Molecular surveillance and genomic characterization of enterovirus D68 in southern Portugal, 2024–2025

Enterovirus D68 (EV-D68) is a re-emerging respiratory pathogen that is increasingly associated with severe respiratory disease and occasional neurological complications, yet its circulation in Portugal remains poorly documented. In the present study, we investigated the presence of EV-D68 in respiratory samples collected in southern Portugal (Algarve region) from October 2024 to February 2025. Screening of 150 respiratory samples from patients (115 from children and 35 from adults), all previously positive for rhinovirus/enterovirus, revealed eight EV-D68-positive cases, corresponding to a detection rate of 5.44%, spanning a broad age range from infancy to the elderly. Clinical manifestations ranged from mild respiratory illness to severe conditions, including pneumonia and acute respiratory distress syndrome. Neurological manifestations, including complex febrile seizures and encephalomyelitis, were also observed in EV-D68-positive patients. Some cases involved coinfections, while others presented with EV-D68 as the sole detected respiratory pathogen. Genomic characterization from one isolate revealed a VP1 fragment belonging to clade B3, and a complete genome from another isolate clustered within clade A2. Phylogenetic analysis confirmed clustering with contemporary European isolates. These findings provide preliminary evidence of EV-D68 detection in southern Portugal and document the presence of clades A2 and B3 among the characterized samples. The results illustrate the heterogeneous clinical presentations observed in affected patients and highlight the value of continued molecular monitoring and genomic characterization of EV-D68.