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Isolation of Birnavirus from Japanese Pearl Oyster <i>Pinctada fucata</i>

1998; Springer Science+Business Media; Volume: 64; Issue: 2 Linguagem: Inglês

10.2331/fishsci.64.342

1444-2906

Satoru Suzuki, Maki Kamakura, Riichi Kusuda,

Marine Bivalve and Aquaculture Studies

Since 1994, mass mortality of the Japanese pearl oyster Pinctada fucata has widely occurred in the southwestern part of Japan.The cause of mass mortality was investigat ed in terms of change of current, decrease in feed plankton, increase in seawater temperature, genetic fac tors, and water pollution.However, the cause of mortality has not been identified.Virus infection is thought to be one of the causes because it has been reported in the past that herpes virus-like particles1) and birnaviruses2) were found in several bivalves.Pathogenicity of birnavirus against hard clam Meretrix lusoria has been reported.3)The authors recently isolated the birnaviruses from Agemaki (jack knife clam) Sinonovacura consticta which originated in Ariake Bay, Saga Prefecture, Japan, as well as in an area on the Korean coast.4)The strain AGJ-90 that was isolated from the Ariake Bay samples showed patho genicity against Agemaki when the shells were exposed to physiological stress.4)The birnavirus genome was detected in high ratios by PCR, suggesting that the birnavirus is widely distributed in Agemaki.4)Therefore, we investigat ed the existence of viruses in Japanese pearl oyster.In this report, we describe the isolation and detection of birnavi rus from Javanese vearl oyster.Japanese pearl oyster samples were collected in the Uwajima and Uchiumi fishing grounds in Ehime Prefec ture, Japan, in October 30, 1996.Three shells taken from the same group were pooled per sample and 6 different samples were employed for analysis.Mainly liver and geni tal organs were used for the experiment.In order to eliminate virus contamination from seawater or digestive systems, gill, mantle lobe and rectum were not included in a sample.Birnavirus genome was surveyed by using 2-step PCR targeting in the VP2/NS junction region of genome segment A. We previously established this method for de tecting birnavirus genome.5)The cDNA fragment obtained by nested-PCR was directly sequenced by the dideoxy-ter minator method as previously described.4)Virus isolation was performed with cultured CHSE-214 and RSBK-2 cells.Liver and genital organs were pooled and weighed; 9-fold volume of Hanks' balanced salt solution was added and homogenated with a stomacker.The homogenates were filtrated through a 220-nm pore filter and 25 and 50 ,u l were inoculated to the cells cultured in a 24-well culture * Samples are from: Uwajima (#1 , non-symptomatic; #2, symptomatic) and Uchiumi (#3, non-symptomatic; #4, symptomatic; #5, symptomatic; #6, nom symptomatic).Main symptom is a red colouring of mantle cavity and adduc tor muscle.