See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/23713266 Novel Human Rotavirus Genotype G5P[7] from Child with Diarrhea, Cameroon Article  in  Emerging Infectious Diseases · February 2009 DOI: 10.3201/eid1501.080899 · Source: PubMed CITATIONS READS 55 47 10 authors, including: Mathew D Esona Krisztian Banyai US Department of Health and Human Services, Centers for Disease Control and Prev… Hungarian Academy of Sciences 314 PUBLICATIONS   2,699 CITATIONS    696 PUBLICATIONS   7,137 CITATIONS    SEE PROFILE SEE PROFILE Nicola Page Maryam Aminu National Institute for Communicable Diseases Ahmadu Bello University 161 PUBLICATIONS   1,493 CITATIONS    101 PUBLICATIONS   406 CITATIONS    SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Diarrhoeal surveillance in hospitalized children <5 in South Africa View project African Rotavirus Network View project All content following this page was uploaded by Nicola Page on 04 July 2014. The user has requested enhancement of the downloaded file. Novel Human The StudyDuring ARN surveillance conducted from 1998 Rotavirus Genotype through 2004, a total of 215 rotavirus-positive stool sam-ples could not be typed by standard reverse transcription– G5P[7] from Child PCR genotyping methods. Among untypeable samples, we with Diarrhea, identifi ed a G5P[7] strain (designated 6784/2000/ARN), which represented a rare G genotype and a new P genotype Cameroon specifi city in humans. This strain was isolated from a stool specimen from a child with gastroenteritis in Kumba, Cam- eroon. Because G5 and P[7] genotype specifi cities are com- Mathew D. Esona,1 Annelise Geyer,1 mon in pigs, we studied the entire genomic composition of Krisztian Banyai, Nicola Page,1 Maryam Aminu,1 this strain to determine if it was an example of a strain that George E. Armah,1 Jennifer Hull, arose through direct interspecies transmission from a par- Duncan A. Steele,1 Roger I. Glass, ticular animal host, or by reassortment with heterologous and Jon R. Gentsch rotavirus strains. Gene fragments of the 11 gene segments of strain We report characterization of a genotype G5P[7] hu- man rotavirus (HRV) from a child in Cameroon who had 6784/2000/ARN were amplifi ed by using consensus prim- diarrhea. Sequencing of all 11 gene segments showed ers for structural protein 4 (VP4), VP6, and VP7 (5–8) and similarities to >5 genes each from porcine and human ro- newly designed consensus primers for VP1, VP2, VP3, taviruses. This G5P[7] strain exemplifi es the importance of nonstructural protein 1 (NSP1), NSP2, NSP3, NSP4, and heterologous animal rotaviruses in generating HRV genetic NSP5 (Table 1). The fragments were sequenced by using diversity through reassortment. the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Dye-labeled products Group A rotaviruses are a major cause of severe diar- were sequenced in an ABI 3130 sequencer (Applied Bio-rheal disease in infants, young children, and a variety systems). Similarity and phylogenetic relationships were of animals. In humans, rotavirus gastroenteritis results in inferred by using aligned nucleotide and deduced amino deaths and hospitalizations; most deaths have occurred in acid sequences by the neighbor-joining method and p-dis- developing countries (1). tance algorithm of MEGA4 software (9). Rotavirus surveillance and strain characterization, in Similarity matrices and phylogenetic trees based on support of rotavirus vaccine development programs, have nucleotide and amino acid sequences were constructed detected many new human rotavirus (HRV) genotype spec- and compared with cognate gene sequences of human and ifi cities and highlighted the importance of mechanisms such animal rotaviruses. Except for the 2 gene segments, which as reassortment and zoonotic transmission in the evolution encode neutralizing antigens VP7 and VP4, respectively, of rotaviruses (2). However, more comprehensive analy- and are commonly encountered in porcine rotaviruses, ses of gene fragments (3) or entire genes (4) are needed to the remaining 9 gene segments of 6784/2000/ARN were clarify the origin of rotavirus gene segments for common grouped in a common phylogenetic clade in which refer- and uncommon strains. To elucidate the possible origin of ence human strains of the Wa genogroup and related por- the novel G5P[7] HRV strain from the African Rotavirus cine rotaviruses also clustered (online Appendix Figure, Surveillance Network (ARN), we determined its genomic available from www.cdc.gov/EID/content/15/1/83-appF. composition and compared its gene sequences with rotavi- htm). However, VP1, NSP3 (likely), and NSP5 genes rus sequences in GenBank. were more closely related to cognate gene