赤点石斑鱼诺达病毒的纯化及其衣壳蛋白的Western-blot分析

采用差速离心,蔗糖(10%～40%,W/V)密度梯度离心和氯化铯(30%～40%,W/W)等密度梯度离心法,对赤点石斑鱼诺达病毒大亚湾株(RGCN)进行了纯化,测定计算其浮密度为1.3102～1.3243g·cm-3。通过Westernblot法检测到的病毒的结构蛋白的分子量为37和31kDa,而以31kDa的蛋白为主。     

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA‐dependent RNA polymerase gene

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single‐stranded positive‐sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open‐reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA‐dependent RNA polymerase was obtained.     

赤点石斑鱼病毒性神经坏死症的组织病理和电镜观察

用逆转录-聚合酶链式反应（RT-PCR）检测患病赤点石斑鱼苗，呈Beta诺达病毒阳性。光学显微镜下观察到病鱼的脑、视网膜、脊髓有空泡．在脑部，空泡主要分布在端脑、间脑和小脑。受感染的细胞明显收缩、致密变化和嗜碱性。包涵体常为圆形，大小不一。透射电镜下，在感染细胞的细胞质可观察到含有病毒粒子的致密体。病毒粒子呈等面体，无外膜，直径为25～28nm，随机分布在细胞质或在致密体内排列成品格状。致密体大小不一。偶尔观察到较大致密体的外膜已破裂，病毒粒子被释放到细胞质。     

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。     

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.     

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.     

斜带石斑鱼神经坏死病毒CP基因 shRNA干扰载体的构建及效果评价

利用Invitrogen公司的在线生物学软件分析斜带石斑鱼神经坏死病毒CP基因,设计针对CP基因不同位置的小发卡RNA(short hairpin RNA,shRNA)干扰序列[其结构特征为正链(19 nt)-环(4 nt)-负链(19 nt)]。化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到干扰载体pENTRTM/U6中,构建shRNA干扰载体pshRNA-124、pshRNA-896和pshRNA-NNV。然后,用脂质体转染法分别将3种shRNA干扰载体和pEGFP-CP基因共转染导入黑头呆鱼(FHM)肌肉细胞,荧光显微镜观察细胞荧光强度,分析荧光抑制效率,Real-time RT-PCR检测CP基因mRNA的表达水平变化。结果表明,在pEGFP-CP与shRNA干扰载体共转染组,pshRNA-124、pshRNA-896、pshRNA-NNV的荧光抑制效率分别为47%、68%、51%。3种shRNA干扰载体都有干扰效果,均能干扰绿色荧光蛋白的表达,其中pshRNA-896干扰效率最好。Real-time RT-PCR检测表明,干扰质粒pshRNA-124、pshRNA-896、pshRNA-NNV对pEGFP-CP基因的沉默效率分别约为60%、96%和55%,与对照组相比差异显著(P<0.05)。研究表明,靶向斜带石斑鱼神经坏死病毒CP基因的shRNA干扰载体构建成功,为进一步运用RNA干扰技术进行CP基因的功能研究奠定了基础。     

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.     

Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue

A nodavirus was isolated from diseased yellow grouper, Epinephelus awoara , larvae cultured in southern Taiwan. The histopathology and RT–PCR results confirmed that it was a fish nodavirus; its coat protein gene sequence was similar to that of red spotted grouper nervous necrosis virus (RGNNV) and it is named yellow grouper nervous necrosis virus (YGNNV). A new nodavirus-susceptible cell line, grouper brain (GB) was established and characterized from the brain tissue of yellow grouper. The GB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum at temperatures between 24 and 32 °C, and have been subcultured more than 80 times, becoming a continuous cell line. The GB cell line consists of fibroblast-like cells and some epithelioid cells. The cell line yielded titres of YGNNV up to 10^8.5 TCID₅₀ mL^–1. The GB cells effectively replicated the virus at 28 °C, which could be purified to homogeneity by caesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 25–30 nm in diameter. The cytoplasm of infected cells was filled with aggregates of virus particles. These results indicate that the GB cell line is a significant tool for the study of fish nodaviruses.     

