Infection, Genetics and Evolution 16 (2013) 62–77

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Infection, Genetics and Evolution

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Sequence analysis of the whole genomes of five African human G9 rotavirus strains

Martin M. Nyaga a, Khuzwayo C. Jere a,b, Ina Peenze a, Luwanika Mlera b, Alberdina A. van Dijk b,Mapaseka L. Seheri a, M. Jeffrey Mphahlele a,⇑a Medical Research Council/Diarrhoeal Pathogens Research Unit, Department of Virology, University of Limpopo (Medunsa Campus) and National Health Laboratory Service,P.O. Box 173, Medunsa 0204, Pretoria, South Africab Biochemistry Division, North-West University, Private Bag X6001, Noordrug, Potchefstroom 2520, South Africa

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 July 2012Received in revised form 25 November 2012Accepted 12 January 2013Available online 29 January 2013

Keywords:Rotavirus G9P[6] and G9P[8] strainsWhole genome454� PyrosequencingSequence analysisReassortmentAfrica

1567-1348/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.meegid.2013.01.005

⇑ Corresponding author. Tel.: +27 12 521 4569/422E-mail addresses: martmune@yahoo.com (M.M. N

com (K.C. Jere), ina.peenze@ul.ac.za (I. Peenze), lmAlbie.VanDijk@nwu.ac.za (A.A. van Dijk), mapaseka.jeffrey.mphahlele@ul.ac.za, jeffmphahlele@yahoo.com

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diar-rhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and charac-terised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequenceof the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and oneG9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe(southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8]from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] fromKenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped asG9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1)genome segments. All the genome segments of the five strains grouped strictly according to their geno-type Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9study strains from southern Africa clustered most closely with rotaviruses from the same geographicalorigin and with those with the same G and P types. The highest nucleotide identity of genome segmentsof the study strains from eastern and central Africa regions on a Wa-like backbone was not limited torotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] andG11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirusgenotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like geneticbackbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Group A rotaviruses (RVAs) are the most important viral agentsthat cause severe dehydrating gastroenteritis in children under theage of five globally (Parashar et al., 2009). The World Health Orga-nization (WHO) global rotavirus surveillance network estimatesthat the annual rotavirus-associated mortality is approximately453,000 worldwide, of which 95% occur in children younger thanfive years (Tate et al., 2012).

ll rights reserved.

7; fax: +27 12 521 5794.yaga), khuzwayojere@yahoo.lera@yahoo.com (L. Mlera),

seheri@ul.ac.za (M.L. Seheri),(M.J. Mphahlele).

Rotaviruses belong to the Reoviridae family and contain 11dsRNA genome segments. Each genome segment encodes either astructural (VP1 to VP4, VP6 and VP7) or a non-structural protein(NSP1 to NSP6) (Estes and Kapikian, 2007). The virion consists ofthree concentric capsid layers. The inner core is composed of VP2encasing VP1, VP3 and the dsRNA genome. The middle layer com-posed of VP6 which is the most abundant protein in the virion andplays a major role in determining the rotavirus serogroups A–H(Matthijnssens et al., 2012). The outer capsid is composed of VP4and VP7. Both VP4 and VP7 elicit neutralising antibodiesindependently. VP4 defines rotavirus P (protease-sensitive) andVP7 defines G (glycoprotein) serotypes and genotypes (Estesand Kapikian, 2007). A wide rotavirus strain diversity has beenreported, however, whole genome sequence analysis ofgroup A rotaviruses has revealed that rotaviruses with either a

M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77 63

I1-R1-C1-M1-A1-N1-T1-E1-H1 (Wa-like) or I2-R2-C2-M2-A2-N2-T2-E2-H2 (DS-1-like) genotype constellations are capable ofsuccessfully spreading and causing rotavirus disease in humanpopulation worldwide (Matthijnssens and van Ranst, 2012).

