The mechanistic basis of ZIKV-116 neutralization. (A) Pre-/post-attachment inhibition assay. Serial dilutions of mAb were added to the Vero cells before (pre, black line) or after (post, red line) virus adsorption to the cells, and infection was determined by FFA. Wells containing mAb were compared with wells containing no mAb to determine the relative infection. (B) Attachment blockade assay. ZIKV (H/PF/2013) was incubated with ZIKV-116 or isotype control (human IgG1) for 1 h, followed by addition to pre-chilled Vero cells for 1 h. Bound ZIKV RNA was measured by qRT-PCR. GAPDH was used for an internal control, and viral RNA fold change was compared with control cells incubated in the absence of mAb. Data are the mean of three independent experiments performed in duplicate or quadruplicate. Error bars indicate SD. (C and D) Fusion blockade assay. Pre-chilled Vero cells were incubated with ZIKV (multiplicity of infection of 80) for 1 h on ice. Subsequently, 100 µg/ml of ZIKV-116 IgG, control ZV-2 IgG (nonneutralizing anti-ZIKV mAb), or medium was added for 1 h on ice, and the pH was shifted to 5.8 to trigger virus fusion with plasma membrane. After pH normalization, cells were incubated at 37°C for 24 h, and the number of infected cells was determined by flow cytometry. 25 mM NH₄Cl was included in the medium throughout the procedure. Representative flow cytometric histogram (C). The data averaged from two independent experiments are shown as means with SD (D). (E and F) Representative BLI profile of ZIKV-116 to recombinant H/PF/2013 DIII at neutral (pH 7.4) and acidic pH (pH 5.5). The experimental curves (black lines) were fitted using a 1:1 Langmuir binding analysis (red lines), and the results are representative of three independent experiments. Statistical significance was determined by two-tailed t test (D) or two-way ANOVA with Sidak's post-test (B). ****, P < 0.0001.