Reversion of PB2-627E to -627K during replication of an H5N1 Clade 2.2 virus in mammalian hosts depends on the origin of the nucleoprotein

H5N1 highly pathogenic avian influenza viruses (HPAIV) of clade 2.2 spread from Southeast Asia to Europe. Intriguingly, in contrast to all common avian strains specifying glutamic acid at position 627 of the PB2 protein (PB2-627E), they carry a lysine at this position (PB2-627K), which is normally found only in human strains. To analyze the impact of this mutation on the host range of HPAIV H5N1, we altered PB2-627K to PB2-627E in the European isolate A/Swan/Germany/R65/2006 (R65). In contrast to the parental R65, multicycle growth and polymerase activity of the resulting mutant R65-PB2_K627E were considerably impaired in mammalian but not in avian cells. Correspondingly, the 50% lethal dose (LD₅₀) in mice was increased by three orders of magnitude, whereas virulence in chicken remained unchanged, resulting in 100% lethality, as was found for the parental R65. Strikingly, R65-PB2_K627E reverted to PB2-627K after only one passage in mice but did not revert in chickens. To investigate whether additional R65 genes influence reversion, we passaged R65-PB2_K627E reassortants containing genes from A/Hong Kong/156/97 (H5N1) (carrying PB2-627E), in avian and mammalian cells. Reversion to PB2-627K in mammalian cells required the presence of the R65 nucleoprotein (NP). This finding corresponds to results of others that during replication of avian strains in mammalian cells, PB2-627K restores an impaired PB2-NP association. Since this mutation is apparently not detrimental for virus prevalence in birds, it has not been eliminated. However, the prompt reversion to PB2-627K in MDCK cells and mice suggests that the clade 2.2 H5N1 HPAIV may have had a history of intermediate mammalian hosts.