Simian Rotaviruses Possess Divergent Gene Constellations That

JOURNAL OF VIROLOGY, Feb. 2010, p. 2013–2026 Vol. 84, No. 40022-538X/10/$12.00 doi:10.1128/JVI.02081-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Simian Rotaviruses Possess Divergent Gene Constellations ThatOriginated from Interspecies Transmission and Reassortment�

Jelle Matthijnssens,1 Zenobia F. Taraporewala,2† Hongyan Yang,2‡ Shujing Rao,3 Lijuan Yuan,4Dianjun Cao,2,4 Yasutaka Hoshino,2 Peter P. C. Mertens,5 Gerry R. Carner,3 Monica McNeal,6

Karol Sestak,7 Marc Van Ranst,1 and John T. Patton2*Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for

Medical Research, University of Leuven, Leuven, Belgium1; Laboratory of Infectious Diseases, National Institute of Allergy andInfectious Diseases, National Institutes of Health, Bethesda, Maryland 208922; Clemson University, Department of Entomology,

South Carolina 296343; Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine andInfectious Diseases, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240614;

Department of Arbovirology, Institute for Animal Health, Ash Road, Pirbright, Woking,Surrey GU24 0NF, United Kingdom5; Division of Infectious Diseases,

Cincinnati Children’s Hospital Medical Center, Cincinnati,Ohio 452296; and Tulane National Primate Research Center and

Tulane University School of Medicine,New Orleans, Louisiana 701187

Received 1 October 2009/Accepted 13 November 2009

Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basicresearch and vaccine development. To explore the origins of simian RVs, the complete genome sequences ofstrains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed thegenotype constellations of each virus to be determined and the phylogenetic relationships of the simian strainswith each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely trans-mitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes havegenotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes ofrhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, thesequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related tothose of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these virusesfrom a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear tohave originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV,RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, ourdata indicate that simian RVs are of diverse ancestry with genome constellations that originated largely byinterspecies transmission and reassortment with nonhuman animal RVs.

Group A rotaviruses (RVs) are a major cause of acute dehy-drating diarrhea in infants and children under the age of 5 yearsworldwide. These infections lead to approximately 527,000 deathseach year, the vast majority occurring in developing countries(33). RVs are also responsible for gastroenteritis in many otheranimal species, notably mammals and birds (16, 38). RVs aremembers of the family Reoviridae and possess a genome consist-ing of 11 segments of double-stranded RNA (dsRNA). The pro-totypic genome of a group A RV encodes six structural proteins(VP) and six nonstructural proteins (NSP) (5). The mature RVvirion is a nonenveloped triple-layered icosahedral particle. Theinner most protein layer is formed by the core lattice protein VP2.

Attached to the interior surface of the VP2 layer near the fivefoldaxes are complexes of the viral RNA-dependent RNA poly-merase VP1 and the RNA capping enzyme VP3. Collectively,VP1, VP2, VP3, and the dsRNA genome form the core of thevirion (5, 11). The core is surrounded by VP6, the sole constituentof the intermediate protein layer of the virion. The antigenicproperties of VP6 are used in classifying RV isolates into groups.The outer protein layer of the virion is composed of trimers of theVP7 glycoprotein penetrated by spikes of the VP4 attachmentprotein (50). The properties of VP7 and VP4 form the basis of adual classification system defining RV G types (glycosylated) andP types (protease sensitive), respectively. At present, 23 Ggenotypes and 31 P genotypes have been recognized in theliterature based on sequence analyses (17, 39, 42, 45, 47).Recently, a comprehensive sequence-based classification sys-tem was established for the RVs which, together with a uni-form nomenclature, allows each genome segment of the virusto be assigned to a particular genotype. In the comprehensiveclassification system, the acronym Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx defines the genotypes of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 encoding genome segments(17, 18).

* Corresponding author. Mailing address: Laboratory of InfectiousDiseases, NIAID, NIH, 50 South Drive, Room 6314, Bethesda, MD20892-8026. Phone: (301) 594-1615. Fax: (301) 496-8312. E-mail:[email protected].

† Present address: Center for Biologics Evaluation and Research, Foodand Drug Administration, 1401 Rockville Pike, 200N, Rockville, MD20852.

‡ Present address: National Eye Institute, National Institutes ofHealth, 6 Center Dr., MSC 0610, Bethesda, MD 20852.

� Published ahead of print on 25 November 2009.

