ABSTRACT: 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 the acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required coadaptations 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 3,385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R and 124I but a frequency of 97.5% for the dual combination. 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 1996/1997, carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S and124T, characteristic of low-pathogenic strains, prevented virus rescue, while the single substitutions attenuated the virus in both 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 coadaptation to increased acid sensitivity promoted the early evolution of H5 Goose/Guangdong-like HPAIV strains and is still required for their zoonotic potential.IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required coadaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic of low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less-acidic endosomes, e.g., within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted the emergence of H5 Goose/Guangdong-like HPAIV strains and is required for their zoonotic potential.
