Flow cytometry analysis
Lung tissue was harvested at days 0, 3, 5, 7 and 21 days post-infection (dpi). These were prepared as single-cell preparations suitable for flow cytometry analysis by collagenase and DNase digest as described25. Selected cell subpopulations were identified using an optimised panel of fluorescently conjugated monoclonal antibodies (Biolegend, BD Pharmingen) directed against cell-surface antigen markers (including I-A/I-E, F480, Ly6G/C, B220, CD11c, CD103) as described in supplementary Figure S1. A pooled cell preparation for each biological replicate was used to assess background fluorescence (non-stained control) and non-specific staining (fluorescence minus one control). Data from all stained and non-stained tissues was acquired on a BD FACS Canto (Becton Dickinson, New Jersey, USA), with RFP signal from fluorescently tagged RSV also collected. Cell subpopulation analysis and RSV co-localisation was completed used FlowJo software (Tree star, Ashland, OR), with gating strategies presented in Figure S1.