CorresDondence

A Proposed System for the Nomenclature of Hepatitis C Viral Genotypes

PETER SIMMONDS,' ALFRED0 ALBERTI,~ HARVEY J. ALTER,:^ FERRUCCIO BONINO,* DANIEL W. BRADLEY,5 CHRISTIAN BRECHOT," JOHANNES T. BROUWER,~ SHIU-WAN CHAN,' W U A K I c H A Y M , 8 DING-SHINN CHEN,' QUI-LIM CHOO," h!L4SSIMO COLOMBO," H. THEO M. CUYPERS," TAKAYAsU DATE,13 GEOFF M. DUSHEIKO,l*

JUAN I. ESTEBAN,15 OSCAR FAY.'" S . J . HADZTYANNIS,17 JANG IIAN,18 ANGELOS HATZAKIS," EDDIE c. HOLMES,~~

GEORGE KU0,24 JOHNSON Y.N. LAU,'" P. NICO LELIE,'~ GEERT MAERTENS,27 FIONA MCOMISH,~~ TATSUO MIYAMURA," MASASHI MIZOKAMI,:~~) !XI0 NOMOT0,31 ALFRED M. PRINCE,32 HENK w. REESINK,33

CHARLIE RICE,34 MICHAEL ROGGENDORF,"5 SOLKO w. SCHALM,7 TOSHIO SHIKATA,37 KUNITADA SHIMOTOHN0,36 LIEVEN sTUYVER,27 CHRISTIAN TREP0,38 AMY WEINER, lo PENG L. YAPz8 AND MICKEY s. URDEA22

HAK HOTTA,~' MICHAEL HOUGHTON, l o BRUCE I R V I N E , ~ ~ MICHINORI KOHARA,~~ JANICE A. KOLBERG,~~

To the Editor:

We are researchers who have published analyses of nucleic acid sequence variation of hepatitis C virus (HCV) and associated virological and clinical signifi- cance. We are concerned that our investigations are hampered by the lack of a consensus nomenclature for variants of HCV and that this leads to confusion when results from different laboratories are compared. Fur- thermore, because there are no consistently applied criteria by which new genotypes are defined, investi- gators assign new type descriptions to novel sequence variants on an ad hoc basis without agreement from

'Department of Medical Microbiology, Ilniversity of Edinburgh Medical School, Edinburgh EH8 9AG, U K 'Clinica Medica 2. lstituto di Medicina Clinica, Universita di Padova, 35123 Padova, Italy: "Immunology Section, Department of Transfution Medicine, Warren G. Magnusson Clinical Center, National Institutes of Health, Bethesda, MD 20892; 4Laboratory of the Department of Gastroenterology, Ospedale Molinette, 10126 l'orino, Italy, 5Hepatitis Branch, Division of Viral Diseases. Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333. "Liver Cancer and Molecular Virology, INSERM U370, Faculte de Medecme, Necker Enfants-Malades, 75730 Paris Cedex 15, France; 'University Hospital Rotterdam, Dijkzigt Hospital, 3015 GD Rotterdam, The Netherlands; 'Department of Gastroen- terology, Toranomon Hospital, Tokyo, Japan, "Hepatitis Research Center, Taiwan University Hospital, Taipei, Taiwan; '"Non-A/Non-B Research, Chiron Corp., Emeryville, CA 94608; "Cattedra di Medicina Interna 1, Universita Degli Studi di Milano, Istituto di Medicina Interna, 20122 Milano, Italy; "Department Development Research, Virus Diagnostics, CLB, Central Labo- ratory of the Netherlands Red Cross Blood Transfusion Service, 1066 CX Amsterdam, The Netherlands; 13Department of Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa 920-02. Japan; "Department of Medicine, Royal Free Hospital School of Medicine, University of London. London NW3-2PF, U K l6Liver Research Unit, Department de Medicina Interna, Hospital General Universitari, Servei d'Hepatologia, 08035 Barcelona, Spain; IBFacultad de Ciencias Bioquimicas y Farmaceuticas, Centro de Tec- nologia en Salud Publica, Universidad Nacional de Rouario. 2000 Rosario. Republica Argentina; "Internal Medicine, Hippokraton General Hospital, 115 27 Athens, Greece; lRMolecular Biology. Chiron Corp.; ISNational Ret- rovirus Reference Center, Department of Hygiene and Epidemiology, Athens University Medical School, 115 27 Athens, Greece. '"Department of Zoology.

