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.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

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.     

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.     

Apparent digestibility of selected ingredients in diets for juvenile grouper, Epinephelus coioides (Hamilton)

Apparent digestibility coefficients (ADCs) for dry matter (ADC_dm) and crude protein (ADC_cp) of selected feed ingredients were determined in vivo for grouper using passive faeces collection (Guelph System). A reference diet (RF) and test diets (consisted of 70% RF and 30% test ingredient) with 1% Cr₂O₃ as an inert indicator were used. An RF contained 45% protein, 10% fat and 15.7 kJ g^?1 metabolizable energy. Three isonitrogenous and isocaloric diets, each contained a test ingredient (white fish meal, white cowpea meal and ipil‐ipil leaf meal), were used in a growth study based on ADC_cp of feed ingredients. An RF without Cr₂O₃ was a control. The ADC values of experimental diets were also determined. In grouper, the ADC_dm of white cowpea meal, defatted soybean meal, wheat flour and shrimp meal (74–76%) were significantly lower than that of squid meal (99%), but comparable with those of the fish meals (84–89%). No significant difference was observed between the ADC_dm of ipil‐ipil leaf meal, rice bran and wheat flour (56–73%). The ADC_cp of white cowpea meal and defatted soybean meal were similar to those of the fish meals, squid meal and shrimp meal (94–99%). The ADC_cp of wheat flour was comparable with that of ipil‐ipil leaf meal (79–83%). Rice bran had the lowest ADC_cp value of 43%. Based on specific growth rate (SGR), the growth of fish fed white cowpea meal‐based diets was similar to that of the control fish (3.2–3.3% day^?1). Also, no significant difference was observed between the ADC_dm (68–72%) and ADC_cp (88–91%) of white cowpea meal‐based diet and the control diet. The results suggest that ADC values can be used as indicators to determine the nutritional value of feed ingredients. White cowpea meal can be incorporated as a protein source in practical diet for grouper at 20.5% of the diet with no adverse effect on growth.     

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.     

Characterization and virus susceptibility of a skin cell line from red-spotted grouper (Epinephelus akaara)

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10^3.5 TCID₅₀ ml^?1, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10^0.5 TCID₅₀ ml^?1). The titers of RGV and SVCV in GCO were 10^6.0 TCID₅₀ ml^?1 and 10^8.0 TCID₅₀ ml^?1, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection.     

Growth and survival of the grouper Epinephelus coioides (Hamilton) at different loading rates in tanks

The effect of increasing fish loading (decreasing water exchange rate, while holding fish stocking density constant) on growth, survival and feed conversion ratio was determined for two size groups of the orange‐spotted grouper Epinephelus coioides (Hamilton). Fingerlings (124.3–145.8 g initial body weight) and subadults (990.6–1147.1 g initial body weight) were reared in 1‐m³ circular tanks for 14 weeks. For the fingerlings experiment, three loading rates of 0.5, 2 and 6 kg L^?1 min^?1 were used, while in the experiment with subadults, the loading rates were 1, 3 and 6 kg L^?1 min^?1. The mean growth rate of fingerlings ranged from 1.60 to 2.14 g fish^?1 day^?1, and survival was high (95–100%); however, there were no significant differences (P > 0.05) as a result of the different loading rates. Similar results were obtained in the subadults experiment, in which the growth rate (3.10–4.90 g fish^?1 day^?1) and survival (86.7–100%) were not significantly (P > 0.05) affected by the different loading rates. In both experiments, the feed conversion ratios were also not affected significantly by the loading rates. These results show that water exchange in aerated, flowthrough tanks can be reduced to as low as 6 kg L^?1 min^?1 without adversely affecting growth, survival and feed conversion of fingerling and subadult groupers.     

Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides

Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8–10, respectively, using Nα-benzoyl-l-arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6–8. Trypsin activity decreased with an increasing NaCl concentration (0–0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(l-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.     

Cryopreservation of sperm from natural and sex-reversed orange-spotted grouper (Epinephelus coioides)

The shortage of males and/or sperm has been an impediment to the aquaculture of orange-spotted grouper (Epinephelus coioides). This study reversed orange-spotted grouper females into males using hormone implants. A cryopreservation protocol for sperm was developed using normal males, and then using similar procedures the cryopreservation of sperm from sex-reversed males was compared. Immature, young and mature female fish were injected with 4 mg kg⁻¹ BW 17α methyltestosterone as implants and the gonad development stage was monitored over a 120-day period. All treated females converted into functional males within 120 days of the experimental period. Younger females (2Y) were all males within 30 days, although not all were capable of fertilizing fresh ova until day 60. The time after injection to sex reversal in immature fish was 50% shorter than in older females. Postthaw fertilization (81%, 82%) and hatching (45%, 47%) of cryopreserved sperm from natural males were the highest in trehalose (15–20%) with 150 mmol NaCl treatment; however, it was less than the control (89% fertilization and 69% hatch). There was no difference in the postthaw fertilization and the hatch percentages between sex-reversed male sperm (64% and 46% respectively) compared with natural male sperm (59% and 49%). The findings of this study suggest the potential use of sex-reversed males and cryopreserved sperm for commercial production of orange-spotted grouper seed for aquaculture.     

