Decoding non-human mammalian adaptive signatures of 2.3.4.4b H5N1 to assess its human adaptive potential

The 2.3.4.4b clade highly pathogenic avian influenza H5N1 infected diverse non-human mammalian species, gained mammal-to-mammal transmission potential, and caused sporadic human infections. However, whether non-human mammals enable the human adaptation of 2.3.4.4b H5N1 to establish human infections is unclear. Gain-of-function research restrictions may hinder the assessment of 2.3.4.4b H5N1 human adaptations. Here, we tracked the evolution of 2.3.4.4b H5N1 that infected non-human mammals and evaluated their ability to gain human adaptations. The non-human mammal 2.3.4.4b H5N1 partly acquired classical human-adapting mutations, which are identical to the residues of H1N1pdm09 and seasonal human H3N2 viruses, while showing a few species-specific adaptations that might be potential barriers for successful human infections. The polymerase complex proteins, PA and PB2, acquired human adaptations in non-human mammals, with fox-infected viruses showing more positive selection in the polymerase complex. The human-adapting Q591K/R substitution in PB2 appeared only in the 2.3.4.4b clade but not in previously circulating H5N1 strains. Despite minimal changes in hemagglutinin (HA), A160T and T199I mutations near the receptor binding site of HA in dairy cattle viruses indicate the rapid HA glycan surface evolution affecting virus entry and immune evasion. The unbiased quantitative assessment of virus adaptations indicated that 2.3.4.4b H5N1 circulating in bears, cattle, dolphins, and foxes might show better human adaptive potential. Thus, 2.3.4.4b H5N1 appears to be acquiring human adaptations due to natural selection pressure in non-human mammals. Overall, our study delineates human adaptation and infection risk of specific non-human mammalian circulating 2.3.4.4b H5N1 strains.IMPORTANCEThe 2.3.4.4b clade H5N1 virus emerged as a panzootic strain, leading to the unprecedented deaths of domestic and wild birds and diverse non-human mammalian species. Intriguingly, the 2.3.4.4b H5N1 transmitted to diverse mammalian species and gained mammal-to-mammal transmission, suggesting its pandemic potential. The H5N1 outbreaks in dairy cattle and sea lions are devastating, and they contributed to sporadic human infections. This indicates the ability of non-human mammal hosts, like dairy cattle, as potential sources for human transmission. However, the signatures of non-human mammal adaptations of 2.3.4.4b H5N1 and how these adaptations drive the human adaptive potential of 2.3.4.4b H5N1 are unclear. In this study, we show the specific molecular patterns of H5N1 proteins that determine species-specific adaptations in non-human mammals. We identified that 2.3.4.4b H5N1 circulating in non-human mammals is rapidly evolving with critical adaptations in PA, PB2, and HA and gaining human adaptive potential in specific non-human mammalian species.