We report characterization of a genotype G5P[7] human rotavirus (HRV) from a child in Cameroon who had diarrhea. Sequencing of all 11 gene segments showed similarities to
Group A rotaviruses are a major cause of severe diarrheal disease in infants, young children, and a variety of animals. In humans, rotavirus gastroenteritis results in deaths and hospitalizations; most deaths have occurred in developing countries (
Rotavirus surveillance and strain characterization, in support of rotavirus vaccine development programs, have detected many new human rotavirus (HRV) genotype specificities and highlighted the importance of mechanisms such as reassortment and zoonotic transmission in the evolution of rotaviruses (
During ARN surveillance conducted from 1998 through 2004, a total of 215 rotavirus-positive stool samples could not be typed by standard reverse transcription–PCR genotyping methods. Among untypeable samples, we identified a G5P[7] strain (designated 6784/2000/ARN), which represented a rare G genotype and a new P genotype specificity in humans. This strain was isolated from a stool specimen from a child with gastroenteritis in Kumba, Cameroon. Because G5 and P[7] genotype specificities are common in pigs, we studied the entire genomic composition of this strain to determine if it was an example of a strain that arose through direct interspecies transmission from a particular animal host, or by reassortment with heterologous rotavirus strains.
Gene fragments of the 11 gene segments of strain 6784/2000/ARN were amplified by using consensus primers for structural protein 4 (VP4), VP6, and VP7 (
| Primer* | Sequence (5′ → 3′)† | Gene‡ | Nucleotide position, strand | Amplicon size, bp | Strain | Reference |
|---|---|---|---|---|---|---|
| MDEVP1F | AAT CAC AAT CTG CAG TTC AAA | VP1 | 68–89, + | 337 | Ku | This article |
| MDEVP1R | AAT GAA TCA GTG TAT TCT TCG | VP1 | 405–384, – | Ku | This article | |
| MDEVP2F | CTG ACA AAG TGC TAT CAC A | VP2 | 156–175, + | 300 | Ku | This article |
| MDEVP2R | AGG TAA TTG TCT TGG TTC | VP2 | 456–438, – | Ku | This article | |
| MDEVP3F | TTG CTA GAT TGT CAA ATC GTG | VP3 | 597–618, + | 327 | Ku | This article |
| MDEVP3R | AAT AAG ATG GAG CTG AAC C | VP3 | 924–905, – | Ku | This article | |
| MDENSP1F | GAG ACC RTC AAC TCC TAC YAA | NSP1 | 120–141, + | 344 | Wa | This article |
| MDENSP1R | ATT GTA AYG TTA TTG GCA T | NSP1 | 464–445, – | Wa | This article | |
| MDENSP2F | GCT TGC TTT TGT TAT CCT | NSP2 | 58–76, + | 327 | Ku | This article |
| MDENSP2R | ATT TTC CAA ATG TCT AAC AG | NSP2 | 385–365, – | Ku | This article | |
| MDENSP3F | GCC ACT TCA ACA TTA GAA | NSP3 | 101–119, + | 303 | Ku | This article |
| MDENSP3R | TAC ACT AAA ACA AGC ATT AAG | NSP3 | 404–383, – | Ku | This article | |
| MDENSP5F | AGC GCT ACA GTG ATG TCT CT | NSP5 | 10–29, + | 337 | Ku | This article |
| MDENSP5R | CCA TTT GAT CGC ACC CA | NSP5 | 347–330, – | Ku | This article | |
| JRG30 | GGC TTT TAA AAG TTC TGT T | NSP4 | 1–19, + | 737 | Wa | This article |
| JRG31 | ACC ATT CCT TCC ATT AAC | NSP4 | 738–721, – | Wa | This article | |
| Con3 | TGG CTT CGC TCA TTT ATA GAC A | VP4 | 11–32, + | 876 | Ku | ( |
| Con2 | ATT TCG GAC CAT TTA TAA CC | VP4 | 887–868, – | Ku | ( | |
| 9con1-L | TA GCT CCT TTT AAT GTA TGG TAT | VP7 | 37–59, + | 896 | Wa | Modified from ( |
| VP7-Rdeg | GAC GGV GCR ACT ACA TGG T | VP7 | 933–914, – | Wa | Modified from ( | |
| VP6-F | GAC GGV GCR ACT ACA TGG T | VP6 | 747–766, + | 379 | ( | |
| VP6-R | GTC CAA TTC ATN CCT GGT G | VP6 | 1126–1106, – | ( |
*F, forward; R, reverse. †R, A or G; Y, C or T; V, A, C, or G; N, A, C, G, or T. ‡VP, structural protein; NSP, nonstructural protein.
