JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2009, p. 3569–3576 Vol. 47, No. 110095-1137/09/$12.00 doi:10.1128/JCM.01471-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Characteristics of German G8P[4] Rotavirus StrainGER1H-09 Suggest that a Genotyping and Subclassification

Update Is Required for G8�

C. Pietsch,1* L. Petersen,2 L. Patzer,2 and U. G. Liebert1

Institute of Virology, Leipzig University, Leipzig, Germany,1 and Department of Pediatrics,Hospital St. Elisabeth and St. Barbara, Halle/Saale, Germany2

Received 30 July 2009/Returned for modification 20 August 2009/Accepted 31 August 2009

A rare G8P[4] rotavirus, designated GER1H-09, was detected in a stool sample from an infant sufferingfrom repeated episodes of emesis for 2 days without diarrhea. Sequencing of all genomic RNA segmentswas performed, and complete coding sequences were determined. The VP7 amino acid sequence revealeda close phylogenetic relationship to human G8P[6] and G8P[8] isolates from Slovenia and Africa.GER1H-09 shared typical amino acid residues within variable regions VR3 to VR7 with those strains, andtheir subclassification as lineage G8-II rotaviruses is proposed. The variability in VR3 was identified asthe likely reason for the failure in genotyping G8-II rotaviruses by commonly used multiplex PCR.Furthermore, the sequences of associated structural and nonstructural proteins showed high amino acididentities to DS-1-like rotaviruses. The genotype composition of GER1H-09 (G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) suggests the occurrence of reassortment events between G8 genotypes and human DS-1-likeG2P[4] rotaviruses.

Rotaviruses (RVs) are the main etiological agents of gas-troenteritis worldwide. They occur in humans and animals,and the annual death rate is �600,000 children (26). Likeother members of the family Reoviridae, the rotaviral ge-nome consists of 11 double-stranded RNA segments en-closed by a triple-layered nucleocapsid. Seven RV groups (Ato G) are determined by the antigenetic properties of themiddle layer capsid protein (VP6) (7). RV infections inhumans are mainly caused by group A RVs (18). They arefurther classified into different P and G genotypes basedupon the main neutralization antigens, namely, the spikeprotein (VP4) and the major outer capsid glycoprotein(VP7) (7). The most prevalent genotypes, G1P[8], G2P[4],G3P[8], G4P[8], and G9P[8], are responsible for approxi-mately 90% of worldwide human RV infections (30). RVepidemiology is in a constant state of diversification, mainlydriven by frequent reassortment events (17). For discoveryof reassortment events, genotyping of all genomic RNAsegments, including the other structural proteins (VP1-3,VP6) and the nonstructural proteins (NSP1-5), is mandatory(22, 23). With regard to the high incidence of RV infectionsworldwide, two oral live-attenuated vaccines have been li-censed. Rotarix is a monovalent vaccine derived from ahuman G1P[8] strain that has been attenuated by serialpassages (29). RotaTeq is a human-bovine reassortant RVvaccine and contains the human genotypes G1, G2, G3, G4,and P[8] (37). The postmarketing surveillance of circulatingRV genotypes is crucial for vaccine efficacy studies. A closelook at rotavirus genotypes in Germany has already revealed

the circulation of uncommon human G10 and G12 rotavi-ruses in prior studies (19, 28, 35). Here we add to thealready known diversity by the description of a recent G8genotype variant derived from a stool sample from a Ger-man infant. The subclassification of G8 genotypes into lin-eages I and II is proposed.

CASE REPORT

An 11-month-old girl was admitted to hospital (Departmentof Pediatrics, Hospital St. Elisabeth and St. Barbara, Saxony-Anhalt, Germany) on 21 January 2009 because of vomiting for2 days. No diarrhea was reported, and on admission the childwas febrile (38.8°C) and slightly dehydrated (�5% of bodyweight). Laboratory investigations showed normal values forroutine chemistry, especially acid-base metabolism, and thechild was treated by intravenous fluid for 24 h. Stool analyticswere unremarkable for adeno-, astro-, and norovirus. The in-fant had not been vaccinated against RV. She had been livingin an urban area and did not attend any day care facility forchildren. No gastroenteritis of close contact persons was re-ported by her parents. Neither the patient nor the family mem-bers had a recent travel history. For religious reasons, neitherpork nor bovine meat had been consumed.

