Journal of Medical Virology 81:937–951 (2009)

Genomic Characterization of Human Rotavirus G8Strains From the African Rotavirus Network:Relationship to Animal Rotaviruses

M.D. Esona,1*{ A. Geyer,2{ N. Page,3{ A. Trabelsi,4{ I. Fodha,4{ M. Aminu,5{ V.A. Agbaya,6{ B. Tsion,7{

T.K. Kerin,1 G.E. Armah,8{ A.D. Steele,9{ R.I. Glass,10 and J.R. Gentsch1

1Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, NCIRD, CDC,Atlanta, Georgia2MRC/DPRU, University of Limpopo, Pretoria Campus, South Africa3Viral Gastroenteritis Unit, National Institute for Communicable Disease, Sandringham, South Africa4Laboratory of Bacteriology-Virology, University Hospital Sahloul, Sousse, Tunisia5Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria6Laboratoire de Bacteriologie/Virologie, Institut Pasteur de Cote-d’Ivoire/DVE, Abidjan, Cote-d’Ivoire7Ethiopian Institute of Virology, Addis Ababa, Ethiopia8Noguchi Memorial Research Institute, Accra, Ghana9PATH, NW, Seattle, Washington10Fogarty International Center, National Institutes of Health, Bethesda, Maryland

Global rotavirus surveillance has led to thedetection of many unusual human rotavirus(HRV) genotypes. During 1996–2004 surveillancewithin the African Rotavirus Network (ARN), sixP[8],G8 and two P[6],G8 human rotavirus strainswere identified. Gene fragments (RT-PCR ampli-cons) of all 11-gene segments of these G8 strainswere sequenced in order to elucidate theirgenetic and evolutionary relationships. Phyloge-netic and sequence analyses of each gene seg-ment revealed high similarities (88–100% nt and91–100% aa) for all segments except for gene 4encoding VP4 proteins P[8] and P[6]. For moststrains, almost all of the genes of the ARN strainsother than neutralizing antigens are related totypical human strains of Wa genogroup. The VP7,NSP2, and NSP5 genes were closely related tocognate genes of animal strains (83–99% and 97–99% aa identity). This study suggests that theARN G8 strains might have arisen through VP7 orVP4 gene reassortment events since most of theother gene segments resemble those of commonhuman rotaviruses. However, VP7, NSP2 (likely),and NSP5 (likely) genes are derived potentiallyfrom animals consistent with a zoonotic intro-duction. Although these findings help elucidaterotavirus evolution, sequence studies of cognateanimal rotavirus genes are needed to conclu-sively determine the specific origin of thosegenes relative to both human and animal rotavi-rus strains. J. Med. Virol. 81:937–951,2009. � 2009 Wiley-Liss, Inc.

KEY WORDS: rotavirus; characterization; clas-sification; evolution; reassort-ment

INTRODUCTION

Group A rotaviruses are the most important etiolog-ical agents of acute gastroenteritis in infants and youngchildren, as well as the young of a variety of animalsworldwide [Estes and Kapikian, 2007]. Globally,approximately 600,000 children die every year fromrotavirus and most of these deaths occur in children inthe poorest countries of Africa and South Asia [Parasharet al., 2006].

Rotaviruses are members of the Reoviridae family.Complete virus particles have a triple-layered capsidstructure (central core, inner capsid and outer capsid)encasing a genome consisting of 11 segments of double-stranded RNA (dsRNA) which encode six structural

Institution where work was completed: National Center forImmunization and Respiratory Diseases, Centers for DiseaseControl and Prevention, Atlanta, GA.

The findings and conclusions in this report are those of theauthors and do not necessarily represent the views of the CDC.

{Members of the African Rotavirus Network.

*Correspondence to: M.D. Esona, 1600 Clifton Road, NE, MailStop G-04, Atlanta, GA 30333. E-mail: mdi4@cdc.gov

Accepted 9 December 2008

DOI 10.1002/jmv.21468

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2009 WILEY-LISS, INC.

(VP1–VP4 and VP6–VP7) and six non-structural(NSP1–NSP5/NSP6) proteins [Estes and Kapikian,2007]. Rotavirus is classified based mainly on the VP6,VP4, and VP7 antigens. The middle capsid consists oftrimers of the highly immunogenic protein VP6, whichspecifies rotavirus group and subgroup (SG) antigens.There are seven groups (A–G) of rotaviruses and withingroup A, which is the most important agent of viraldiarrhea in children, four subgroups (SGI, SGII,SGIþ II, and non-SGIþ II) have been documented[Greenberg et al., 1983; Lopez et al., 1994; Estes andKapikian, 2007]. The outer capsid consists of the VP7glycoprotein, in which VP4 spikes are embedded. Thesetwo outer capsid proteins are the two rotavirus-serotypeantigens and are encoded by segments 4 (VP4 protease-sensitive protein and P serotype antigen) and segment 7,8, or 9 depending on the strain (VP7 glycoprotein and Gserotype antigen). The VP7 and VP4 proteins areencoded by separate gene segments and form the basisfor the binary classification of group A rotavirus into Gand P types, respectively [Estes and Kapikian, 2007].Both proteins play key roles in rotavirus genetic andantigenic diversity and are independently involved[Hoshino et al., 1988] in the development of protectiveimmunity and the neutralization response to rotavirus.

Studies of rotavirus genetic diversity have beencarried out in a variety of ways using both serologicand molecular approaches. After methods to cultivateand plaque human rotavirus were developed, neutral-ization tests were used to define four distinct antigenictypes (serotypes) [Wyatt et al., 1983], now designatedserotypes G1, G2, G3, and G4, that were later shown tocorrespond to determinants located on the VP7 protein.Subsequently, serotype-specific monoclonal antibodieswere developed [Coulson et al., 1987; Taniguchi et al.,1987; Padilla-Noriega et al., 1993], and used to showthat these four serotypes were globally common,representing at least 90% of the strains in circulation[Gentsch et al., 2005]. Characterization of these commonstrains defined shared or distinct genetic and antigenicfeatures depending on the serotypes. For example,almost all serotype G2 strains contained a SGI VP6protein and a VP4 serotype now designated P1B[4],while serotype G1, G3, and G4 rotaviruses had a SGIIVP6 and were defined as VP4 serotype P1A[8] [Gorzigliaet al., 1988; Gentsch et al., 2005; Santos and Hoshino,2005]. Serotype G2 and the other 3 strains also differ inoverall genetic relatedness; radioactive probes madefrom the genome of serotype G1 hybridized strongly toother serotype G1, G3, and G4 isolates, but not at all togene segments of serotype G2 isolates and vice versa.These results demonstrated that the segments ofserotypes G1, G3, and G4 were closely related to eachother but highly divergent from serotype G2 and viceversa. These distinct gene constellations were referredto as genogroups Wa (serotypes G1, G3, G4) and DS-1(serotype G2) [Flores et al., 1982; Nakagomi andNakagomi, 1993]. Another minor genogroup of HRV,completely distinct from the Wa and DS-1 gene families(designated AU-1) was reported a few years later

[Nakagomi and Nakagomi, 1989; Nakagomi et al.,1990; Ward et al., 1990]. Rotaviruses in the AU-1genogroup belonged to SGI but were identified asserotype G3 and had long electrophoretic patterns.Finally, G2 strains have partial gene duplication insegment 11, the gene encoding NSP5 protein, causing itto migrate more slowly in polyacrylamide gels than thecognate gene of G1, G3, and G4 strains, and these arereferred to as short and long electropherotypes, respec-tively [Matsui et al., 1990]. Based on the epidemiologicevidence that only these 4 strains were common causesof gastroenteritis, their VP7 antigens were targeted inmodified Jennerian vaccination strategies.

More extensive surveillance and characterizationstudies using hybridization, sequencing and serologicmethods have defined enormous strain diversity andhelped understand the evolution and circulation ofrotavirus strains. Altogether, 14 rotavirus G (glycopro-tein) serotypes and 19 G genotypes and 14 P serotypesand 27 P genotypes have been defined to date, of which11 G serotypes and 12 G genotypes and 12 P serotypesand 15 P genotypes have been recovered from humans[Gentsch et al., 2005; Rahman et al., 2005; Martellaet al., 2006, 2007; Estes and Kapikian, 2007; Khamrinet al., 2007; Steyer et al., 2007; Matthijnssens et al.,2008a]. These strains have included apparent single andmulti-segment reassortants between animal and com-mon or uncommon human rotavirus strains as well asseveral examples of direct interspecies transmission ofanimal strains into humans [Nakagomi and Nakagomi,1989, 1996, 2002; Nakagomi et al., 1990; Palombo et al.,1996, 2000; Ramachandran et al., 1998; Holmes et al.,1999]. While these studies have identified huge diver-sity among rotaviruses, more conclusive studies on eachgene segment are needed to better understand theevolution, serotypes and genomic relatedness of rotavi-ruses [Maunula and von Bonsdorff, 2002; Matthijnssenset al., 2006b; Rahman et al., 2007].

