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.     

Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus

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.     

Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10–12 with a 4 log₁₀ drop in infectivity. Purified GNNV was analysed by SDS–PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 10⁶ and 0.50 × 10⁶ Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart.     

Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer , muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.     

Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China

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.     

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.     

虎纹蛙病毒主要衣壳蛋白基因的克隆及其序列

从新分离感染虎纹蛙的病毒培养细胞中提取病毒DNA作模板，用分别对应于蛙病毒－3型（FV3）主要衣壳蛋白（Major Capsid Protein,MCP）基因读码框两侧的寡核苷酸片段作引物进行PCR扩增，得到预期大小基因片段，进一步将此基因片段插入到pGEM-T载体中，进行全长片段的序列测定和分析。结果表明，编码虎纹蛙病毒的MCP基因的读码框核苷酸数为1392bp，编码463个氨基酸；基因的核苷酸序列与其他脊椎动物虹彩病毒的MCP基因序列比较结果显示，该病毒与蛙病毒属的FV3的同源性（98％)明显高于囊肿病毒属的FLDV－1（52％），并且与虹彩病毒科其他成员的MCP基因序列均有所不同，说明该病毒株是虹彩病毒科蛙病毒属的新成员。     

Isolation and characterization of a regulator of G protein signaling (RGS16) gene homologue in yellow grouper Epinephelus awoara

Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAP) which act as modulators of G protein-coupled receptors. We isolated a RGS16 homologue in yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of yellow grouper RGS16 full-length cDNA was 700 bp and contained an open reading frame of 537 bp, encoding a putative protein of 178 amino acids. The encoded protein shows 47–61% identities to other homologues. RT-PCR analysis demonstrated that RGS16 was expressed in yellow grouper spleen and up-regulated in kidney heart, liver, and anterior kidney by lipopolysaccharide. This study will help towards validating the specific function of RGS in marine fish.     

中国大菱鲆虹彩病毒主要衣壳蛋白基因的PCR扩增及序列分析

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。     

Anaesthetic efficacy and physiological responses to clove oil-anaesthetized kelp grouper Epinephelus bruneus

The efficacy of clove oil as an anaesthetic and at producing a physiological response (plasma cortisol and glucose) was evaluated in the kelp grouper, Epinephelus bruneus . To acquire complete anaesthesia in less than 3 min and recovery in <10 min, three doses of clove oil were tested at 18, 22 and 26 °C. Although higher anaesthetic doses resulted in shorter induction times and longer recovery times, and a lower temperature resulted in longer anaesthesia induction and slower recovery, we found the optimal dose and administering temperature of clove oil to be 250–300 mg L⁻¹ at water temperature of 18 °C, 150–200 mg L⁻¹ at water temperature of 22 °C and 50–100 mg L⁻¹ at water temperature of 26 °C respectively. Following the administration of 150 mg L⁻¹ of clove oil at 22 °C, the plasma cortisol level was highest (4.24 ± 1.571 μg dL⁻¹) after 12 h and the plasma glucose was highest (92.7 ± 9.61 mg dL⁻¹) after 2 h. These results should be useful to the aquaculture industry, where anaesthesia is necessary for a range of activities.     

Identification and expression analysis of ghrelin gene in common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

ABSTRACT:   A ghrelin gene has been cloned and sequenced in common carp Cyprinus carpio . Ghrelin cDNA is composed of 461 bp [with a 36-bp 5'-untranslated region (UTR) and a 113-bp 3'-UTR], which translates into a protein of 103 amino acid residues. Carp ghrelin (preproghrelin) contained a predicted signal peptide of 26 amino acid residues, the ghrelin domain ( Gly ²⁷– Val ⁴⁵) and C-terminal peptide ( Gly ⁴⁶– Phe ¹⁰³). Homology analysis of the ghrelin domain of carp with that of other known ghrelin in vertebrates showed good similarity to teleost ghrelin (50–81.8%). Hydropathy analysis based on the deduced amino acid sequence of ghrelin domains in teleosts showed a similar profile. Carp ghrelin clustered with ghrelin of goldfish Carassius auratus and other teleosts, away from mammalian, reptilian, avian, amphibian and chondrichthian ghrelin, by phylogenetic analysis. Genomic organization of carp ghrelin gene was composed of four exons and three introns, which was the same as that of other teleosts and human ghrelin genes. The carp ghrelin gene was expressed in unstimulated tissues such as foregut, hindgut, spleen and brain. In spleen cells, expression of the ghrelin gene increased upon stimulation with lipopolysaccharide (LPS), phytohemagglutinin (PHA) or imiquimod. The identification of carp ghrelin gene and the analysis of the modulation of its expression in immune-activated conditions will allow a more complete analysis of the roles of ghrelin in teleosts.     

Cage culture of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) at different stocking densities in a shallow eutrophic lake

Postlarvae of Litopenaeus vannamei were acclimated and stocked in lake-based cages at the following stocking densities: 10, 20, 30 and 40 shrimp m⁻². Another set of shrimp was stocked in concrete tanks as reference samples at 30 shrimp m⁻². Significant differences were observed among stocking densities throughout the 95-day culture. The final weight at harvest decreased with increasing stocking density: mean weights of 23.3, 15.8, 13.0, 10.9 and 14.6 g for the 10, 20, 30, 40 shrimp m⁻² and reference tanks were observed respectively. There were no significant differences in survival throughout the culture period, ranging between 69% and 77%. Daily growth rates (range: 0.11–0.24 g day⁻¹) and specific growth rates (range: 3.54–4.34%) also differed significantly among stocking densities, both increasing with decreasing stocking density. The feed conversion ratio in the cages did not differ among the stocking densities, ranging from 1.53 to 1.65. The relationship between stocking density and mean individual weight at harvest followed the equation y =81.06 x ^−0.54 ( R ²=0.938) and that of stocking density and production (in g m⁻²) is y =58.01 x ^−0.46 ( R ²=0.834).     

