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1 Division of Gastroenterology/Hepatology, Department of Medicine, University of Kansas Medical Center, 4035 Delp, MS 1023, Kansas City, KS 66160, USA
2 The First People’s Hospital of Yunnan Province, Kunming, China
3 Gunagzhou Blood Center, Guangzhou City, Guangdong Province, China
4 Department of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
5 Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand
6 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
7 Department of Microbiology, Kobe University School of Medicine, Chuo-ku, Kobe, Hyogo 650, Japan
8 Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken, Japan
9 Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
10 Department of Medicine, Johns Hopkins University, Baltimore, MD, USA
11 Department of Zoology, Oxford University, Oxford, UK
12 Institut National de Santé Publique du Quebec, Laboratoire de Santé Publique du Quebec, Sainte-Anne-de-Bellevue, Quebec, Canada
Correspondence
Ling Lu
llu{at}kumc.edu
Kenrad E. Nelson
kenelson{at}jhsph.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this study are DQ835760–DQ835770.
Supplementary tables showing PCR primers and amplification strategy, and origin of the retrieved sequences reanalysed are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HCV is transmitted efficiently through blood transfusion and injection drug use (Murphy et al., 2000). Transmission is also associated with other parenteral exposures, such as tattooing, ear or body piercing, surgery, acupuncture, haemodialysis, occupational needle-stick injuries and other health-care procedures (Alter, 1994; Alter et al., 1990). Although the efficiency of HCV transmission through sexual or household contact is not yet understood entirely (Luksamijarulkul et al., 2000; Romanowski et al., 2003), the observation of closely related HCV sequences obtained from infected couples or sexual partners could be interpreted as evidence of HCV sexual transmission (Capelli et al., 1997; Chayama et al., 1995; Halfon et al., 2001; Healey et al., 1995; Kao et al., 1992, 2000; Komine et al., 1999; Nakayama et al., 2005; Quer et al., 2003; Rice et al., 1993; Romanowski et al., 2003; Ross et al., 1999; Thaikruea et al., 2004; Yagura et al., 2002).
Currently, in most industrialized countries, HCV antibody screening and mini-pool nucleic acid amplification testing (NAT) are mandatory for virtually all blood donations collected. However, in many developing countries, HCV transmission through blood transfusion remains a critical health problem. For example, in Thailand, resources preclude the general implementation of NAT and, in some rural areas, HCV antibody screening of blood donors is not performed consistently (Luksamijarulkul et al., 2004; Wiwanitkit, 2005). The seroprevalence of HCV among the general population in Thailand has been estimated to be approximately 5 % (3.5 million of the total 63 million population) (Songsivilai et al., 1997; Wiwanitkit & Suyaphan, 2002). The group with the highest risk of HCV infection in Thailand is injection drug users, 95 % of whom have been found to be HCV-infected (Luksamijarulkul & Plucktaweesak, 1996). Another important risk group in Thailand is female sex workers, who have been reported to have an infection rate of 9.5 % (Luksamijarulkul & Deangbubpha, 1997). Minority hill-tribe populations in northern Thailand have also been reported to have a high HCV prevalence of approximately 8 % (Wiwanitkit & Suyaphan, 2002). The seroprevalence of HCV among Thai blood donors has increased since 1991 (Nantachit et al., 2003; Songsivilai et al., 1997) and a number of unique HCV subtypes have been identified in Thailand (Thaikruea et al., 2004).
HCV is classified in the genus Hepacivirus of the family Flavirividae. It has a single-stranded, positive-sense RNA genome of about 9600 nt in length. The genome contains a single open reading frame (ORF) that encompasses nearly the entire genome range. Flanked by non-coding regions (NCRs) at both 5' and 3' ends, this ORF encodes three structural (core, E1, E2) and seven non-structural (P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. HCV has been classified by phylogenetic analysis into six major genotypes; variants within each genotype are grouped into a number of subtypes. Recently, HCV nomenclature has been revised and 71 subtypes in total were defined. Among these subtypes, 17 have been assigned to genotype 6 (subtypes 6a–6q). Geographically, genotype 6 infections are restricted exclusively to south-eastern Asia or immigrants from this region (Lu et al., 2006).
