Genotyping of Type A Human Respiratory Syncytial Virus Based on Direct F Gene Sequencing

Abstract

Background and objectives: The human respiratory syncytial virus (hRSV) is among the important respiratory pathogens affecting children. Genotype-specific attachment (G) gene sequencing is usually used to determine the virus genotype. The reliability of the fusion (F) gene vs. G gene genotype-specific sequencing was screened. Materials and Methods: Archival RNA from Saudi children who tested positive for hRSV-A were used. Samples were subjected to a conventional one-step RT-PCR for both F and G genes and direct gene sequencing of the amplicons using the same primer sets. Phylogeny and mutational analysis of the obtained sequences were conducted. Results: The generic primer set succeeded to amplify target gene sequences. The phylogenetic tree based on partial F gene sequencing resulted in an efficient genotyping of hRSV-A strains equivalent to the partial G gene genotyping method. NA1, ON1, and GA5 genotypes were detected in the clinical samples. The latter was detected for the first time in Saudi Arabia. Different mutations in both conserved and escape-mutant domains were detected in both F and G. Conclusion: It was concluded that a partial F gene sequence can be used efficiently for hRSV-A genotyping.

Keywords: F gene; G gene; Saudi Arabia; children; genotyping; hRSV; respiratory diseases.

Figure 1

Phylogenetic trees of the nucleotide sequences of partial fusion (F) and attachment (G) genes of human respiratory syncytial virus A (hRSV-A) strains from Saudi Arabia in comparison to relevant published strains. (a) F gene; (b) G gene. The maximum likelihood tree was constructed using MEGA 5.2 freeware. Strains detected in the current study are shown in blue color. Only bootstrap values above 70% are shown.

Figure 2

Deduced amino acid sequences of partial fusion proteins from different Saudi hRSV-A strains. Highlighted are the signal peptide (1–22) (blue box), heptad repeat domain C (75–97), cleavage site-1 (CS-1) and cleavage site-2 (CS-2) at Arg109 (NSRARR↓E) and Arg136 (KKRKRR↓F) (red color), p27 (110–136) (green box), fusion peptide (FP) (137–155), and heptad repeat domain A (153–end of the current sequence). The N-glycosylation sites, NXT/S, where X is not a proline, are underlined.

Figure 3

Deduced amino acid sequences of the partial attachment protein (G) from different Saudi hRSV-A strains. The N-glycosylation sites, NXT/S, where X is not a proline, are underlined. Duplicated regions in the ON1 strains are shown in the boxes.