Post-pandemic re-establishment of respiratory virus seasonality and molecular epidemiology of Rhinovirus/Enterovirus in Northern Italy: a three-year surveillance study (2023–2026)

Objectives: Following the COVID-19 pandemic, respiratory virus circulation progressively returned towards patterns observed in the pre-pandemic period. However, long-term epidemiological data describing the re-establishment of respiratory viral seasonality and the molecular diversity of Rhinovirus/Enterovirus (RV/EV) remain limited. Methods: To investigate the circulation patterns of respiratory viruses and characterize the RV/EV in Northern Italy, a retrospective observational study was conducted on samples collected between April 2023 and April 2026 at Brescia Civic Hospital. Respiratory viruses were detected by real-time PCR, and RV/EV-positive samples were characterized through next-generation sequencing. Results: A total of 6,919 respiratory virus-positive samples were identified. SARS-CoV-2 remained the most frequently detected pathogen, followed by RV/EV, influenza A/B (FLU A/B), adenovirus, respiratory syncytial virus (RSV), parainfluenza virus, bocavirus, and human metapneumovirus. Distinct seasonal peaks were observed for FLU A/B and RSV during winter months, whereas RV/EV circulated continuously throughout the year. Molecular characterization of 224 RV/EV-positive samples revealed substantial genetic diversity. Pediatric patients accounted for the majority of sequenced cases. Conclusions: Three years after the end of the COVID-19 pandemic, respiratory viruses have re-established their characteristic seasonal circulation patterns. Nevertheless, RV/EV remain the dominant non-influenza respiratory pathogens and display considerable genetic heterogeneity. Continuous molecular surveillance is essential for early detection of emerging respiratory viral threats.