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1 Division of Gastroenterology-Hepatology and Nutrition, Department of Medicine, University of Utah, 30N 1900E, Salt Lake City, UT 84132, USA
2 Institut National de Santé Publique du Québec, Laboratoire de Santé Publique du Québec, Sainte-Anne-de-Bellevue, QC, Canada
3 The First People's Hospital of Yunnan Province, Kunming, PR China
4 Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, PR China
5 Department of Immunology and Physiopathology, University of Medicine and Pharmacy in Ho Chi Minh City, Ho Chi Minh City, Vietnam
6 Department of Microbiology, Hebei Medical University, Shijiazhuang, PR China
7 Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan
Correspondence
Ling Lu
ling.lu{at}hsc.utah.edu
Kenji Abe
kenjiabe{at}nih.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this paper. 
The GenBank/EMBL/DDBJ sequence accession numbers for the sequences reported in this study are EF632069–632071.
A supplementary table showing the primers used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The infection can demonstrate no symptoms but manifests a typical form of hepatitis in the majority of the infected individuals. In approximately 80 % of the patients, the infection progresses chronically and therefore constitutes one of the major causes for liver cirrhosis and hepatocellular carcinoma (Hoofnagle, 2002; Zoulim et al., 2003). The main transmission routes are injection drug use, unsafe medical practices and blood transfusion prior to the introduction of screening assays. Transmission is also associated with other parenteral exposures such as sexual contacts (Lu et al., 2007a).
HCV is classified in the Hepacivirus genus of the Flaviviridae family. It possesses a single-stranded RNA genome of positive polarity. The genome is approximately 9600 nt and contains a single open reading frame (ORF) of nearly full genome size. The ORF encodes a large polyprotein precursor that is cleaved into three structural (core, E1 and E2) and seven non-structural (P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins (Major & Feinstone, 1997). Phylogenetically, HCV is classified into six genotypes. Genotypes 1, 2, 3, 4 and 6 are further divided into a number of subtypes. Different HCV genotypes have unique patterns of geographical distribution and are associated with differences in response to interferon therapy (Robertson et al., 1998). The genotype 6 viruses show the greatest genetic diversity. They contain 17 (6a–6q) confirmed and two (6r, 6s) provisionally assigned subtypes and many additional variants (Simmonds et al., 2005; Murphy et al., 2007). These variants may be candidates for as yet unassigned novel subtypes. Previously, we characterized the complete genome sequences for 11 assigned subtypes and two novel variants (km41 and gz52557), using samples from patients in south-east Asia or immigrants from this region. These studies have completed a full panel of genomes for subtypes 6a–6q (Li et al., 2006; Lu et al., 2006, 2007a, b). In the present study, we further describe the complete genomes of three novel HCV variants. These variants qualify for a new subtype, designated 6t.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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PCR and sequencing.
Each of the complete genomic sequences was amplified from 100 µl serum, essentially as described previously (Li et al., 2006). Briefly, RNA was extracted using TriPure (Roche) and cDNA was synthesized by use of random primers (Promega) and AMV (avian myeloblastosis virus) reverse transcriptase (Roche). The cDNAs were amplified using conventional PCR (Roche) with strategies illustrated in Fig. 1 and primers listed in Supplementary Table S1 (available with the online version of this paper). Standard procedures were adopted to avoid nested RT-PCR false positives. The fragments amplified were sequenced directly.
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) and pairwise nucleotide similarities were estimated using MEGA3 (Kumar et al., 2004). Potential genetic recombination events were examined using the RDP2 software (Martin et al., 2005) and the bootscanning approach. All these procedures were performed as described previously (Lu et al., 2007a). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These resulted in three genomes of 9407, 9460 and 9445 nt. The 5'UTRs have 324, 338 and 338 nt followed by single ORFs of 9051 nt. The 3'UTRs have 32, 71 and 56 nt, including the poly(U) tracts of 29 and 27 nt for the TV241 and TV249 isolates. The three sequences shared common sizes in the ten protein-encoding regions. These included the core (573 nt or 191 aa), E1 (576 nt or 192 aa), E2 (1092 nt or 364 aa), P7 (189 nt or 63 aa), NS2 (651 nt or 217 aa), NS3 (1893 nt or 631 aa), NS4A (162 nt or 54 aa), NS4B (783 nt or 261 aa), NS5A (1356 nt or 452 aa) and NS5B regions (1776 nt or 591 aa) (see Table 1). These sizes are identical to those of the QC227/6o and QC216/6p isolates (Lu et al., 2007b).
