Infection, Genetics and Evolution 28 (2014) 537–560

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Infection, Genetics and Evolution

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Full genome characterization of human Rotavirus A strains isolatedin Cameroon, 2010–2011: Diverse combinations of the G and P genesand lack of reassortment of the backbone genes

http://dx.doi.org/10.1016/j.meegid.2014.10.0091567-1348/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding authors.E-mail addresses: valentinengum@yahoo.com (V.N. Ndze), bkrota@hotmail.com

(K. Bányai).

Valentine Ngum Ndze a,⇑, Mathew Dioh Esona b, Eric Akum Achidi c, Kamga Hortense Gonsu a,Renáta Dóró d, Szilvia Marton d, Szilvia Farkas d, Marxcel Bong Ngeng e, Akum Felix Ngu f,Marie Therese Obama-Abena a, Krisztián Bányai d,⇑a Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Cameroonb Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USAc Faculty of Sciences, University of Buea, Cameroond Institute for Veterinary Medical Research, Hungarian Academy of Sciences, Budapest, Hungarye Medical Diagnostic Laboratory, Regional Hospital Maroua, Cameroonf Regional Hospital Laboratory Bamenda, Cameroon

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

Article history:Received 25 June 2014Received in revised form 9 October 2014Accepted 11 October 2014Available online 17 October 2014

Keywords:Rotavirus A genomicsPhylogenyIon Torrent PGMCameroon

Over the past few years whole genome sequencing of rotaviruses has become a routine laboratorymethod in many strain surveillance studies. To study the molecular evolutionary pattern of representa-tive Cameroonian Rotavirus A (RVA) strains, the semiconductor sequencing approach was used followingrandom amplification of genomic RNA. In total, 31 RVA strains collected during 2010–2011 in threeCameroonian study sites located 120 to 1240 km from each other were sequenced and analyzed.Sequence analysis of the randomly selected representative strains showed that 18 RVAs were Wa-like,expressing G1P[6], G12P[6], or G12P[8] neutralization antigens on the genotype 1 genomic constellation(I1-R1-C1-M1-A1-N1-T1-E1-H1), whereas 13 other strains were DS-1-like, expressing G2P[4], G2P[6],G3P[6], and G6P[6] on the genotype 2 genomic constellation (I2-R2-C2-M2-A2-N2-T2-E2-H2). Nointer-genogroup reassortment in the backbone genes was observed. Phylogenetic analysis of theCameroonian G6P[6] strains indicated the separation of the strains identified in the Far North region(Maroua) and the Northwest region (Bamenda and Esu) into two branches that is consistent withmultiple introductions of G6P[6] strains into this country. The present whole genome based molecularcharacterization study indicates that the emerging G6P[6] strain is fully heterotypic to Rotarix, thevaccine introduced during 2014 in childhood immunization program in Cameroon. Continuous strainmonitoring is therefore needed in this area and elsewhere to see if G6s, besides genotype G1 to G4, G8,G9 and G12, may become a new, regionally important genotype in the post vaccine licensure era in Africa.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Group A rotaviruses (Rotavirus A, RVA) constitute the mostimportant etiological agents of acute gastroenteritis in infantsand young children worldwide (Estes and Kapikian, 2007). Globalmortality estimates show that about 453,000 children <5 years ofage die worldwide annually due to rotavirus (RV) disease, withmost of the deaths occurring in developing countries of Africa

and Asia (Tate et al., 2012). Rotaviruses (family Reoviridae, genusRotavirus) are icosahedral viruses with a genome consisting of 11segments of dsRNA (Attoui et al., 2012) in a triple-layered virion,encoding six structural proteins (VP1-VP4, VP6 and VP7) and fiveor six non-structural proteins (NSP1-NSP6) (Estes and Kapikian,2007).

VP7 and VP4 are the components of the outer capsid, each car-rying neutralization-specific epitopes (Estes and Kapikian, 2007).The middle inner capsid is composed of VP6 and surrounds thecore shell, which is composed of VP2 (McClain et al., 2010).Packaged within the core shell are the viral RNA-dependent RNApolymerase (VP1) and RNA capping enzyme (VP3), as well as the

538 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

11 dsRNA genome segments (Estes and Kapikian, 2007). Therotavirus NSPs have various functions in the replication andmorphogenesis, and in evasion of the host immune response(Hu et al., 2012).

Matthijnssens et al., 2008b have recently established anextended classification and nomenclature of RVA to include all11 genome segments, defining genotypes for each genome seg-ment. The nomenclature Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hxrepresents, respectively, the genotypes of the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5-encoding gene segments,with ‘x’ indicating the numbers of the corresponding genotypes(Matthijnssens et al., 2008b). For viruses with segmented genomes,this approach of classification could also be used to determinewhether or not certain rotavirus genes co-segregate during reas-sortment events (gene linkage) or whether certain gene constella-tions play a role in rotavirus host range restriction or virulence(Matthijnssens et al., 2008b). Thus far, at least 27 G, 37 P, 16 I, 9R, 9 C, 8 M, 16 A, 9 N, 12 T, 15 E and 11 H genotypes have beenidentified from humans and animals, based on the 11 RVA genes,with many new genotypes identified in the past five years (Abeet al., 2009; Schumann et al., 2009; Solberg et al., 2009; Ursuet al., 2009; Esona et al., 2009b; Matthijnssens et al., 2011a; Pappet al., 2012; Trojnar et al., 2013). In humans, RVA strains of six Ggenotypes (G1, G2, G3, G4, G9, and G12), and two P genotypes(P[8] and P[4]) predominate worldwide, although genotype P[6]RVA strains have been recognized as a common cause of diarrheaon several continents and have sometimes been described as aregionally predominant strain (Steele and Ivanoff, 2003; Gentschet al., 2005; Santos and Hoshino 2005; Bányai et al., 2012). A largenumber of rare or regionally common strains have been identifiedduring surveillance in anticipation of vaccines introduction includ-ing genotypes G5, G6, G8, G9, G10, and G12 and genotypes P[1],P[3], P[6], P[9], P[11], P[14], P[19] and P[25] (Esona et al., 2004;Gentsch et al., 2005; Rahman et al., 2005; Bányai et al., 2007,2009a; Castello et al., 2009; Cunliffe et al., 2009; Payne et al.,2009; Bányai et al., 2010; Esona et al., 2010; Martella et al.,2010; Matthijnssens et al., 2010; Iturriza-Gómara et al., 2011;Matthijnssens et al., 2011b; Bányai et al., 2012). Studies from Africareported high prevalence of genotypes G8 and P[6] in variouscombinations suggesting that both of these genotypes should beconsidered common in Africa. (Cunliffe et al., 1999; Steele andIvanoff, 2003; Armah et al., 2010).

Currently, RVA strains are being analyzed and compared to oneanother by partial or complete sequencing of all 11 gene segmentsas this approach allows direct determination of genetic relation-ships (Rahman et al., 2003; Matthijnssens et al., 2006a,b, 2008a).Furthermore, sequencing of RVA genomes is critical to theunderstanding of phylogenetic analyses and to the elucidation ofthe patterns of virus evolution. For example, the comprehensiveclassification system has already revealed genetic relationshipsamong RVAs from different host species, including evidence thathuman RVAs belonging to the Wa-like genogroup have a commonorigin with porcine rotaviruses, while those belonging to theDS-1-like genogroup have a common origin with bovine RVAs(Matthijnssens et al., 2008a). Furthermore, a better understandingof the diversity of viral proteins, other than the neutralizationantigens, is also considered important to help evaluate the roleand extent of both homotypic and heterotypic immunity evokedby various vaccines.

Considering that most full genome studies were performedon uncommon RVA strains, limited data on medically impor-tant strains have been available from Africa, in particular, thecentral African sub-region. In this study we report the fullgenome analysis of several epidemiologically major and minorhuman RVA strains having circulated in Northern Cameroonduring 2010–2011.

2. Materials and methods

2.1. Rotavirus strains

The epidemiology of RVA infections during 2010–2011 inNorthern Cameroon has been described elsewhere (Ndze et al.,2012, 2013). Briefly, stool samples were collected from children<5 years of age who presented with acute diarrhea at the RegionalHospital Maroua and at the Domayo Djama integrated health cen-ter in the Far North region; and at the Regional hospital Bamendaand at the Esu integrated health centre in the North West region.Routine typing of the VP7 (G) and VP4 (P) antigens was carriedout by multiplex semi-nested PCR targeting G1–G4, G8, G9, G10and G12 VP7 type and P[4], P[6], P[8], P[9] P[10] and P[11] VP4type specificities. The amplified gene fragment polymorphismwas evaluated by agarose gel electrophoresis (Gentsch et al.,1992; Das et al., 1994; Ndze et al., 2013).

2.2. Random primed reverse transcription-polymerase chain reaction

This study included 31 RVA positive stool specimens analyzedpreviously by a semi-nested PCR (Ndze et al., 2012). In brief, thegenomic RNA was extracted using the Viral RNA Mini kit (QIAGEN)according to the manufacturer’s recommendation. The RNA samplewas subsequently denatured at 97 �C for 5 min in the presence of10 lM random hexamer tailed by a common PCR primer sequence(Djikeng et al., 2008). Reverse transcription was performed with1 U AMV reverse transcriptase (Promega), 400 lM dNTP mixture,and 1X AMV RT buffer at 42 �C for 45 min following a 5 min incu-bation at room temperature. Then, 5 ll cDNA was added to 45 llPCR mixture to obtain a final volume of 50 ll and a concentrationof 500 nM for the PCR primer, 200 lM for dNTP mixture, 1.5 mMfor MgCl2, 1X Taq DNA polymerase buffer, and 0.5 U for Taq DNApolymerase (Thermo Scientific). The reaction conditions consistedof an initial denaturation step at 95 �C for 3 min, followed by 40cycles of amplification (95 �C for 30 s, 48 �C for 30 s, 72 �C for2 min) and terminated at 72 �C for 8 min.

