ZIKV attachment and entry assay

A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were precooled at 4°C prior to the addition of ice-cold virus inoculum. Inocula were allowed to adsorb to cells at 4°C for 1 h. Inocula were then removed, and cells were washed with ice-cold 1× PBS before fresh prewarmed cDMEM was added to cells. Cells were returned to 37°C until the appropriate time points to evaluate viral entry, genome replication, and virion production. For viral attachment assays, intracellular viral RNA was harvested using the Qiagen RNeasy kit with DNase I digestion following ice-cold 1× PBS washes.