**4. Discussion**

Both of the designed generic primers were successful in amplification of the three di fferent genotypes of hRSV-A, ON1, NA1, and GA5, that were detected among Saudi archival RNA samples. It is also assumed that the generic F primer set could detect all hRSV-A strains, since it showed complete identities to most of the published hRSV-A strains except for a single mismatch detected occasionally. Genotyping based on partial gene sequencing of both F and G genes showed similar results, which denotes that the designed F gene could be used e fficiently in genotyping of hRSV-A strains.

The Saudi strains in the current study are classified as NA1, ON1, and GA5 genotypes. Both NA1 and GA5 showed higher virulence to the children in comparison to ON1 and other hRSV-A strains [24–26]. Two out of the thirteen strains were related to GA5, and this represents the first record of this subtype in Saudi Arabia. In Saudi Arabia, only a few studies have been conducted on hRSV genotypes; however, the hRSV-A type, mostly the NA-1 subtype, is the dominant genotype [32,33]. The hRSV-A ON1 genotype was detected for the first time in Canada in 2010 [23]. A Saudi strain in the GenBank database was found to be closely related to the ON1 strain isolated in 2009 in Riyadh and also closely related to ON1 strains detected in the current study from Al-Taif. This finding reveals that the ON1 strains have circulated in Saudi Arabia since 2009. This assumption is confirmed by the global data that suggested that the ancestor of ON1 emerged during 2008–2009 [34].

It is known that the F protein is focused on the signal peptide, p27, transmembrane domain, and ø antigenic site [35]. Similarly, the highest variability among amino acids among Saudi strains was detected in the signal peptide, followed by the p27 domain of the fusion protein. The fusion protein of hRSV strain harbors six N-glycosylation sites [9,36], which were confirmed to be conserved among the current Saudi strains, except for strain Taif-103 (A/ON1), which has lost one of the N-glycosylations at N120. It is known that N-glycosylation is important for the folding and transport of viral proteins and hence virus infectivity [37]. It is worth mentioning here that the three N-glycosylation sites—N116, N120, and N126—are found in close proximity to the proteolytic cleavage site [38,39]. It seems that the T122A amino acid substitution that resulted in a loss of the N-glycosylation site in Taif-103 does not affect the fusion or the proteolytic a ffinity of the protein, as confirmed previously [38].

The deduced amino acid sequence of the G gene revealed the presence of L298P, V303A, and Y304H amino acid substitution in all ON1 Saudi strains. Y273N was detected in Taif 23 and L274P was detected in the Taif-24 and Taif-103 strains. Such substitutions are considered noteworthy as they are present next to aa 265–273 (antigenic site) [40]. The P310L was recorded in the majority of the Saudi strains. Positively selected residues reported by Botosso et al., were found to be conserved in the Saudi ON1 and NA1 strains, except at the residues T237D and L274P [41] that constituted escape-mutant screened monoclonal antibodies [42,43]. The N273 N-glycosylation site was lost in the Saudi NA1 and most of the ON1 strains, which was also observed in NA1 strains from Japan and China [11,20]. Absence of the N250 site in all Saudi strains except for Saudi GA5 strains was expected, since it is a specific N-glycosylation site for both GA5 and SAA1 of hRSV-A [11]. The variation of the number of N-glycosylation sites was found to be associated with altered antigenicity [44].