Serotype G8 human rotavirus strains were firstrecovered from young children with gastroenteritis inIndonesia [Matsuno et al., 1985]. The prototype strain(69M), genotype P4[10],G8 is SGI and has a ‘‘super-short’’ RNA electropherotype. Additional G8 strainsidentified later had different characteristics; in FinlandG8 strains with long RNA electropherotype and SGIspecificity were isolated [Gerna et al., 1990]. Other G8strains have been reported in the United Kingdom[Beards and Graham, 1995], India [Jagannath et al.,2000; Kang et al., 2002], Australia [Bishop et al., 2001],Brazil [Santos et al., 1998], Japan [Okada and Matsu-moto, 2002], Malawi [Cunliffe et al., 1999, 2000], Ghana[Armah et al., 2001], Nigeria [Adah et al., 2001], Egypt[Holmes et al., 1999], Kenya [Nakata et al., 1999],Guinea-Bissau [Fischer et al., 2003], South Africa[Steele et al., 1999], and Democratic Republic of Congo[Matthijnssens et al., 2006b]. In the Malawi study,serotype G8 was one of the most common strains incirculation [Cunliffe et al., 1999, 2000]. Human rotavi-rus G8 strains have been associated with a variety ofVP4 genotypes including P[1], P[2], P[4], P[5], P[6], P[8],

J. Med. Virol. DOI 10.1002/jmv

938 Esona et al.

P[10], P[11], and P[14] [Qian and Green, 1991; Gernaet al., 1994; Cunliffe et al., 1999; Fukai et al., 1999;Fischer et al., 2000; Jagannath et al., 2000; Adah et al.,2001; Kang et al., 2002; Okada and Matsumoto, 2002;Matthijnssens et al., 2006b], and are apparent reassor-tants with animal strains as indicated in part by theclose relationships of their VP7 and VP4 genes withanimal rotaviruses. They also include apparent multi-genic reassortants (e.g., those with relationships withbovine rotaviruses) as well as strains that are reassor-tants in serotype antigen genes only (e.g., P[6]G8 andP[4]G8 strains) with all other genes from DS-1 gen-ogroup strains as in the Malawi study [Holmes et al.,1999; Chang et al., 2000; Cunliffe et al., 2000; Palomboet al., 2000; Matthijnssens et al., 2006b]. To date, moststudies of G8 rotavirus and other unusual strains haveused nucleotide sequencing only to examine the genesfor the two outer capsid proteins VP4 and VP7. Recently,a classification system has been proposed for rotaviruswhich is based on complete or almost complete (ORFs)sequences of all 11 RNA segments and which turns out tobe very helpful in recognizing natural reassortmentevents and inter-species transmissions. To obtain moredetailed understanding of the entire genome of theAfrican G8 strains in the present study, fragments of all11 gene segments of 6 P[8],G8 strains with long electro-pherotypes and SGII specificity and 2 P[6], G8 strainswith undetermined electropherotypes and SGI VP6genes were sequenced and then compared to otherrotavirus sequences from the GenBank database toelucidate the genetic and evolutionary relationships ofthese strains.

MATERIALS AND METHODS

Sample Collection

From 1996 to 2004, a total of 215 rotavirus-positivenon-typeable stool samples collected from the AfricanRotavirus Network (ARN) member countries weretransported on dry ice from South Africa to CDC,Atlanta, USA. From these, 8 rotavirus-positive sampleswere originally untypeable with our routine multiplexRT-PCR in South Africa and CDC, Atlanta, USA,because only primers specific for G-genotypes G1-G4and G9 [Gouvea et al., 1990; Das et al., 1994] and for P-genotypes only primers specific for P[4], P[6], P[8], P[9],and P[10] [Gentsch et al., 1992] were used. These strainswere subsequently determined as genotype G8 (Table II)by nucleotide sequencing using primers 9con1-L andVP7-Rdeg [Das et al., 1994; Iturriza-Gomara et al.,2001].

Viral RNA Extraction

Viral RNA from each of the eight specimens wasextracted from a 10% stool suspension (made from 0.1 gor 100 ml stool in 2 ml of a 1:1 Vertrel/Water solution)using either a commercial RNA extraction kit (BIO 101,Vista, CA) or a NucliSens automated extractor (BIO-MERIEUX, Durham, NC) according to the protocol

specified by the manufacturer and method describedpreviously [Boom et al., 1990].

Polyacrylamide Gel Electrophoresis (PAGE)

A portion of the extracted dsRNA was used for thedetermination of the electropherotypes of these 8rotavirus positive specimens using a previouslydescribed method [Herring et al., 1982].

RT-PCR and Nucleotide Sequencing of theVP1–VP4, VP6–VP7, and NSP1–NSP5 Genes

The extracted dsRNA of each strain was denatured at978C for 5 min and RT-PCR was carried out using a OneStep RT-PCR kit (Qiagen, Inc., Valencia, CA) accordingto manufacturer’s instructions. Previously publishedforward and reverse primers [Gentsch et al., 1992; Daset al., 1994; Iturriza-Gomara et al., 2001, 2002; Esonaet al., 2009] used for the amplification of the differentgene segment (partial) are given in Table I. Theseoligonucleotides were designed based on the sequencesof published strains Wa and Ku found in GenBank. Afterdenaturation of the dsRNA, reverse transcription (RT)of each gene from each sample was carried out for 30 minat 508C, followed by 15 min at 958C to inactivate thereserve transcriptase and activate the DNA polymerase.The cDNA was then subjected to 30 cycles of PCR in aGeneAmp PCR System 9700 thermal cycler (AppliedBiosystems, Inc., Foster City, CA) using the followingconditions: 30 sec at 948C; 30 sec at 508C; 45 sec at 728C,followed by a 7 min extension at 728C and a 48C coolingstep. Amplicons were run in a 1% agarose gel and thedesired band excised and purified with the QIAquick Gelextraction kit (Qiagen, Inc.) according to the manufac-turer’s protocol. Cycle sequencing of each amplicon wasperformed with the same consensus primers used forRT-PCR, using a Big Dye Terminator cycle sequencingReady kit (Applied Biosystems, Inc.). Cycle sequencingproducts were purified using Centri-sep spin columns(Princeton Separations, Inc., Adelphia, NJ), dried in aDNA speed VacR (Savant Instruments, Inc., Holbrook,NY) and reconstituted in 15 ml Hi-Di formamide.Automated separation and base-calling of cycle sequenc-ing products was performed using an ABI 3130sequencer (Applied Biosystems). Overlapping sequencefragments were assembled using the Sequencher pro-gram (Gene Codes Corporation, Inc., Ann Arbor, MI).

An alignment of the sequences was then performedusing the PILEUP program within the University ofWisconsin Genetics Computer Group software suiteVersion 11.1 [Devereux et al., 1984]. Phylogeneticrelationships were inferred using aligned nucleotidesequences by the neighbor-joining method and theprograms DNADist and Neighbor of PHYLIP, Version3.57 from the University of Washington in Seattle[Kimura, 1980]. Branch lengths in consensus trees weredetermined using the maximum-likelihood quartet-puzzling method, using the Tree-Puzzle version 5.0programs and nucleotide substitution model [Tamuraand Nei, 1993; Strimmer and vonHaeseler, 1996]. Trees

J. Med. Virol. DOI 10.1002/jmv

Characterization of Human Rotavirus G8 Strains 939

obtained were saved as puzzle.tree or puzzle.tree.newick files and then opened using TreeView programversion 1.6.6 [Page, 1996] for proper view and editing. Tofurther evaluate the relationships between the sequen-ces of the African isolates and those acquired from theGenBank, nucleotide and amino acid distance matrixeswere prepared using the OldDistances program [Dever-eux et al., 1984]. Also, conserved regions in thesequences of the individual strains were done usingthe PRETTY program [Devereux et al., 1984].

Accession numbers and strains used for each gene:The accession numbers and strains used for this studyare in Appendix. Accession numbers in bold facecharacters represent the gene sequences of these ARNstrains.

RESULTS

dsRNA Electrophoretic Pattern

Using analysis by PAGE and silver staining, six of theeight ARN G8 rotavirus strains had long RNA migrationpatterns (electropherotypes) typical of human group Arotaviruses, while we were unable to determine RNAmigration profiles for the remaining two G8 strains(data not shown).

Sequence Analysis

The partial nucleotide and deduced amino acidsequences for the 11 gene segments encoding VP1–VP4, VP6–VP7, and NSP1–NSP5, of the eight Africanrotavirus G8 strains were determined (Table II). Phy-

logenetic analysis for each gene segment, which con-sisted of the African G8 strains together with thecorresponding gene sequences of selected rotavirusstrains available in GenBank, were drawn based ontheir nucleotide sequences (Fig. 1A–K). In addition,multiple sequence alignments for gene segments wereconstructed and nucleotide and amino acid similaritymatrices were prepared (Table III). The gene segmentsof the African G8 strains presented in this study havebeen classified using both the current nomenclature andthe proposed full genome-based classification system[Matthijnssens et al., 2008a] (Table II). A genotype foreach segment was assigned according to percentnucleotide identities, as genetic diversity betweenstrains are more evenly distributed across the gene atthe nucleotide level [Matthijnssens et al., 2008a].

Analysis of Partial VP1 Sequence

A comparison of the partial VP1 nucleotide anddeduced amino acid sequences among the Africanstrains demonstrated relatedness in the range of 92–100% and 91–100% respectively. A very high nucleotideand amino acid identity (�99%) was shared betweenstrains 6787/2000/ARN, 6810/2004/ARN, 6854/2002/ARN, and 6862/2000/ARN. When the nucleotide andamino acid homologies of the African strains werecompared with other strains from the GenBank data-base, all of them had relatively high identity (nucleotide;93–96% and amino acid; 89–94%) with human rotavi-rus P[8],G1 prototype strain Wa, and a human rotavirusP[8],G3 strain, P (nucleotide; 91–94% and amino acid;

J. Med. Virol. DOI 10.1002/jmv

TABLE I. Primers Used for Amplification and Sequencing of Rotavirus Genes

F, forward; R, reverse; VP, structural protein; NSP, nonstructural protein; Nt, nucleotide.aR, A or G; Y, C or T; V, A, C or G; N, A, C, G or T.

