Updated A(H1N1)pdm09 influenza virus ferret infection model permits refined antiviral assessment using aerosol inhalation challenge

Seasonal influenza viruses continue to pose a significant threat to human health. As influenza viruses exhibit sustained genetic drift, it is imperative that animal studies utilize challenge strains that reflect contemporary, currently circulating viruses when evaluating pathogenicity, viral tropism, transmissibility, and antiviral sensitivity to better inform public health responses. Ferrets are considered the gold-standard small animal model for assessing currently circulating influenza viruses. Seasonal influenza A(H1N1)pdm09 viruses replicate well in both the upper and lower respiratory tract of ferrets, providing an important model for developing improved vaccination and therapeutic strategies; however, many of these studies have relied on a 2009 virus isolate. Utilising representative influenza A(H1N1)pdm09 virus strains from 2009 to 2022, we explored virus replication kinetics and lung pathogenesis in ferrets following intranasal inoculation with these contemporary strains. Our results revealed strain specific differences, with greater lung viral loads and pathogenesis following inoculation with A/Sydney/5/2021 compared to other strains. Efficient transmissibility of A/Sydney/5/2021 virus to naïve recipients was also observed following both contact and airborne exposure to infected donor ferrets. To refine this updated model, we performed side-by-side evaluation of oseltamivir antiviral efficacy following traditional intranasal or aerosol inhalation influenza challenges. Pre-treatment with oseltamivir demonstrated greater reductions in viral shedding from the upper respiratory tract than post-infection treatment of ferrets infected by aerosol inhalation, while the intranasal route showed reduced oseltamivir efficacy independent of the timing of antiviral treatment. These findings provide the basis for using an updated A(H1N1)pdm09 challenge virus for ferret studies as an alternative to the commonly used, but now less relevant 2009 early pandemic viruses. It also highlights how different methods of virus inoculation can influence outcomes of ferret antiviral studies, with an aerosol challenge model able to demonstrate differences between therapeutic and prophylactic treatments, which were not apparent with an intranasal challenge model.