Genomic Surveillance and Evolution of Influenza Virus Across Two Consecutive Seasons, 2024–2026, in Southern Greece

Continuous genomic surveillance of influenza virus is critical for monitoring viral evolution, detecting emerging variants, and informing vaccine strain selection. While partial-gene Sanger sequencing has traditionally underpinned national surveillance programs, whole-genome sequencing (WGS) enables simultaneous characterization of all eight genomic segments, providing resolution not achievable through conventional approaches. This is the first published, systematic, whole-genome-based influenza surveillance study reported from Greece. We sequenced and characterized influenza viruses circulating in Southern Greece (S. Greece) during two consecutive influenza seasons, 2024–2025 and 2025–2026, using Oxford Nanopore Technologies (ONT) sequencing. A total of 40 samples were analyzed, with complete or near-complete genome assembly achieved in the vast majority of them. During 2024–2025, co-circulation of A(H1N1)pdm09 (subclades C.1.9.3 and D.3.1), A(H3N2) (subclades J.1.1 and J.2.2), and B/Victoria (C.5 subclades, predominantly C.5.7) influenza viruses was confirmed, with the detection of an oseltamivir-resistant H1N1 variant carrying NA H275Y as a clinically significant finding. Extension of surveillance into 2025–2026 revealed a complete inter-seasonal lineage transition in A(H1N1)pdm09 from subclade C.1.9.3 to D.3.1/D.3.1.1, and the rapid emergence and geographic dissemination of A(H3N2) subclade K across multiple regions of Southern Greece. Phylogenetic analysis characterized the HA subclade composition across the two seasons and positioned the Greek strains within the European and global context. Sequences of subclade K strains from S. Greece were phylogenetically concordant with the updated 2026–2027 vaccine strains, in line with ECDC and WHO surveillance data. Whole-genome analysis confirmed the absence of virulence-associated polymerase markers in all isolates with fully assembled polymerase segments, and identified conserved internal segment substitution patterns co-segregating with circulating subclades. These findings provide the first published whole-genome evolutionary dataset for influenza in Southern Greece, contributing insight into subtype and clade-level evolutionary dynamics and demonstrating the public health value of sustained genomic surveillance at a national reference laboratory level.