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Several years ago, Nakagomi et al. provided evidence byRNA-RNA hybridization assays that RVs originating from dif-ferent animal species could be resolved into genogroups basedupon the existence of unique species-specific genome constel-lations (29–31). More recently, the concept that RVs prefer-entially retain certain species-related genome constellationshas been further supported by whole-genome sequencing (8,24). For human RVs, two major genogroups (Wa-like geno-group 1 and DS-1-like genogroup 2) and one minor genogroup(AU-1-like genogroup 3) have been described (8, 17, 30). Al-though these genogroups are generally species specific, it isbelieved that the human AU-1 genogroup is of feline origin(31) and that the human Wa and DS-1 genogroups sharecommon ancestor with porcine and bovine RVs, respectively(17). Another recent study based on full genome sequencedata has indicated that the rarely seen human G3P[3] RVs areof feline or canine origin (46). Two additional sequence-basedstudies have indicated that human RVs with P[14] specificitymay have originated after interspecies transmission from rab-bit RVs and RVs from hosts belonging to the order Artiodac-tyla (i.e., hoofed mammals with even toes, including ruminantsand pigs) (19, 20). These examples indicate that interspeciestransmission of entire RV gene constellations from one hostspecies to another may contribute significantly to viral evolu-tion. In addition to interspecies transmission, complete ge-nome sequencing of RVs have revealed multiple examples ofnaturally occurring inter- and intragenogroup reassortment(17, 19, 21–23, 37, 41).

The simian RV strains, notably RRV and the SA11 deriva-tives (e.g., SA11-Cl3 and SA11-4F), have been used extensivelyas models in the study of all aspects of RV biology, includingcharacterizing genome replication and virion assembly, delin-eating high-resolution structures of viral proteins and thevirion capsid, and describing the functions of viral proteins.Moreover, the RRV strain was used to create a set of human-simian reassortant viruses that formed the basis of the firstcommercially licensed RV vaccine (Rotashield; Wyeth Labo-ratories) (10). Serological analyses have indicated that simianRVs are probably endemic in wild nonhuman primate (NHP)species in Africa (32). However, whether or not unique geno-groups or preferred genome constellation exist for the simianRVs has not been determined, because of the lack of compre-hensive genetic data. Most simian RVs isolated to date (e.g.,rhesus macaque viruses RRV [43] and TUCH [25] and thepig-tailed macaque virus PTRV [9]) have been recovered frommonkeys kept in captivity in the United States. An importantexception is the SA11 isolate, which was recovered from avervet monkey in South Africa (15). Simian RV infectionsoccur mostly in young monkeys, similar to human RV infec-tions in children (32, 40).

To gain further insight into the origins and properties ofsimian RVs, we sequenced and contrasted the genomes ofPTRV, RRV, and TUCH with other RVs, including SA11-H96(G3P[2]), the only previously fully sequenced simian RV (41).Our results reveal that these four simian RVs are of divergentancestry and have evolved by combinations of interspeciestransmission and reassortment with RVs naturally occurring inother animal species. Thus, the simian RVs do not possess acommon genome constellation nor define a unique genogroup.Although frequently used as disease models, the simian RVs

show limited genetic similarity with the human RVs (geno-groups 1 and 2) responsible for most human disease.

MATERIALS AND METHODS

Cells and viruses. Monkey kidney (MA104) cells were propagated in 199medium containing 10% fetal bovine serum. The cell culture-adapted RV strainsPTRV (G8P[1]), RRV (MMU18006; G3P[3]), and TUCH (G3P[24]) were ac-tivated by treatment with 10 �g of trypsin (T0303-1G; Sigma-Aldrich)/ml andgrown in MA104 cells infected with �1 PFU of virus. Infected cells were main-tained in serum-free 199 medium until the cytopathic effects were complete.

Sequencing of the complete genome of PTRV. Genomic dsRNA was extractedfrom PTRV using TRIzol (Invitrogen). The dsRNA of PTRV was converted intocDNA using the FLAC (full-length amplification of cDNA) method (14). Briefly,anchor primers were ligated onto the ends of the dsRNA using T4 RNA ligase.Afterward, the dsRNA segments were resolved by electrophoresis on a 1%agarose gel, and each was recovered by using a QIAquick gel extraction kit(Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reversetranscriptase (New England Biolabs) and then PCR amplified with ExTaq DNApolymerase (Takara). The cDNAs were cloned into the pGEM-T Easy vector(Promega).

Sequencing of the open reading frames of the RRV and TUCH genomes.Genomic dsRNA was recovered from infected cell lysates using TRIzol-LS(Invitrogen) and denatured by incubation with 50% dimethyl sulfoxide. TheRNA was used as a template for the preparation of viral cDNAs using a OneStepRT-PCR kit (Invitrogen) and appropriate segment-specific primers (availableupon request). Reverse transcription-PCR products were gel purified and se-quenced with an ABI Prism BigDye v3.1 terminator cycle sequencing kit anddetected with an Applied Biosystems 3730 DNA analyzer. In a few cases, theterminal segment-specific primers used in preparing RRV cDNAs extended intothe open reading frame, preventing the de novo identification of some N- andC-terminal residues of proteins. The numbers of masked terminal residues are asfollows: VP1 (two N-terminal residues: two C-terminal residues [2:2]), VP2 (4:0),VP4 (7:1), VP6 (2:0), NSP3 (2:0), and NSP5 (3:0).

