Multistep growth curves of influenza A virus.

MDCK or A549 cells (2 × 10⁵) were seeded in 24-well plates and inoculated the next day with 50 TCID₅₀ (MDCK) or 10⁵ TCID₅₀ (A549) of IAV. MDCK cells were washed once with phosphate-buffered saline (PBS) before the inoculum was added into MEM containing penicillin, streptomycin, l-glutamine, and vitamins plus 0.1% bovine serum albumin (M0.1B) for 1 h at 37°C. After 1 h, the cells were washed once with PBS and 1.0 ml of M0.1B with 1 μg/ml TPCK-trypsin was added to each well. A549 cells were washed once in DMEM containing penicillin, streptomycin, l-glutamine, and vitamins plus 0.1% bovine serum albumin and NEAA (D0.1B) prior to inoculation with virus diluted in D0.1B. After 1 h at 37°C, the A549 cells were washed once with D0.1B and 1.0 ml of D0.1B with 0.5 μg/ml TPCK-trypsin was added to each well. Culture supernatants from either cell type were collected at 24 and 48 h postinfection (hpi), and the amount of infectious virus was quantified by titration on MDCK cells. The results are the averages of two to three experiments, each performed in duplicate.