Lung tissue analysis for RSV titre
A portion of lung tissue was snap frozen in liquid nitrogen for analysis of viral load by real-time quantitative PCR (qPCR). RNA lysis buffer was added to frozen lung tissue and homogenised using a microfuge pestle on ice. Samples were centrifuged at 11 000 rpm for 5 mins and total RNA extracted from resulting supernatants using Purelink RNA extraction kit (Ambion, New Haven, USA). A minimum of 2 mg used as a template for cDNA synthesis using random primers and M-MLV reverse transcriptase (Promega, Southampton, UK). The presence of RSV was measured using a commercially available TaqMan real-time PCR kit designed to determine RSV titre by interpolation to a standard curve (RSVSpp, GeneSig, Southampton, UK). The PCR reaction was analysed using Applied Biosystems ABI 5400 thermocycler.