*2.4. CellTiter-Glo Activity Assay*

NSCs (5000 cells/well) were seeded into 1536-well black, clear bottom plates containing 20-point, 2-fold serial dilutions of ProTides or DMSO vehicle controls and were either mock-infected, infected with ZIKV H/PAN/2016/BEI-259634 at the multiplicity of infection (MOI) of 10, or infected with ZIKV PRVABC59/Human/2015/Puerto Rico at MOI of 10. Cell viability was measured using the CellTiter-Glo assay (Promega, Madison, WI, USA) 72 h post-infection. An EnVision plate reader (PerkinElmer) was used for readouts of luminescence intensity. All data were normalized to DMSO vehicle controls and were expressed as % relative luminescence intensity. Normalized activity and toxicity data were plotted against ProTide concentration and fit to a sigmoidal dose-response curve with variable slope using Prism 6 (GraphPad Software) to generate EC50 and CC50 curves.

#### *2.5. Bright-Field Microscopy*

GSC 387 cells (5000 cells/well) were seeded into 1536-well black, clear bottom plates with wells containing either 10 or 30 μM sofosbuvir, 10 or 30 μM 2- -*C*-methyluridine aryloxyl phosphoramidate ProTide, or DMSO vehicle controls, and were either mock-infected or infected with ZIKV H/PAN/2016/BEI-259634 at MOI of 10. Cell foci images were acquired 72 h post-infection in bright-field using an ImageXpress Micro automated microscope (Molecular Devices, San Jose, CA, USA).
