Prevalence and genetic characterisation of respiratory syncytial viruses circulating in Bulgaria during the 2014/15 and 2015/16 winter seasons

Abstract

The respiratory syncytial virus (RSV) is a leading cause of acute respiratory illnesses (ARI) in infants and young children. The objectives of this study were to investigate the RSV circulation among children aged <5 years in Bulgaria, to identify the RSV-A and RSV-B genotypes and to perform an amino acid sequence analysis of second hypervariable region (HVR2) of the G gene. During the 2014/15 and 2015/16 winter seasons, nasopharyngeal specimens of 610 children aged <5 years with ARI were tested using Real Time RT-PCR for influenza viruses, RSV, metapneumovirus, parainfluenza viruses, rhinoviruses and adenoviruses. Viral respiratory pathogens were detected in 429 (70%) out of 610 patients examined and RSV was the most frequently identified virus (26%) followed by influenza A(H1N1)pdm09 virus (14%) (p < .05). RSV was the most prevalent pathogen in patients with bronchiolitis (48%) and pneumonia (38%). In the 2014/15 season, RSV-A dominated slightly (53%), while in the next season RSV-B viruses prevailed more strongly (66%). The phylogenetic analysis based on the G gene indicated that all 21 studied RSV-A strains belonged to the ON1 genotype; the vast majority (96%) of the RSV-B strains were classified into BA9 genotype and only one - into BA10 genotype. All Bulgarian RSV-A and RSV-B sequences contained a 72-nt and a 60-nt duplication in the HVR2, respectively. The study showed the leading role of this pathogen as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, a shift from RSV-A to RSV-B subgroup dominance and relatively low genetic divergence in the circulating strains.

Keywords: Acute respiratory infection; amino acid substitution; genetic characterization; respiratory syncytial virus.

Figure 1.

Weekly distribution of RSV detected during the 2014/15 and 2015/16 seasons in Bulgaria.

Figure 2.

Age distribution of children infected with respiratory viruses.

Figure 3.

Phylogenetic trees based on the G gene sequences of RS-A (a) and RSV-B (b) strains. Phylogenetic trees were constructed using maximum-likelihood method with 1000 bootstrap replicates running within MEGA 6.06 software. Only bootstrap values ≥70% are displayed at the branch nodes. The scale bar indicates the number of nucleotide substitutions per site. The GenBank name of the strains, the country and the year of isolation are shown in the phylogenetic trees. The genotypes are denoted by lines on the right side. The ON67–1210A and BA/100/04 strains, prototype for the ON1 and BA9 genotypes, respectively, are indicated in bold. Bulgarian RSV strains detected during the 2014/15 and 2015/16 seasons are indicated in green and blue, respectively.

Figure 4.

Deduced amino acid alignments of the second variable region of the G-protein from RSV-A (a) and RSV-B (b) strains. The alignment (a) is shown relative to the sequence of reference RSV-A strain A2 (GenBank accession number M11486) and the alignment (b) is shown relative to the sequence of prototype BA strain BA4128/99B (GenBank accession number AY333364). The amino acid numbers correspond to G protein positions 212 to 298 of the strain A2 (a) and to G protein positions 213 to 315 of the strain BA4128/99B (b). Identical residues are identified as dots. Dashes indicate gaps corresponding to the nucleotide insertions; asterisks indicate the stop codons. The outlined rectangles represent the two copies of the duplicated 23-amino acid region in RSV-A strains (a) and the two copies of the duplicated 20-amino acid region in RSV-B strains (b). Genotype names are shown on the right. Light grey shading highlights the predicted N-glycosylation sites. Black circles denote the predicted O-glycosylation sites of the A2 reference strain (a) and BA prototype strain BA4128/99B (b); predicted O-glycosylation sites of Bulgarian strains are indicated by unfilled circles.