Abstract
To explore an off-season enterovirus D68 (EV-D68) upsurge in the winter season of 2019/2020, we adapted a whole-genome sequencing approach for Nanopore Sequencing for 20 hospitalized patients with accompanying respiratory or neurological presentation. Applying phylodynamic and evolutionary analysis on Nextstrain and Datamonkey respectively, we report a highly diverse virus with an evolutionary rate of 3.05 × 10(-3) substitutions per year (entire EV-D68 genome) and a positive episodic/diversifying selection with persistent yet undetected circulation likely driving evolution. While the predominant B3 subclade was identified in 19 patients, one A2 subclade was identified in an infant presenting with meningitis. Exploring single nucleotide variations using CLC Genomics Server showed high levels of non-synonymous mutations, particularly in the surface proteins, possibly highlighting growing problems with routine Sanger sequencing for typing enteroviruses. Surveillance and molecular approaches to enhance current knowledge of infectious pathogens capable of pandemic potential are paramount to early warning in health care facilities.