Gill damage to juvenile orange‐spotted grouper Epinephelus coioides (Hamilton, 1822) following exposure to suspended sediments

Juvenile Epinephelus coioides were exposed to three nominal concentrations (8, 32, 128 mg wet sediments L^?1) of suspended sediments (SS) from Port Shelter (PS), Mirs Bay (MB) and Victoria Harbour (VH) for 10 and 30 days using semi‐static system. Sediments from VH contained higher concentrations of Cu and polycyclic aromatic hydrocarbons (PAHs) than sediments from PS and MB. Gill damages including lamellar blood sinus dilation and vascular congestion were prevalent after just 10 days of exposure to SS. Fish exposed to SS at the highest concentration of 128 mg L^?1 showed higher incidence of lamellar aneurism. Hyperplasia in the base of lamellae was recorded in fish that had been exposed to contaminated SS from VH. Significant increases in the density of chloride cells and mucous cells were found on the gills of fish that had been exposed to 128 mg L^?1 of SS from PS. Clogging of gills by SS produced hypoxic‐like responses in fish. Polluted sediments from VH produced addictive or synergistic effects between SS and chemical contaminants on fish.     

Application of logistic regression analysis to predict cannibalism in orange‐spotted grouper Epinephelus coioides (Hamilton) fry

Cannibalism is frequently observed in larviculture of orange‐spotted grouper Epinephelus coioides. Previously, based on measurements of morphometric characters, a linear equation of total length (TL) of prey to cannibals was proposed: TL_prey = 0.80 TL_cannibal – 1.50. To verify the reliability of the equation, experiments were performed with pairs of fish with different TLs. Cannibalism occurred only when the cannibal‐prey size ratios were equal to or larger than that predicted by the equation. To predict the probability of cannibalism among the grouper of known TLs, a logistic regression model was proposed. The logistic regression model is: The logistic regression model showed the following: when either TL_prey or TL_cannibal is constant, the probability of cannibalism increases with increase in the cannibal‐prey size ratios; if given a constant cannibal‐prey size ratio, probability of cannibalism is lower in early stages than in later stages. The prediction contrasts with that calculated from the linear equation where the minimum cannibal‐prey size ratios decrease with size of the cannibal. However, the prediction matches observed rearing experiences: cannibals prefer smaller prey to larger ones and the cannibalism rate declines as fry age.     

Temporal genetic heterogeneity of juvenile orange-spotted grouper (Epinephelus coioides, Pisces: Serranidae)

Juveniles of orange-spotted grouper ( Epinephelus coioides ), a tropical serranid species, are heavily harvested for aquaculture seeds from nursing grounds in several Southeast Asian countries. Because juveniles of similar sizes are present in a nursery area throughout the year, we aimed to determine whether more than one genetically distinct population contributes to juvenile aggregations. We examined the temporal genetic heterogeneity of juvenile aggregations collected at four different times of the year at a nursery area in coastal waters of the Andaman Sea in Trang province, Thailand. Also, we examined the differences between these temporal samples and an outgroup collected from the Gulf of Thailand (Chantaburi). The genetic variation at six polymorphic microsatellite loci within each sample was moderate, with observed heterozygosities across all loci ranging from 0.551 to 0.629 and number of alleles per locus ranging from 7.0 to 8.33. Results indicated substantial genetic differences between the two geographically distant samples, Trang and Chantaburi (F_st=0.040–0.050, P <0.005), and between the July sample and the remaining samples from Trang (F_st=0.096–0.106, P <0.005). The observed temporal genetic heterogeneity of E. coioides juveniles may reflect high variability in the reproductive success of each spawning event and the existence of spatially isolated groups of spawners.     

Establishment and characterization of macrophage cell line from thymus of Catla catla (Hamilton, 1822)

A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L‐15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha‐naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage‐specific enzymes and cytokines.     

Establishment of a new cell line from the heart of giant grouper,Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

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.     

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.     

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.     

Alternative vegetable lipid sources in diets for grouper, Epinephelus coioides (Hamilton): effects on growth, and muscle and liver fatty acid composition

The aim of this study was to investigate the effects of different oils on growth performance and lipid metabolism of the grouper, Epinephelus coioides. Five experimental fish meal‐based isonitrogenous and isolipidic diets were formulated containing either 5.5%‐added fish oil (FO), soybean oil (SBO), corn oil (CO), sunflower oil (SFO) or peanut oil (PO). Each diet was fed to triplicate groups of 20 fish (initial body weight 13.2±0.02 g) grown in seawater at 28.0–30.5 °C for 8 weeks. Fish were fed twice a day to visual satiety. No significant differences in the survival, weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio or hepatosomatic index were found between fish fed the FO or vegetable oils (VO) diets. Dietary lipid sources did not affect whole‐body composition among grouper fed the various diets. Muscle of fish fed the FO diet had significantly higher levels of 14:0, 16:0, 16:1n‐7, 20:5n‐3[eicosapentaenoic acid (EPA)] and docosahexaenoic acid (DHA)+EPA (except for PO fed fish) compared with those of fish fed VO diets. However, the levels of 18:1n‐9, 18:2n‐6 and DHA/EPA ratios in the muscle of fish fed FO diet were significantly lower than those of fish fed the VO diets. The liver of fish fed the FO diet had significantly higher levels of 18:0, 20:5n‐3, 22:6n‐3, n‐3 highly unsaturated fatty acids and DHA+EPA than those of fish fed the VO diets, whereas increases in 18:1n‐9, 18:2n‐6 and mono‐unsaturated fatty acid levels were observed in the liver of fish fed the VO diets.     

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。