Figure 4.

Neutralization and Antiviral Assays. A. Reporter virus neutralization scheme. Vero cells were seeded in an opaque, white 96 well plate at 1.5 × 10⁴ cells per well the day before. Sera samples were two-fold serially diluted, starting at 1:50 and then mixed with equal volume of virus and incubated for 1 h at 37°C. The sera:virus mixture was then used to inoculate Vero cells in a 96 well plate. After 4 h incubation at 37°C, plates were washed twice with PBS and then NanoGlo substrate was added and luciferase levels read by plate reader. B. NT₅₀ values of a panel of mouse sera tested with the stable reporter viruses. For ZIKV and YFV NT₅₀ tests, the C38 virus was used in both instances. C. Antiviral assay scheme. Huh7 cells (1.5 × 10⁴ cells per well) were seeded the previous day. NITD008 was two-fold serially diluted starting at 10 µM. The dilutions were mixed with virus and plated on the Huh7 cells in a white, opaque 96 well plate and incubated at 37°C for 48 h. Following a 3X PBS wash, results were read by plate reader after addition of NanoGlo substrate. D-E. EC₅₀ results from testing NITD008 against the panel of stable reporter viruses in both graphical and table formats, respectively.