In one of our previous studies, partial sequences representing six HCV subtypes (6a, 6f, 6i, 6j, 6m and 6n) were obtained from samples of blood donors in Chiang Mai, northern Thailand. Despite the enormous importance of genetic diversity for our understanding of HCV infection and evolution, only eight of the 17 subtypes of genotype 6 have had their whole genomes sequenced to date (subtypes 6a, 6b, 6d, 6e, 6g, 6h, 6k and 6n; Li et al., 2006; Lu et al., 2006; Simmonds et al., 2005). As a step towards a comprehensive understanding of HCV diversity, we have sequenced the entire genomes of HCV isolates of subtypes 6f, 6i, 6j and 6m, and report the data in this paper. By using phylogenetic analysis, we found that subtype 6i was genetically similar to 6j and that subtype 6m was similar to 6n. In addition to the four prototypic isolates (Th602, Th553, B4/92 and D86/93) that represent subtypes 6i, 6j, 6m and 6n, we also sequenced the complete genomes of HCV isolates obtained from two HCV-infected couples (Thaikruea et al., 2004), enabling us to study the possibility of interspousal HCV transmission.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. From the remaining four individuals, partial sequences had been determined previously (Apichartpiyakul et al., 1994; Doi et al., 1996; Tokita et al., 1995), which have been used for classifying HCV subtypes 6i, 6j, 6m and 6n, respectively (Simmonds et al., 2005). These individuals included a healthy voluntary blood donor (B4/92), an injection drug user (D86/93) and two patients receiving kidney transplantation (Th553 and Th602).
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). Briefly, RNA was extracted by using Tripure (Roche), cDNA was synthesized by using avian myeloblastosis virus reverse transcriptase (Roche) and random primers (Promega), and overlapping fragments were amplified by using conventional PCR (Roche) with the strategies illustrated in Fig. 1 and primers listed in Supplementary Table S1 (available in JGV Online). Standard procedures were adopted to avoid nested RT-PCR false positives. These included at least a negative control, a positive control and a water blank, which were tested during each batch of RNA extraction, reverse transcription and cDNA amplification. All of these steps and the final product-resolving step were completed in different spaces with considerable distance and floor separation. After amplification, the fragments were sequenced directly by using methods described previously (Lu et al., 2006). All sequence information was analysed by using GCG version 10.0 (Wisconsin Sequence Analysis Package; Genetic Computer Group), PHYML (Guindon & Gascuel, 2003) and MEGA3 (Kumar et al., 2004) software. Initially, multiple alignments were performed with PILEUP and adjustments to the alignments were performed with PRETTY. Phylogenetic trees were reconstructed by using the maximum-likelihood method under the HKY+I+
substitution model (gamma distribution approximated by using six rate categories; Hasegawa et al., 1985). The transition/transversion ratio, proportion of invariable sites and gamma-distribution shape parameter were estimated from the data. The base frequencies were adjusted to maximize the likelihood. Bootstrap resampling was performed by using 500 neighbour-joining replicates. For comparison between isolates, pairwise nucleotide similarities were calculated by using the MEGA3 software. For the detection and analysis of potential genetic recombination events among all available genotype 6 sequences, the RDP2 software (Recombination Detection Program, version 2) was used (Martin et al., 2005). The program was run by using default settings with the following adjustments: (i) window size was 40 nt, (ii) the option of linear sequences was chosen, (iii) six different methods (RDP, GENECONV, MaxChi, Bootscan, Chimaera and SiScan) were run simultaneously against the multiple sequence alignment, and (iv) listing events detected by more than two methods. In addition, the complete genomes were analysed by using the bootscanning approach implemented in the hepatitis C virus subtyping tool available from http://www.bioafrica.net/virus-genotype/.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Their genome lengths and genomic organizations are described in Table 2. Pairwise comparisons of nucleotide sequences over the entire genome and within the ten protein-encoding regions showed that C-0159 was related closely to C-0667, whilst C-0208 was related closely to the reference strain km42. The genome-wide nucleotide similarity between C-0159 and C-0667 was 82.7 %; it was 81.3 % between C-0208 and km42 (Table 3). It has been proposed that HCV genotypes differ by 31–35 % and HCV subtypes by 20–25 % of nucleotides over the entire genome length (Simmonds et al., 1994, 2005). This is consistent with our analysis, which included 189 retrievable complete HCV genomic sequences from GenBank (data not shown). We found that the highest inter-subtype nucleotide similarities were 79.7–80.0 % between subtypes 1a and 1c and 79.5 % between subtypes 6a and 6b. Based on the above criteria, C-0159 and C-0667 qualify as belonging to one subtype, whereas C-0208 and km42 qualify as belonging to another. Phylogenetic analysis was performed with complete genome sequences from 30 reference isolates that represented various HCV genotypes and subtypes; the analysis showed that C-0159 clustered with C-0667 and that C-0208 clustered with km42, with each cluster having a bootstrap score of 100 % (Fig. 2).
