Molecular detection and characterization of respiratory syncytial virus B genotypes circulating in Pakistani children

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections in young children, but very little is known about its epidemiology and circulating genotypes in Pakistan. This study analyzed the epidemiological and molecular characteristics of RSV B genotypes in Pakistani children below 5 years with acute respiratory tract infections (ARIs) during three consecutive winter seasons from 2010 to 2013. A total of 1941 samples were analyzed for RSV infection by real time PCR and 24% (472/1941) samples were found positive out of which 22.3% (105/472) were sub-typed as RSV-B. The frequency of outpatient cases was higher (62.5%; 295/472) as compared to hospitalized patients (37.5%; 177/472). Patient ages ranged from 2 month to 5 years with a mean age of 1.48 ± 1.2 (years) and a median age of 1 year. Children below one year made up the highest percentage of enrolled subjects and male to female ratio of RSVB positive cases was nearly equivalent (1:1.1). The most common clinical symptoms were cough (96%), fever (80%) and sore throat (50%). All Pak RSVB strains ascribed to the BA genotype showing 91.9-97.1% and 86.2-95.3% homology at the nucleotide and amino acid levels respectively in comparison to BA prototype strain. On phylogenetic analysis, three genotypes of Pakistan RSV B viruses were observed; BA-9 and BA-10 which have been reported previously from other regions, and a third novel genotype assigned as BA-13 which formed a distinct cluster with protein length of 319 AA and showed 9-11 unique AA substitutions. All the RSV B isolates had two potential N-glycosylation sites in HVR2 of G protein and with heavy O-glycosylation of serine and threonine residues (G scores of 0.5-0.7). This study highlights the diversity of RSVB viruses and the significance of RSV as a dominant viral etiologic agent of pediatric ARI. It also emphasizes the need for continued molecular surveillance for early detection of prevalent and newly emerging genotypes to understand epidemiology of RSV infections in various regions of Pakistan.