Work

Influenza A virus H5Nx genotypes assigned by genetic similarity are incongruent with global phylogenies

Year: 2026

Type: preprint

Abstract: Influenza A virus H5N1 and related subtypes (H5Nx) have broad host ranges and pose a pandemic risk. Since early 2022, H5 clade 2.3.4.4b has predominated globally, causing unprecedented mortality in wild birds, poultry, and mammals, with spillovers to humans. Accurate viral classification is essential for tracking H5Nx variant emergence and spread, but reassortment among genome segments with distinct evolutionary histories complicates genotyping. H5Nx genotype assignment tools, GenoFlu in North America and Genin2 in Europe, assign segments to clusters by genetic similarity to reference sequences and define genotypes as constellations of segment clusters. However, genetic similarity does not necessarily imply recent shared ancestry due to independently re-occurring mutations and variability in the evolutionary rate. Furthermore, similarity is defined relative to references that may inadequately represent diversity. We evaluated the congruence of genetic similarity-based H5Nx clusters and genotypes with phylogenies of global sequences across eight segments. H5Nx genomes from 41,366 samples were assigned GenoFlu and Genin2 genotypes, and maximum likelihood phylogenies were inferred for each segment. CD-HIT and phylogenetic TreeCluster clusters were also generated for a range of distance thresholds. Clustering methods were evaluated based on phylogenetic purity, monophyly, and diversity. Reassortment within predominant GenoFlu and Genin2 genotypes was evaluated based on their phylogenetic cluster spans. Expectedly, GenoFlu was largely unable to assign genomes from outside of North America, South America, and Antarctica from 2022 to 2025, whereas Genin2 assignments struggled outside of Europe, Africa, and Western Asia. Only 14.2% of GenoFlu clusters were monophyletic, while Genin2 clusters were more variable across segments (overall 49.3% monophyletic). Despite CD-HIT yielding genetic distance clusters of similar size to TreeCluster, they were less monophyletic (44%) and encompassed greater phylogenetic diversity. We identified several genotypes, such as GenoFlu B3.13, D1.1, and D1.3, with segments that had diversified into separate subclusters. Similarly, GenoFlu genotypes B3.5 and A3, and Genin2 genotypes EA-2020-C, EA-2021-AB, and EA-2022-CH may erroneously combine multiple distinct reassortants. Genetic similarity-based H5Nx genotyping tools are rapid, stable, and sufficient for broad categorization, but may group viruses with distinct evolutionary histories and fail to assign divergent viruses, risking biased surveillance, modeling, and risk assessment.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.23.756838