940 Esona et al.

89–94%). The lowest nucleotide and amino acid sim-ilarities among Wa-, DS-1-, and AU-1-like group Arotaviruses used in this study, was with DS-1 and DS-1-like rotavirus strains which shared 74–76% and 79–84% identity, respectively (data not shown). Consistentwith their high sequence identity to strain Wa, phylo-genetic analysis showed that the African strainsclustered within the R1 gengroup which is the pre-viously identified Wa-like VP1 genotype containingstrains Wa, P, RMC321 and other recently publishedhuman rotavirus strains from Bangladesh and Belgium[Wyatt et al., 1983; Varghese et al., 2006; Rahman et al.,2007; Matthijnssens et al., 2008a] (Fig. 1A). Within thiscluster, the VP1 gene of African P[6]strain 6809/2000/ARN/Hu fell in a separate sublineage supported by abootstrap value of 80%.

Analysis of Partial VP2 Sequence

Distance matrix analysis indicated that the Africanstrains shared nucleotide and amino acid homologies inthe range of 95–100% and 98–100%, respectively, witheach other. Complete nucleotide and amino acid sim-ilarity (100%) was shared between strains 6736/2004/ARN, 6780/2000/ARN, and 6782/2000/ARN and alsobetween 6787/2000/ARN, 6810/2004/ARN, 6854/2002/ARN, and 6862/2000/ARN. However, when the nucleo-tide and amino acid homologies of the African strainswere compared with strains belonging in the threeknown human genogroups, all of them were more closelyrelated to strains in the Wa genogroup than to those inthe DS-1 and AU-1 genogroups. They had a maximumidentity (nucleotide; 94% and amino acid; 95–97%) withhuman rotavirus prototype P[8],G1 strain Ku, followedby strain Wa (nucleotide; 91–92% and amino acid; 94%).The African strains were distantly related to DS-1-likerotavirus strains exhibiting nucleotide and amino acidhomologies in the range of 76–77% and 88–89%respectively (data not shown). Consistent with the highsequence relatedness to strains of the Wa genogroup,the VP2 of all African strains clustered within the C1genogroup or the Wa-like VP2 genes consisting mainlyof old and newly published Wa genogroup strains(Fig. 1B).

Analysis of Partial VP3 Sequence

The partial VP3 nucleotide and deduced amino acidsequences of the African strains revealed they shared anoverall high nucleotide (88–100%) and amino acid (96–100%) sequence identity to each other. Absolute nucleo-tide and amino acid similarities were shared amongstrains 6736/2004/ARN, 6780/2000/ARN, 6782/2000/ARN, 6809/2000/ARN, 6854/2002/ARN, and 6862/2000/ARN. Among strains available in the GenBank data-base, these same six African strains had maximumnucleotide identity (96%) with human rotavirus proto-type strain Wa and shared a maximum amino acididentity (99%) with Wa and another human rotavirusprototype strain ST-3. Two African strains (6787/2000/ARN and 6810/2004/ARN) had maximum nucleotide

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Characterization of Human Rotavirus G8 Strains 941

(91–94%) and amino acid (97%) homologies to porcinestrain OSU and human strain ST-3. The lowest nucleo-tide and amino acid similarities observed within thehuman rotavirus strains use in this analysis was withprototype strain DS-1 and two human G8 strains DRC86and DRC88 isolated from Democratic Republic of Congoin 2003 [Matthijnssens et al., 2006b] with which theyshared 71–74% and 83–85% identity respectively (datanot shown). Phylogenetic analysis confirmed theserelationships. Within the Wa-like (M1) genogroup, thesix African strains with maximum homology to the VP3of Wa, clustered more closely with these genes than toother VP3 genes of genotype M1. In contrast, one of theAfrican strains (6787/2000/ARN) clustered more closelywith genotype M1 strain OSU than to other strains(Fig. 1C).

Analysis of Partial VP4 Sequence

In order to determine the P genetic variant of the eightAfrican strains, the portion of VP4 gene (nucleotide; 11–887) encoding the full-length VP8* was sequenced. TheAfrican strains shared nucleotide and amino acidhomologies of 71–100% and 74–100% respectively.Absolute nucleotide and amino acid identity was seenamong strains 6736/2004/ARN, 6787/2000/ARN, 6810/2004/ARN, 6854/2002/ARN, and 6862/2000/ARN. Whenthe nucleotide and amino acid identities of the Africanstrains were compared with other strains from Gen-Bank database, six of them had a maximum identity(nucleotide; 96–97% and amino acid; 97–98%) withhuman rotavirus G8P[8] strain DRC88 isolated fromDemocratic Republic of Congo. They were also closely

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. A–F: Phylograms indicating the genetic relationship ofpartial nucleotide sequences of VP1 (A), VP2 (B), VP3 (C), VP4 (D), VP6(E), and VP7 (F) of human serotype G8 rotaviruses from the AfricanRotavirus Network with some of the known human and animalrotavirus strains from GenBank database. The trees were drawn toscale and rooted with sequences of avian rotavirus strain PO-13.Significant bootstrap values (>80%) are indicated at the branch nodes.Av, Avian; Po, porcine; Bo, bovine; Ov, ovine; Lp, lapine; Ca, canine; Hu,human; Eq, equine; Mu, murine; Rm, Rhesus Macaques; and Si,simian. Accession numbers of all strains used are shown in Appendix.African G8 strains are in bold face characters. G–K: Phylograms

indicating the genetic relationship of partial nucleotide sequences ofNSP1 (G), NSP2 (H), NSP3 (I), NSP4 (J), and NSP5 (K) of humanserotype G8 rotaviruses from the African Rotavirus Network with someof the known human and animal rotavirus strains from GenBankdatabase. The trees were drawn to scale and rooted with sequences ofavian rotavirus strain PO-13. Significant bootstrap values (>80%) areindicated at the branch nodes. Av, avian; Po, porcine; Bo, bovine; Ov,ovine; Lp, lapine; Ca, canine; Hu, human; Eq, equine; Mu, murine; Rm,rhesus macaques; and Si, simian. Accession numbers of all strains usedare shown in Appendix. ARN G8 strains are in bold face characters.

942 Esona et al.

related to human rotavirus prototype strain Ku withwhich they shared an identity of �94% (nucleotide) and�96% (amino acid). The remaining two African strainsshared maximum nucleotide and amino acid identity of�95% with three human rotavirus strains DRC86(G8P[6]) also isolated from the Democratic Republic ofCongo, US1205 (G9P[6]) isolated from the United Statesand strain MW467 (G8P[6]) isolated from Malawi(Table IIIA). Phylogenetic analysis shows the Africanstrains clustered into two groups (Fig. 1D). One groupwhich consists six strains (6736/2004/ARN, 6787/2000/ARN, 6854/2002/ARN, 6810/2004/ARN, 6862/2000/ARN, and 6780/2000/ARN) clustered with humanrotavirus genotype P[8] strains Wa, Ku, DRC88 and anumber of recently published genotype P[8] strains.The second group consisted of strains 6782/2000/ARNand 6809/2000/ARN clustered with human genotypeP[6] strains MW467, M37, US1205, and DRC86. TheARN P[6] strains are in a distinct subcluster from theporcine P[6] strain Gottfried.

Analysis of Partial VP6 Sequence

A comparison of the partial VP6 nucleotide anddeduced amino acid sequences of the African strains

showed they shared nucleotide and amino acid homol-ogies of 82–100% and 92–100% respectively. Six strains(6736/2004/ARN, 6780/2000/ARN, 6782/2000/ARN,6787/2000/ARN, 6854/2002/ARN, and 6862/2000/ARN)were closely related or identical to each other innucleotide (�96%) and amino acid (�97%) sequences.These six strains were closely related (nucleotideidentity of�96%) to recently published human rotavirusSGII strains, rj6906, US0408, B4633-03, Dhaka25-02,Dhaka12-03, and Matlab13-03 [Araujo et al., 2007;Kerin et al., 2007; Rahman et al., 2007]. The remainingtwo strains (6809/2000/ARN and 6810/2004/ARN)shared a moderately high nucleotide (89–92%) and avery high amino acid (97–98%) identity with fourhuman rotavirus SGI strains DRC88, DRC86, US1205,and US8908 isolated from Democratic Republic of Congoin 2003 and United States between 1996 and 2002,respectively (Table IIIC). Phylogenetic analysis dividesthe African strains into two separate clusters (Fig. 1E).Consistent with distance analyses strains 6809/2000/ARN and 6810/2004/ARN formed a phylogenetic clusterwith human rotavirus SGI (DS-1-like or I2 genotype)strains, while the remaining six strains clustered withWa-like or genotype I1 (SGII) VP6 genes.

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. (Continued )

Characterization of Human Rotavirus G8 Strains 943

J. Med. Virol. DOI 10.1002/jmv

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Analysis of Partial VP7 Sequence

Comparison of the VP7 nucleotide and deduced aminoacid sequences of the African strains shows they sharednucleotide and amino acid homologies in the range of98–100% among themselves, and a somewhat lowerrelatedness to previously identified G8 strains fromhuman and animals (92–98%). In contrast, ARN strainsshared a much lower nucleotide (61–85%) and aminoacid (63–89%) relatedness to other serotypes(Table IIIB). Phylogenetic analysis of the VP7 geneshowed the African G8 and other published G8 strains intwo separate clusters (Fig. 1F). The first cluster (G8d) isdivided into two separate lineages supported by boot-strap value of 93%. The first lineage (LI) consisted ofpublished human and animal strains, while the secondlineage (LII) consisted of only the African strains.The second cluster (G8c) is also made up of mostlypublished animal and human strains. Branched closelyto this cluster is a guanacos rotavirus G8 strain GRV-Chubut and bovine rotavirus G8 strain Cody1801. Thesetwo G8 clusters (G8d and G8c) are amongst the four G8lineages reported previously [Fukai et al., 2004].

