ZIKV Envelope Protein Quantification

Serial dilutions of the samples were performed in DMEM – 2% FBS. Diluted samples were applied onto 90% confluence Vero cells monolayers for 2 h at 37°C, 5% CO₂. Inocula were removed after viral adsorption and were replaced by a mixed solution of DMEM – 4% FBS and PBS – 1% carboxymethyl cellulose in a 1:1 ratio. 3 days later, cells were fixated in PBS – 4% paraformaldehyde for 15 min at room temperature (RT), and permeabilized with PBS – 0.1% Triton for 3 min at RT. PBS – 1% bovine serum albumin – 0.1% Tween 20 was applied on cells for 30 min at RT in order to block non- specific antigenic sites. ZIKV envelope protein (E) immune-staining was performed using the mouse anti-E [clone 4G2; home-purified from the ATCC hybridoma (Monel et al., 2017 (link))] antibody as primary antibody and the HRP conjugated goat anti-mouse IgG antibody (Biorad) as secondary. Finally, after addition of freshly prepared peroxidase substrate (Vector Vip; Vector Laboratories) for 5–15 min, purple foci of infection were manually counted.