Prevalence and genetic diversity of respiratory syncytial viruses circulating in Bulgaria, 2023-2025

Neli Korsun, Ivelina Trifonova, Yordanka Uzunova, Petar Velikov, Diyana Pavlova, Iliana Grigorova, Ivan Ivanov, Daniel Ivanov, Silviya Voleva, Iva Christova

Background Respiratory syncytial virus (RSV) is a major cause of acute respiratory infections (ARIs) with a considerable disease burden among children under 5 years of age. This study aimed to characterize the prevalence of RSV, genetic diversity of circulating strains, and amino acid variability in viral proteins, as well as identify mutations within the binding sites of monoclonal antibodies (mAbs) approved for RSV prophylaxis. Methods Nasopharyngeal specimens from 5,170 patients of all ages with ARI were tested for 13 respiratory viruses using multiplex real-time PCR. Representative RSV-positive samples were subjected to whole-genome sequencing, followed by phylogenetic and amino acid sequence analyses. Results RSV was positive in 303 (5.9%) patients, of whom 61 (20.1%) had co-infections with other respiratory viruses. Among children under 5 years of age, RSV was the most frequently detected pathogen in cases of bronchiolitis (30.6%) and pneumonia (12.6%). RSV-positive samples from the 2024-2025 season were genotyped, and predominance of RSV-A over RSV-B was observed. Phylogenetic analysis identified eight and seven genetic lineages within RSV-A and RSV-B, respectively, with A.D.1.11 and B.D.1.1 being the most prevalent. No substitutions were detected in the F protein antigenic sites of RSV-A (Ø, I-V), including the binding sites for nirsevimab (site Ø), palivizumab (site II), and clesrovimab (site IV). Conversely, RSV-B strains exhibited substitutions in antigenic sites Ø (n=3), I (n=2), and V (n=2). Conclusions RSV is a leading cause of severe respiratory disease in early childhood and demonstrates substantial genetic diversity among circulating strains. The absence of resistance-associated substitutions in the F protein indicates that currently circulating RSV strains remain susceptible to approved mAbs.