sequences of porcine strains (Gottfried, PRICE, CMP034, and OSU) Author affi liations: Centers for Disease Control and Prevention, At- than to HRVs and shared an nt identity of 92%–99%. lanta, Georgia, USA (M.D. Esona, J. Hull, J. Gentsch); University VP2, VP3, VP6, NSP1, NSP2, and NSP4 genes showed of Limpopo, Pretoria, South Africa (A. Geyer); Association of Public a stronger genetic relationship with human strains of the Health Laboratories, Washington, DC, USA (K. Banyai); National Wa genogroup (90%–99% nt identities) than with known Institute for Communicable Diseases, Johannesburg, South Africa porcine rotaviruses (Table 2). (N. Page); Ahmadu Bello University, Zaria, Nigeria (M. Aminu); No- Sequence analysis of the VP7 gene demonstrated that guchi Memorial Research Institute, Accra, Ghana (G.E. Armah); 6784/2000/ARN had 85%–91% nt and 92%–100% aa iden- Program for Appropriate Technology in Health, Seattle, Washing- tities with representative G5 rotaviruses from humans and ton, USA (D.A. Steele); and National Institutes of Health, Bethesda, animals, respectively. Although the VP7 gene was highly Maryland, USA (R.I. Glass) divergent from other human G5 isolates detected in South DOI: 10.3201/eid1501.080899 1Member of the African Rotavirus Network. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 83 DISPATCHES Table 1. Primers used for amplification and sequencing of rotavirus genes Nucleotide Amplicon Primer* Sequence (5 3 )† Gene‡ position, strand 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 (5) Con2 ATT TCG GAC CAT TTA TAA CC VP4 887–868, – Ku (5) 9con1-L TA GCT CCT TTT AAT GTA TGG TAT VP7 37–59, + 896 Wa Modified from (6) VP7-Rdeg GAC GGV GCR ACT ACA TGG T VP7 933–914, – Wa Modified from (7) VP6-F GAC GGV GCR ACT ACA TGG T VP6 747–766, + 379 (8) VP6-R GTC CAA TTC ATN CCT GGT G VP6 1126–1106, – (8) *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. America and Asia, it was identical to a human serotype G5 serotype has also been reported in children with severe diar- rotavirus isolated in Cameroon (10) and clustered with 2 rhea in Paraguay, Cameroon, Argentina, Vietnam, and the porcine strains from Argentina (online Appendix Figure). People’s Republic of China (2,13,14), which suggests that Genetic analysis of the VP8* portion of the VP4 gene of G5, although uncommon overall in humans, is found world- strain 6784/2000/ARN had higher similarity (90% nt and wide. Partial molecular analyses showed that human G5 89% aa) with porcine genotype P[7] strains, e.g., OSU and strains are reassortants with various genetic compositions. JL94, than with strains of other genotypes (39%–85% nt Some human G5 strains from Brazil, China (LL36755), and 55%–72% aa). This fi nding suggests that 6784/2000/ and Vietnam (KH210) contain a genotype P[6] VP4 gene, ARN also belongs to genotype P[7]. but their other genes have not been characterized (12–14). Although we did not sequence the minimum 500 bp/ The novel 6784/2000/ARN strain characterized here shares gene, we propose a tentative genotype classifi cation based a VP6 subgroup II specifi city and a long RNA electropho- on ≈300–350 nucleotides sequenced by using the scheme retic pattern with prototype human G5 strain IAL-28 but of Matthijnssens et al. (11). VP1-, VP2-, VP3-, VP6-, differs in subgroup and electropherotype from the Camer- NSP1-, NSP2-, NSP3-, NSP4-, and NSP5-encoding gene oon isolate MRC3105 (10). Strain 6784/2000/ARN has a segments of strain 6784/2000/ARN form a close phyloge- P[7] VP4 genotype and represents a human strain with this netic cluster with human and animal rotavirus strains of the VP4 specifi city. Wa-like genogroup, respectively, in R1, C1, M1, I1, A1, Detection of G5 rotaviruses with different genetic com- N1, T1, E1, and H1 genotypes (11). Nucleotide sequences positions from children in Cameroon raises questions about deposited in GenBank are FM179285 (VP1), FM179286 the origin of these strains. MRC3105 not only represents a (VP2), FM179287 (VP3), FM179288 (VP4), FM179289 reassortant strain between porcine rotaviruses and HRVs (VP6), FM179290 (NSP1), FM179291 (NSP2), FM179292 but also may have obtained gene segments from isolates (NSP3), FM179293 (NSP4), FM179294 (NSP5), and of human Wa and DS-1 genogroups, as suggested by un- EF218667 (VP7). usual combinations in its RNA profi le, subgroup specifi c- ity, and P type (10). In contrast, 6784/2000/ARN seems to Conclusions have obtained its outer capsid combination from a porcine Serotype G5 rotaviruses, which are common in pigs rotavirus, and its overall genomic composition showed ge- but also detected in horses and cattle, were identifi ed in the netic exchange between a porcine parental