MATERIALS AND METHODS

RV antigen testing. The stool specimen was processed as described previously(28) and was screened for RV group A antigen by IDEIA (Dako Ltd., Ely,Cambridgeshire, United Kingdom). Stool suspensions (10%) in phosphate-buff-ered saline were prepared from parts of the specimen for further moleculartesting.

RNA extraction and reverse transcription-PCR amplification. Viral RNA wasextracted from an aliquot of the stool suspension by a NucliSens easyMAGsystem (bioMerieux, Boxtel, The Netherlands). The 11 genomic RNA segmentswere reverse transcribed and amplified by specific forward and reverse primers asdescribed previously (28). Nucleotide sequences of entire open reading frames

* Corresponding author. Mailing address: Institute of Virology,Leipzig University, Johannisallee 30, 04103 Leipzig, Germany. Phone:49 3419714333. Fax: 49 3419714309. E-mail: corinna.pietsch@medizin.uni-leipzig.de.

� Published ahead of print on 9 September 2009.

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were determined, and phylogenetic analyses of deduced amino acid sequenceswere performed as described previously (28).

Nucleotide sequence accession numbers. The complete coding nucleotide se-quences of the 11 gene segments of strain GER1H-09 were obtained and de-

posited in GenBank under the following accession numbers: VP1, GQ414540;VP2, GQ414541; VP3, GQ414542; VP4, GQ414543; VP6, GQ414544; VP7,GQ414545; NSP1, GQ414546; NSP2, GQ414547; NSP3, GQ414548; NSP4,GQ414549; and NSP5/NSP6, GQ414550.

FIG. 1. Comparison of amino acid residues of variable VP7 regions VR3 to VR7 (13). The G8-II RVs are represented by GER1H-09, SI-885,CAM/6782, and CAM/6809. RV strains 6736, 6780, 6787, 6810, 6854, and 6862 were identical to strain SI-885 in all variable regions depicted. Identicalamino acid residues are indicated with dashes. The seven amino acid positions characteristic of G8-II are shaded in gray. G8-I strains representative ofthe spectrum of different amino acids within the variable regions were chosen for the alignment. The nucleotide sequence of primer aAT8 and the locationof the respective primer binding site within VR3 of strain GER1H-09 are given below the amino acid sequences. Differences in primer and targetsequences are shaded in gray. Nucleotide positions are indicated below the sequences. GenBank accession numbers for the sequences used in amino acidcomparison were as follows: SI-885, ABJ91296; 6809, ABQ43350; 6782, ABQ43345; Cody I-801, AAA87710; OVR762, ABU49783; 69 M, ABV53232;U25.96, AAB95420; 678, AAA57566; EGY1805, AAD04928; Sun9, BAD27262; AU109, BAF48398; RUBV0161, ABQ00953; DRC88, AAY55959;MW23, CAB92921; A5, BAA00856; C-8008, AAA87707; UP30, AAD33921; 19744, AAG31659; and EGY2295, AAD04930.

TABLE 1. Amino acid identity rates for strain GER1H-09 genomic RNA segments in comparison to those of published RV isolatesa

Strain Genotype of genomic RNA segments

Amino acid identity (%)

VP7(G)

VP4(P)

VP6(I)

VP1(R)

VP2(C)

VP3(M)

NSP1(A)

NSP2(N)

NSP3(T)

NSP4(E)

