FIG 4.

Cytokine induction by first- and third-wave viruses and the role of NS gene segments in virus replication in HAE cells. (a) Human nasal MucilAir cell cultures (HAE; Epithelix) were infected in triplicate with RG A/195 and A/687 virus at an MOI of 1 for 16 h. The basal medium was harvested, and levels of the cytokine IP-10 were measured using mesoscale discovery (MSD) plates. Statistical analysis was performed with an unpaired t test. (b) 293T cells transiently transfected with a beta interferon promoter luciferase reporter plasmid were infected with RG viruses A/195 (dark blue), A/195 with A/687 segment 8 (light green), A/687 (dark green), and A/687 with the A/195 segment 8 (light blue) at an MOI of 3. Infection with NDV was used as a positive control. Statistical analysis entailed a one-way ANOVA, with Tukey's multiple-comparison test (and associated adjusted P values). (c) 293T cells transiently transfected with an beta interferon promoter luciferase reporter and pCAGGs NS1 plasmids A/195 (blue), A/687 (green), and H3N2 (purple). Positive controls were stimulated with NDV. Statistical analysis was performed with a one-way ANOVA and Tukey's multiple-comparison test.