2.3. Nucleotide sequencing using Ion Torrent PGM

Ten to 100 ng cDNA obtained by the random RT-PCR wassubjected to enzymatic fragmentation using the reagents suppliedin the NEBNext� Fast DNA Fragmentation & Library Prep Set forIon Torrent™ kit (New England Biolabs) according to themanufacturer’s instruction. The adaptor ligation was performedusing reagents from the same kit, whereas barcoded adaptorswere retrieved from the Ion Xpress™ Barcode Adapters (LifeTechnologies). The barcoded library DNA samples were columnpurified using the Gel/PCR DNA Fragments Extraction kit (Geneaid)kit. The eluted library DNA was then run on 2% precast gel.Products between 300 and 350 bp were directly used withoutfurther purification in the PCR mixture of the NEBNext� FastDNA Fragmentation & Library Prep Set for Ion Torrent™ kit(New England Biolabs). Library amplification was made using thefollowing profile: denaturation at 98 �C for 30 s, followed by 12amplification cycles (98 �C for 10 s, 58 �C for 30 s, 72 �C for 30 s)and terminated at 72 �C for 5 min. Following amplification of thelibrary DNA the products were purified by the Gel/PCR DNAFragments Extraction kit (Geneaid). The DNA was eluted in 50 llnuclease free water and quantified fluorometrically on a Qubit�

2.0 equipment using the Qubit� dsDNA BR Assay kit (Invitrogen).Approximately equimolar aliquots of the individually barcodedproducts were mixed in a single tube and this library mixturewas used in subsequent emulsion PCR. This step was carried outaccording to the manufacturer’s protocol using the Ion PGM

V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 539

Template kit on an OneTouch v2 instrument. Enrichment of thetemplated beads (on an Ion One Touch ES machine) and furthersteps of pre-sequencing set-up were performed according to the200 bp protocol of the manufacturer. The sequencing protocolrecommended for Ion PGM™ Sequencing Kit on a 316 chip wasstrictly followed. Raw sequence data were mapped onto referenceavian origin RVA sequences obtained from the GenBank in the CLCGenomics Workbench (http://www.clcbio.com/). After visualinspection of the sequence alignments, a single consensussequence was finalized for each strain.

2.4. Phylogenetic analysis

Sequences were aligned using the MUSCLE program withinMEGA version 6 software (Tamura et al., 2013). Once aligned, theDNA Model Test program implemented in MEGA (Tamura et al.,2013) was used to identify the optimal evolutionary models thatbest fit the sequence datasets. Using corrected Akaike InformationCriterion (AICc), the following models were found to best fit thesequence data for the indicated genes: GTR+G+I (for all VP7 andVP4 trees), TN93+G+I (NSP1, NSP2, NSP3, VP2, VP3, and VP6),T92+G (NSP4), GTR+G+I (NSP5), and T92+G+I (VP1). Using the cor-responding substitution models, maximum likelihood trees wereconstructed in PhyML (Guindon et al., 2010) with aLRT statisticsas support. Nucleotide and amino acid distance matrixes were pre-pared using the p-distance algorithm in MEGA version 6 (Tamuraet al., 2013).

2.5. GenBank accession numbers

Nucleotide sequences of the 31 Cameroonian strainswere deposited in GenBank under the following accessionnumbers: KM660080–KM660420 (NSP1, KM660080–KM660108,KM660109, KM660110; NSP2, KM660111–KM660141, NSP3,KM660142–KM660172; NSP4, KM660173–KM660203; NSP5,KM660204–KM660232, KM660233, KM660234; VP1, KM660235–KM660263, KM660264, KM660265; VP2, KM660266–KM660293,KM660294, KM660295, KM660296; VP3, KM660297–KM660326,KM660327; VP4, KM660328–KM660356, KM660357, KM660358;VP6, KM660359–KM660389; VP7, KM660390–KM660418, KM660419,KM660420).

3. Results

3.1. Genotype constellation of 31 Cameroonian human RVA strains

A total of 31 RVA strains from the 2010–2011 surveillanceperformed in three Cameroonian study sites were selected forwhole genome sequencing based on their respective genotypeconstellations. The list of strains is shown in Table 1. Of interest,five G6 strains have been previously misidentified as G12 by amultiplex semi-nested RT-PCR with published primers (Ndzeet al., 2013). These five strains are designated now as genotypeG6P[6].

The sequence analysis of the 31 RVAs showed that 18 strainswere Wa-like, expressing G1P[6], G12P[6], and G12P[8] neutraliza-tion antigens on the genotype 1 genomic constellation, i.e.I1-R1-C1-M1-A1-N1-T1-E1-H1, whereas 13 other strains wereDS-1-like, expressing G2P[4], G2P[6], G3P[6] and G6P[6] on thefollowing backbone: I2-R2-C2-M2-A2-N2-T2-E2-H2 (Table 1). Nointergenogroup reassortment in the backbone genes was observed.On the contrary, Table 2 shows a high degree of RVA reassortmentof the P and G antigens, which apparently preceded the detectionof co-circulating strains in the study areas.

3.2. Phylogenetic analysis of the genes encoding neutralizationantigens

Genotype G1. The two Cameroonian G1 strains shared 99.9%nucleotide (nt) and 99.7% amino acid (aa) identities among them-selves. Both strains shared lower (nt, 85.5–99.2%; aa, 91.5–98.7%)similarity with reference human and porcine G1 RVA strainsdescribed in other countries, sharing the greatest nt and aa homol-ogies with a Bangladeshi strain (RVA/Human-wt/BGD/Agroj23/2002/G1P[8]). Phylogenetic analysis of the Cameroonian G1 RVAstrains classified them into lineage I together with other contem-porary strains from Asia and Europe (Fig. 1, panel A1).

Genotype G2. The three selected Cameroonian G2 strains (onefrom Maroua, two from Bamenda) shared nt and aa identities inthe range 99.5–100% among themselves and with maximum aminoacid homologies with Asian G2 strains; RVA/Human-wt/CHN/CH-128/2001-2003/G2P[X], RVA/Human-wt/IND/253/XXXX/G2P[4],RVA/Human-wt/TWN/TE65/1993/G2P[x] (sequence identity ranges:97.6–99.0%). Phylogenetic analysis showed that the Cameroonian G2strains clustered together with other strains from Africa (Kenya,South African) within lineage II and were also closely related toTaiwanese G2 strains from the 1990s (Fig. 1, panel A2).

Genotype G3. The five Cameroonian G3 RVA strains, collected inEsu and Maroua, shared high nucleotide and amino acid identitiesin the range 98.9–100% and 100%, respectively with one another.Strains from the same region were more closely related to eachother than to strain in the other study area. Furthermore, moderateto high nt (P80.3%) and aa (P89.1%) similarity values were foundwith other human G3 strains and low similarities were seen(nt, 680%; aa, 691.2%) when compared to animal origin (e.g.equine, lapine, canine and simian) G3 strains. The CameroonianG3 strains formed a separate cluster within lineage I and weremost closely related with G3 strains from USA, Thailand and Japan(Fig. 1, panel A3).

Genotype G6. The VP7 gene of genotype G6 RVA strains detectedfor the first time in Cameroon shared >95% nucleotide and aminoacid similarity identities among themselves. Compared with otherG6 rotaviruses the five Cameroonian strains shared similar rangesof sequence identities with contemporary African and European G6rotavirus strains, whereas a lower identity was seen with a varietyof other human and animal origin strains detected worldwide(range: nt, 76.7–86.6%; aa, 87.8–93.0%). The phylogenetic analysisshowed that our Cameroonian G6 RVA strains formed two minorclusters, both classified into lineage I (Fig. 1, panel A4).

Genotype G12. The VP7 gene of all 15 Cameroonian G12 strainsshowed high to maximum nt and aa similarity (P98.9%) amongthemselves and remarkably great nt (96.3–99.2%) and aa (96.7–100%) similarities with other G12 strains reported from othercountries. More specifically, phylogenetic analysis showed thatall characterized Cameroonian G12 strains clustered in the modernG12 lineage III and these strains were closely related to G12 strainsfrom India and Sri Lanka (Fig. 1, panel A5).

Genotype P[4]. Analysis of the two P[4] strains showed nt and aasimilarity identities of 100% among themselves and very highnt and aa homology identities ranging from 86.5–97.5% to89.3–99.0%, respectively with other P[4] strains collected world-wide. Phylogenetic analysis showed that the Cameroonian P[4]strains belonged to a cluster within lineage III and were mostclosely related to modern P[4] strains, including those detectedin Bangladesh, Italy or the USA (Fig. 1, panel B1).

Genotype P[6]. The 14 P[6] strains demonstrated remarkablyhigh to maximum nt and aa similarities in the range of 96.4–100% among themselves and 79.9–98.8% nt and aa similarities withtypical human P[6] strains and several porcine or porcine-derivedhuman P[6] strains. The Cameroonian P[6] strains constituted astatistically well supported cluster within the lineage I P[6]

Table 1List of RVA strains with complete genome characterized in Cameroon during 2010–2011.

STRAIN VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5

RVA/Human-wt/CMR/MA92/2011/G1P[6] G1 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA130/2011/G1P[6] G1 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ ES283/2010/G12P[6] G12 P[6]* I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA01/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA02/2010/G12P[8] G12 P[8] I1 R1* C1* M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA11/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA16/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA24/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA33/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA46/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA52/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA60/2010/G12P[8] G12 P[8] I1 R1* C1* M1 A1* N1 T1 E1 H1

RVA/Human-wt/CMR/ MA70/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA75/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA88/2011/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ MA127/2011/G12P[8] G12* P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ ES290/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/ BA356/2010/G12P[8] G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1

RVA/Human-wt/CMR/BA366/2010/G2P[4] G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/BA368/2010/G2P[4] G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/MA104/2011/G2P[4] G2 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ MA109/2011/G3P[6] G3 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ MA114/2011/G3P[6] G3 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ MA155/2011/G3P[6] G3 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ ES276/2011/G3P[6] G3 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ ES293/2011/G3P[6] G3 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ MA202/2011/G6P[6] G6 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ MA228/2011/G6P[6] G6 P[6] I2 R2 C2 M2 A2* N2 T2 E2 H2

RVA/Human-wt/CMR/ ES298/2011/G6P[6] G6 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/BA346/2010/G6P[6] G6 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

RVA/Human-wt/CMR/ BA369/2010/G6P[6] G6 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2

The sequenced fragments of these genes were shorter than the open reading frame of the particular gene, therefore they were omitted from the phylogenetic analysis.

Table 2G–P type association of 31 RVA strains isolated in Cameroon (2010–2011) highlight-ing likely reassortment events.

G types P types Total Likely reassortment

P[4] P[6] P[8]

G1 2 2 2G2 2 1 3 1G3 5 5 5G6 5 5 5G12 1 15 16 1Total 2 14 15 31 14

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genotype and all these strains were closely related to historicChinese P[6] strains (Fig. 1, panel B2). Of interest, whereas a

historic Cameroonian RVA strain showed very close relationshipto a bat-origin P[6] strain, contemporary Cameroonian strainscarrying the P[6] VP4 genotype did not show this close geneticrelationship with bat RVAs.

