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Acute respiratory infections (ARI) are associated with high morbidity and mortality rates at the global level. Approximately 20% of the deaths attributable to pneumonia globally occur in Pakistan with nearly one half the infections among children younger than 5 years of age. The objectives of this study were to define the molecular epidemiology of respiratory viruses in Pakistani children and to identify viral genotypes causing respiratory infections. In this study, we analyzed 1941 respiratory samples collected over three winter seasons and tested for common respiratory pathogens using real time PCR. We found 76% prevalence rate for viral pathogens with Influenza and RSV predominant among 0.01-6 and 12-24 months age groups respectively. Fever and cough were identified as significant clinical parameters along with malnutrition and maternal breastfeeding. Furthermore, phylogenetic analysis of representative Influenza A/H3N2 viruses showed that recent viruses belonged to subgroup 3C.3 and carried important amino acid substitutions in the H3 hemagglutinin, while no substitutions attributable to neuraminidase resistance were detected. We genotyped the Respiratory Syncytial viruses subtypes and analyzed the two surface glycoproteins G and F of over100 isolates. All RSV-A strains carried 297 amino acids long G protein and clustered in the NA1/GA2 genotype. We also reported a novel RSV B genotype BA-13 and two other genotype variants (BA-9 and BA-10). We analysed the fusion protein F in RSV A isolates at the binding sites for the monoclonal antibodies Palivizumab and Motavizumab specifically and found that the N276S substitution in over 90% of RSVA and all RSVB viruses. This work highlights the need for respiratory disease surveillance as it will support the development of appropriate public health surveillance programs to inform health planning.
