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1.
Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125–247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201–212 was confirmed on the basis of its function associated with NES activity.  相似文献   

2.
Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.  相似文献   

3.
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.  相似文献   

4.
加州鲈肌肉生长抑制素(MSTN)cDNA的克隆和序列分析   总被引：1，自引：2，他引：1  
肌肉生长抑制素是抑制肌肉生长和发育的生长调控因子。对运用RT-PCR和RACE技术从加州鲈成鱼肌肉总RNA中扩增得到的MSTN cDNA全序列进行了序列分析。结果表明,加州鲈MSTN cDNA全长为1626bp,其开放阅读框为1 134bp,共编码377个氨基酸,前面的22个氨基酸为信号肽,中间有四个氨基酸(RARR)为蛋白水解加工位点;该基因总共有13个半胱氨酸残基,后面9个在蛋白水解加工位点之后的C端生物活性区,与其它脊椎动物比较,它们的位置完全一致,对该基因的结构和功能非常重要。与GenBank中已知的条纹狼鲈、金鲷、斑马鱼、虹鳟、斑点叉尾鮰、人、猪、鸡、鸽MSTN的ORF相比较,核苷酸序列同源性为63%~94.4%,氨基酸同源性为61.4%~96%,特别是在C端生物活性区氨基酸同源性为88.1%~100%,高度的保守性反映了该基因受到了高度的进化限制以及功能的重要性。加州鲈MSTN基因的克隆为研究该基因打靶和鱼类肌肉发育调控机理奠定了基础。  相似文献   

5.
Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.  相似文献   

6.
为了早期快速诊断近年来流行于广东省养殖大口黑鲈(Micropterussalmoides)中的病毒性溃疡综合征,本研究用基因组步移的方法获得了大口黑鲈溃疡综合征病毒(Largemouthbassulcerativesyndromevirus,LBUSV)主要衣壳蛋白(MCP)基因,该基因编码区全长1392bp。通过序列比较分析,在MCP基因内确定了一段241bp的特异性较强的片段作为靶序列,设计并合成引物,经过优化PCR反应条件,建立了可以快速检测大口黑鲈溃疡综合征病毒的PCR方法。实验表明,在PCR进行到30个循环反应时可以检测到的质粒最小浓度是104拷贝数/μL,相当于104个病毒粒子。利用该方法,从天然感染LBUSV的大口黑鲈脾脏组织DNA可扩增出241bp的片段,而健康大口黑鲈和感染了传染性脾肾坏死病毒样病毒的大口黑鲈脾脏组织则没有扩增条带。本研究建立的PCR检测方法具有检测快速、成本低、准确性高的特点,适用于大范围早期病害诊断的推广应用。  相似文献   

7.
鱼类诺达病毒及其所导致的疾病   总被引：1，自引：1，他引：1  
黄剑南 《水产学报》2006,30(6):831-836
In recent years, piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide. Affected fish exhibit a range of neurological signs, such as erratic swimming behaviour with the associated microscopic lesions of necrosis and vacuolation of the central nervous tissues and retina. Numerous roundshaped, unenveloped and 25-30 nm in diameter virus particles were found in the cytoplasm of affected retinal and nerve cells. Nodaviruses have a bipartite genome of positivesense RNA,with RNA1 encoding the RNAdependent RNA polymerase and RNA2 encoding the capsid protein. Both RNA are capped, but not polyadenylated. The family Nodaviridae comprises two genera: Alphanodavirus and Betanodavirus, members of which primarily infect insects and fish, respectively. Therefore, betanodavirus is also named piscine nodavirus. At present, piscine nodaviruses are divided into four genotypes based on partial sequences of the coat protein gene. ELISA and RT-PCR amplification have been developed as specific diagnostic methods for the d etection of the virus. Antibodies to striped jack (Pseudocaranx dentex) nervous necrosis (SJNNV) were found in 65% of plasma samples collected from wild and domestic brood stocks of striped jack, suggesting that the virus is very prevalent. Viral antigens were detected in eggs, larvae, and ovaries of hatcheryreared and wild spawner fish, suggesting both horizontal and vertical modes of transmission of the virus. Selection of nodavirusfree spawners using ELISA for detection of antigens and RT-PCR techniques have successfully reduced incidences of the virus infections in juvenile sea bass (Dicentrarchus labrax),striped jack and barfin flounder (Verasper moseri). The SSN1 and GF cell lines have been successfully used in isolating piscinenodaviruses.Although there are many papers describing the molecular characteristics of betanodavirus, our knowledge of the genomic attributes of these viruses is still limited. Vaccination studies are being undertaken by a number of researchers and need to be fostered. In particular, the use of passive immunization of broodfish with homologous and heterologous, high titre antisera are worthy of investigation.  相似文献   

