2.4. Virus Replication Inhibition Assay

Confluent A549 or MDBK cells in 24-well plates were pretreated with either DMSO control or indicated inhibitors (Gefitinib, U73122, and LY294002) for 2 h before infection. Cells were then infected with BoHV-1 (MOI of 1) in the presence of the DMSO control or indicated inhibitors at the concentrations specified. After inoculation for 2 h, the cells were washed three times using PBS, and fresh DMEM medium containing either DMSO control or indicated inhibitors was added. After infection for the time length indicated, the cell cultures were collected, and viral titers were determined in MDBK cells. The results were expressed as TCID₅₀/mL calculated using the Reed–Muench method.