Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel)

Abstract. Mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles, Oplegnathus fasciatus (Temminck & Schlegel), have occurred in Nagasaki Prefecture. Light and electron microscopic examinations showed that the only consistent histopathological feature was extensive nervous necrosis in the spinal cord, spinal ganglia and brain. Numerous non-enveloped virus particles, icosahedral in morphology and measuring about 34 nm in diameter, were found in the cytoplasm of affected neurones and glial cells. Such nervous necrosis is believed to be the major cause of the mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles.     

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.     

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.     

赤点石斑鱼病毒性神经坏死症的组织病理和电镜观察

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

Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel)

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 .     

Microbiota of yellow grouper (Epinephelus awoora Temminck & Schlegel, 1842) fed two different diets

The rapidly growing yellow grouper industry has experienced relatively severe bacterial disease problems in China. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this background, we used nested polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) and sequence analysis to investigate microbiota in the skin, gills and intestines, including adherent bacteria and non‐adherent bacteria in yellow grouper fed with natural diet and complete feed. A total of 21 bacterial species were identified using phylogenetic analysis. The γ‐Proteobacteria group (81.0%, 17 species) dominated the bacterial communities in yellow grouper completely. Others belonged to Firmicutes (9.5%, two species), Actinobacteria (4.75%, one species) and Verrucomicrobia (4.75%, one species). The higher similarities (above 91%) of the DGGE band patterns in skin, gill and intestinal‐non‐adherent bacteria between two groups of fish indicated that existed more stable microbial communities existed in these specifically ecological niches in yellow grouper. However, considerable differences existed between two intestinal‐adherent bacteria (IAB) samples; that is, compared with natural diet fed yellow grouper, higher bacterial apparent species richness and possibly less abundance existed in IAB in fish fed with complete diets, probably indicating that the community structures in IAB were affected easily and significantly by diet.     

赤点石斑鱼Epinephelus akaara(Temminck et Schlegel)亲鱼强化饲育与产卵的试验研究

本文采用虾塘越冬的赤点石斑鱼亲鱼在产卵前一个月,水温在15℃-20℃之间,如何对其进行强化饲育的试验研究。结果表明：海区网箱强化饲育的亲鱼与室内水泥池强化饲育的亲鱼．虽然都能自然产卵,但是差异很大。     

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.     

Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass,Dicentrarchus labrax (L.)

Mass mortalities of the European sea bass, Dicentrarchus labrax (L.), occurred in different ongrowing units in Greece. A presumptive diagnosis of viral nervous necrosis (VNN) was made on the basis of the light microscopic observations of a vacuolating encephalopathy and retinopathy. Positive peroxidase reaction with VNN antiserum confirmed nodavirus as the causative agent. Usually recorded in larvae and juvenile stages of different marine fish species, VNN is described in adult sea bass and sea bass reared in floating cages in the Mediterranean for the first time. The horizontal transmission of the disease has been strongly suspected. Histological analyses clearly demonstrate that cells other than nerve cells are also infected. Some epidemiological aspects of the disease are described and their implications for the establishment of prophylactic guidelines are discussed.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.     

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.     

赤点石斑鱼人工育苗的初步研究

<正> 赤点石斑鱼Epinephelus akaara(Temminck et SchIegel)广东俗称红斑,分布于我国南海和东海南部,是我国南方沿海地区主要网箱养殖鱼类之一。由于还未掌握人工育苗方法,在以往的网箱养殖生产中,全靠天然鱼苗。研究解决赤点石斑鱼人工繁殖与育苗问题,已列为重要攻关课题之一。各有关水产单他相继开展对此课题的研究。许波涛等1983     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

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.     

Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole

In the present study, we implanted 2‐year‐old female red‐spotted grouper, Epinephelus akaara, with a non‐steroidal aromatase inhibitor (AI), fadrozole, in the breeding season and examined changes in gonadal histology, serum sex steroids, aromatase activities and P450 aromatase (P450arom) gene expression in gonads after AI implantation. Aromatase inhibitor at doses from 0.1 to 10.0 mg kg^?1 BW induced a sex inversion and completion of spermatogenesis up to the functional male phase, but doses of 1.0 and 10.0 mg kg^?1 BW AI produced more males than 0.1 mg kg^?1 BW AI. Serum estradiol‐17β (E₂) levels decreased, but 11‐ketotestosterone (11‐KT) levels increased significantly in all the AI‐implanted groups, whereas testosterone (T) levels increased significantly only in the 1.0 mg kg^?1 BW AI‐implanted group. Aromatase activities and P450arom gene expression in gonads were inhibited significantly in the AI‐implanted groups, which was in accordance with the decrease in serum E₂ levels. These results suggested the optimal dose of AI to induce sex inversion to be 1.0 mg kg^?1 BW. Furthermore, the sex inversion induced by AI may be attributed to the inhibition of P450arom gene expression and aromatase activity and the resultant decrease in the biosynthesis of endogenous E₂. Meanwhile, the elevated 11‐KT levels were also associated closely with the occurrence of sex inversion in protogynous red‐spotted grouper.     

Gill filament necrosis in farmed Japanese eels, Anguilla japonica (Temminck & Schlegel), infected with Herpesvirus anguillae

A herpesviral gill disease accompanied by mass mortality occurred in Japanese eels, Anguilla japonica (Temminck & Schlegel), reared in warm water ponds from 1993 to 1995. Diseased fish displayed marked haemorrhage and congestion within gill filaments and destruction at the tips of affected filaments with necrosis and inflammation in the central connective tissue and the central sinus. Electron microscopy revealed herpesvirus particles in infected fibrocytes within the filamental connective tissue. The isolate was identified as Herpesvirus anguillae by a neutralization test. Infectivity experiments with the isolates revealed that the virus was pathogenic.     

Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage

Abstract Viral nervous necrosis caused by sevenband grouper nervous necrosis virus (SGNNV) has occurred in grow-out stages (0-3 years old) of sevenband grouper, Epinephelus septemfasciatus, since the 1980s. In the present study, based on histopathological features of the central nervous system (CNS) in naturally diseased fish, pernasal infection experiments using grow-out fish were performed and pernasal infection was established as a putative invasion route of SGNNV. The definite SGNNV-targeted cells were determined by histopathological studies including indirect fluorescent antibody test and electron microscopy. Nerve cells in the olfactory lobe were most extensively necrotized with vacuolation followed by infiltration of microglia and macrophages. Purkinje cells and Golgi cells were extensively infected in the cerebellum. Megalocells and small nerve cell nuclei were also infected in the preoptic area, thalamus, medulla oblongata and spinal cord. Only a few small nerve cells were infected in the olfactory bulb and optic tectum. The retina of some diseased fish displayed vacuolated bipolar cells of the inner nuclear layer and in the ganglion cell layer. These SGNNV-infected nerve cells displayed viroplasmic inclusions containing virions, vacuoles and myelin-like structures. Based on observed histopathological changes, the lesion of the CNS was characterized by encephalitis but not encephalopathy.     

Establishment and characterization of two new cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel)

Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets ofmicrosatellite markers of flounder. Optimal growth temperature was 20 degrees C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.