Viral acute respiratory infections (ARIs) are a leading cause of infectious morbidity and mortality and have serious health, economic, and social consequences. Among the known respiratory viruses (more than 200), respiratory syncytial virus (RSV) is one of the most common etiological agents of upper and lower respiratory tract infections (LRTIs), with the greatest disease burden in infants, young children, the elderly, and immunocompromised adults. It is also a leading cause of serious LRTI (bronchiolitis and pneumonia) in children aged < 5 years. According to epidemiological data from 2019, 33 million episodes of acute LRTI, 3.6 million hospital admissions, and 101,400 death cases in children aged 0–60 months have been attributed to RSV (Li et al., 2022). RSV belongs to the family Pneumoviridae and the genus Orthopneumovirus (Lefkowitz et al., 2018). The genome of this virus is a non-segmented, single-stranded, negative-sense RNA approximately 15.2 kb in length. It encodes 11 proteins, namely NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L, each with specific functions (Collins and Karron, 2013). The surface glycoproteins G and F play crucial roles in the initial stage of viral infection. The G protein is accountable for attaching the virus to host cells. On the other hand, the F protein makes it possible for the viral ribonucleoprotein to enter the cell’s cytoplasm as a result of the fusion of the viral envelope with the host cell membrane (McLellan et al., 2013). During the fusion process, RSV F undergoes dramatic pre-fusion-to-post-fusion conformational changes. The active pre-fusion conformation includes six major antigenic sites (Ø and I–V) and produces more potent neutralizing antibodies. Only four sites (I, II, III, and IV) are present in the inactive post-fusion conformation (Chang et al., 2022). The F protein is the major viral antigen and is relatively conserved, whereas the G protein contains two hypervariable regions (HVRs) in the superficial ectodomain and exhibits greater genetic diversity. The highly variable C-terminal region of the G gene (HVR2) is commonly used in molecular, epidemiological, and evolutionary studies (Gimferrer et al., 2019; Sáez-López et al., 2019; Kim et al., 2023). The G and F proteins are heavily glycosylated with N-linked and O-linked sugars, which limit antibody access to the antigenic еpitopes, thereby facilitating RSV evasion from pre-existing immunity (Holmes, 2013).

RSV has a single serotype divided into two major subgroups, RSV-A and RSV-B, each comprising multiple genotypes (Mufson et al., 1985). Based on the genetic variability of the G gene, RSV-A is divided into 14 genotypes (GA1–GA7, SAA1, NA1–NA4, CB-A, and ON1), whereas RSV-B comprises 37 genotypes (GB1–GB6, GB12, GB13, SAB1–SAB4, URU1, URU2, CB1, THB, BA1–BA14, BA-C, BA-CCA, BA-CCB, JAB1, NZB1, and NZB2; Gaymard et al., 2018; Muñoz-Escalante et al., 2021). Goya et al. (2020) proposed a new classification in which the number of genotypes was reduced to three for RSV-A (GA1–GA3) and seven for RSV-B (GB1–GB7), with two additional levels of classification defined as sub-genotypes and lineages (Goya et al., 2020). Variations in the temporal and geographical distributions of individual RSV genotypes and the periodic replacement of predominant genotypes were observed (Esposito et al., 2015; Sáez-López et al., 2019; Kamau et al., 2020; Lee et al., 2021; Tabatabai et al., 2022; Kim et al., 2023). Viruses of different genotypes can co-circulate during a single epidemic within the same community. Genotyping of circulating RSV is essential for monitoring the emergence and clinical burden of new genotypes and for developing therapeutic and preventive strategies. Furthermore, analyzing the amino acid composition of RSV surface glycoproteins enables tracing of the evolutionary dynamics of the pathogen and understanding the molecular mechanisms underlying its genetic and antigenic variability.

During the COVID-19 pandemic, various public health and social distancing measures (e.g., mandatory mask-wearing, working from home, school closures, limited social gatherings, travel restrictions, quarantine, and patient isolation) were implemented to prevent the spread of SARS-CoV-2; these measures resulted in greatly reduced transmission of RSV and other seasonal respiratory viruses (Agca et al., 2021; Groves et al., 2021). After two seasons of low circulation, in the winter of 2022–2023, RSV activity returned to levels similar to those in the seasons preceding the pandemic (Jia et al., 2022; Guo et al., 2023; Pierangeli et al., 2023). In Bulgaria, RSV has been detected using real-time RT-PCR since 2010 within the national influenza surveillance system. This study aimed to explore the circulation patterns of RSV and other respiratory viruses during the COVID-19 pandemic, describe the epidemiological and clinical characteristics of RSV infection and the genetic diversity of RSV strains in the first RSV post-pandemic season, and perform an amino acid sequence analysis of the RSV surface and internal proteins.

