*2.6. Plaque Assay*

Vero cells were used for the titering experiments for drug-treated and vehicle-treated hNSC cultures infected with ZIKV H/PAN/2016/BEI-259634. Vero cells were seeded at a density of 30,000 cells per well in 96-well plates and incubated in 5% CO2 at 37 ◦C in high-glucose DMEM with 1% FBS and 1% penicillin/streptomycin for 24 h before the addition of the viral inoculum from ZIKV-infected hNSCs. Inocula were collected from ZIKV H/PAN/2016/BEI-259634-infected NSCs (MOI 0.1) in the presence of 10 μM or 30 μM 2- -*C*-methyluridine aryloxyl phosphoramidate ProTide, 10 μM or 30 μM sofosbuvir, mock-infected cultures, infected cultures without compound treatment, and infected cultures with the

DMSO vehicle at 48 h post-infection. Collected inocula were diluted 10-fold in high-glucose DMEM containing 1% FBS and 1% penicillin-streptomycin in 96-well plates. These inocula were added to the pre-prepared Vero cells for 1 h in a volume of 100 μL and at a temperature of 37 ◦C, covered with an agarose overlay, and lastly incubated for 72 h at 37 ◦C and 5% CO2. Fixation with 4% formaldehyde (final concentration) for 24 h was then performed for the samples. Following this, the overlay was removed, and crystal violet staining was performed to count the ZIKV plaques.