A web based tool, RotaC (Maes et al., 2009) that analyses thenucleotide sequence of all 11 RNA genome segments, has beenused to characterise whole genomes of rotaviruses by assigninggenotypes to each genome segment. As of April 2011, 27G, 35P,16I, 9R, C9, 8M, 16A, 9N, 12T, 14E and 11H had been identifiedusing the complete genome constellation criteria proposed bythe Rotavirus Classification Working Group (RCWG) (Matthijnssenset al., 2011). In humans, G1, G2, G3, G4 and G9 strains are the ma-jor rotavirus circulating genotypes, accounting for over 88% of allstrains analysed worldwide (Bányai et al., 2012; Santos and Hosh-ino, 2005). Out of the four, G9 rotaviruses were the last to emergeand their origin is unclear. Besides humans, G9 rotaviruses haveonly been identified from pigs (Matthijnssens et al., 2010). Reportson both human and porcine G9 strains show they were detected asearly as 1980 (Cao et al., 2008). Since then, G9 rotavirus strainshave been characterised at higher frequencies and are now consid-ered the fifth most widespread genotype (Armah et al., 2010; Cun-liffe et al., 1999, 2001; Das et al., 1994; Nakagomi et al., 1990; Pageet al., 2010; Ramachandran et al., 2000; Urasawa et al., 1992). InAfrica, G9 rotaviruses were not common before the middle of the1990s. However, they have been characterised more often sincethen. For example, the African Rotavirus Surveillance Network(AfrRSN) recently reported that G9 rotaviruses were characterisedmore frequently compared to the G1, G2, G3 and G4 strains insome African countries like Kenya (Mwenda et al., 2010). Despitethis finding, the currently licensed rotavirus vaccines (RotaTeq™and Rotarix™) do not contain G9 genotypes in their formulation(Vesikari, 2008).

A common origin of animal and human rotaviruses ascertainedby the close similarity between the genome segments of G9 rotavi-rus strains circulating in these species has been reported (Jereet al., 2011a, 2011b; Santos et al., 1999). Evolutionary mechanismsthat include genome reassortment and rearrangement are specu-lated to be the cause for such close genetic relationships. Further-more, phylogenetic analysis over a wide range of porcine andhuman G9 strains reported from Japan and China in the middleof the1980s revealed that interspecies transmission might have oc-curred between rotaviruses circulating in pigs and people. It ishowever, thought that G9 rotaviruses were transmitted from pigsto people due to wider diversity of G9 strains circulating in porcinespecies compared to those circulating in humans (Teodoroff et al.,2005).

Table 1GenBank accession numbers of the complete consensus nucleotide sequence of the genomsegment; NSP, non-structural protein; VP, viral protein.

Study strains GenBank accession numbers

S1 (VP1) S2 (VP2) S3 (VP3) S4 (VP4) S6 (V

RVA/Human-wt/CMR/MRC-DPRU1424/2009/G9P[8]

JN605404 JN605405 JN605406 JN605407 JN60

RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8]

JN605415 JN605416 JN605417 JN605418 JN60

RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8]

JN605426 JN605427 JN605428 JN605429 JN60

RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6]

JN605437 JN605438 JN605439 JN605440 JN60

RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8]

JN605448 JN605449 JN605450 JN605451 JN60

To date, the whole genome sequences of only a few rotaviruseshave been fully characterised globally. Although G9 rotaviruseshave been associated with P[6], P[7] P[8], P[11], P[19] and P[23]genotypes globally, the only two wholly sequenced G9 strains fromAfrica has been associated with only P[6] and P[8] genotypes(Ghosh and Kobayashi, 2011; Ghosh et al., 2012; Jere et al.,2011a, 2011b; Potgieter et al., 2009). Since most of the studieson G9s from different African countries only involved analysis ofthe two outer capsid proteins (VP7 and VP4) and their respectiveencoding genome segments (Page et al., 2010), whole genomeanalysis of more G9 rotaviruses are necessary to understand theircomplete genome constellation better. This study is the first tocompare the whole genomes of G9 rotaviruses associated withP[6] and P[8] genotypes from eastern, central and southern Africanregions. The information gained in this study could be useful forformulation of strain-specific and regional vaccines in future thatcould be used especially in African countries where G9 rotavirusesare being detected frequently.

2. Materials and methods

2.1. Ethics approval and study design

This study was based on samples that were partially character-ised previously as part of the AfrRSN training. Five rotavirusstrains, representing G9 genotypes collected between 1999 and2010 from southern Africa (RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] and RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa and RVA/Human-wt/ZWE/MRC-DPRU1723/2010/G9P[8] from Zimbabwe), eastern Africa (RVA/Hu-man-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya), and cen-tral Africa (RVA/Human-wt/CMR/MRC-DPRU/1424/2009/G9P[8]from Cameroon) were selected. The study was approved by Med-unsa Research Ethics Committee (MREC/P/196/2010:PG).

