Vero and Vero-E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS, Invitrogen). HEK-293T cells were maintained in the same medium supplemented with 1% (v/v) non-essential amino acids. SW-13 cells were maintained in Leibovitz’s L-15 medium (Life Technologies) with 2mM L-glutamine and 10% (v/v) FBS. C6/36 and U4.4 were maintained in Leibovitz’s L-15 medium (Life Technologies) supplemented with 10% FBS (v/v), 1% (v/v) non-essential amino acids and 1% (v/v) tryptose phosphate broth (Sigma). All cell lines were supplemented with 5 units/mL penicillin and 5 μg/mL streptomycin (Life Technologies). Mammalian cell lines were maintained in a humidified atmosphere at 37 °C with 5% CO2. C6/36 and U4.4 cells were maintained at 28 °C without CO2.

The Zika virus strain used in this study is the prototype African MR-766 strain, derived from a previously described infectious clone54. Rescue of WT Zika virus was carried out by transfection of the infectious clone in HEK-293T cells (TransIT-LT1, Mirus Bio) according to the manufacturer’s instructions. 3-4 days post-transfection (p.t.), the cell culture supernatant was clarified by centrifugation. Virus stocks were then prepared in Vero cells, by infecting cells with a multiplicity of infection (MOI) of 0.01 plaque-forming units (PFU)/cell. 5-6 days post-infection (p.i.) the cell culture supernatant was clarified by centrifugation, and virus titers determined by plaque assay in Vero-E6 cells.

The yellow fever virus 17D strain was derived from pACNR/FLYF plasmid containing the full-length infectious YF17D genome under a SP6 promoter55.

West Nile virus (Israeli strain IS-98) was generated from the “two-plasmid” system infectious clone previously described56.

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