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). These isolates have the same ORF and encoded protein sizes as isolates C-0159 and C-0667 (Table 2). Pairwise comparisons revealed that C-0159 and Th602 had a genome-wide nucleotide similarity of 95.3 %, whilst C-0667 and Th553 had a genome-wide similarity of 96.8 % (Table 3). Phylogenetic analysis demonstrated that Th602 clustered most closely with C-0159, and Th553 clustered most closely with C-0667, with each cluster having a bootstrap value of 100 % (Fig. 2). In previous studies, partial sequences of Th602 had been classified into subtype 9b and partial sequences of Th553 classified into subtype 9c (Tokita et al., 1995). In the recent consensus HCV nomenclature proposal, 9b and 9c correspond to subtypes 6i and 6j, respectively (Simmonds et al., 2005). With the addition of the complete genomic sequences of Th602 and Th553, it is possible to classify C-0159 definitively as subtype 6i, and C-0667 as subtype 6j. Subtypes 6i and 6j have genome-wide nucleotide similarities of 82.7–83.4 %. Although this range includes the typical range of values for a single HCV subtype, 6i and 6j still qualify as two distinct subtypes. This is because the two clusters (6i and 6j) are maintained consistently and significantly in phylogenetic analyses of different genomic regions (trees not shown), complying with the criteria for HCV subtype classification (Simmonds et al., 2005). Furthermore, when 6i and 6j sequences were tested against the complete genome sequences of all available genotype 6 subtypes by using a variety of different statistical approaches, no recombination events were confirmed within the ORFs or between the isolates whose sequences were determined in this study (data not shown). This provides strong evidence that subtypes 6i and 6j are not recombinants of other genotype 6 subtypes.
Characterization of the complete sequences for B4/92 and D86/93
Similarly, C-0208 grouped with km42 and the two isolates appeared to represent another single subtype (Fig. 2). For verification, we determined the entire genomic sequences for B4/92 and D86/93, which were the prototype isolates of subtypes 6m and 6n (Simmonds et al., 1996, 2005). The ORF and encoded protein sizes of the two isolates were similar to those of C-0208 and km42, respectively (Table 2). Pairwise comparison revealed that C-0208 and B4/92 had a genome-wide nucleotide similarity of 97.4 %, whilst that between D86/93 and km42 was 93.1 % (Table 3). Phylogenetic analysis showed that B4/92 clustered with C-0208, and D86/93 clustered with km42, with each cluster having a bootstrap value of 100 % (Fig. 2). The pairwise nucleotide similarities of the two clusters were 81.3–81.7 %. Therefore, subtypes 6m and 6n are related closely to each other, but are distinct, a pattern similar to that observed between subtypes 6i and 6j. As before, the distinction of the two subtypes was further confirmed by phylogenetic analyses of different genomic regions and by the lack of evidence for recombination between the complete genomic sequences from all available genotype 6 subtypes (data not shown).
Characterization of the complete sequences for C-0044, C-0046, C-0185 and C-0192
In order to study the possible transmission of HCV between spouses and because no complete genome sequences have been reported for subtype 6f, four isolates from two infected couples were sequenced completely (C-0044, C-0046, C-0185 and C-0192). The sequences of C-0044 and C-0046 were 9454 nt long; those of C-0185 and C-0192 were 9449 nt long. C-0185, C-0192 and C-0208 had the same ORF and encoded protein lengths. However, the C-0044 and C-0046 genomes had smaller E2 regions (1089 nt or 363 aa) and larger NS5A regions (1365 nt or 455 aa) (Table 2). Phylogenetic analysis demonstrated that C-0044 resembled C-0046 closely; both were found on a branch designated subtype 6f. C-0185 resembled C-0192 closely, and they both grouped closely with another subset containing C-0208 and B4/92. The two pairs form a clade of closely related sequences, designated subtype 6m (Fig. 2). In phylogenetic analyses of various genomic regions and in recombination tests against all available complete genotype 6 genome sequences, both C-0044 and C-0046 remained closely related and distinct from all other subtypes. Pairwise comparison showed that C-0044 and C-0046 had a genome-wide nucleotide similarity of 98.1 %, whilst C-0185 and C-0192 had a similarity of 97.8 %. When the complete C-0208 sequence was compared, the nucleotide similarity to C-0185 was 96 % and that to C-0192 was 95.5 % (Table 3).