Similarity matrices and phylogenetic trees based on nucleotide and amino acid sequences were constructed and compared with cognate gene sequences of human and animal rotaviruses. Except for the 2 gene segments, which encode neutralizing antigens VP7 and VP4, respectively, and are commonly encountered in porcine rotaviruses, the remaining 9 gene segments of 6784/2000/ARN were grouped in a common phylogenetic clade in which reference human strains of the Wa genogroup and related porcine rotaviruses also clustered (
| Strains† | Nucleotide/amino acid identity, % | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| VP1 | VP2 | VP3 | VP4 | VP6 | VP7 | NSP1 | NSP2 | NSP3 | NSP4 | NSP5 | |
| Ku/G1P[8]/Hu | 88/94 | 90/94 | 61/55 | 90/98 | 74/79 | 85/88 | 94/94 | 89/93 | 94/96 | ||
| DRC88/G8P[8]/Hu | 79/85 | 76/77 | 69/73 | – | 76/93 | 74/81 | 74/75 | 85/93 | 84/90 | 79/83 | 90/93 |
| OSU/G5P[7]/Po | – | – | 86/90 | 80/90 | 86/94 | 86/89 | – | 89/95 | 88/96 | ||
| RMC321/G9P[19]/Hu | 82/93 | 84/94 | – | – | 80/91 | – | 81/87 | 88/91 | 89/93 | 88/95 | 97/98 |
| Tb-chen/G2P[4]/Hu | 79/83 | 77/79 | 69/72 | 62/56 | 77/91 | – | 77/76 | 88/92 | 85/90 | 81/83 | 89/92 |
| AU-1/G3P[9]/Hu | 78/84 | 77/84 | 74/76 | 59/56 | 76/91 | 78/85 | 69/72 | 81/89 | 82/93 | 78/82 | 93/97 |
| ST-3/G4P[6]/Hu | – | – | 91/94 | – | – | – | 94/94 | – | 97/97 | 88/91 | – |
| 69M/G8P[10]/Hu | – | – | 71/75 | – | – | – | 77/76 | – | 82/88 | – | 94/95 |
| T152/G12P[9]/Hu | – | – | – | 60/57 | 76/91 | 75/82 | 70/71 | – | – | – | – |
| R14a/G9P[8]/Hu | – | – | – | – | – | – | – | – | – | – | |
| Wa/G1P[8]/Hu | 85/94 | 93/93 | 62/55 | 88/98 | 74/78 | 85/88 | 88/91 | 97/97 | 95/95 | ||
| DS-1/G2P[4]/Hu | 79/84 | – | 70/75 | 62/56 | 79/92 | 72/74 | 76/76 | 88/94 | 83/89 | 81/84 | – |
| 30/96/G3P[14]/Lp | 79/85 | 79/85 | 73/82 | 61/56 | – | – | – | 86/95 | 83/90 | 79/83 | 91/96 |
| PRICE/Po | – | – | – | – | – | – | – | – | – | – | |
| RRV/G3P[3]/Si | – | – | – | 69/72 | – | – | – | – | 82/92 | 79/82 | – |
| PO-13/G18P[17]/Av | 67/61 | 63/56 | 62/57 | 39/20 | 70/77 | 65/58 | – | 56/46 | 60/58 | – | 63/52 |
| KJ75/G5P[5]/Bo | – | – | – | – | – | 86/93 | – | 89/94 | – | – | – |
| US1205/G9P[6]/Hu | – | – | – | 61/57 | 76/91 | 77/84 | – | – | – | 80/83 | – |
| EW/G16P[16]/Mu | – | – | – | 62/60 | – | – | – | – | – | 62/62 | – |
| KUN/G2P[4]/Hu | – | – | – | – | – | – | – | – | – | 81/83 | 90/93 |
| CU-1/G3P[3]/Ca | – | – | – | – | – | – | – | – | – | 78/84 | – |
| FRV-1/G3P[9]/Fe | – | – | – | – | – | – | – | – | – | 79/82 | – |
| EHP/G16P[20]/Mu | – | – | – | 65/66 | – | – | – | – | – | 62/61 | – |
| SA-11/G3P[1]/Si | 77/85 | 79/84 | – | 71/72 | – | – | – | – | – | – | 94/96 |
| CMP034/G2P[27]/Po | – | – | – | – | – | – | – | – | – | – | |
| YM/G11P[7]/Po | 88/94 | – | – | 85/88 | 81/90 | 82/90 | – | – | – | – | – |
| Gottfried/G4P[6]/Po | – | – | 61/57 | 88/98 | – | – | – | – | – | – | |
| JL94/G5P[7]/Po | – | – | – | – | – | – | – | – | – | – | |
| rj6906/03/Hu | – | – | – | – | – | – | – | – | – | – | |
| MRC3105/G5P[8]/Hu‡ | – | – | – | – | – | – | – | – | – | – | |
| CC117/G5/Po | – | – | – | – | – | 91/97 | – | – | – | – | – |
| C134/G5/Po | – | – | – | – | – | 90/97 | – | – | – | – | – |
| LL4260/G5P[6]/Hu | – | – | – | – | – | 90/94 | – | – | – | – | – |
| KH210/G5P[6]/Hu | – | – | – | – | – | 89/93 | – | – | – | – | – |
| IAL-28/G5P[8]/Hu | – | – | – | – | – | 85/92 | – | – | – | – | – |
