Progressive Adaptation of H6N1 Avian Influenza Virus in Taiwan Enhances Mammalian Infectivity, Pathogenicity and Transmissibility

Interspecies transmission of avian influenza viruses remains a significant public health concern. H6 viruses have gained attention following the first human infection by a chicken-origin H6N1 virus (A/Taiwan/02/2013, Hu/13), which highlighted their zoonotic potential. To understand the evolutionary trajectory and mammalian adaptation of this Taiwan lineage, we compared two avian isolates (A/Chicken/Taiwan/CF19/2009, Ck/09; A/Chicken/Taiwan/2267/2012, Ck/12) and Hu/13 in vitro and in vivo. Hu/13 exhibited enhanced replication in MDCK cells, with larger plaques and higher viral titers than Ck/09 and Ck/12. In BALB/c mice, Hu/13 caused high pathogenicity and mortality, while Ck/09 induced minimal morbidity. Hu/13 replicated efficiently in respiratory tissues, eliciting robust cytokine responses and severe pulmonary lesions, with Ck/12 showing intermediate virulence. In ferrets, Hu/13 demonstrated efficient transmission, infecting all direct-contact and one airborne-contact ferret, whereas Ck/09 failed to transmit. Histopathology confirmed escalating lung pathology from Ck/09 to Ck/12 and Hu/13. Whole-genome sequencing identified adaptive mutations in Hu/13 during ferret replication, though no canonical mammalian-adaptive changes (e.g., PB2-E627K or HA-Q226L) were detected. These findings demonstrate progressive mammalian adaptation, replication efficiency, and transmissibility within the Taiwan H6N1 lineage, underscoring its zoonotic risk and emphasizing the need for enhanced surveillance to monitor mammalian-adaptive mutations, informing pandemic preparedness and public health strategies.