Analysis of Partial NSP1 Sequence

A comparison of the partial NSP1 nucleotide anddeduced amino acid sequences of the African strainsshowed they shared homologies in the range of 91–100%and 92–100% with each other, respectively. Absolutenucleotide and amino acid identity was shared amongstrains 6736/2004/ARN, 6787/2000/ARN, 6810/2004/ARN, 6854/2002/ARN, and 6862/2000/ARN. Sequenceidentities between the NSP1 sequences of Africanstrains and those of other rotaviruses ranged from 73%to 96% and 78% to 96% at the nucleotide and amino acidlevel respectively, with the identities to three humanrotaviruses (M37, ST-3, and R14a) being the highest. Anintermediate level of nucleotide (73–75%) and aminoacid (78–82%) similarities was observed between theseAfrican G8 strains and two other African G8 strains(DRC88 and DRC86) published earlier (data not shown).By phylogenetic analysis, the African strain NSP1 genefragments clustered within A1 genogroup which consistof Wa-like human rotavirus strains, particularly ST-3,M37, Dhaka25-02, and B4633-03 (Fig. 1G).

Analysis of Partial NSP2 Sequence

A comparison of the partial NSP2 nucleotide anddeduced amino acid sequences of the African strainsshowed they shared nucleotide and amino acid homol-ogies in the range of 89–98% and 95–100% respectively.Absolute amino acid identity was shared betweenstrains 6780/2000/ARN and 6736/2004/ARN. When thenucleotide homologies of the African strains werecompared with other strains from GenBank, six of theAfrican strains had maximum identity (�94%) withbovine rotavirus strains KJ75, while the remaining twoshared an identity of 90–94% with human rotavirusstrains Ku and RMC321. Although the KJ75 strain was

detected in cattle, it is indeed a bovine-porcine reassor-tant strain with its NSP2 gene coming from a porcineparental rotavirus strain. Hence, it is likely thatthe NSP2 of these six African strains might be of porcineorigin. Every African strain shared relatively highamino acid identities (92–100%) with all humanrotavirus NSP2 genes from the GenBank database.The African strain 6862/2000/ARN had an absoluteamino acid identity with lapine rotavirus strain 30/96(data not shown). In phylogenetic analysis, the humanrotavirus strains clustered together in three separategroups (Fig. 1H). Group I which correspond to N1genogroup in the new full-genome based classificationsystem, consisted of the African strains, referencestrains Wa and Ku, bovine strain KJ75 and a fewrecently published human rotavirus strains. Withingroup I or N1 genogroup, African strain 6787/2000/ARN/Hu fell into separate sublineage with bootstrap supportof 97%. Group II or N2 genogroup consisted mainly ofrotavirus strain DS-l and DS-1-like strains, while strainAU-1 represented the third group or N3 genogroup.

Analysis of Partial NSP3 Sequence

The NSP3 gene fragment of African G8 strains sharednucleotide and amino acid homologies in the range of97–100% among themselves and had maximum identityof 96–99% with five human rotaviruses strains Wa, ST-3, IGV-F, I321 and one porcine rotavirus strain PRICEavailable in the GenBank database. The African strainsshared relatively high amino acid similarity amongthemselves (97–100%) and with strains from theGenBank (90–100%) (data not shown). Phylogeneticanalysis based on nucleotide sequences demonstratedthat all African strains clustered within the T1 or Wa-like NSP3 genotype which included several referencestrains (Wa, ST-3, Ku, and I321) and a number ofrecently published human and animal strains (Fig. 1I).Closely branched from this cluster are human(RMC321), bovine (KJ75), and porcine (OSU) strains.

Analysis of Partial NSP4 Sequence

The African isolates exhibited nucleotide and aminoacid identities of 93–100% and 98–100%, respectivelywith each other. Strains 6736/2004/ARN, 6810/2004/ARN, 6854/2002/ARN, and 6862/2000/ARN were iden-tical. Comparative analysis of the NSP4 nucleotide andamino acid of these African G8 strains with cognatesequences of Group A rotavirus strains representing fiveof the eight distinct NSP4 genotypes suggested that theAfrican strains belonged to the E1 genogroup or NSP4genotype B (Wa-like strains) [Cunliffe et al., 1997; Horieet al., 1997; Ciarlet et al., 2000; Mori et al., 2002; Lin andTian, 2003; Matthijnssens et al., 2008a] (Table IIID).Phylogenetic analysis of the NSP4 genes for all Africanstrains along with the reference human and animalstrains revealed that all the African strains clusteredtogether, forming two distinct lineages within a broadercluster that includes the reference human strains Wa,Ku, and ST-3. Also included in this cluster are a porcine

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Characterization of Human Rotavirus G8 Strains 945

(OSU), an equine (H-1) and a few recently publishedhuman strains (Dhaka25-02, Dhaka12-03, andDhaka16-03). This cluster was designated NSP4 geno-type B (Wa-like strains) or E1 genogroup. Closerelationship between the NSP4 genes of human, porcineand equine rotavirus strains have been reportedpreviously [Ciarlet et al., 2000]. The remaining strainsalso formed distinct NSP4 genotypes in the phylogenetictree (Fig. 1J).

Analysis of Partial NSP5 Sequence

The African G8 strains shared nucleotide and aminoacid identity in the range of 99–100% amongst them-selves and had maximum identity in the range of 96–99% (nucleotide) and 98–99% (amino acid) with porcinerotavirus strains CMP034, OSU and human strainsDhaka25-02, Dhaka12-03 Dhaka16-03, B4633-03, andRMC321. The African strains had lower nucleotide (94–95%) and amino acid (95–98%) relatedness to prototypehuman strains KU and Wa. The lowest nucleotide andamino acid similarities observed within the humangroup A rotavirus was with DS-1-like strains (DRC86and DRC88) with which they shared 90% and 95–96%identity respectively (data not shown). Phylogeneticanalysis revealed that African strains clustered withinthe H1 genogroup with NSP5 genes from both humansand animals to which the African strains sharedmaximum nucleotide and amino acid similarity(Fig. 1K).

DISCUSSION

Rotavirus surveillance and strain characterizationhas highlighted the importance of mechanisms such asreassortment between animal and human rotavirusesand interspecies transmission in the evolution of mostrotavirus strains. Examples include; (1) detection of>40G and P genotype combinations in human rotavirusesincluding many neutralization antigens that are highlyprevalent in animals, (2) demonstration that highlyprevalent human rotaviruses have one or more genesthat could have been derived from animal rotavirusesthrough reassortment, (3) detection of rotaviruses inhumans that are virtually indistinguishable in each oftheir 11 gene segments to animal rotavirus isolatessuggesting that these strains arose through trans-mission of complete animal rotaviruses to humans[Ramig and Ward, 1991; Ramig, 1997; Nakagomi andNakagomi, 2000; Maunula and von Bonsdorff, 2002;Matthijnssens et al., 2006a]. While numerous studieshave demonstrated the importance of these mechanismsin evolution of rotaviruses, relatively few have lookedcomprehensively at rotavirus genomes. Expansion ofstudies that examine all 11 rotavirus genome segmentsthrough whole genome sequencing [Rahman et al.,2007] or the sequencing of gene fragments [Maunulaand von Bonsdorff, 2002] are needed to better under-stand the evolution of common and uncommon humanrotaviruses. In order to gain a better understanding ofthe possible origin of rotavirus G8 strains isolated in this

study, partial or complete sequences of all 11 genomicsegments encoding six structural (VP1-VP4, VP6-VP7)and five non-structural (NSP1–NSP5) proteins of sixAfrican P[8],G8 (6736/2004/ARN, 6780/2000/ARN,6787/2000/ARN, 6810/2004/ARN, 6854/2002/ARN, and6862/2000/ARN) and two P[6],G8 (6782/2000/ARN and6809/2000/ARN) strains were analyzed.

Comparative sequence analyses showed that eachgene of African P[8],G8 strains had a close evolutionaryrelationship to prototype P[8],G1 human rotavirusstrain Wa (i.e., were Wa-like) with the exception of theVP7 gene that was closely related to bovine G8 and VP6gene of strain 6810/2004/ARN that was closely related toa DS-1-like strain. Thus, the VP7 gene is of potentialanimal origin. Similar results were observed with theAfrican P[6],G8 strains with gene 9 of potential animalorigin, while the VP6 gene of strain 6809/2000/ARN wasclosely related to a DS-1-like strain. While at least 8genes of the African strains could be classified as Wa-like, the NSP2 and NSP5 genes were more closelyrelated to corresponding sequences of animal rotavirusmembers of the Wa-like lineage than to human strainssuch as Wa. These results suggest a potential animalorigin for NSP2 and NSP5 genes as well. Analyses oflarger numbers of these genes from human and animalrotavirus strains to examine their frequency in thesespecies may help solidify their true origin. Overall, ourresults suggest that these African strains could havearisen through reassortment of animal rotavirus neu-tralization antigen genes, and perhaps NSP2 and NSP5genes into human rotavirus Wa-genogroup strainscirculating in the local population. Also, the possibilitythat the NSP2 and NSP5 genes are not derived from arecent interspecies transmission, but are circulating inthe natural human population for a longer period cannotbe excluded. However, classification of these African G8strains into Wa-like and/or DS-1-like lineages corre-sponded to the classification and nomenclature of thestructural and non-structural protein-encoding genesproposed recently [Maunula and von Bonsdorff, 1998,2002; Matthijnssens et al., 2008a,b].