Genotype P[8]. The predominant RVA VP4 genotype inCameroon was P[8]. Sequence analysis of 15 P[8] strains showedthat the Cameroonian P[8] strains had 97.5–100% nt and98.2–100% aa identities among themselves. These strains alsodemonstrated high to very high nt (range: 86.5–99.3%) and aa(range: 86.7–99.6%) identities with other P[8] strain from aroundthe world. The Cameroonian P[8] RVAs shared great aa (P98.2%)identities with several modern US G1P[8] RVAs and some historicMalawian G1P[8] RVA strains. These strains formed two clusterswithin lineage P[8] III together with other African P[8] strains from

A1:G1RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/BGD/Agroj23/2002/G1P[8]RVA/Human/NCA/9J/2010/G1P[8]RVA/Human/NCA/25J/2010/G1P[8]

RVA/Human-wt/AUS/CK00083/2008/G1P[8]RVA/Human-wt/BEL/BE00017/2006/G1P[8]RVA/Human-wt/USA2007719825/2007/G1P[8]RVA/Human-wt/VNM/VN-355/2003/G1P[x]

RVA/Human-wt/URY/Mvd9815/1996-99/G1P[x]RVA/Human-wt/URY/Mvd9806/1996/G1P[8]RVA/Human-wt/URY/Mvd9814/1996-99/G1P[x]RVA/Human-wt/ITA/PA73R-/04/2004/G1P[8]RVA/Human-wt/ITA/PAH191/96/1996/G1P[8]RVA/Human-wt/CHN/Chi-83/2002/G1P[x]RVA/Human-wt/KOR/CAU136/2006/G1P[8]

RVA/Human-wt/KOR/CAU219/2006/G1P[8]RVA/Human-wt/JPN/J-6219/05/2005/G1P[x]RVA/Human-wt/ITA/PA8/01/2001/G1P[8]RVA/Human-wt/ITA/PA19/01/2001/G1P[8]

RVA/Human-wt/ITA/PA258/97/1997/G1P[8]RVA/Human-wt/ITA/PA32/90/1990/G1P[8]

Lineage I

RVA/Human-wt/ITA/PAF166/94/1994/G1P[8]RVA/Human-wt/ITA/PA164/99/1999/G1P[8]RVA/Human-wt/ITA/PA33/99/1999/G1P[8]RVA/Human-wt/ITA/PA9/03/2003/G1P[8]RVA/Human-tc/CRI/Cos-70/1995/G1PxRVA/Vaccine/USA/Rotarix-RIX4414/1988/G1P1A[8]RVA/Human-wt/URY/Mvd9606/1996/G1P[8]RVA/Human-wt/URY/Mvd9614/1996-99/G1P[x]RVA/Human-wt/URY/Mvd9616/1996-99/G1P[x]

Lineage II

RVA/Human-wt/ITA/PA10/90/xxxx/G1P[8]RVA/Human-wt/ITA/PA78/89/xxxx/G1P[8] Lineage V

RVA/Human-wt/JPN/87Y1397/xxxx/G1P[x]RVA/Human-wt/JPN/89H452/xxxx/G1P[x]RVA/Human-tc/KOR/Kor-64/1988/G1P[x]

Lineage IV

RVA/Human-tc/ISR/Isr-56/1995/G1P[x]RVA/Human-tc/BRA/Brz-6/1995/G1P[x]

RVA/Vaccine/USA/RotaTeq-WI79-9/1992/G1P75RVA/Human-tc/USA/Wa/1974/G1P1A[8]

Lineage III

Lineage VIRVA/Human-wt/JPN/AU19/xxxx/G1P[x] RVA/Pig-tc/VEN/C60/xxxx/G1P[x]

RVA/Pig-tc/VEN/C95/xxxx/G1P[x] Lineage VIIOutgroupRVA/Human-tc/USA/DS-1/1976/G2P[4]

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0.1 Fig. 1. Panels A–K. Maximum likelihood phylogenetic trees obtained from nucleotide sequences of all 11 genes [VP7 (A1, G1; A2, G2; A3, G3; A4, G6, A5, G12), VP4 (B1, P[4];B2, P[6]; B3, P[8]), VP1 (C), VP2 (D), VP3 (E), VP6 (F), NSP1 (G), NSP2 (H), NSP3 (I), NSP4 (J) and NSP5 (K)] of RVA from Cameroon with known human and animal rotavirusstrains from GenBank database. The trees were drawn to scale. The aLRT values P80% are shown with an asterisk. The strains labeled with filled circles indicate the Cameroonisolates sequenced in this study. The scale bar at the bottom of the trees indicates genetic distance.

V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 541

A2:G2

RVA/Human-wt/JPN/J-4787/2001-2003/G2P[4]RVA/Human-wt/JPN/KO-2/XXXX/G2P[X] RVA/Human-wt/CHN/CH-107/2001-2003/G2P[X]RVA/Human-wt/VN/VN-19/2001-2003/G2P[X]RVA/Human-wt/CHN/CH-128/2001-2003/G2P[X]RVA/Human-wt/CHN/CH-130/2001-2003/G2P[X]RVA/Human-wt/IRA/CIT-220RV/1997-1999/G2P[4]

RVA/Human-wt/IND/Sc27/XXXX/G2P[X] RVA/Human-wt/IND/253/XXXX/G2P[4] RVA/Human-wt/THA/CMH019/2003/G2P[X]

RVA/Human-wt/CAM/MA104/2011/G2P[4]RVA/Human-wt/CAM/BA366/2010/G2P[4]RVA/Human-wt/CAM/BA368/2010/G2P[4]

RVA/Human-wt/TWN/TE65/1993/G2RVA/Human-wt/TWN/TD69/1992/G2RVA/Human-wt/JPN/PAK458/XXXX/G2 RVA/Human-wt/ZAF/NG4585/99/1999/G2P[6]RVA/Human-wt/CHN/XJ00-486/2000/G2P[6]

RVA/Human-tc/IND/N1/XXXX/G2 RVA/Human-wt/PA83/2007/KC178812

RVA/Human-wt/IND/SC-4/XXXX/G2 RVA/Human-wt/ZAF/TN1529/99/1999/G2RVA/Human-wt/ZAF/4476PT/97/1997/G2

RVA/Human-wt/JPN/JAPAN0022/XXXX/G2 RVA/Human-wt/JPN/CHIN-5/XXXX/G2 RVA/Human-wt/JPN/92H102/XXXX/G2 RVA/Human-wt/JPN/JAPAN085/XXXX/G2 RVA/Human-wt/JPN/JAPAN076/XXXX/G2

RVA/Human-wt/KEN/KY3303/99/1999/G2RVA/Human-tc/IND/S2/XXXX/G2P[4]

RVA/Human-wt/AUS/94A/XXXX/G2 RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]RVA/Human-wt/JPN/PAK426/XXXX/G2 RVA/Human-wt/JPN/JAPAN137/XXXX/G2

RVA/Human-wt/JPN/TMC-II/XXXX/G2 RVA/Human-wt/JPN/KUNJPN/XXXX/G2

Lineage II

RVA/Human-wt/ZAF/514GR/87/1987/G2P[4]RVA/Human-wt/ZAF/410GR/85/1985/G2P[4]RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-wt/TWN/TA3/1981/G2RVA/Human-wt/TWN/TW6/1981/G2

Lineage I

Lineage IIIRVA/Human-wt/AUS/95A/XXXX/G2 RVA/Human-wt/AUS/92A/XXXX/G2

OutgroupRVA/Human-tc/USA/P/1974/G3P[8]

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0.1 Fig. 1 (continued)

542 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

A3:G3

RVA/Horse-wt/xxx/4775G7/xxxx/G3P[12]

RVA/Horse-wt/xxx/4698G5/xxx/G3P[12]RVA/Horse-wt/xxx/ERV316/xxxx/G3P[x]

RVA/Horse-wt/xxx/4616G11/xxxx/G3P[x]RVA/Horse-wt/GBR/H-2/1976/G3P[3]RVA/Horse-wt/IRL/03V04954/2003/G3RVA/Horse-wt/ARG/E30/1993/G3P[12]RVA/Horse-wt/ARG/E384/2004/G3P[12]RVA/Horse-wt/JPN/JE29/xxxx/G3P[x]

Lineage V

RVA/Rabbit-tc/ITA/30/96/1996/G3P[14]RVA/Human-wt/BEL/B4106/2000/G3

Lineage IV RVA/Human-tc/USA/HCR3A/1984/G3

RVA/Dog-tc/ITA/RV198-95/1995/G3P[3]RVA/Human-wt/ISR/Ro1845/1985/G3P[3]

Lineage III

RVA/Human-wt/THA/CMH079/2005/G3P[10]RVA/Human-wt/THA/CMH222/2001/G3P[3]

RVA/Human-wt/AUS/RCH272/2012/G3P[14]RVA/Horse-wt/ARG/E3198/2008/G3P[3]

RVA/Human-wt/IND/UP/H2/2009/G3RVA/Human-tc/ITA/PA260-97/1997/G3

RVA/Simian-tc/USA/RRV/1975/G3P[3]RVA/Rabbit-wt/CHN/N5/1992/G3P[14]

Lineage II

RVA/Human-wt/xxx/02-92/1992/G3P[x]RVA/Human-tc/USA/CC425/1997/G3P[9]RVA/Human-tc/JPN/AU-1/1982/G3P[9]

RVA/Vaccine/USA/WI78-8/1992/G3P[5]RVA/Human-wt/JPN/4643/xxxx/G3P[x]RVA/Human-wt/THA/CMH054/xxxx/G3P[x]RVA/Human-wt/USA/VU08-09-22/2008/G3P[8]RVA/Human-wt/USA/VU08-09-19/2008/G3P[8]

RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]

Lineage I

OutgroupRVA/Mouse-tc/USA/ETD_822/G16P[16]*

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V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 543

Cameroon, Democratic Republic of the Congo, Malawi, Tunisia andIvory Coast, whereas the strain RVA/Human-wt/CMR/BA356/2010/G12P[8] formed a different cluster together with other historic P[8]strains from Cameroon (Fig. 1, panel B3).