NSP5(H) NSP6

Wa G1 P�8� I1 R1 C1 M1 A1 N1 T1 E1 H1 76.6 89.1 91.9 89.6 89.5 81.6 68.9 89.2 82.4 85.1 80.5 79.3DS-1 G2 P�4� I2 R2 C2 M2 A2 N2 T2 E2 H2 74.8 96.7 98.7 96.8 98.5 95.6 94.0 95.5 97.4 97.1 93.5 94.5TB-Chen G2 P�4� I2 R2 C2 M2 A2 N2 T2 E2 H2 75.4 98.1 98.2 96.8 98.8 97.3 93.0 95.8 98.4 96.0 98.0 97.8AU-1 G3 P�9� I3 R3 C3 M3 A3 N3 T3 E3 H3 83.4 65.7 94.7 94.5 93.7 83.2 57.2 87.6 88.7 85.1 85.5 NC69 M G8 P�10� I2 R2 C2 M2 A2 N2 T2 E2 H2 94.7 72.5 99.2 96.9 96.4 90.0 90.7 96.8 94.5 96.5 91.0 88.0DRC86 G8 P�6� I2 R2 C2 M2 A2 N2 T2 E2 H2 93.8 76.9 99.7 98.8 99.4 98.3 91.4 96.5 99.6 96.0 98.5 97.8DRC88 G8 P�8� I2 R2 C2 M2 A2 N2 T2 E2 H2 94.1 90.8 100 98.4 99.3 98.0 91.4 96.5 99.6 94.8 98.5 96.7OVR762 G8 P�14� I2 R2 C2 M2 A11 N2 T6 E2 H3 96.0 65.6 98.9 97.3 96.7 93.5 54.5 97.7 82.1 97.1 86.0 NCGR10924/99 G9 P�6� I2 R2 C2 M2 A2 N2 T2 E2 H2 83.4 77.4 99.4 98.8 99.3 98.6 91.6 96.8 99.6 97.7 99.5 98.9L26 G12 P�4� I2 R2 C2 M1 A2 N1 T2 E2 H1 79.1 96.2 97.9 96.5 98.6 79.6 93.2 89.9 97.4 95.4 81.5 81.5N26-02 G12 P�6� I2 R2 C2 M2 A2 N1 T2 E6 H2 79.1 76.9 99.2 98.7 99.4 98.3 91.9 89.5 99.3 85.1 98.0 NCRV161-00 G12 P�6� I2 R2 C2 M2 A2 N2 T2 E1 H2 78.8 77.2 100 98.7 99.5 98.4 91.9 96.5 99.3 86.2 99.0 98.9

a Genomic RNA segments of GER1H-09 were compared to those of isolates bearing a similar genotype pattern and to the prototype strains Wa, DS-1, and AU-1of the different RV genogroups (22). Table elements representing genotype G8-P�4�-I2-R2-C2-M2-A2-N2-T2-E2-H2 are shown in bold. NC, amino acid identities werenot calculated because different open reading frames result in longer and shorter NSP6 proteins. GenBank accession numbers for sequences used in comparison toGER1H-09 were as follows: Wa VP1, ABF67546; VP2, CAA33074; VP3, AAQ02692; VP4, AAA47290; VP6, P03530; VP7, AAA47342; NSP1, AAA02910; NSP2,AAA47301; NSP3, CAA57193; NSP4, AAA47309; NSP5, AAK15269; NSP6, AF306494; DS-1 VP1, ABU87834; VP2, ABI58292; VP3, ABU87836; VP4, ABV53252;VP6, ABI58293; VP7, ABV53256; NSP1, AAA47332; NSP2, ABV53255; NSP3, BV53254; NSP4, AAG09190; NSP5, P23048; NSP6, EF672583; TB-Chen VP1,AAV65743; VP2, AAV65742; VP3, AAV65744; VP4, AAV65734; VP6, AAV65735; VP7, AAV65736; NSP1, AAV65737; NSP2, AAV65738; NSP3, AAV65739; NSP4,AAV65740; NSP5, AAV65741; NSP6, AY787651; AU-1 VP1, ABF67540; VP2, ABF67543; VP3, ABF67544; VP4, BAA01747; VP6, ABF67545; VP7, BAA23292;NSP1, BAA08200; NSP2, ABF67541; NSP3, ABF67542; NSP4, BAA24413; NSP5, BAB83813; 69 M VP1, ABU50963; VP2, ABU87823; VP3, AAQ21043; VP4,ABV53228; VP6, ABU87825; VP7, ABV53232; NSP1, ABV53229; NSP2, ABV53231; NSP3, CAA57184; NSP4, ABV53233; NSP5, P23047; NSP6, EF672562; DRC86VP1, AAY55975; VP2, AAY55974; VP3, AAY55973; VP4, AAY55972; VP6, AAY55971; VP7, AAY55970; NSP1, AAY55969; NSP2, AAY55968; NSP3, AAY55967;NSP4, AAY55966; NSP5, AAY55965; NSP6, DQ005115; DRC88 VP1, AAY55964; VP2, AAY55963; VP3, AAY55962; VP4, AAY55961; VP6, AAY55960; VP7,AAY55959; NSP1, AAY55958; NSP2, AAY55957; NSP3, AAY55956; NSP4, AAY55955; NSP5, AAY55954; NSP6, DQ005104; OVR762 VP1, ABU49748; VP2,ABU49755; VP3, ABU49762; VP4, ABU49769; VP6, ABU49776; VP7, ABU49783; NSP1, BU49790; NSP2, ABU49797; NSP3, ABU49804; NSP4, ABU49811; NSP5,ABU49818; GR10924/99 VP1, ACJ06213; VP2, ACJ06214; VP3, ACJ06215; VP4, ACJ06216; VP6, ACJ06218; VP7, ACJ06220; NSP1, ACJ06217; NSP2, ACJ06221;NSP3, ACJ06219; NSP4, ACJ06223; NSP5, ACJ06222; NSP6, FJ183362; L26 VP1, ABU87842; VP2, ABU87843; VP3, AAQ21045; VP4, ABV53268; VP6, ABA34215;VP7, ABV53272; NSP1, ABV53269; NSP2, ABA34232; NSP3, ABA34239; NSP4, CAC43306; NSP5, ABA34252; NSP6, DQ146698; N26-02 VP1, ABA34189; VP2,ABA34196; VP3, ABA34203; VP4, ABA34209; VP6, ABA34214; VP7, ABA34221; NSP1, ABA34226; NSP2, ABA34231; NSP3, ABA34238; NSP4, ABA34245; NSP5,ABA34251; RV161-00 VP1, ABF67552; VP2, ABF67553; VP3, ABF67554; VP4, ABF67555; VP6, ABF67556; VP7, ABF67557; NSP1, ABF67547; NSP2, ABF67548;NSP3, ABF67549; NSP4, ABF67550; NSP5, ABF67551; and NSP6, DQ490544.

