Loss of MDA5 function does not affect RSV replication, whereas affecting RSV-induced IFN-regulated transcripts. (A) Number of RSV reads in RNA-seq data during RSV infection. RSV-infected (MOI: 1) A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg). (B) Immunoblotting showing efficiencies of MDA5 protein expression, after transient transfection of A549 cells with the indicated siRNA, or without transfection (MOCK). Cells were either left uninfected or infected with RSV, as indicated. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C) “Response to type I interferon” genes (GO term GO0034340) versus all genes, from A. (D) Heatmap representation of RNA-seq data showing the expression change between MDA5 siRNA and nonspecific siRNA control of IFN-regulated gene over the course of RSV infection. Mean of triplicate expression ratios from triplicate infections were shown for each time point in A, C, and D. (E) RSV transcripts in primary nasal epithelial cells from the patient (open bar), parents (hatched bars), and two normal healthy controls (solid bars), normalized to father at 6 h, after RSV-GFP infection (MOI: 0.2). (F) Percent GFP⁺ of gated live RSV-infected cells from (E). Data show means ± SD from four experiments (E and F). *, P < 0.05, by Kruskal-Wallis test (C and D); other comparisons were nonsignificant.