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. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: Arch Virol. 2008 Jul 5;153(8):1621–1629. doi: 10.1007/s00705-008-0155-1

Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments

Jelle Matthijnssens 1,*, Max Ciarlet 2, Mustafizur Rahman 1,3, Houssam Attoui 4, Krisztián Bányai 5, Mary K Estes 6, Jon R Gentsch 7, Miren Iturriza-Gómara 8, Carl Kirkwood 9, Vito Martella 10, Peter PC Mertens 4, Osamu Nakagomi 11, John T Patton 12, Franco M Ruggeri 13, Linda J Saif 14, Norma Santos 15, Andrej Steyer 16, Koki Taniguchi 17, Ulrich Desselberger 18, Marc Van Ranst 1
PMCID: PMC2556306  NIHMSID: NIHMS68906  PMID: 18604469

Abstract

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al., 2008; J. Virol. 82:3204-3219). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied, genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the eleven gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.

Introduction

Rotaviruses are a common cause of severe, acute gastroenteritis in infants and young children worldwide. By the age of 5 years, 95% of children will have experienced at least one rotavirus infection, with or without evidence of gastroenteritis symptoms. It is estimated that, globally, 1 in 5 cases will be presented to a doctor, 1 in 65 will require hospitalization, and 1 in 293 will die [27, 28, 39].

Rotaviruses have a genome of 11 segments of double stranded RNA encoding 6 structural viral proteins [VP] (VP1, VP2, VP3, VP4, VP6, VP7) and 6 nonstructural [NS] proteins (NSP1-NSP6). With the exception of genome segment 11 which encodes 2 proteins (NSP5 and NSP6) the genome segments are monocistronic. Full gene-protein assignments have been achieved for several rotavirus strains ([8], http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/Rotavirus.htm.). The genome is enclosed in a triple layered particle: the inner layer consisting of VP2 encloses the genome and two minor structural proteins, VP1 and VP3, thus forming the ‘core’; the middle layer consisting of VP6 surrounds the core, forming a double layered particle (DLP); the outer layer, consisting of VP7 and spike-like projections of VP4, enwraps the DLP to form the triple-layered particle (TLP) or infectious virion [8].

Rotaviruses (Rotavirus genus) are classified into the Reoviridae family, along with the genera: Orthoreovirus, Orbivirus, Rotavirus, Coltivirus, Aquareovirus, Cypovirus, Fijivirus, Phytoreovirus, Oryzavirus, Seadornavirus, Idnoreovirus and Mycoreovirus [21]. The Rotavirus genus includes at least seven serogroups (A to G) that may be distinguished on the basis of their antigenic relationships and the pattern of migration of the dsRNA segments in polyacrylamide gel electrophoresis [4]. Group A rotaviruses are important enteric pathogens in humans and in a wide range of mammalian and avian species [8].

Historically, VP6 was the first rotavirus protein used for classification. Both VP2 and VP6 are highly immunogenic protein in the virion [4, 41]. After infection, antibodies to VP6 are easily detected [4, 41], and the most sensitive immunologic diagnostic assays are based on detection of this protein. VP6 bears different epitopes which allow to differentiate different subgroup (SG) specificities of group A rotaviruses: SG I, SG II, SG I + II, or SG non-I, non-II viruses can be distinguished according to reactivities with two monoclonal antibodies (MAbs) [4]. More recently, based on molecular characterization, only 2 groups (termed genogroups) were distinguished among group A human rotaviruses (genogroup I: SGI; genogroup II: SGII, SGI + II , and SG non-I, non-II.) [13]. In 1985, a classification scheme for rotaviruses was proposed to allow for the presence of multiple “groups” of rotaviruses and for the existence of “serotypes” which crossed species [10] and later in 1989, a binary classification system reminiscent of the one used for the classification of influenza viruses was established, derived from immunological reactivities and gene structures of the two outer capsid proteins, VP4 and VP7, that independently elicit neutralizing antibodies [4]. Thus, rotavirus strains are classified into VP4 or P serotypes (P for protease-sensitive) and VP7 or G (G for glycoprotein) serotypes [8]. Classification of rotaviruses into VP4 or VP7 serotypes is performed by cross-neutralization assays using hyperimmune sera raised to prototype viruses and/or to laboratory-engineered mono-reassortants. Since antigenic characterization is time-consuming and requires virus collections and proper immunological reagents that are not available in all the laboratories, and due to the increasing ease of sequencing, the antigenic classification has slowly been replaced by a classification system of rotaviruses into VP4 or VP7 genotypes, performed by sequence analyses and based on identities between sequences of cognate rotavirus gene segments. So far, 19 G genotypes (14 G serotypes) and 27 different P genotypes (14 P serotypes, 1A, 1B, and 2 to 14) have been identified [19]. G serotype designations largely coincide with G genotype designations. In contrast, a dual nomenclature has been adopted for the VP4 antigenic and genetic classification [8]. The P serotype (when known) is denoted by an arabic number (sometimes followed by a capital letter) and the P genotype is denoted immediately after the P serotype number by an arabic number within squared brackets. Rotavirus strains belonging to 11 G types (G1-G6, G8-G12) and 12 P types (P1A[8], P1B[4], P2A[6], P2C[6], P3[9], P4[10], P5A[3], P6[1], P8[11], P11[14], P12[19], P[25]) have been isolated from humans [4, 7, 9, 19, 25, 33, 36]. During the late 1990s, sequence analyses of the rotavirus enterotoxin NSP4 gene from human and animal rotavirus strains revealed the presence of six (A to F) distinct NSP4 genotypes [3, 5, 8, 11, 15, 22].