This study investigated the circulation patterns of RSV and other respiratory viruses among patients presenting with ARI symptoms in Bulgaria during the COVID-19 pandemic, with an emphasis on the first season after COVID-19 restrictions were lifted. The genetic characteristics of the RSV responsible for the 2022–2023 outbreak were also explored. Extensive non-pharmaceutical preventive interventions targeting SARS-CoV-2 have had a tremendous impact on viral respiratory infection epidemiology, such as the low prevalence and diminished genetic diversity of circulating viruses and the monthly and age distribution of cases (Garg et al., 2022; Chow et al., 2023). Viral interference, the behavior of individuals, and societal and health system factors may have also contributed to the significant reduction in respiratory virus circulation during the pandemic (Abu-Raya et al., 2023). During the first two winters, the activity of RSV and other seasonal respiratory viruses plummeted abruptly worldwide, including in Bulgaria (Hönemann et al., 2023). An unusual summer spike in RSV-associated activity was observed in 2021 during the first relaxation of COVID-19-related public health measures. The low RSV exposure during the winter of 2020–2021 probably resulted in a significant decline in population RSV-specific immunity and an atypical inter-seasonal resurgence of RSV infections (Reicherz et al., 2022; Bardsley et al., 2023). In the fall of 2022, with the complete withdrawal of containment measures, RSV returned to the seasonally high levels of activity observed in the preceding years. The sudden upsurge in RSV cases in the summer of 2021 and autumn of 2022, as well as the earlier start of the 2022–2023 epidemic, has been registered in many countries (Redlberger-Fritz et al., 2021; Goya et al., 2023; Hönemann et al., 2023; Munkstrup et al., 2023). The detection rate of RSV in the 2022–2023 outbreak was 13.7% vs. 3.2% and 6.6% during the previous two seasons, respectively (p < 0.05). Among all the studied respiratory viruses, RSV was the most frequently identified etiologic agent of ARIs during the 2022–2023 season and the second most commonly detected respiratory virus after SARS-CoV-2 during the previous two seasons. Temporal and geographic variations exist in the incidence of RSV infection, depending on the study population, detection methods, climate, and other factors (Gimferrer et al., 2019; Valley-Omar et al., 2022; Bimouhen et al., 2023; Kim et al., 2023). During the COVID-19 pandemic, the intensity of the applied anti-epidemic measures has played a crucial role in determining the spread of seasonal respiratory viruses. In this study, RSV was co-detected with other respiratory viruses in 68 (20.5%) patients, including SARS-CoV-2 in 14 patients. A high rate (23.3%, 55/236) of co-infections was found among hospitalized RSV-positive patients. The proportion of RSV co-infections varied between studies (Trento et al., 2010; Holmes, 2013; Gimferrer et al., 2019), and no co-infections with other respiratory viruses were detected in a study conducted in Washington, United Kingdom (Goya et al., 2023). Currently, there is no conclusive evidence linking the presence of co-infections with disease severity (Goka et al., 2014).

The strong dominance of RSV-B during the 2022–2023 epidemic can be explained by the low transmission of RSV in the previous two seasons, which could have caused a genetic bottleneck, resulting in reduced viral diversity (Chow et al., 2023). Significant declines in RSV genetic diversity during the implementation of COVID-19-related restrictions have also been observed in other countries (Eden et al., 2022). Our findings were consistent with reports from other European countries, where the 2022–2023 outbreak was also driven by RSV-B (Redlberger-Fritz et al., 2021; Munkstrup et al., 2023; Pierangeli et al., 2023). During the period preceding the pandemic, year-to-year fluctuations in the incidences of RSV-A and RSV-B were observed in Bulgaria (Korsun et al., 2021). Herd immunity against the RSV subgroup, which was dominant in the country in the preceding year, is likely the reason for the dominance of other subgroups in the following season. A consistent shift in the predominance of RSV-A to RSV-B at different time intervals (1–3 years) has been reported worldwide (Hall, 2001; Gimferrer et al., 2019).

