The infectivity of residual virus was titrated in quadruplicate on 96-well microtitre plates containing 100 μL of confluent FRhK-4 cells. 100 μL of serial 10-fold of dilutions of virus in maintenance medium starting from 10−1 to 108 was added into FRhK-4 cells. The infected cells were incubated at 37°C for 4 days. Appearance of CPE was recorded daily. TCID50 was determined according to the Reed and Muench method [19].