The complete ORF sequences of all eleven genome segments of 53 rotavirus strains have been determined (human Wa, DS-1, AU-1, D, KU, P, TB-Chen, ST3, IAL28, Se584, 69M, WI61, A64, L26, T152, B1711, B3458, B10925-97, 111/05-27, B4633-03, RV161-00, RV176-00, N26-02, Dhaka12-03, Dhaka16-03, Matlab13-03, Dhaka25-02, Dhaka6, DRC86, DRC88, Hun5, MG6, PA169, KTM368, B4106, simian SA11, RRV, TUCH, bovine B383, UK, RF, WC3, BRV033, porcine A131, A253, YM, OSU, Gottfried, avian PO-13, ovine OVR762, guanaco Chubut, Río Negro, and lapine 30/96) [12, 17-19, 34, 38].

The simian rotavirus SA11 strain is considered the type species of group A rotaviruses, and its genome segments range in size from 3,302 (segment 1) to 667 (segment 11) base pairs (bp) [8, 35]. Sequences from different rotavirus strains show that each RNA segment starts with two 5′guanines followed by the 5′end non-coding sequences [= untranslated region, UTR] (9-49 nucleotides [nt]), an open reading frame (or two in the case of genome segment 11), the 3′ UTRs (17-182 nt), and ends with two 3′ cytidines. In addition, the plus-stranded RNA is capped at the 5′ end with m7GpppG(m)Gpy, but there are no polyadenylated sequences at the 3′ end [8].

The overall genetic relatedness among homologues genome segments has been assessed by RNA-RNA hybridizations performed under high stringency conditions [23] and, more recently, by direct sequence comparisons. RNA-RNA hybridization has provided molecular evidence to show close interspecies relationships between human and animal strains, or confirms the existence of naturally occurring rotavirus reassortant strains. Three human genogroups, represented by reference rotavirus strains Wa, DS-1, and AU-1 have been established. Several animal genogroups have also been identified but the extent of their relationship among each other and to human strains has not been completely elucidated [24]. However, inter-genogroup reassortments may generate chimeric viruses with mixed constellations of dsRNA segments that are not easily assignable to a defined genogroup. In addition, viruses partially or completely divergent in their genome constellation from the prototype viruses may not be readily characterized. Sequence analysis of human and animal rotavirus strains has revealed the existence of a number of unusual rotavirus strains and has revealed that some strain have a puzzling genome composition derived from repeated reassortment events that could not be tracked effectively using nucleic acids hybridization techniques [2, 14, 16, 18].

Sequence-based studies targeting all the rotavirus gene segments appear more appropriate to generate conclusive data and support studies on rotavirus evolution. When rotavirus strains are analyzed and compared to one another by partial or complete sequences of all 11 gene segments, as initiated by Maunula and von Bonsdorff [20], the genetic relationships among all rotavirus strains can be determined [12, 17-19, 29, 34, 38]. Recently, using comparison of sequenced genes, evidence was presented that the human rotavirus strains belonging to the Wa-like genogroup might have a common origin with porcine rotaviruses, while the human DS-1-like genogroup might have a common origin with bovine rotaviruses [19]. Sequence comparison of rotavirus genomes is critical to the assignment of genotypes and elucidation of rotavirus evolutionary patterns [19]. One method that is used to study typical evolutionary distances between virus strains is pairwise sequence identity profiles [1], and this method resolves virus genotypes as well distinguished frequency peaks and on this biological basis provides the rationale for classification. This approach has been used successfully to classify astroviruses [26], sapoviruses [37], noroviruses [42] and papillomaviruses [6].