8.
Four dietary protein sources were bio-assayed for amino acid availability, as estimated by true digestibility, when fed to striped bass Morone saxatilis . Diets were formulated to contain either herring fish meal, soybean meal, corn gluten meal or peanut meal as the sole source of dietary protein. A fifth diet, containing no protein, was fed to estimate the level of endogenous amino acids for calculation of true digestibility. The five dietary treatments were randomly assigned to ten tanks of striped bass having an average weight of 150 g per fish. All fish received the assigned diet fed at a rate 1.5% of the biomass per day for a period of 10 d. Fecal samples were collected from anesthetized fish by gentle, manual stripping of the lower digestive tract. Diets and feces were analyzed for dry matter, chromium, nitrogen and amino acid concentrations. There were no statistical differences (P > 0.05) among the protein sources for apparent dry matter digestibility or availability of arginine, threonine, valine and nonessential amino acids with the exception of cysteine. Corn gluten meal had a significantly lower availability coefficient for lysine, and peanut meal had significantly lower availability coefficients for histidine, isoleucine, leucine, and lysine when compared to herring fish meal and soybean meal. Statistically there were no differences between soybean meal and herring fish meal for any nutrient tested. These data suggest that in terms of amino acid availability and overall protein quality, soybean meal could be used to spare herring fish meal in striped bass diets, with corn gluten meal being equally as useful when supplemented with lysine or complemented with other proteins.  相似文献   

9.
采用电子显微镜观察和细胞培养等技术, 从湖北黄陂某养殖场患病大口黑鲈(Micropterus salmoides)体内发现并分离到一株蛙病毒。患病大口黑鲈的临床症状主要表现为体表出血、溃疡, 肝脏发白。将病鱼内脏组织匀浆超微滤液接种鳜脑细胞系(mandarin fish brain, MFB)细胞能产生典型细胞病变效应(cytopathic effect, CPE), 病毒滴度达到 108.36±0.15 TCID50/mL。细胞培养病毒的超薄切片电镜观察结果显示, 细胞质中存在大量直径约为 150 nm 左右的正六边形病毒粒子, 呈晶格排列。细胞培养病毒的人工感染大口黑鲈试验结果显示, 7 d 内试验鱼死亡率高达 100%, 其临床症状与自然发病鱼相似。采用大口黑鲈病毒(largemouth bass virus, LMBV)的特异性 PCR 检测方法对患病鲈组织样品和细胞培养病毒样品进行检测, 均能扩增出 241 bp 的单一目的条带。进一步根据 GenBank 中 LMBV 主衣壳蛋白(major capsid protein, MCP)基因序列设计特异性引物, 均能从上述样品中扩增出 1392 bp 的 MCP 基因开放阅读框(open reading frame, ORF)全长。将 MCP 氨基酸全序列进行比对, 结果显示其与 Santee-Cooper 蛙病毒、孔雀鱼病毒 6 型及大口黑鲈溃疡综合征病毒的 MCP 氨基酸序列同源性高达 100%。系统进化结果显示, 与感染鱼类的虹彩病毒科蛙病毒属病毒, 如鳜鱼蛙病毒、Santee-Cooper 蛙病毒、孔雀鱼病毒 6 型和大口黑鲈溃疡综合征病毒等聚成一支。这些结果证明, 该分离株为虹彩病毒科蛙病毒属的成员, 暂命名为大口黑鲈蛙病毒(largemouth bass ranavirus, LMBRaV)湖北株 LMBRaV-HB001。病毒敏感细胞系筛选试验结果表明, 病毒 LMBRaV-HB001 感染鲤上皮瘤细胞(epithelioma papulosum cyprinid, EPC)、草鱼性腺细胞(grass carp ovary, GCO)、大鲵肌肉细胞(giant salamander muscle, GSM)和鲫脑组织细胞(gibel carp brain, GiCB)均能产生典型 CPE, 病毒滴度可达 108.0 TCID50/mL 以上。本研究首次在湖北省养殖大口黑鲈体内分离与鉴定了 LMBRaV 病毒, 建立了病毒的细胞培养方法, 为进一步研究该病毒的传播、诊断和防控技术提供了重要参考。  相似文献   