The cDNAs of PTRV, RRV, and TUCH were sequenced with an ABI PrismBigDye v3.1 terminator cycle sequencing kit (Applied Biosystems Group). Thedye terminator was removed by using Performa DTR cartridges (Edge Biosys-tems), and sequences were determined with an Applied Biosystems 3730 DNAanalyzer. Sequence files were analyzed and assembled by using Sequencher 4.5software (Genes Code Corp).

Phylogenetic and structural analyses. Phylogenetic and molecular evolution-ary analyses were conducted by using the MEGA software (version 4) (44).Genetic distances were calculated by using the Kimura-2 correction parameter atthe nucleotide level, and phylogenetic trees were constructed by using the neigh-bor-joining method with 500 bootstrap replicates. VP7 and VP8* images weregenerated by using the UCSF Chimera-Molecular Modeling System (35).

Assignment of newly identified genotypes. The genotypes of the genome seg-ments for PTRV, RRV, and TUCH were assigned based on the recommenda-tions of the Rotavirus Classification Working Group (RCWG) (18), using theonline RotaC genotyping tool (http://rotac.regatools.be/).

Sequence submission. The nucleotide sequences of the VP1, VP2, VP3, VP4,NSP1, VP6, NSP3, NSP2, VP7, NSP4, and NSP5 genes of RV strains RRV,TUCH, and PTRV are available in GenBank under the accession numbersEU636924 to EU636934, EF583010 to EF583013 and FJ816611 to FJ816617, andFJ422131 to FJ422141, respectively.

RESULTS

PTRV is a bovine-like virus. PTRV was recovered from astool sample of a young female pig-tailed macaque with diar-rhea housed at the Medical Lake breeding facility of the Na-tional Primate Center, University of Washington, Seattle, in1990. Previous analysis established that PTRV (G8P[1]) has a“long” electropherotype, a subgroup I VP6, and a genotype ANSP4 (9), all characteristics typical of bovine RVs. Likewise,RNA-RNA hybridization assays have indicated that PTVRand bovine RVs are closely related, since eight to nine of theirRNA segments formed heteroduplexes with electrophoreticmigration patterns similar to those of homoduplexes (9).

Complete genome sequencing identified the following geno-

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type constellation for PTRV: G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. This constellation is typical of P[1] bovine RVs,suggesting that PTRV may have originated by interspeciestransmission from a bovine to a simian host (Table 1). Phylo-genetic analyses confirmed that all 11 gene segments of PTRVcluster with those of bovine RVs (Fig. 1). Interestingly, manyof the PTRV gene segments (VP1, VP2, VP3, VP6, VP7,NSP1, NSP3, and NSP5) also cluster with several human P[14]and nonbovine animal RV strains: 111/27-05, B10925-97,Hun5, MG6, PA169 (human), RC/18-08 (sable antelope),Chubut (lama guanaco), and OVR762 (ovine) (Fig. 1). Thisclustering pattern is not surprising, since it was recently shownthat these P[14] human and animal strains share a largelyconserved consensus genotype constellation with P[1] bovineRVs (Table 1) (19). Thus, although PTRV may have origi-nated from interspecies transmission of a bovine RV to apig-tailed macaque, it is possible that RVs infecting othermembers of the Bovidae family or the Artiodactyla order mayhave been the source of PTRV.

To determine whether PTRV exhibits similarities to bovineRVs at the protein level, we contrasted the surface-exposedresidues of their outer capsid glycoprotein VP7 and spike pro-tein VP4. Such analysis was made possible by the recent de-scription of the atomic structure for the VP7 trimer (1) and theearlier description of the atomic structure for the VP8* glob-ular head of the VP4 spike protein (4). The location of struc-ture-based antigenic domains on the VP7 trimer (7-1a, 7-1b,and 7-2) (Fig. 2 and 3) and the VP8* monomer (8-1, 8-2, 8-3,and 8-4) (Fig. 3 and 4) were predicted based on the charac-terization of antibody-escape mutants and the identification of

serotype-specific amino acid changes. The VP7 antigenic do-mains 7-1a, 7-1b, and 7-2 include the previously describedantigenic regions A (residues 87 to 101) and D (residue 291);C (residues 208 to 221), E (residues 189 to 190) and F (residues233 to 242); and B (residues 142 to 152), respectively (36).Comparison of PTRV VP7 with those of the G8 bovine strainsTokushima 9503 (P[11]) and O-agent (P[1]), and the ovinestrain OVR762 (P[14]) identified a single difference (A87V) ofa surface-exposed residue (Fig. 3C). PTRV VP7 differs fromthat of the G8 human strain 69M (P[10]) by one additionalresidue (T171A). Only the A87V change maps definitely withinan antigenic domain (7-1a). The location and limited numberof changes, combined with their conservative nature, suggeststhat the antigenicity of PTRV VP7 is identical to, or nearly so,with that of several G8 bovine RVs. This hypothesis is sup-ported by the results of plaque-reduction assays which haveshown that antiserum raised against PTRV (G8P[1]) displaysa high level of neutralizing activity to 69M (G8P[10]) (9). Asmight be anticipated from these data, the amino acid iden-tity of PTRV VP7 with the VP7 proteins of Tokushima 9503,O-agent, OVR762, and 69M is extremely high (�98%) (Ta-ble 2).

