A dual motif in the hemagglutinin of H5N1 Goose/Guangdong-like HPAIV is conserved from their early evolution and increases both membrane fusion pH and virulence

Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required co-adaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R or 124I each, in dual combination however at 97.5%. This highly conserved dual motif is still retained in contemporary H5 HPAIV including the novel H5NX reassortants carrying neuraminidases of different subtypes like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/97 carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S/124T, characteristic in low-pathogenic strains, prevented virus rescue while the single substitutions attenuated the virus both in chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less acidic endosomes, e.g. within the respiratory tract, resulting in an extended organ tropism. Taken together, HA co-adaptation to increased acid sensitivity promoted early evolution of H5 Goose/Guangdong-like HPAIV and is still required for their zoonotic potential.