Based on paired sequences from a single patient with a separation time of 13 years, the rate of HCV evolutionary change was estimated to be 1.92x10–3 substitutions per site per year for the Hutchinson strain (Ogata et al., 1991). By using this molecular clock, we estimated the age of the common ancestor of the pairs of sequences sampled from the two infected couples. A nucleotide dissimilarity of 1.8 % between C-0044 and C-0046 corresponds to a common ancestor existing about 4.9 years ago. Likewise, a nucleotide divergence of 2.2 % between C-0185 and C-0192 corresponds to a common ancestor about 5.7 years ago. These estimates are a reasonable reflection of the known epidemiological data: couple C-0044 and C-0046 had been married for 10 years and couple C-0185 and C-0192 had been married for 24 years.
Co-analyses with retrieved partial HCV sequences
The 11 complete genomic sequences of HCV characterized in this study were analysed phylogenetically in four different genomic regions together with many other sequences retrieved from the Los Alamos HCV database (Kuiken et al., 2005). The four regions included a 302 nt partial core region, a 453 nt partial E1 region and two partial NS5B regions. One of the latter spanned 305 nt in the middle of the NS5B region and the other spanned 366 nt close to the 3' end. Based on the new consensus numbering system proposed for HCV sequences (Kuiken et al., 2006), these four regions corresponded to nt 376–676, 843–1295, 8282–8586 and 8808–9173, respectively, of the complete H77 genome (GenBank accession no. NC_004102
[GenBank]
). Phylogenetic trees based on the core (Fig. 3a), E1 (Fig. 3b) and 366 nt NS5B (Fig. 3d) regions demonstrated that the four subtypes 6f, 6i, 6j and 6m each clustered with between two and 22 retrieved sequences, all from Thailand (Mellor et al., 1995, 1996; Simmonds et al., 1996; Sugiyama et al., 1995; Thaikruea et al., 2004; Theamboonlers et al., 2002; Tokita et al., 1995). The tree of the 305 nt NS5B region (Fig. 3c) identified 10 sequences that were from Myanmar, which borders Thailand (Shinji et al., 2004). A similar pattern of geographical distribution is therefore strongly suggested. In order to give a better interpretation of the phylogenies, detailed information about the origin of the reference HCV sequences is summarized in Supplementary Table S2 (available in JGV Online). Although no recombination was detected by using the RDP2 software, clusters representing the four HCV subtypes 6f, 6i, 6j and 6m were positioned in different parts of the phylogenies. This probably reflects a lack of phylogenetic resolution when subgenomic regions are used to estimate trees; although the sequences within each subtype group with high bootstrap support, the bootstrap values for the grouping together of different subtypes are typically much lower (Figs 3, 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 4; Supplementary Table S2, available in JGV Online). Historically, subtype 6f has been identified from an original HCV variant BB7 (Apichartpiyakul et al., 1994) and then as a novel genotype VII (Sugiyama et al., 1995), a new genotype 7c (Tokita et al., 1995) and the genotype NG(II) (Mellor et al., 1995, 1996). In a subsequent analysis, related variants were reclassified as subtype 6f and this designation has since been used widely (Simmonds et al., 1996). In our previous studies, 13 isolates of this subtype were determined from haemodialysis patients (GenBank accession numbers AY765240
[GenBank]
–AY765252
[GenBank]
) and eight isolates were sequenced from blood donors and a spouse (Thaikruea et al., 2004). The latter included C-0044 and C-0046, which were sequenced completely in this study. Initially, subtype 6i was identified from a new HCV variant, D10/93 (Apichartpiyakul et al., 1994), and then as a novel genotype VIII (Sugiyama et al., 1995), a novel genotype 9b (Tokita et al., 1995) and variants having a restriction fragment-length polymorphism (RFLP) pattern identical to that of subtype 1b (Mellor et al., 1996). In subsequent analysis, the related variants were reclassified as subtype 6i (Simmonds et al., 1996). C-0159 was the sole isolate of this subtype from our previous report (Thaikruea et al., 2004) and was sequenced completely here. For subtype 6j, TH33 was the first identified isolate (Sugiyama et al., 1995). Th553 was initially characterized as belonging to genotype 9c (Tokita et al., 1995). EUTH1 was found to have an RFLP pattern identical to that of subtype 1b (Mellor et al., 1996) and then reclassified into subtype 6j (Simmonds et al., 1996). C-0667 was classified into this subtype (Thaikruea et al., 2004) and has been sequenced completely here. Of subtype 6m, B4/92 was the first isolate (Apichartpiyakul et al., 1994), followed by EUBUR1 (Mellor et al., 1996). They both represented the prototypic sequences for reclassifying subtype 6m (Simmonds et al., 1996). Recently, 11 isolates were characterized among blood donors from Myanmar (Shinji et al., 2004) and three isolates (C-0185, C-0208 and C-0229) were from our previous report (Thaikruea et al., 2004); they all belong to subtype 6m.
