This study was approved by the IRB Committee of King Abdullah International Medical research center (KAIMRC), National Guard Health affaires, Riyadh, KSA (reference number # RC08-112). All the samples were taken as part of standard care.
A total of 541 stool samples were collected from ongoing prospective study assessing the rotavirus epidemiology and genotypes. These isolates were taken from pediatric patients with acute diarrhea in the city of Riyadh between 2011 and 2012, which represents the main urban center in Saudi Arabia. Each stool sample was immediately frozen undiluted at −20°C until use.
Epidemiological data and data analysis
Epidemiological data including age, sex, and symptoms (diarrhoea and/or vomiting and their number of episodes), feeding type, date of onset, and date of sample collection were collected using Epic. Info vr.4 and molecular genotyping data were entered into Excel worksheets. Descriptive analysis, frequencies and percentages were calculated using SPSS vr. 20 statistical software.
ELISA screening for rotavirus
All stool samples were screened using ImmunoCard Stat Rotavirus kit® which detects the presence of rotavirus antigen in stool based on enzyme-linked immunosorbent assay (ELISA) method. Briefly, the stool specimen was diluted 1:15 in sample diluents and thoroughly mixed. The suspension was introduced (50 μl) to the sample port of the device and rotavirus detection was carried out following the manufacturer’s instructions. In addition to the internal (test) control, we included two external controls from the previously tested (known) samples.
All positive ELISA samples were confirmed by RT-PCR. In brief, RNA was extracted from approximately 1 mg of stool sample using MagNA Pure Compact RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) and following the manufacturer’s instructions. Extracted RNA was immediately stored at - 80°C until further use.
Reverse transcription (RT) and PCR amplification reactions
RT reactions were carried out in a final volume of 60 μl using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) on a Tetrad 2 Peltier thermal cycler from BIO-RAD. A master mix was prepared containing 5× RT buffer, 40 U/μl RNAse inhibitor, 20U/μl Transcriptor reverse transcriptase, 10 mM each Deoxynucleotide Mix, 600 μM Random Hexamer Primer, RotaA.ext. Fwd primer 5′-TTT AAA ACG AAG TCT TCR ACA TGG AKG TYC TGT A-3′ and RotaA.ext. Rev primer 5′-TAA TTG GTG ATC TAC CAA TTC CTC CAG TTT G-3′ . RNA sample (10 μl) was heated at 99°C for 10 min, placed immediately on ice, and 50 μl of master mix was then added to each tube. The temperature cycling was 50°C for 50 min, 94°C for 7 min and cDNA was amplified using RotaA.ext. forward and reverse primers . The temperature cycling was 95°C for 10 min, 40 cycles of (94°C for 1 min, gradient from 45°C to 65°C for 3 min, 72°C for 1 min), and final extension at 72°C for 10 min.
Genotyping by PCR amplification and sequencing
ELISA positive rotavirus samples (n = 171) were further confirmed by RT-PCR using primers set used for rotavirus genotyping VP4 [P] and VP7 [G] genotypes. Genotyping was performed using the following oligonucleotide primer pairs (Eurofins MWG Operon) targeting the VP4 and VP7 gene regions: VP4-F 5′-TAT GCT CCA GTN AAT TGG-3′, VP4-R 5′-ATT GCA TTT CTT TCC ATA ATG-3′, VP7-F 5′-ATG TAT GGT ATT GAA TAT ACC AC-3′ and VP7-R 5′-AAC TTG CCA CCA TTT TTT CC-3′[13,14]. Thermal cycling for the VP4 region was 95°C for 10 min, 35 cycles of (94°C for 1 min, 50°C for 1 min, 72°C for 1 min), then 72°C for 10 min. Thermal cycling for the VP7 region was 95°C for 10 min, 40 cycles of (94°C for 1 min, gradient from 45°C to 65°C for 3 min, 72°C for 1 min), then 72°C for 10 min. Amplified products were analyzed on 1.5% agarose ethidium bromide-stained gels for genotyping. All PCR products of the appropriate size were purified using 3 M sodium acetate and absolute ethanol. Purified products were confirmed by sequinning using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied biosystems™ Austin, TX, USA). Thermal cycling was 96°C for 1 min, 40 cycles of (96°C for 10 min, 50°C for 5 sec, 60°C for 4 min). Amplified products for VP4 (663 bp) and VP7 (880 bp) were purified a second time using XTerminator™ and SAM™ solutions (Applied Biosystems™ Foster City, CA, USA). Purified products were sequenced using 3730xl DNA Analyzer (Applied Biosystems™, Hitachi, Tokyo, Japan).
Assignment of the genotypes and phylogenetic analysis
All isolates sequences were analyzed in comparison to VP4 and VP7 of the international rotavirus sequences available at NCBI sequence database using BALSTn-2 and RotaC 2.0  automated genotyping tool for Group A rotaviruses. Molecular phylogenetic analysis was carried out using Maximum Likelihood method based on Tamura-Nei model . The tree was drawn to scale with branch lengths measured in the number of substitutions per site and was bootstrapped with 1000 replicates. The analysis involved 635 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 681 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 .