2.5. Plaque-Forming and Focus-Forming Assays

The virus titer was determined by either plaque-forming assay (PFA) or focus-forming assay (FFA), depending on the ability of the virus to cause cytopathic effect (CPE). PFAs were performed for Paraiba_01/2015 and ZIKV ic, and FFAs were performed for GFP-50C/FrSh and nLuc-50C/FrSh. Briefly, WHO Vero cells were seeded in 24-well plates to reach 80–100% confluence on the day of the assay. Virus samples were ten-fold serially diluted in MEM supplemented with 1% FBS, and 100 µL of each dilution were used to infect the cells in duplicates. After 1 h incubation at 37 °C and 5% CO₂ with periodic rocking of the plates, cells were overlaid with 0.8% methylcellulose in DMEM containing 2% FBS and 2 mM l-Glutamine and maintained at 37 °C and 5% CO₂ for 4–5 days. Cells were fixed with cold methanol, and either stained with crystal violet for PFA or subjected to immunostaining using Anti-Flavivirus Group Antigen Antibody 4G2 (EMD Millipore, Burlington, MA, USA), goat anti-mouse HRP-conjugated secondary antibody (Jackson Immuno, West Grove, PA, USA) and True Blue peroxidase substrate (KPL, Silver Spring, MD, USA) for FFA.