Accordingly, the establishment of a classification system encompassing all rotavirus gene segments and sorting individual genes into defined clusters/genotypes based on reliable percentage identity cut-off values is required in order to adopt a universal, standardized nomenclature suitable for reliable data analysis and to exchange readily and unequivocally information on the various rotavirus strains. Recently, phylogenetic analyses were performed and pairwise sequence identity profiles constructed for each of the 11 genome segments of many group A rotaviruses. Based on these analyses, a modified classification system was proposed for VP4, VP7, and NSP4, and a novel classification system for VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 [19] to be used for international standardization and implementation. The system provides an excellent framework to further analyze rotavirus interspecies evolutionary relationships [19], gene reassortment events (genetic shift), functional gene linkage in reassortant progeny, and emergence of new rotaviruses by interspecies transmission.

Review of Nomenclature System

A summary of the calculated cut-off values for the 11 rotavirus RNA segments is shown in Table 1 [19]. The nucleotide cut-off percentages define different genotypes for all genes. To designate the complete genetic make up of a virus, the schematic nomenclature was proposed: Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, representing the genotypes of respectively the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding gene segments, with x indicating the number of the genotype. A nomenclature overview of a large number of strains is provided in Table 2. It should be noted that no classification was obtained for NSP6, since the NSP6 ORF does completely fall into the NSP5 ORF. The phylogenetic analysis (at the nucleotide level) of the NSP6 ORF would be nearly identical to the NSP5 phylogenetic tree. Moreover, not all strains possess a NSP6 ORF.

Table 1.

Nucleotide percentage identity cut-off values defining genotypes for 11 rotavirus gene segments.

Gene product Percentage identity
cut-off values		 Genotypes Name of genotypes
VP7 80% 19G Glycosylated
VP4 80% 27P Protease sensitive
VP6 85% 11I Inner capsid
VP1 83% 4R RNA-dependent RNA polymerase
VP2 84% 5C Core protein
VP3 81% 6M Methyltransferase
NSP1 79% 14A Interferon Antagonist
NSP2 85% 5N NTPase
NSP3 85% 7T Translation enhancer
NSP4 85% 11E Enterotoxin
NSP5 91% 6H pHosphoprotein
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Table 2.

Application of the proposed classification and nomenclature to the structural and non-structural protein encoding genes for known human, bovine, porcine, simian, avian, equine, lapine, murine and ovine rotavirus strains. Green, red, and orange were used for Wa-like, DS-1-like and AU-like gene segments, respectively, while yellow, blue, and purple were used for the avian PO-13-like rotavirus gene segments, some typical porcine VP4, VP7, and VP6 genotypes, and the SA-11-like gene segments, respectively. It should be noted that the genotypes of more than 60% of the isolates in this table are partial, requiring additional work.

