Genomic signatures and host adaptation of H5N1 clade 2.3.4.4b: A call for global surveillance and multi-target antiviral strategies

Fig. 1. Geographical distribution of H5N1. (A) Global geographical distribution of the top 15 countries with circulating H5 subtype influenza viruses between 2020 and 2024. Data were obtained from GISAID on December 25, 2024. (B) Clade progression of H5 subtype influenza viruses from January 2020 to December 2024. Data were obtained from GISAID on December 25, 2024. (C) Geographical distribution of H5N1 infection cases in dairy cows and human infection cases associated with dairy cows in the United States. Data were obtained from the USDA and U.S. CDC on January 5, 2025.

Fig. 2. Phylogenetic analysis of HA and NA amino acid sequences of H5N1 clade 2.3.4.4b in North America. (A) Maximum likelihood phylogenetic analysis of 48 hemagglutinin (HA) proteins and (B) 51 neuraminidase (NA) proteins. Identified sequences detected from avians and mammals (other than human) are labeled with green rhombus and triangle filled with blue separately. Red circles represent sequences detected in human samples.

Fig. 3. The signature of receptor binding site and cleavage sites on HA. (A) Structural modeling of HA trimers of A/Texas/37/2024 (PDB ID: 9DIP) that interacted with receptor analog LSTa. HA1 is shown in wheat, and HA2 is in light blue. Enlarged view highlights interactions between the receptor binding sites (RBS) of HA and LSTa and the lower panel showed the enlarged view of the multi-basic cleavage sites (MBCS). Side chains of amino acids were shown in light pink sticks. (B) Sequence alignment of the binding receptor site and multi-basic cleavage site (MBCS) in representative strains of human-infecting H5N1 clade 2.3.4.4b viruses. Logo plot of amino acid substitution of all 65 strains of human-infected viruses collected globally from 2020 to 2024 visually represents the variability observed within these regions. (C) Mutation frequencies of all detected B3.13, D1.1 and human-infected clade 2.3.4.4b H5N1 viruses on the RBS and MBCS.

Fig. 4. Comparative genomic heatmap in the genome of the circulating H5N1 in North America. (A) HA, NA and PB2 amino acid substitutions of H5N1 viruses isolated from human, avian and other mammalian cases in North America. Heatmap depicts the frequency of each amino acid substitution, with green gradient indicating prevalence of the mutation across analyzed sequences. Specific mutations on each site (with H3/N2 numbering for HA and NA based on A/Aichi/2/1968 (H3N2) and PB2 numbering according to alignments with A/Goose/Guangdong/1/1996 (H5N1)) are labeled directly on the heatmap grid. (B) and (C) heatmaps demonstrated the substitution of PB1, NS1, NS2, PA, M1, M2, and NP across the analyzed H5N1 clade 2.3.4.4b sequences.

Fig. 5. Catalytic pockets and mutations of resistance to antivirals against AIVs. (A) Catalytic site of N1 neuraminidase in complex (PDB ID: 3CL0, 3CKZ) with oseltamivir and zanamivir. Baloxavir acid binding to PB2 protein (PDB ID: 6FS6). All interacting amino acid side chains are highlighted in light pink. (B) Structural basis of M2 proton channel – Amantadine complexes (PDB ID: 6BKK) binding pattern and comparison between the H7N9 HA2-Arbidol binding sites (PDB ID: 5T6S) and H5N1 HA2-Arbidol binding sites (PDB ID:9DIP). (C) Antiviral resistance mutation frequencies of all detected B3.13, D1.1 and human-infected clade 2.3.4.4b H5N1 viruses targeting Neuraminidase (NA), Polymerase acidic (PA), and Matrix 2 (M2).