4.3. Plaque Assay and TCID50

All viruses were titrated using CV-1 cells. The virus was released from infected cells through three cycles of freezing and thawing. For the plaque assay, the supernatant was serially diluted 10-fold in serum-free medium and 200 μL of the diluted solution was added to CV-1 cells in 24-well plates. The cells were incubated for two hours, after which the inoculums were replaced with overlay media containing 0.75% carboxy methyl cellulose and incubated at 37 °C for 5–7 days. Subsequently, the cells were fixed using 4% paraformaldehyde. They were then stained with 250 μL of a 1% crystal violet solution containing 20% ethanol for plaque counting. In the TCID₅₀ assay, CV-1 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.1, and samples were collected at 2, 4, 8, 12, 18, 24, and 48 h post-infection. The viral lysates were serially diluted 10-fold in standard serum-free medium, and 100 μL of the diluted solution was added to CV-1 cells seeded in 96-well plates. Eight wells were infected for each dilution and further incubated for 5–7 days. Virus titers were calculated using the Reed–Muench method and expressed as Log10 (TCID₅₀ /mL).