Genotypes
Strain name Host VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5
M37 Human G1 P[6] A1 E1
Wa Human G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
KU Human G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
Dhaka16-03 Human G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
D Human G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
K8 Human G1 P[9] A1 H3
RV4 Human G1 N1 E1
DS-1 Human G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2
TB-Chen Human G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2
NR1 Human G2 P[4] I2 A2 N2 T2 E2 H2
S2 Human G2 P[4] I2 R2 C2 M2 T2 E2
RV5 Human G2 P[4] N2 E2
RMC/G66 Human G2 P[4] I1 E2 H2
KUN Human G2 P[4] E2 H2
E210 Human G2 P[4] I1 E2
AU85 Human G2 P[4] E2 H1
AU102 Human G2 P[4] E2 H1
1076 Human G2 P[6] I2 E2
IS2 Human G2 I2 N2 T2 H2
PA260/97 Human G3 P[3] E8
RV3 Human G3 P[6] I1 E1
YO Human G3 P[8] I1 R1 C1 M1 E1
RMC437 Human G3 P[8] I1 E1 H1
P(rice?) Human G3 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
Ai-75 Human G3 P[8] E1
CH32 Human G3 P[8] E1
CHW2 Human G3 P[8] E1
MO Human G3 P[8] E1
M Human G3 T1 E1 H1
Au-1 Human G3 P[9] I3 R3 C3 M3 A3 N3 T3 E3 H3
“02-92” Human G3 P[9] E3
B4106 Human G3 P[14] I2 R2 C2 M3 A9 N2 T6 E5 H3
RMC61 Human G4 P[4] I1 E1 H1
ST3 Human G4 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1
Hosokawa Human G4 P[8] I1 R1 C1 M1
RMC100 Human G4 P[8] I1 A1 N1 T1 E1 H1
Hochi Human G4 P[8] M1 A1 E1
VA70 Human G4 P[8] E1
Odelia Human G4 P[8] E1
IAL28 Human G5 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
B1711 Human G6 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2
Se584 Human G6 P[9] I2 R2 C2 M2 A3 N2 T1 E2 H3
Hun5 Human G6 P[14] I2 R2 C2 M2 A11 N2 T6 E2 H3
MG6 Human G6 P[14] I2 R2 C2 M2 A11 N2 T6 E2 H3
PA169 Human G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
111/05-27 Human G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
B10925-97 Human G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
MP409 Human G8 P[1] A11 T6 E2
DRC86 Human G8 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H2
DRC88 Human G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2
69M Human G8 P[10] I2 R2 C2 M2 A2 N2 T2 E2 H2
B37 Human G8 N2 H2
US1205 Human G9 P[6] I2 E2
WI61 Human G9 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
B3458 Human G9 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
F45 Human G9 P[8] N2
116E Human G9 P[11] I1 A1 E1
RMC321 Human G9 P[19] I5 A1 N1 T1 E1 H1
RMC/G7 Human G9 P[19] I5 A1 E1 H1
Mc323 Human G9 P[19] H1
Mc345 Human G9 P[19] H1
I321 Human G10 P[11] I2 A1 N2 T1 E2
A64 Human G10 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
Dhaka6 Human G11 P[25] I1 R1 C1 M1 A1 N1 T1 E1 H1
KTM368 Human G11 P[25] I5 R1 C1 M1 A1 N1 T1 E1 H1
L26 Human G12 P[4] I2 R2 C2 M1/M2 A2 N1 T2 E2 H1
Dhaka12-03 Human G12 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1
Matlab13-03 Human G12 P[6] I1 R1 C1 M1 A1 N1 T2 E1 H1
N26-02 Human G12 P[6] I2 R2 C2 M2 A2 N1 T2 E6 H2
RV176-00 Human G12 P[6] I2 R2 C2 M2 A2 N2 T2 E6 H2
RV161-00 Human G12 P[6] I2 R2 C2 M2 A2 N2 T2 E1 H2
B4633-03 Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
Dhaka25-02 Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
T152 Human G12 P[9] I3 R3 C3 M3 A12 N3 T3 E3 H6
RUBV3 Bovine G3 P[3] I2 E2 H3
KJ44 Bovine G5 P[1] A1 N1 T1 E1 H1
KJ75 Bovine G5 P[5] A1 N1 T1 E1 H1
NCDV-Lincoln Bovine G6 P[1] I2 R2 C2 M2 N2 T6 E2
BRV033 Bovine G6 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
RF Bovine G6 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
Uk(tc) Bovine G6 P[5] I2 R2 C2 M2 A3 N2 T7 E2 H3
WC3 Bovine G6 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3
B641 Bovine G6 P[5] A3
O Bovine G8 P[1] A11 N2
A5 Bovine G8 P[1] A14
B223 Bovine G10 P[11] I2 A13 E2
A44 Bovine G10 P[11] A3
Hg18 Bovine G15 P[21] E2
993-83 Bovine G18 P[17] I4
Ty-1 Avian G7 P[17] I4 N4 E4
Ty-3 Avian G7 P[17] I4 H11
PO-13 Avian G18 P[17] I4 R4 C4 M4 A4 N4 T4 E4 H4
Ch-1 Avian G19 P[17] I10 H10
P21-5 Porcine G1 P[27] E9
34461-4 Porcine G2 P[23] I5 E1 H1
CMP034 Porcine G2 P[27] I5 E9 H1
A131 Porcine G3 P[7] I5 R1 C2 M1 A1 N1 T1 E1 H1
CRW-8 Porcine G3 P[7] I5
4F Porcine G3 P[19] I5
A411 Porcine G3 I5 A1 N1 T1 E1 H1
Gottfried Porcine G4 P[6] I1 R1 C1 M1 A8 N1 T1 E1 H1
OSU Porcine G5 P[7] I5 R1 C1 M1 A1 N1 T1 E1 H1
A34 Porcine G5 P[23] E1
134/04-15 Porcine G5 P[26] I5 E1
344/04-1 Porcine G5 P[27] E1
HP140 Porcine G6 P[13] I2 E1 H1
HP113 Porcine G6 P[13] I2 E1 H1
A253 Porcine G11 P[7] I5 R1 C2 M1 A1 N1 T1 E1 H1
YM Porcine G11 P[7] I5 R1 C1 M1 A8 N1 T1 E1 H1
RU172 Porcine G12 P[7] I5 E1 H1
H2 Equine G3 P[12] I2 A10 E2
FI14 Equine G3 P[12] I6 A10 E2
H1 Equine G5 P[7] I5 A8 E1
R-22 Equine G10 P[11] I2
L338 Equine G13 P[18] I6 M6 A6
FI23 Equine G14 P[12] I2 A10 E2
EDIM (EW) Murine G16 P[16] I7 A7 E7
EC Murine G16 P[16] E7
EB Murine G16 P[16] E7
EHP Murine G16 P[20] A7 E7
SA11g4“O” (5N) Simian G3 P[1] I2 R2 C5 M5 A5 N5 T5 E2 H5
SA11-5S Simian G3 P[1] I2 R2 C5 M5 A5 N5 T5 E2 H5
SA11-30/19 Simian G3 P[1] I2 R2 C5 M5 A5 N5 T5 E2 H5
SA11-30/1A Simian G3 P[1] I2 R2 C5 M5 A5 N5 T5 E2 H5
SA11-H96 Simian G3 P[2] I2 R2 C5 M5 A5 N5 T5 E2 H5
SA11-Both Simian G3 P[2] I2 R2 C5 M5 A5 N2 T5 E2 H5
RRV Simian G3 P[3] I2 R2 C3 M3 A9 T3 E3 H6
TUCH Simian G3 P[24] I9 R3 C3 M3 A9
OVR762 Ovine G8 P[14] I2 R2 C2 M2 A11 N2 T6 E2 H3
Lp14 Ovine G10 P[15] I2 E2 H3
30/96 Lapine G3 P[14] I2 R2 C2 M3 A9 N2 T6 E5 H3
C-11 Lapine G3 P[14] A9 E5
ALA Lapine G3 P[14] A9 E5 H3
160/01 Lapine G3 P[22] E5
FRV64 Feline G3 P[3] A9 E3
Cat2 Feline G3 P[9] A3
FRV1 Feline G3 P[9] E3
CU-1 Canine G3 P[3] E3
RV198/95 Canine G3 P[3] E8
RV52/96 Canine G3 P[3] E8
K9 Canine G3 P[3] A9
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Recommendations