2.2. Extraction of rotavirus dsRNA from the stool specimens

The dsRNA was extracted from stool samples following estab-lished methods described previously (Jere et al., 2011a; Potgieteret al., 2009). Briefly, 900 ll of TRI-REAGENT-LS, (Molecular Re-search Centre, Cincinnati, OH) was added to 300 ll of a 10% stoolsuspension (ratio of 3:1), mixed and incubated for 5 min at roomtemperature. Then, 267 ll of chloroform was added, followed bycentrifugation at 4 �C for 15 min at 16,000g. The supernatant wasadded to 650 ll of isopropanol for precipitation of RNA. The RNAwas collected by centrifugation at room temperature for 30 min

e segments of the five African G9 rotavirus strains analysed in this study. S, genome

P6) S9 (VP7) S5 (NSP1) S8 (NSP2) S7 (NSP3) S10(NSP4)

S11(NSP5)

5408 JN605409 JN605410 JN605411 JN605412 JN605413 JN605414

5419 JN605420 JN605421 JN605422 JN605423 JN605424 JN605425

5430 JN605431 JN605432 JN605433 JN605434 JN605435 JN605436

5441 JN605442 JN605443 JN605444 JN605445 JN605446 JN605447

5452 JN605453 JN605454 JN605455 JN605456 JN605457 JN605458

Table 2Comparison of the whole genome constellation of the G9 rotavirus study strains and all other fully characterized G9 strains available in GenBank.

1Study strains on a Wa-like genetic backbone.2Study strains on a DS-1-like genetic backbone (the common names of the study strains are in bold), Colours were added to visualise patterns of genome constellations as follows: green (Wa-like), red (DS-1-like), purple (sometypical animal strains), blue (P6) and other undetermined genotypes were not coloured. S, genome segment; VP, viral structural protein; NSP, viral non-structural protein.

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Fig. 1. Phylogenetic trees based on full-length ORF nucleotide sequences of rotavirus genome segments encoding structural proteins ((A)–(F) representing genome segments1–4, 6 and 9 encoding VP1-4, VP6-7, respectively) and non-structural proteins ((H)–(K) representing genome segments 5, 7, 10 and 11 that encode NSP1 to NSP5,respectively). Fig. 1(F) shows the six different G9 lineages (I–VI). All known G9 rotavirus nucleotide sequences with full ORF available in the GenBank were included asreferences for the study strains and are shown in bold text underlined in every phylogram for each of the genome segments. Each cluster was assigned a designation letterwithin the classification based on its function as described by the RCWG. The accession numbers of all reference strains are indicated in supplementary data 4. The studystrains are shown with black squares, and blocks in shaded dashed lines indicate the published strains that shared greatest bootstrap values with the study strains at 1000replicates as shown by numbers adjacent to the nodes from 70% to 100% in all trees. The scale bar indicates the genetic distance as formulated by MEGA 4.0. The arrows pointsat the geographical location where study strains were identified; C, central Africa; E, east Africa; S, southern Africa.

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at 16,000g. The dsRNA pellet was then re-suspended in 95 ll elu-tion buffer (Qiagen, Hilden, Germany). The ssRNA was removedby adding lithium chloride (Sigma, St. Louis, MO) to a concentra-tion of 2 M and incubation at 4 �C for 16 h, followed by centrifuga-tion at 16,000g for 30 min. The integrity of the dsRNA was analysedon 1% Tris–borate-Ethylenediaminetetraacetic acid (TBE) agarosegel stained with ethidium bromide.

2.3. Oligo-ligation

Purified dsRNA was ligated to an anchor primer, PC3-T7 loop(50-GGATCCCGGGAA TTCGGTAATACGACTCACTATATTTTTATAGTGA

GTCGTATTA-OH-3’), using a T4 RNA ligase (Takara) at 37 �C for16 h (Potgieter et al., 2009). The oligo-ligated dsRNA was purifiedwith a MinElute gel extraction kit (Qiagen, Hilden, Germany).

