Human G9P[8] rotavirus strains circulating in Cameroon, 1999–2000: Genetic relationships with...
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Human G9P[8] rotavirus strains circulating in Cameroon, 1999–2000: Genetic relationships with other G9 strains and detection of a new G9 subtype M.D. Esona a,g,⇑ , S. Mijatovic-Rustempasic a , K. Foytich a , S. Roy a , K. Banyai b , G.E. Armah c , A.D. Steele d , E.M. Volotão e , M.M. Gomez e , M.F.M. Silva e , R. Gautam a , O. Quaye a , K.I. Tam a , J.C. Forbi f , M. Seheri g , N. Page g,h , J. Nyangao i , V.N. Ndze j , M. Aminu k , M.D. Bowen a , J.R. Gentsch a a Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, USA b Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary c College of Health Sciences/Noguchi Memorial Research Institute Accra, University of Ghana, Legon, Ghana d PATH, 1455 Leary Way, NW, Seattle, WA, USA e Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil f Virology Laboratory, Innovative Biotech, Keffi/Abuja, Nigeria g MRC/UL Diarrhoeal Pathogens Research Unit, Department of Virology, Medunsa Campus, University of Limpopo, South Africa h National Institute for Communicable Diseases, Viral Gastroenteritis Unit, Private Bag X4, Sandringham 2131, South Africa i Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenya j Faculty of Medicine and Biomedical Sciences, University of Yaoundé I., Yaoundé, Cameroon k Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria article info Article history: Received 27 February 2013 Received in revised form 17 May 2013 Accepted 3 June 2013 Available online 14 June 2013 Keywords: Rotavirus A Genotype P[8]G9 Genomic phylogenetic analysis Structural proteins Non-structural proteins abstract Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and geno- type G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collected during the 1999–2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P[8] strains (15/89 = 16.8%) were identified whose genomic configu- rations was subsequently determined by complete or partial gene sequencing. In general, all Cameroo- nian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6– 100% nucleotide identity amongst themselves and 81.2–99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P[8]-I1–R1–C1–M1–A1–N1–T1–E1–H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constel- lation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3 0 end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999–2000. Published by Elsevier B.V. 1. Background Childhood mortality has been declining worldwide as a result of socioeconomic development and implementation of prevention and survival interventions (Claeson et al., 2000). Group A rotavirus- es (RV-A) are the main etiologic agent of acute gastroenteritis in infants and young children worldwide (Estes and Kapikian, 2007) and an estimated 453,000 children aged <5 years die from rotavirus diarrhea each year, with >85% of these deaths occurring in low-income countries of Africa and Asia (Parashar et al., 2009; Tate et al., 2011). Rotaviruses belong to the family Reoviridae, and the rotavirus genome consists of 11 double-stranded RNA gene segments that encode six structural (VP) and six non-structural proteins (NSP). Based on the two genes that encode the outer cap- sid proteins, VP4 (P-type) and VP7 (G-type), a widely used binary classification system was established for RV-A (Estes and Kapikian, 2007). This system has been recently standardized and extended to all 11 genes (Matthijnssens et al., 2008b). To date, at least 27 G, 35 1567-1348/$ - see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.meegid.2013.06.005 ⇑ Corresponding author. Address: 1600 Clifton Rd, NE, Atlanta, GA 30333, USA. Tel.: +1 404 639 4379. E-mail address: [email protected] (M.D. Esona). Infection, Genetics and Evolution 18 (2013) 315–324 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
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Transcript of Human G9P[8] rotavirus strains circulating in Cameroon, 1999–2000: Genetic relationships with...
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Infection, Genetics and Evolution 18 (2013) 315–324
Contents lists available at SciVerse ScienceDirect
Infection, Genetics and Evolution
journal homepage: www.elsevier .com/locate /meegid
Human G9P[8] rotavirus strains circulating in Cameroon, 1999–2000:Genetic relationships with other G9 strains and detection of a new G9subtype
1567-1348/$ - see front matter Published by Elsevier B.V.http://dx.doi.org/10.1016/j.meegid.2013.06.005
⇑ Corresponding author. Address: 1600 Clifton Rd, NE, Atlanta, GA 30333, USA.Tel.: +1 404 639 4379.
E-mail address: [email protected] (M.D. Esona).
M.D. Esona a,g,⇑, S. Mijatovic-Rustempasic a, K. Foytich a, S. Roy a, K. Banyai b, G.E. Armah c, A.D. Steele d,E.M. Volotão e, M.M. Gomez e, M.F.M. Silva e, R. Gautam a, O. Quaye a, K.I. Tam a, J.C. Forbi f, M. Seheri g,N. Page g,h, J. Nyangao i, V.N. Ndze j, M. Aminu k, M.D. Bowen a, J.R. Gentsch a
a Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, USAb Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungaryc College of Health Sciences/Noguchi Memorial Research Institute Accra, University of Ghana, Legon, Ghanad PATH, 1455 Leary Way, NW, Seattle, WA, USAe Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazilf Virology Laboratory, Innovative Biotech, Keffi/Abuja, Nigeriag MRC/UL Diarrhoeal Pathogens Research Unit, Department of Virology, Medunsa Campus, University of Limpopo, South Africah National Institute for Communicable Diseases, Viral Gastroenteritis Unit, Private Bag X4, Sandringham 2131, South Africai Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenyaj Faculty of Medicine and Biomedical Sciences, University of Yaoundé I., Yaoundé, Cameroonk Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria
a r t i c l e i n f o a b s t r a c t
Article history:Received 27 February 2013Received in revised form 17 May 2013Accepted 3 June 2013Available online 14 June 2013
Keywords:Rotavirus AGenotype P[8]G9Genomic phylogenetic analysisStructural proteinsNon-structural proteins
Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and geno-type G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight intothe degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collectedduring the 1999–2000 rotavirus season in two different geographic regions in Cameroon (Southwest andWestern Regions). By RT-PCR, 15 G9P[8] strains (15/89 = 16.8%) were identified whose genomic configu-rations was subsequently determined by complete or partial gene sequencing. In general, all Cameroo-nian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6–100% nucleotide identity amongst themselves and 81.2–99.5% nucleotide identity with global G9 strains.The full genome classification of all Cameroonian strains was G9-P[8]-I1–R1–C1–M1–A1–N1–T1–E1–H1but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinctlineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constel-lation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly(18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide andamino acid alignments revealed that the 30 end of this gene is highly divergent from other G9 VP7 genessuggesting that it arose through extensive accumulation of point mutations. The results of this studydemonstrate that diverse G9 strains circulated in Cameroon during 1999–2000.
Published by Elsevier B.V.
1. Background
Childhood mortality has been declining worldwide as a result ofsocioeconomic development and implementation of preventionand survival interventions (Claeson et al., 2000). Group A rotavirus-es (RV-A) are the main etiologic agent of acute gastroenteritisin infants and young children worldwide (Estes and Kapikian,
2007) and an estimated 453,000 children aged <5 years die fromrotavirus diarrhea each year, with >85% of these deaths occurringin low-income countries of Africa and Asia (Parashar et al., 2009;Tate et al., 2011). Rotaviruses belong to the family Reoviridae,and the rotavirus genome consists of 11 double-stranded RNA genesegments that encode six structural (VP) and six non-structuralproteins (NSP). Based on the two genes that encode the outer cap-sid proteins, VP4 (P-type) and VP7 (G-type), a widely used binaryclassification system was established for RV-A (Estes and Kapikian,2007). This system has been recently standardized and extended toall 11 genes (Matthijnssens et al., 2008b). To date, at least 27 G, 35
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316 M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324
P, 16 I, 9 R, 9 C, 8 M, 16 A, 9 N, 12 T, 14 E and 11 H genotypeshave been identified based on the eleven rotavirus A genes (Eso-na et al., 2010b; Matthijnssens et al., 2011). In humans, at leastfive RV-A G types (G1–G4 and G9), and two common P types(P[8] and P[4]) circulate worldwide (Banyai et al., 2012; Gentschet al., 2005; Santos and Hoshino, 2005). G9 strains emerged in1990s, and there has been a global description of the appearanceand dominance of this genotype (Gentsch et al., 2005; Lairdet al., 2003; Matthijnssens et al., 2009; Santos and Hoshino,2005). Genotype G9 strains with a Wa-like or a DS-1-like geno-mic configuration or a mixture thereof have been detected spo-radically in localized outbreaks (Page et al., 2010). In Cameroon,the first molecular identification of genotype G9 in human sam-ples was reported in a study conducted by Steele and colleaguesin 2003 (Steele and Ivanoff, 2003).