The sequences representing the four HCV subtypes 6f, 6i, 6j and 6m are of great interest, because all were identified exclusively in a contiguous geographical region: Thailand and its neighbouring country Myanmar. Common sources of infection were therefore suggested. Epidemiologically, HCV infection has been characterized as having epidemic and endemic patterns. The epidemic pattern is found with HCV genotypes 1a, 1b, 2a, 2b, 2c and 3a. They have spread worldwide over the past 50–70 years as a result of efficient transmission through blood transfusion, injection drug use and unsafe medical practices (Pybus et al., 2001). However, some variants of other genotypes, for example the genotype 4 variants in west-central Africa and genotype 6 in south-eastern Asia, are genetically more diverse, but restricted to a few geographical regions (Ndjomou et al., 2003; Simmonds, 2004). They have been suggested to be endemic with a long-term circulation, a low level of infection and possibly unique patterns of transmission. Eleven genotype 6 isolates that have been sequenced completely in the current study are representatives of such endemic HCV strains.
Subtype classification
A criterion has been proposed that HCV genotypes should differ by 31–33 % and subtypes by 20–25 % of nucleotides over the entire genome length. Moreover, it was found that the nucleotide differences between various subtypes of genotype 6 ranged between 21 and 29 %, with a mean difference of 27 % (Simmonds et al., 1994, 2005). In this study, pairwise comparison of complete sequences revealed that subtypes 6i (C-0159) and 6j (C-0667) only differed by 17.3 % of nucleotides, whereas 6m (C-0208) and 6n (km42) differed by 18.7 %. When the four subtypes were proposed, only partial sequences were available (Simmonds et al., 1996). However, after the representative (C-0159, C-0667, C-0208 and km42) and prototypic (Th602, Th553, B4/92 and D86/93) isolates were sequenced completely, smaller ranges of differences have been obtained. Optimally, precise classification of viral sequences should be based on entire genome sequences. However, complete HCV sequences have been always difficult to obtain, for many reasons. The use of modified procedures in this study has enabled the complete sequencing of 11 isolates, representing four HCV subtypes, each obtained from a serum sample of 100 µl. It has also been recommended recently that accurate HCV classification should be based on extensive phylogenetic analysis, preferably upon analysis of the complete coding region (Simmonds et al., 2005). By following this standard, 6i, 6j, 6m and 6n stand firmly as four different subtypes. This is because they remain consistently and significantly distinct in phylogenetic analyses of entire genome sequences and of partial subgenomic sequences, and in recombination tests against all other genotype 6 subtypes.
Genetic heterogeneity among related viral variants derives from the accumulation of mutations during long-term virus evolution. Although the number of mutations varies among different genomic regions and according to viral and host selection forces, the process of accumulation of mutations driving virus evolution is continual. Although such differences are continual variables, they do tend to show clear discontinuities between genotypes, subtypes and isolates (Simmonds et al., 1996). The classification of HCV into genotypes and subtypes is most useful when the classification scheme matches the epidemiological processes that generate discontinuities in genetic variation – when genetic diversity thresholds are used to define subtype boundaries, they should be flexible enough to accommodate important epidemiological information. The current HCV classification system has distinguished six HCV genotypes and a great number of subtypes. However, some HCV variants may be characterized poorly at the subtype level, and incomplete sampling could in part explain current discontinuities in HCV genetic diversity. As more such variants are identified and sequenced completely, the continual range of subtypes may become wider, with discontinuities being filled by the novel variants observed.
