A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of approximately 500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry.

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degrees C, but had a reduced growth rate below 18 degrees C. The growth rate of FEC cells in medium containing 15% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.

Background: Turbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China. Results: The genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae). Conclusions: The determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.