During the study period, RSV affected all age groups across the population, with much more frequent involvement in children aged < 5 years, especially those under 2 years. In line with many reports, the rate of RSV infection was highest in the youngest age group (up to 1 year) and decreased with increasing age due to the development of immunity after repeated infections (Jepsen et al., 2018; Jallow et al., 2023). Administration of the recently approved Abrysvo vaccine during pregnancy could protect infants against LRTI from birth to 6 months of age (EMA; Saso and Kampmann, 2016). According to the literature, the burden of RSV infection is high in older adults who are at risk of severe infection (Savic et al., 2023). In contrast to a previous study (Nguyen-Van-Tam et al., 2022), the study older population aged ≥65 years was minimally affected by RSV infection (4.66% vs. 1.3%, respectively). For this age group, licensed vaccines (Arexvy and Abrysvo) have already been designed to protect against LRTI (such as bronchitis and pneumonia) caused by RSV [European Medicine Agency (EMA), 2023a,b]. Our previous study showed that RSV infections follow a seasonal pattern, occurring predominantly in winter and early spring, with very few cases occurring during the summer (Korsun et al., 2021). During the 2022–2023 season, RSV was in circulation from November to late March, with peak activity in December 2022, which is consistent with data from other European countries (Jepsen et al., 2018; Gimferrer et al., 2019; Pierangeli et al., 2023). From 2020 to 2021, the typical seasonality of RSV was disrupted because of the applied anti-COVID-19 measures. Information on the monthly distribution of RSV infections is important for the implementation of prophylactic measures, including vaccines and monoclonal antibodies, among high-risk populations and for strengthening infection control to prevent nosocomial infections.

Similar to the pre-pandemic seasons, respiratory viruses were more frequently detected among inpatients (63.5%) than outpatients (49.4%; p < 0.05; Korsun et al., 2021). RSV was the major pathogen associated with hospitalization in children, and 59.8% (198/331) of all RSV cases occurred in hospitalized children aged < 5 years. RSV is well known as the leading etiological agent of LRTI in the pediatric population and the predominant cause of bronchiolitis and pneumonia (Hall, 2001). During the 2022–2023 season, RSV was the most common cause of bronchiolitis (40%) and pneumonia (25.4%) in children aged < 5 years. RSV-associated cases of LRTI were observed more frequently in children younger than 2 years of age, indicating a higher susceptibility of this age group to RSV infection. According to a previous report, boys are more likely to develop severe disease, and male infants are twice as likely to be hospitalized than female infants (Hall, 2004). In our study, no statistically significant differences were found in the incidence and hospitalization rates among males and females < 5 years old with confirmed RSV infection.

Whole-genome sequencing and phylogenetic analysis of 47 RSV-B sequences indicated that the GB5.0.5a and GB5.0.6a genotypes (both equivalent to the BA strains) were responsible for the 2022–2023 outbreak. The 2022–2023 RSV-B surge was also driven by GB5.0.5a in the United States (Adams et al., 2023), Austria (Redlberger-Fritz et al., 2021), Italy (Tramuto et al., 2023), and other countries (Jallow et al., 2023). Molecular analysis of RSV G genes revealed the presence of a 60-nucleotide duplication in HVR2, which is a landmark characteristic of BA strains initially described in Buenos Aires, Argentina, in 1999. Following its appearance, the BA genotype rapidly evolved and displayed significant diversification into at least 14 new genotypes (Ábrego et al., 2017; Lee et al., 2021; Kim et al., 2023). Since 2006, RSV-B strains harboring this partial duplication have become globally predominant and have completely replaced all previously circulating RSV-B genotypes (Trento et al., 2010). The worldwide spread of RSV-B strains carrying this 60-nucleotide duplication indicates that this unique insertion may enhance viral attachment to host cells and improve fitness, thereby facilitating transmission (Hotard et al., 2015).

