Whole-Genome Analysis of Influenza A(H1N1)pdm09 Viruses in Cameroon (2019–2024) Using Nanopore Sequencing

Background Since 2019, Cameroon has reported a high number of seasonal influenza cases caused by the A(H1N1)pdm09 subtype, which remained the predominant global strain as of 2024. Methods To characterize the evolutionary dynamics of circulating A(H1N1)pdm09 viruses, whole-genome sequencing was conducted using Oxford Nanopore Technologies, with multiplexing native barcode expansion kits. DNA repair and end preparation were performed using the NEBNext Ultra II End-Repair/dA-tailing kit. Phylogenetic trees for HA genes segments were inferred using the maximum likelihood (ML) method implemented in IQ-TREE v3.0.1(http://www.iqtree.org) under the TVM + F + I + R2 substitutions models. Additionally, Mutation analysis was performed across all eight gene segments using MEGA with A/Wisconsin/67/2022 (A/H1N1pdm09) serving as the reference strain. Identified amino acid substitutions were annotated and their potential phenotypic effects were evaluated using FluSurver (http://flusurver.bii.a-star.edu.sg. accessed on August10, 2025). Results All Cameroonian A(H1N1)pdm09 strains from 2019 to 2024 belonged to subclade 6B.1A.5a.2a. Phylogenetic analysis revealed annual divergence from Northern Hemisphere vaccine strains, suggesting a mismatch with locally circulating variants. Several mutations of functional interest were identified, including A3L, A214T, F12V, R159K, A267V, A241E, T137A, I42L, V7I, I84V and I33V across the HA, PA, M2, NS1 and PB1 genes, many of which are associated with increased virulence. Similarly, amino acid substitutions at sites V13I, S200N, L339S, S37T, V80M, and I163V in the NA protein were observed in the NA protein, relative to the 2024 vaccine strain A/Wisconsin/67/2022. Overall, the number of amino acid mutations between circulating strains and the vaccine strain was notably high, indicating that local viruses may be evolving away from the vaccine strain selected for the 2023–2024 season. Conclusions These findings underscore the ongoing genetic evolution of the influenza A(H1N1)pdm09 virus in Cameroon and highlight the importance of local genomic data into the selection of WHO vaccine candidate strains for the Northern Hemisphere.