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1.
Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.  相似文献   

2.
为评估石斑鱼养殖过程虹彩病毒病发生的风险,实验结合Delphi法和层次分析法(analytic hierarchy process,AHP)构建新加坡石斑鱼虹彩病毒(SGIV)发生的风险评估模型.风险因素评估指标体系包括1个目标层(石斑鱼虹彩病毒病发生风险),5个准则层(水质、石斑鱼健康状况、饲养管理、养殖模式和养殖环...  相似文献   

3.
This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV‐K1), red sea bream iridovirus (RSIV‐Ku), grouper nervous necrosis virus (GNNV‐K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV‐K1, RSIV‐Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP‐B1 was predominantly localized in the nuclei, EFPF‐B2 was distributed throughout the cytoplasm and nucleus, and granular 35L‐DsRed was localized with secreted vesicles. The expression of EFPF‐B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP‐B1 or 35L‐DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum‐dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.  相似文献   

4.
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5.
Grouper iridovirus causes high mortality rates in cultured groupers, and effective treatment for grouper iridovirus infection is urgently required. Illicium verum Hook. f. is a well-known medicinal plant with a variety of biological activities. The aim of this study was to analyse the use of I. verum extracts to treat grouper iridovirus infection. The safe working concentration of each I. verum extract was identified both in vitro and in vivo as follows: I. verum aqueous extract (IVAE) ≤ 500 μg/ml; I. verum ethanol extract (IVEE) ≤ 250 μg/ml; shikimic acid (SKA) ≤ 250 μg/ml; trans-anethole (TAT) ≤ 800 μg/ml; 3,4-dihydroxybenzoic acid (DDBA) ≤ 400 μg/ml; and quercetin (QCE) ≤ 50 μg/ml. The inhibitory activity of each I. verum extract against grouper iridovirus infection was analysed using aptamer (Q2)-based fluorescent molecular probe (Q2-AFMP) and RT-qPCR. All of the I. verum extracts displayed dose-dependent antiviral activities against grouper iridovirus. Based on the achieved per cent inhibition, IVAE, IVEE, DDBA and QCE were associated with the greatest antiviral activity (all > 90%). Together, our results indicate that I. verum extracts have effective antiviral properties, making it an excellent potential source material for the development of effective treatment for grouper iridovirus infection.  相似文献   

6.
应用同源PCR技术，从被一种球状病毒感染的患病大菱鲆（Scophthalmus maximus）脾脏和肾脏组织中扩增出了一段长度为620bp的DNA片断。序列测定和Blast分析表明，该DNA片断与鱼类虹彩病毒主要衣壳蛋白（MCP）C末端编码区的DNA序列高度相似，由此证实感染养殖大菱鲆的这种球状病毒为一种鱼类虹彩病毒，暂命名为大菱鲆红体病虹彩病毒（TRBIV）。多序列比对和分析发现，TRBIV MCP C末端的205个氨基酸序列与GenBank中20种虹彩病毒相应序列的相似性分别为99．47％（韩国大菱鲆虹彩病毒）、97％～98％（待指定病毒属的7种病毒），以及50％以下（蛙病毒属、淋巴囊肿病毒属、虹彩病毒属的12种病毒），由此绘制出了包含TRBIV在内的21种虹彩病毒的系统发育树。研究结果表明，感染中国养殖大菱鲆的TRBIV属于虹彩病毒科待指定病毒属，位于该属ISKNV亚群和RSIV亚群之间，是该病毒属的一个新成员。  相似文献   

7.
Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.  相似文献   

8.
Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F1 hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.  相似文献   

9.
Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.  相似文献   

10.
Molecular characterization was carried out on an iridovirus isolated from yellow grouper, Epinephelus awoara . The major capsid protein (MCP) gene was located, sequenced and compared with homologous genes from other iridoviruses. The nucleotide sequence is 1392 bases long and contains a single open reading frame beginning at an ATG codon from the 5' end and terminating at a TAA codon at the 3' end. The open reading frame encodes a protein of 463 amino acids with a predicted molecular weight of 50 272 Da. Pairwise amino acid alignments detected a high degree of sequence identity between grouper iridovirus (GIV) MCP and the homologous genes of other iridoviruses. The MCP gene of GIV was most similar to the MCP gene from frog virus 3 (FV3) with 70% nucleotide and 73% amino acid sequence identity. The predicted molecular weight of the protein of this gene is comparable with the apparent weight obtained by SDS–PAGE. Pathogenicity of the GIV was investigated in yellow grouper by intraperitoneal injection of 107 and 104 TCID50 virus. Cumulative mortalities reached 100% within 11 and 25 days post-infection, respectively, while no grouper died in the control group. The molecular studies demonstrated that GIV is a member of the genus Ranavirus .  相似文献   