These conclusions are supported by findings ofsignificant genetic relatedness of some human andanimal rotavirus genes [Dunn et al., 1994; Nakagomiand Kaga, 1995; Rao et al., 1995; Kojima et al., 1996;Ciarlet et al., 2000; Cunliffe et al., 2000; Masendycz andPalombo, 2001; Fukai et al., 2004; Varghese et al., 2004;Shah et al., 2006; Subodh et al., 2006]. Our resultssuggest that both the human and animal G8 strainscould have originated from a common human or animalrotavirus ancestor of the Wa-like or DS-1-like (VP6 geneof African strains 6782/2000/ARN and 6809/2000/ARN)lineage by single or multiple gene segment reassortmentevent in nature [Browning et al., 1992; Holmes et al.,1999]. Further, it is plausible that this process isongoing continuously as a means of generating diversityin both human and animal rotaviruses.

Earlier reports indicated that among human rotavi-ruses, NSP4 genotype A is associated with VP6 SGI typeand NSP4 genotype B is associated with VP6 SGII type

J. Med. Virol. DOI 10.1002/jmv

946 Esona et al.

[Kirkwood and Palombo, 1997; Iturriza-Gomara et al.,2003]. Such results are consistent with RNA-RNAhybridization studies demonstrating that except forneutralizing antigen genes, reassortment between thetwo common HRV genogroups is detected infrequently.However, some exceptions have been reported; includ-ing HRV with SGI specificity but NSP4 genotype B type[Varghese et al., 2004]. In this study, six of the ARNstrains had the common linkage VP6 SGII/NSP4genotype B. However, consistent with the results ofVarghese and co-workers the other two ARN strainsthat were isolated in different countries and had differ-ent genotypes (P[8],G8 and P[6],G8) were VP6 SGI/NSP4 genotype B, confirming that such linkages are notabsolute. A similar observation among porcine rotavi-ruses has been documented [Ghosh et al., 2007].

In conclusion, it was found that for most strains,almost all of the genes other than those coding for theneutralizing antigens are related to typical humanstrains of Wa genogroup, suggesting that the strainsevolved in part through VP7 and VP4 gene reassortmentevents. The parent strains (such as P[8],G1 and P[6],G1)are unknown but might be locally circulating rotavirusthat have not been identified. The results also show theclose relationship between human and animal NSP2and NSP5 genes. In addition, the uniformity of the NSP5gene from different years and different countries,suggest that this gene might be critically important forthe reassortment to occur and potentially for thecontinuation of this reassortant virus in the humanspecies. This study is of evolutionary importance andemphasizes the need for rotavirus studies in ruralpopulations that focus on sequencing of both humanand animal rotavirus genes, in order to understandbetter the extent to which reassortment contribute todiversity in both species.

ACKNOWLEDGMENTS

The post doctoral fellowship of Dr. Mathew DiohEsona was provided through the Rotavirus VaccineProgram, a collaboration between the Program forAppropriate Technology in Health, The World HealthOrganization and the Centers for Disease Control andPrevention. Our sincere thanks also go to all the staff ofthe MRC/Diarrhoeal Pathogens Research Unit, Univer-sity of Limpopo and The Gastroenteritis and Respira-tory Viruses Laboratory Branch at CDC, Atlanta, USAfor their immense assistance.

APPENDIX: ACCESSION NUMBERS ANDSTRAINS USED IN THIS STUDY

NSP1: 6736/2004/ARN (FJ425104); 6780/2000/ARN (FJ425105); 6782/2000/ARN (FJ425108); 6787/2000/ARN (FJ425109); 6809/2000/ARN (FJ425110);6810/2004/ARN (FJ425111); 6854/2002/ARN(FJ425106); 6862/2000/ARN (FJ425107); EHP(U08423); KU (AB022769); DRC86 (DQ005119);DRC88 (DQ005108); OSU (D38153); RMC321

(AF506292); TB-chen (AY787647); 30/96 (DQ205225);AU-1 (D45244); ST-3 (U11492); 69M (D38151); L338(D38158); EW (U08428); Gottfried (U08431); T152(AB097459); R14A (DQ199658); I321 (U08418); Wa(L18943); M37 (U11491); DS-1 (L18945); H-1(U23728); B4633-03 (DQ146644); Dhaka16-03(DQ492675); Dhaka25-02 (DQ146655); Matlab13-03(DQ146677); Dhaka12-03 (DQ146666).

NSP2: 6736/2004/ARN (FJ425112); 6780/2000/ARN (FJ425113); 6782/2000/ARN (FJ425114); 6787/2000/ARN (FJ425115); 6809/2000/ARN (FJ425116);6810/2004/ARN (FJ425117); 6854/2002/ARN(FJ425118); 6862/2000/ARN (FJ425119); RMC321(AF506293); PO-13 (AB009625); KU (AB022770);DRC86 (DQ005115); DRC88 (DQ005104); TB-chen(AY787648); 30/96 (DQ205227); Wa (L04534); NCDV(L04530); DS-1 (L04529); AU-1 (DQ490534); KJ75(DQ494402); B4633-03 (DQ146645); Dhaka25-02(DQ146656); Matlab13-03 (DQ146678); Dhaka12-03(DQ146667); L26 (DQ146696); ST-3 (EF672615); Wi61(EF672622).

NSP3: 6736/2004/ARN (FJ425120); 6780/2000/ARN (FJ425121); 6782/2000/ARN (FJ425122); 6787/2000/ARN (FJ425123); 6809/2000/ARN (FJ425124);6810/2004/ARN (FJ425125); 6854/2002/ARN(FJ425126); 6862/2000/ARN (FJ425127); KU(AB022771); Wa (X81434); DRC86 (DQ005117);DRC88 (DQ005106); OSU (X81431); TB-chen(AY787649); 30/96 (DQ205228); ST-3 (X81436); 69M(X81425); S2 (X81428); PO-13 (AB009626); RMC321(AF541920); PRICE (X81432); IGV-F (AF190172); I321(X81433); AU-1 (DQ490535); RRV (DQ391186); NCDV(X81429); DS-1 (EF136660); B4633-03 (DQ146646);Dhaka16-03 (DQ492677); Dhaka25-02 (DQ146657);Matlab13-03 (DQ146679); Dhaka12-03 (DQ146668);RV176-00 (DQ490559); L26 (DQ146697); KJ(DQ494404); Wi61 (EF672621).

NSP4: 6736/2004/ARN (FJ425128); 6780/2000/ARN (FJ425129); 6782/2000/ARN (FJ425130); 6787/2000/ARN (FJ425131); 6809/2000/ARN (FJ425132);6810/2004/ARN (FJ425133); 6854/2002/ARN(FJ425134); 6862/2000/ARN (FJ425135); KU(AB022772); Wa (AF093199); DS-1 (AF174305);DRC86 (DQ005116); DRC88 (DQ005105); OSU(D88831); TB-chen (AY787650); 30/96 (DQ205230); ST-3 (U59110); US1205 (AF079358); 116E (U78558); EW(U96335); S2 (U59104); RMC321 (AF541921); I321(AF165066); AU-1 (D89873); KUN (D88829); CH-1(AB065287); B223 (AF144805); H-2 (AF144801); RRV(L41247); CU-1 (AF144806); FRV-1 (D89874); M37(U59109); H-1 (AF144800); EHP (U96336); EC(U96337); Dhaka16-03 (DQ492678); Dhaka25-02(DQ146658); Dhaka12-03 (DQ146669); RV176-00(DQ490560); T152 (DQ146705).

NSP5: 6736/2004/ARN (FJ425136); 6780/2000/ARN (FJ425137); 6782/2000/ARN (FJ425138); 6787/2000/ARN (FJ425139); 6809/2000/ARN (FJ425140);6810/2004/ARN (FJ425141); 6854/2002/ARN(FJ425142); 6862/2000/ARN (FJ425143); KU(AB022773); Wa (AB091726); DRC86 (DQ005115);

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Characterization of Human Rotavirus G8 Strains 947

DRC88 (DQ005104); OSU (X15519); SA11 (M28347);TB-chen (AY787651); 30/96 (DQ205231); AU-1(AB008656); 69M (M33607); PO-13 (AB009628);RMC321 (AY033396); CMP034 (DQ916134); KUN(AB091727); B4633-03 (DQ146648); Dhaka16-03(DQ492679); Dhaka25-02 (DQ146659); Matlab13-03(DQ146681); Dhaka12-03 (DQ146670); ST-3(EF672618); Wi62 (EF672625); KJ75 (DQ492679); L26(DQ146698).

VP1: 6736/2004/ARN (FJ425144); 6780/2000/ARN(FJ425145); 6782/2000/ARN (FJ425146); 6787/2000/ARN (FJ425147); 6809/2000/ARN (FJ425148); 6810/2004/ARN (FJ425149); 6854/2002/ARN (FJ425150);6862/2000/ARN (FJ425151); KU (ab022765); Wa(AF044358); DS-1 (AF044360); DRC86 (DQ005125);DRC88 (DQ005114); TB-chen (AY787653); UK(X55444); 30/96 (DQ205221); PO-13 (AB009629);RMC321 (AY601114); YM (X76486); SA11 (X16830); P(AF044368); S2 (AF106303); Gottfried (M32805); AU-1(DQ490533); B4633-03 (DQ146638); Dhaka16-03(DQ492669); Dhaka25-02 (DQ146649); Matlab13-03(DQ146671); Dhaka12-03 (DQ146660).