Analysis of PTRV VP8* showed that it included only threesurface-exposed residues (D111N, P157S, and H202Y) differ-ing from VP8* of the P[1] bovine O-agent strain (G8) (Fig.5C). Likewise, these were the only differences noted betweenPRTV VP8* and VP8* of SA11-5N, a reassortant virus with abovine VP4 gene. In addition to the D111N, P157S, andH202Y replacements, VP8* of the P[1] bovine strain NCDV(G6) contains one additional difference (N185T). Only the

TABLE 1. Genotypes of simian RVs and related virus strains

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FIG. 1. Phylogenetic neighbor-joining trees based on the open reading frame sequences of the RV genes for VP1, VP2, VP3, and VP4 (A);VP6 and VP7 (B); and NSP1, NSP2, NSP3, NSP4, and NSP5 (C). Bootstrap values (500 replicates) above 50 are shown. Sequences used in buildingthe trees were downloaded from GenBank; sequence alignments will be provided on request. Only certain genotypes are shown in detail; othersare represented only by their genotype number. The annotation defines species of origin/strain name. Simian RV strains are in boldface. Thephylogenetic cluster comprising simian and feline/canine/human strains is boxed in pale blue. The cluster comprising PTRV and the Bovidae/artiodactyl RV strains is boxed in pale yellow. Any simian RV not already boxed, is highlighted in green. The SA11-H96 strain is surrounded bya yellow oval.

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D111N change is located within a defined antigenic domain(8-3); thus the antigenicity of the PTRV VP8* protein can beexpected to closely mirror that of several P[1] bovine RVs. Thisprediction is in agreement with the results of plaque reduction

assays which have shown that antiserum raised against PTRV(G8P[1]) exhibits a high level of neutralizing activity to NCDV(G6P[1]) (9). The results are also consistent with analysesshowing that the amino acid identity of PTRV VP8* is greater

FIG. 1—Continued.

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FIG. 1—Continued.

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FIG. 2. Amino acid alignment of the VP7 protein of simian and related RV strains. Antigenic regions A to F are identified by heavy lines abovesequences. Antigenic domains 7-1a (red), 7-1b (orange), and 7-2 (purple) are highlighted by color shading. Assignment of specific residues toantigenic domains was based on sequencing of neutralization escape mutants (triangles) and analysis of G-type specific sequence variations(squares) (1, 24). Surface-exposed residues identified in Fig. 3 that differ between certain virus strains are colored cyan.

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than 96% with the VP8* proteins of O-agent, SA11-5N, andNCDV (Table 3).

Along with antigenic domains, the VP8* proteins of mostanimal RVs and a few human strains contain a sialic acid-binding site that facilitates virus attachment to the host cell (4).Amino acid residues that form the sialic-acid binding site ofPTRV VP8* are identical to those of many P[1] bovine RVsbut differ from those of other simian RVs (Table 4). SincePTRV grows to high titers in macaques and spreads efficiently

within macaque colonies, the sialic acid binding site character-istic of P[1] bovine viruses can be presumed to successfullyrecognize carbohydrates displayed on macaque enterocytes(13). Collectively, analysis of the PTRV genome sequence andstructural antigenic domains suggests that PTRV originated byinterspecies transmission of a G8P[1] bovine virus into amacaque.

RRV and TUCH. The RRV (MMU18006) strain was iso-lated in 1975 from a juvenile rhesus macaque with diarrhea

FIG. 3. Structure-based analysis of the antigenic domains of the VP7 trimer of simian RVs. (A) Cryoelectron microscopy image reconstructionof the SA11 RV virion, taken with permission from Pesavento et al. (34). The multimeric VP4 attachment protein and VP7 trimer are identified.(B) Color code for the VP7 antigenic domains and residues differing among VP7 proteins in panel C. Sequence alignments identifying antigenicdomains and amino acid differences are shown in Fig. 2. (C) Surface renderings of the RRV VP7 trimer (PDB 3FMG) from the perspective ofthe virion exterior, with residues color-coded to reveal antigenic domains. Colored in cyan are the approximate positions of the surface-exposedresidues of the PTRV (G8) VP7 trimer that differ from those of the G8 VP7 trimers of O-agent (O), OVR762 (OVR), Tokushima 9503 (Toku),and 69M and the surface-exposed residues of the RRV (G3) VP7 trimer that differ from those of the G3 VP7 trimers of SA11-H96, TUCH, K9,RO1845 (RO), A79-10 (A79), CU-1 (CU), and Cat97 (CAT). Only residues in one of the trimers have been labeled. VP7 GenBank accessionnumbers: O-agent, DQ838596; OVR762, EF554153; 69M, EF672560; K9, EU708928; RO1845, EU708895; A79-10, EU708939; CU-1, EU708917;Cat97, EU708950.