At least seven major phylogenetic lineages and eleven minorlineages within G9 VP7 genes have been described (Phan et al.,2007; Wu et al., 2011). A molecular evolutionary analysis studyutilizing Bayesian inference supported the idea that one singlesub-lineage introduced in the 1980s was responsible for all theworldwide spread of G9 in the 1990s (Matthijnssens et al.,2010).
In order to gain insight into the degree of genetic variability ofG9P[8] strains circulating in Cameroon, Central Africa, sequencedetermination and phylogenetic analysis of all eleven genome seg-ments from G9P[8] RV-A strains detected in two different geo-graphic regions of Cameroon (Southwest and Western Regions)was performed in order to infer the genetic relationship of Came-roonian strains with G9P[8] worldwide. The results of these studiesrevealed a new G9 genetic variant circulating in Cameroon duringthe 1999–2000 rotavirus seasons.
2. Material and methods
2.1. Fecal samples, strains and nomenclature
Fifteen diarrheic stool specimens collected from children<5 years of age, genotyped as G9P[8] (Esona et al., 2010a), were ob-tained during the 1999–2000 rotavirus season in two different geo-graphic regions in Cameroon (Southwest and Western Regions).The strains and nomenclature are shown in Table 1.
Table 1Characteristics of the Cameroon G9P[8] strains.
GenotypesVP7 Lineage VP4 Lineage
Length of gene sequenced (nucleotide)a 840 834Presence of ORFb P P
Strain nameRVA/Human-wt/CMR/6735/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6778/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6779/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6788/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6791/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6796/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6807/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6806/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6795/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6777/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6790/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6792/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6793/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6805/1999/G9P[8] G9 III P[8] P[8]-3RVA/Human-wt/CMR/6794/1999/G9P[8] G9 III P[8] P[8]-3
a Length of gene sequenced in nucleotides.b P and C denotes partial or complete ORF, respectively.
2.2. Viral RNA extraction, amplification, and sequencing
Viral RNA from each of the 15 specimens was extracted from a10% stool suspension made from 0.1 g or 100 ll stool in 2 ml of a1:1 Vertrel/Water solution using either a commercial RNA extrac-tion kit (NucliSens automated extractor, BIOMERIEUX, Durham,NC) according to the protocol specified by the manufacturer or asilica binding method described previously (Boom et al., 1990).
Previously published forward and reverse primers (Das et al.,1994; Gentsch et al., 1992; Iturriza-Gomara et al., 2001, 2002; Ker-in et al., 2007; Matthijnssens et al., 2006; Mijatovic-Rustempasicet al., 2011) were used for the amplification of the different genesegments. The extracted dsRNA of each strain was denatured at97 �C for 5 min and RT-PCR was carried out using a one step RT-PCR kit (Qiagen, Inc., Valencia, CA) according to manufacturer’sinstructions. Reverse transcription (RT) of each gene from eachsample was carried out for 30 min at 42 �C, followed by 15 minat 95 �C to inactivate the reverse transcriptase and activate theTaq polymerase. The cDNA was then subjected to 35 cycles ofPCR in a GeneAmp PCR System 9700 thermal cycler (Applied Bio-systems, Inc., Foster City, CA) using the following conditions: 30 sat 94 �C; 30 s at 42 �C; 45–90 s at 72 �C (depending upon the ex-pected size of the amplified gene fragment), followed by a 7 minfinal extension at 72 �C. Amplicons were analyzed by gel electro-phoresis in 1% SeaKem agarose gels (Thermo Fisher Scientific,Inc., Waltham, MA) then excised and purified with the QIAquickGel Extraction kit (Qiagen, Inc., Valencia, CA) following the manu-facturer’s instructions.
DNA cycle sequencing of each amplicon was performed withthe same consensus primers used for RT-PCR, using a Big Dye Ter-minator cycle sequencing Ready kit v1.1 (Applied Biosystems, Inc.,Foster City, CA). Previously published primers homologous tointernal regions of each gene segment were also used (Mijatovic-Rustempasic et al., 2011). Cycle sequencing products were purifiedusing Centri-sep spin columns (Princeton Separations, Inc.,Adelphia, NJ), dried in a DNA speed VacR (Savant Instruments,Inc., Holbrook, NY) and reconstituted in 15 ml Hi-Di formamide.Automated separation and base-calling of cycle sequencingproducts was performed using an ABI 3130xl sequencer (AppliedBiosystems, Foster City, CA). Overlapping sequence fragments wereassembled and edited using Sequencher 4.8 (Gene CodesCorporation, Inc., Ann Arbor, MI).
VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP51191 1917 1367 1187 1043 948 930 525 591C P P P P C C C C
I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1I1 R1 C1 M1 A1 N1 T1 E1 H1
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RVA/Human wt/CMR/6777/1999/G9P[8] RVA/Human-wt/CMR/6794/1999/G9P[8]
/ /C / / /G
A B RVA/Human-wt/CMR/6777/1999/G9P[8] RVA/Human-wt/CMR/6790/1999/G9P[8]
94
RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6792/1999/G9P[8]98 RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6794/1999/G9P[8] [ ]
RVA/Human-wt/CMR/6777/1999/G9P[8]82 RVA/Human-wt/CMR/6795/1999/G9P[8] RVA/Human-wt/CMR/6796/1999/G9P[8]
96
RVA/Human-wt/USA/2007719825/2007/G1P[8]
RVA/Human wt/BRA/rj11772/2005/G9P[8]
[ ] RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human wt/CMR/6788/1999/G9P[8] RVA/Human-wt/BRA/rj11772/2005/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]89 RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]/ / / / / /G
RVA/Human-wt/HUN/Hun9/1998-2001/G9P[8] RVA/Human-wt/BRA/rj1528/98/1998/G9P[8] RVA/Human-wt/MUS/MR4710/00/2000/G9P[8]
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/COD/DRC88/2003/G8P[8]73 RVA/Human-wt/MUS/MR4730/2000/G9P[4]
RVA/Human-wt/CMR/6807/1999/G9P[8] RVA/Human-wt/COD/DRC88/2003/G8P[8]
RVA/Human-wt/MWI/OP498/1999/G3P[8]
[ ] RVA/Human-wt/CMR/6792/1999/G9P[8]RVA/Human wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/MWI/OP351/1998/G1P[8]97RVA/Human-wt/CMR/6793/1999/G9P[8] RVA/Human-wt/GHA/GH3574/2000/G9P[X]
RVA/H /CMR/6 8/1999/G9P[8] RVA/Human-wt/MWI/OP601/1999/G1P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8] P[8]-3RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]Lineage III
[ ]
RVA/Human-wt/CMR/6805/1999/G9P[8]9484
[ ] RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/IND/ISO-3/2001/G9P[X] RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human wt/CMR/6796/1999/G9P[8]
[ ] RVA/Human-wt/THA/CMH214/2001/G9P[X]
RVA/Human wt/CMR/3298CM/1999/G9P[8] RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-wt/CMR/3298CM/1999/G9P[8] RVA/Human-wt/NGA/Bulumkutu/XXXX/G9P[X]RVA/H t/BRA/ j1609/98/1998/G9P[8]
98
RVA/Human-wt/CMR/6791/1999/G9P[8]7198
RVA/Human-wt/BRA/rj1609/98/1998/G9P[8] RVA/Human-wt/IRL/CIT-254RV/1997/G9P[8]
RVA/Human-wt/TUN/6854/2002/ARN/2002/G8P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]
7198 RVA/Human-wt/CHN/XJ99-468/1999/G9P[6]
RVA/Human-wt/BRA/ac11824/06/2006/G9P[8] RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CIV/6736/2004/ARN/2004/G8P[8]
[ ] RVA/Human-wt/KEN/KY6923/02/2002/G9P[X]RVA/Human-tc/USA/US1205/1997/G9P[6]
98
RVA/Human-wt/CMR/6779/1999/G9P[8]87RVA/Human-tc/USA/US1205/1997/G9P[6]
RVA/Human-wt/MWI/MW69/1997/G9P[6]RVA/H t/ZAF/6601LC/98/1998/G9P[6] RVA/Human-wt/CMR/6780/2000/ARN/2000/G8P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
95 RVA/Human-wt/ZAF/6601LC/98/1998/G9P[6] RVA/Human-wt/VNM/608VN/XXXX/G9P[X]
99[ ]
RVA/Human-tc/XXX/KU/XXXX/G1P[8]90 RVA/Human-wt/THA/CMP003/XXXX/G9P[19] RVA/Human-tc/THA/MC345/1989/G9P[19]
RVA/Human-wt/JPN/F45/1987/G9P[8]
RVA/Human tc/USA/WI61/1983/G9P[8]
95 P[8]-2[ ]
RVA/Pig-wt/JPN/Hokkaido-14/2001/G9P[23]RVA/Human-wt/JPN/K-1/1995/G9P[X] Lineage VI RVA/Human-tc/USA/WI61/1983/G9P[8]
RVA/Human-wt/MWI/OP530/1999/G4P[8]P[8] 4
RVA/Human-wt/JPN/K-1/1995/G9P[X] RVA/Pig-wt/JPN/JP35-7/2000/G9P[6]
Lineage VI
Li IIRVA/H t /IND/116E/1985/G9P[11]8380
RVA/Human-wt/MWI/OP354/1998/G4P[8]91P[8]-4Lineage II RVA/Human-tc/IND/116E/1985/G9P[11]
Lineage V RVA/Human-wt/USA/OM67/2000/G9P[8]
83
RVA/Human-tc/JP/Hochi/1983/G4P[8]
RVA/Human-tc/JPN/ITO/1981/G3P[8]95
P[8]-1Lineage IV RVA/Human-wt/CHN/97SZ37/XXXX/G9P[X]
RVA/Human-tc/USA/WI61/1983/G9P[8] RVA/Human-tc/JPN/ITO/1981/G3P[8]
RVA/Human-tc/JPN/Hosokawa/1983/G4P[8]
P[8] 1[ ] RVA/Human-wt/JPN/AU32/1986/G9P[8]RVA/Human-wt/JPN/F45/1987/G9P[8]
Lineage I74
RVA/Human-tc/USA/DS-1/1976/G2P[4] RVA/Human-wt/JPN/F45/1987/G9P[8]
RVA/Human-tc/GBR/ST3/1975/G4P[6]
0.020.02
Fig. 1. A–K Maximum likelihood phylogenetic trees built in PhyML with aLRT statistics as support show the genetic relationships of nucleotide sequences of VP7 (A), VP4 (B),VP1(C), VP2 (D), VP3 (E), VP6 (F), NSP4 (G), NSP1 (H), NSP2 (I), NSP3 (J) and NSP5 (K) of human G9P[8] rotaviruses from Cameroon with known human and animal rotavirusstrains from GenBank database. The trees were drawn to scale. Only aLRT values of 70% and greater are shown. The strains labeled with filled squares indicate the CameroonG9P[8] isolates sequenced in this study. The scale bar at the bottom of the trees indicates genetic distance.
M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324 317
2.3. Computational analysis
Sequences were aligned using the MUSCLE program withinMEGA version 5 (Tamura et al., 2011). Once aligned, the JModel-Test 2 program (Posada, 2008) was used to identify the optimalevolutionary model that best fitted the sequence datasets. Usingcorrected Akaike Information Criterion (AICc) the following mod-els; TPM3uf + I + G (NSP1), TIM2 + I + G (NSP2), GTR + I + G (NSP3,VP1, VP2, VP6), HKY + G (NSP4), TVM + G (NSP5), TIM3 + I + G(VP3), TPM1uf + G (VP4), and TPM3uf + G (VP7) were found tobest fit the sequence data for the different genes. Using thesemodels, maximum likelihood trees were constructed usingPhyML 3.0 along with approximate likelihood-ratio test (aLRT)statistics for branch support (Guindon et al., 2010). Nucleotideand amino acid distance matrixes were prepared using the p-dis-tance algorithm of MEGA version 5 software (Tamura et al.,2011).
Using the crystal structure of the RRV VP7 protein (PDB acces-sion number 3fmg; (Aoki et al., 2009), amino acid substitutionsfound in Cameroonian strain RVA/Human-wt/CMR/6788/1999/G9P[8] were mapped spatially onto the 3D protein structure usingthe PyMOL Molecular Graphics System, version 1.5.0.1 (Schroding-er, 2010).
3. Results
3.1. The genotype configuration of Cameroonian G9P[8] strains
The names and characteristics of the Cameroonian strainsanalyzed in this study, and lengths of each gene are presentedin Table 1. The accession numbers of each gene of the Cameroo-nian G9 strains and those from the GenBank are in the appendix.Accession numbers in bold face characters represent the gene se-quences of these Cameroon G9 strains. Nucleotide sequencesanalysis based on VP7–VP4–VP6–VP1–VP2–VP3–NSP1–NSP2–NSP3–NSP4–NSP5 genes from all Cameroonian samples analyzedrevealed a consensus genotype constellation of G9-P[8]-I1–R1–C1–M1–A1–N1–T1–E1–H1, respectively, according to the classifi-cation proposed by Matthijnssens and colleagues (Matthijnssenset al., 2008b).