VP2: 6736/2004/ARN (FJ425152); 6780/2000/ARN(FJ425153); 6782/2000/ARN (FJ425154); 6787/2000/ARN (FJ425155); 6809/2000/ARN (FJ425156); 6810/2004/ARN (FJ425157); 6854/2002/ARN (FJ425158);6862/2000/ARN (FJ425159); KU (AB022766); Wa(X14942); DRC86 (DQ005124); DRC88 (DQ005113);SA11 (L33364); TB-chen (AY787652); UK (X52589);PO-13 (AB009630); RMC321 (AY601115); 30/96(DQ205222); RF (X14057); AU-1 (DQ490536); B4633-03 (DQ146639); Dhaka16-03 (DQ492670); Dhaka25-02(DQ146650); Matlab13-03 (DQ146672); Dhaka12-03(DQ146661).

VP3: 6736/2004/ARN (FJ425160); 6780/2000/ARN(FJ425161); 6782/2000/ARN (FJ425162); 6787/2000/ARN (FJ425163); 6809/2000/ARN (FJ425164); 6810/2004/ARN (FJ425165); 6854/2002/ARN (FJ425166);6862/2000/ARN (FJ425167); KU (AB022767); Wa(AY267335); DRC86 (DQ005123); DRC88 (DQ005112);OSU (AY277921); TB-chen (AY787654); UK(AY300923); 30/96 (DQ205223); ST-3 (AY277919);116E (AY028978); 69M (AY277916); L338 (AY277922);DS-1 (AY277914); PO-13 (AB009631); AU-1(DQ490537); B4633-03 (DQ146640); Dhaka16-03(DQ492671); Dhaka25-02 (DQ146651); Matlab13-03(DQ146673); Dhaka12-03 (DQ146662); L26(AY277918); WI-61 (AY277917).

VP4: 6736/2004/ARN (FJ425168); 6780/2000/ARN(FJ425169); 6782/2000/ARN (FJ425170); 6787/2000/ARN (FJ425171); 6809/2000/ARN (FJ425172); 6810/2004/ARN (FJ425173); 6854/2002/ARN (FJ425174);6862/2000/ARN (FJ425175); KU (AB222784); Wa(L34161);ST-3 (L33895); DRC86 (DQ005122); DRC88(DQ005111); SA11 (d16346); TB-chen (AY787644); UK(M22306); 30/96 (DQ205224); AU-1 (D10970); US1205(AF079356); 116E(L07934); L338 (D13399); EW(U08429); Gottfried (M33516); YM (M63231); PO-13(AB009632); RMC321 (AF523677); T152 (AB077766);I321 (L07657); PA169 (D14724); TUCH (AY596189);

160/01 (AF526374); EHP (U08424); 993/83 (D16352);EB (U08419); Lp14 (L11599); BAP-2 (U62151); MDR-13(L07886); H-2 (L04638); B223 (D13394); K8 (D90260);K9 (13400); M37 (L20877); MW467 (AJ427321); B641(M63267); RRV (AY033150); DS-1 (AB118025); B4633-03 (DQ146641); Dhaka16-03 (DQ492672); Dhaka25-02(DQ146652); Hun9 (AJ605320); MD28 (AB297792).

VP6: 6736/2004/ARN (FJ425176); 6780/2000/ARN(FJ425177); 6782/2000/ARN (FJ425178); 6787/2000/ARN (FJ425179); 6809/2000/ARN (FJ425180); 6810/2004/ARN (FJ425181); 6854/2002/ARN (FJ425182);6862/2000/ARN (FJ425183); KU (AB022768); Wa(K02086); DRC86 (DQ005121); DRC88 (DQ005110);OSU (AF317123); TB-chen (AY787645); UK (X53667);US1205 (AF079357); 116E (U85998); Gottfried(D00326); YM (X69487); S2 (Y00437); PO-13 (D16329);RMC321 (AF531913); I321 (X94618); H-1 (AF242394);NCDV (AF317127); AU-1 (DQ490538); T152(DQ146702); DS-1 (EF619345); US0408 (EF426119);US8908 (EF426140); rj6906/03 (DQ498165); B4633-03(DQ146642); Dhaka25-02 (DQ146653); Matlab13-03(DQ146675); Dhaka12-03 (DQ146664); RV176-00(DQ490555).

VP7: 6780/2000/ARN (EF218668); 6782/2000/ARN(EF218669); 6787/2000/ARN (EF218672); 6809/2000/ARN (EF218674); 6810/2004/ARN (EF218675); 6854/2002/ARN (EF218676); 6862/2000/ARN (EF218677);6736/2004/ARN (EF218678); KU (AB222787); Wa(K02033); DS-1 (AB118023); DRC86 (DQ005120);DRC88 (DQ005109); OSU (X04613); SA11 (K02028);TB-chen(AY787646); 30/96(DQ205229); AU-1(D86271);US1205 (AF060487); 116E (L14072); L338 (D13549);EW (U08430); Gottfried (X06759); YM (M23194); PO-13(X04613); RMC321 (AF501578); NCDV (M12394); CH-2(X56784); Hg18 (AF237666); BAP-2 (U62153); T152(AB071404); I321 (L07658); B223 (X52650); UK(X00896); MW333 (AJ278257); FI23 (M61876); CH3(D25229); L26 (M58290); R291 (AY855064); GRV-chubut (AF545860); 94H109 (AB045375); HMG035(AF359359); MG8.01 (AF207061); MW23 (AJ278254);NGRBg8 (AF361439); A5 (D01054); UP30 (AF143690);69M (EF672560); DG8 (AF034852); Cody 1801(U14999); 1290 (EU488721); EGY2295 (AF104104);678 (L20883); BG8.01 (AF207060); HAL1166 (L20882).

REFERENCES

Adah MI, Wade A, Taniguchi K. 2001. Molecular epidemiology ofrotaviruses in Nigeria: Detection of unusual strains with G2P[6]and G8P[1] specificities. J Clin Microbiol 39:3969–3975.

Araujo IT, Heinemann MB, Mascarenhas JDP, Assis RMS, Fialho AM,Leite JPG. 2007. Molecular analysis of the NSP4 and VP6 genes ofrotavirus strains recovered from hospitalized children in Rio deJaneiro, Brazil. J Med Microbiol 56:854–859.

Armah GE, Pager CT, Asmah RH, Anto FR, Oduro AR, Binka F, SteeleD. 2001. Prevalence of unusual human rotavirus strains inGhanaian children. J Med Virol 63:67–71.

Beards G, Graham C. 1995. Temporal distribution of rotavirus G-serotypes in the West Midlands region of the United Kingdom,1983–1994. J Diarrhoeal Dis Res 13:235–237.

Bishop RF, Masendycz PJ, Bugg HC, Carlin JB, Barnes GL. 2001.Epidemiological patterns of rotaviruses causing severe gastroen-teritis in young children throughout Australia from 1993 to 1996. JClin Microbiol 39:1085–1091.

J. Med. Virol. DOI 10.1002/jmv

948 Esona et al.

Boom R, Sol CJA, Salimans MMM, Jansen CL, WertheimvandillenPME, Vandernoordaa J. 1990. Rapid and simple method forpurification of nucleic-acids. J Clin Microbiol 28:495–503.

Browning GF, Snodgrass DR, Nakagomi O, Kaga E, Sarasini A, GernaG. 1992. Human and bovine serotype g8 rotaviruses may be derivedby reassortment. Arch Virol 125:121–128.

Chang KO, Parwani AV, Saif LJ. 2000. Comparative sequence analysisof the VP7 genes of G6, G8 and G10 bovine group A rotaviruses andfurther characterization of G6 subtypes. Arch Virol 145:725–737.

Ciarlet M, Liprandi F, Conner ME, Estes MK. 2000. Species specificityand interspecies relatedness of NSP4 genetic groups by compara-tive NSP4 sequence analyses of animal rotaviruses. Arch Virol145:371–383.

Coulson BS, Unicomb LE, Pitson GA, Bishop RF. 1987. Simple andspecific enzyme-immunoassay using monoclonal-antibodies forserotyping human rotaviruses. J Clin Microbiol 25:509–515.

Cunliffe NA, Woods PA, Leite JPG, Das BK, Ramachandran M, BhanMK, Hart CA, Glass RI, Gentsch JR. 1997. Sequence analysis ofNSP4 gene of human rotavirus allows classification into two maingenetic groups. J Med Virol 53:41–50.

Cunliffe NA, Gondwe JS, Broadhead RL, Molyneux ME, Woods PA,Bresee JS, Glass RI, Gentsch JR, Hart CA. 1999. Rotavirus G and Ptypes in children with acute diarrhea in Blantyre, Malawi, from1997 to 1998: Predominance of novel P[6]G8 strains. J Med Virol57:308–312.

Cunliffe NA, Gentsch JR, Kirkwood CD, Gondwe JS, Dove W,Nakagomi O, Nakagomi T, Hoshino Y, Bresee JS, Glass RI,Molyneux ME, Hart CA. 2000. Molecular and serologic character-ization of novel serotype G8 human rotavirus strains detected inBlantyre, Malawi. Virology 274:309–320.

Das BK, Gentsch JR, Cicirello HG, Woods PA, Gupta A, RamachandranM, Kumar R, Bhan MK, Glass RI. 1994. Characterization ofrotavirus strains from newborns in New-Delhi, India. J ClinMicrobiol 32:1820–1822.

Devereux J, Haeberli P, Smithies O. 1984. A comprehensive set ofsequence-analysis programs for the vax. Nucleic Acids Res 12:387–395.

Dunn SJ, Cross TL, Greenberg HB. 1994. Comparison of the rotavirusnonstructural protein nsp1 (ns53) from different species bysequence-analysis and northern blot hybridization. Virology203:178–183.

