Assays

A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were uninfected, mock-infected, or infected with ZIKV variants the next day at an MOI of 5 FFU/cell, and virus inocula prepared in DMEM were allowed to adsorb at 37°C for 2 h with rocking. Uninfected samples are seeded cells that remained untouched throughout the infection time course, whereas mock-infected samples were inoculated with DMEM. Following adsorption, the inocula were removed, and cells were washed ...

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 ...

A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. Extracellular RNA was extracted from the clarified supernatants using the QIAmp viral RNA minikit to quantify extracellular viral copy numbers. Cells were washed with ice-cold stringent wash buffer (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) for 3 min to remove cell surface-associated viruses. After ice-cold 1× PBS washes, ...

A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. The clarified supernatants were used for plaque assays to quantify extracellular virions. Cells were washed with ice-cold stringent wash buffer as described above and then detached with 0.05% trypsin-EDTA (Fisher Scientific). Cells were resuspended in 1 mL cDMEM and centrifuged at 300 × g for 5 min at 4°C. Cell pellets were ...

Vero cells were seeded at a density of 1.85 × 105 cells per well in 6-well plates. The next day, supernatants from infected samples were serially diluted in DMEM containing 1% FBS. Vero cells were incubated with dilutions in duplicate at 37°C for 1 h with rocking. Following incubation, cells were overlaid with 1% low-melt agarose (Fisher Scientific), 5% sodium bicarbonate (Fisher Scientific), and 5% FBS in 2× DMEM (Fisher Scientific). Plaques were visualized 4 to 5 days later with a 1% low-melt ...

Cells were washed with 1× PBS and lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing freshly added phosphatase inhibitors (1:100; VWR), protease inhibitors (1:100; Sigma-Aldrich), and 250 nM okadaic acid (1:1,000; Fisher Scientific). Cell lysates were collected and stored at −80°C. Lysates were subsequently thawed on ice and sonicated in a water bath containing ice slurry for three 30-s bursts on ...