3.2. Analysis of VP7 nucleotide sequences
Phylogenetic analyses of the eleven genes determined the ge-netic relationships of the Cameroonian strains with a global collec-tion of rotavirus genotypes. Phylogenetic analysis based on VP7
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RVA/H t/CMR/6794/1999/G9P[8] RVA/H t/CMR/6735/1999/G9P[8]C D RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
99
99 RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]95
94C D
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8]
99 RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human wt/CMR/6806/1999/G9P[8]
97 RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]
98
RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]100
RVA/Human wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/H t/CMR/6777/1999/G9P[8]96
RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]9189 RVA/Human-wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]97 RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]99
89
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
/ /C / / /G
99
100 RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6805/1999/G9P[8]
99 RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]89
100
RVA/Human wt/CMR/6805/1999/G9P[8]
RVA/Human-tc/ken/B10/1987/G3P[2] RVA/Mouse-tc/USA/ETD 822/ XXXX/G16P[16]
RVA/Bat-wt/KEN/KE4852/07/2007/G25P[6]98 RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Human-tc/USA/DS-1/1976/G2P[4] RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Human-tc/USA/DS-1/1976/G2P[4]
98
RVA/Mouse-tc/USA/ETD 822/XXXX/G16P[16]
RVA/Guanaco-wt/ARG/Chubut/1999/G8P[14]
94 RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Human-tc/JPN/AU-1/1982/G3P[9] RVA/Guanaco wt/ARG/Chubut/1999/G8P[14]
RVA/Horse-tc/GBR/L338/1991/G13P[18] RVA/Simian/ZAF/SA-11-H96/1958/G3P[2]
0.050.05
RVA/Human-wt/CMR/6788/1999/G9P[8]E RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]99
E
RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
99
99 RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human wt/CMR/6796/1999/G9P[8]
100 RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]100
100[ ]
RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/H t/CMR/6792/1999/G9P[8]96 RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]
96
RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Horse-tc/GBR/L338/1991/G13P[18]
90 RVA/Horse tc/GBR/L338/1991/G13P[18]
RVA/Simian-tc/ZAF/SA11-H96/1958/G3P[2]
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Mouse-tc/USA/ETD 822/ XXXX/G16P[16]
0.1
Fig. 1 (continued)
318 M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324
nucleotide sequences showed that all G9 strains detected inCameroon during 1999–2000 clustered together in three distinctsub-clusters with lineage III G9 strains isolated worldwide(Fig. 1A). Nucleotide and amino acid identities among Cameroo-nian strains ranged between 88.6–100% and 84.6–100%,respectively. The strain RVA/Human-wt/CMR/6788/1999/G9P[8]showed a maximum nucleotide identity of 90% with Cameroonianstrains RVA/Human-wt/CMR/6806/1999/G9P[8], RVA/Human-wt/CMR/6805/2000/G9P[8] and RVA/Human-wt/CMR/6796/2000/G9P[8], while its nucleotide identity with global G9 strains rangesfrom 81% to 89% (data not shown). Comparison of the amino acidsequence of strain RVA/Human-wt/CMR/6788/1999/G9P[8] to ref-erence and contemporary human G9 genotypes from the GenBankrevealed a low identity in the range of 81–85% as well as one tonumerous substitutions in the nine major VP7 variable regionsVR-1–VR-9 (Green et al., 1989) described for this protein (Fig. 2);VR-1 (I16L), VR-3 (A43 V), VR-4 (A68T), VR-5/antigenic epitope A(S90A, Q92E, G94A, and D100E), VR-8/antigenic epitope C (I208L,T210P, T212P, A213P, T214P, E217 K, and S221L), and VR-9/anti-genic epitope F (D238E, T241P, T243P, and T245P) (Green et al.,1989; Kirkwood et al., 2003). The VR-2, VR-6 and VR-7 were highlyconserved amongst both contemporary and older G9 strains. Substi-tutions in the three major variable regions; VR-5/antigenic epitopeA, VR-8/antigenic epitope C and VR-9/antigenic epitope F weremapped to the VP7 crystal structure of the RRV strain (G3P[3])
available in the Protein Data Bank (Fig. 3). Out of the 15 substitutionsidentified in these three regions, 11 were radical in nature. Radicalchanges are associated with changes in size, charge and polarity(Zhang, 2000). Out of the 11 radical changes 9 were associated withchanges in polarity with the strain 6788 being non-polar when com-pared to the consensus G9 strains. In VR-5/antigenic epitope Amutation in site 94 is associated with neutralization escape mutants(Aoki et al., 2009). Similar sites associated with neutralization es-cape mutants were observed in VR-8/antigenic epitope C (positions213, 217, 221) and in VR-9/antigenic epitope F (position 238).Comparative analysis of this strain with representative strainsbelonging to the major G9 VP7 lineages revealed a high similarityin the 50 end of the gene and a lower similarity downstream of thecentral part of the gene (data not shown).
3.3. Analysis of VP4 nucleotide sequences
Evolutionary analysis of VP4 nucleotide sequences from Came-roonian strains and representatives of genotype P[8] from the Gen-Bank database demonstrated that strains from both regions ofCameroon grouped in four different sub-clusters of lineage P[8]-III together with other rotaviruses from around the world(Fig. 1B). Nucleotide (amino acid) identities between Cameroonianstrains ranged between 95.4% and 100% (95.7–100%). StrainRVA/Human-wt/CMR/6735/1999/G9P[8] showed a close genetic
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RVA/Human-wt/CMR/6795/1999/G9P[8]RVA/Human-wt/CMR/6805/1999/G9P[8]F G
RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]99F G
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8] RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6796/1999/G9P[8]100
RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
[ ]
RVA/Human-wt/CMR/6794/1999/G9P[8]87 RVA/Human wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8]95 RVA/Cow-lab/GBR/PP-1/1976/G3P[7]
RVA/Human-wt/CMR/6735/1999/G9P[8] RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6735/1999/G9P[8]98 RVA/Human-wt/CMR/6793/1999/G9P[8]
RVA/Human wt/CMR/6791/1999/G9P[8]
96 RVA/Human wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]98 RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8] RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]99
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8][ ]
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/H /CMR/6 9/1999/G9P[8]99
99 RVA/Human wt/CMR/6790/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]86
RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
99 RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
86
91 RVA/Human-tc/USA/Wa/1974/G1P[8]
RVA/Pi t /MEX/YM/1983/G11P[7]9989
[ ]
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/H t/CMR/6778/1999/G9P[8]
91
RVA/Pig-tc/MEX/YM/1983/G11P[7]
RVA/Human-wt/NPL/KTM368/2004/G11P[25]
9996
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Pig-wt/THA/CMP034/2000/G2P[27]89
RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Pig wt/CAN/CE M 06 0003//2006/G2P[27]
RVA/Human-wt/BGD/N26/2000/G12P[6]
RVA/Human tc/JPN/AU 1/1982/G3P[9]82
93RVA/Pig-wt/CAN/CE-M-06-0003//2006/G2P[27]
RVA/Human-tc/KEN/B10/1987/G3P[2]
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Cat-wt/JPN/FRV1/1985/G3P[9]7293
RVA/Bat-wt/KEN/KE4852/07/2007/G25P[6]
RVA/Rhesus-tc/USA/TUCH/2002/G3P[24]90 RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Human-wt/BEL/B4106/2000/G3P[14]90
RVA/Rhesus-tc/USA/TUCH/2002/G3P[24]
RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Human wt/BEL/B4106/2000/G3P[14]
RVA/Horse-wt/ARG/E30/1993/G3P[12]80 97
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Mouse-tc/USA/ETD 822/XXXX/G16P[16]
RVA/Guanaco-wt/ARG/Chubut/1999/G8P[14]
RVA/Human-tc/USA/DS-1/1976/G2P[4] RVA/Mouse tc/USA/ETD 822/XXXX/G16P[16]
RVA/Human-wt/ECU/Ecu534/2006/G20P[28] RVA/Human-tc/USA/KC814/1998/G3P[9]
RVA/Mouse wt/USA/ETD 8222/XXXX/G16P[16] RVA/Human-wt/THA/CMH120/2004/G3P[9] RVA/Mouse-wt/USA/ETD 8222/XXXX/G16P[16]
0.050.05
Fig. 1 (continued)
M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324 319
relationship with strains isolated in Acre-Brazil during 2005–2006rotavirus season. Strain RVA/Human-wt/CMR/6778/1999/G9P[8]grouped in a separate cluster together with a previously detectedAfrican P[8] strain. Two Cameroonian strains, RVA/Human-wt/CMR/6779/1999/G9P[8] and RVA/Human-wt/CMR/6788/1999/G9P[8], showed complete identity with P[8] strains detected inTunisia (North Africa) in 2002 and Cote D’Ivoire (West Africa) in2004 (data not shown).