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RESULTS

Analysis of VP7 sequence. An amino acid sequence align-ment of VP7 of GER1H-09, 49 of the complete G8 sequences,and VP7 of other G types available at GenBank was per-formed. Amino acid identities of GER1H-09 VP7 were above92% in comparison to the G8 lineage but below 85% in com-parison to other G genotypes (Table 1). The highest identitieswere obtained in comparison to the human RV strain SI-885from Slovenia (99%), strain 6810 from Ethiopia (99%), strains6862 and 6854 from Tunisia (99%), strain 6736 from IvoryCoast (99%), and strains 6787, 6780, 6782, and 6809 fromCameroon (98%) (data not shown) (6, 33). A characteristicpattern of residues V43, I44, A45, Q73, E100, A122, and S145within GER1H-09 VP7 variable regions VR3, VR4, VR5,VR6, and VR7 was identical to that for the same Slovenianand African RVs (13). All other G8 isolates showed differentresidue patterns within the variable regions. Except for bovinestrain Cody I-801, which shared two residues, the other G8strains had only one or none of the distinctive amino acids incommon (Fig. 1). Two main clusters resulted in VP7 phyloge-netic analysis (Fig. 2a) and were provisionally classified aslineage G8-I and lineage G8-II. Lineage G8-II consisted of

GER1H-09, SI-885, and the mentioned African strains. Nucle-otide identities of GER1H-09 were below 86% in comparisonto G8-I RVs but above 97% in comparison to G8-II strains(data not shown). The primer binding site within VR3 ofprimer aAT8, frequently used to assign the G8 genotype bymultiplex nested PCR (11), was altered at four nucleotidepositions in all strains of the G8-II lineage (Fig. 1).

