Loss of MDA5 function does not affect influenza virus replication, or influenza-induced IFN production or cytotoxicity, in respiratory epithelial cells or fibroblasts. (A) Influenza transcripts, normalized to father control at 24 h. Primary nasal epithelial cells from the patient, parents, and two normal healthy controls were infected with influenza (MOI: 0.02). (B) Influenza virus quantitated by infectious plaque assay, after infection (MOI: 1) of SV40-transformed fibroblasts having the indicated genotypes: empty squares, IFIH1^−/−; empty inverted triangles, IFIH1^K365E/-; solid triangles, IFIH1^K365E/WT; empty circles, STAT1^−/−; solid circles or solid squares, healthy controls. (C) IFN-β released into supernatants as measured by ELISA after infection with influenza strain A/Puerto Rico/8/1934 (MOI: 0.37–54). Decreasing MOI of influenza virus correspond to the bars proceeding from left to right for each cell line. Sendai Virus (SeV) was included as positive control of IFN induction as the right-most bar for each cell line. Genotypes of SV40-transformed fibroblasts are as indicated, with each cell line separated by vertical dotted lines. (D) LDH released into samples from C. Data show means ± SD from four experiments (A) and three independent experiments (B) that are representative of 11 experiments with varying MOIs (0.1–30) and three different influenza strains (A/Netherlands/602/2009, A/California/4/2009, and A/Puerto Rico/8/1934), and three independent experiments (C and D) that are representative of 6 and 7 experiments, respectively, in which the MOI were varied.