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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 ~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. KEY WORDS: Fish virus · Iridovirus · Ranavirus · Iridoviridae · Grouper · Epinephelus tauvina Resale or republication not permitted without written consent of the publisherDis Aquat Org 53: [1][2][3][4][5][6][7][8][9] 2003 significant economic losses in some Singapore marine net-cage farms. The pathogen was suggested as an iridovirus based on histopathological and morphological evidence. However, the virus was not isolated by cell culture, and no biochemical data are available to confirm the virus as a member of the family Iridoviridae (Chua et al. 1994). In 1998, an outbreak of the same disease occurred in fry and adult brown-spotted groupers. The grouper fry were imported from other SE Asian countries and cultured in fish farms in Singapore. The outbreak lasted several weeks and resulted in more than 90% mortality. The present work describes isolation of the viral pathogen in cell culture, investigation of virus infectivity and pathogenicity, and characterization of the virus based on biochemical, structural and molecular properties. MATERIALS AND METHODSCell lines and maintenance. Three local tropical marine-fish cell lines and 3 commercial fish cell lines were used. Grouper (GP) embryo cells from brownspotted grouper Epinephelus tauvina (Chew-Lim et al. 1994), Asian seabass fry (SF) cells from Lates calcarifer (Chang et al. 2001), and Asian seabass (SB) embryo cells (Chong et al. 1987) were cultured in Eagles' mi...

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