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that was born in the California National Primate ResearchCenter (43). The TUCH strain was isolated in 2002 from anasymptomatic juvenile rhesus macaque housed at the TulaneNational Primate Research Center in Louisiana (25). Previousstudies have established that RRV (G3P[3]) and TUCH(G3P[24]) have “long” electropherotypes, subgroup I VP6s,and genotype C NSP4s (6, 12). Complete genome sequencingidentified the genotype constellations for RRV (G3-P[3]-I2-R2-C3-M3-A9-N2-T3-E3-H6) and for TUCH (G3-P[24]-I9-R3-C3-M3-A9-N1-T3-E3-H6). Thus, seven genes of RRV andTUCH share common genotypes (Table 1). Comparison of theRRV and TUCH genotype constellations with other com-pletely sequenced RV genomes revealed an unexpected find-

ing. As shown in Table 1, RRV and TUCH share eight andseven genotypes, respectively, with several G3P[3] strains ofcanine (A79-10, CU-1, and K9), feline (Cat97), and humancanine/feline-like (HCR3A, RO1845) RVs. In addition, twoother human strains, which are believed to be of feline origin,AU-1 (G3P[9]) and T152 (G12P[9]), also share five to sixgenotypes with RRV and TUCH (Table 1). These data suggestthat RRV and TUCH are related to one another and have ashared ancestry with canine/feline RVs. Moreover, these datareveal that overall RRV and TUCH are more closely related tothe human AU-l/feline-like RVs than to human Wa- or DS-1-like RVs.

To probe the origins of RRV and TUCH in greater detail,

FIG. 4. Amino acid alignment of the VP8* protein of simian and related RV strains. Antigenic domains 8-1 (red), 8-2 (orange), 8-3 (purple),and 8-4 (green) are highlighted by color shading. Assignment of specific residues to antigenic domains was based on sequencing of neutralizationescape mutants (triangles) and analysis of P-type specific sequence variations (squares) (24). Surface-exposed residues identified in Fig. 5 that differbetween certain virus strains are colored cyan.

TABLE 2. Identity and similarity matrix of the VP7 amino acid residues of select RVsa

a Serotype G3 (orange) and G8 (blue) RVs are color coded; UK and RF are G6 RVs. Yellow shading identifies a simian RV with a VP7 protein that has �89%sequence identity with the VP7 protein of another virus. CLUSTALW (v1.83) multiple sequence alignment and the identity and similarity matrix were generated usingMacVector 10.6.

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FIG. 5. Comparative analyses of the VP8* core of the RV VP4 protein. (A) Alpha-carbon traces of RRV VP8*-core monomers (white) anda VP5�-core dimer (yellow) core fit into a density map of a VP4 dimer as determined by cryoelectron-microscopy image reconstruction. The imagewas taken with permission from Monnier et al. (26). (B) Color code for the VP8* antigenic domains and residues differing among VP8* proteinsin panel C. Sequence alignments identifying antigenic domains and amino acid differences are shown in Fig. 4. (C) Surface renderings of front (left)and back (right) orientations of the RRV VP8*-core monomer (PDB 1KQR) (4), color coded to reveal antigenic domains. The front orientationapproximates that of the leftward VP8* globular head in panel A. The sialic acid binding site of the front orientation is defined by the yellowelemental stick model of a sialoside. Colored in cyan are the approximate positions of the surface-exposed residues of PTRV (P[1]) VP8* that differfrom those of the P[1] VP8* proteins of O-agent, SA11-5N, and NCDV, and the surface-exposed residues of RRV (P[3]) that differ from thoseof the P[3] VP8* proteins of GRV and K9. Residues that differ have been labeled. VP4 GenBank accession numbers: O-agent, DQ838596;SA11-5N, DQ838602; NCDV, AB119636; GRV, AB055967; K9, EU708926.

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phylogenetic dendrograms were constructed for each of the 11viral gene segments (Fig. 1A to C). The analysis showed thatthe VP3 (M3), VP7 (G3), NSP1 (A9), NSP3 (T3), NSP4 (E3),and NSP5 (H6) genes of RRV and TUCH clustered, albeitonly distantly, within the same genotype as canine, feline, andcanine/feline human RVs. The dendrograms also showed thatthe RRV VP4 (P[3]) and TUCH VP1 (R3) genes clustered,again distantly, within the same genotype as the canine/feline/human RVs. For the VP2 gene, both RRV and TUCH, to-gether with AU-1 and T152, clustered within the C3 genotype,whereas the canine/feline/human RVs were contained withinthe M2 genotype, suggesting the possible reassortment of anRRV/TUCH ancestor with a human DS-1-like (genogroup 2)RV (Fig. 1A). Other genes of RRV and TUCH that are not ofthe canine/feline/human genotype may have also originated byreassortment. These include the VP4 (P[24]), VP6 (I9), andNSP2 (N1) genes of TUCH, which have genotypes unrelatedto canine/feline/human RVs (Fig. 1). In particular, the NSP2gene of TUCH is most closely related to the Wa-like strainIAL28 (92% nucleotide identity), suggesting reassortment witha human Wa-like (genogroup 1) virus. On the other hand, theonly representatives of the P[24] and I9 genotypes are the VP4and VP6 genes, respectively, of the TUCH virus, rendering theorigins of these genes obscure. RRV was found to possess VP1(R2), VP6 (I2), and NSP2 (N2) genes with genotypes charac-teristic of RVs infecting Bovidae or related hosts. Thus, somegenes of RRV may have originated by reassortment betweenan ancestor and a bovine or other ruminant RV.

