FFU assay for determining the titers of ZIKV stocks

A549 cells were seeded in 96-well plates at a density of 2 × 104 cells per well. The following day, virus stocks were serially diluted in DMEM containing 2% FBS (2% DMEM), and cells were inoculated with dilutions in triplicate at 37°C for 2 h. After the incubation, cells were overlaid with DMEM supplemented with 1% carboxymethylcellulose (Fisher Scientific), 2% FBS, 10 mM HEPES, and 1× penicillin/streptomycin. At 48 hpi, the methylcellulose overlay and medium were removed, and cells were fixed with 4% paraformaldehyde (Fisher Scientific) for 20 min at room temperature. Following fixation, cells were washed with 1× PBS, and anti-ZIKV envelope antibody, ZV-13, diluted 1:250 in permeabilization/wash/block buffer (2.5% NGS, 2.5% normal donkey serum [Sigma-Aldrich], 0.2% BSA, and 0.1% Triton X-100 [Fisher Scientific] in 1× PBS) was added. Primary antibody was incubated for 2 h with rocking at room temperature. Plates were then washed, and donkey anti-mouse HRP secondary antibody (Jackson ImmunoResearch) diluted 1:3,000 in permeabilization/wash/block buffer was added and incubated with rocking for 1 h in the dark. Following washes, TrueBlue peroxidase substrate (VWR, Radnor, PA, USA) was added, and the mixture was incubated until foci of infected cells were observed. Distilled water (dH2O) was added to stop the enzymatic reaction and removed prior to imaging. Foci of infected cells were detected via a BioSpot plate reader (Cellular Technology Limited, Cleveland, OH, USA) and manually counted using the multipoint tool in Fiji (version 2.1.0, NIH, USA) to determine viral titers (113).