3.4. Analysis of VP1, VP2, VP3 and VP6 nucleotide sequences
Phylogenetic analysis based on VP1, VP2, VP3 and VP6 nucleo-tide sequences demonstrated that each gene of Cameroonianstrains detected during the 1999–2000 rotavirus season groupedin several separate clusters together with strains isolated world-wide (Fig. 1C–F). The Cameroonian strains showed a close geneticrelationship with cognate gene sequences of previously reportedG1P[8], G3P[8], and G4P[8] strains detected in the USA, Bangladeshand Belgium (McDonald et al., 2009; Mijatovic-Rustempasic et al.,2011; Rahman et al., 2007). Nucleotide (amino acid) identity valuesamong Cameroonian strains ranged from 91.8–100% (94–100%),90.7–100% (86.8–100%), 89.6–100% (93.9–100%), and 96–100%(99.2–100%) for VP1, VP2, VP3 and VP6, respectively. Completenucleotide and amino acid similarity (100%) was shared betweenstrains RVA/Human-wt/CMR/6735/1999/G9P[8] and RVA/Human-wt/CMR/6795/1999/G9P[8] (VP1 gene); RVA/Human-wt/CMR/6735/1999/G9P[8] and RVA/Human-wt/CMR/6779/1999/G9P[8],RVA/Human-wt/CMR/6805/1999/G9P[8] and RVA/Human-wt/CMR/6806/1999/G9P[8], RVA/Human-wt/CMR/6790/1999/G9P[8]and RVA/Human-wt/CMR/6792/1999/G9P[8] (VP2 gene); RVA/Hu-man-wt/CMR/6788/1999/G9P[8] and RVA/Human-wt/CMR/6805/1999/G9P[8] (VP3 gene); and RVA/Human-wt/CMR/6806/1999/G9P[8] and RVA/Human-wt/CMR/6807/1999/G9P[8] (VP6 gene).However, when the nucleotide and amino acid homologies of the
VP1–VP3 and VP6 gene sequences of the Cameroonian strains werecompared with cognate gene sequences of strains belonging to pre-viously identified VP1–VP3 and VP6 genotypes, all of them weremore closely related to strains in the R1, C1, M1 and I1 genotypes,respectively. Further comparison showed that within each geno-type, the Cameroonian strains had maximum nucleotide (aminoacid) identities of 84.8–98.6% (93.1–99.4%) for VP1, 89.8–98.8%(89.8–99.8%) for VP2, 88.3–99.2% (91.4–99.5%) for VP3 and 89.4–99.5% (97.5–100%) for VP6.
3.5. Analysis of NSP4 nucleotide sequences
Phylogenetic analysis based on NSP4 nucleotide sequencesshowed that the Cameroonian strains grouped into four differentsub-clusters together with strains belonging to genotype E1(Fig. 1G). The nucleotide (amino acid) identity among Cameroonianstrains ranged between 90.9%-100% (94.3%-100%). Analysis of de-duced amino acid sequences of Cameroonian strains and rotavirus-es detected worldwide demonstrated that seven of the fifteenstrains detected in Cameroon showed an amino acid substitutionwithin the enterotoxin domain (aa 114–135) (Ball et al., 2005).Three strains exhibited changes at position H131Y and the otherfour at position N133S. Amino acid substitutions were observedwithin previously described antigenic sites (Ball et al., 2005; Bor-gan et al., 2003): ASIV (aa 1–24) at position L7 V; ASIII (aa 112–133) at positions H131Y and N133S; ASII (aa136–150) at positionsI136A , P138S, V139A , V141I and I142V and ASI (151–169 aa) atpositions E160N, S161N, and L169I when aligned with other E1genotype strains (Fig. 4).
3.6. Analysis of NSP1- NSP3 and NSP5 nucleotide sequences
Phylogenetic analysis of NSP1- NSP3 and NSP5 nucleotide se-quences, demonstrated that for each gene, the Cameroonian strains
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RVA/Human-wt/CMR/6805/1999/G9P[8]100 RVA/Human-wt/CMR/6777/1999/G9P[8]H I
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-wt/CMR/6796/1999/G9P[8]
10096 RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]93H I
RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]
87 RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]
87
RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]98 RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6805/1999/G9P[8]98
RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]100
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8] RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
98 RVA/Human-tc/USA/Wa/1974/G1P[8]
RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]99
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]
RVA/Horse-tc/GBR/H1/1975/G5P[7]
97
100 RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
99
RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Cow-wt/ARG/B383/1998/G15P[11]
RVA/Cow-tc/THA/A5-13/XXXX/G8P[1]96100
97 RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
94
RVA/Cow-tc/THA/A5-13/XXXX/G8P[1]
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Human-wt/HUN/Hun5/1997/G6P[14]
96100 RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Simian-tc/ZAF/SA11-H96/1958/G3P[2]
RVA/Human-tc/JPN/AU-1/1982/G3P[9]85 95 RVA/Human-wt/HUN/Hun5/1997/G6P[14]
RVA/Human-tc/THA/T152/1998/G12P[9]
RVA/Human-tc/ITA/PA260-97/1997/G3P[3]
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Mouse-tc/USA/ETD 822/XXXX/G16P[16]
8599
95
RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Horse-wt/ARG/E30/1993/G3P[12]
RVA/Human-wt/BEL B4106/2000/G3P[14]
98 RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Horse-tc/GBR/L338/1991/G13P[18]86
RVA/Human-wt/BEL B4106/2000/G3P[14]
RVA/Simian/ZAF/SA-11-H96/1958/G3P[2]
RVA/Mouse-tc/USA/ETD 822/XXXX/G16P[16]
RVA/bat-wt/KEN/Ke4852/07/2007/G25P[6]
RVA/Mouse-tc/USA/ETD 822/XXXX/G16P[16]
0.1
0.05
RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]KJ RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6735/1999/G9P[8]97
RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
7787
KJ
RVA/Human-wt/CMR/6735/1999/G9P[8]
RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6793/1999/G9P[8]
97
100
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
87
93 RVA/Human-wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6795/1999/G9P[8]
RVA/Human-wt/CMR/6794/1999/G9P[8]
100 RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-wt/CMR/6796/1999/G9P[8]
93
RVA/Human-wt/CMR/6794/1999/G9P[8]
RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human-wt/CMR/6805/1999/G9P[8]
RVA/Human-wt/CMR/6794/1999/G9P[8]74
RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human-wt/CMR/6778/1999/G9P[8]
RVA/Human-tc/USA/Wa/1974/G1P1A8100
RVA/Human-wt/CMR/6793/1999/G9P[8]
RVA/Human-wt/CMR/6735/1999/G9P[8] RVA/Human-tc/USA/Wa/1974/G1P1A8
RVA/Human-wt/CMR/6796/1999/G9P[8]
RVA/Human-wt/CMR/6779/1999/G9P[8]80
100 RVA/Human-wt/CMR/6806/1999/G9P[8]
RVA/Human-wt/CMR/6807/1999/G9P[8]100 RVA/Human-wt/CMR/6779/1999/G9P[8]
RVA/Human-wt/CMR/6791/1999/G9P[8]
RVA/Human-wt/CMR/6788/1999/G9P[8]998780 RVA/Human-wt/CMR/6807/1999/G9P[8]
RVA/Human-wt/CMR/6792/1999/G9P[8]
RVA/Human-wt/CMR/6777/1999/G9P[8] RVA/Human-wt/CMR/6788/1999/G9P[8]
RVA/Human-tc/CHN/R 479/2004/G4P[6]
RVA/Cow-tc/USA/WC3/1981/G6P[5]
99
88
RVA/Human-wt/CMR/6777/1999/G9P[8]
RVA/Human-wt/CMR/6790/1999/G9P[8]
RVA/Human-tc/JPN/AU-1/1982/G3P[9] RVA/Cow-tc/USA/WC3/1981/G6P[5]
RVA/Cow-wt/JPN/AzuK-1/2006/G21P[29]
88 RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Bat-wt/KEN/KE4852/07/2007/G25P[6]