3.3. Phylogenetic analysis of the structural genes of the inner capsidand the core

VP1. The Cameroonian RVA VP1 genes were classified intogenotypes R1 or R2. The majority of Cameroonian Wa-like R1

genotype strains clustered with modern variants of genotype R1strains, showing the closest relationship with South Africanstrains (RVA/Human-wt/ZAF/3133WC/2009/G12P[4], RVA/Human-wt/ZAF/3176WC/2009/G12P[6]; nt and aa identities, P97%). A singleR1 strain, RVA/Human-wt/CMR/BA356/2010/G12P[8], clusteredwith several historical strains, including Wa and the G1P[8] strainused to develop the Rotarix vaccine. Cameroonian genotype R2strains were all closely related to modern clusters of DS-1-like R2strains, sharing nt and aa similarities between 90% and 100%among themselves and comparable similarities with several

A4:G6 RVA/Human-wt/BFA/3-BF/2010/G6P[6]RVA/Human-wt/BFA/240-BF/2010/G6P[6]RVA/Human-wt/BFA/8-BF/2010/G6P[6]RVA/Human-wt/BFA/48-BF/2010/G6P[6]RVA/Human-wt/ITA/CEC06/2011/G6P[6]RVA/Human-wt/BFA/2-BF/2010/G6P[6]RVA/Human-wt/BFA/50-BF/2010/G6P[6]RVA/Human-wt/BFA/38-BF/2010/G6P[6]RVA/Human-wt/BFA/52-BF/2010/G6P[6]RVA/Human-wt/BFA/21-BF/2010/G6P[6]RVA/Human-wt/BFA/51-BF/2010/G6P[6]RVA/Human-wt/BFA/17-BF/2010/G6P[6]

RVA/Human-wt/CMR/MA202/2011/G6P6RVA/Human-wt/CMR/MA228/2011/G6P6

RVA/Human-wt/COG/12-G0868/2012/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P6

RVA/Human-wt/CMR/BA356/2010/G6P[8]RVA/Human-wt/CAM/BA369/2010/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]

RVA/Human-xx/FRA/R353/XXXX/G6P[6]RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-tc/HUN/HUN7/1998/G6P[9]

RVA/Human-wt/ITA/PG05/2011/G6P[9]RVA/Human-wt/TUN/17237/2008/G6P[9]RVA/Human-wt/ITA/PA43/2003/G6P[9]RVA/Human-wt/ITA/PA17/2003/G6P[9]

RVA/Human-tc/USA/Se584/1998/G6P[9]RVA/Human-wt/ITA/PA27-GV1/1993/G6P[9]

Lineage I

RVA/Human-wt/HUN/Hun3/1995/G6P[9]RVA/Cow-wt/SVN/SI-B17/2004/G6P[11]RVA/Cow-wt/HUN/BoRo4/2010/G6P[x] RVA/Buffalo-tc/ITA/10733/2001/G6P[3]RVA/Rabbit-tc/NLD/K11027/2011/G6P[11]

Lineage III

RVA/Cow-wt/ARG/B1190ER/2000/G6P[11]RVA/Cow-wt/ARG/B3702BA/2008/G6P[11]RVA/Cow-wt/ARG/B3538BA/2008/G6P[11]

RVA/Cow-wt/BRA/BRA1758/2011/G6P[11]RVA/Cow-wt/BRA/BRA1752/2011/G6P[11]RVA/Cow-wt/ARG/B2932/2006/G6P[5]RVA/Cow-wt/ARG/B1541/2001/G6P[11]

RVA/Cow-wt/ARG_B175_D_BA/1997/G6P[11]RVA/Cow-wt/ARG/B3206_BA/2006/G6P[5]RVA/Cow-wt/ARG/B611_BA_1999/G6P[11]RVA/Cow-wt/ARG/B609_BA_1999/G6[11]

Lineage VI

RVA/Cow-tc/JPN/KN-4/XXXX/G6P[11] RVA/Cow-wt/CAN/FMV1078090/2009/G6P[x]RVA/Cow-wt/CAN/FMV1089635/2009/G6P[x]RVA/Cow-wt/CAN/FMV1075018/2009/G6P[x]

RVA/Cow-wt/HUN/HUN-5136/2009/G6P[x]RVA/Human-wt/SVN/SI-R56/07/2007/G6P[11]

Lineage V

RVA/Cow-tc/USA/NCDV/1967/G6P[1]RVA/Cow-tc/FRA/RF/1982/G6P[1]RVA/Vaccine/USA/RotaTeq-WI79-4/1992/G6P1A[8]

RVA/Cow-tc/VEN/BRV033/1990/G6P1Lineage IV

RVA/Human-tc/AUS/MG6/1993/G6P[14]RVA/Antelope-wt/ZAF/RC18/G6P[14]

RVA/Human-wt/HUN/BP1879/2003/G6P[14]Lineage II

RVA/Goat-tc/BGD/GO34/1999/G6P[1]Lineage VIII RVA/Cow-wt/IND/RUBV319/G6P[11]

Lineage VII RVA/Human-wt/IND/HP140/G6P[13]

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0.05 Fig. 1 (continued)

544 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

African strains (RVA/Human-wt/COD/DRC86/2003/G8P[6], RVA/Human-wt/COD/DRC88/2003/G8P[6], RVA/Human-wt/MWI/1473/2001/G8P[4]) (Fig. 1, panel C).

VP2. Molecular analysis of the Cameroonian VP2 genes classifiedthese strains into genotypes C1 (Wa-like) and C2 (DS-1-like).Nucleotide and amino acid identities ranged between 80% and

100% for the Wa-like Cameroonian strains and between 81% and100% for the DS-1-like Cameroonian strains, respectively. Thesestrains also had nucleotide and amino acid homologies of P80%and P91%, respectively with other African RVA strains. Phyloge-netic analysis showed that the Cameroonian C1 strains were clo-sely related to a historic US G4P[8] strain detected in 1980 and

A5:G12

RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8] RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA60/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]

RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]

RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/BA356/2010/G12P[6]

RVA/Human-wt/IND/14B2/xxxx/G12RVA/Human-wt/LKA/05SLC030/2005/G12P[8]

RVA/Human-wt/LKA/05SLC013/2005/G12P[8]RVA/Human-wt/BGD/Dhaka25/2002/G12P[8]

RVA/Human-wt/IND/ISO22/xxxx/G12RVA/Human-wt/IND/ISO1/xxxx/G12

RVA/Human-wt/BGD/RV176/2000/G12P[6]RVA/Human-wt/BGD/N26/2002/G12P[6]RVA/Human-wt/BEL/B4633/2003/G12P[8]

RVA/Human-wt/SAU/MD844/xxxx/G12RVA/Human-wt/NPL/05N054/2005/G12

RVA/Human-wt/IND/13B2/xxxx/G12RVA/Human-wt/IND/17B1/xxxx/G12RVA/Human-wt/USA/US6588/xxxx/G12P[8]

RVA/Human-wt/LKA/KUH407/2006/G12P[8]RVA/Human-wt/USA/US6597/xxxx/G12P[6]

RVA/Human-wt/KOR/CAU195/xxxx/G12RVA/Human-wt/KOR/CAU214/xxxx/G12

RVA/Human-wt/IND/ISO5/xxxx/G12RVA/Human-wt/IND/ISO21/xxxx/G12

RVA/Human-wt/IND/36B2/xxxx/G12RVA/Human-wt/ZAF/SA4958JHB/2004/G12P[6]RVA/Human-wt/ZAF/SA3315JHB/2004/G12P[6]RVA/Human-wt/THA/MS040/2007/G12RVA/Human-wt/THA/MS051/2007/G12RVA/Human-wt/IND/ISO14/xxxx/G12

RVA/Human-wt/ZAF/SA731DGM/2005/G12P[6]RVA/Human-wt/BGD/Dhaka12/2003/G12P[6]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-wt/THA/MS064/2005/G12RVA/Human-wt/IND/mani-125/2005-2008/G12

RVA/Human-wt/ZAF/SA4727DGM/2004/G12P[6]RVA/Human-wt/BGD/SK327/2006/G12P[6]RVA/Human-wt/BGD/SK277/2005/G12P[6]

RVA/Human-wt/IND/HRV52/xxxx/G12RVA/Human-wt/IND/ISO29/xxxx/G12RVA/Human-wt/IND/ISO16/xxxx/G12

RVA/Human-wt/DEU/GER172/2008/G12P[6]RVA/Human-wt/IND/mani-48/2005-2008/G12

Lineage III

RVA/Human-tc/ARG/Arg720B/G12RVA/Human-wt/ARG/Arg721/1999/G12RVA/Human-tc/ARG/Arg721M/G12

RVA/Human-tc/ARG/Arg720B/G12RVA/Human-tc/ARG/Arg720A/G12

RVA/Human-wt/BRA/HC91/xxxx/G12RVA/Human-wt/THA/T152/xxxx/G12RVA/Human-wt/JPN/CP727/xxxx/G12RVA/Human-wt/JPN/K12/xxxx/G12

Lineage II

Lineage IRVA/Human-wt/PHL/L26/G12Lineage IV RVA/Pig-wt/IND/RU172/G12

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V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 545

C2 strains were most closely related to a Malawian strain (RVA/Human-wt/MWI/1473/2001/G8P[4]) forming a common branchwith that African strain (Fig. 1, panel D).

VP3. The VP3 gene of the Cameroonian strains had nt homolo-gies P77% and moderate to maximum aa identities rangingbetween 80% and 100%. When compared to other African strains,

B1:P[4]

RVA/Human-wt/CMR/BA368/2010/G2P[4]RVA/Human-wt/CMR/BA366/2010/G2P[4]

RVA/Human-wt/BGD/MMC88/2005/G2P[4]RVA/Human-wt/BGD/MMC6/2005/G2P[4]

RVA/Human-wt/BGD/DH392/2004/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]

RVA/Human-wt/USA/LB2764/2006/G2P[4]RVA/Human-wt/DEU/GER1H-09/2009/G8P[4]

RVA/Human-wt/JPN/KO-2/XXXX/G2P[4]RVA/Human-WT/USA/LB2772/2006/G2P[4]RVA/Human-wt/USA/LB2744/2006/G2P[4]

RVA/Human-wt/ZAF/3203WC/2009/G2P[4]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-tc/IND/107E1B/XXXX/G3P[4]RVA/Human-tc/IND/IS-2/XXXX/G2P[4]

RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

Lineage III

RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-tc/AUS/RV-5/1984/G-P[4]

Lineage I

RVA/Human-wt/MWI/1473/G8P[4]RVA/Human-tc/PHL/L26/1987/G12P[4]

RVA/Human-xx/KEN/AK26/1982/G-P[4]Lineage II

OutgroupRVA/Human-tc/USA/Wa/G1P[8]

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546 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

nucleotide and amino acid similarity identities ranged between77% and 98%. Similarly, most of Cameroonian Wa-like M1 genotypestrains clustered with modern variants of genotype M1 strains,showing the closest relationship with two modern South Africanstrains (RVA/Human-wt/ZAF/3133WC/2009/G12P[4], RVA/Human-wt/ZAF/3176WC/2009/G12P[6]; nt and aa identities,P97%). On the other hand M2 strains were more closely relatedto other African strains from Malawi, DRC and South Africa(Fig. 1, panel E).

