V.L. N. S. Raghuram1, Wajihul Hasan Khan2, Shama Parveen2, Shobha Broor1
1Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
2Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.
Human respiratory syncytial virus (HRSV) is the commonest etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. RSV strains have been classified into two broad groups, A and B. The G protein of the virus has the highest antigenic and genetic variability between and within the groups. The second hyper variable region reflects overall G protein gene variability and has been analyzed in molecular epidemiological studies. BA genotype of group B RSV has 60bp duplication in G protein gene as compared to the prototype strain (CH18537). BA viruses were first reported in 2003 from Argentina. Subsequently they have been reported from different parts of the world including India. For the analysis of G genes, stored aliquots of 39 nasopharyngeal aspirate samples (NPAs) which were positive for group B HRSV by N gene PCR were selected. A Semi-nested PCR was carried out to amplify IInd hypervarible region of G gene. Out of 39 NPAs, fourteen amplicons were sequenced. Analysis was carried out for phylogentic relatedness and predictive amino acid changes were studied in comparison with 113 partial G gene sequences downloaded from Genbank including group B and BA genotype reference strains using MEGA 5.0 and BioEdit softwares. Of thesefull length G genes of 9 strains were amplified and sequenced. Phylogenetic analysis of these full length sequences were also carried out. Full length G genes were also analyzed for Potential O-linked and N-linked glycosylation sites using NetOGlyc 3_1 server and NetNGlyc 1 servers. HRSV group B strains have earlier been classified in to 12 reported genotypes. Out of 14 sequenced partial G genes one sequence did not have 60bp duplication and has fallen in to a new genotype. Rest thirteen sequences exhibited 60bp duplication in IInd hyper variable region and belonged to BA genotype. The BA genotype of RSV has been previously classified in to 6 lineages, (BAI-BAVI), however in this study using additional sequences from genebank and study sequences 10 lineages of BA genotype have been identified. All BA sequences from this study closely clustered with BA/100/04 sequence of previously described BAIV lineage which is now falling in to BAX lineage. A 6bp deletion in Ist hypervariable region was also identified in all 9 full length G gene sequences. All the nine sequenced full length G genes had a putative 310 amino acid length of the G protein due to the 6 base pair deletion which is different from earlier reported 312 aa predicted length of G protein. Sequence length polymorphism was identified in the study sequences due to changes in stop codon positions. G protein of BA viruses has around 100 O-glycosylation sites, only 55-67 sites have the potential for glycosylation. Using the program Net-O-Glyc, 12-18 and 45-49 serine and threonine residues among the full length G gene sequences compared in this study were predicted. Putative O-glycosylation sites are mainly concentrated at I and II hyper variable regions of the G protein Potential glycosylation sites in the duplicated region are less in comparison to its counterpart region. Only 5 N glycosylation sites were predicted using NetNGlyc software. Read more…