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Highly pathogenic avian influenza (HPAI) H5 viruses, of the A/goose/Guangdong/1/1996 lineage, have exhibited substantial geographic spread worldwide since the first detection of H5N1 virus in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health and can cause lethal human disease following virus exposure. Here, HPAI H5N6 and H5N2 viruses (isolated between 2014 and 2017) of the 2.3.4.4 clade were assessed for their capacity to replicate in human respiratory tract cells, and to cause disease and transmit in the ferret model. All H5N6 viruses possessed increased virulence in ferrets compared to the H5N2 virus; however, pathogenicity profiles varied among the H5N6 viruses tested, from mild infection with sporadic virus dissemination beyond the respiratory tract, to severe disease with fatal outcome. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not with the H5N2 virus. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the identification of mammalian adaptation markers in key genes associated with pathogenesis supports these findings.
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