An in vitro method to determine phosphorus digestibility of rainbow trout Oncorhynchus mykiss (Walbaum) feed ingredients

An in vitro method was developed to assess the digestibility of phosphorus in 12 plant and animal feed ingredients for rainbow trout Oncorhynchus mykiss (Walbaum). The method simulates the gastrointestinal tract of the rainbow trout with regard to pH and gastrointestinal enzymes. Phosphorus solubility was measured after acid digestion (pH 3) with and without gastric enzymes, after alkaline digestion (pH 9) with and without intestinal enzymes, and after a two-step process involving acid and alkaline digestion. Commercially available digestive enzymes from mammals were compared with digestive enzymes from rainbow trout. Correlating in vitro digestibility with in vivo digestibility showed that acid digestion with both commercial enzymes ( r ²=0.98, P  < 0.05) and trout enzymes ( r ²=0.94, P  < 0.05) predicted the in vivo digestibility of animal feed ingredients. Alkaline digestion with both enzyme systems (commercial r ²=0.79; trout r ²=0.74, P  < 0.05) or without ( r ²=0.82, P  < 0.05) enzymes predicted the in vivo digestibility of ingredients from animal byproducts but not those from plant products. The in vitro digestibility with two enzyme steps (acid and alkaline) predicted in vivo digestibility of plant and animal ingredients ( r ²=0.79 for commercial enzymes and r ²=0.74 for trout enzymes) better than did one-step acid or alkaline digestion.     

In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Mouse monoclonal antibodies (MAbs) were produced by using yellow grouper nervous necrosis virus (YGNNV) as an immunogen, isolated from infected yellow grouper, Epinephelus awoara (Temminck & Schlegel), and propagated in GB cells. In enzyme linked immunosorbent assay (ELISA), 43 hybridoma clones secreting MAbs strongly reacted with the purified virus. Ten of them showed a higher neutralization index (NI) value between 6.5 and 4.5 (log₁₀ NI) than the other 33 MAbs against YGNNV infection in cell culture. All 10 MAbs belonged to the IgG isotype with a κ light chain and recognized the 42 kDa coat protein of YGNNV by Western blot analysis. Immunohistochemical results demonstrated that the viral signals co-located with pathological lesions observed in retina, brain and spinal cord. These results indicate that the MAbs are useful for confirmative diagnosis of YGNNV infection.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

石斑鱼虹彩病毒ORF050的分子特征和功能初步分析

新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)是导致石斑鱼养殖产业严重经济损失的主要病毒病原之一。SGIV 是大分子DNA病毒,包含162个基因开放阅读框,其中ORF050是一个肿瘤坏死因子受体类似物,可能在SGIV的免疫逃避中发挥作用。本研究克隆了SGIV ORF050基因,并构建了全长基因的真核表达重组质粒和四个半胱氨酸富集结构域(CRD)分别缺失的突变体。RT-PCR和药物抑制实验结果表明,SGIV ORF050是病毒的一个立即早期基因。亚细胞定位结果表明,该基因在细胞质内均匀地弥散性分布,并在细胞核周围聚集;第一个CRD缺失后,基因的定位发生明显的变化,即呈点状分布在胞质中,推测第一个CRD对其功能有影响。在过表达SGIV ORF050的鱼类细胞中观察SGIV感染引起的CPE,发现与对照相比没有明显区别;荧光定量PCR检测SGIV 主要衣壳蛋白MCP的转录表达水平,也没有明显变化,提示该基因对SGIV在宿主细胞内的复制增殖可能没有影响。荧光定量PCR检测过表达ORF050的细胞在SGIV感染后宿主TNF/TNFR的转录水平,结果显示在感染10 h后TNF1、TNF2和TNFR2的表达量升高了2～3倍,而TNFR1的表达量没有明显变化,说明SGIV可能通过ORF050来调节细胞TNF和TNFR的表达,从而逃避宿主的免疫攻击。     

The potential of liposomes as a nutrient supplement in first-feeding marine fish larvae

The ingestion rate (ng liposome larva^–1 h^–1) of extruded [1–¹⁴C] palmitic acid-labelled liposomes containing physiological saline (PHS) or cod fish extract (CFE), was tested in 5-day-old gilthead seabream Sparus aurata and white grouper Epinephelus aeneus larvae. A follow-up study compared the assimilation of radioactive free fatty acid (FFA) label of these two liposome treatments into six phospholipid and neutral lipid fractions as well as the nonlipid fraction in 5-day-old seabream. In seabream larvae, there was a 50-fold ( P  < 0.05) increase in the net consumption rate when fed CFE liposomes (2305.8 ng liposome larva^–1 h^–1) compared with liposomes containing physiological saline (42.7 ng liposome larva^–1 h^–1). A similarly significant ( P  < 0.05) but less marked pattern was also observed in the grouper larvae where the CFE treatment larvae ingested 238.5 ng liposome larva^–1 h^–1 compared with 54.3 ng liposome larva^–1 h^–1 in larvae fed the PHS liposomes. In seabream larvae ingesting CFE and PHS liposomes, radioactivity was found in all larval fractions analysed. However, marked treatment differences ( P  > 0.05) in assimilation were found only in the triacylglycerol fraction (3.4 and 0.6 dpm larva^–1 h^–1, respectively) and nonlipid fraction (11.2 and 15 dpm larva^–1 h^–1, respectively).