Esona MD, Geyer A, Banyai K, Page N, Aminu M, Armah GE, Hull J,Steele AD, Glass RI, Gentsch JR. 2009. Novel human rotavirusgenotype G5P[7] from child with diarrhea, Cameroon. Emerg InfectDis 15:83–86.

Estes M, Kapikian A. 2007. Rotaviruses. In: Knipe DM, Howley PM,Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors.Fields virology, 5th edition. Philadelphia, PA: Kluwer/Lippincott,Williams and Wilkins. pp 1917–1974.

Fischer TK, Steinsland H, Molbak K, Ca R, Gentsch JR, Valentiner-Branth P, Aaby P, Sommerfelt H. 2000. Genotype profiles ofrotavirus strains from children in a suburban community inGuinea-Bissau, Western Africa. J Clin Microbiol 38:264–267.

Fischer TK, Page NA, Griffin DD, Eugen-Olsen J, Pedersen AG,Valentiner-Branth P, Molbak K, Sommerfelt H, Nielsen NM. 2003.Characterization of incompletely typed rotavirus strains fromGuinea-Bissau: Identification of G8 and G9 types and a highfrequency of mixed infections. Virology 311:125–133.

Flores J, Perez I, White L, Perez M, Kalica AR, Marquina R, Wyatt RG,Kapikian AZ, Chanock RM. 1982. Genetic relatedness amonghuman rotaviruses as determined by RNA hybridization. InfectImmun 37:648–655.

Fukai K, Sakai T, Hirose M, Itou Y. 1999. Prevalence of calf diarrheacaused by bovine group A rotavirus carrying G serotype 8specificity. Vet Microbiol 66:301–311.

Fukai K, Onoda H, Itou T, Sato M, Miura Y, Sakai T. 2004. Genetic andserological characterization of novel serotype G8 bovine groupA rotavirus strains isolated in Japan. J Vet Med Sci 66:1413–1416.

Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, DasBK, Bhan MK. 1992. Identification of group-A rotavirus gene-4types by polymerase chain-reaction. J Clin Microbiol 30:1365–1373.

Gentsch JR, Laird AR, Bielfelt B, Griffin DD, Banyai K, RamachandranM, Jain V, Cunliffe NA, Nakagomi O, Kirkwood CD, Fischer TK,Parashar UD, Bresee JS, Jiang B, Glass RI. 2005. Serotypediversity and reassortment between human and animal rotavirus

strains: Implications for rotavirus vaccine programs. J Infect Dis192:S146–S159.

Gerna G, Sarasini A, Zentilin L, Dimatteo A, Miranda P, Parea M,Battaglia M, Milanesi G. 1990. Isolation in Europe of 69M-like(serotype-8) human rotavirus strains with either subgroup-I orsubgroup-II specificity and a long RNA electropherotype. Arch Virol112:27–40.

Gerna G, Sears J, Hoshino Y, Steele AD, Nakagomi O, Sarasini A,Flores J. 1994. Identification of a new VP4 serotype of humanrotaviruses. Virology 200:66–71.

Ghosh S, Varghese V, Samajdar S, Bhattacharya SK, Kobayashi N,Naik TN. 2007. Evidence for independent segregation of the VP6-and NSP4-encoding genes in porcine group A rotavirus G6P[13]strains. Arch Virol 152:423–429.

Gorziglia M, Hoshino Y, Nishikawa K, Maloy WL, Jones RW, KapikianAZ, Chanock RM. 1988. Comparative sequence-analysis of thegenomic segment-6 of 4 rotaviruses each with a different subgroupspecificity. J Gen Virol 69:1659–1669.

Gouvea V, Glass RI, Woods P, Tanguchi K, Clark HF, Forrester B, FangZY. 1990. Polymerase chain-reaction amplification and typing ofrotavirus nucleic-acid from stool specimens. J Clin Microbiol28:276–282.

Greenberg H, McAuliffe V, Valdesuso J, Wyatt R, Flores J, Kalica A,Hoshino Y, Singh N. 1983. Serological analysis of the subgroupprotein of rotavirus, using monoclonal-antibodies. Infect Immun39:91–99.

Herring AJ, Inglis NF, Ojeh CK, Snodgrass DR, Menzies JD. 1982.Rapid diagnosis of rotavirus infection by direct detection of viralnucleic-acid in silver-stained polyacrylamide gels. J Clin Microbiol16:473–477.

Holmes JL, Kirkwood CD, Gerna G, Clemens JD, Rao MR, Naficy AB,Abu-Elyazeed R, Savarino SJ, Glass RI, Gentsch JR. 1999.Characterization of unusual G8 rotavirus strains isolated fromEgyptian children. Arch Virol 144:1381–1396.

Horie Y, Masamune O, Nakagomi O. 1997. Three major alleles ofrotavirus NSP4 proteins identified by sequence analysis. J GenVirol 78:2341–2346.

Hoshino Y, Saif LJ, Sereno MM, Chanock RM, Kapikian AZ. 1988.Infection immunity of piglets to either VP3 or VP7 outer capsidprotein confers resistance to challenge with a virulent rotavirusbearing the corresponding antigen. J Virol 62:744–748.

Iturriza-Gomara M, Isherwood B, Desselberger U, Gray J. 2001.Reassortment in vivo: Driving force for diversity of human rotavirusstrains isolated in the United Kingdom between 1995 and 1999. JVirol 75:3696–3705.

Iturriza Gomara M, Wong C, Blome S, Desselberger U, Gray J. 2002.Rotavirus subgroup characterisation by restriction endonucleasedigestion of a cDNA fragment of the VP6 gene. J Virol Methods105:99–103.

Iturriza-Gomara M, Anderton E, Kang G, Gallimore C, Phillips W,Desselberger U, Gray J. 2003. Evidence for genetic linkage betweenthe gene segments encoding NSP4 and VP6 proteins in common andreassortant human rotavirus strains. J Clin Microbiol 41:3566–3573.

Jagannath MR, Vethanayagam RR, Reddy BSY, Raman S, Rao CD.2000. Characterization of human symptomatic rotavirus isolatesMP409 and MP480 having ‘long’ RNA electropherotype andsubgroup I specificity, highly related to the P6[1],G8 typebovine rotavirus A5, from Mysore, India. Arch Virol 145:1339–1357.

Kang G, Green J, Gallimore CI, Brown DWG. 2002. Molecularepidemiology of rotaviral infection in South Indian children withacute diarrhea from 1995–1996 to 1998–1999. J Med Virol 67:101–105.

Kerin TK, Kane EM, Glass R, Gentsch JR. 2007. Characterization ofVP6 genes from rotavirus strains collected in the United Statesfrom 1996 to 2002. Virus Genes 35:489–495.

Khamrin P, Maneekarn N, Peerakome S, Chan-it W, Yagyu F, OkitsuS, Ushijima H. 2007. Novel porcine rotavirus of genotype P[27]shares new phylogenetic lineage with G2 porcine rotavirus strain.Virology 361:243–252.

Kimura M. 1980. A simple method for estimating evolutionary rates ofbase substitutions through comparative studies of nucleotide-sequences. J Mol Evol 16:111–120.

Kirkwood CD, Palombo EA. 1997. Genetic characterization ofthe rotavirus nonstructural protein, NSP4. Virology 236:258–265.

J. Med. Virol. DOI 10.1002/jmv

Characterization of Human Rotavirus G8 Strains 949

Kojima K, Taniguchi K, Kobayashi N. 1996. Species-specific andinterspecies relatedness of NSP1 sequences in human, porcine,bovine, feline, and equine rotavirus strains. Arch Virol 141:1–12.

Lin SL, Tian P. 2003. Detailed computational analysis of a compre-hensive set of group a rotavirus NSP4 proteins. Virus Genes26:271–282.

Lopez S, Espinosa R, Greenberg HB, Arias CF. 1994. Mappingthe subgroup epitopes of rotavirus proteinVP6. Virology 204:153–162.

Martella V, Ciarlet M, Banyai K, Lorusso E, Cavalli A, Corrente M, EliaG, Arista S, Camero M, Desario C, Decaro N, Lavazza A,Buonavoglia C. 2006. Identification of a novel VP4 genotype carriedby a serotype G5 porcine rotavirus strain. Virology 346:301–311.

Martella V, Ciarlet M, Banyai K, Lorusso E, Arista S, Lavazza A,Pezzotti G, Decaro N, Cavalli A, Lucente MS, Corrente M, Elia G,Camero M, Tempesta M, Buonavoglia C. 2007. Identification ofgroup a porcine rotavirus strains bearing a novel VP4 (P) genotypein Italian swine herds. J Clin Microbiol 45:577–580.

Masendycz PJ, Palombo EA. 2001. Genetic relatedness of VP1 genes ofAustralian and Taiwanese rotavirus isolates. FEMS Microbiol Lett198:147–150.

Matsui SM, Mackow ER, Matsuno S, Paul PS, Greenberg HB. 1990.Sequence-analysis of gene-11 equivalents from short and supershort strains of rotavirus. J Virol 64:120–124.

Matsuno S, Hasegawa A, Mukoyama A, Inouye S. 1985. A candidate fora new serotype of human rotavirus. J Virol 54:623–624.

Matthijnssens J, Rahman M, Martella V, Xuelei Y, De Vos S, De LeenerK, Ciarlet M, Buonavoglia C, Van Ranst M. 2006a. Full genomicanalysis of human rotavirus strain B4106 and lapine rotavirusstrain 30/96 provides evidence for interspecies transmission. J Virol80:3801–3810.

Matthijnssens J, Rahman M, Yang XL, Delbeke T, Arijs I, Kabue JP,Muyembe JJT, Van Ranst M. 2006b. G8 rotavirus strains isolated inthe Democratic Republic of Congo belong to the DS-1-likegenogroup. J Clin Microbiol 44:1801–1809.

