Fecal specimens were collected from children <5 years of age who sought medical care for acute diarrheal illness, defined as >3 liquid stools over a 24-h period. Stool specimens were collected in sterile containers at the microbiology laboratory in Saint Camille Medical Center, in Ouagadougou, Burkina Faso. A 10% (wt/vol) stool suspension was prepared, and 3 aliquots were frozen at −20°C for additional analysis. All ROTAV-positive fecal specimens collected during the ROTAV season (December 2009–March 2010) at the Saint Camille Medical Center and the Biomolecular Research Center Pietro Annigoni, were analyzed. The samples were part of a larger epidemiologic study conducted during May 2009–March 2010 to identify the enteropathogens causing gastroenteritis in children (26).

Clinical information was obtained by reviewing the clinical records of case-patients, as described (26). In brief, information was obtained regarding age, sex, place of residence, ethnicity, signs and symptoms and their duration (e.g., fever [temperature >38°C], nausea, vomiting, loss of appetite, and number of loose stools during the past 24 h), hydration and nutrition status, and whether the children had been given antimicrobial and antiparasitic drugs. All children were clinically evaluated by general practitioners in accordance with a local adaption of the World Health Organization strategy for diarrheal management (28). Dehydration was classified as follows, according to the World Health Organization guidelines: severe dehydration, some dehydration, or no dehydration.

We used the ProSpect Rotavirus R240396 (Oxoid, Kamstrupvej, Denmark) enzyme immunoassay kit, according to the manufacturer’s instructions, to detect group A human ROTAV in fecal specimens. The results were determined visually and confirmed by absorbance readings.

Viral RNA was extracted from 10% stool suspensions by using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A total of 60 μL of viral RNA was collected and stored at −70°C until further use.

Reverse transcription (RT) was performed essentially as described (29). Twenty-eight μL of dsRNA was mixed with 2.5 μg of random hexadeoxynucleotides (pd[N]₆ primer; GE Healthcare, Uppsala, Sweden), denatured at 97°C for 5 min, and chilled on ice for 2 min. The suspension was then added to 1 RT-PCR bead (GE Healthcare) with RNase-free water to a final volume of 50 μL. The RT reaction was performed for 30 min at 42°C for cDNA synthesis.

All ROTAV-positive specimens were quantified and subgrouped by using the LUX (light-upon-extension) real-time quantitative PCR (qPCR) as described (30). This qPCR uses labeled primers with different fluorophores for each VP6 subgroup, and external plasmid standards were used for quantification (30). The quantified ROTAV was detected in a range from ≈5.0 × 10⁴ to 7 × 10¹¹ gene equivalents per gram of feces; median concentration was 7.8 × 10⁹.

G and P genotyping for ROTAV was performed by using seminested type-specific multiplex PCRs that were able to detect 7 G types (G1, G2, G3, G4, G8, G9, G10) and 6 P types (P[4], P[6], P[8], P[9], P[10], P[11]) (31–33). The PCR products were later visually examined on a 2% agarose gel stained with ethidium bromide and observed in ultraviolet light. The G and P types were determined by the specific sizes of the amplicons on agarose gels.

Sequencing of the PCR amplicons for the VP7 gene of unknown G types was conducted with primers VP7-F and VP7-R (33). All G6P[6] strains were further sequenced for VP4 and VP6 genes by using primers Con-2/Con-3 (32) and GEN_VP6F/GEN_VP6R (7), respectively. Furthermore, to understand the evolution of these G6P[6] ROTAV strains, we randomly selected 4 strains and sequenced the nonstructural protein (NSP) genes (NSP1, NSP2, NSP3, NSP4, and NSP5) by using primers GEN_NSP1F/GEN_NSP1R, MAX-NSP2F/ MAX-NSP2R, MAX-NSP3F/MAX-NSP3R, GEN_NSP4F/GEN_NSP4R, and MAX-11F/ MAX-11R, respectively (7). The following thermal cycling conditions were used: an initial denaturation step at 94°C for 5 min followed by 40 cycles of amplification (30 s at 94°C, 30 s at 50°C, and 1 min 30 s at 72°C), with a final extension of 7 min at 72°C.

Nucleotide sequencing was performed by Macrogen Inc. (Seoul, South Korea). The sequencing reaction was based on BigDye chemistry, and the sequencing primers were the same primers as those used in the PCR.

All chromatograms were visually inspected. Those containing multiple ambiguous base positions were considered to potentially represent multiple ROTAV strains and were analyzed by using the RipSeq mixed DNA interpretation software (iSentio Ltd., Bergen, Norway) (34).

Multiple sequence alignment of the obtained nucleotide sequences was performed by using the ClustalW algorithm (www.ebi.ac.uk/Tools/msa/clustalw2/) with default parameters on the European Bioinformatics Institute server. Phylogenetic analysis of the aligned file was performed by using MEGA5 (www.megasoftware.net) with the neighbor-joining method. Phylogenetic distances were measured by using the Kimura 2-parameter model. The statistical significance of the phylogenetic tree was supported by bootstrapping with 1,000 replicates. The sequenced ROTAV strains and GenBank accession numbers are shown in Technical Appendix Table 1; the nucleotide sequences for sequenced genes can be found in GenBank by using accession nos. JN116505-JN116556 and JQ255029-JQ255033.

Categorical data were analyzed by using the χ² test or Fisher exact test with 2-tailed significance. Interval data were analyzed by running an independent t-test with 2-tailed significance in SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).

