Ferrets

Experiments were performed in a containment level 2 laboratory. Outbred female ferrets (28–32 weeks old) weighing 750–1000 g were used (Highgate Farms). Ferrets were confirmed to be seronegative for influenza virus A virus NP protein by competitive ELISA (ID.vet) [19]. For virus inoculation, ferrets were lightly anesthetized with ketamine (22 mg/kg) and xylazine (0.9 mg/kg).

Ferrets were immunized with 5, 20 or 80 µg of saRNA expressing H1 in a 50-µL volume in the hindleg muscle at 0 and 4 weeks and challenged at 6 weeks after the first immunization. Animals were infected with 10⁶ PFU pandemic H1N1 virus (A/California/07/2009) in PBS (100 µL per nostril).

The effect of vaccine on direct contact transmission was assessed by employing an experimental setup cohousing naïve donor and immunized sentinel ferrets [20]. All 12 direct contact sentinel ferrets were pre-exposed to 10⁶ PFU pandemic H1N1 virus (A/California/07/2009) 4 weeks prior to immunization (week −4). Animals were then immunized with 20 µg saRNA expressing H3N2 or rabies virus glycoprotein as a control antigen in a 50-µL volume at 0 and 6 weeks and exposed to infected donor animals 12 weeks after the first immunization. Naïve donor ferrets were inoculated intranasally with 5 × 10⁵ PFU A/Japan/WRAIR1059P/2008(H3N2) diluted in PBS (100 μL per nostril). On day 1 post inoculation of donor, two immunized direct contact sentinels were co-housed with each donor animal.

In both studies, all animals were nasal-washed daily, while conscious, by instilling 2 mL PBS into the nostrils, and the expectorate was collected in modified 250 mL centrifuge tubes. Ferrets were weighed daily post-infection and body temperature was measured daily via subcutaneous IPTT-300 transponder (Plexx B.V, Netherlands). Bedding litter in the cage was changed daily. At 14 days post infection (DPI), ferrets were injected with a non-reversible anaesthetic consisting of ketamine (≥25 mg/kg) and xylazine (≥5 mg/kg), before sacrifice by intracardiac injection of sodium pentobarbitone (≥1000 mg/kg), at which time blood was collected by cardiac puncture.