Updated A(H1N1)pdm09 influenza virus ferret infection model permits refined antiviral assessment using aerosol inhalation challenge
4 affiliations
- 1Victorian Infectious Diseases Reference Laboratory, at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- 2Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- 3Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
- 4Section of Anatomic Pathology, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Melbourne, Victoria, Australia
Abstract
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 pre-treatment schedules, which were not apparent with an intranasal challenge model. Author summary: Small mammalian models serve a critical role for assessing the efficacy of existing and novel antiviral drugs against influenza A virus. However, drug efficacy studies in the ferret model frequently use an A(H1N1)pdm09 virus challenge strain that was isolated over 15 years ago, and inoculate animals in a way that does not reflect how humans are typically exposed to influenza virus. In this study, we first examined a panel of contemporary A(H1N1)pdm09 viruses and identified a strain from 2021 that is well-suited to serve as an updated challenge virus for these studies. Using this 2021 strain, we performed a head-to-head comparison of two inoculation routes, and found that the antiviral effects of oseltamivir in ferrets were more apparent early after infection following an aerosol inhalation-based inoculation method compared to the standard intranasal inoculation route. Collectively, these findings support that continued refinement of preclinical mammalian models used for antiviral drug assessments can lead to higher relevance and reliability of future studies aiming to explore anti-influenza interventions.
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BibTeX
@article{stannard2026updated,
title = {{Updated A(H1N1)pdm09 influenza virus ferret infection model permits refined antiviral assessment using aerosol inhalation challenge}},
author = {Harry L. Stannard and Nicole Brock and Xiangjie Sun and Joanna A. Pulit-Penaloza and Troy J. Kieran and Ashwin Muraleetharan and Clyde Dapat and Smitha Rose Georgy and Taronna R. Maines and Ian G. Barr and Patrick C. Reading and Saira Hussain and Jessica A. Belser},
journal = {PLOS Pathogens},
year = {2026},
month = sep,
doi = {10.1371/journal.ppat.1014604},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014604},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Updated A(H1N1)pdm09 influenza virus ferret infection model permits refined antiviral assessment using aerosol inhalation challenge
AU - Harry L. Stannard
AU - Nicole Brock
AU - Xiangjie Sun
AU - Joanna A. Pulit-Penaloza
AU - Troy J. Kieran
AU - Ashwin Muraleetharan
AU - Clyde Dapat
AU - Smitha Rose Georgy
AU - Taronna R. Maines
AU - Ian G. Barr
AU - Patrick C. Reading
AU - Saira Hussain
AU - Jessica A. Belser
PY - 2026
DA - 2026/09/09
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014604
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014604
AB - 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 pre-treatment schedules, which were not apparent with an intranasal challenge model. Author summary: Small mammalian models serve a critical role for assessing the efficacy of existing and novel antiviral drugs against influenza A virus. However, drug efficacy studies in the ferret model frequently use an A(H1N1)pdm09 virus challenge strain that was isolated over 15 years ago, and inoculate animals in a way that does not reflect how humans are typically exposed to influenza virus. In this study, we first examined a panel of contemporary A(H1N1)pdm09 viruses and identified a strain from 2021 that is well-suited to serve as an updated challenge virus for these studies. Using this 2021 strain, we performed a head-to-head comparison of two inoculation routes, and found that the antiviral effects of oseltamivir in ferrets were more apparent early after infection following an aerosol inhalation-based inoculation method compared to the standard intranasal inoculation route. Collectively, these findings support that continued refinement of preclinical mammalian models used for antiviral drug assessments can lead to higher relevance and reliability of future studies aiming to explore anti-influenza interventions.
SN - 1553-7374
ER -