Quantification of extracellular and intracellular virions

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 resuspended in 0.5 to 1 mL cDMEM and stored at −80°C. To quantify intracellular virions, cell pellets were thawed at 37°C to lyse cells, and cellular debris removed by centrifuging at 3,200 × g for 5 min at 4°C. The clarified supernatants were used for plaque assays.