A group of wild broodstock of Senegalese sole Solea senegalensis was introduced into a culture facility in southwest Spain for endocrinological studies, and fish were kept under standard culture conditions . External signs of disease (dark coloration, hyperactivity, uncoordinated swimming and behavior) appeared suddenly, and the group experienced almost 100% mortality thereafter. The few survivors from this group, kept in a different facility, developed similar signs the next year. The cause of the disease was unclear, although viruslike structures were observed in ultrathin sections of kidney from one diseased fish, and it was possible to isolate a birnavirus from all the visceral and skin homogenates from this and other specimens inoculated onto samples of the CHSE-214 (chinook salmon embryo) cell line . Further serologic, phenotypic, and biochemical characterizations of the agent indicated its similarity to infectious pancreatic necrosis virus (IPNV) . Western blot assays using the anti-IPNV monoclonal antibody AS 1 reacted positively with the VP2 protein of the new agent . A complementary DNA fragment was amplified from each of the IPNV reference strains tested and from the virus isolated from S. senegalensis . In all cases, the fragments amplified were of 339 base pairs, as expected . The results show that the new isolate is an aquatic birnavirus .

Infectious pancreatic necrosis virus (IPNV) was infected in CHSE-214 cells, and viral antigens in the cells stained with indirect immunofluorescence method were monitored by flow cytometer during early multiplication stage of the virus. Viral antigen could first be detected at 2 h post infection (p.i.), and the larger numbers of labeled CHSE-214 cells were obtained at 8 and 24 h p.i. in both VR-299 and Sp strains of IPNV examined. The detection method is fast and sensitive and yields quantitative results on single cell basis.

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