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

The recent 2.3.4.4b clade highly pathogenic avian influenza H5N1 infected diverse non-human mammalian species globally, 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 preclude assessing human adapting mutations of 2.3.4.4b H5N1. 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 diverse non-human mammals. The human adapting Q591 mutation 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 show higher human adaptive potential than other hosts. 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.