Virus culture
Mice were infected with a genetically modified strain of RSV derived from the RSV wild-type clone MP224, designed to express red-fluorescent protein (RFP) when replicating22,24 (gift from Professors Mark Peeples and Peter Collins, Nationwide Children’s Hospital, Columbus, Ohio, USA and National Institute of Allergy and Infectious Diseases, Bethseda, Maryland, USA respectively). Briefly, virus was added to HeLa monolayers grown in flasks until the cytopathic effect reached approximately 50% (48 h). The remaining media slurry and HeLa monolayer was harvested and centrifuged 500 g. The supernatant was filtered by centrifugation to concentrate viral particles and remove remaining cellular debris. Plaque assay analysis was used to determine the titre of RSV with RSV plaque formation being indicated by fluorescence .