Plaque-forming units (PFU) assay

The PFU assay was performed according to a previously described protocol [47] with some modifications. Twenty-four-well plates containing Vero cell monolayers were inoculated with 0.3 mL of each ten-fold dilution of samples in DMEM without FBS, and placed for 1 hour in a homogenizing platform for viral adsorption. Subsequently, the inocula were removed, and 1 mL of a semi-solid medium (1% w/v carboxymethylcellulose in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin solution) was added. After 72 h, the overlay medium was removed, and the cell monolayers were fixed for 1 hour with 10% w/v paraformaldehyde, and stained with a 1% w/v methylene blue solution. At the end of the experiment, viral lysis plaques were counted, the average quantities observed in replicates were calculated, and the titers were expressed as plaque-forming units per mL.