strain and a hu- 1990s in children from Brazil who had diarrhea (12). This man strain of the Wa genogroup. We hypothesize that these 84 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 Novel Human Rotavirus Genotype G5P[7] in Cameroon Table 2. Nucleotide/amino acid identities of rotavirus 6784/2000/ARN gene segments with cognate gene sequences of 36 known human and animal rotavirus sequences from GenBank* Nucleotide/amino acid identity, % Strains† VP1 VP2 VP3 VP4 VP6 VP7 NSP1 NSP2 NSP3 NSP4 NSP5 Ku/G1P[8]/Hu 88/94 96/96 90/94 61/55 90/98 74/79 85/88 91/94 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 90/89 80/90 86/94 86/89 – 89/95 88/96 98/100 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 – – – – – – 97/97 – – – – Wa/G1P[8]/Hu 85/94 93/93 95/96 62/55 88/98 74/78 85/88 88/91 97/97 90/95 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 – – – – – – – – 98/98 – – 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 – – – – – – – – – – 99/100 YM/G11P[7]/Po 88/94 – – 85/88 81/90 82/90 – – – – – Gottfried/G4P[6]/Po 92/96 – – 61/57 88/98 – – – – – – JL94/G5P[7]/Po – – – 90/89 – – – – – – – rj6906/03/Hu – – – – 98/99 – – – – – – MRC3105/G5P[8]/Hu‡ – – – – – 100/100 – – – – – 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 boldface. †Species of origin. Hu, human; Po, porcine; Lp, lapine; Si, simian; Av, avian; Bo, bovine; Ca, canine; Fe, feline; Mu, murine; Eq, equine. ‡VP7 gene of MRC3105 was derived from a porcine rotavirus. 2 G5 isolates with identical VP7 genes in different HRV rotavirus in which a few genes are derived from human genetic backgrounds might be independent progenies of a strains. Introduction of animal rotavirus genes into the ge- porcine G5 rotavirus that was co-circulating with human netic background of common HRVs has resulted in global DS-1–like and Wa-like strains at the time of identifi cation spread of various genotype specifi cities, including G9 and of the G5 isolate in southwestern Cameroon. Additional G12. In these emerging human strains, DS-1 and Wa geno- sequencing of common porcine and human strains is re- groups served as parental strains to carry the new antigenic quired to elucidate mechanisms involved in generation of variants on the background of old genotype specifi cities. genetic diversity during reassortment of rotaviruses from Further, human G5 strains whose overall genomic com- 2 species. position is Wa-like have a wide geographic distribution Although G5P[7] strains might be common in pigs, and were considered clinically important HRVs in South strain 6784/2000/ARN is a novel representative of this America during the 1990s. Surveillance is needed to de- antigen combination in humans. Similarities of some of termine if G5P[7] strains on a Wa-like genetic background its gene segments with those of porcine rotavirus strains will spread to other African countries. suggest that ARN G5P[7] is an animal–human reassortant Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 1, January 2009 85 DISPATCHES Acknowledgments 6. Das BK, Gentsch JR, Cicirello HG, Woods PA, Gupta A, Ramachan- We thank the staff of the Medical Research Council/Diar- dran M, et al. Characterization of rotavirus strains from newborns in rhoeal Pathogens Research Unit, University of Limpopo, and of the New Delhi, India. J Clin Microbiol. 1994;32:1820–2. 7. Iturriza-Gomara M, Isherwood B, Desselberger U, Gray J. Reassort- Gastroenteritis and Respiratory Viruses Laboratory Branch, Cen- ment in vivo: driving force for diversity of human rotavirus strains ters for Disease Control and Prevention (CDC), for assistance. isolated in the United Kingdom between 1995 and 1999. J Virol. 2001;75:3696–705. DOI: 10.1128/JVI.75.8.3696-3705.2001 Dr Esona is an associate research fellow in the Gastroen- 8. Iturriza-Gómara M, Wong C, Blome S, Desselberger U, Gray J. teritis and Respiratory Viruses Laboratory Branch at CDC. His Rotavirus subgroup characterisation by restriction endonuclease primary research interest is the molecular epidemiology of enteric digestion of a cDNA fragment of the VP6 gene. J Virol Methods. 2002;105:99–103. DOI: 10.1016/S0166-0934(02)00087-3 viruses. 9. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolu- tionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–9. DOI: 10.1093/molbev/msm092 References 10. Esona MD, Armah GE, Geyer A, Steele AD. Detection of an unusual human rotavirus strain with G5P[8] specifi city in a Cameroonian 1. Estes M, Kapikian A. Rotaviruses. In: Knipe DM, Howley PM, Grif- child with diarrhea. J Clin Microbiol. 2004;42:441–4. DOI: 10.1128/ fi n DE, Martin MA, Lamb RA, Roizman B, et al., editors. 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