SA11. The RV strain SA11 was initially isolated in SouthAfrica in 1958 from a healthy vervet monkey and, after adap-tation to cell culture, this and derivative reassortant strainshave been used extensively as prototypes in the study of RVbiology. SA11, like RRV and TUCH, was determined by se-rotyping to represent a G3 virus (25). A previous comparisonof the complete genomes of several SA11 laboratory isolatesshowed that some represented reassortants formed in vitro bythe introduction of a bovine VP4 (SA11-4F) or NSP2 (SA11-both) gene into the SA11 genetic background (41). SA11-H96,the strain believed to most closely resemble the original SA11isolate, has a genotype constellation of G3-P[2]-I2-R2-C5-M5-A5-N5-T5-E2-H5. Phylogenetic analyses indicated that for sev-

eral genes [VP2 (C5), VP3 (M5), VP4 (P[2]), NSP1 (A5),NSP2 (N5), NSP3 (T5), and NSP5 (H5)], SA11-H96 is the onlyrepresentative of its genotype (Fig. 1 and Table 1). For theremaining genes (VP1, VP6, VP7, and NSP4), SA11-H96forms a distinct subcluster inside the R2, I2, G3, and E2 ge-notypes, respectively (Fig. 1). These findings indicate thatSA11-H96 is not closely related genetically to any other knownRV, including those that are of simian or human origin.

RRV, TUCH, and SA11-H96 VP7 and VP8* proteins. Geno-typing and phylogenetic analysis indicate that the RRV andTUCH strains are related to one another and have originatedindependently of the SA11-H96 strain. Thus, it was surprisingto find that the exterior surface-exposed residues of the RRVVP7 show greater similarity to the corresponding residues ofSA11-H96 VP7 than TUCH VP7. Specifically, only three sur-face-exposed residues (D123N, T212A, and A213T) differ be-tween the RRV and SA11-H96 VP7 proteins, while sevenresidues differ between the RRV and TUCH VP7 proteins(Fig. 3). This parallels data showing that the amino acid iden-tity of RRV VP7 with SA11-H96 VP7 is greater than that withTUCH VP7 (96 and 91%, respectively) (Table 2). Analyses ofsurface-exposed residues and amino acid identities also showthat RRV VP7 is more similar to the VP7 proteins of theG3P[3] canine/feline/human RVs (e.g., Cat97, K9, andRO1845) than to TUCH VP7 (Fig. 3C and Table 2). Notably,residues that differ between RRV VP7 and the VP7 proteins ofthe G3P[3] canine/feline/human RVs exclude the 7-1a domainand only one or two residues at the periphery of the 7-1adomains differ between the RRV and SA11-H96 VP7 proteins(D123N) and the RRV and TUCH VP7 proteins (D123N andS126T) (Fig. 3C). The conserved nature of the 7-1a domainsuggests that it is chiefly responsible for the antigenic proper-ties that define the G3 serotype. Indeed, the 7-1A domaincontains the antigenic epitope for the VP7-specific monoclonalantibody (MAb 159) that is used as a reference standard inidentifying G3 RVs. The limited number of differences be-tween the RRV and SA11-H96 VP7 proteins suggests thattheir antigenicity should be quite similar, which has been dem-onstrated previously (43). Whether the increased number ofdifferences between the G3 VP7 proteins of RRV and TUCH

TABLE 3. Identity and similarity matrix of the VP8* amino acid residues of select RVsa

a Genotype P1 (orange) and P�3� (blue) RVs have been color coded; SA11-H96 and TUCH are P�2� and P�24� viruses, respectively. Yellow shading identifies a simianRV with a VP8* protein that has � 89% sequence identity with the VP8* protein of another virus. CLUSTALW (v1.83) multiple sequence alignment and the identityand similarity matrix were generated using MacVector 10.6.

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measurably decreases the production of cross-reacting neutral-izing antibodies in infected animals is unclear.