Genotyping using all 11 genomic RNA segments. All 15complete amino acid sequences of different VP4 P[4] strainsavailable at GenBank were included in an alignment. Thehighest identity was obtained in a comparison of GER1H-09 tothe G2 strain KO-2 from Japan (99%). P[4] reference strainDS-1 showed 96% identity (Table 1). In the phylogenetic anal-ysis, GER1H-09 clustered together with KO-2 and with strainsfrom India (SC185 and NR1) and Bangladesh (MMC84,MMC6, DH392, and SK138) (Fig. 2b). Since only partial VP4sequences are available for many RVs, a second amino acidalignment representing just the VP8 part of VP4 was performed.Consistent with the full-length alignment, KO-2 showed the high-est amino acid identity to RV strain GER1H-09 (data not shown).

Table 1 shows a comparison of the amino acid sequences ofthe 10 viral proteins, VP1, VP2, VP3, VP6, and NSP1 to NSP6,

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FIG. 2. Phylogenetic dendrograms at the amino acid level for G8 VP7 (a); P[4] VP4 (b); VP1, VP2, VP3, and VP6 (c); and NSP1 to NSP6 (d).Only bootstrap values (1,000 replicates) above 65% are shown. G8 strains are labeled by filled circles (b to d). Strain GER1H-09 of this study istagged by a solid tailed arrow. In panel a, lineages G8-I and G8-II have tentatively been assigned. In each phylogenetic dendrogram, the strainname is prefixed by the country of origin (ARG, Argentina; AUS, Australia; BAN, Bangladesh; BEL, Belgium; BRA, Brazil; CAM, Cameroon;DRC, Democratic Republic of Congo; EGY, Egypt; ETH, Ethiopia; FIN, Finland; GBI, Guinea Bissau; GER, Germany; HUN, Hungary; INA,Indonesia; IND, India; ISR, Israel; ITA, Italy; IVC, Ivory Coast; JAP, Japan; KEN, Kenya; MAL, Malawi; NIG, Nigeria; PHI, Philippines; PRC,People’s Republic of China; RSA, Republic of South Africa; SKO, South Korea; SLO, Slovenia; SPA, Spain; SWI, Switzerland; THA, Thailand;TUN, Tunisia; UK, United Kingdom; and USA, United States) as well as the viral host (Av, avian; Bo, bovine; Gu, guanaco; Hu, human; Ov, ovine;and Si, simian) and is followed by the G genotype (b to d). Strain U25.96 was detected in water samples of a river, which is indicated instead ofthe host prefix.

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of strain GER1H-09 to those of several published RV strainsused for reference. The phylogenetic relationships based oncomplete amino acid alignments revealed that all genes exceptfor the VP7 gene were highly similar to DS-1-like RV genes(Fig. 2c and d). For 6 of the 10 proteins (VP1, VP2, VP3, VP6,NSP3, and NSP5), the closest relatives were isolates from Ban-gladesh (N26-02 and RV161-00) and Africa (DRC86, DRC88,and GR10924/99) (Fig. 2c and d). NSP1 clustered with humanstrain L26 from the Philippines and with DS-1 from the UnitedStates (Fig. 2d). The identity of the GER1H-09 NSP2 aminoacid sequence was highest in comparison to the ovine G8P[14]strain OVR762 from Spain (Table 1).

Following the recommendations for the classification ofgroup A RVs (23), the genotype G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 was assigned.

DISCUSSION

Rotaviruses with G8 genotype specificity were first detectedin the late 1970s in human stool samples from Indonesia (15,20). They are highly prevalent in some African countries (4)and have been recovered sporadically from humans and ani-mals worldwide (6, 8, 20, 21, 25, 38). In Europe, evidence of G8has been found in the United Kingdom, Finland, Italy, Swit-zerland, Spain, Hungary, France, and Slovenia (1, 2, 3, 5, 9, 10,16, 31, 32, 33). In addition to humans, G8 RVs affect a widerange of animal hosts, including cattle, pigs, horses, guanacos,monkeys, and sheep (2, 12, 14, 16, 27, 31). Accordingly, variousP type associations of G8 have been reported, including P[1],P[4], P[5], P[6], P[7], P[8], P[10], P[11], and P[14] (9, 15, 24, 32,34) (GenBank accession no. FJ87036). In this study, we de-tected a G8P[4] rotavirus in a stool sample derived from an11-month-old German infant. The determined full coding se-quences of all genomic RNA gene segments and the completegenotyping of strain GER1H-09 make comprehensive data ona G8P[4] strain available.