2.4. Sequence-independent cDNA synthesis and amplification

The method described by Potgieter et al. (2009) and modified byJere et al. (2011a) was used. In brief, 30 mM methyl mercuryhydroxide (Alfa Aesar, Massachusetts, MA) was used to denaturethe dsRNA, which was then reverse transcribed to synthesisecDNA. 0.1 M Sodium hydroxide (Sigma, St. Louis, MO) was addedto remove RNA, followed by addition of equal amount of 0.1 M

Fig. 1. (continued)

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Tris/HCl, pH 7.5 (Sigma, St Louis, MO) and annealing at 65 �C for30 min. Amplification of the cDNA was done using primer PC2(50-CCGAATTCCCGGGAT CC-OH3’) under the following reactionconditions: one cycle at 72 �C for 2 min to fill ends, followed by25 PCR cycles each of 94 �C for 2 min, 65 �C for 30 s, and 72 �Cfor 4 min. Finally, an extension step was done at 72 �C for 5 min.The amplified cDNA was evaluated on a 1% TBE agarose gel thatwas stained with ethidium bromide.

2.5. 454� Pyrosequencing, genotype assignment and phylogeneticanalyses

The PCR product was purified using a QIA�quick PCR purifica-tion kit (Qiagen, Hilden, Germany). Pyro-sequencing was done onthe purified PCR product using the 454� GS FLX Titanium technol-ogy (Roche, Mannheim, Germany) (Inqaba Biotec [Pty] Ltd., Preto-

ria, South Africa) as previously reported (Jere et al., 2011a).Pyrosequence data was analysed using the DNASTAR Lasergene�

Core Suite version 8.1.2 as described by Jere et al. (2011a).RotaC (http://rotac.regatools.be) (Maes et al., 2009) was used to

assign genotypes to all 11 genome segments of all the five G9 rota-virus study strains using the consensus nucleotide sequences thatwere generated. For phylogenetic analyses, BioEdit software (Hall,1999) was used to align the nucleotide sequence of the studystrains with all other G9 rotavirus strains of which the whole gen-ome sequences was known and other relevant sequences availablefrom GenBank. MEGA software, version 4.0, was used to constructphylogenetic trees, whereas the genetic distances were calculatedusing the Kimura-2 correction parameter (Tamura et al., 2007). Thephylogenetic trees were constructed by the neighbour-joiningmethod. The bootstrap probabilities of each node were calculatedusing 1000 replicates.

Fig. 1. (continued)

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3. Results

3.1. Genotype assignment and classification of the study strains

The study strains were named based on the guidelines for theuniformity of rotaviruses proposed by the RCWG (Matthijnssenset al., 2011). The five study strains were named as follows: RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8], RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6], RVA/Human-wt/ZWE/MRC-DPRU1723/2010/G9P[8], RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] and RVA/Human-wt/CMR/MRC-DPRU/1424/2009/G9P[8] (Table 1). The consensus nucleotide and deduced aminoacid sequences of all the genome segments of the study strains

were determined (Supplementary data 1). These sequences weresubmitted to GenBank under the accession numbers listed in(Table 1). Based on the genotypes assigned to each genome seg-ments of the five study strains, their complete genotype constel-lations were determined as follows: MRC-DPRU1424, G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (Wa-like); MRC-DPRU1723, G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (Wa-like); MRC-DPRU4677, G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (Wa-like); MRC-DPRU2427,G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (Wa-like) and MRC-DPRU9317, G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2 (DS-1-like)(Table 2). The genotypes assigned to the genetic backbone forfour of the five study strains (those with G9P[8] genotypes) weretypical Wa-like genotype constellations. However, the G9P[6]

Fig. 1. (continued)

68 M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77

study strain had a typical DS-1-like genotype constellation withthe exception of the genome segment 8 (NSP2) which exhibiteda Wa-like genotype.

3.2. Sequence and phylogenetic analyses of the genome segments ofthe study strains

Phylogenetic and pairwise identity analyses of the open read-ing frames (ORFs) of the consensus nucleotide sequence of all ele-ven genome segments of the five study rotavirus strains andthose retrieved from the GenBank was performed to determinehow closely the African G9 strains were related to each otherand also to other G9 strains that were characterised elsewhere

(Fig. 1(A)–(K) and Supplementary data 2 (A)–(K)). Rotavirusstrains with the closest related genome segments to the studystrains have been summarized in (Table 3), and the comparisonof amino acid sequence alignment of the Wa-like and DS-1-likestudy strains for the whole rotavirus genome in (Supplementarydata 3 (A)–(K)).

