A Dual Motif in the Hemagglutinin of H5N1 Goose/Guangdong-Like Highly Pathogenic Avian Influenza Virus Strains Is Conserved from Their Early Evolution and Increases both Membrane Fusion pH and Virulence

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. 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.

Avian influenza overview December 2023–March 2024

“…Both A(H5N1) sequences were assigned to clade 2.3.2.1c and carried the previously detected HA amino acid substitutions T108I (H5 numbering, the combination S107R-T108I has been associated with increased virulence in chickens and mice and increased pH of fusion) (Wessels et al, 2018), S133A (increased pseudovirus binding to α2-6 human-type receptors) (Yang et al, 2007) and S155N (associated with increased virus binding to α2-6 human-type receptors) (Wang et al, 2010), of which the latter is present in most viruses of this clade. Furthermore, they also carried D94N (associated with increased virus binding to α2-6) (Su et al, 2008), and one (A/Cambodia/i0125001G/2024, EPI_ISL_18823967) had N154D (associated with decreased virulence in mice) (Jin et al, 2019).…”

Avian influenza overview March–June 2024

“…The mutation analysis of the HA segment for the eight A(H5Nx) sequences revealed a previously reported profile (EFSA, ECDC and EURL, 2024) in which seven out of eight carried both T108I, which in combination with S107R has been associated with increased virulence in chickens and mice, and increased pH of fusion (Wessels et al, 2018), and S133A, which is linked to increased pseudovirus binding to α2-6 human-type receptors (Yang et al, 2007). Furthermore, all the A(H5Nx) viruses had a deletion at position 328 (a polybasic cleavage motif), which is seen in the vast majority of clade 2.3.2.1c, compared to A/Goose/Guangdong/1/96.…”

Comparative Antigenicity and Pathogenicity of Two Distinct Genotypes of Highly Pathogenic Avian Influenza Viruses (H5N8) From Wild Birds in China, 2020–2021

“…Bioinformatics analysis identified many mutations that would increase virulence in mice, such as R114 and I115 (H3 numbering) of the HA gene (Wessels et al, 2018); D30, M43, and A215 of the M1 gene (Fan et al, 2009;Nao et al, 2015); S42, E55, E66 (SX1/2020, Y173/2020, and Y175/2020), M106, and F138 (except SX1/2020, Y173/2020, and Y175/2020) of the NS1 gene (Jiao et al, 2008;Ayllon et al, 2014;Li et al, 2018); the NS1 C-terminal ESEV motif of the PDZ domain at position aa227-230 (Jackson et al, 2008;Soubies et al, 2010;Zielecki et al, 2010); combination of V89, D309, K339, G477, V495, E627, and T676 of the PB2 gene (Li et al, 2009); V3 (except NQ1/2021) and G622 of the PB1 gene (Feng et al, 2016;Elgendy et al, 2017); and D383 of the PA gene (Song et al, 2015;Suttie et al, 2019).…”