VP6. A comparison of the complete VP6 nucleotide and deducedamino acid sequences of the Cameroonian strains showed theyshared moderate to very high nucleotide and amino acid homolo-gies with each other in the range of 79–100% and 92–99%, respec-tively; with 698% and P92%, respectively with other Africanstrains. Phylogenetic analysis classified the Cameroonian strainsinto VP6 genotypes I1 (Wa-like) and I2 (DS-1-like). The Cameroo-nian I2 strains formed two clusters. Generally, most of the strainsfrom Maroua clustered into genotype I1 and were closely relatedto a Bangladeshi G12P[6] strain (RVA/Human-wt/BGD/Matlab13/2003/G12P[6], (nt, P99%; aa, 100%) (Fig. 1, panel F).

Phylogenetic analysis of the non-structural genes

Both nt and aa sequences of the NSP1, NSP2, NSP3, NSP4 andNSP5 genes of Cameroonian Wa-like and DS-1-like strains showed

very high to maximum sequence identities range (within Wa-likestrains: nt, 92–100%; aa, 94–100%; within DS-1-like strains nt,87–100%; aa, 93–100%) and low to maximum identities with otherAfrican Wa-like and DS-1-like strains (nt, 75–99%; aa, 69–100%).

Panels G to K of Fig. 1 shows the ML trees for the NSP1, NSP2,NSP3, NSP4, and NSP5 genes of the representative RVA strains fromCameroon. Both Wa-like and DS-1-like NSP1 and NSP3 genes of allCameroonian RVA strains, respectively, clustered together withintheir respective homotypic branch and showed close relatednessto contemporary RVA strains detected worldwide (Fig. 1, panel Gand I). All Cameroonian RVA strains were classified into NSP2genotypes N1 or N2, with the N2 strains forming two clusters. Ofinterest, three G6P[6] strains from the North West region formeda separate cluster with a closely related Belgian G6 strain (RVA/Human-wt/BEL/B1711/2002/G6P[6]), while the G6P[6] strainsfrom Maroua formed another N2 cluster, where all other Cameroo-nian N2 strains belonged to (Fig. 1, panel H). Phylogenetic analysisof the enterotoxin NSP4 gene of the Cameroonian strains revealedmultiple clusters within both genotypes (i.e. E1 and E2). The Came-roonian E1 strains formed two clusters, whereas the E2 strainsformed four clusters. The strain RVA/Human-wt/CMR/BA356/2010/G12P[8] clustered with some historic E1 strains includingthe Rotarix vaccine strain re-isolated from a US child in 2009.Other Cameroonian E1 strains were closely related to more recentstrains detected worldwide. Four G6P[6] strains with E2 NSP4

B2:P[6] RVA/Human-wt/IND/mani-476/2008/GxP[6] RVA/Human-wt/DEU/GER172/2008/G12P[6] RVA/Human-wt/ZAF/PRU9317/1999/G9P[6] RVA/Human-wt/BGD/SK277/2005/G12P[6] RVA/Human-wt/BGD/SK423/2005/G12P[6] RVA/Human-wt/BGD/Dhaka12/2003/G12P[6] RVA/Human-wt/USA/Se585/xxx/G12P2A[6] RVA/Human-wt/USA/US1205/xxxx/G9P[6] RVA/Human-wt/ZAF/GR10924/1999/G9P[6] RVA/Human-wt/KOR/CAU214/xxx/G12P[6] RVA/Human-wt/IND/GRAVP424/xxx/GxP[6] RVA/Human-wt/CMR/6809/2000/G8P[6] RVA/Bat-wt/KEN/4852/2007/GxP[6]

RVA/Human-wt/BGD/RV176/2000/G12P[6] RVA/Human-wt/BGD/Matlb13/2003/G12P[6] RVA/Human-wt/BGD/MMC24/2005/G9P[6] RVA/Human-wt/CHN/XJ99-468/1999/G9P[6] RVA/Human-wt/CHN/XJ00-486/2000/G2P[6]

RVA/Human-wt/CMR/MA109/2011/G3P[6] RVA/Human-wt/CMR/MA114/2011/G3P[6] RVA/Human-wt/CMR/MA155/2011/G3P[6] RVA/Human-wt/CMR/ES293/2011/G3P[6] RVA/Human-wt/CMR/ES276/2011/G3P[6] RVA/Human-wt/CMR/MA104/2011/G2P[6] RVA/Human-wt/CMR/MA202/2011/G6P[6] RVA/Human-wt/CMR/MA92/2011/G1P[6] RVA/Human-wt/CMR/MA130/2011/G1P[6] RVA/Human-wt/CMR/MA228/2011/G6P[6] RVA/Human-wt/CMR/ES298/2011/G6P[6] RVA/Human-wt/CMR/BA346/2010/G6P[6] RVA/Human-wt/CMR/BA369/2010/G6P[6]

RVA/Human-wt/VEN/M37/xxxx/GxP[6] RVA/Human-wt/AUS/RV3/xxxx/GxP[6] RVA/Human-tc/GBR/ST3/1975/G4P[6]

RVA/Pig-wt/ITA/221/04-7/xxxx/GxP[6] RVA/Pig-wt/ITA/221/04-13/xxxx/GxP[6] RVA/Pig-wt/ITA/134/04-8/xxxx/GxP[6] RVA/Pig-wt/ESP/51/04/xxxx/GxP[6] RVA/Pig-wt/ESP/51/03/xxxx/GxP[6] RVA/Pig-wt/ESP/51/02/xxxx/GxP[6] RVA/Pig-wt/ITA/134/04-11/xxxx/GxP[6] RVA/Pig-wt/ITA/134/04-10/xxxx/GxP[6]

RVA/Human-wt/IND/mcs/13-07/2007/G9P[6] RVA/Human-wt/VNM/KH228/2004/G4P[6] RVA/Human-wt/VNM/KH210/2004/G5P[6]

Lineage I

RVA/Human-wt/HUN/BP720/P[6] RVA/Human-wt/HUN/BP1227/P[6] Lineage V RVA/Pig-wt/JPN/JP3-6/P[6] RVA/Pig-wt/JPN/JP29-6/P[6] Lineage III

RVA/Human-wt/HUN/BP1198/P[6] RVA/Human-wt/HUN/BP1338/P[6] Lineage IV

Lineage II RVA/Pig-wt/x/Gottfried/P[6] Outgroup RVA/Human-tc/USA/Wa/GP[8]

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V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 547

genotype formed a single cluster with G8 strains described fromDemocratic Republic of the Congo. The NSP4 genes of two G3P[6]strains shared genetic relatedness to unusual human G6P[14]strains detected in Italy and Hungary and the ovine G8P[14] strain,

RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]. An additional fourstrains appeared to form a separate cluster within E2 (Fig. 1, panelJ). Similarly, the NSP5 gene analysis classified our strains into H1 orH2 genotypes, with the H2 composed of three clusters. None of the

B3:P[8]

RVA/Human-wt/CMR/MA33/2010/G12P[8] RVA/Human-wt/CMR/MA24/2010/G12P[8] RVA/Human-wt/CMR/MA46/2010/G12P[8] RVA/Human-wt/CMR/MA52/2010/G12P[8] RVA/Human-wt/CMR/MA70/2010/G12P[8] RVA/Human-wt/CMR/MA75/2010/G12P[8] RVA/Human-wt/CMR/MA88/2011/G12P[8] RVA/Human-wt/CMR/MA16/2010/G12P[8] RVA/Human-wt/CMR/MA01/2010/G12P[8] RVA/Human-wt/CMR/MA127/2011/G12P8 RVA/Human-wt/CMR/MA02/2010/G12P[8] RVA/Human-wt/CMR/MA11/2010/G12P[8]

RVA/Human-wt/CMR/ES290/2010/G12P[8] RVA/Human-wt/CMR/ES283/2010/G12P[8]

RVA/Human-wt/USA/200719825/2007/G1P[8] RVA/Human-wt/CMR/6777/1999/G9P[8]

RVA/Human-wt/BRA/rj11772/2005/G9P[8] RVA/Human-wt/COD/DRC88/2003/G8P[8]

RVA/Human-wt/CMR/6735/1999/G9P[8] RVA/Human-wt/HUN/Hun9/1998-2001/G9P[8]

RVA/Human-wt/MWI/OP498/1999/G3P[8] RVA/Human-wt/MWI/OP351/1998/G1P[8] RVA/Human-wt/MWI/OP601/1999/G1P[8]

RVA/Human-wt/CMR/BA356/2010/G12P8 RVA/Human-wt/CMR/6806/1999/G9P[8] RVA/Human-wt/CMR/6807/1999/G9P[8]

RVA/Human-wt/TUN/6854/2002/ARN/2002/G8P[8] RVA/Human-wt/CIV/6736/2004/ARN/2004/G8P[8]

RVA/Human-wt/CMR/6780/ARN/2000/G8P[8]

Lineage III

RVA/Human-tc/XXX/KU/XXXX/G1P[8] RVA/Human-wt/JPN/F45/1987/G9P[8]

RVA/Human-tc/USA/WI61/G9P[8] Lineage II

RVA/Human-wt/MWI/OP354/G4P[8] RVA/Human-wt/MWI/OP530/G4P[8]

Lineage IV

RVA/Human-tc/JP/Hochi/G4P[4] RVA/Human-tc/JPN/ITO/1981/G3P[8]

RVA/Human-tc/JPN/Hosokawa/1983/G4P1A[8] Lineage I

Outgroup RVA/Human-tc/USA/DS-1/G2P[4]

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548 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

H2 strains were closely related to the reference DS-1 strain;instead they were more closely related to RVA strains identifiedduring the past decade or so. Within genotype H1, most of theCameroonian RVA strains formed a common branch and showedclose relatedness to the H1 NSP5 gene of several contemporaryG12 strains; the divergent Cameroonian G12 strain, RVA/Human-wt/CMR/BA356/2010/G12P[8], was closely related to modern H1strains from South Africa (RVA/Human-wt/ZAF/3133WC/2009/

G12P[4], RVA/Human-wt/ZAF/3176WC/2009/G12P[6]) (Fig. 1,panel K).