3.2.1. Phylogenetic analyses of the southern African rotavirus strainson a Wa-like genetic backbone; RVA/Human-wt/ZWE/MRC-DPRU1723/2010/G9P[8] and RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8]

Phylogenetic analyses of all 11 genome segments of strain MRC-DPRU1723/G9P[8] and MRC-DPRU4677/G9P[8] clustered with

Fig. 1. (continued)

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rotavirus strains that exhibit Wa-like genotype constellations(Fig. 1(A)–(K)). Pairwise percentage identities showed that all gen-ome segments of strain MRC-DPRU1723/G9P[8] from South Africawas most closely related to rotavirus strain RVA/Human-wt/ZAF/

2371WC/2008/G9P[8]. The most related genome segments were;7 (NSP3), 8 (NSP2), 9 (VP7) and 10 (NSP4) that were 100% identicalto RVA/Human-wt/ZAF/2371WC/2008/G9P[8]. Genome segment 6(VP6) shared the least nucleotide identity of 99.4%.

0.1

Fig. 1. (continued)

70 M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77

Analyses of rotavirus strain MRC-DPRU4677 from Zimbabwe re-vealed that the highest nucleotide identity of ten of its genomesegments (99.5% to 100%) was with those of strain RVA/Human-

wt/ZAF/2371WC/2008/G9P[8]. Genome segment 3 (VP3) of strainMRC-DPRU4677 was most closest related to strain RVA/Human-wt/ZAF/3176WC/2009/G12P[6] at 99.2% (Table 3).

Fig. 1. (continued)

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3.2.2. Phylogenetic analyses of the southern Africa rotavirus strain on aDS-1-like backbone; RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6]

Phylogenetic analysis of rotavirus strain MRC-DPRU9317 fromSouth Africa showed that nine of its genome segments clusteredwith those of other DS-1-like genotype constellations. Only genomesegment 8 (NSP2) and 9 (VP7) clustered with those of the strainsexhibiting Wa-like genotype constellation. With the exception ofgenome segment 8 (NSP2), which shared highest nucleotide iden-

tity of 94.9% with those of strain RVA/Human-wt/BEL/BE00094/2009/G1P[8]. The other 10 segments were most closely related tostrain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] (Table 3).

3.2.3. Phylogenetic analyses of the eastern African rotavirus strainRVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] on a Wa-likegenetic backbone

Phylogenetic and pairwise nucleotide sequence analysis ofrotavirus strain MRC-DPRU2427 from Kenya revealed that most

Fig. 1. (continued)

72 M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77

of its genome segments were most closely related to those ofnon-G9P[8] strains. Eight genome segments, namely; 1 (VP1), 2(VP2), 3 (VP3), 4 (VP4), 6 (VP6), 7 (NSP3), 8 (NSP2) were mostclosely related to those of G12P[6] and G1P[8] strains. Only gen-ome segments 5 (NSP1) and 11 (NSP5) shared highest nucleotideidentity with those of characterised G9P[8] strains. Interestingly,genome segment 9 (VP7) was most closely related (nucleotideidentity of 97.7%) with a DS-1-like strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] (Table 3).

3.2.4. Phylogenetic analyses of the central Africa rotavirus strain on aWa-like genetic backbone; RVA/Human-wt/CMR/MRC-DPRU1424/2009/G9P[8]

Strain MRC-DPRU1424 from Cameroon was the most diverseWa-like study strain when phylogenetic and pairwise nucleotidesequence analyses were performed. Its genome segments 2 (VP2)and 8 (NSP2) were most closely related to strain RVA/Human-wt/ZAF/2371WC/2008/G9P[8] (98.7% and 100%, respectively).All its other genome segments were closely related those of to

Fig. 1. (continued)

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rotavirus strains that were characterised from different geo-graphical locations globally. For instance, genome segments 1(VP1), 6 (VP6) and 7 (NSP3) were most closely related tostrains from Belgium, whereas genome segments 3 (VP3) and 5(NSP1) were most closely related to strains from theUSA. Genome segment 4 (VP4) was more similar to that of astrain from the Democratic Republic of Congo; andgenome segment 10 (NSP4) to that of unusual strain RVA/Hu-man-wt/Dhaka6/BGD/2001/G11P[25] from Bangladesh (Table 3).

