2.5. Viral Inhibition Assays

For the post-infection assays, confluent monolayers of Vero or A549 cells (96-plate format, 5.0 × 10⁴ cells/well, quadruplicates) were infected with 25 plaque-forming units (PFU)/well of ZIKV Paraiba/2015 or MR-766 strains, or 50 PFU/well of PRVABC59 strain, for 2 h at 37 °C. The use of 50 PFU/well of PRVABC59 was due to differences of plaque size formation of ZIKV PRVABC59 as compared to ZIKV Paraiba/2015 and MR-766. After viral adsorption, cells were treated with 100 µL of infection media (DMEM supplemented with 2% FBS and 1% PSG) containing 3-fold dilutions (starting concentration of 100 µM) of the different compounds, or 0.1% DMSO vehicle control. For the pre-infection assay, Vero cells (96-plate format, 5.0 × 10⁴ cells/well, quadruplicates) were treated with the same dilutions of the different compounds or 0.1% DMSO vehicle control 12 h before virus infection. For the co-infection assays, viruses were incubated with the compounds or 0.1% DMSO vehicle control and used to infect confluent monolayers of Vero cells (96-plate format, 5.0 × 10⁴ cells/well, quadruplicates). In all cases, after viral absorption, virus inoculum was removed, and cells were washed with infection media before adding fresh infection media containing 1% Avicel (Sigma-Aldrich). Infected cells were incubated at 37 °C for 36 h for ZIKV Paraiba/2015 or 48 h for MR-766 and PRVABC59 strains. After viral infections, cells were fixed for immunostaining with 4% formaldehyde for 1.5 h at room temperature, the overlays removed, and the cells washed three times with 1x PBS. Cells were then permeabilized with 0.4% Triton X-100 in PBS for 15 min at room temperature. Then, plates were blocked with 2.5% bovine serum albumin (BSA) in PBS (blocking solution) for 1 h at 4 °C, followed by incubation with 1 µg/mL of the pan-flavivirus E protein monoclonal antibody (mAb) 4G2 (BEI resources; NR-50327, Manassas, VA, USA). Viral plaques were visualized using Vectastain ABC kit and DAB Peroxidase Substrate kit (Vector Laboratories Inc., Burlingame, CA, USA), following the manufacturer’s instructions. Stained plaques were analyzed using the CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH, USA). Individual wells from three independent experiments conducted in quadruplicates were used to calculate the average and standard deviation (SD) of viral inhibition using Microsoft Excel software. Virus titers were calculated as plaque-forming units (PFU)/mL. Non-linear regression curves and the half maximal effective concentration (EC₅₀) were determined (GraphPad Software, San Diego, CA, USA, Version 8.2.1).