RVA/Horse-tc/GBR/L338/1991/G13P[18]8486 RVA/Bat-wt/KEN/Ke4852/07/2007/G25P[6]
RVA/Simian-tc/ZAF/SA11-H96/1958/G3P[2]
RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Horse-wt/ARG/E30/1993/G3P[12]
RVA/Human-tc/USA/DS-1/1976/G2P[4]
8486
RVA/Human-tc/JPN/AU-1/1982/G3P[9]
RVA/Human-tc/USA/DS-1/1976/G2P[4]88 RVA/Human-tc/USA/DS-1/1976/G2P[4]
RVA/Simian-tc/ZAF/SA11-H96/1958/G3P[2]
RVA/Horse-tc/GBR/L338/1991/G13P[18]
RVA/Mouse-tc/USA/ETD 822/ XXXX/G16P[16]
RVA/Human-tc/THA/T152/1998/G12P[9]
RVA/Mouse-tc/USA/ETD 822/ XXXX/G16P[16]
0.1 0.01
Fig. 1 (continued)
320 M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324
grouped in small separate sub-clusters of genotypes A1, N1, T1,and H1, respectively, together with other strains from around theworld (Fig. 1H–K). Nucleotide (amino acid) identity values amongCameroonian strains ranged from 87.8–99.8% (82.9–99.7%), 86.8–100% (88–100%), 92.8–100% (93.1–100%) and 97.6–100% (98.5–100%) for NSP1, NSP2, NSP3 and NSP5, respectively. Completenucleotide and amino acid identity was shared between strainsRVA/Human-wt/CMR/6777/1999/G9P[8] and RVA/Human-wt/CMR/6790/1999/G9P[8] (NSP2, NSP3 and NSP5 genes) and RVA/Human-wt/CMR/6806/1999/G9P[8] and RVA/Human-wt/CMR/6807/1999/G9P[8] (NSP3 and NSP5 genes), while only strainsRVA/Human-wt/CMR/6779/1999/G9P[8] and RVA/Human-wt/CMR/6791/1999/G9P[8] were completely identical in their NSP5gene sequences. However, when the nucleotide and amino acidhomologies of the NSP1–NSP3 and NSP5 gene sequences of theCameroonian strains were compared with similar gene sequencesof strains belonging to already identified NSP1–NSP3 and NSP5genotypes, all of them were more closely related to strains in theA1, N1, T1, and H1 genotypes, respectively. Within each of thesegenotypes, the Cameroonian strains shared maximum nucleotide(amino acid) identity of 75.4–99.6% (68.7–98.8%), 81.2–99.5%
(84.5–100%), 85.9–99.6% (87.9–100%), and 94.6–99.8% (93.9–100%), respectively.
4. Discussion
The genetic variability of RV-A strains is the result of accumula-tion of single nucleotide mutations (genetic drift) and suddenchanges in the RV-A genome (genetic shift), primarily by reassort-ment and recombination events (Estes and Kapikian, 2007; Mat-thijnssens et al., 2008c; McDonald et al., 2009; Ramig, 1997).Since the proposal that RV-A classification should be based on all11 RV-A gene segments (Matthijnssens et al., 2008b), the numberof studies reporting RV-A full genome sequences has increased (Ba-nyai et al., 2011; Esona et al., 2010b, 2011; Matthijnssens et al.,2008a; McDonald et al., 2009, 2011; Mijatovic-Rustempasic et al.,2011). Previous studies have shown that the predominance of aspecific G type is related to the emergence of atypical VP7 lineages(Banyai et al., 2009; Parra, 2009; Parra et al., 2005). The results ob-tained in this study revealed multiple amino acids changes in 6 ofthe 9 variable regions (Green et al., 1989; Kirkwood et al., 1993)when comparing Cameroon G9 strain RVA/Human-wt/CMR/6788/
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12 20 25 32 37 53 65 76RVA/Human-wt/CMR/6788/1999/G9P[8] F L I S L V L L N S L T S A M D F F L L L I V I V S P F V K T Q N Y T A Y T N S S Q Q E T FRVA/Human-tc/IND/116E/1985/G9P[11] . . . . I I . . . . V . . . . . . . . . I . . V . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/CMR/3298CM/1999/G9P[8] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/BRA/ac11824/06/2006/G9P[8] . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/JPN/AU32/1986/G9P[8] . . . . I I . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . A . L . . . . . .RVA/Human-wt/GHA/GH3574/2000/G9PX . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Pig-wt/JPN/Hokkaido-14/2001/G9P[23] . . . . I . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . A . . . . . . . .RVA/Pig-wt/JPN/JP35-7/2000/G9P[6] . . . . I I . . . . . . . V . . . . . . . . . . . . . L . . . . . . A . . A . . . . . . A .RVA/Human-wt/JPN/K-1/1995/G9P[X] . . . . I . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/KEN/KY6923/02/2002/G9P[X] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-tc/THA/MC345/1989/G9P[19] . . . . I . . . . . . . . . . . . . . . . . . . . . . S . . . . . H . . . A . . . . . . P .RVA/Human-wt/MUS/MR4710/00/2000/G9P[8] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/MAW/MW69/1997/G9P[6] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/USA/OM67/2000/G9P[8] . . . . I . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . A . . . . . . . .RVA/Human-wt/BRA/rj1528/98/1998/G9P[8] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/ZAF/66010LC/98/1998/G9P[6] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-tc/USA/US1205/1997/G9P[6] . . . . I . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .RVA/Human-tc/USA/WI61/1983/G9P[8] . . . . I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . L D . .RVA/Human-wt/CHN/XJ99-468/1999/G9P[6] . . . . I . . . . . . . . V . . . . . . . . . . A . . . . . . . . . . . . A . . . . . . . .
VR-1 (9-20) VR-2 (25-32) VR-3 (37-53) VR-4 (65-76)
VR-5/antigenic site A (87-100)05114123191100178
RVA/Human-wt/CMR/6788/1999/G9P[8] T E A A T E I A D T E W K E K E Y T D I A S F S I D P Q L M K Y D S T L E LRVA/Human-tc/IND/116E/1985/G9P[11] I . . S . Q . G . . . . . G . . . . . . . . . . . . . . . V . . N . . . . .RVA/Human-wt/CMR/3298CM/1999/G9P[8] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/BRA/ac11824/06/2006/G9P[8] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/JPN/AU32/1986/G9P[8] A . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . K .RVA/Human-wt/GHA/GH3574/2000/G9PX . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Pig-wt/JPN/Hokkaido-14/2001/G9P[23] . . . S . Q . G . A . . . D . . . . N . . . . . . . . . . . . . . . . . . .RVA/Pig-wt/JPN/JP35-7/2000/G9P[6] . . . S . Q . G . P . . . N . . . . N . . . . . . . . . . . . . . . . . . .RVA/Human-wt/JPN/K-1/1995/G9P[X] . . . S . Q . G . . . . . N . . . . . . . . . . . . . . . . . H . . . . . .RVA/Human-wt/KEN/KY6923/02/2002/G9P[X] . . . S . Q . G . A . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-tc/THA/MC345/1989/G9P[19] . . . S . Q . G . . . . . D . . . . . . . . . . . . . K . . . . . . . . . .RVA/Human-wt/MUS/MR4710/00/2000/G9P[8] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/MAW/MW69/1997/G9P[6] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/USA/OM67/2000/G9P[8] . . . S . Q . G . . . . . D . . . A . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/BRA/rj1528/98/1998/G9P[8] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/ZAF/66010LC/98/1998/G9P[6] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-tc/USA/US1205/1997/G9P[6] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-tc/USA/WI61/1983/G9P[8] A . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .RVA/Human-wt/CHN/XJ99-468/1999/G9P[6] . . . S . Q . G . . . . . D . . . . . . . . . . . . . . . . . . . . . . . .