Matthijnssens J, Ciarlet M, Heiman E, Arijs I, Delbeke T, McDonaldSM, Palombo EA, Iturriza-Gomara M, Maes P, Patton JT, RahmanM, Van Ranst M. 2008a. Full genome-based classification ofrotaviruses reveals a common origin between human Wa-like andporcine rotavirus strains and human DS-1-like and bovinerotavirus strains. J Virol 82:3204–3219.

Matthijnssens J, Ciarlet M, Rahman M, Attoui H, Banyai K, Estes MK,Gentsch JR, Iturriza-Gomara M, Kirkwood CD, Martella V,Mertens PPC, Nakagomi O, Patton JT, Ruggeri FM, Saif LJ,Santos N, Steyer A, Taniguchi K, Desselberger U, Van Ranst M.2008b. Recommendations for the classification of group A rota-viruses using all 11 genomic RNA segments. Arch Virol 153:1621–1629.

Maunula L, von Bonsdorff CH. 1998. Short sequences define geneticlineages: Phylogenetic analysis of group A rotaviruses based onpartial sequences of genome segments 4 and 9. J Gen Virol 79:321–332.

Maunula L, von Bonsdorff CH. 2002. Frequent reassortments mayexplain the genetic heterogeneity of rotaviruses: Analysis ofFinnish rotavirus strains. J Virol 76:11793–11800.

Mori Y, Borgan MA, Ito N, Sugiyama M, Minamoto N. 2002. Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, whichdiffer greatly from mammalian rotavirus NSP4 glycoproteins indeduced amino acid sequence, in suckling mice. J Virol 76:5829–5834.

Nakagomi O, Kaga E. 1995. Distinctness of NSP1 gene of humanrotavirus AU-1 from NSP1 gene of other human genogroups. ResVirol 146:423–428.

Nakagomi T, Nakagomi O. 1989. RNA-RNA hybridization identifies ahuman rotavirus that is genetically related to feline rotavirus.J Virol 63:1431–1434.

Nakagomi O, Nakagomi T. 1993. Interspecies transmission of rota-viruses studied from the perspective of genogroup. MicrobiolImmunol 37:337–348.

Nakagomi O, Nakagomi T. 1996. Molecular epidemiology of humanrotaviruses: Genogrouping by RNA-RNA hybridisation. Arch VirolSuppl 12:93–98.

Nakagomi T, Nakagomi O. 2000. Human rotavirus HCR3 possesses agenomic RNA constellation indistinguishable from that of felineand canine rotaviruses. Arch Virol 145:2403–2409.

Nakagomi O, Nakagomi T. 2002. Genomic relationships amongrotaviruses recovered from various animal species as revealed byRNA-RNA hybridization assays. Res Vet Sci 73:207–214.

Nakagomi O, Ohshima A, Aboudy Y, Shif I, Mochizuki M, Nakagomi T,Gotliebstematsky T. 1990. Molecular-identification by RNA-RNAhybridization of a human rotavirus that is closely related torotaviruses of feline and canine origin. J Clin Microbiol 28:1198–1203.

Nakata S, Gatheru Z, Ukae S, Adachi N, Kobayashi N, Honma S, MuliJ, Ogaja P, Nyangao J, Kiplagat E, Tukei PM, Chiba S. 1999.Epidemiological study of the G serotype distribution of group arotaviruses in Kenya from 1991 to 1994. J Med Virol 58:296–303.

Okada N, Matsumoto Y. 2002. Bovine rotavirus G and P types andsequence analysis of the VP7 gene of two G8 bovine rotaviruses fromJapan. Vet Microbiol 84:297–305.

Padilla-Noriega L, Wernereckert R, Mackow ER, Gorziglia M, LarraldeG, Taniguchi K, Greenberg HB. 1993. Serologic analysis of humanrotavirus serotypes p1a and p2 by using monoclonal-antibodies. JClin Microbiol 31:622–628.

Page RDM. 1996. TreeView: An application to display phylogenetictrees on personal computers. Comput Appl Biosci 12:357–358.

Palombo EA, Bugg HC, Masendycz PJ, Coulson BS, Barnes GL, BishopRF. 1996. Multiple-gene rotavirus reassortants responsible for anoutbreak of gastroenteritis in central and northern Australia. J GenVirol 77:1223–1227.

Palombo EA, Masendycz PJ, Bugg HC, Bogdanovic-Sakran N, BarnesGL, Bishop RF. 2000. Emergence of serotype G9 human rotavirusesin Australia. J Clin Microbiol 38:1305–1306.

Parashar UD, Gibson CJ, Bresee JS, Glass RI. 2006. Rotavirus andsevere childhood diarrhea. Emerg Infect Dis 12:304–306.

Qian Y, Green KY. 1991. Human rotavirus strain 69M has a uniqueVP4 as determined by amino-acid-sequence analysis. Virology182:407–412.

Rahman M, Matthijnssens J, Nahar S, Podder G, Sack DA, Azim T, VanRanst M. 2005. Characterization of a novel P[25],G11 human groupA rotavirus. J Clin Microbiol 43:3208–3212.

Rahman M, Matthijnssens J, Yang XL, Delbeke T, Arijs I, Taniguchi K,Iturriza-Gomara M, Iftekharuddin N, Azim T, Van Ranst M. 2007.Evolutionary history and global spread of the emerging G12 humanrotaviruses. J Virol 81:2382–2390.

Ramachandran M, Gentsch JR, Parashar UD, Jin S, Woods PA, HolmesJL, Kirkwood CD, Bishop RF, Greenberg HB, Urasawa S, Gerna G,Coulson BS, Taniguchi K, Bresee JS, Glass RI. 1998. Detection andcharacterization of novel rotavirus strains in the United States.J Clin Microbiol 36:3223–3229.

Ramig RF. 1997. Genetics of the rotaviruses. Annu Rev Microbiol51:225–255.

Ramig RF, Ward RL. 1991. Genomic segment reassortment inrotaviruses and other reoviridae. Adv Virus Res 39:163–207.

Rao CD, Das M, Ilango P, Lalwani R, Rao BS, Gowda K. 1995.Comparative nucleotide and amino-acid-sequence analysis of thesequence-specific RNA-binding rotavirus nonstructural proteinNSP3. Virology 207:327–333.

Santos N, Hoshino Y. 2005. Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementa-tion of an effective rotavirus vaccine. Rev Med Virol 15:29–56.

Santos N, Lima RCC, Pereira CFA, Gouvea V. 1998. Detection ofrotavirus types G8 and G10 among Brazilian children withdiarrhea. J Clin Microbiol 36:2727–2729.

Shah K, Kirkwood CD, Bhave M, Palombo EA. 2006. Genetic variationof NSP1 and NSP4 genes among serotype G9 rotaviruses causinghospitalization of children in Melbourne, Australia, 1997–2002. JMed Virol 78:1124–1130.

Steele AD, Parker SP, Peenze I, Pager CT, Taylor MB, Cubitt WD.1999. Comparative studies of human rotavirus serotype G8 strainsrecovered in South Africa and the United Kingdom. J Gen Virol80:3029–3034.

Steyer A, Poljsak-Prijatelj M, Barlic-Maganja D, Jamnikar U, MijovskiJZ, Marin J. 2007. Molecular characterization of a new porcinerotavirus P genotype found in an asymptomatic pig in Slovenia.Virology 359:275–282.

Strimmer K, vonHaeseler A. 1996. Quartet puzzling: A quartetmaximum-likelihood method for reconstructing tree topologies.Mol Biol Evol 13:964–969.

Subodh S, Bhan MK, Ray P. 2006. Genetic characterization of VP3gene of group A rotaviruses. Virus Genes 33:143–145.

J. Med. Virol. DOI 10.1002/jmv

950 Esona et al.

Tamura K, Nei M. 1993. Estimation of the number of nucleotidesubstitutions in the control region of mitochondrial-DNA inhumans and chimpanzees. Mol Biol Evol 10:512–526.

Taniguchi K, Urasawa T, Morita Y, Greenberg HB, UrasawaS. 1987. Direct serotyping of human rotavirus in stools by anenzyme-linked-immunosorbent-assay using serotype 1-specific, 2-specific, 3-specific, and 4-specific monoclonal-antibodies to VP7. JInfect Dis 155:1159–1166.

Varghese V, Das S, Singh NB, Kojima K, Bhattacharya SK,Krishnan T, Kobayashi N, Naik TN. 2004. Molecular characteriza-tion of a human rotavirus reveals porcine characteristics in most ofthe genes including VP6 and NSP4. Arch Virol 149:155–172.

Varghese V, Ghosh S, Das S, Bhattacharya SK, Krishnan T, KarmakarP, Kobayashi N, Naik TN. 2006. Characterization of VP1, VP2 andVP3 gene segments of a human rotavirus closely related to porcinestrains. Virus Genes 32:241–247.

Ward RL, Nakagomi O, Knowlton DR, McNeal MM, Nakagomi T,Clemens JD, Sack DA, Schiff GM. 1990. Evidence for naturalreassortants of human rotaviruses belonging to different gen-ogroups. J Virol 64:3219–3225.

Wyatt RG, James HD, Pittman AL, Hoshino Y, Greenberg HB, KalicaAR, Flores J, Kapikian AZ. 1983. Direct isolation in cell-culture ofhuman rotaviruses and their characterization into 4 serotypes. JClin Microbiol 18:310–317.

J. Med. Virol. DOI 10.1002/jmv

Characterization of Human Rotavirus G8 Strains 951