In vitro characterization of the recombinant viruses encoding different-length NS1 protein.

(A) A549 or PK-15 cells were infected at an MOI of 0.001 TCID50 per cell. After washed with PBS, fresh medium containing 0.1 µg/ml TPCK-trypsin was added. Supernatants were sampled at 12, 24, 36 and 48 h p.i. and virus titers were determined as log10TCID50/ml in MDCK cells. The statistical analysis was performed using Student's t test. (B) Virus plaque assay was performed by inoculating confluent MDCK cells in 6-well plates with appropriate dilutions of viruses. After washed with PBS, cells were overlaid with DMEM (without phenol red)-0.8% agarose mixture containing 1% bovine serum albumin and 1 µg/ml TPCK-trypsin. After incubated at 37°C for 4 days, a second agar overlay containing 1∶10,000 neutral red was added.