*2.3. Viruses*

Viral strains used in this study were provided through BEI Resources, NIAID, NIH: Zika virus H/PAN/2016/BEI-259634, NR-50210 (GenBank accession number KX198135), Zika virus PRVABC59/Human/2015/Puerto Rico, NR-50240 (GenBank accession number KU501215).

Vero cells were used to expand viral cultures for 2 to 3 serial passages in order to amplify the titers. Centrifugation of infected cell supernatants was performed to remove cell debris, and then viral stocks were concentrated through use of a sucrose cushion. Neural maintenance medium base [50% DMEM–F-12 medium–GlutaMAX, 50% Neurobasal-A medium, 1× N-2 supplement, 1× B-27 supplement (Life Technologies)] supplemented with 1% DMSO (Sigma) and 5% fetal bovine serum (FBS) (Gibco) was used to resuspend concentrated viral stocks, and aliquots were stored at −80 ◦C. Plaque assay on Vero cells was used to determine viral titers, which were greater than or equal to <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>8</sup> PFU/mL.