10.
Progressive research has been recently made in dissecting the molecular biology of Betanodavirus life cycle, the causative pathogen of viral encephalopathy and retinopathy in economic important marine fish species. Establishment of betanodavirus infectious clone allows the manipulation of virus genome for functional genomic study, which elucidates the biological event of the viral life cycle at molecular level. The betanodavirus strategizes its replication by expressing anti‐apoptosis/antinecrotic proteins to maintain the cell viability during early infection. Subsequently utilizes and controls the biological machinery of the infected cells for viral genome replication. Towards the late phase of infection, mass production of capsid protein for virion assembly induces the activation of host apoptosis pathway. It eventually leads to the cell lysis and death, which the lysis of cell contributes to the accomplishment of viral shedding that completes a viral life cycle. The recent efforts to dissect the entire betanodavirus life cycle are currently reviewed.  相似文献   

11.
克隆了凡纳滨对虾脂肪酸结合蛋白基因全长cDNA并进行了序列分析。该基因由1042bp的碱基组成,开放阅读框长411bp,编码由136个氨基酸组成的蛋白,基因两翼分别存在113bp(5'端)和518bp(3'段)的非翻译区。聚类分析表明,凡纳滨对虾脂肪酸结合蛋白氨基酸序列与斑节对虾脂肪酸结合蛋白紧密聚为一支,之后聚类顺序依次为刀额新对虾、意大利蜜蜂、斑马鱼、大西洋鲑、鸡、猪和人。通过半定量RT-PCR对该基因在不同组织的表达分析表明,该基因在抗IHHNV对虾肠、胃、肝胰腺和肌肉组织中表达较高,在心肌中表达较低,在眼柄中不表达。比较该基因在抗IHHNV对虾和IHHNV易感对虾心、肝胰腺、肠、胃、眼柄和肌肉组织表达发现,该基因在两种对虾心、肠、胃和肌肉组织中的表达无明显差异,但在肝胰腺中抗IHHNV对虾的表达量明显高于IHHNV易感对虾的表达量,说明该基因参与抗性对虾抑制IHHNV感染的免疫过程。  相似文献   

12.
Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red‐spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture‐based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 μg mL?1 (3 × 105 TCID50 per mL), whereas for capture‐based ELISA, the sensitivity for the antigen in solution was 17 μg mL?1 (35 × 105 TCID50 per mL). The capture‐based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 108 TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv‐specific IgM is required, or for use in samples where PCR is difficult to perform.  相似文献   

13.
Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT‐PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock‐infected group. VERv presence was confirmed by rRT‐PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.  相似文献   

14.
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15.
Nutritional amino acid requirements of varying size classes of largemouth bass were estimated using A/E ratios. Nutrient and amino acid contents of roe and carcass of the different size classes were determined and compared to results of selected, classic works related to the concept of ideal protein and relationship between the contents of individual, essential amino acids and the total contents of essential amino acids – A/E ratios. Protein content in the roes of the largemouth bass were higher in comparison to the carcass, but the content of lipids of the roes and of the carcass didn’t present significant difference (P<0.05). Largemouth bass showed higher muscle protein content in comparison to other species. Although some authors report variation in the contents of some amino acids in the carcass of selected species, differences observed in this study regarding carcass amino acid contents of tilapia, speckled catfish and largemouth bass were not significant (P<0.05), values of the ratio A/E followed the same trend. Results, herein presented, indicate that the amino acid profile of largemouth bass could be used as complementary tool for balancing amino acids in formulated feed for the species, and in the validation of amino acid requirements determined in performance studies.  相似文献   

16.
Betanodavirus infection was diagnosed in larvae of farm‐raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07–93.88%) and closely related to red‐spotted grouper nervous necrosis virus (RGNNV).  相似文献   

17.
Yellowtail ascites virus and related strains isolated from marine fish have been shown to be similar to infectious necrosis virus (IPNV) in terms of biological and serological characteristics. This paper explores the relationship of aquatic birnaviruses at the genetic level. The junction region on the genome segment A coding viral capsid protein VP2 and viral protease NS was amplified by PCR in six marine strains. Analysis of nucleotide and the deduced amino acid sequences revealed that the six marine strains have amino acid variations in the possible amino terminus of NS when compared to IPNV. The six marine strains form a new genogroup which is distinguished from three serotypes ofPNV.  相似文献   