| H-1/G5P[7]/Eq | – | – | – | – | 81/90 | 86/94 | – | – | – | 85/93 | – |
*ARN, African Rotavirus Surveillance Network; VP, structural protein; NSP, nonstructural protein; –, not included or not sequenced. High and moderate nucleotide/amino acid percentage identities are in
Sequence analysis of the VP7 gene demonstrated that 6784/2000/ARN had 85%–91% nt and 92%–100% aa identities with representative G5 rotaviruses from humans and animals, respectively. Although the VP7 gene was highly divergent from other human G5 isolates detected in South America and Asia, it was identical to a human serotype G5 rotavirus isolated in Cameroon (
Although we did not sequence the minimum 500 bp/gene, we propose a tentative genotype classification based on ≈300–350 nucleotides sequenced by using the scheme of Matthijnssens et al. (
Serotype G5 rotaviruses, which are common in pigs but also detected in horses and cattle, were identified in the 1990s in children from Brazil who had diarrhea (
Detection of G5 rotaviruses with different genetic compositions from children in Cameroon raises questions about the origin of these strains. MRC3105 not only represents a reassortant strain between porcine rotaviruses and HRVs but also may have obtained gene segments from isolates of human Wa and DS-1 genogroups, as suggested by unusual combinations in its RNA profile, subgroup specificity, and P type (
Although G5P[7] strains might be common in pigs, strain 6784/2000/ARN is a novel representative of this antigen combination in humans. Similarities of some of its gene segments with those of porcine rotavirus strains suggest that ARN G5P[7] is an animal–human reassortant rotavirus in which a few genes are derived from human strains. Introduction of animal rotavirus genes into the genetic background of common HRVs has resulted in global spread of various genotype specificities, including G9 and G12. In these emerging human strains, DS-1 and Wa genogroups served as parental strains to carry the new antigenic variants on the background of old genotype specificities. Further, human G5 strains whose overall genomic composition is Wa-like have a wide geographic distribution and were considered clinically important HRVs in South America during the 1990s. Surveillance is needed to determine if G5P[7] strains on a Wa-like genetic background will spread to other African countries.
Phylograms showing genetic relationships between known human and animal rotavirus strains from GenBank and partial nucleotide sequences of structural protein 1 (VP1), VP2, VP3, VP4, VP6, and VP7 genes and nonstructural protein 1 (NSP1), NSP2, NSP3, NSP4, and NSP5 genes of genotype G5P[7] rotavirus isolated from a child with diarrhea in Cameroon. Trees were drawn to scale and rooted with sequences of avian rotavirus strain PO-13. Significant bootstrap values (>80%) are indicated at branch nodes. Scale bars show 0.01 substitutions per nucleotide. Strain 6784/2000/ARN/Hu is in
We thank the staff of the Medical Research Council/Diarrhoeal Pathogens Research Unit, University of Limpopo, and of the Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention (CDC), for assistance.
Dr Esona is an associate research fellow in the Gastroenteritis and Respiratory Viruses Laboratory Branch at CDC. His primary research interest is the molecular epidemiology of enteric viruses.