Focus-forming assays (FFAs) were performed with Vero cells as described previously (Govero et al., 2016 (link)). Supernatant samples containing ZIKV were serially diluted and added to Vero cell monolayers in 96-well plates. The virus was allowed to infect for 2–4 h, and then 100 µl of a 1:1 solution of 2× DMEM with 8% FBS and 2% methylcellulose was added to cells. Plates were incubated for 48 h and then fixed by the addition of 2% paraformaldehyde (PFA). Cells were then incubated with 500 ng/ml of the flavivirus cross-reactive mouse monoclonal antibody E60 (Oliphant et al., 2006 (link)) for 2 h at room temperature. After incubation for 1 h with a 1:5,000 dilution of horseradish peroxidase (HRP)–conjugated goat anti–mouse IgG (Sigma-Aldrich), foci were detected by addition of TrueBlue substrate (KPL). Foci were analyzed with a CTL Immunospot instrument.