other groups. There are at least four different systems of nomenclature that differ in the degree of sequence divergence required to assign a new virus type and in the range of sequence variants compared. In many cases, it is not possible to equate particular variants in one classification with those in another because different regions of the genome have been compared.

The clinical consequences of sequence variability are unknown. However, it is likely that major antigenic differences exist between genotypes. Antibody elicited by infection with HCV to the envelope proteins E l and E2 might fail to cross-neutralize heterologous virus geno- types. This would have important consequences for

Iiniversity of Oxford, South Parks Road, Oxford, UK; ZIDepartment of Microbi- ology, Kobe University School of Medicine, Kobe 650, Japan; 22Nucleic Acid Systems, Chiron Corp.; "Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan; 241mmunoche~t ry , Chiron Corp.: "Section of Hepatobiliary Diseases, Department of Medicine, University or Florida. Gainesville, Florida 32610; "Department of Virus Serology, Diag- nostics Division, CLB, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service; "71nnogenetics NV/SA, B-9052 Zwijnaarde Ghent Belgium; '"Scotland and Southeast Scotland Blood Transfusion Service, Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh EH3 9HB, UK, ZsDepartment of Virology 11, National Institute of Health, Tokyo 162, Japan; 30Second De- partment of Internal Medicine, Nagoya City University Medical School, Nagoya 467. Japan; 31Department of Microbiology, The Institute of Medical Science at The University of Tokyo, Tokyo 108, Japan; S"Laboratory of Virology and Para- sitology, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NewYork 10021; "3Red Cross Blood Bank, 1066 CXAmsterdam, The Netherlands; 34Department of Molecular Microbiology, Washington University School of Medicine at The Washington University Medical Center, St. Louis, Missouri 631 10; 35Universitiitsklimikum Essen, Robert-Koch-Haus, Institut fur Virologie, World Health Organization Collaborating Centre for Reference and Research on Neurological Zoonoses, Medizinische Einrichtungen der Univer- sitiit, D-4300 Essen 1, Federal Republic of Germany; 37Department ofPathology, Nihon University School of Medicine; "Virology Division, National Cancer Center Research Institute, Tokyo 104, Japan; and 39Research Unit on Hepatitis, AIDS and Human Retrovirus, INSERM U271, Institut National de la Sant6 et de la Recherche Medicale, 69424 Lyon Cedex 03, Franm.

H EPATOLOGY 1994; 19: 132 1 - 1324. 3 1/8/54774

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TABLE 1. Sequence similarity (%) between different genotypes of HCV in the NS-6 region Alternative names"

No. of sequences compared in each

Proposedname Ch Si Ok En Ts group

la l b l c 2a 2b 2c 3a 3b 4a 5a 6a

I l a I PT I I1 l b I1 K1 I NC NC NC NC NC I11 2a I11 K2a I1 I11 2b IV K2b I1 I11 NC NC NC NC IV 3 V NC NC IV NC NC NC NC NC 4 NC NC NC V NC NC NC NC NC NC NC NC NC

12 19 2 6

10 4

13 2 3 4 1

96 80 94 83 77 95 62 63 66 92 64 66 67 79 93 63 63 64 81 79 92 67 66 65 62 64 62 95 67 71 69 63 66 62 78 99 66 62 65 61 63 62 64 68 88 69 70 69 65 66 63 65 69 65 96 66 65 62 65 65 64 64 62 66 67 NA la l b l c 2a 2b 2c 3a 3b 4a 5a 6a

NC, not classified; NA, not available. Comparisons were made of nucleotide sequences between positions 7975 and 8196 (numbered as in [21). "Nomenclature according to existing schemes: Ch, Houghton et al. (15); Cha, et al. (17); Si, Chan et al. (12) and Simmonds et al. (16); Ok,

Okamoto et al. (10) and Mori et al. (14); En, Enomoto et al. (11) and Tsukiyama-Kohara et al. (13).

future vaccines for HCV. Second, some studies have suggested significant biological differences between dif- ferent genotypes, both in terms of disease progression and, more particularly, in the sensitivity of different variants to treatment with interferons.

