kidney attc ccl22 which had been subcultured previously was trypsinized and spread on a plate followed by counting the cell colonies formed then equal amount of dmem dulbecco 39 s modified eagle 39 s medium and ham 39 s f 12 were mixed and 10 fetal bovine serum was added to the resulting mixture to prepare a tissue culture medium the mdbk cells were inoculated to the medium at a concentration of 6 u00d7 10 4 cells ml and then the resulting cell solution was added to a 96 well flat bottomed plate in an amount of 100 u03bcl per well meanwhile bvdv atcc vr534 which had been stored in a u2212 80 u00b0 c freezer at a pre determined concentration was transferred to a biological safety cabinet and allowed to thaw at room temperature the virus was diluted with the tissue culture medium in an sufficient amount for the virus in each well to induce 85 to 95 cell death 6 days after the virus infection specified pyrimidinedione derivatives samjin pharmaceuticals were prepared as test compounds and a conventional drug for treating hepatitis c ribavirin sigma was used as a comparative drug each of the test compounds was dissolved in dmso to prepare a sample.