4. Discussion

Human G9 group A rotaviruses are one of the five leading rota-virus G-types that cause severe diarrhoea in young children glob-ally (Bányai et al., 2012). Worldwide studies on the origin andspread of human G9 rotaviruses based on partial sequencing ofgenome segments 9 (VP7, G-typing) and 4 (VP4, P-typing) have re-vealed a high degree of genetic diversity amongst G9 genotypes(Martinez-Laso et al., 2009; Phan et al., 2007). To obtain a more

Fig. 1. (continued)

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comprehensive understanding of the genetic diversity and the ori-gin of some of the African G9 rotavirus strains, we determined andanalysed the consensus whole genome sequence of five G9 rotavi-ruses from eastern, central and southern Africa: one G9P[6] strain(MRC-DPRU9317/G9P[6]) and four G9P[8] strains (MRC-DPRU1424/G9P[8]; MRC-DPRU1723/G9P[8]; MRC-DPRU4677/G9P[8]; MRC-DPRU2427/G9P[8]). The G9P[6] strain was collectedin 1999 and the four G9P[8] strains a decade later between 2009and 2010.

The genotype constellation for the genome segments of the fourG9P[8] strains (MRC-DPRU1424/G9P[8], MRC-DPRU1723/G9P[8],

MRC-DPRU4677/G9P[8] and MRC-DPRU2427/G9P[8]) was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 which is typical of Wa-likestrains (Matthijnssens and van Ranst, 2012). The genotype constel-lation for the genome segments of the G9P[6] strain (MRC-DPRU9317/G9P[6]) was G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2.The genome constellation of RVA strain MRC-DPRU9317 that con-sists of a Wa-like genome segment 8 encoding NSP2 and a DS-1-like genetic backbone could suggest that this strain emergedthrough intergenotype reassortment between Wa-like and DS-1-like rotaviruses. Unlike intergenotype reassortment between gen-ome segments that encode outer capsid proteins, intergenotype

Fig. 1. (continued)

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reassortment involving genome segments that encoding the otherprotein proteins is rare (Heiman et al., 2008; Ward et al., 1990). Thestudy strain MRC-DPRU9317/G9P[6] is the first reassortant G9rotavirus strain to be characterised with a DS-1-like genetic back-bone and Wa-like genome segment 8 (NSP2). The other two reas-sortant rotavirus strains that are known to contain a Wa-like(genotype 1) genome segment 8 (NSP2) on a DS-1-like (genotype2) backbone are G12 rotaviruses. These strains are RVA/Human-wt/PHL/L26/1987/G12P[4] that was isolated in 1987 in the Philip-

pines and RVA/Human-wt/BGD/N26-02/2002/G12P[6] isolated in2002 in Bangladesh (Rahman et al., 2007).

Phylogenetic and distance matrix analyses of the consensusnucleotide sequence of the ORFs of all the genome segments ofthe five G9 study strains showed that they each grouped strictlyaccording to the Wa-like or DS-1-like genotype clusters. Withineach respective genotype, the ORFs of the genome segments ofthe study strains, especially those from southern African region,clustered closely with rotaviruses that were characterised from

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MR

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U46

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010/

G9P

[8]

RV

A/H

um

an-w

t/ZW

E/M

RC

-D

PRU

1723

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0/G

9P[8

]R

VA

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man

-wt/

KEN

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C-D

PRU

2427

/20

10/G

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]R

VA

/Hu

man

-wt/

CM

R/M

RC

-DPR

U14

24/

2009

/G9P

[8]

1(V

P1)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.1%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.7%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.9%

)R

VA

/Hu

man

-wt/

ZAF/

3176

WC

/200

9/G

12P[

6](9

7.6%

)R

VA

/Hu

man

-wt/

BEL

/BE0

0094

/G1P

[8]

(98.