Few P[2] and P[24] RVs have been identified, ruling out acomparative analysis of their VP4 proteins. On the other hand,several P[3] RVs have been described, most of which, like theRRV strain, are G3 viruses that have been isolated from feline,canine, or human hosts (Table 1). Sequence alignments revealthat the RRV P[3] VP4 protein is most closely related to theP[3] VP4 protein of the goat RV isolate GRV (Fig. 4 and datanot shown). However, these proteins are somewhat evolution-arily distant, as illustrated by the fact that their VP8* compo-nents differ in 13 surface-exposed residues (Fig. 5C) and havean amino acid identity of 92% (Table 3). The number ofsurface-exposed residues that differ increases to more than 26when RRV VP8* is compared to the VP8* proteins to someother G3P[3] viruses (e.g., K9, Cat97, HCR3A, A79-10, CU-1,and RO1845) (Table 1, Fig. 5C, and data not shown). Parallelwith this increase, the amino acid identity of RRV VP8* withVP8* proteins of other G3P[3] viruses falls to below 87%(Table 3). Thus, although RRV and several other viruses (e.g.,GRV, K9, Cat97, HCR3A, A79-10, CU-1, and RO1845) havebeen assigned P[3] genotypes based on their nucleotide se-quences, the VP8* proteins of these viruses show significantvariation in amino acid sequence. Since some of the variationmaps to the antigenic domains of VP8*, it is possible that P[3]viruses may display antigenic differences that influence thespecificities of neutralization antibodies formed in infectedanimals (Fig. 4 and 5C).

Interestingly, the amino acid composition of the VP8* sialicacid binding sites varies markedly among G3P[3] RVs, withthose of the caprine GRV and human CMH22 strains exhib-iting greatest similarity to the RRV sialic acid binding site(Table 4). Unexpectedly, the GRV and CMH22 sialic acidbinding sites were found to be identical to that of the TUCHP[24] VP8* and thus are even more closely related than thesialic acid binding sites of GRV and CMH222 are with theTUCH site. The GRV and CMH222 sialic acid binding sites

also show notable similarity with that of SA11-H96. The sig-nificance of the amino acid variation observed for the sialicacid binding is not clear but may be a determinant affectinghost range and cell tropism. However, our analysis establishesthat based on amino acid composition, there is no single typeof sialic acid binding site for simian RVs.

DISCUSSION

A principal goal in the isolation and characterization ofNHPs RVs has been the desire to identify surrogates of humanRVs that in large animal models will mimic the growth, pa-thology, and immune responses that are characteristic of hu-man infections. To date, only five simian RVs have been de-scribed (PTRV, RRV, SA11-H96, TUCH, and YK-1); all havebeen used to one extent or another to probe the biology of thevirus. Of these viruses, only the SA11-H96 isolate has beenfully sequenced (41). To further understand the genetic originand relatedness of the simian RVs to each other and to humanand other animal RVs, the complete genomes of three addi-tional strains—PTRV, RRV, and TUCH—were determinedand phylogenetically analyzed.

Analyses of PTRV, isolated from a colony of pig-tailed ma-caques at the University of Washington, revealed that its ge-nome was entirely of bovine/artiodactyl origin. Serologicalanalyses showed that this virus was endemic in the colony fromat least 1987 until 1994 (9). This implies that an ancestor ofPTRV crossed the host species barrier successfully and wasable to spread and persist in the new NHP host without un-dergoing reassortment. The successful introduction of thePTRV ancestor into the simian host illustrates once again thatthe species barrier for RVs is far from absolute. Notably, anearlier analysis of human RVs belonging genogroup 2 (DS-1-like) suggests that these viruses have a common origin withBovidae RVs. In addition, the unusual human G6P[14] strainsare suspected of originating from a virus infecting the Bovidaeor other member of the Artiodactyla (2, 17, 19).

TABLE 4. Residues comprising the VP8* sialic acid binding site of simian and related RVs

a Residue numbers are based on the sequence of RRV VP4. VP4 accession numbers: BRV033, U62155; C486, Y00127; NCDV, AB119636;O-agent, DQ838596; RF, U65924; Arg/Rio/Negro, FJ347125; OVR762, EF554151; RC/18-08, FJ495129; GRV, AB055967; CMH222,DQ288661; K9, EU708926; HCR3A, EU708904.

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RRV was one of the first animal RVs used in the develop-ment of human vaccine candidates (48). By generating reas-sortants with human RVs strains, a tetravalent RRV-based(serotype G1, G2, G3, and G4) vaccine (Rotashield; WyethLaboratories) was produced that was licensed and commer-cially used in infants and young children in the United States in1998 to 1999 (7). Unfortunately, the vaccine was withdrawnfrom the market due to an association with an increased risk ofintussusception in vaccinees (3, 27, 28). Our genomic analysessuggest that RRV may originate from the reassortment of afeline/canine ancestral RV with a Bovidae RV. The genes ofRRV encoding VP1 (genotype R2), VP6 (genotype I2), andNSP2 (genotype N2) are closely related to those of BovidaeRVs (Fig. 1A and C); thus, these genes were probably intro-duced into the RRV genome by reassortment relatively shortlybefore the isolation of the virus in 1975. However, the genes ofRRV and the feline/canine RVs that share the same geno-types—VP3 (M3), VP4 (P[3]), VP7 (G3), NSP1 (A9), NSP3(T3), NSP4 (E3), and NSP5 (H6)—are phylogenetically ratherdistantly related. Thus, the interspecies transmission of RRVfrom a feline or canine host to a NHP was most likely not arecent event.

