2.5. Viral Titration

Serial dilution of the viral stock was performed (10-1 to 10-6). A volume of 0.2 mL of the viral stock dilutions was applied to a monolayer of Vero E6 cells maintained in a 12-well plate. The cells were incubated for 1 h, at 37 °C, in an atmosphere with 5% CO₂. After that incubation period, the virus’s medium was removed, and 1.0 mL of DMEM containing 1.4% carboxymethylcellulose (CMC) and 1% fetal bovine serum (FBS) was added. The cells were incubated at 37 °C, in an atmosphere with 5% CO² for 72 h. Then, 1.0 mL of 10% formaldehyde was applied per well, and cells were incubated at room temperature for at least 3 h. The wells containing the cells were washed with water until the complete removal of the medium. Then 0.5 mL of 1% crystal violet, diluted in 20% methanol, was added. Cells were incubated for 5 to 10 min, at room temperature, followed by washing with water until the removal of the crystal violet excess. The formed plaques were counted, and the calculation was performed to determine the viral titer.