2%)

2(V

P2)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.2%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.8%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.6%

)R

VA

/Hu

man

-wt/

BEL

/BE0

0098

/200

9/G

1P[8

](9

7.0%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

8.7%

)3

(VP3

)R

VA

/Hu

man

-wt/

ZAF/

GR

1092

4/19

99/

G9P

[6]

(99.

5%)

RV

A/H

um

an-w

t/ZA

F/31

76W

C/2

009/

G12

P[6]

(99.

2%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

5%)

RV

A/H

um

an-w

t/B

GD

/Mat

lab1

3/20

03/

G12

P[6]

(99.

2%)

RV

A/H

um

an-w

t/U

SA/2

0087

4730

7/20

08/G

9P[8

](9

7.6%

)4

(VP4

)R

VA

/Hu

man

-wt/

ZAF/

GR

1092

4/19

99/

G9P

[6]

(99.

2%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

7%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

8%)

RV

A/H

um

an-w

t/B

EL/B

3458

/200

3/G

1P[8

](9

7.4%

)R

VA

/Hu

man

-wt/

CO

D/D

RC

88/2

003/

G8P

[8]

(97.

4%)

5(N

SP1)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.1%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.5%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.7%

)R

VA

/Hu

man

-wt/

USA

/200

8747

307/

2008

/G9P

[8]

(97.

4%)

RV

A/H

um

an-w

t/U

SA/2

0097

2704

7/20

09/G

9P[8

](9

8.5%

)6

(VP6

)R

VA

/Hu

man

-wt/

ZAF/

GR

1092

4/19

99/

G9P

[6]

(98.

7%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

5%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

4%)

RV

A/H

um

an-w

t/ZA

F/31

76W

C/2

009/

G12

P[6]

(96.

2%)

RV

A/H

um

an-w

t/B

EL/B

E000

94/2

009/

G1P

[8]

(98.

9%)

7(N

SP3)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.4%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.6%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](1

00%

)R

VA

/Hu

man

-wt/

BEL

/BE0

0094

/200

9/G

1P[8

](9

6.2%

)R

VA

/Hu

man

-wt/

BEL

/BE0

0094

/200

9/G

1P[8

](9

9.1%

)8

(NSP

2)R

VA

/Hu

man

-wt/

BEL

/BE0

0094

/200

9/G

1P[8

](9

4.9%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](1

00%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](1

00%

)R

VA

/Hu

man

-wt/

BEL

/BE0

0094

/200

9/G

1P[8

](9

4.1%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](1

00%

)9

(VP7

)R

VA

/Hu

man

-wt/

ZAF/

GR

1092

4/19

99/

G9P

[6]

(99.

2%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(99.

6%)

RV

A/H

um

an-w

t/ZA

F/23

71W

C/2

008/

G9P

[8]

(100

%)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

7.7%

)R

VA

/Hu

man

-wt/

ZAF/

GR

1092

4/19

99/

G9P

[6]

(98.

7%)

10(N

SP4)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.4%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.6%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](1

00%

)R

VA

/Hu

man

-wt/

BG

D/M

atla

b13/

2003

/G

12P[

6](9

7.2%

)R

VA

/Hu

man

-wt/

Dh

aka6

/BG

D/2

001/

G11

P[25

](9

9.1%

)11

(NSP

5/6)

RV

A/H

um

an-w

t/ZA

F/G

R10

924/

1999

/G

9P[6

](9

9.4%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.6%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

9.6%

)R

VA

/Hu

man

-wt/

ZAF/

2371

WC

/200

8/G

9P[8

](9

7.8%

)R

VA

/Hu

man

-wt/

ZAF/

3176

WC

/200

9/G

12P[

6](9

8.6%

)

76 M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77

the same geographical area irrespective of their G-type for mostgenome segments. This supports previous reports that G9 strainsfrom Africa cluster mostly within geographical areas (Armahet al., 2010; Page et al., 2010). However, that was not the case withthe central and eastern African study strains. Genome segments ofthe central African G9 study strains that contained a Wa-like ge-netic backbone clustered closest to cognate genome segments ofG1, G8, G9, G11 and G12 strains, whereas most of the genome seg-ments of the DS-1-like study strain clustered closest to cognategenome segments of only G2 and G8 strains characterized fromdifferent parts of the world. This difference in genetic clusteringmight reflect preferred genotype constellations as described byHeiman et al. (2008).