The TUCH strain has been characterized in an attempt todevelop a reliable NHP model for studies on RV protection.Pathogen-free macaques, which were challenged with TUCH,remained clinically normal, but shed large quantities of RVantigen in their feces, which resolved by the end of a 14-dayobservation (25). This model has been used to study extra-intestinal spread of RVs, and the CD4� and CD8� lymphocyteresponses of macaques after RV challenge (40, 51). Genomicsanalysis surprisingly revealed that the majority of the genes (7of 11) of the TUCH strain are shared with feline/canine RVsand RRV (Table 1 and Fig. 1). These data suggest that TUCHis also a descendant of the hypothetical ancestor from whichthe feline/canine and RRV strains derive. Most likely, one ormore reassortment events have taken place starting from thehypothetical ancestor, replacing the NSP2, VP4, and VP6 genesegments, yielding the TUCH strain. The NSP2 gene segmentof TUCH (genotype N1) may have originated from a Wa-likehuman RV, whereas the origin of its VP4 (P[24]) and VP6 (I9)remains unknown. As is the case for RRV, the phylogeneticrelationships between TUCH on one hand, and RRV and thefeline/canine RRV strains on the other hand, is rather distant,suggesting that the interspecies transmission from a feline/canine host to a simian host must have happened a long timeago. Indeed, this long time period would explain why so manyamino acid differences are noted between the VP7 proteins ofRRV and TUCH. Interestingly, RRV can cause disease inmacaques, whereas TUCH cannot, raising the possibility thatone of the unique genes (e.g., the P[3] VP4) of RRV repre-sents a virulence determinant. Although TUCH does not causedisease in macaques, it does grow to high titers and is capableof spread. Thus, the determinants of the virus that dictateproductive growth in the animal are not necessarily the samedeterminants affecting disease characteristics.

No common genotype constellation has been established forsimian RVs, which up to this point, can be blamed on the lackof comprehensive sequence data. Our analysis indicates thatthe PTRV strain is unlikely to represent a typical simian ge-notype constellation, since it most likely originated after the

interspecies transmission of a Bovidae or artiodactyl RV to acolony of pig-tailed macaques prior to 1987, where it was ableto spread, evolve, and sustain itself. The genetic constellationsof the RRV and TUCH are also unlikely to represent typicalsimian RVs genogroups, since these strains appear to haveundergone reassortment with bovine, human, and/or unknownRV strains relatively recently. However, based on the observedgenetic divergence, the proposed interspecies transmission ofRRV and TUCH ancestors from a canine/feline host to asimian host, or vice versa, must have occurred quite a whileago. In its new host, the virus must not only have survived butalso evolved. If macaques were the host species in which thisancestor virus established itself and spread, then the genomesof RRV and TUCH could be representatives of a simian geno-group, in which a few gene segments were more recently re-placed by reassortment. This hypothesis is supported by theobservation that RRV and TUCH (with their related genotypeconstellations) were independently isolated from differentstates in the United States (California and Louisiana, respec-tively). Additional support for this hypothesis comes from theRV strain YK-1, isolated from an immunodeficient pig-tailedmacaque in the Yerkes National Primate Research Center,Emory University in Atlanta, which possesses identical geno-types as RRV for VP7, VP4, and NSP4 (49). However, basedon current information, SA11-H96 is the most likely represen-tative of a typical simian RV genogroup, since none of the genesegments of SA11 are closely related to other known RVstrains.

In summary, interspecies transmission and reassortment hascontributed significantly to the emergence of the simian RVstrains PTRV, RRV, and TUCH. Epidemiological and se-quence data of simian RVs collected from animals living in thewild will be crucial to further elucidate the ancestry of theseviruses. None of the simian RVs characterized thus far areclosely related to the strains of human RVs (Wa-like geno-group 1 and DS-1-like genogroup 2) that are responsible forthe vast majority of cases of rotaviral diarrheal disease occur-ring in infants and young children. The attractiveness of simianRVs as model agents of human disease would be greatly im-proved if future searches were to identify simian genogroup 1or 2 viruses that emulated human disease in juvenile animals.

ACKNOWLEDGMENTS

We are grateful to Sarah McDonald for critical revisions of themanuscript.

J.M. was supported by a postdoctoral fellowship from the Fund forScientific Research (FWO) Flanders. J.T.P., Z.F.T., D.C., and H.Y.were supported by the Intramural Research Program of the NationalInstitutes of Health (NIH), National Institute of Allergy and InfectiousDiseases. The sequencing of PTRV was partially supported by startupfunds to L.Y. from Virginia Polytechnic Institute and State University.K.S. was supported by NIH R21RR024871 grant awarded to the Na-tional Center for Research Resources.

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