4. Discussion

Classical molecular epidemiological studies of rotaviruses havebeen based on the characterization of the neutralization antigens,

C:VP1

RVA/Human-wt/CMR/MA202/2011/G6P6RVA/Human-wt/CMR/MA104/2011/G2P4RVA/Human-wt/CMR/BA366/2010/G2P4RVA/Human-wt/CMR/BA368/2010/G2P4RVA/Human-wt/CMR/ES298/2011/G6P6RVA/Human-wt/CMR/BA346/2010/G6P6RVA/Human-wt/CMR/BA369/2010/G6P6RVA/Human-wt/CMR/MA228/2011/G6P6RVA/Human-wt/CMR/ES293/2011/G3P6RVA/Human-wt/CMR/ES276/2011/G3P6RVA/Human-wt/CMR/MA155/2011/G3P6RVA/Human-wt/CMR/MA109/2011/G3P6RVA/Human-wt/CMR/MA114/2011/G3P6

RVA/Human-wt/COD/DRC86/2003/G8P[6]RVA/Human-wt/COD/DRC88/2003/G8P[8]RVA/Human-wt/USA/LB2772/2006/G2P[4]RVA/Human-tc/GER/GER1H-09/2009/G8P4

RVA/Human-wt/BGD/MMC6/2005/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human/CHN/TB-Chen/1996/G2P[4]RVA/Human-wt/MWI/1473/2001/G8P[4]

RVA/Human-tc/IND/69M/1980/G8P410RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Cow-wt/CHN/CHLY/xxxx/G6P[x]RVA/Rhesus-tc/USA/PTRV/1990/G8P[1]

RVA/Cow-tc/VEN/BRV003/1990/G6P[1]

R2

R3 RVA/Human-tc/JPN/AU-1/G3P[9] RVA/Pig-tc/USA/Gottfried/1983/G4P[6]

RVA/Human-tc/USA/D/1974/G1P[8]RVA/Human-tc/USA/Wa/1974/G1P[8]RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Human-wt/CMR/BA356/2010/G12P[6]RVA/Human-tc/GBR/ST3/1975/G4P[6]RVA/Human-tc/IND/116E/1988/G9P[11]

RVA/Human-wt/ZAF/3133WC/2009/G12P[4]RVA/Human-wt/ZAF/3176WC/2009/G12P[6]

RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CAM/MA130/2011/G1P6RVA/Human-wt/CMR/MA11/2010/G12P8RVA/Human-wt/CMR/MA16/2010/G12P8RVA/Human-wt/CMR/ES283/2010/G12P6RVA/Human-wt/CMR/ES290/2010/G12P8RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P8RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]

R1

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V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 549

VP7 and VP4 (Estes and Kapikian, 2007). From 1980s onward elec-tropherotyping and silver staining of the genomic RNA (Herringet al., 1982; Dolan et al., 1985; Gouvea et al., 1990), RNA–RNAhybridization of the whole genome (Nakagomi et al., 1987, 1989,1990), subgrouping of the VP6 protein by monoclonal antibody

based enzyme immunoassay (Greenberg et al., 1983; Beardset al., 1984) and subsequently sequencing and phylogenetic analy-sis of the NSP4 and VP6 genes (Maunula and von Bonsdorff, 2002;Ball, 2005; Bányai et al., 2009b; De Grazia et al., 2011) were pref-erentially used to supplement routine strain characterization stud-

D: VP2

RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/MA228/2011/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]RVA/Human-wt/CMR/MA104/2011/G2P[4]

RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/ZAF/3203WC/2009/G2P[4]RVA/Human-wt/BGD/RV176/2000/G12P[6]RVA/Human-wt/USA/LB2744/2006/G2P[4]RVA/Human-wt/DEU/GER1H-09/2009/G8P[4]RVA/Human-wt/USA/LB2764/2006/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]

RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

RVA/Rhesus-tc/USA/PTRV/1990/G8P[1]RVA/Human-wt/IND/69M/1980/G8P[10]

RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Cow-tc/VEN/BRV03/1990/G6P[1]

RVA/Cow-tc/FRA/RF/1982/G6P[1]

C2

C3 RVA/Human-tc/JPN/AU1/G3P[9]C5 RVA/Simian-tc/ZAF/SA11-H96/G3P[2]

RVA/Pig-tc/USA/Gottfried/1983/G4P[6]RVA/Human-wt/CHN/R479/2004/G4P[6]

RVA/Human-tc/IND/116E/1988/G9P[11]RVA/Human-tc/USA/Rotarix/2009/G1P[8]RVA/Human-tc/USA/Wa/1974/G1P[8]

RVA/Human-wt/USA/DC1359/1980/G4P[8]RVA/Human-wt/CMR/BA356/2010/G12P8RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P6

RVA/Human-wt/CMR/ES283/2010/G12P6RVA/Human-wt/CMR/ES290/2010/G12P8RVA/Human-wt/CMR/MA01/2010/G12P[8].RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P8

C1

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550 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

ies to obtain a more complex picture about genetic and phenotypicfeatures of circulating RVA strains. Contemporary studies often usewhole genome sequencing that is based on either conventional

chain termination sequencing methods, or, novel massively paral-lel sequencing techniques with or without the need of prior cloningof amplified cDNA (Potgieter et al., 2009; McDonald et al., 2009,

E: VP3

RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/MA228/2011/G6P[6]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/MA104/2011/G2P[4]RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]

RVA/Human-wt/COD/DRC86/2003/G8P[6]RVA/Human-wt/COD/DRC88/2003/G8P[8]RVA/Human-wt/USA/LB2744/2006/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]RVA/Human-wt/BGD/MMC6/2005/G2P[4]

RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/ZAF/3203WC/2009/G2P[4]

RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]RVA/Human-tc/USA/DS-1/1976/G2P[4] RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]

RVA/Cow-tc/FRA/RF/1982/G6P[1]RVA/Cow-tc/VEN/BRV003/1990/G6P[1]

RVA/Rhesus-tc/USA/PTRV/1990/G8P[1]RVA/Cow-tc/USA/WC3/1981/G6P[5]

RVA/Human-tc/IND/69M/1980/G8P[10]

M2

RVA/Human-wt/CMR/MA60/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/BA356/2010/G12P[8]

RVA/Human-wt/ZAF/3176WC/2009/G12P[6]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-tc/GBR/ST3/1975/G4P[6]RVA/Human-wt/BGD/Dhaka16/2003/G1P[8]

RVA/Human-tc/USA/Wa/1974/G1P[8]RVA/Human-tc/IDN/57M/1980/G4P[10]

RVA/Pig-tc/USA/Gotffried/1983/G4P[6]RVA/Human-tc/IND/116E/1988/G9P[11]

M1

M3RVA/Human-tc/JPN/AU-1/1982/G3P[9]

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V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 551

2011, 2012; Esona et al., 2011, 2013; Ianiro et al., 2013a; Ghoshet al., 2013a, 2013b; Mullick et al., 2013; Papp et al., 2013;Steyer et al., 2013; Wang et al., 2013; Dennis et al., 2014;Giammanco et al., 2014; Gómez et al., 2014; Theamboonlerset al., 2014; Dóró et al., 2014a). Considering the easy access to

whole genome sequencing services, it is likely that this methodutilizing fast and simple sample processing algorithms will becomeroutine method in future RVA surveillance studies.

Earlier studies from Cameroon reported the circulation ofG1–G5, G8, G9, and G12 VP7 types in combination with P[4] and

F: VP6

RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA228/2011/G6P[6]RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/MA104/2011/G2P[4]

RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/BGD/RV161/2000/G12P[6]

RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]

RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]RVA/Human-wt/BGD/MMC6/2005/G2P[4]RVA/Human-wt/IND/ISO97/XXXX/G9P[4] RVA/Human-wt/COD/DRC86/2003/G8P[6]RVA/Human-wt/COD/DRC88/2003/G8P[8]

RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-wt/BGD/N26/2002/G12P[6]RVA/Human-tc/IND/69M/1980/G8P[10]

RVA/Rhesus-tc/USA/PTRV/1990/G8P[1] RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]

RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Cow-tc/VEN/BRV03/1990/G6P[1]

RVA/Cow-tc/FRA/RF/1982/G6P[1]RVA/Human-tc/PHL/L26/1987/G12P[4]RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

I2

I3 RVA/Human-tc/JPN/AU-1/G3P[9]I9 RVA/Rhesus-tc/USA/TUCH/G3P[24]

I15 RVA/Bat-wt/KEN/KE4852/2007/G25P[6] RVA/Pig-tc/MEX/YM/G11P[7]

RVA/Pig-wt/CHN/JL94/G5P[7] I5 RVA/Pig-tc/USA/Gottfried/1983/G4P[6]

RVA/Human-tc/IND/116E/1988/G9P[11]RVA/Human-tc/USA/Wa/1974/G1P[8]RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Pig-tc/JPN/YO/1974/G3P[8]RVA/Human-wt/BEL/B4633/2003/G12P[8]RVA/Human-tc/GBR/ST3/1975/G4P2A[6]RVA/Human-wt/USA/US6597/2002/G12P[6]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-wt/CMR/BA356/2010/G12P8RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/BGD/Matlab13/2003/G12P[6]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/MA60/2010/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]

I1

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552 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

P[6]–P[10] VP4 types during a surveillance period encompassing1999–2011 (Esona et al., 2004, 2009a, 2009b, 2010, 2013; Armahet al., 2010; Mwenda et al., 2010; Ndze et al., 2013). In this report,we used the semiconductor sequencing platform, Ion Torrent PGM,to obtain whole genome sequence information for 31 RVA strains

collected in 2010–2011 in three Cameroonian study sites, located120–1240 km from each other. The analyzed strains belonged toone of the major human RVA genogroups, Wa-like and DS-1-like(Nakagomi et al., 1989), also referred to as genotype 1 and 2 con-stellation, respectively. Both Wa-like and DS-1-like Cameroonian

G: NSP1

RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]RVA/Human-wt/CMR/BA356/2010/G12P[8]

