Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines. Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness. Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented. Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six–ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four–nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences. Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.

Respiratory syncytial virus (RSV) and rhinovirus (RV)—the two primary causative viruses of bronchiolitis in children—have been studied extensively in terms of their associations with disease severity and serious late disease outcomes. In this study, we explored the differences in the clinical values of IFN-γ and IL-10 serum levels in RSV and RV bronchiolitis in the Bulgarian childhood population. Eighty-eight children with acute bronchiolitis, aged two months to two years, who were admitted to the General Pediatrics Clinic of University Hospital “Prof. St. Kirkovich”, Stara Zagora, Bulgaria served as this study’s subjects. The degree of wheezing and respiratory failure were classified. Naso-pharyngeal swabs were collected from all participants, and molecular identification of viruses was performed using real-time PCR. Serum samples were used to determine IFN-γ and IL-10 quantities using ELISA kits, and data are presented as the median IQR (25–75%). The total serum IL-10 levels were significantly enhanced in RSV-infected children compared to those infected with RV (14.4 (12.2–24.0) vs. 8.9 (7.2–12.5); p < 0.001) and the other viral bronchiolitis groups (14.4 (12.2–24.0) vs. 6.65 (3.9–15.3); p = 0.003). The highest IL-10 levels (14.4 (12.8–27.9)) were found in RSV-positive patients with first-degree respiratory failure. Almost identical serum IFN-γ levels were determined for RSV- and RV-positive bronchiolitis patients (3.2 (1.6–6.8) and 2.8 (1.1–7.3); p = 0.781). Variance analysis of IL-10 serum levels revealed statistically significant differences among the patient groups depending on the type of viral infection, concerning respiratory failure (p = 0.005) and wheezing severity (p = 0.017). Our findings show that the IL-10 levels and the type of virus have a combined effect on disease severity. These data might contribute to patients’ personalized/individualized therapy and the prevention of recurrent wheezing later in life.

Нuman bocaviruses (hBoVs) are often associated with acute respiratory infections (ARIs). Information on the distribution and molecular epidemiology of hBoVs in Bulgaria is currently limited. The objectives of this study were to investigate the prevalence and genetic characteristics of hBoVs detected in patients with ARIs in Bulgaria. From October 2016 to September 2019, nasopharyngeal/oropharyngeal swabs were prospectively collected from 1842 patients of all ages and tested for 12 common respiratory viruses using a real-time RT-PCR. Phylogenetic and amino acid analyses of the hBoV VP1/VP2 gene/protein were performed. HBoV was identified in 98 (5.3) patients and was the 6^th most prevalent virus after respiratory-syncytial virus (20.4), influenza A(H1N1)pdm09 (11.1), A(H3N2) (10.5), rhinoviruses (9.9), and adenoviruses (6.8). Coinfections with other respiratory viruses were detected in 51 of the hBoV-positive patients. Significant differences in the prevalence of hBoVs were found during the different study periods and in patients of different age groups. The detection rate of hBoV was the highest in patients aged 04 years (6.9). In this age group, hBoV was the only identified virus in 9.7, 5.8, and 1.1 of the children diagnosed with laryngotracheitis, bronchiolitis, and pneumonia, respectively. Among patients aged 5 years, hBoV was detected as a single agent in 2.2 of cases of pneumonia. Phylogenetic analysis showed that all Bulgarian hBoV strains belonged to the hBoV1 genotype. A few amino acid substitutions were identified compared to the St1 prototype strain. This first study amongst an all-age population in Bulgaria showed a significant rate of hBoV detection in some serious respiratory illnesses in early childhood, year-to-year changes in the hBoV prevalence, and low genetic variability in the circulating strains.