This study reports increased Enterovirus D68 (EV-D68) circulation in 2024, re-establishing its biennial circulation cycle after its interruption during the COVID-19 pandemic. A total of 1,395 respiratory and cerebrospinal fluid (CSF) samples, positive for rhinovirus/enterovirus, collected from January to November 2024 were screened. EV-D68 was the predominant enterovirus detected (72.6% of EV-positive samples), with cases peaking in October, consistent with historical seasonal patterns. Demographically, children under 5 years were predominantly infected with EV-D68 (41.6% of cases). Phylogenetic analysis revealed the co-circulation of two EV-D68 subclades: B3 (71%) and A2 (29%). Subclade B3 was primarily associated with pediatric infections (median age: 5 years), while A2 was more common in adults (median age: 42 years). Comparative genomic analysis of the 2024 B3 genomes, along with genomes from 2018 and 2022, identified the emergence of four amino acid substitutions, including three in nonstructural proteins (3C: I597V; 3D: I950V, T2173A) and one in the structural protein VP2 (T145S). The six positive enterovirus CSF samples diagnosed in 2024 included six different types: EV-D68, E9, E30, E18, CV-A9, and CV-B1. Notably, the 2024 EV-D68 outbreak did not coincide with a reported increase in acute flaccid myelitis (AFM) cases. This study highlights the importance of EV-D68 genomic surveillance for monitoring EV-D68 evolution, given its association with severe respiratory disease and neurological complications. Enhanced surveillance is also critical for the early detection of the emergence of enteroviruses, such as EV-C105, identified in this study.IMPORTANCEEnteroviruses (EVs), a genus within the Picornaviridae family, are small, single-stranded RNA viruses linked to a wide spectrum of diseases, including neurological conditions. Despite their prevalence, they remain understudied. EV-D68 and EV-A71 have raised global public health concerns due to outbreaks of acute flaccid myelitis (AFM) and encephalomyelitis in North America and Europe. EVs exhibit high genetic variability, and viral evolution has been associated with changes in neurovirulence. Notably, EV-D68 epidemics in 2014, 2016, and 2018 coincided with spikes in AFM cases. However, AFM reports from 2019 to 2022 were low, even with a significant increase in EV-D68 infections in 2022. We developed an EV-D68 genomic surveillance workflow to investigate genotype associations with severe disease. Our previous work linked amino acid substitutions in 2018 strains to increased disease severity. This study analyzes EV-D68 evolution in 2024 and documents the return of its biennial circulation pattern following disruption during the COVID-19 pandemic.
Keywords: EV-D68; enterovirus.