In a recent collaborative study (11, nucleotide se- quence comparisons were made in the NS-5 region between HCV variants present in a worldwide panel of HCV-infected individuals and published sequences. Phy- logenetic analysis revealed clustering of sequences into six major groups, some of which contained two or three distinct clusterings of more closely related variants (subgroups). Sequence similarities between members of the different groups ranged from 55% to 72% (mean, 64.5%), whereas similarities of the more closely related variants in the subgroups ranged from 75% to 86% (mean, 80%). Individual isolates within each of these clusters showed 88% sequence similarity.

Although different regions of the viral genome show differing degrees of sequence divergence, the same three nonoverlapping ranges of sequence variation are ob- served for all the coding regions (with the exception of certain hypervariable regions of the envelope proteins, where the high degree of variability obscures phyloge- netic relationships). Comparison of eight complete ge- nomic sequences gives sequence similarities of 67.1% to 68.6% between variants in two major groups (12 painvise comparisons) and 77.0% to 78.9% between those in subgroups (nine pairwise comparisons). These clear groupings give rise to the proposed nomenclature.

Proposal. We propose a classification of HCV based on nucleotide sequence similarity, with the major groupings of sequence variants designated HCV types and the more closely related groups observed within some types termed subtypes (Table I). HCV types should be numbered in order of discovery with arabic numerals to avoid confusion with previous schemes. Subtypes

should be identified by lower-case letter, again in order of discovery. Thus the genotype of the variant first cloned by Choo et al. (2) is called subtype la, and the complete genomic sequences that show an overall sequence similarity of 77.0% to 78.9% with subtype l a are designated l b (3-8). The more divergent complete genomic sequences HC-J6 and HC-JS (67.1% to 68.6% sequence similarity with subtypes la and lb) are designated subtypes 2a and 2b, respectively (9, 10).

This nomenclature closely follows the schemes origi- nally described by Enomoto et al. (11) on the basis of sequence comparisons of the NS-5 region (PT [la], K1 [lbl, K2a [2al and K2b [2bl) and by Chan et al. (121, who provided evidence for another HCV type (subtype 3a) on the basis of formal phylogenetic analysis of the NS-5, NS-3, core and 5' noncoding regions. A similar scheme assigned subtypes la and l b to group I and types 2a and 2b to group I1 (13). Subtype 3a and another genotype, genotype 3b, were described by Mori et al. (14) as types V and VI, respectively. This nomenclature was an exten- sion of that proposed by Okamoto et al. (9), which desig- nated subtypes la, lb, 2a, and 2b types I, 11, I11 and IV, respectively. A similar scheme assigned subtype la and l b to groups I and 11, respectively; type 2 (a and b) to group 111; and type 3 to group IV (15). Evidence for further genotypes of HCV has been provided by Sim- monds et al. (16) and Stuyver et al. (18) and confirmed by means of analysis of the core region (16). Another geno- type (groupv) was reported by Cha et al. (17) (now called 5a) on the basis of sequence'comparisons in 5' noncoding region, core and NS-5 regions. These assignments have been confirmed by formal phylogenetic analysis of se- quences in the NS-5 region (1). Recently, evidence of the existence of many of these genotypes has been obtained by means of sequence comparisons in the E l gene (19).