RVA/Human/BGD/Matlab13/2003/G12P[6]RVA/Human-wt/BGD/Dhaka25/2002/G12P[8]RVA/Human-tc/ZAF/3133WC/2009/G12P[4]RVA/Human-wt/ZAF/3176WC/2009/G12P[6]RVA/Human-wt/BGD/Dhaka6/2001/G11P[25]RVA/Human-wt/BEL/B4633/2003/G12P[8]RVA/Human-wt/USA/US6597/2005/G12P[6]

RVA/Human-tc/GBR/ST3/1974/G4P2A[6]RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Hunam-tc/IND/116E/1988/G9P[11]RVA/Human-tc/USA/Wa/1974/G1P[8]

RVA/Cow-wt/KOR/KJ13/XXXX/G8P[7] RVA/Cow-wt/KOR/KJ75/XXXX/G5P[5] RVA/Cow-wt/KOR/KJ44/XXXX/G5P[1]

A1

RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-wt/DEU/GER1H/2009/G8P[4]

RVA/Human-tc/IND/69M/1980/G8P[10]RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-wt/COD/DRC86/2003/G8P[6]RVA/Human-wt/COD/DRC88/2003/G8P[8]RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/ZAF/3203WC/2009/G2P[4]RVA/Human-wt/USA/LB2772/2006/G2P[4]RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-wt/BGD/MMC6/2005/G2P[4]RVA/Human-wt/BGD/MMC88/2005/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]

RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA104/2011/G2P[4]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]

A2

RVA/Pig-tc/MEX/YM/1983/G11P[7] RVA/Pig-tc/USA/Gottfried/G4P[6] A8

RVA/Human-tc/JPN/AU-1/1982/G3P[9]RVA/Simian-tc/USA/PTRV/1990/G8P[1]

RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Cow-tc/VEN/BRV03/1990/G6P[1]

A3* * *

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0.2 Fig. 1 (continued)

V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 553

RVA strains carried numerous neutralization antigen combinationsincluding the G1P[6], G12P[6], G12P[8] on the Wa-like backbone,and G2P[4], G2P[6], G3P[6], and G6P[6] on the DS-1-like backbonegene constellation. One important finding was the conserved

backbone gene constellation within both Wa-like and DS-1-likestrains. No intergenogroup reassortment was observed; however,the pattern of backbone gene constellations in some cases wasconsistent with evidence for intragenogroup reassortment. The

H: NSP2

RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA60/2010/G12P[8]

RVA/Human-wt/CMR/BA356/2010/G12P[8]RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/BGD/Dhaka16/2003/G1P[8]RVA/Human-wt/BGD/Dhaka25/2002/G12P[8]

RVA/Human-wt/BGD/Dhaka12/2003/G12P[6]RVA/Human-wt/ZAF/3176WC/2009/G4P[26]

RVA/Human-wt/BEL/B4633/2003/G12P[8]RVA/Human-tc/IND/116E/1988/G9P[11]RVA/Human-wt/USA/LB2771/1975/G1P[8]RVA/Cat-tc/AUS/Cat2/1984/G3P[9]RVA/Human-tc/USA/Wa/1974/G1P[8]

RVA/Human-tc/GBR/ST3/1975/G4P[6]RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Pig-tc/USA/Gottfried/1983/G4P[6]RVA/Human-tc/USA/P/1974/G3P[8]RVA/Human-tc/PHL/L26/1987/G12P[4]

N1

RVA/Human-wt/DEU/GER1H-09/2009/G8P[4]RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]RVA/Human-tc/IND/69M/1980/G8P[10]RVA/Human-tc/USA/DS-1/1976/G2P[4]

RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]RVA/Cow-tc/VEN/BRV003/1990/G6P[1]

RVA/Cow-tc/FRA/RF/1982/G6P[1]RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Vaccine/USA/RotaTeq-SC2-9/1992/G2P[5]

RVA/Simian-wt/USA/PTRV/1990/G8P[1]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]

RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-tc/COD/DRC86/2003/G8P[6]RVA/Human-tc/COD/DRC88/2003/G8P[8]

RVA/Human-wt/BGD/MMC86/2005/G2P[4]RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-tc/MW1/1473/2001/G8P[4]RVA/Human-wt/ITA/PA83/2007/G2P[4]RVA/Human-wt/ZAF/3203WC/2009/G2P[4]

RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/MA104/2011/G2P[4]RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]

N2

N3 RVA/Human-tc/JPN/AU-1/1982/G3P[9]N5 RVA/Simian-tc/ZAF/SA11-H96/G3P[2]*

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0.05 Fig. 1 (continued)

554 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

majority of backbone genes of the analyzed strains were monophy-letic within both genogroups. This monophyly was seen in therespective genotypes of the VP2, VP3, several VP4 and VP7 genes,as well as the NSP1 and NSP3 genes, whereas both NSP4 and

NSP5 genes were polyphyletic in both genogroups. In this respect,the VP1, VP6 and NSP2 genes showed a mixed pattern. This partic-ular pattern of gene clusters suggested moderate reassortmentactivity among the co-circulating 2010/2011 Cameroonian RVA

I: NSP3

RVA/Human-wt/CMR/MA16/2010/G12P[8]

RVA/Human-wt/CMR/MA60/2010/G12P[8]

RVA/Human-wt/CMR/MA11/2010/G12P[8]

RVA/Human-wt/CMR/MA02/2010/G12P[8]

RVA/Human-wt/CMR/MA52/2010/G12P[8]

RVA/Human-wt/CMR/MA70/2010/G12P[8]

RVA/Human-wt/CMR/MA75/2010/G12P[8]

RVA/Human-wt/CMR/MA127/2011/G12P[8]

RVA/Human-wt/CMR/MA88/2011/G12P[8]

RVA/Human-wt/CMR/MA01/2010/G12P[8]

RVA/Human-wt/CMR/MA33/2010/G12P[8]

RVA/Human-wt/CMR/MA46/2010/G12P[8]

RVA/Human-wt/CMR/MA24/2010/G12P[8]

RVA/Human-wt/CMR/BA356/2010/G12P[8]

RVA/Human-wt/CMR/ES283/2010/G12P[6]

RVA/Human-wt/CMR/ES290/2010/G12P[8]

RVA/Human-wt/CMR/MA92/2011/G1P[6]

RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/BEL/B4633/2003/G12P[8]

RVA/Human-wt/BGD/Dhaka12/2003/G12P[6]

RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-wt/ZAF/3176WC/2009/G12P[6]

RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Human-tc/GBR/ST3/1975/G4P[6]

RVA/Human-tc/USA/Wa/1974/G1P[8]

RVA/Cow-wt/KOR/KJ75/XXXX/G5P[5]

RVA/Cow-wt/KOR/KJ44/XXXX/G5P[1]

RVA/Cow-wt/KOR/KJ16/XXXX/G8P[7]

T1

RVA/Human-tc/IND/116E/1988/G9P[11]

T7 RVA/Cow-tc/GBR/UK/1973/G6P[5]

RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]

RVA/Vaccine/USA/RotaTeq-SC2-9/1992/G2P[5] T6 RVA/Human-tc/IND/69M/1980/G8P[10]

RVA/Human-tc/USA/DS-1/1976/G2P[4]

RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

RVA/Human-wt/BGD/RV161/2000/G12P[6]

RVA/Human-wt/BGD/MMC6/2005/G2P[4]

RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]

RVA/Human-wt/ZAF/3203WC/2009/G2P[4]

RVA/Human-wt/USA/LB2772/1974/G2P[4]

RVA/Human-wt/DEU/GER1H-09/2009/G8P[4]

RVA/Human-wt/COD/DRC86/2003/G8P[6]

RVA/Human-wt/COD/DRC88/2003/G8P[8]

RVA/Human-wt/BEL/B1711/2002/G6P[6]

RVA/Human-wt/MWI/1473/2001/G8P[4]

RVA/Human-wt/ZAF/GR10924/1999/G9P[6]

RVA/Human-wt/CMR/BA366/2010/G2P[4]

RVA/Human-wt/CMR/BA368/2010/G2P[4]

RVA/Human-wt/CMR/MA104/2011/G2P[4]

RVA/Human-wt/CMR/MA228/2011/G6P[6]

RVA/Human-wt/CMR/ES293/2011/G3P[6]

RVA/Human-wt/CMR/MA202/2011/G6P[6]

RVA/Human-wt/CMR/ES276/2011/G3P[6]

RVA/Human-wt/CMR/BA346/2010/G6P[6]

RVA/Human-wt/CMR/BA369/2010/G6P[6]

RVA/Human-wt/CMR/ES298/2011/G6P[6]

RVA/Human-wt/CMR/MA109/2011/G3P[6]

RVA/Human-wt/CMR/MA155/2011/G3P[6]

RVA/Human-wt/CMR/MA114/2011/G3P[6]

T2

T3 RVA/Human-tc/JPN/AU-1/1982/G3P[9]*

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0.1 Fig. 1 (continued)

V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 555

strains, which mainly affected the neutralization protein encodinggenes and the non-structural protein encoding genes. The analysisof G12P[8] RVA strains, which predominated in the study period,

was consistent with the clonal spread of a single strain, althoughone G12P[8] strain, detected in Bamenda (BA356/2010), appearedto segregate from the majority G12P[8] variant. Nonetheless, the

J: NSP4 RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]

RVA/Human-wt/ZAF/3203WC/2009/G2P[4]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]RVA/Human-wt/BGD/MMC6/2005/G2P[4]RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/USA/LB2772/1974/G2P[4]

RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]

RVA/Human-wt/MWI/MW333/1998-1999/G8P[x]RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-wt/COD/DRC86/2003/G8P[6]RVA/Human-wt/COD/DRC88/2003/G8P[8]

RVA/Human-wt/CMR/MA228/2011/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/BA369/2010/G6P[6]

RVA/Human-tc/IND/69M/1980/G8P[10]RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/CMR/MA104/2011/G2P[4]RVA/Human-wt/CMR/BA366/2010/G2P[4]RVA/Human-wt/CMR/BA368/2010/G2P[4]

RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/HUN/Hun5/1997/G6P[14]RVA/Human-wt/ITA/111-05-27/2005/G6P[14] RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]

RVA/Cow-tc/VEN/BRV003/1990/G6P[1]RVA/Cow-tc/FRA/RF/1982/G6P[1]

RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Vaccine/USA/RotaTeq-WI79-9/1992/G1P[5]RVA/Vaccine/USA/RotaTeq-SC2-9/1992/G2P[5]