18.
A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus , maintained in fresh water at 30 °C . Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 °C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.  相似文献   

19.
Three experiments were conducted that were designed to evaluate our ability to predict essential amino acid (EAA) needs of hybrid striped bass using the quantified lysine requirement and whole‐body amino acid concentrations. In the first experiment, six diets containing various amino acid profiles were fed to triplicate groups of fish initially weighing 7.7 g per fish. At the end of the 8‐week experiment, no significant differences were detected in growth rates or feed efficiencies (FE) between fish fed a practical diet containing 510 g kg?1 herring fish meal (FM) and fish fed a purified diet containing the amino acid profile of herring fish meal (CAA‐FM). Growth responses of fish fed purified diets containing 100 (HSB), 110 (HSB110), 120 (HSB120) or 140 g 100 g?1 (HSB140) of the amino acid profile of hybrid striped bass whole‐bodies were significantly lower than those of fish fed diet FM. In the second experiment, triplicate groups of fish (5.6 g per fish) were fed diets containing various energy : protein (E : P) ratios (34.8, 41.2, 47.5 and 53.9 kJ g?1 protein) and one of two amino acid profiles (CAA‐FM and HSB120) in a 4 × 2 factorial design. Carbohydrate concentration was varied to achieve the desired energy concentrations. At the end of the 8‐week experiment, weight gain and FE were significantly higher in fish fed diets formulated to simulate the amino acid profile of herring fish meal (CAA‐FM) compared with fish fed diets formulated to contain 120 g 100 g?1 of the amino acid profile of hybrid striped bass whole‐bodies (HSB120). Weight gain, FE and survival data indicated the optimum dietary E : P was 41.2 kJ g?1 protein. Dietary treatments in the final experiment included three amino acid profiles and four levels of lipid in a 3 × 4 incomplete factorial design. Dietary amino acid treatments included the amino acid profile of herring fish meal (CAA‐FM) or 120 g 100 g?1 of the predicted EAA requirement profile for hybrid striped bass (HSB120). The amino acid profile of the remaining dietary treatment (PRED+) was similar to that of the HSB120 treatment, but contained additional threonine, isoleucine and tryptophan. Diets CAA‐FM and HSB120 contained either 90, 130, 170 or 210 g kg?1 lipid, whereas diet PRED+ contained 130 g kg?1 lipid. Dietary treatments were fed for 10 weeks to triplicate groups of fish initially weighing 81.0 g per fish. Weight gain and FE were not significantly affected by dietary amino acid profile. Feed efficiency was significantly reduced in fish fed diets containing 210 g kg?1 lipid compared with fish fed diets containing 90–170 g kg?1 lipid. Intraperitoneal fat (IPF) ratio and hepatosomatic index (HSI) values generally increased as dietary lipid concentrations increased. Total liver lipid concentrations were significantly reduced in fish fed diets containing 210 g kg?1 lipid compared with those of fish fed 90–130 g kg?1 lipid. Results of this study indicate an appropriate dietary amino acid profile can be predicted for hybrid striped bass using the quantified lysine requirement and whole‐body amino acid concentrations. Further, the optimum E : P appears to be 40 kJ g?1 protein.  相似文献   

20.
European sea bass (Dicentrarchus labrax) and gilthead sea bream(Sparus aurata) are amongst the most important finfish speciescultured in the Mediterranean region. Production of these species isnowadays a well-controlled process, but knowledge of their nutritionalrequirements is still very limited. Nevertheless, a considerable amountof data has been accumulated in recent years, and the purpose of thispaper is to review the recent advances on the nutritional requirementsof sea bass and sea bream. The optimum protein to energy ratio of thediets of sea bass and sea bream seem to be higher than for salmonids,and there is some evidence that high dietary lipid levels have nobeneficial effects on fish performances. Although the essential aminoacid requirements were estimated by the ideal protein method, data basedon the dose-response method is only available for a few amino acids.Essential fatty acid requirements were estimated for sea bream juvenilesbut data is lacking for sea bass. Vitamin and mineral requirements ofthese species are practically unknown. Although the importance ofbroodstock nutrition on gonadal development, spawning and egg quality isrecognized, few studies were done to elucidate these aspects. The recentdevelopment of microparticulate diets for larvae will contribute to theaccurate evaluation of their nutritional requirements.  相似文献   

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