Subtyping Influenza A Viruses: Genomic Perspectives on Taxonomy

Introduction: Influenza A viruses (IAVs) are pathogens found in natural reservoirs. These viruses are able to cross species barriers, which can lead to disease outbreaks in humans. The diversity of IAVs poses a major challenge for the development of effective vaccines. Traditionally, the classification of IAVs was based on phenotypic characteristics. However, with advances in sequencing technology, the focus has shifted to genomic classification, highlighting the need for an integrated approach to understanding IAV diversity. Methods: In this study, we retrieved all amino acid sequences of IAV hemagglutinin (HA) and neuraminidase (NA) available in January 2023 from the GenBank database. These sequences were clustered according to different thresholds (percentage of different amino acids) using the CD-HIT software. Results: Our analysis included 151,716 HA and 111,312 NA records. The results showed that sequences assigned to the same subtype can be genetically more diverse than sequences assigned to different subtypes. For example, #ACD40325 (HA of subtype H13) and #BBB38910 (HA of subtype H16) differed in 117 out of 567 amino acids (20.6 % of the different amino acids), while #QKY65691 and #QTC20480 (both HA of subtype H1) differed in 133 out of 567 amino acids (23.6 % of the different amino acids). This pattern was also observed in the NA sequences, with certain sequences of subtype N5 and N8 being closer to each other than different representatives of subtype N2. The phylogenetic analysis showed that the current classifications of subtypes based on phenotypic characteristics do not always correspond to the differences in the amino acid sequences. Discussion: The traditional classification of IAVs using serologic tests that measure cross-reactivity between antibodies and viral antigens is increasingly challenged by the growing genomic data. The results of this bioinformatic analysis are consistent with experimental data showing that different subtypes can exhibit cross-reactivity, while some strains within the same subtype do not exhibit cross-reactivity, complicating the subtype classification. In addition, the formation of antibodies against different viral epitopes makes the immune response and vaccine development even more complex. Conclusion: The canonical approach to identifying new IAV subtypes relies on serologic tests that detect antibodies against specific viral antigens. However, recently discovered IAV subtypes have been described solely on the basis of nucleotide sequences. To clearly distinguish all taxa from each other and to ensure that diversity within a taxon is not greater than diversity between taxa, quantitative criteria, possibly based on nucleotide or amino acid distances, would allow clear differentiation between taxa of the same level. Establishing quantitative criteria for taxonomy based on nucleotide or amino acid sequences will increase the precision of classification of IAV subtypes and thus contribute to better surveillance and prevention strategies.