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). When compared to the 19 reference sequences, the nucleotide similarities were 71.7–79.4 % (mean 75.4 %) over the entire genome and 71.0–78.8 % (mean 75.0 %) over the entire ORF. Higher similarities were observed in comparisons to 6o (mean 78.8 %) and 6q (mean 79.3 %), while there were lower similarities to 6a (mean 71.9 %) and 6b (mean 73.2 %). Within the 10 protein-encoding regions, the core (mean 87.1 %) and NS5B (mean 77.8 %) presented the highest similarities, while P7 (mean 66.8 %) and NS2 (mean 68.6 %) presented the lowest. Isolate 6q/QC99 showed the highest similarities to VT21, TV241 and TV249 in five genomic regions: E2 (mean 73.3 %), NS2 (mean 73.3 %), NS3 (mean 80.5 %), NS4A (mean 76.9 %) and NS5A (mean 75.8 %). Isolate 6p/QC216 displayed the highest similarities (mean 90.2 % and 78.8 %, respectively) in core and E1, 6e/GX004 in P7 (mean 76.9 %) and NS5B (mean 82.0 %) and 6d/VN235 in NS4B (mean 80.3 %). VT21, TV241 and TV249 were closely related yet subtypically distinct from the 19 reference sequences, by a criterion described previously (Simmonds et al., 1996).
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). VT21, TV241 and TV249 formed a close cluster in the tree with full (100 %) bootstrap support. The branch leading to the three isolates appeared equivalent to the branches directing the 6a–6q and km41 and gz52557 genomes. Consequently, VT21, TV241 and TV249 were assigned to subtype 6t, following the recently described 6r and 6s subtypes (Murphy et al., 2007). Within genotype 6, four major groups were formed. From the lower part of the tree (Fig. 2a), the first group included subtypes 6a and 6b. The second group included 6h, 6j, 6i, 6m, 6n, 6l, 6k and km41. The third group included 6g and gz52557. The fourth group included 6f, 6d, 6c, 6p, 6o, 6e, 6q and 6t. A second phylogeny was reconstructed using the predicted amino acid sequences. The tree topology was analogous to that obtained from the nucleotide sequences (Fig. 2b). Finally, the nucleotide sequences from the 10 protein-coding regions were analysed separately. Phylogenetic trees consistently showed the three 6t isolates to be distinct from the 19 reference sequences (data not shown).
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Phylogenetic analysis with database sequences
VT21, TV241 and TV249 were compared to genotype 6 variants for which partial genome sequences were available. Segments studied included 308 nt of core, 421 nt of E1 and 340 nt of NS5B regions, corresponding to nt positions 352–659, 869–1289 and 8276–8615 of the H77 genome (NC_004102
[GenBank]
), respectively. Trees estimated from these segments showed new findings.
Firstly, five reported isolates grouped with VT21, TV241 and TV249. They showed bootstrap values of 88, 96 and 98 % for sequences of core, E1 and NS5B regions, respectively (Fig. 3a–c). Among them, D49 (GenBank accession nos DQ155472
[GenBank]
and DQ155530
[GenBank]
) was from a blood donor in Vietnam with partial sequences available in the core and NS5B regions (Noppornpanth et al., 2006). QC131 (GenBank accession nos EF115932
[GenBank]
and EF116155
[GenBank]
), QC145 (EF115934
[GenBank]
and EF116157
[GenBank]
), QC240 (EF115948
[GenBank]
and EF116171
[GenBank]
) and QC351 (EU127994
[GenBank]
and EU127995
[GenBank]
) were from Vietnamese immigrants in Quebec, Canada with partial sequences available in the E1 and NS5B regions (Murphy et al., 2007). The finding of five additional isolates grouping with VT21, TV241 and TV249 in patients originating from Vietnam consolidates the designation of this new subtype.
