*2.1. Cells and Virus Infection*

A549, HEK293T, Vero and U251 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Waltham, MA, USA) supplemented with 100 U/mL penicillin and streptomycin, 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)(Gibco; Waltham, MA, USA), 2 mM glutamine (Gibco; Waltham, MA, USA), 10% heat-inactivated fetal bovine serum (FBS; Gibco; Waltham, MA, USA) at 37 ◦C in 5% CO2. Primary human fetal astrocytes (HFAs) were prepared as previously described [28] from 15–19 week aborted fetuses with written consent approved under the protocol 1420 by the University of Alberta Human Research Ethics Board (Biomedical). HFAs were grown in Minimum Essential Media (MEM) (1 g/<sup>L</sup> Glucose, 15 mM HEPES, Gibco; Waltham, MA, USA) supplemented with 10% FBS, L-glutamine, MEM non-essential amino acids, sodium pyruvate, and 1 g/mL glucose at 37 ◦C in 5% CO2. PLCal and PRVABC59 strains of ZIKV were kindly provided by Dr. David Safronetz (Public Health Agency of Canada). The Zika virus (strain H/PF/2013, French Polynesia) was kindly provided by Dr. Michael Diamond (Washington University School of Medicine, St. Louis, MO, USA). The Zika virus (strain MR766) was generated from a molecular clone [29] kindly provided by Dr. Matthew J. Evans (Icahn School of Medicine at Mount Sinai, New York, NY, USA). All virus manipulations were

performed according to level-2 containment procedures. Virus stocks were generated in C6/36 cells and titrated by plaque assay using Vero cells.
