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 .