2.8. Focus Forming Assay (FFA)

Supernatants from infected cultures were analyzed by FFA, performed as previously described [18,33] with some modifications. Semi-solid medium (MEM with final concentrations of carboxymethylcellulose 0.75%, FBS 2% and PS 1%) was added 2 h post-infection. The fixation process was performed 72 hpi with paraformaldehyde 1% overnight at 4 °C. Cell monolayers were washed 3× with PBS 1×, and blocked with 5% milk in PBS 1x (blocking solution) at room temperature for 30 min. Cells were permeabilized with a perm wash buffer (PBS 1×, BSA 0.1%, Triton^® X-100 0.1%) by washing 3×. Then, Oropouche immune ascitic fluid was used (1:1000 in blocking solution) as the primary antibody. Antimouse IgG (whole molecule)–Peroxidase antibody was added (1:2000 in blocking solution). Foci were developed with TrueBlue Peroxidase Substrate and counted visually. All determinations were performed in triplicate.