The distribution of G and P types is shown in Table 1. We sequenced all unknown G types for which the VP7 amplicon could be generated and found that they belong to genotype G6 with P[6] specificity. Of the 100 ROTAV-positive specimens, 13 contained G6P[6], including 2 with mixed infections with other G types. We detected 5 G types: G1 (23% incidence rate), G2 (2%), G3 (9%), G6 (13%), and G9 (53%). Among the P genotypes, P[8] was most prevalent (61%), followed by P[6] (39%); P[4] was rarely detected (2%). The G-P combinations detected were G9P[8] (48%), G6P[6] (11%), G1P[6] (11%), G3P[6] (8%), G1P[8] (5%), and G2P[4] (2%). Mixed G or P type infections were found in 9% of the total specimens. Among the ROTAV-positive specimens, 6% and 1% could not be assigned a G and P type, respectively (Table 1).

In the first part of the ROTAV season (December 2009 and early January 2010), we observed a dominance of G9P[8] strains (Figure 1, panel A). Thereafter, the G9P[8] strains completely disappeared and other G types began to circulate (Figure 1, panel A). All G types observed during this period were associated with P[6] genotype. Moreover, at the end of the ROTAV season, the G6P[6] and G3P[6] strains emerged, and most of the mixed infections were detected during this period. Furthermore, we observed a temporal shift in subgroup specificity (Figure 1, panel B). We subgrouped the VP6 gene by using our qPCR assay, and of the 100 ROTAV-positive specimens, 39 belonged to SGI, 59 belonged to SGII, and 2 specimens contained a mix of both subgroups. In the middle of the ROTAV season, we observed a shift of subgroups, with SGI dominating toward the end of the ROTAV season and SGII dominating in the beginning (Figure 1, panel B).

All P[8] ROTAVs were associated with VP6 SGII, and all P[6] ROTAVs were associated with VP6 SGI. Children infected with ROTAV SGII had a mean age of 11.8 months (median 11.1 months), compared with a mean age of 15.0 months (median 13.0 months) for children infected with ROTAV SGI (p < 0.05). These observations correlate with the shift of VP6 subgroups and P types in the middle of the ROTAV season; children infected later were older than those who were infected early (data not shown). We compared clinical signs and symptoms and found no significant differences, but we did find higher prevalence of vomiting (p = 0.33), fever (p = 0.052), and severe dehydration (p = 0.09) among SGII-infected children (Table 2). We compared the 3 most prevalent G, P, and VP6 constellations (Table 2) and found no significant differences in age profiles; however, we found that ROTAVs of the G9P[8]SGII constellation were more frequent than G1P[6]SGI- and G6P[6]SGI-specific strains among younger children (p = 0.069). We also found that G9P[8]SGII infections, compared with SGI-associated G and P type infections, induced significantly more cases of severe dehydration (p<0.05). The ROTAV viral load, quantified by using the LUX qPCR, did not correlate with disease severity (data not shown).

At first, we observed a high number of G3/G9P[6] mixed infections as revealed by multiple bands in the multiplex PCR (data not shown). We sequenced the VP7 gene of these strains (n = 6) and found all 6 to have high nucleotide homology with G3 ROTAVs and to cluster together in a new sublineage (Figure 2, panel A). This sublineage had 96.0%–96.3% nt homology to the most similar G3 strain found in GenBank (Bethesda/DC1730). Furthermore, the G9 primer used in this study (33) was highly homologous (16 [80%] of 20 nt) to these G3 strains but less so to other G3 strains available in GenBank (maximum 14 [70%] of 20 nt) (Figure 2, panel B). Furthermore, the 14 bases from the 3′ end of the primer showed a complete match to the G3 strains from Burkina Faso, except at position 9, thereby facilitating unspecific primer annealing and polymerase extension. The VP7 gene of 6 G9P[8] samples was sequenced for verification which proved them to be G9. These findings led us to conclude that G9 ROTAVs appearing as mixed infections with G3P[6], as determined by multiplex G-typing PCR, were nonspecific amplifications resulting in G9 artifact.

We further characterized the unusual G6P[6] strains (n = 11) by sequencing the VP7, VP4, VP6, and NSP1–5 genes. All characterized VP4 and VP7 sequences clustered together, demonstrating high homology (Figure 3; Figure 4, panel A). The VP6 genes clustered in different lineages; 4 strains clustered with the B1711 strain, which is the only previously described human ROTAV G6P[6] strain (22), and 2 strains clustered closely with bovine strains (Figure 4, panel B). Phylogenetic analysis of the VP6 genes revealed that all strains clustered within lineage I2, which is typical of strains belonging to the DS-1 genogroup (35). However, the nucleotide difference between the 2 VP6 clusters was 7.7%–7.8%, strongly indicating different origins of the VP6 genes. To further characterize the G6P[6] strains from Burkina Faso, we sequenced the NSP1–5 genes for 4 of the G6P[6] strains (>80% nt coverage). All NSP genes of these 4 strains were highly homologous (>98.6% nt similarity); thus, we chose a representative isolate (272/BF) for comparison with reference strains (comparison results are shown in Technical Appendix Table 2).

The representative isolate, 272/BF, had a high level of identity with G6 (B1711) and G8 (DRC88) strains belonging to the DS-1 genogroup. The G6P[6] strains were classified as G6-P[6]-I2-A2-N2-T2-E2-H2, strongly suggesting that G6P[6] strains from Burkina Faso belong to the human DS-1 genogroup.