Background: Enterovirus D68 (EV-D68) is an important respiratory pathogen occasionally linked to acute flaccid myelitis. While recombination drives enterovirus evolution, recombinant EV-D68 strains have been rarely documented.
Methods: As part of 2025 genomic surveillance in Maryland, 115 EV-D68 genomes were sequenced using an amplicon-based approach. Consensus genomes were aligned with global references and analyzed with IQ-TREE3 and SimPlot to assess phylogeny and recombination.
Results: Complete genomes were obtained from 78% (90/115) of specimens, all belonging to subclade A2. Five genomes formed a distinct cluster with discordant phylogenies across genomic regions: P1 grouped with A2, whereas P2-P3 clustered with B3. SimPlot and BootScan analyses identified a recombination breakpoint near the 2A/2B junction (∼nt 3,700), consistent with an A2(P1)/B3(P2-P3) recombinant. BAM alignment review excluded co-infection.
Conclusions: We report a novel EV-D68 A2/B3 recombinant circulating locally in 2025, highlighting the need for continued whole-genome surveillance.