E2

RVA/Human-wt/BGD/N26/2002/G12P[6]RVA/Human-tc/JPN/AU-1/1982/G3P[9] E3

RVA/Pig-wt/THA/CMP034/2000/G2P[27]RVA/Cow-wt/KOR/KJ44/XXXX/G5P[1]RVA/Cow-wt/KOR/KJ75/XXXX/G5P[5]RVA/Cow-wt/KOR/KJ13/XXXX/G8P[7]

RVA/Pig-wt/USA/OSU/1976/G5P[7]RVA/Pig-tc/USA/Gottfried/1975/G4P[6]

RVA/Human-tc/USA/Wa/1974/G1P[8]RVA/Human-tc/USA/Rotarix/2009/G1P[8]

RVA/Human-tc/GBR/ST3/1975/G4P[6]RVA/Human-wt/CMR/BA356/2010/G12P[8]

RVA/Human-wt/IND/116E/1988/G9P[11]RVA/Human-wt/USA/LB2771/2006/G1P[8]

RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/BGD/RV161/2000/G12P[6]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]

RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/MA88/2011/G12P[8]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/MA60/2010/G12P[8]

E1

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0.05 Fig. 1 (continued)

556 V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560

shared gene combination in RVA strains from different geographicareas that express various neutralization antigens is an interestingfinding and future whole genomic studies of Cameroonian RVAs

might help elucidate if any conserved monophyletic branches inthe backbone gene constellations are favorable for viral fitness.Past whole genomic characterization of African rotavirus strains

RVA/Human-wt/CMR/MA16/2010/G12P[8]RVA/Human-wt/CMR/MA60/2010/G12P[8]RVA/Human-wt/CMR/MA02/2010/G12P[8]RVA/Human-wt/CMR/ES283/2010/G12P[6]RVA/Human-wt/CMR/ES290/2010/G12P[8]RVA/Human-wt/CMR/MA11/2010/G12P[8]RVA/Human-wt/CMR/MA52/2010/G12P[8]RVA/Human-wt/CMR/MA75/2010/G12P[8]RVA/Human-wt/CMR/MA70/2010/G12P[8]RVA/Human-wt/CMR/MA88/2011/G12P[8]

RVA/Human-wt/CMR/MA92/2011/G1P[6]RVA/Human-wt/CMR/MA130/2011/G1P[6]

RVA/Human-wt/CMR/MA127/2011/G12P[8]RVA/Human-wt/CMR/MA46/2010/G12P[8]RVA/Human-wt/CMR/MA01/2010/G12P[8]RVA/Human-wt/CMR/MA33/2010/G12P[8]RVA/Human-wt/CMR/MA24/2010/G12P[8]

RVA/Human-wt/BEL/B4633/2003/G12P[8]RVA/Human-wt/BGD/Matlab13/2003/G12P[6]

RVA/Human-tc/GBR/ST3/1975/G4P[6]RVA/Human-wt/BGD/Dhaka25/2002/G12P[8]RVA/Human-wt/BGD/Dhaka6/2001/G11P[25]RVA/Human-wt/ZAF/3133WC/2009/G12P[4]RVA/Human-wt/ZAF/3176WC/2009/G12P[6]RVA/Human-wt/CMR/BA356/2010/G12P[8]

RVA/Pig-tc/USA/Gottfried/1975/G4P[6]RVA/Cow-wt/KOR/KJ44/XXXX/G5P[1]RVA/Cow-wt/KOR/KJ13/XXXX/G8P[7]RVA/Cow-wt/KOR/KJ75/XXXX/G5P[5]RVA/Human-tc/IND/116E/1988/G9P[11]

RVA/Human/JPN/KU/1974/G1P[18]RVA/Human/USA/Rotarix/2009/G1P[8]

RVA/Human-tc/USA/Wa/1974/G1P[8]RVA/Human/USA/LB2771/2006/G1P[8]

H1

H5 RVA/Simian-tc/ZAF/SA-11/1958/G3P[2] H6 RVA/Human/THA/T152/1998/G12P[9]

RVA/Human-tc/JPN/AU-1/G3P[9]RVA/Simian-tc/USA/PTRV/1990/G8P[1]

RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]RVA/Cow-tc/VEN/BRV03/1990/G6P[1]

RVA/Cow-tc/FRA/RF/1982/G6P[1]RVA/Cow-tc/USA/WC3/1981/G6P[5]RVA/Vaccine/USA/RotaTeq-WI79-9/1992/G1P[5]RVA/Vaccine/USA/RotaTeq-SC2-9/1992/G2P[5]

H3

RVA/Human-wt/CMR/BA369/2010/G6P[6]RVA/Human-wt/CMR/BA346/2010/G6P[6]RVA/Human-wt/CMR/ES298/2011/G6P[6]

RVA/Human-wt/MWI/1473/2001/G8P[4]RVA/Human-wt/ZAF/3203WC/2009/G2P[4]

RVA/Human-wt/BEL/B1711/2002/G6P[6]RVA/Human-wt/ITA/PA83/2007/2007/G2P[4]

RVA/Human-wt/CHN/TB-Chen/1996/G2P[4]RVA/Human-wt/CMR/MA104/2011/G2P[4]

RVA/Human-wt/CMR/MA202/2011/G6P[6]RVA/Human-wt/BGD/MMC6/2005/G2P[4]

RVA/Human-wt/ZAF/GR10924/1999/G9P[6]RVA/Human-wt/USA/LB2772/2006/G2P[4]

RVA/Human-wt/CMR/BA368/2010/G2P[4]RVA/Human-wt/CMR/BA366/2010/G2P[4]

RVA/Human/CMR/MA228/2011/G6P[6]RVA/Human-wt/CMR/ES276/2011/G3P[6]RVA/Human-wt/CMR/ES293/2011/G3P[6]RVA/Human-wt/CMR/MA155/2011/G3P[6]RVA/Human-wt/CMR/MA114/2011/G3P[6]RVA/Human-wt/CMR/MA109/2011/G3P[6]

RVA/Human-wt/COD/DRC88/2003/G8P[8]RVA/Human-wt/COD/DRC86/2003/G8P[6]

RVA/Human-tc/USA/DS-1/1976/G2P[4]RVA/Human-tc/IDN/69M/1980/G8P[10]

H2

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0.02

K: NSP5

Fig. 1 (continued)

V.N. Ndze et al. / Infection, Genetics and Evolution 28 (2014) 537–560 557

indicated extensive intergenogroup reassortment among Wa-likeand DS-1-like strains and greater antigenic diversity among VP4genotypes (including P[4], P[6], and P[8]), although those studies

had analyzed strains from different locations and years (Esonaet al., 2009b, 2011, 2013; Jere et al., 2011). In addition, evidencehas been published that many human RVA strains detected in

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Africa had acquired neutralization and/or backbone genes from ananimal species through interspecies transmission coupled withreassortment (Esona et al., 2009a; Esona et al., 2009b; Esonaet al., 2011; Heylen et al., 2014; Seheri et al., 2014). In contrastwith those findings, in this study we found limited evidence forzoonotic origin of strains or selected genes (e.g., bovine-like E2NSP4 gene).

One unexpected, and perhaps the most intriguing finding wasthe detection of G6P[6] strains on the DS-1-like backbone geneconstellation. In our previous paper, G12 strains were reported toemerge in the three study areas of Cameroon (Ndze et al., 2013).Here we demonstrated that five out of six strains originallydescribed as G12P[6] were, in fact, misidentified G6P[6] strains.Although cross reactivity between the G12 primers included inthe original typing primer cocktail used in genotyping multiplexnested PCR assay was demonstrated by our group (László et al.,2012), we were surprised at seeing that such a great proportionof suspect G12P[6] strains are G6P[6] strains. These strains weredetected in all three study sites, where, altogether 13 G12P[6]strains were initially described. We speculate that if this propor-tion of G6P[6]s among randomly selected strains represents thetrue prevalence of this particular genotype, we need to reconsiderthe relative importance of G12P[6] and G6P[6] strains in the threestudy sites during the 2010–2011 season. G6P[6] strains have beenreported to emerge in other west and central African countries,including Burkina Faso in 2010 and Democratic Republic of theCongo in 2012 (Nordgren et al., 2012; Pukuta et al., 2014), suggest-ing a relatively rapid spread of this genotype in Western and Cen-tral Africa. Furthermore, molecular analysis of the VP7 (and someof the backbone genes) of Cameroonian G6P[6] strains indicatedthe separation of the strains identified in the Far North region(Maroua) and the Northwest region (Bamenda and Esu) into twobranches that is consistent with multiple introductions of G6P[6]strains into this country. The first documentation of a G6P[6]strains has been published by Belgian researchers who reported acase from 2002 that was related to a probable importation fromMali (Rahman et al., 2003). Subsequently, G6P[6] strains wereidentified sporadically in several European countries, includingFrance and Italy, but never reported to occur at great frequency(de Rougemont et al., 2009; Ianiro et al., 2013b). The recent AfricanG6P[6] RVA strains were not very closely related to the probablyimported Belgian strain or the French strain, but seemed to bemonophyletic with a 2011 Italian G6P[6] strain. In light of currentreports about emergence of G6P[6]s in Africa it seems possible thatthis genotype circulated in this region without prior detection,probably due to the low surveillance activity of RVAs in manyAfrican countries and a previously limited transmission potentialof these strains in human populations.

5. Conclusions

The emergence and explosive spread of G6P[6] RVA strains inwest and central African countries over the past few years mustbe better understood and should be closely monitored in the futurefollowing the routine administration of rotavirus vaccines in thesecountries. Countries worldwide using Rotarix have frequentlyreported transient increase of heterotypic strains among vacci-nated children. Our whole genome based molecular characteriza-tion study indicates that the emerging G6P[6] strain is also fullyheterotypic to Rotarix. Whereas several studies reported higheffectiveness of Rotarix and RotaTeq even against heterotypic wildtype strains (reviewed in Dóró et al., 2014b; Leshem et al., 2014),continuation of strain monitoring is needed in this area andelsewhere to see if G6s, besides genotype G1 to G4, G8, G9 andG12, may become an eighth regionally important VP7 genotypein the post vaccine licensure era in Africa.

Acknowledgements

This research was funded by a grant from the HungarianScientific Research Fund (OTKA, T100727) and the Momemtumprogram awarded by the Hungarian Academy of Sciences.Assistance with NGS related bioinformatics is acknowledged toBalázs Horváth. We are also grateful to Anett Horváth, WilfredNgwoh Tang, and Zang Magdaleine for the excellent technicalassistance.

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