In order to render the classification system useful and to explore its biological significance in full, a set of recommendations is herein summarized, which is already followed by many, when investigating the VP4, VP7 or NSP4 genotype(s) of a new rotavirus strain but now comprises all genes [19]. The new classification system [19] is based on the nt sequences of complete open reading frames (ORF), and therefore the nt sequence of the entire ORFs of all genes of a new strain should preferentially be obtained in order to unequivocally assign it to one of the known/established genotypes or to a new genotype. As the UTRs of group A rotavirus genes are relatively short (see above) and terminal nt sequences are partially conserved, they are, at this stage, excluded from classification attempts within group A rotaviruses. In addition, it is much less defined what drives the evolution of these gene portions.

Classification strategy

Complete ORF analysis

Once the complete ORF of a rotavirus gene under investigation has been determined, it will be compared to other complete ORFs of cognate genes available in the GenBank database through a BLAST (Basic Local Alignment Search Tool) –search (http://www.ncbi.nlm.nih.gov/BLAST/). If pairwise nucleotide identities between the gene of the novel strain under investigation (strain X) and strains belonging to an established genotype A are above the cut off value of that gene segment (Table 1), strain X can be assigned to genotype A. The exact relationship between the gene of strain X and cognate genes of all established genotypes, has to be obtained phylogenetically. When all the pairwise nucleotide identities between a gene of the new strain Y, and the cognate genes of all the established genotypes are below the cut-off value for that gene segment (Table 1), strain Y may be the prototype of a new genotype (Figure 1).

Figure 1.

Figure 1

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Flowchart summarizing the guidelines for genotype classification provided in this manuscript.

