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 v3.0.1under the LG + F + G4 substitution model. 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. 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 functionally relevant mutations were identified in the viral genes, including A3L, A214T, and F12V in HA; R159K and A267V in PA; A241E and T137A in M2; I42L and V7I in NS1; and I84V and I33V in PB1. Many of these mutations have been associated with increased virulence. In addition, amino acid substitutions were observed in the NA protein at V13I, S200N, L339S, S37T, V80M, and I163V, 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. CLINICAL TRIAL NUMBER: Not applicable.