Tissue Culture Infectious Dose 50 (TCID50) assay

TCID50 assay was conducted to determine infectious virus titre using a method that has been previously described [35–37]. For each determination, a 96-well plate culture of confluent BHK-2 was inoculated with a 150 μl mixture of 100 μl medium and 50 μl of serial 10-fold dilutions of clarified cell culture supernatant media. Each row included 6 wells of the same dilution and two negative controls. Plates were incubated at at 37°C and 5% CO₂ for 5 days. Each well was observed for the presence or absence of cytopathic effect (CPE). The determination of TCID50/ml was performed using the Reed and Muench method [38].