Isolation of viruses from wild Japanese flounder Paralichthys olivaceus

For a survey on fish pathogenic viruses among wild Japanese flounder Paralichthys olivaceus, 274 fish (body weight: 29-2240 g) were collected in 9 coastal areas of Japan in 1999 and 2000. Aquabirnaviruses were isolated, using RTG-2 and/or SSN-1 cells, from 111 fish (40.5%) caught in almost all the coastal areas and the representative isolate was identified as YTAV (yellowtail ascites virus) by cross-neutralization tests. Besides aquabirnaviruses, VHSV (viral hemorrhagic septicemia virus) was isolated, using EPC, SSN-1 and/or FHM cells, from 18 fish (6.6%) collected in 2 areas mainly in winter and spring. In a pathogenicity test, 4 isolates of aquabirnaviruses did not cause any mortality or morbidity in young Japanese flounder (average body weight 8.7 g) by intramuscular injection at 10(3.0)-10(7.6) TCID50/fish. Inoculation of an isolate of VHSV at 10(2.0) and 10(6.0)TCID(50)/fish produced mortalities of 60% and 100%, respectively, in flounder (24.8 g). Common external signs of flounder experimentally infected with VHSV were dark coloration of the body with hemorrhagic fins and abdominal swelling, and the internal signs included ascites and extensive hemorrhage in the muscle and/or viscera.