*2.2. Samples*

A total of 59 archived RNA/DNA samples that showed positive real-time RT-PCR results for hRSV-A, determined in our previous study [28], were used as targets for amplification of F and G genes. The RNA samples were stored at −80 ◦C. All samples were collected previously from children with respiratory distress from hospital of Paediatrics, Taif, Saudi Arabia, during the period from January to May 2012. The viral load in the samples (*n* = 56) samples were found to be weak, with a range of 1 × 10<sup>2</sup> to 1 × 104.5 copies/mL [29].

#### *2.3. One-Step Conventional RT-PCR*

An F gene-specific oligonucleotide primer set: Fus-For (1–29) 5-ATGGAGTTGCCAATCCTCA AAGCAAATGC-3 and Fus-Rev (622–643) 5-ATATGCTGCAGCTTTGCTTGTT-3, that flanks F2 and the first part of F1 (FP and HRA), was designed based on hRSV-A-GZ08-0 (KP218910). G gene genotype-specific primers for ON1/NA1 and GA5 that flank HVR2 in the C-terminal region of the G gene were also designed and used for amplification of the partial G gene based on the result of F gene sequencing. G gene-specific primer sets were as follows: ON1/NA1 (365-384)-For 5-CTGAGTC AACCCCACAATCC-3, ON1/NA1-Rev (24–43 G–F intergene UTR) 5-ATTTGGTCATG GCTTTTTGC-3 based on based on hRSV-A-GZ08-0 (KP218910) and GA5-For (374–395) 5-TCCT GCAATCTACAACAGTCAA -3 and GA5-Rev-(867–889) 5-CTGTTATGTTGGATGGAGATG-GA-3

based on RSVA/Homo sapiens/ITA/120/2009 (KF826832) were designed. Reverse transcription was conducted using GoTaq ® 1-Step RT-PCR (Promega, Southampton, UK) that was based on GoScript ™ Reverse Transcriptase. Briefly, the program was adjusted for reverse transcription at 45 ◦C for 45 min, followed by an initial denaturation step at 95 ◦C for 5 min and 35 cycles of 95 ◦C for 30 s, 50 ◦C for 30 s, and 72 ◦C for 1 min. This was followed by a final 10 min elongation step at 72 ◦C. The RT-PCR amplicons were subjected to 1.5% agarose gel electrophoresis.

#### *2.4. Direct Sequencing and Gene Sequence Analysis*

Amplicons with the expected size were excised from the gel and then purified using a gel-extraction kit (Koma Biotek, Seoul, Korea). The purified amplicons were used as templates for direct sequencing using the same primers as those used for the F and G genes' amplification. Sequencing was performed commercially using the BigDye v.3.1 Applied Biosystems (Foster City, CA, USA) kit according to the manufacturer's protocol. Raw sequences were visualized and analyzed using MEGA 5.2. Homology BLASTn searches from di fferent strains were performed using highly similar sequences (megablast) against published hRSV sequences in the GenBank databases using default algorithm parameters. The gene sequences of both F and G genes were deposited in GenBank under the accession numbers: F (KU924011-KU924023) and G (MK182708-MK182720). CLUSTAL W multisequence analysis was performed and the phylogenetic tree was constructed using the maximum likelihood statistical method with 1000 bootstrap replicates and Tamura–Nei model substitution model. Nucleotide variability among di fferent strains was calculated using the CLUSTALW available in GenomeNet Database [30].

#### *2.5. Deduced Amino Acid Sequence and Sequence Analysis*

Deduced amino acid sequences of both F and G amino acid sequences were compared using MEGA 5.2. Amino acid substitutions at di fferent functional and structural protein fragments were analysed. The polyphen (polymorphism phenotyping) prediction webtool [31] was used to screen the potential e ffect of amino acid substitutions among strains on the function.