Phylogenetically, VP7 nucleotide and amino acid sequencesof strain GER1H-09 clustered with those of human strainsdetected in Africa and Slovenia (6, 33). These RVs shared apattern of seven characteristic amino acid substitutions withinvariable regions VR3, VR4, VR5, VR6, and VR7. A certainlink to bovine strain Cody I-801 does probably exist, as it sharesresidues I44 and A45 in variable region VR3. Based on thephylogenetic dendrogram and the amino acid substitutionswithin variable regions, subclassification of G8 RVs into twodifferent lineages, G8-I and G8-II, is proposed. This was nec-essary because the former subclassification of G8 genotypes (8)did not comprise any of the G8-II RVs and was based onnucleotide sequence alignments. The probable animal hostorigin of the new G8-II genotypes remains unclear, as thecluster encompasses only human G8 strains.

The phylogenetic analysis of the other structural and non-structural proteins of GER1H-09 identified the genotype G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This composition sus-tains the hypothesis of reassortment events between G8genotypes and human DS-1-like RVs (21).

The altered VR3 of VP7 may also hinder G typing of theproposed G8-II isolates by common genotyping using multi-plex nested PCR (6, 11, 33). Due to the changes in the primeraAT8 binding site, G8-II RVs may regularly escape genotyping

and may be classified as untypable by multiplex nested PCR.Hence, the worldwide prevalence of G8-II strains is obviouslyunderestimated. A valuable effort to facilitate rotavirus geno-typing was the recent development of a new reverse hybridiza-tion line assay (36). However, correct typing of G8-II rotavi-ruses might fail, as three to five mismatches within the probebinding area of the two G8-specific probes present on thereverse hybridization strip are predicted (data not shown). Dueto the high degree of rotavirus diversity and variability, con-tinual determination of RV sequences appears mandatory toupdate primer and probe sequences.

In conclusion, the detection of G8P[4] RV strain GER1H-09represents a VP7 variant distinct from most G8 genotypesavailable. Furthermore, phylogenetic data support the assump-tion of reassortment between G8 and common DS-1-like RVs.Since G8-II RVs escape conventional VP7 genotyping by mul-tiplex nested PCR, monitoring of the RV genotype diversity bynucleotide sequencing is apparently indispensable.

