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  • 1
    ISSN: 1365-2761
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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 108.5 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.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2761
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2761
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2761
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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 °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.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1365-2761
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
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