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; Lwin et al., 2007). Using E1 gene sequences for analysis, NK046 (GenBank accession no. EF158183
[GenBank]
) clustered with CMBD-14 (DQ179116
[GenBank]
) and CMBD-86 (DQ179143
[GenBank]
), which were isolated from Thailand (Fig. 3b). Of the CMBD-14 isolate, a partial sequence was also available in the NS5B region (GenBank accession no. DQ179117
[GenBank]
), and this sequence grouped with the NK046 isolate (EU158186) (Fig. 3c). Although these two clusters do not meet the minimal requirements for the provisional designation of new subtypes (Simmonds et al., 2005), they are strong candidates.
The analysis of partial genome sequences revealed the existence of additional novel genotype 6 variants. The tree reconstructed with core gene sequences showed five branches that do not associate with any one of the described subtypes (Fig. 3a). Clockwise from the outgrouped ED43 isolate, these branches denote variants North 5 (GenBank accession no. AY190347
[GenBank]
), VT887 (AB162873
[GenBank]
), MYAN-C184 (AB269339
[GenBank]
), IG57272 (AY734479
[GenBank]
) and N103 (AY921163
[GenBank]
), respectively. The tree reconstructed with E1 gene sequences had seven such branches (Fig. 3b). Clockwise, these branches identify QC81 (GenBank accession no. EF115928
[GenBank]
), IG57272 (AY734480
[GenBank]
), QC112 (EF115929
[GenBank]
), QC251 (EF115952
[GenBank]
), QC56 (EF115927
[GenBank]
), QC269 (EF115955
[GenBank]
) and QC148 (EF115935
[GenBank]
), respectively. Partial sequences in the NS5B gene were also available for the latter seven isolates: QC81 (GenBank accession no. EF116151
[GenBank]
), IG57272 (AY734481
[GenBank]
), QC56 (EF116150
[GenBank]
), QC112 (EF116152
[GenBank]
), QC148 (EF116158
[GenBank]
), QC251 (EF116175
[GenBank]
) and QC269 (EF116178
[GenBank]
). They are identified in the same clockwise manner (Fig. 3c). These novel variants may represent additional new subtypes of genotype 6. However, the phylogenetic relationships of the core region sequences appeared to be not equivalent to that observed for the E1 and NS5B regions, nor to that observed for the full genome sequences. For example, in the core phylogeny, subtypes 6a and 6b do not group together. Neither do 6c and 6d. One possibility is that the core phylogeny may not represent what was observed in the E1 and NS5B phylogenies, and that some of the novel variants identified in the core phylogeny could be of the same lineage as that observed in the E1 and NS5B trees. For these reasons, we may only consider the seven variants with consistent divergence in the E1 and NS5B phylogenies as distinct novel genotype 6 variants.
Finally, trees reconstructed with partial E1 and NS5B region sequences (Fig. 3b and c) consistently showed three clusters that group closely to those of assigned subtypes. Close to 6e, a fork was structured by two variants, QC191 (GenBank accession nos EF115943
[GenBank]
and EF116166
[GenBank]
) and QC323 (EF115974
[GenBank]
and EF116197
[GenBank]
). Near to 6k, km41 and km45, two branches split. One led to the variants QC226 (GenBank accession nos EF115947
[GenBank]
and EF116170
[GenBank]
) and QC327 (EF115975
[GenBank]
and EF116198
[GenBank]
), the other led to QC273 (GenBank accession nos EF115957
[GenBank]
and EF116180
[GenBank]
). From the trunk where subtype 6q grew, three branches deviated. They directed QC150 (GenBank accession nos AY754611
[GenBank]
and AY754612
[GenBank]
), QC197 (AY754629
[GenBank]
and AY754630
[GenBank]
) and QC266 (EF115954
[GenBank]
and EF116177
[GenBank]
). These variants show greater genetic distance with their neighbours than with isolates of single subtypes, but smaller distance than that observed between isolates of different subtypes.
