Plaque Assay Protocol for Virus Titers

For plaque assays corresponding to titers presented in S1C Fig, plaque assays were carried out as described in Hanley et al. (2010) with the exception that A549 cells were used with overlay medium of F-12K media supplemented with 10% FBS, pen-strep, and 0.8% methylcellulose [58 (link)].To determine virus titer and for the plaque assays presented in S8C Fig, HEp-2 cells were plated in 24 well plates. When cells were confluent, virus was added as an inoculum for 1 h at 37°C. Overlay medium of DMEM, FBS, pen-strep, and avicel (FMC Biopolymer) was then added on top of inoculum, and cells were incubated for 6 days at 37°C before fixation with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature. Cells were then blocked for 30 minutes at 37°C with 5% BSA (Sigma Aldrich). Cells were stained with an antibody specific for RSV F protein for 30 minutes at 37°C, followed by an anti-human horse radish peroxidase secondary antibody (Jackson Immunoresearch, 1:250 dilution) for 30 minutes at 37°C. Finally, cells were incubated with TrueBlue peroxidase substrate (SeraCare) for 10 minutes at RT. Plaques were then counted to determine titer. Biological replicates were performed, where n = 4.