VR-6 (119-132) VR-7/antigenic site B (141-150)
208 224 235 245RVA/Human-wt/CMR/6788/1999/G9P[8] L T P N P P P F E K V A T L E K L H K L E V T P N P C PRVA/Human-tc/IND/116E/1985/G9P[11] T . T . T A T . . E . . . N . . . . . . D . . T . T . TRVA/Human-wt/CMR/3298CM/1999/G9P[8] I . T . T A T . . E . . . S . . . . . . D . . I . T . TRVA/Human-wt/BRA/ac11824/06/2006/G9P[8] I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-wt/JPN/AU32/1986/G9P[8] T . T . T A T . . E . . A S . . . . . . D . . T T T . TRVA/Human-wt/GHA/GH3574/2000/G9PX I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Pig-wt/JPN/Hokkaido-14/2001/G9P[23] T . T . T A T . . E . . . N . . . . . . D . . T . T . TRVA/Pig-wt/JPN/JP35-7/2000/G9P[6] T . T . T A T . . E . . . G . . . . . . D . . T . T . TRVA/Human-wt/JPN/K-1/1995/G9P[X] T . T . A A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-wt/KEN/KY6923/02/2002/G9P[X] I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-tc/THA/MC345/1989/G9P[19] T . T . T A T . . E . . . S . . . . Q . D . . T . T . TRVA/Human-wt/MUS/MR4710/00/2000/G9P[8] I . T . T A T . G E . . . S . . . . . . D . . T . T . TRVA/Human-wt/MAW/MW69/1997/G9P[6] I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-wt/USA/OM67/2000/G9P[8] T . T . T A T . . E . . A S . . . . . . D . . T . T . TRVA/Human-wt/BRA/rj1528/98/1998/G9P[8] I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-wt/ZAF/66010LC/98/1998/G9P[6] I . T . T V T . . E . . . S . . . . . . D . . T . T . TRVA/Human-tc/USA/US1205/1997/G9P[6] I . T . T A T . . E . . . S . . . . . . D . . T . T . TRVA/Human-tc/USA/WI61/1983/G9P[8] T . T . T A T . . E . . A S . . . . . . D . . T T T . TRVA/Human-wt/CHN/XJ99-468/1999/G9P[6] I . T . T A T . . E . . . S . . . . . . D . . T . T . T
VR-8/antigenic site C (208-224) VR-9/antigenic site F (235-245)
Fig. 2. Comparison of the deduced amino acid sequence of gene segment 9 of strain RVA/Human-wt/CMR/6788/1999/G9P[8] to a selection of older and contemporary G9sequences from the GenBank. Only amino acids which differ are shown. Variable regions designated VR-1–VR-9 are shown.
D100E
Q92E
G94A
T245P
T243P
A213P T212P T210PE217K
S90A
S221L
D238E
T241P
I208L
T214P
Fig. 3. Substitutions in strain RVA/Human-wt/CMR/6788/1999/G9P[8] highlighted on the crystal structure of RRV VP7 protein (3fmg). The molecule is colored in green.Residues corresponding to previously describe major antigenic sites A, C and F are indicated in red, blue and orange spheres, respectively. Arrows indicate substitutions atamino acid positions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324 321
1999/G9P[8] to both contemporary and older G9 strains from theGenBank database. This suggests that this Cameroon G9 strainmight represent a new genetic variant of VP7 gene G9 genotype.
The relatively low overall amino acid homology with other G9strains together with numerous changes in important antigenic re-gions raises questions on whether this strain may be antigenically
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RVA/Human-tc/USA/WI61/1983/G9P1A[8] MDKFADLNYT LSVITLMNDT LHSI MIDKLTTREI EQVELLKRIH DN IKPVDV IDMSKEFNQ K NIKTLDEWESG KNPYEPL
RVA/Human-wt/CMR/6735/1999/G9P[8] .......... .......... .... .......... .......... .S ...... ......... . ........... ......I
RVA/Human-wt/CMR/6778/1999/G9P[8] ......V... .......... .... .......... .......... .. ...... ......... . ........N.. ......T
RVA/Human-wt/CMR/6779/1999/G9P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/CMR/6788/1999/G9P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/CMR/6791/1999/G9P[8] .......... .......... .... .......... .......... .S ...... ......... . ........... ......I
RVA/Human-wt/CMR/6796/1999/G9P[8] .......... .......... .... .......... .......... .. ..S..I V........ . .........N. ......I
RVA/Human-wt/CMR/6807/1999/G9P[8] .......... .......... .... .......... .........Y .. ...A.I V........ . ........... ......I
RVA/Human-wt/CMR/6806/1999/G9P[8] .......... .......... .... .......... .........Y .. ...A.I V........ . ........... ......I
RVA/Human-wt/CMR/6794/1999/G9P[8] .......... .......... .... .......... .......... .. ..S..I V........ . .........N. ......I
RVA/Human-wt/CMR/6795/1999/G9P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... ......I
RVA/Human-wt/CMR/6777/1999/G9P[8] .......... .......... .... .......... .......... .S ...... ......... . ........... ......I
RVA/Human-wt/CMR/6790/1999/G9P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/CMR/6792/1999/G9P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/CMR/6793/1999/G9P[8] .......... .......... .... .......... .......... .S ...... ......... . ........... ......I
RVA/Human-wt/CMR/6805/1999/G9P[8] .......... .......... .... .......... .........Y .. ...A.I V........ . ........... ......I
RVA/Human-wt/BGD/Dhaka6/2001/G11P[25] .......... .......... .... .......... .........Y .. .....I V........ . ........... ......I
RVA/Human-wt/BGD/Dhaka16/2003/G1P[8] .......... .......... .... .......... .........Y .. .....I V........ . ........... ......I
RVA/Human-wt/BGD/Dhaka12/2003/G12P[6] .......... .......... .... .......... .......... .. .....I V........ . .V.......N. ......I
RVA/Human-wt/BGD/Dhaka25/2002/G12P[8] .......... .......... .... .......... .......... .. .....I ......... . .........N. ......I
RVA/Human-tc/USA/Wa/1974/G1P1A[8] .......... .......... .... .......... .......... .. ...... ......... . ........... .......
RVA/Human-tc/XXX/KU/XXXX/G1P[8] .......... .......... .... .......... .......S.. .. ..S... ......... . ........... R......
RVA/Human-tc/GBR/ST3/1975/G4P2A[6] .......... .....S.... .... .......... ..I....... .. ....N. ....M.... . ..........R .......
RVA/Horse-tc/GBR/H1/1975/G5P[7] .......... .N........ .... ..E....... .......... .K A....A ......... . ..R........ .......
RVA/Pig-tc/USA/OSU/1977/G5P9[7] .......... .......... .... .......... .......... .K A.S..A ......... . ..R........ .......
RVA/Human-wt/IND/RMC321/1990/G9P[19] .......... .......... .... .......... .......... .K A....A ......... . ..R......N. .......
RVA/Pig-tc/MEX/YM/1983/G11P9[7] .......... .......... .... .......... .......... .K A....A ......... . ..R......N. .......
RVA/Cow-tc/KOR/KJ75/2004/G5P[5] .......I.. .......... .... .......... .......... .K V....A ......... . ..R........ .......
RVA/Human-wt/CMR/6787/2000/ARN/2000/G8P[8] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/ETH/6810/2004/ARN/2000/G8P[6] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/TUN/6862/2000/ARN/2000/G8P[6] .......... .......... .... .......... .......... .. A..... ......... . ........... .......
