Phylodynamics and Molecular Mutations of the Hemagglutinin Affecting Global Transmission and Host Adaptation of H5Nx Viruses

Abstract

Highly pathogenic avian influenza (HPAI) H5 viruses have circulated globally causing incidental human infection with a substantial pandemic threat. The present study investigated the molecular evolution and phylodynamics of hemagglutinin (HA) in avian and human-isolated H5Nx viruses globally circulating since 2000. We investigated the dynamics of amino acid substitution in the HA sequences of avian and human H5Nx viruses and performed a phylogenetic analysis. Our study found that the H5Nx lineages dominantly expanded since 2000 and diverged into multiple sublineages with unique genetic mutations. P185S mutation in HA became a molecular characteristic of dominant H5Nx viruses throughout clades 2.3.4.1 to 2.3.4.4 (2.3.4.1-4). The key mutations, ΔE130 and I155T, and potential N-linked glycosylation at residues 128, 144, and 159 in the HA gene of human-isolated viruses possibly contributed to both the individual and population levels of the H5 evolution and the host adaptation. Our analysis detected heterogeneity in amino acid sites under positive selection in the HA gene of clades 2.3.4.1-4. Accumulated mutations in the HA protein may potentially affect not only the genetic and antigenic diversity of HPAI H5Nx viruses but also increase the functional compatibility with NA subtypes. Given the global spread and incessantly occurring HA mutations of H5Nx viruses, our results emphasize the importance of early identification of HA mutations as well as the need for a comprehensive assessment of H5Nx variants in terms of pandemic preparedness.

Figure 1

Phylogenetic relationships of the H5Nx HAs between 1996 and 2022. The MCC tree of the H5Nx HAs is reconstructed using the Bayesian evolutionary interference method. The phylogenetic branches with specific subtypes are color-labeled in orange (H5N1), strawberry (H5N2), wine red (H5N3), brown (H5N5), purple (H5N6), light blue (H5N8), and green (H5N9). The genetic clusters containing the same H5 subtype are collapsed to simplify the tree representation. The HAs of human-isolated H5Nx viruses are shown with red asterisks. The geographic regions that have confirmed isolated H5 viruses from clades 2.3.2.1, 2.3.4.1-3, and 2.3.4.4 are presented on the right.

Figure 2

Bayesian coalescent LTT plots of H5Nx HAs by clade over time. The LTT plots display the temporal patterns of lineage diversification through time based on the phylogeny inferred by the Bayesian coalescent approach. The solid lines and blue shading indicate the mean and the 95% confidence interval of the effective population size in the log scale at the designated time interval (years). The dashed line under each curve represents the temporal change of the effective population size with an estimated constant rate of diversification without extinction within the lineages. The vertical, bold-dashed lines represent the divergence time of each clade estimated by the molecular clock model. The pink shade explains the period with a dramatic increase in the effective population size. The bar plot under the curve in the complete Gs/Gd lineage explains the number of the HAs used for the phylogenetic analysis.

Figure 3

The temporal dynamics and molecular interactions of amino acid substitutions around the HA globular head region of H5Nx viruses. The proportion of amino acid substitutions around the HA globular head region of H5Nx viruses is calculated by (a) the number of H5Nx subtypes or by (b) the 6 time periods: (a) ∼2004 (n = 147), (b) 2005–2008 (n = 533), (c) 2009–2012 (n = 425), (d) 2013–2016 (n = 1082), (e) 2017–2020 (n = 842), and (f) 2021∼ (n = 860). Using the HA structure of VN1194, molecular interactions in the HA globular head region of (c) human-isolated H5N6, A/Changsha/1/2014 (CS1, subclade 2.3.4.4h), (d) human H5N1 HA (VN1194, subclade 1.1), and (e) human H5N6 HA of A/Sichuan/26221/2014 (SC26221, subclade 2.3.4.4a) are analyzed. The amino acid residues of Y95, W153, H183, and Y195 (pale yellow) constitute the conserved floor in the H5 HA RBS. The hydrophobic residues of I155, A131, and L133 are colored gray, the oxygen atoms are red, and the nitrogen atoms are blue. The potential NLG sites at N128 and N158 are colored orange and residues T131 and T155 are colored cyan. S132, S133, and S185 side chains are colored green.