Vero 76 cells

A placental-derived, Asian lineage ZIKV strain R103451 (Zika Virus, R103451, NR-50355, BEI Resources, NIAID, NIH, Manassas, VA, USA) was passaged once and expanded upon acquisition in Vero-76 cells (CRL-1587, ATCC, Manassas, VA, USA) and stored in sheep serum at −80 °C. Virus was quantitated by PFU assay, in which ZIKV was mixed as eight 10-fold serial dilutions in commercial cell culture media (Modified Eagle’s Media, MEM, Gibco), inoculated into duplicate wells of a 12-well plate containing 95% confluent Vero-76 cells, and incubated at 37 °C with 5% CO₂ for 1 h. The inoculum was removed and 1 mL of 0.05% methylcellulose overlay was added to each well. After incubation for 72 h, wells were rinsed and stained with 0.1% Coomassie blue (Thermo Fisher Scientific, Waltham, MA, USA) in 50% methanol, 43% ethanol, and 7% acetic acid for visualization. Plaques were counted and the PFU was calculated as previously described [52 (link)].

The Asian lineage ZIKV strain R103451 (NR-50355, BEI Resources, NIAID, NIH, Manassas, VA, USA) was passaged once and expanded upon acquisition in Vero-76 cells (CRL-1587, ATCC, Manassas, VA, USA) and stored in sheep serum at −80 °C. Virus was quantitated by plaque forming unit (PFU) assay, in which ZIKV mixed as eight 10-fold serial dilutions in commercial cell culture media (Modified Eagle’s Media, MEM, Gibco-Thermo Fisher Scientific, Gaithersburg, MD, USA), inoculated into duplicate wells of a 12-well tissue culture plate containing 95% confluent Vero-76 cells and incubated at 37 °C with 5% CO₂ for 1 h. Inoculum was removed and 1 mL of 0.05% methylcellulose overlay was added to each well. After incubation for 72 h, wells were rinsed and stained with 0.1% Coomassie blue (Thermo Fisher Scientific, Waltham, MA, USA) in 50% methanol, 43% ethanol, and 7% acetic acid for visualization. Plaques were counted and the PFU was calculated as previously described [50 (link)].