions aimed at controlling diseases in fish farming practices.