Sequence comparison within the 5'UTR
Previously, we found that the 5'UTR sequences of QC131, QC145 and QC240 (GenBank accession nos EF115712
[GenBank]
, EF115714
[GenBank]
and EF115728
[GenBank]
, respectively, corresponding to nt 96–291) were identical to that of genotype 1b isolate HCV-BK (Murphy et al., 2007). The 5'UTR sequences of VT21, TV241, TV249 and QC351 were also compared with HCV-BK. In this region (nt 96–291) VT21 and QC351 were also identical to BK, but TV241 and TV249 showed two and one nucleotide differences from BK. Thus, in this region of the 5'UTR targeted by genotyping assays, genotype 6t isolates do not show a distinct sequence profile that could distinguish them from genotype 1 sequences. When sequences outside this range were compared, more nucleotide changes were observed for the VT21, TV241 and TV249 isolates (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The three variants described in this study meet these criteria for a confirmed novel subtype of genotype 6, namely 6t. When the genotype nomenclature was updated, 17 subtypes (6a–6q) were designated within genotype 6. Recently, two additional subtypes, 6r and 6s, have been provisionally assigned from partial sequences (Murphy et al., 2007). Following this order, we designed these three variants as subtype 6t.
Analyses of partial sequences revealed that five additional isolates also clustered into subtype 6t. Among them, D49 was isolated from a blood donor in Vietnam (Noppornpanth et al., 2006). Four variants, QC131, QC145, QC240 and QC351, were isolated among Vietnamese immigrants in Quebec, Canada (Murphy et al., 2007). This result not only verified the existence of subtype 6t but also indicated that the 6t variants may be exclusively endemic in Vietnam. Since relatively few patients were investigated, studies are needed to further characterize the geographical distribution, the frequency of infection among the general population, the clinical manifestations and the response of these variants to antiviral therapies. The fact that this subtype was identified outside of Vietnam, i.e. in Canada, suggests that it may also be present in other parts of the world. Since most laboratories use the 5'UTR for HCV genotyping purposes, 6t isolates are likely to be missed due to the similarity of its 5'UTR sequence to that of genotype 1b. Therefore, the worldwide prevalence and distribution of genotype 6t, and of genotype 6 subtypes other than 6a and 6b for that matter, can only be established by analysis of appropriate coding region sequences.
Analysis of partial sequences from the core, E1 and NS5B regions indicated the existence of numerous additional genotype 6 variants. Two types of relationships were observed. The first type corresponds to isolates showing genetic diversities that are great enough to classify them into new subtypes. Among them, NK046, CMBD-14 and CMBD-86 formed one cluster and DH012, MYAN-3E-3 and MYAN-TC-148 constituted a second cluster (Shinji et al., 2004; Lwin et al., 2007). Single variants IG57272, QC56, QC81, QC112, QC148, QC251 and QC269, characterized in both E1 and NS5B, may be analogous to km41 and gz52557 (Lu et al., 2006). These may represent an additional nine new subtypes, although the confirmation requires more isolates to be identified and longer fragments or complete genomes to be sequenced (Simmonds et al., 2005). Given these subtypes are established, there is a potential for at least 31 subtypes under genotype 6, including 6a–6t, km41 and gz52557, plus nine unassigned variants. With such a great genetic diversity and complexity, one may wonder what roles they have played during virus evolution and transmission. Geographically, these variants are endemic in the tropical south-east Asian countries. Zoonotic factors may have been involved in the virus biological processes (Lu et al., 2007b). The correlation between these factors and the great genetic diversity of HCV remains to be determined. One trivial question may concern how to assign the potentially new subtypes after the use of 6u to 6z. The current HCV genotype nomenclature has been well established and has resolved the inconsistencies observed in the literature. A nomenclature to identify subtypes past ‘z’ is required to avoid recreating the confusion that now seems to have disappeared.
The second type of relationship observed is that of variants showing genetic distances greater than those within subtypes, but smaller than those between subtypes. Among them, QC191 and QC323 were related to subtype 6e, QC226, QC273 and QC327 to 6k, and QC150, QC197 and QC266 to 6q. The constant accumulation of mutations is thought to have driven continual development of HCV genetic diversity. However, the genetic diversity is a variable that has shown clear discontinuities between HCV genotypes, subtypes and isolates. The discontinuities are possibly due to the missing detection of some HCV variants or incomplete sampling (Lu et al., 2007a). It is likely that this second group of variants may partially fill the discontinuities between subtypes and isolates when their complete genomes are sequenced.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 19 September 2007;
accepted 24 October 2007.
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, Missing nucleotides; ., nucleotides identical to that of BK.