Separation Of Rna Viruses In A Class Of Aquatic Research, And Clams Cell Pathological Changes

Since last decade, culture of the hard clam Meretrix meretrix Linnaeus is economically important industry in JiangSu province. Mass mortalities at clam farms which occur every summer and autumn, have caused serious losses to the industry. After we studied the epidemiology and histopathology, abundant spherical-shaped virus particles were found in the moribund hard clams.Electron microscopic examinations of ultrathin sections reveal the presence of spherical virus particles in the intercellular substance and cytoplasm of the gill, digestive diverticula, mantle, and digestive tract of the moribund hard clam Meretrix meretrix Linnaeus. And the virions are approximately 45 to 55 nm in diameter , having no envelope, no ambit between the core and nucleocapsid. In the infected cells with the virus, the cellular organelles presented obvious pathological changes: the endoplasmic reticulum swelled and mostly turn into vesicles, the nuclear chromatin condensed, the mitochondira swelled and dissolved , the muscle fiber was disarray, the number of lysosome arised , and so on.The virus was purified by differential centrifugation, potassium tartrate cushion ultracentrifugation, CsCl density gradient centrifugation. We also compared the sucrose, potassium tartrate-glycerol, CsCl density gradient ultracentrifugation, and the result showed that the CsCl density gradient ultracentrifugation is suitable for virus purification. With the negative staining, the virions are isometric symmetry, and approximately 60 to 75 nm in diameter. And there are many obvious capsomers on the surface of the virions. The virions having buoyant density of 1.554 to 1.582g/ml in the isopycnic CsCl gradient by the metage mothod.To ensure the virus systematic status, two step (RT)-PCR was used in the study. The first step is a reverse transcription (RT) using the M-MLV Rtase cDNA Kit (TaKaRa). The second step is PCR using two Primer (PrA and PrB) which reported by Blake (1995) for detection the aquatic Birnavirus. The results showed that PrA did not identify the 1180-bp fragment,but the PrB identified the 524bp fragment. So we suggested the spherical-shaped virus could be the Aquatic Birna-Like virus.The infetious virus, which was isolated from the moribund hard clam Meretrix meretrix Linnaeus in the outbreak area, were used to reinfect the cultured hard clam Meretrix meretrix Linnaeus. The artificial infection experimental results showed that the mortality of the hard clam both reached 100% after virus injection and virus soak. The mortality of the hard clam in the control was only 10%. Our preliminary suggestion is that the aquatic Birna-like virus could be the pathogen of the massive death.