Surface glycoproteins G and F are under selective immune pressure and undergo constant evolution. Therefore, they were subjected to molecular analysis. In Bulgarian RSV-B, amino acid substitutions were identified at 18 G protein positions, of which 16 were located in HVR1/2 and six N-linked glycosylation motifs, including two new ones, compared to the NH1182 strain discovered 20 years ago in the early years after the appearance of the BA genotype. Duplication of 20 amino acids in the RSV-B strain resulted in additional amino acid substitutions in HVR2. Some substitutions identified in the G protein (S247P, V271A, I281T, T290I, and T312I) have been described in other European countries and elsewhere (Kim et al., 2023; Tramuto et al., 2023). The RSV F protein is a vaccine antigen and a target of the monoclonal antibody product palivizumab (Synagis), which is administered as a passive immunoprophylaxis to high-risk infants, as well as several other monoclonal antibodies and small molecules that are under clinical development (Kopera et al., 2023). Key variations in the F protein antigenic sites could affect the antigenicity and susceptibility of RSV to prophylactic and therapeutic agents targeting these regions. In our study, the F protein showed 13 substitutions and six N-linked glycosylation sites, confirming its lower genetic variability than the G protein (Sun et al., 2022). No amino acid variations were found at antigenic sites II (target of palivizumab and motavizumab) or IV (target of 101F and MAb19; Lu et al., 2019). More variation was found in the antigenic sites Ø (target of nirsevimab) and V (target of suptavumab). This variability is likely a result of neutralization escape, given that these regions prompt the production of antibodies with high neutralizing activity (Ruckwardt et al., 2019; Tabor et al., 2020; Adhikari et al., 2022; Sun et al., 2022). The antigenic site Ø is located at the apex of the pre-fusion trimer and forms much of the variability of the F protein (~25%; McLellan et al., 2013). The variations L172Q, S173L, S190N, and K191R were located at site V (aa 148–194; target of the monoclonal antibody AM14), and the substitution F45L was located at site I (aa 27–45; target of human antibody MPE8; Hause et al., 2017). The substitutions A103V, L172Q, S173L, K191R, I206M, and Q209R have also been reported in other recent studies (Lu et al., 2019; Adhikari et al., 2022; Chen et al., 2022; Sun et al., 2022). The N-linked glycosylation sites of the F protein were relatively conserved, whereas those of the G protein were more variable. The acquisition or removal of N-linked glycosylation can affect viral antigenicity and facilitate immune evasion (Feng et al., 2022). The continuous accumulation of amino acid changes and extensive glycosylation of RSV G and F proteins with N-and O-linked sugars allow viruses to escape neutralization by pre-existing antibodies. Consequently, the continued emergence of genetically altered RSV has enabled this pathogen to cause repeated infections in the same individual and annual epidemics. Full-length sequencing of other structural proteins showed a high degree of similarity with the reference strain. In agreement with a previous study, a small number of amino acid changes were identified in the internal proteins (Jallow et al., 2023). The L56I, K570R, I715V, R1759K, and A1787E substitutions were located in the conserved enzymatic regions RdRp, Cap, and MT of L protein, which are potential targets for inhibitor development (Gilman et al., 2019). A recent study in Austria reported the L protein variations K570R, V1479A, and R1759K, which were also observed in Bulgarian strains (Redlberger-Fritz et al., 2021).

It is worth noting that this study had a few limitations. In the first two seasons of the COVID-19 pandemic, the number of clinical samples examined was notably smaller than that in the 2022–2023 season. This is because the national ARI surveillance system was disrupted during this period, as resources and specialists were redirected toward COVID-19 diagnosis, treatment, and contact tracing. A significant portion of the National Laboratory’s “Influenza and ARI” work involved testing for SARS-CoV-2. The reporting forms for clinical samples were often incomplete and lacked the necessary clinical information. This made it impossible to perform an extensive epidemiological and clinical analysis of respiratory infections other than SARS-CoV-2 during the acute phase of the pandemic. In addition, due to the limited detection of RSV-A, we did not sequence viruses in this subgroup and analyze their genetic characteristics. Finally, as the objectives of the study focused on RSV infection, other respiratory infections caused by RVs, AdVs, PIVs, and BoVs were not analyzed in detail. Despite these limitations, our study provides a comprehensive overview of RSV circulation patterns during the COVID-19 pandemic. Policymakers can utilize this information to devise effective strategies for controlling the transmission of RSV and preventing future epidemics. We identified complete genome sequences of one or more respiratory viruses in addition to the RSV sequence in some strains, a unique finding not present in other publications.

This study found a high incidence of viral respiratory infections, particularly RSV, during the first season after anti-COVID-19 restrictions were lifted. The high activity of RSV is likely a result of diminished population immunity and the accumulation of vulnerable individuals, particularly children, due to prolonged low exposure to natural infections. We identified RSV as the primary cause of severe respiratory illnesses in young children. This study emphasizes the need for ongoing local and global surveillance of this pathogen. Phylogenetic and molecular analyses of RSV play crucial roles in identifying new epidemic strains, tracking the evolutionary and epidemiological patterns of viruses, and evaluating the impact of genetic variation on the transmissibility, virulence, and effectiveness of preventative vaccines and medications.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The studies involving humans were approved by Institutional Review Board and Ethics Committee of the NCIPD (IRB Number 00006384). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

NK: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Methodology. IT: Data curation, Formal analysis, Investigation, Methodology, Visualization, Validation, Writing – review & editing. IM: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. IA: Methodology, Resources, Writing – review & editing. IU: Data curation, Formal analysis, Writing – review & editing. II: Data curation, Formal analysis, Writing – review & editing. PV: Data curation, Formal analysis, Writing – review & editing. TT: Data curation, Formal analysis, Writing – review & editing. IC: Formal analysis, Supervision, Writing – review & editing, Data curation.