(A) Schematic of RSV A2 or RSV-GFP infection (1 × 10⁶ pfu) of hPCLSs prepared from donor lungs of infants and adults (n = 3 donors) followed by staining and confocal imaging at 2 dpi. (B) Representative images of RSV-GFP (green) and RFX3 (red) staining in infant hPCLSs. Left panel shows an RSV infected airway on hPCLS. Arrow indicates an airway infected by RSV. Dotted lines mark basement membrane. Blue color is nucleus staining and the overlay of red, green, and blue shows as pink color. (C) Representative images of RSV F (green) and RFX3 (red) staining in infant and adult hPCLSs. Arrows indicate RSV-infected cells. Dotted lines mark basement membrane. Blue color is nucleus staining and the overlay of red, green, and blue shows as pink color. (D) Schematic of RSV infection of differentiated bronchial epithelium in day 21 (D21) ALI cultures of neonatal and adult TA BSCs. RSV strain A2 was applied apically (MOI 2, 4 × 10⁵ PFU) for 1 hour. Assays (E–J) were performed at 2 and 4 dpi. (E) Representative cross-section images of antibody staining for major bronchial epithelial cell types prior to infection. Blue color is nucleus staining and the overlay of red, green, and blue shows as pink color. (F) Representative top views of double staining for RSV F(green) protein and ZO1 (red). (G) Relative abundances of RSV F⁺ cells and relative levels of RSV L gene by RT-qPCR. (H) Assays of RSV binding at 4°C for 1 hour followed by quantification of relative RSV L gene levels by RT-qPCR. (I) Assays of primary RSV transcript and genome replication after infection at 37°C for 6 hours by RT-qPCR. (J) Assays of JNJ-678 (100 nM) treatment at 6 hpi followed by quantification of relative RSV L gene levels by RT-qPCR at 2 dpi. Each dot represents 1 donor. Bar graphs represent mean ± SEM. Statistical significance was calculated by 2-way ANOVA followed by Dunn’s test in G and by 2-tailed Student’s t test in H–J. Dotted lines mark basement membrane. **P < 0.01, ***P < 0.001. Scale bars: 50 μm.