APPENDIX

GenBank accession numbers used in phylogenetic analyses were asfollows. For Fig. 2a, the following sequences were used: 678,AAA57566; 1290, ACC96235; 6736, ABQ43354; 6780, ABQ43344;6782, ABQ43345; 6785, ABQ43346; 6787, ABQ43348; 6809,ABQ43350; 6810, ABQ43351; 6854, ABQ43352; 6862, ABQ43353; 69M, ABV53232; A5, BAA00856; AU109, BAF48398; B17, ABF48491;B37, AAA47344; BRV16, BAB83660; C-8008, AAA87707; Cody I-801,AAA87710; DRC86, AAY55970; DRC88, AAY55959; EGY1850,AAD04928; EGY2295, AAD04930; GB5882, AAM97652; GR570/85,AAD33919; GRV Chubut, AAQ11983; GRV Rio Negro, AAQ11982;HAL1166, AAA57565; HMG035, AAK66976; KAG74, BAB83655;KJ11, ACI48150; KJ13, ACI48151; KY6914, ACJ73899; MG8.0,AAF23774; MP409, AAD38366; MW23, CAB92921; MW333,CAB92924; NGRBg8, AAL56244; OVR762, ABU49783; PR/1300/04,ACF93690; PTRV, ACL36060; QEH14262, AAD33920; R291,AAW65719; SA4948JHB, FJ386447; SI-885, ABJ91296; Sun9,BAD27262; Tokushima9503, BAB18911; U25.96, AAB95420; andUP30, AAD33921. For Fig. 2b, the following sequences were used:107E1B, AAA18376; CHW17, BAA77540; DH392, ACJ54806; DS-1,BAC82358; IS-2, CAA57766; KO-2, AAK94068; L26, AAA47335;MMC6, ACJ54807; MMC84, ACJ54808; MW333, CAB92923; NR1,AAQ09556; PO-13, BAA24149; RV-5, AAA47333; SC185,CAC14875; SK138, ACJ54802; and TB-Chen, AAV65734. For Fig. 2cand d, the following sequences were used: 69 M VP1, ABU50963; VP2,ABU87823; VP3, AAQ21043; VP6, ABU87825; NSP1, ABV53229;NSP2, ABV53231; NSP3, CAA57184; NSP4, ABV53233; NSP5,P23047; NSP6, EF672562; AU-1 VP1, ABF67540; VP2, ABF67543;VP3, ABF67544; VP6, ABF67545; NSP1, BAA08200; NSP2,ABF67541; NSP3, ABF67542; NSP4, BAA24413; NSP5, BAB83813;B10925-07 VP1, ABU49745; VP2, ABU49752; VP3, ABU49759; VP6,ABU49773; NSP1, ABU49787; NSP2, ABU49794; NSP3, ABU49801;NSP4, ABU49808; NSP5, ABU49815; B1711 VP1, ABU49742; VP2,ABU49749; VP3, ABU49756; VP6, ABU49770; NSP1, ABU49784;NSP2, ABU49791; NSP3, ABU49798; NSP4, ABU49805; NSP5,ABU49812; NSP6, EF554092; B3458 VP1, ABI60856; VP2, ABI60857;VP3, ABI60858; VP6, ABI60859; NSP1, ABV66090; NSP2,ABV66084; NSP3, ABV66078; NSP4, ABV66094; NSP5, ABV57763;NSP6, EF990713; B4106 VP1, AAU43799; VP2, AAU43798; VP3,AAU43797; VP6, AAU43795; NSP1, AAU43793; NSP2, AAU43792;NSP3, AAU43791; NSP4, AAU43790; NSP5, AAU43789; B4633-03VP1, ABA34185; VP2, ABA34192; VP3, ABA34199; VP6, ABA34210;NSP1, ABA34222; NSP2, ABA34227; NSP3, ABA34234; NSP4,ABA34241; NSP5, ABA34247; NSP6, DQ146648; DRC86 VP1,AAY55975; VP2, AAY55974; VP3, AAY55973; VP6, AAY55971;NSP1, AAY55969; NSP2, AAY55968; NSP3, AAY55967; NSP4,AAY55966; NSP5, AAY55965; NSP6, DQ005115; DRC88 VP1,AAY55964; VP2, AAY55963; VP3, AAY55962; VP6, AAY55960;NSP1, AAY55958; NSP2, AAY55957; NSP3, AAY55956; NSP4,