In order to avoid the appearance of duplications in the literature and identical numbering of different new genes, a Rotavirus Classification Working Group (RCWG) including molecular virologists, infectious disease physicians, epidemiologists and public health specialists has been formed (Table 3), who would be prepared and responsible to help validate the new sequence and to assign an appropriate successive genotype number, in close consultation with the ReoviridaeStudy Group of the ICTV.

Table 3.

Current members of the RCWG

Name E-mail Telephone number
Houssam Attoui houssam.attoui@bbsrc.ac.uk +44 1483 231 194
Krisztián Bányai bkrota@hotmail.com +36 1 467 4082
Max Ciarlet max_ciarlet@merck.com +1 267 305 7424
Ulrich Desselberger ud207@medschl.cam.ac.uk +44 1223 763403
Mary K. Estes mestes@bcm.edu + 1 713 798 3585
Jon R. Gentsch Jrg4@cdc.gov +1 404 639 2860
Miren Iturriza-Gómara Miren.iturriza@hpa.org.uk +44 208 327 6225
Carl Kirkwood Carl.kirkwood@mcri.edu.au +613 8341 6439
Vito Martella v.martella@veterinaria.uniba.it +39 80 467 9805
Jelle Matthijnssens jelle.matthijnssens@uz.kuleuven.be +32 16 332166
Peter P.C. Mertens Peter.mertens@bbsrc.ac.uk +44 1483 231 017
Osamu Nakagomi onakagom@nagasaki-u.ac.jp +81 95 819 7061
John T. Patton jpatton@niaid.nih.gov +1 301 594 1615
Mustafizur Rahman rahman.mustafizur1@gmail.com +32 16 332166
Franco M. Ruggeri Franco.Ruggeri@iss.it +39 06 49902980
Linda J. Saif saif.2@osu.edu +1 330263 3742
Norma Santos nsantos@micro.ufrj.br +55 21 2560 8344
Andrej Steyer andrej.steyer@mf.uni-lj.si +386 1 543 74 62
Koki Taniguchi kokitani@fujita-hu.ac.jp +81 562 93 2467
Marc Van Ranst marc.vanranst@uz.kuleuven.be +32 16 347909
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Partial ORF analysis

If only the partial ORF sequence of a rotavirus genome segment is available, assigning it to a certain genotype is less certain because the genotypic diversity across the ORF is not a constant value. Some regions of the ORF may be highly variable, while others may be more conserved. Since the cut-off percentage values for each of the 11 genome segments has been calculated based on entire ORFs, applying these cut off percentages to only a part of the ORF, might lead to erroneous conclusions. Only under certain circumstances when all three of the following restrictions are obeyed, a partial gene sequence might be used to assign a rotavirus gene to an established genotype:

  1. At least 50% of the ORF sequence should be determined.

  2. At least 500 nt of the ORF should be determined.

  3. Identity between strain X and a strain belonging to an established genotype A should be at least 2% above the appropriate cut-off sequence (Table 1), before strain X can be assigned to genotype A (Figure 1).

Remarks

To assign a genotype to a new ORF sequence, whether complete or partial, the comparison should only be done to strains for which the genotype has been established based on the entire ORF analysis, and not with other partial ORF sequences. Due to intra-segmental recombination [30-32, 40] or to different rates of diversification throughout a genome segment, classification assignments based on partial ORFs may yield misleading or incorrect results.

Practical Recommendations

Any sequence of a rotavirus RNA segment that had been analyzed as described above, and found to clearly belong to one of the established genotypes, can be assigned to that genotype. When a potential new genotype is found, the complete ORF should be determined and can be sent (in confidence and out of competition) to a member of the RCWG (Table 3). The sequence will be analyzed and, if appropriate, a new successive genotype number will be assigned. The new number can be used for publication. Most reputable journals will request to provide the GenBank accession number of the new sequence, and this should be obtained as soon as possible and the correct new genotype be assigned to it. The whole process of the RCWG: (i) receiving the sequence, (b) appropriate phylogenetic analysis, (c) presentation to the RCWG members, and (d) approval by the RCWG, should not take more than 6 weeks. An annual report of the RCWG will be published summarizing all newly assigned genotypes.

Acknowledgments

We would like to thank all the colleagues of the Laboratory of Clinical & Epidemiological Virology, Department of Microbiology & Immunology, Rega Institute for Medical Research, University of Leuven, Belgium, for their helpful comments and discussions. J.M. was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen). JTP was supported by the Intramural Research Program of the NIH, National Institutes of Health, USA.

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