Assignment of Sequences Within the Scheme. The ideal material for sequence comparisons is the complete

HEPATOLOGY Vol. 19, No. 5, 1994 CORRESPONDENCE 1323

viral genomes, and this should remain the goal of any nucleotide sequencebased system of virus classifi- cation. However, this aim is not practical. Identification of new HCV variants and their assignment as new geno- types has often been based on comparisons of short sub- genomic fragments of sequence amplified by means of polymerase chain reaction or by cDNA cloning. This approach is acceptable for the following reasons. (a) Se- quence relationships derived from analysis of one part of the coding region are equivalent to those produced in others (12, 17) (apart from hypervariable regions in genes for envelope proteins), and these match those pro- duced by sequence analysis of complete viral genomes (12, 10). (b) There is plentiful comparative sequence data for specific subgenomic regions of HCV, but there are complete genomic sequences only for subtypes la , lb, 2a and 2b. In the NS-5 region, sequences of six geno- types (and eight subtypes) between nucleotide positions 7975 to 8196 (numbered as in C21, have been identified (1). The region may be readily amplified from plasma of infected individuals with previously published conserved primers (1 1); techniques for further amplification using nested internal primers have been published elsewhere (1 1. Sequence data for the core region have been obtained from types 1 through 4. For both core and NS-5 regions, the ranges of sequence similarities between different types and subtypes are known and may act as guides for the assignment of new sequences (1). In the future it may also be possible to assign new genotypes from other regions as sequence data accumulates. For example, se- quences in the E l region for a wide range of genotypes were recently published (19).

Consequently, we suggest that identification and classification of new genotypes should be divided into at least two stages.

(a) Provisional identification could initially be based on measurement of percent sequence similarities be- tween the new sequence and those previously classified. For example, in the part of NS-5 amplified by primers described by Enomoto et al. ( 11 1, sequence similarities of less than 72% with any known sequence would be evidence for the existence of a new HCV type, whereas maximum sequence similarities of 75% to 86% with some members of the data set would provide evidence of a new subtype (Table 1).

(b) Identification could be confirmed by (i) formal phylogenetic analysis of the new sequence with those already obtained (the branching order within the re- sulting unrooted tree will indicate the relationship of the variants with existing genotypes); and (ii) observation of equivalent phylogenetic relationships using nucleotide sequences from at least one other coding region of the genome.

(c) We propose that a committee be instituted to receive submissions from researchers with new se- quence data. Type numbers and subtype letters will be assigned in order of discovery, irrespective of their geographic origin, according to the above criteria. Because of delays associated with publication, re- searchers with new sequence variants should be invited

to assign type or subtype status to them but not to specify actual type numbers or subtype letters until acceptance has been agreed on by the committee. This should help prevent proliferation of typing schemes.

(d) We also propose that the committee make freely available the consensus sequences in NS-5 and other regions for the currently assigned types and subtypes of HCV to the research community. This would enable more rapid provisional identification of new genotypes.

According to the criteria described above, the known variants of HCV can be classified as follows.

Confirmed. (a) Complete genomes: subtype la (2, 20, 21), subtype l b (4 ,5 ,6 ,7 ,8) , subtype 2a (9) and subtype 2b (10); and (b) partial genomes: subtype 3a (core, NS-3, NS-5; [121), subtype 2c (El , NS-5; [16, 171) and subtype 4a (core, NS-5 [161).

Provisional. Subtypes lc, 3b, 5a and 6a (sequence data from NS-5 only in the coding region; 11, 14, 16, 171).

In summary, we believe that the typehubtype nomen- clature more closely reflects the observed two-tiered nature of HCV sequence variation found in all coding regions of the genome (with the exception of parts of E2). Most variants analyzed to date show little evidence of recombination, as manifested by the presence of se- quences of one genotype in one part of the genome, and of a different genotype elsewhere. Should such recom- binant viruses be found, the procedure for assigning genotypes would have to be modified. Classification of viruses on the basis of sequence relationships rather than by biologcal properties has advantages of objec- tivity and clarity and does not depend on often complex biologcal assays such as the cross-neutralization, the c.ytopathogenicity in tissue culture and pathogenicity in experimental animals. Furthermore, without the general use of a satisfactory virus culture system for HCV, and with a host range restricted to human beings and higher primates, most of the biological assays that have been used are laborious, expensive or impossible.

It is probable that additional grouping of HCV based on these properties will arise in the future, but until then the proposed nomenclature should serve our requirements.

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