Sexual and interspousal HCV transmission
Although injection drug use and other parenteral exposures are the most efficient modes of HCV transmission, many studies have suggested that the virus can be transmitted by sexual contact. High prevalence of HCV antibody has been detected among commercial sex workers (Mesquita et al., 1997), homosexual men (Osmond et al., 1993), active heterosexuals (Daikos et al., 1994) and patients attending clinics for sexually transmitted diseases (Thomas et al., 1995). It has also been suggested that persons with multiple partners or those at risk of acquiring sexually transmitted diseases have a higher risk for HCV seroconversion than those in long-term, monogamous partnerships (Terrault, 2002). An explosive AIDS epidemic in northern Thailand was attributed primarily to heterosexual transmission of human immunodeficiency virus and to some unique traditions and cultural practices (Beyrer et al., 2005; Morrison, 2004; Nantachit et al., 2003; Weniger et al., 1991). Transmission of HCV between sexual partners may be common in some populations in this region; this could partly explain the limited geographical distribution of subtypes 6f, 6i, 6j and 6m. Four blood donors, C-0159, C-0192, C-0208 and C-0667, each had a history of sex with female sex workers on one to eight occasions and each had had five to ten female partners. These qualify as risk factors associated significantly with HCV infection among blood donors in Thailand (Thaikruea et al., 2004).
Sexual exposure has also been suggested to be crucial for interspousal transmission of HCV, with a longer duration of marriage being a more evident risk factor (Akahane et al., 1994). However, studies on heterosexual monogamous spouses or partners of patients with hepatitis C have revealed only infrequent interspousal HCV transmission (Stroffolini et al., 2001). Although confounded with other risk factors in some cases, a number of interspousal HCV transmissions have been indicated or confirmed by analyses of partial sequences from the core, E1, E2, NS3 and NS5B regions. These included the short span of hypervariable region 1 (HVR1) to best trace recent HCV transmission events. Nucleotide similarities obtained from the infected couples ranged between 96.3 and 100 %. Within the E1–E2 junction region, including HVR1, the similarities varied from 89.6 to 96.6 % (Capelli et al., 1997; Chayama et al., 1995; Halfon et al., 2001; Healey et al., 1995; Kao et al., 1992, 2000; Komine et al., 1999; Nakayama et al., 2005; Quer et al., 2003; Rice et al., 1993; Romanowski et al., 2003; Ross et al., 1999; Thaikruea et al., 2004; Yagura et al., 2002). Notwithstanding, there is a paucity of complete viral sequences isolated from HCV-infected couples. HCV genotypes that have been analysed for interspousal transmission include 1a, 1b, 2a, 2b and 3a, but there are no reports on other genotypes (Table 4). In our previous study, closely related HCV sequences were identified from five HCV-infected couples (Thaikruea et al., 2004) and HCV genomic sequences from two of these couples were determined completely in this study. By using the rate of accumulation of mutations, the level of HCV genetic divergence was in accordance with the timescale of the cohabitation of the couples studied. The present study provides the first report of complete viral sequences that is consistent with the hypothesis of sexual transmission of HCV. However, genetic data from cohabiting couples cannot by itself determine the actual transmission route definitively. An alternative common source of infection for the couples might be their close but less efficient household contacts or other unrecognized parenteral or blood-to-blood exposures (Alter et al., 1990). Moreover, risk factors other than monogamous contact might be the actual cause, as both of the female spouses claimed histories of surgery, blood transfusion and body piercing. One of them had also had body suture (Table 1). Therefore, it is likely that the direction of HCV transmission was from the female spouses to their husbands. On the other hand, the couples may have acquired HCV infection individually from different sources, with the paired viral sequences appearing highly similar. Infections before marriage are also speculative, but the possibilities of these are small. Couple C-0044 and C-0046 had been married for 10 years, whereas the viruses were estimated to have evolved for approximately 5 years. Couple C-0185 and C-0192 had been married for 24 years, but the accumulation of mutations suggested a common virus ancestor existing 5.7 years ago. Both estimates are consistent with interspousal transmission.
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; Doi et al., 1996; Thaikruea et al., 2004). However, as little information was available regarding the B4/92 sample, the true relationship between the two donors cannot be ascertained in this study.
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ACKNOWLEDGEMENTS |
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Received 29 September 2006;
accepted 25 January 2007.
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