Viral pathogens in the etiology of acute respiratory infections in Bulgaria during the 2024–2025 season and genetic diversity of circulating influenza viruses

Background: Respiratory viruses are major contributors to global morbidity and mortality, with influenza viruses having a significant clinical and epidemiological impact. In this study, we examined the virological and epidemiological aspects of influenza infection in comparison with those of other respiratory infections. We further assessed the genetic diversity of circulating influenza viruses and their susceptibility to antivirals. Methods: A total of 2,981 nasopharyngeal specimens from patients with acute respiratory illness were tested for 13 different respiratory viruses using multiplex real-time PCR. Representative influenza strains underwent whole-genome sequencing, phylogenetic and amino acid analyses. Results: At least one respiratory virus was detected in 1,635 (54.8%) patients, and 182 (6.1%) had co-infections involving two to four viruses, including six cases of SARS-CoV-2 and influenza co-infection. Influenza A(H3N2) and B/Victoria lineage viruses predominated, with positivity rates of 12.1 and 10.9%, respectively, followed by rhinoviruses (9.0%), whereas SARS-CoV-2 circulation remained low (3.7%). Influenza infections were most prevalent among children aged 5-14 years. Phylogenetic analysis identified multiple genetic subclades, including C.1.9, C.1.9.1, C.1.9.3, C.1.9.4 and D.5 for A(H1N1)pdm09; J.2, J.2.1, and J.2.2 for A(H3N2), and C.5.1, C.5.6, C.5.6.1, and C.5.7 for B/Victoria lineage. Numerous amino acid substitutions were observed across viral proteins, including within hemagglutinin antigenic sites compared to vaccine strains. Conclusion: The 2024-2025 influenza season in Bulgaria was characterized by high influenza activity and substantial genetic heterogeneity of circulating strains. These findings underline the importance of continuous molecular surveillance to monitor viral evolution, inform vaccine strain selection, and guide national public health strategies.