IMPAIRED NEURAMINIDASE AND POLYMERASE ACTIVITIES CORRESPOND WITH LIMITED AEROSOL INFECTIVITY OF B3.13 AND D1.1 H5N1 LINEAGES IN HUMAN RESPIRATORY CULTURES
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The ongoing panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses has reached a critical point, marked by unprecedented mammalian spillover and sustained outbreaks in U.S. dairy cattle. While these viruses remain highly lethal in traditional ferret models, human infections—primarily linked to the B3.13 and D1.1 lineages—have been notably mild, typically presenting as conjunctivitis with minimal respiratory involvement. In this study, we address this disconnect by evaluating the infectivity of recent H5N1 isolates using a physiologically relevant air-liquid interface (ALI) culture system that incorporates an aerosol settling chamber. We demonstrate that while direct liquid inoculation leads to efficient replication, aerosolized H5N1 strains exhibit a significant defect in their ability to infect human respiratory epithelium. In contrast, a prototypic H5N1 virus remains highly pathogenic and lethal in ferrets regardless of the inoculation route, showing systemic dissemination to the brain and other organs. Our findings identify two primary viral determinants driving this respiratory restriction: reduced neuraminidase (NA) enzymatic activity and impaired polymerase activity.
Collectively, these results suggest that commonly used mammalian models may overstate current human pandemic risk. This work highlights the critical need for alternative risk- assessment platforms to identify the specific genetic shifts required for these viruses to overcome existing barriers to human adaptation.
IMPORTANCE
Current pandemic risk assessments rely heavily on animal models, particularly ferrets, to evaluate the threat posed by emerging influenza viruses. However, recent H5N1 viruses that have caused predominantly mild human infections have remained highly virulent in these models, creating uncertainty about how well they predict human disease. Using a physiologically relevant human airway model infected through aerosol exposure, we demonstrate that contemporary H5N1 are markedly restricted in their ability to establish respiratory infection despite retaining high virulence in ferrets. These findings highlight the importance of incorporating human airway aerosol models into pandemic risk assessment frameworks to better evaluate the human adaptation and respiratory infection potential of emerging influenza viruses.
