Whole gene analysis of a genotype G29P[6] human rotavirus strain identified in Central African Republic

Rotavirus A (RVA) remains the main causative agent of gastroenteritis in young children and the young of many mammalian and avian species. In this study we describe a RVA strain detected from a 6-month-old child from Central African Republic (CAR). We report the 11 open reading frame sequences of a G29-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 rotavirus strain, RVA/Human-wt/CAR/CAR91/2014/G29P[6]. Nine genes (VP1–VP3, VP6, NSP1–NSP5) shared 90–100% sequence similarities with genogroup 2 rotaviruses. Phylogenetically, backbone genes, except for VP3 and NSP4 genes, were linked with cognate gene sequences of human DS-1-like genogroup 2, hence their genetic origin. The VP3 and NSP4 genes, clustered genetically with both human and animal strains, an indication genetic reassortment human and animal RVA strains has taken place. The VP7 gene shared nucleotide (93–94%) and amino acid (95.5–96.7%) identities with Kenyan and Belgian human G29 strains, as well as to buffalo G29 strain from South Africa, while the VP4 gene most closely resembled P[6]-lineage I strains from Africa and Bangladesh (97%).


Patient and sample collection
In 2014, a 6-month-old child was hospitalized for acute diarrhea with fever and vomiting at the Complexe Pédiatrique, Bangui (CPB), CAR. The patient had no record of exposure to animals. Diarrheic stool specimen was collected and tested at surveillance site laboratory for RVA antigen by using the Rotaclone EIA kit (Premier Rotaclone ™ , Meridian Diagnostics, Cincinnati, OH, USA), according to the manufacturer's instructions. RVApositive stool specimen was shipped on ice packs to the Centers for Disease Control and Prevention (CDC) for genotyping and sequencing analyses.

RVA dsRNA extraction, RT-PCR, sequencing and genotype assignment
RNA was extracted from stool using the QIAamp Viral RNA mini kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). The sequencing templates were prepared using sequence independent whole-genome reverse transcription-PCR (RT-PCR) amplification [7] with slight modifications. The amplified cDNA amplicons were sequenced using the Illumina MiSeq reagent kit v.2, 500 cycles and the standard 250 bp paired-end reads method. Illumina sequence reads were analyzed using CLC Genomics Workbench 11.0 (http:// www. clcbio. com/ produ cts/ clc-genom ics-workb ench/) incorporating a combination of de novo-and referenceguided assemblies to generate contigs and consensus sequences to obtain the complete open reading frame (ORF) sequences of strain CAR91/2014. Genotypes were determined using RotaC 2.0 (http:// rotac. regat ools. be/) [8].

Phylogenetic and genetic analyses
For each gene, multiple alignments were made by using the MUSCLE algorithm implemented in MEGA6 software [9], http:// www. megas oftwa re. net/). Once aligned, the DNA Model Test program implemented in MEGA version 6 was used to identify the optimal evolutionary models that best fit the sequence datasets. Using Corrected Akaike Information Criterion (AICc), the following models were found to best fit the sequence data for the indicated genes: GTR + G + I (VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, and NSP3), GTR + G (NSP4), and HKY + G (NSP5). With these models, maximumlikelihood trees were constructed using MEGA 6 with 1000 bootstrap replicates to estimate branch support. Nucleotide and amino acid distance matrices were prepared using the p-distance algorithm of MEGA 6 software [9].  like strains belonging to R2, C2, M2, I2, A2, N2, T2, E2 and H2 genotypes (data not shown).

Discussion
Here we present the sequence of strain CAR91/2014 which represents the fourth report of the G29 genotype and the first complete sequence for a G29P [6] RVA strain combination detected in humans. A unique feature of this CAR91/2014 strain is that the genes that constitute the genetic backbone belong exclusively to DS-  [6] G29 strains with complete sequences displayed the backbone I2-R2-C2-M2-A11-N2-T6-E2-H3,typical of artiodactyl RVA strains. Phylogenetically, the VP7 gene of the strain CAR91/2014 occupied the basal position of the G29 strains, suggesting that the CAR91/2014 might be the ancestral origin of all the G29 genotype that have been detected so far. Analysis of the genetic backbone of the CAR91/2014 strain shows that all the genes, with the exception of the VP3 and NSP4, appears to be human RVA and are phylogenetically linked to human DS-1-like genogroup 2 G9P [6], G12P [6], equine-like G3P [8], reassortant G1P [8] and G3P [4] strains from Australia, Uganda, Brazil, South Africa, Philippines, Thailand, Japan and Hungary. Phylogenetically, the VP3 and NSP4 genes of strain CAR91/2014 clustered together with both human and animal RVA strains, suggesting that the VP3 and NSP4 genes of ovine, buffalo and bovine strains might have originated from a genetic reassortment between human and animal strains. Unfortunately, the complete genome of the Kenyan G29 strain RVA/ Human-wt/KEN/0279/2010/G29P[X] was not available for comparison. In a nutshell, the closeness of the genetic backbone of strain CAR91/2014 to those of several DS-1-like strains including reassortant G1P [8] and G3P [8] is an indication that any of these strains might have donated these genes to the CAR91/2014 strain.
Analysis of RVA complete-ORF sequences from all 11 genes provides valuable data to better understand the contemporary diversity among RVA strain and helps to expand our knowledge of the genetic diversity and origin of uncommon RVA genotypes such as CAR91/2014.

Limitations
This manuscript describe characterization of a single G29P [6] strain and there might be others in circulation in Central African Republic. Also, rotavirus vaccine has not been introduced in Central African Republic, hence no post vaccine data is included in this study.