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AAY55955; NSP5, AAY55954; NSP6, DQ005104; DS-1 VP1,ABU87834; VP2, ABI58292; VP3, ABU87836; VP6, ABI58293; NSP1,AAA47332; NSP2, ABV53255; NSP3, BV53254; NSP4, AAG09190;NSP5, P23048; NSP6, EF672583; GER126-08 VP1, FJ747613; VP2,FJ747614; VP3, FJ747615; VP6, FJ747617; NSP1, FJ747620; NSP2,FJ747621; NSP3, FJ747622; NSP4, FJ747623; NSP5, FJ747624; NSP6,FJ747624; GER172-08 VP1, FJ747625; VP2, FJ747626; VP3, FJ747627;VP6, FJ747629; NSP1, FJ747631; NSP2, FJ747632; NSP3, FJ747633;NSP4, FJ747634; NSP5, FJ747635; NSP6, FJ747635; GR10924/99VP1, ACJ06213; VP2, ACJ06214; VP3, ACJ06215; VP6, ACJ06218;NSP1, ACJ06217; NSP2, ACJ06221; NSP3, ACJ06219; NSP4,ACJ06223; NSP5, ACJ06222; NSP6, FJ183362; Hun5 VP1,ABU49744; VP2, ABU49751; VP3, ABU49758; VP6, ABU49772;NSP1, ABU49786; NSP2, ABU49793; NSP3, ABU49800; NSP4,ABU49807; NSP5, ABU49814; IAL28 VP1, ABU87838; VP2,ABU87839; VP3, ABU87840; VP6, ABU87841; NSP1, ABV53261;NSP2, ABV53263; NSP3, ABV53262; NSP4, ABV53265; NSP5,ABV53266; NSP6, EF672590; L26 VP1, ABU87842; VP2, ABU87843;VP3, AAQ21045; VP6, ABA34215; NSP1, ABV53269; NSP2,ABA34232; NSP3, ABA34239; NSP4, CAC43306; NSP5, ABA34252;NSP6, DQ146698; N155 VP1, ABY64681; VP2, ABY64682; VP3,ABY64683; VP6, ABY64685; NSP1, ABY64687; NSP2, ABY64688;NSP3, ABY64689; NSP4, ABY64690; NSP5, ABY64691; N26-02 VP1,ABA34189; VP2, ABA34196; VP3, ABA34203; VP6, ABA34214;NSP1, ABA34226; NSP2, ABA34231; NSP3, ABA34238; NSP4,ABA34245; NSP5, ABA34251; OVR762 VP1, ABU49748; VP2,ABU49755; VP3, ABU49762; VP6, ABU49776; NSP1, BU49790;NSP2, ABU49797; NSP3, ABU49804; NSP4, ABU49811; NSP5,ABU49818; P VP1, ABU87846; VP2, ABU87847; VP3, ABU87848;VP6, ABU87849; NSP1, ABV53277; NSP2, ABV53279; NSP3,ABV53278; NSP4, ABV53281; NSP5, ABV53282; PO-13 VP1,BAA24146; VP2, BAA24147; VP3, BAA24148; VP6, BAA03836;NSP1, BAA24150; NSP2, BAA24142; NSP3, BAA24143; NSP4,BAA24144; NSP5, BAA24145; NSP6, AB009628; Ro1845 VP1,ACH97395; VP2, ACH97396; VP3, ACH97397; VP6, ACH97399;NSP1, ACH97401; NSP2, ACH97402; NSP3, ACH97403; NSP4,ACH97404; NSP5, ACH97405; NSP6, EU708900; RV161-00 VP1,ABF67552; VP2, ABF67553; VP3, ABF67554; VP6, ABF67556; NSP1,ABF67547; NSP2, ABF67548; NSP3, ABF67549; NSP4, ABF67550;NSP5, ABF67551; NSP6, DQ490544; SA11 VP1, ABG75819; VP2,AAA47349; VP3, ABG75824; VP6, AAA65638; NSP1, ABG75773;NSP2, ABG75789; NSP3, AAA47295; NSP4, AAC61867; NSP5,P11202; NSP6, M28347; S2 VP1, ABI60844; VP2, ABI60845; VP3,ABI60846; VP6, ABI60847; NSP3, CAA57187; NSP4, AAB81290; ST3VP1, ABU87854; VP2, ABU87855; VP3, AAQ21046; VP6,ABU87857; NSP1, AAA75585; NSP2, ABV53295; NSP3, CAA57195;NSP4, AAB81296; NSP5, ABV53298; NSP6, EF672618; Sun9 VP1,BAG54800; VP2, BAG54801; VP3, BAG54802; VP6, BAG54803;T152 VP1, ABA34191; VP2, ABA34198; VP3, ABA34204; VP6,ABA34216; NSP1, BAC77249; NSP2, ABA34233; NSP3, ABA34240;NSP4, ABA34246; NSP5, ABA34253; NSP6, DQ146706; TB-ChenVP1, AAV65743; VP2, AAV65742; VP3, AAV65744; VP6,AAV65735; NSP1, AAV65737; NSP2, AAV65738; NSP3, AAV65739;NSP4, AAV65740; NSP5, AAV65741; NSP6, AY787651; UK VP1,CAA39085; VP2, CAA36825; VP3, AAQ74387; VP6, P18610; NSP1,AAA18011; NSP2, P03538; NSP3, AAA47317; NSP4, P04513; NSP5,P04515; Wa VP1, ABF67546; VP2, CAA33074; VP3, AAQ02692;VP6, P03530; NSP1, AAA02910; NSP2, AAA47301; NSP3,CAA57193; NSP4, AAA47309; NSP5, AAK15269; and NSP6,AF306494.

ACKNOWLEDGMENTS

We are grateful to Andreas Poge for organizing sample storage andtransports.

We negate any financial or other conflict of interest that might beconstrued to influence the contents of the manuscript, including theresults or interpretation of publication.

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