Characterization and viral susceptibility of a brain cell line from brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål) with persistent betanodavirus infection

A continuous cell line designated BMGB (brown‐marbled grouper brain) was established from the brain tissues of the brown‐marbled grouper Epinephelus fuscoguttatus and characterized. BMGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic protein and keratin and were persistently infected by betanodavirus, as confirmed through immunocytochemistry, polymerase chain reaction and immunoblot analyses. Because few intact virions were present in the BMGB cell culture fluid, the cytopathic effect (CPE) was not observed when the culture fluid was inoculated with GBC1 cells. However, BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocytivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. Because the cells contained several unusual or degraded viral proteins, the persistent infection of betanodavirus in the BMGB cells may have resulted from a mechanism that destroys the viral proteins rather than the result of Mx protein expression. Despite the persistent betanodavirus infection, BMGB cells proliferated in a manner similar to other normal tropic fish cells and supported the propagation of several piscine viruses; however, the yield was lower than that of normal cells. The BMGB cells will be useful for investigating virus and host cell interaction.     

鳜传染性脾肾坏死病毒主衣壳蛋白单克隆抗体的制备及鉴定

为了建立鳜传染性脾肾坏死病毒(ISKNV)疫苗抗原含量的ELISA检测方法,制备了3株抗ISKNV主衣壳蛋白(MCP)的单克隆抗体,鉴定了其生物学特性。将大肠杆菌表达的重组MCP纯化复性后,连续3次免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆、筛选,获得3株能稳定分泌抗ISKNV MCP蛋白的单克隆抗体阳性细胞株,分别命名为5F1、3D9和5B4,均为Ig G1亚型。间接ELISA实验表明,3株单抗可特异性识别ISKNV,与鳜弹状病毒、大鲵虹彩病毒等无交叉反应。将5F1株免疫小鼠后制备腹水,以重组MCP和ISKNV细胞培养物上清液为检测抗原,ELISA检测腹水效价分别为1∶51 200和1∶400。间接免疫荧光(IFA)和Western Blotting鉴定结果显示,5F1能够与ISKNV病毒发生特异性反应,并初步确定5F1单抗株制备的腹水用于IFA的使用浓度为1∶200、Western Blotting的使用浓度为1∶1000。结果证实,成功制备了抗ISKNV MCP的单克隆抗体,可特异性识别ISKNV病毒粒子和MCP蛋白,为建立ISKNV疫苗抗原含量检测方法奠定了基础。     

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％。     

急性病毒性坏死病毒IAP-86基因的克隆、表达及抗凋亡研究

在已经完成的栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)全基因组序列测序与分析的基础上,设计特异性引物,克隆得到了ORF86编码的杆状病毒凋亡抑制蛋白基因(IAP-86)。IAP-86基因与pET32a(+)质粒连接构建得到重组质粒pET32a-IAP86,将重组质粒转化到E.coil BL21(DE3)中,使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,SDS-PAGE检测显示表达蛋白分子量约为40 ku,经Western-blotting和质谱分析证明,该蛋白即为IAP-86融合蛋白,Co2+柱纯化后得到了纯化的IAP-86融合蛋白。将重组的IAP-86蛋白用FITC标记,荧光显微镜下观察,发现重组的IAP-86蛋白最终能够与栉孔扇贝血淋巴细胞的细胞核和细胞质结合。细胞凋亡检测实验发现,重组的IAP-86蛋白能够在一定程度上抑制栉孔扇贝血淋巴细胞凋亡,凋亡抑制率为7%。本实验应用原核表达成功得到了IAP-86蛋白,并证明IAP-86对栉孔扇贝细胞的凋亡有一定抑制作用,这为进一步研究AVNV的侵染机制提供依据。     

Enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfBcl-xL and green fluorescent protein genes

Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.