Virus infectivity was determined by serial dilution of the sample on BHK-21 cells grown in 96-well plates and the virus titer was calculated as a tissue culture infective dose by the Reed-Muench method [38] . A viral suspension titrated at 10 5 to 10 7 TCID 50 per 0.1 mL was used for viral challenge. BHK-21 cells were seeded in 96-well plates in DMEM 24 h before transfection. The cells (about 95% confluent) were transiently transfected with 0.1 lg of a single plasmid expressing 3D, VP4 or 2B specific siRNA. Five hours after transfection, the transfection complex was removed, cells were washed twice with DMEM, and 100 TCID 50 of FMDV per 0.1 mL was added to each well. After 1 h of adsorption, the inoculum was removed and cells were washed twice with DMEM. The infection then proceeded in DMEM supplemented with 10% fetal bovine serum. Cell supernatants were collected at designated time points, and the virus titers were determined three times on BHK-21 cells. Statistical analysis was performed with the Microsoft Excel program (Microsoft, Redmond, WA, USA).