11.
Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.  相似文献   

12.
运用建立化学发光免疫(chemiluminescence immunoassay,CLIA)检测法,对梭鱼血清中主要卵黄蛋白原(Vg)类型(B类型;VgB)进行定量测定。梭鱼VgBCLIA法按两步法操作进行,使用纯化的梭鱼VgB制备特异型抗血清(a-VgB)。优化抗体浓度和培育时间等检验条件后,检验范围设定为3.91～500ng/mL。卵黄发生期的梭鱼雌鱼血清和雌激素处理后的梭鱼稚鱼血清稀释曲线与纯化的梭鱼Vg曲线平行,而梭鱼雄鱼稀释血清中几乎不发生相应的免疫学反应。对天津原种场养殖的10尾梭鱼血清VgB水平的定量检测结果表明,在雌鱼(9尾)血清中检测到VgB但个体间差异较大,变化范围为3.0～2700.1μg/mL,并表现出随着卵巢发育而增长的变化趋势。相对而言,成熟梭鱼雄鱼血清VgB水平则极低(2.7μg/mL),表明在其生长环境中不存在雌激素活性。另外,所有个体的组织学观察也未呈现出明显的性腺异常情况。研究为梭鱼主要雌激素诱导生物标志物(VgB)的量化提供了一种新的手段,较以往的检验方法具有更高的灵敏度,有助于建立多元化的水生环境中雌激素活性的检测体系。  相似文献   

13.
An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 102 to 7.95 × 106 copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.  相似文献   

14.
A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.  相似文献   

15.
A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC50) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL−1, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.  相似文献   

16.
新加坡石斑鱼虹彩病毒(singapore grouper iridovirus,SGIV)是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46(infected cell polypeptides 46)基因的真核表达载体pEGFP-ICP46转染到胖头鲤细胞(fathead minnow cells,FHM)中进行融合表达,用荧光显微镜观察到ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIVICP46的序列,设计并体外化学合成了特异性干扰SGIVICP46的siRNA(siRNA-ICP46),与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24～48h,实验细胞(共转染siRNA-ICP46和pEGFP-ICP46)和阳性对照细胞(共转染siRNA-GFP和pEGFP-ICP46)中的荧光微弱,发荧光的细胞数量较阴性对照(共转染siRNA-Negative和pEGFP-ICP46)少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72h其与阴性对照组已差别不大。说...  相似文献   

17.
This study presents an economic analysis of tiger and humpback grouper at different production scales in Indonesia. The results highlight the non-viability of small-scale tiger grouper farming, with a 5-year projected negative cumulative cash flow of −IDR 18,102,650.00 and a negative net present value (NPV) of −IDR 22,059,576.28. An increased production scale of tiger grouper highlights a marginal viability for medium-scale farms (with a 5-year projected cumulative cash flow of IDR 198,320,673.00, a positive NPV of IDR 105,578,440.42; a benefit cost ratio of 1.25; an internal rate of return (IRR) of 88% and a payback period of 0.99 years), and an economically viable large-scale cage culture (with a 5-year projected cumulative cash of IDR 707,746,923.00; a NPV of IDR 406,801,749.07; a benefit cost ratio of 1.33; an internal rate of return of 157%; and a payback period of 0.57 years). The economic analysis of humpback grouper at different production scales highlighted a positive cumulative cash and NPV, a benefit cost ratio over 2, an internal rate of return over 300% and a payback period <1 year. A sensitivity analysis revealed that increased survival rate up to 80% would increase cumulative cash and NPV of small-scale tiger grouper cage culture. Additionally, improved profitability performance was associated with decreasing major production costs, increasing production and price of the product.  相似文献   

18.
Two iridovirus-susceptible cell lines were established and characterized from grouper Epinephelus awoara kidney and liver tissues. These cell lines have been designated GK and GL, respectively. The cells multiplied well in Leibovitz's L-15 medium, supplemented with 10% foetal bovine serum, at temperatures between 20 and 32 °C, and have been subcultured more than 120 times, becoming continuous cell lines. The cell lines consist of a heterogeneous mixture of fibroblastic and epithelial cells. The viability of cells, stored frozen in liquid nitrogen (−196 °C), was 95% after 1 year. Chromosome morphologies of GK and GL cells were homogeneous. Both cell lines were susceptible to grouper iridovirus, and yielded high titres of up to 108 TCID50 mL−1. In addition, both cell lines effectively replicated the virus, which could be purified to homogeneity by cesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 170±10 nm in diameter, and were hexagonal in shape. Virus-infected cells showed an abundance of virus particles inside the cytoplasm. These results show that the GK and GL cell lines effectively replicate grouper iridovirus, and can be used as a tool for studying fish iridoviruses.  相似文献   

19.
Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.  相似文献   

20.
两种杂交石斑鱼子一代杂种优势的微卫星标记分析   总被引：5，自引：3，他引：2  
使用6对微卫星引物对两种杂交子一代(青龙斑和虎龙斑)及其亲本(斜带石斑鱼、棕点石斑鱼和鞍带石斑鱼)共5个群体进行微卫星分析,计算等位基因频率、有效等位基因数、基因杂合度、多态信息含量、相似指数和Nei氏遗传距离。结果显示,在5种石斑鱼中,棕点石斑鱼的平均有效等位基因数最大(6.849 3),最小的是鞍带石斑鱼(2.608 6)。6对微卫星引物在这5个群体中具有丰富的多态性,平均多态信息含量(PIC)分别为0.666 7、0.751 3、0.441 9、0.664 0、0.542 6。在平均观测杂合度(Ho)中,虎龙斑和青龙斑的最高均为0.940 0,鞍带石斑鱼的最低为0.508 3。遗传距离和遗传相似率结果显示,青龙斑和虎龙斑均与父本鞍带石斑鱼的亲缘关系较近。杂交子一代群体的等位基因基本来自父母本群体双方,可推断杂交子一代的遗传物质来自父母双方,属两性融合生殖,是真正意义上的杂交种。杂交后代的遗传变异水平明显增强,这是杂种优势得以形成的重要遗传物质基础。  相似文献   

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