Analyses of the whole genome of the four G9P[6] group A rota-viruses which have to date been completely sequenced reveal thattwo have a Wa-like genetic constellation; the Indian strain RVA/Human-wt/IND/mcs-13/2007/G9P[6] (Mukherjee et al., 2009) andthe Belgian strain RVA/Human-wt/BEL/BE2001/2007/G9P[6] (Zel-ler et al., 2012), whereas the two strains from South Africa [studystrains MRC-DPRU9317/G9P[6] and RVA/Human-wt/ZAF/GR10924/1999/G9P[6] (Jere et al., 2011a)] are on a DS-1-like ge-netic backbone. Interestingly, all these G9P[6]s are reassortants.The Indian strain, IND/mcs13/2007/G9P[6] is a mono reassortantwhose genome segment 5 (NSP1) is porcine-like; the Belgianstrain, BEL/BE2001/2007/G9P[6] is a multiple reassortant with por-cine-like genome segments 6 (VP6) and 5 (NSP1) and an unusualT7 genome segment 7 (NSP3) which has been detected in humansand pigs. The South African strain ZAF/GR10924/1999/G9P[6]could be a multiple genome reassortant strain between G9, humanP[6] and DS-1-like rotaviruses; and the South African study strainMRC-DPRU9317/G9P[6] is a reassortant with a Wa-like genomesegment 8 (NSP2) on a DS-1-like backbone. With the exceptionof the reassortant genome segment 8 (NSP2) of the study strainMRC-DPRU9317/G9P[6], which was closely related to the Wa-likestudy strain MRC-DPRU2427/G9P[8] from east Africa, the other10 genome segments of the two southern African G9P[6] strainson a DS-1-like genetic backbone (MRC-DPRU9317/G9P[6] andGR10924/G9P[6]) were genetically closely related with nucleotideidentities between 98.7% and 99.6%. They were probably circulat-ing together in the same community as they were collected duringthe same year at the same hospital (Jere et al., 2011a; Page et al.,2010; Potgieter et al., 2009). The finding that the Wa-like genomesegment 8 (NSP2) of the reassortant South African study strainMRC-DPRU9317/G9P[6], which was collected in 1999, was mostclosely related (97.1% nucleotide identity) to that of the Wa-likeKenyan study strain MRC-DPRU2427/G9P[8] collected in 2010seems to indicate that the reassortment between Wa and DS-1-likerotaviruses potentially occurred more than a decade ago and thatsimilar strains could be circulating in the communities. Furtherrotavirus whole genome analysis studies on G9 samples collectedfrom these regions could potentially provide more information.

5. Conclusion

Overall, our whole genome sequence data confirmed and ex-tended the results from previous partial and full genome sequenc-ing data for genome segment 9 (VP7) (Page et al., 2010; Jere et al.,2011a, 2011b) that African G9 rotavirus strains cluster in the gen-ome segment 9 lineage III, and have both DS-1-like (SGI, P[6], andshort RNA profile) and Wa-like characteristics (SGII, P[8] and longRNA profile) conferred by the size of genome segment 11 (NSP5/6).The whole genome sequence data of the five African G9 strainsgenerated in this study brings the total number of fully analysedAfrican G9 rotaviruses to seven. Prior to our work reported here,only one G9P[8], (RVA/Human-wt/ZAF/2371WC/G9P[8]) strain,

M.M. Nyaga et al. / Infection, Genetics and Evolution 16 (2013) 62–77 77

and one G9P[6], RVA/Human-wt/ZAF/GR10924/1999/G9P[6])strain had previously been fully analysed from Africa. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] is the fourthG9P[6] rotavirus to be completely characterised globally and thesecond with a DS-1-like genetic backbone.

Acknowledgements

The study received financial support from the Medical ResearchCouncil and Poliomyelitis Research Foundation (PRF) in South Afri-ca. M.M. Nyaga is grateful for the MSc bursary (10/51) receivedfrom the PRF, South Africa. We also thank the staff of MRC/Diarrho-eal Pathogens Research Unit together with Dr. H.G. O’Neill of theDepartment of Biochemistry, North-West University for valuablein-depth discussions and Dr. M.J. Mwenda of WHO/AFRO, Brazza-ville, Congo for logistics with use of some of the samples.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2013.01.005.

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