The mammalian-adaptive PB2-E627K substitution preserves viral fitness of clade 2.3.4.4b H5N1 HPAIV in birds.

Billington, E.; Riccio, S.; Brown, M. D.; Mollett, B.; Quantrill, J. L.; Johnson, S.; Di Genova, C.; Yang, J.; Sadeyen, J.-R.; Schlachter, A.-L.; Roper, K. J.; Hassan, J.; Clifton, B.; Ralh, K.; Warren, C. J.; Ward, G.; De Silva, D.; Nunez, A.; Coombes, H. A.; Slomka, M. J.; Iqbal, M.; Barclay, W. S.; Peacock, T.; Banyard, A. C.; James, J.

doi:10.64898/2026.10.06.757006

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bioRxiv · 10.64898/2026.10.06.757006

The mammalian-adaptive PB2-E627K substitution preserves viral fitness of clade 2.3.4.4b H5N1 HPAIV in birds.

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Abstract

Since emerging in 2020, clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has disseminated globally, causing substantial infection and mortality in wild birds and poultry. Additionally, numerous spill-over events into mammals have occurred, including mass mortalities and sporadic human infections. Such events can drive the acquisition of mammalian-adaptive mutations, which further increase the risk to humans; the most well characterised mutation is the glutamate to lysine change at amino acid position 627 in the polymerase basic 2 protein (PB2-627K). Across avian influenza viruses, PB2-627K enhances polymerase activity and replication in mammalian cells, increasing zoonotic and pandemic potential. However, bioinformatic analyses indicate that PB2-627K remains rare among clade 2.3.4.4b H5N1 viruses circulating in avian populations. Here, we investigated the impact of PB2-627K on viral fitness in avian hosts to assess the potential for mammalian-adapted viruses to re-establish in birds, mimicking infection of avian species following scavenging on mammalian species where PB2-627K has already emerged. Using a representative H5N1 virus (European genotype AB), PB2-627K increased polymerase activity and replication in human cells without compromising replication in avian cells. A mammalian-derived PB2-627K H5N1 isolate from a fox exhibited comparable replication kinetics and transmission efficiency in chickens and ducks to a closely related PB2-627E virus. Notably, PB2-627K remained genetically stable at consensus level following infection and transmission in both avian hosts. These findings demonstrate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian populations, potentially elevating zoonotic risk and underscoring the importance of surveillance for such mutations in birds.

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Billington, E., Riccio, S., Brown, M. D., Mollett, B., Quantrill, J. L., Johnson, S., Di Genova, C., Yang, J., Sadeyen, J.-R., Schlachter, A.-L., Roper, K. J., Hassan, J., Clifton, B., Ralh, K., Warren, C. J., Ward, G., De Silva, D., Nunez, A., Coombes, H. A., Slomka, M. J., Iqbal, M., Barclay, W. S., Peacock, T., Banyard, A. C., James, J.. 2026-10-06. The mammalian-adaptive PB2-E627K substitution preserves viral fitness of clade 2.3.4.4b H5N1 HPAIV in birds.. https://doi.org/10.64898/2026.10.06.757006

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