Phylogenetic and phylodynamic analysis of respiratory syncytial virus strains circulating in children less than five years of age in Karachi-Pakistan

Respiratory syncytial virus (RSV) is one of the leading causes of infant morbidity and mortality worldwide, especially in Pakistan. To date, few studies have explored RSV epidemiology in different areas of Pakistan. However, none have performed comprehensive phylogenetic and phylodynamic analyses of RSV strains. This study presents a comprehensive genetic and phylodynamic analysis of RSV strains in children less than five years old in Karachi, Pakistan. This study used retrospectively collected nasopharyngeal (swab) samples from 155 children with qPCR-confirmed RSV infection. The samples were used to perform RSV genotyping using PCR employing RSV glycoprotein gene-specific primers. The RSVA and RSVB genotyping was performed using BLAST and Maximum-likelihood (ML) phylogenetic methods. Similarly, the relationship with other RSV strains was analyzed using ML phylogenetic cluster analysis. The RSVA and RSVB mean genetic diversity and coefficient of differentiation were calculated using MEGA7 software. Furthermore, the time to the most common recent ancestor (tMRCA) and effective population size of RSV genotypes A and B were estimated using a Bayesian MCMC analysis. Finally, site selection pressure and glycosylation analyses were performed using FUBAR and NetNGlyc/NetOGlyc tools. Out of 155, 98 and 57 sequences were RSVA and RSVB, respectively. The tMRCA was estimated to be around 2002 and 2005 for RSVA and RSVB, respectively. RSVA sequences formed two NA1 genotype clusters, comprising 95 and three sequences, respectively. RSVB formed three clusters, where 24 and two sequences clustered with BA9 and BA12 genotypes, respectively, while 31 sequences formed a unique cluster. The RSVA and RSVB glycoprotein gene sequences exhibited N- and O- glycosylation and selection pressure at several sites. RSV B exhibited slightly higher (0.042) nucleotide diversity per site (π) as compared to RSVA (0.019). Our results suggest that RSVA and RSVB strains in Pakistan exhibit distinct genotypic clusters and differ in their estimated tMRCA. Additionally, both genotypes showed glycosylation and selection pressure at specific sites, with RSVB exhibiting higher nucleotide divergence per site (π), indicating its potential to undergo further evolutionary changes and adaptation. Overall, this study provides unique insights into RSV molecular epidemiology. The study may also help improve our understanding of RSV evolutionary changes and the emergence of new genotypes in different region