RVA/Human-wt/BGD/RV161/2000/G12P[6] .......... .......... .... .......... .......... .. .....I V........ . .V.......N. ......I
RVA/Human-wt/CMR/6782/2000/ARN/2000/G8P[6] .......... .......... .... .......... .......... .. A..... ......... . ........N.. ......I
RVA/Human-wt/CMR/6780/2000/ARN/2000/G8P[8] .......... .......... .... .......... .......... .. A..... ......... . ........N.. .......
RVA/Human-tc/USA/DS-1/1976/G2P1B[4] .E..T..... ........N. ..T. .......... .........Y .K V.ST.E ......I.. . .VR..E...N. ......K
RVA/Human-wt/COD/DRC86/2003/G8P[6] .E..T..... .......... ..T. .......... .........Y .K V.SNSE ......I.. . .VR..E..... ......R
RVA/Human-wt/COD/DRC88/2003/G8P[8] .E..T..... .......... ..T. .......... .........Y .K V.SNSE ......I.. . .VR..E..... ......R
RVA/Bat-wt/KEN/KE4852/07/2007/G25P[6] .E..T..... ........S. ..T. .......... .......... .K ..AA.E ......I.. . .V...E...N. ......K
RVA/Human-tc/JPN/AU-1/1982/G3P3[9] .E..T..... .......... ..T. .......... .......... .M .....K ....Q.... R QF...N..AE. E.....K
RVA/Human-tc/THA/T152/1998/G12P[9] .E........ .......... ..T. .......... .......... .M V...NK ....Q.... . HF...N..AE. E....SR
RVA/Human-wt/BEL/B4106/2000/G3P[14] ....T..... ........S. ..T. .......... ......R..Y .R VQKT.E ......I.. . .VR......N. .......
RVA/Human-wt/BGD/RV176-00/2000/G12P[6] .......... MN........ ..T. ...R...... .......... .M ...IEK ......... . QF......TD. E.....K
RVA/Mouse-tc/USA/EW/XXXX/G16P[16] .E........ .G....L... ..T. ..E....... .........Y .M V.QNRE ......T.. . AF...HD.GND R.YDDNT
AS IV (aa 1-24) AS III (112-133) AS II (136-150) AS I (151-169)
1 24 112 133 136 150 151 169Strain
Fig. 4. Alignment showing amino acid substitutions inside the four NSP4 antigenic sites of the Cameroon G9 strains.
322 M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324
distinct from typicalG9 strains. Also, the previously described con-served N-glycosylation site found within VR-4 at amino acid resi-dues 69–71 (Green et al., 1989) was found to be conserved in allG9 strains used in this analyses. Definitive conclusions on the pos-sible origin of this variant could not be made by bioinformaticsanalysis of the VP7 gene sequence. However, in this case it remainsunclear what selective pressures on this gene fragment could havedriven this strong diversification, given that other gene segments
of this strain retained their identity with related G9P[8] strainsidentified in the same region and time period. In this context, itis difficult to determine if a single sub-lineage of G9 can be respon-sible for the worldwide spread of G9 rotavirus as proposed recently(Matthijnssens et al., 2010).
At least six different neutralization epitopes (A through F)have been identified in the RVA VP7 protein, with A–C and F de-scribed as the most important (Kirkwood et al., 2003). The strain
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M.D. Esona et al. / Infection, Genetics and Evolution 18 (2013) 315–324 323
RVA/Human-wt/CMR/6788/1999/G9P[8] shows distinct changes inits antigenic regions when compared to G9 strains circulating inthe same region and also globally (Fig. 2). A strong shift in polarity,with strain RVA/Human-wt/CMR/6788/1999/G9P[8] beingstrongly non-polar as compared to other G9 strains suggest possi-ble inaccessibility of epitopes on the VP7 protein of strain RVA/Hu-man-wt/CMR/6788/1999/G9P[8] as the region becomes morehydrophobic in nature. Multiple sites previously identified asimportant in producing neutralization escape mutants show sub-stitutions in strain RVA/Human-wt/CMR/6788/1999/G9P[8] whencompared with global G9 strains. If strain RVA/Human-wt/CMR/6788/1999/G9P[8] is also a neutralization escape mutant it couldbe due in part to changes in polarity at the antigenic epitopes.The amino acid sequences at positions 87–101 and 208–211 (epi-tope region A and C) is said to be conserved within serotypes(Green et al., 1988). However, we observe substitutions withinthese regions in an alignment of a global collection of G9 strains.
Seven of the fifteen strains detected in Cameroon showed anamino acid substitution in the enterotoxin domain (114–135 aa)of NSP4. These changes occur at amino acid positions 131 and133, which are in the region (amino acid 131–140) reported tobe responsible for altered pathogenesis mediated by the NSP4 pro-tein (Zhang et al., 1998). Also observed are amino acid substitu-tions in the four previously described NSP4 antigenic sites (Ballet al., 2005). Antigenic sites AS IV, AS III, AS II and AS I had 1, 1,5, 3 amino acids substitutions, respectively, when compared withstrains in the E1 genotype. NSP4 is a trans-membrane glycoproteinknown to be involved in virus assembly and is capable of inducingdiarrhea in infant mice (Ball et al., 1996; Tian et al., 1996). It is pos-sible these changes may affect the conformation or activity of NSP4and also alter ability of host responses to neutralize enterotoxicfunction of the NSP4 gene segment (Ball et al., 1996; Tian et al.,1995).
Recent studies on origin and spread of the G9 rotavirus have re-vealed a high degree of genetic diversity within this genotypeworldwide (Martinez-Laso et al., 2009; Phan et al., 2007; Wuet al., 2011). These studies have also shown close similarity be-tween human and porcine G9 strains and zoonotic transmissionand convergent evolution were proposed as the possible evolution-ary mechanisms. Studies in Brazil have implicated porcine G9strains in many outbreaks (Leite et al., 2008). Also, reassortmentevents among animal and human strains have continued to be animportant mechanism for rotavirus evolution and emergence indeveloping countries (Banyai et al., 2010; Esona et al., 2010b). Thispresent report confirms the circulation of a diverse G9 genotypeamong children in two regions of Cameroon. However, all of theG9P[8] strains reported here showed close similarity in all genesegments except VP7 with human rotavirus strains of different Ggenotypes including G1P[8], G3P[8] and G4P[8]. Nonetheless, dueto the increased close contact between human and animals in mostdeveloping countries, the full genome sequence data obtained fromany future G9 study from Cameroon maybe entirely different.
Two live oral vaccines from Merck (RotaTeq�) and Glaxo-SmithKline (Rotarix�) have been licensed in more than 100 coun-tries and are being introduced into routine immunizationprograms in several countries worldwide (Glass et al., 2006; Vesik-ari et al., 2007), but has not been introduced in Cameroon. There-fore, it will be important to monitor the circulation of G9s inCameroon before the introduction of these vaccines and, onceintroduced, to monitor the abilities of the vaccines to provide het-erotypic protection against divergent G9 strains.
Furthermore, monitoring temporal changes in all 11 gene seg-ments may help us to comprehend the nature and pattern of rota-virus evolution. This study revealed the presence of a novel VP7genetic variant and diverse G9P[8] strains in Cameroon. Surveil-lance to monitor the strain diversity of circulating RV-A to detect
possible strain replacement following the introduction of universalRV-A vaccine is a priority of the World Health Organization. Suchstudies are important to estimate potential impact of vaccinationprograms on circulating strains including whether escape mutantsof known serotypes or novel strains that evade vaccine immunitywill emerge.
Acknowledgements
We thank the staff of the Gastroenteritis and RespiratoryViruses Laboratory Branch at the Centers for Disease Control andPrevention for invaluable assistance and the anonymous reviewersfor constructive comments. This study was supported by the Cen-ters for Disease Control and Prevention. K.B. was supported by theHungarian Scientific Research Fund (PD76364).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2013.06.005.
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