9 mg/kg) and xylazine

(3 6 mg/kg) then inoculated intrana

9 mg/kg) and xylazine

(3.6 mg/kg) then inoculated intranasally with 500 μl (250 μl per nostril) PFI-2 ic50 of 100 TCID50 2009 influenza virus A/California/04/09 (A/Cal; H1N1). Solutions were prepared on the day of challenge and the titre of the virus confirmed by infectivity assay. Control groups were infected with virus or inoculated with saline. Rectal temperatures were measured daily. Ferrets were monitored twice-daily post-challenge throughout the course of the study for clinical signs of influenza infection (lack of activity, sneezing, nasal discharge, lack of appetite, weight loss and pyrexia). Clinical signs were scored as follows: loss of activity scored 0 for normal activity levels, 1 for reduced activity, and 2 if inactive; nasal discharge scored 0 for no discharge and 1 for a discharge; sneezing scored 0 for no sneezing, and 1 for sneezing; appetite was scored 0 for no loss of appetite, and 1 for loss of appetite. Nasal washes were collected from each ferret following ketamine and xylazine sedation (as above) at days 1–6 and then at days 8, 10 12 and 14 post-challenge. For each nasal wash, 2 ml of PBS were instilled by small multiple volumes into each nasal cavity with expectorate collected into a beaker. The study was terminated at 14 days post-challenge. 244 DI RNA was generated spontaneously during the transfection of 293T cells with plasmids to

make infectious GW-572016 mouse influenza A/PR/8/34 (Dimmock et al., 2008 and Subbarao et al., 2003). The haemagglutinin (HA) protein of the original 244/PR8 virus had a preference for cell receptors comprising α2,3-linked sialyl receptor sequences, so we reconstructed 244 DI virus with the HA protein of a PR8 virus that binds to both α2,6- and α2,3-linked sialyl receptors (Meng et al., 2010), so that DI RNA would be delivered to cells bearing both types of receptor, and thus protect against

infectious viruses which recognise either type of receptor as described previously (Meng et al., 2010). The resulting mixture of 244/PR8 DI virus and infectious helper A/PR8 virus was purified by pelleting through sucrose. Stocks were resuspended in PBS, standardized by haemagglutination SPTLC1 titration, and stored in liquid nitrogen. All DI virus stocks were tested for their ability to protect mice as described previously (Dimmock et al., 2008) prior to their use in ferrets (data not shown). Before inoculation into animals, helper virus infectivity was eliminated with a short burst (50 s) of UV irradiation at 253.7 nm (0.64 mW/cm2). This is referred to as ‘active DI virus’. The UV inactivation target is viral RNA, and UV has little effect on the DI RNA because of its small target size, 395 nt compared with 13,600 nt for infectious virus. The absence of infectivity after UV-irradiation was checked by infectivity assay (see Section 2.4) and by intranasal inoculation into mice (Dimmock et al., 2008).

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