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Short Communication |
1 Institut ‘Cavanilles' de Biodiversitat i Biologia Evolutiva, Universitat de València, 46070 València, Spain
2 Hospital General Universitari, Avinguda Tres Creus s/n, 46014 València, Spain
Correspondence
Fernando González-Candelas
fernando.gonzalez{at}uv.es
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the complete HCV genome sequence described in this study is AJ851228 [GenBank] .
Supplementary material is available in JGV Online.
These authors contributed equally to this work. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Hepatitis C prevalence is far from being distributed evenly in human populations and varies widely depending on the risk population and the world region being studied (Memon & Memon, 2002). Its genome consists of a positive-sense RNA molecule of approximately 9.6 kb, which encodes a polyprotein of about 3000 aa. The nucleotide sequence of the HCV genome is highly variable and, based on genetic distances and phylogenetic analyses, all isolates are currently grouped into six genotypes [formerly denoted as clades by Robertson et al. (1998)], corresponding to the main branches in the phylogenetic tree. The same kind of analysis applied to isolates within each genotype has revealed several clusters of closely related sequences, denoted as subtypes (Simmonds et al., 2005).
Simple estimates of genetic distances for the core, E1 and/or NS5B regions have been proposed to distinguish between types, subtypes and isolates (Simmonds et al., 1993; Stuyver et al., 1994). However, as more branches have been added to the HCV phylogenetic tree, type and/or subtype assignations often have become ambiguous. Moreover, intense sequence characterization of new partial HCV genomes has shown many instances in which genetic distance estimates were in between those corresponding to genotype- and subtype-delimiting ranges (Robertson et al., 1998; Simmonds et al., 2005). Hence, some confusion was generated when this distance-based criterion was used for classification and nomenclature of novel putative genotypes or subtypes (Jeannel et al., 1998; Stuyver et al., 1994; Tokita et al., 1994a, b, 1995, 1996). In fact, recent proposals (Robertson et al., 1998; Simmonds et al., 2005) to establish a more comprehensible classification of HCV recommended that sequence-based classification and/or nomenclature should be based on extensive phylogenetic analysis rather than genetic distance, and preferably should be based on the complete sequence of the coding region for new genotypes. However, in order to assign a given sequence to a particular HCV subtype, Simmonds et al. (2005) recommended consistent phylogenetic grouping based on at least partial genomes, including both the core/E1 region and the NS5B region (nt 869–1296 and 8276–8615, respectively, in the H77 sequence reference; GenBank accession number AF009606
[GenBank]
), for which a considerable amount of sequence data are available. Nonetheless, due to the wide epidemiological use of subtype assignations, three or more examples of independently infected individuals with a new proposed subtype will be required for a definitive, subtype-specific designation (Simmonds et al., 2005).
The most widespread variants of genotype 1 isolated in Western countries can be classified clearly as subtype 1a or 1b. However, further molecular epidemiological research in some West and Central African countries has demonstrated that much greater diversity exists not only within this particular genotype, but also within genotypes 2 and 4 (Candotti et al., 2003; Ndjomou et al., 2003; Njouom et al., 2003; Pasquier et al., 2005; Simmonds et al., 2005). Previous phylogenetic studies have shown that genotypes 1 and 4 form a statistically well-supported cluster (Salemi & Vandamme, 2002) in contrast to the entirely separate and independent branching at the base of the tree of the remaining genotypes. Moreover, phylogenetic analysis of partial sequences of highly variable HCV genomes isolated in West and Central Africa suggest that these two genotypes share a common origin in the region currently known as Cameroon and would have spread subsequently to other regions of Africa and the rest of the world (Ndjomou et al., 2003). Almost certainly, more confident conclusions could be reached if complete genomes rather than partial genomes were used in phylogenetic (Salemi & Vandamme, 2002) and genome recombination studies (Colina et al., 2004; Kalinina et al., 2002), but complete genomes from African isolates are especially scarce: only two complete genomes belonging to genotype 4 (Bukh et al., 1998) and genotype 5a (Chamberlain et al., 1997) have been determined so far.
In the present study, we report the complete genome of an HCV isolate from a patient of African origin. Based on both genetic distance and phylogenetic analyses, this isolate could well be a representative of a new subtype within genotype 1. However, given the recent recommendations for a unified system of nomenclature (Simmonds et al., 2005), no specific subtype designation can be applied to this case as yet.
A serum sample from a native Equatorial Guinean patient was obtained as part of an HCV molecular epidemiology study in the Comunitat Valenciana (Spain). The complete HCV genome (9481 nt; GenBank accession number AJ851228
[GenBank]
) from this patient's serum was obtained from six overlapping RT-PCR fragments, which subsequently were sequenced directly (see details in Supplementary Material, available in JGV Online). The initial amplification design consisted of only three overlapping fragments of 
3000 nt in the first-round PCR, followed by a hemi-nested second-round PCR, but this was achieved for only two of the three fragments. Although this design did not work successfully for one fragment, it could be a suitable approach for sequencing complete genomes, as the modification of conventional protocols required would not be extensive (Lu et al., 2005; Rispeter et al., 1997). Such minor modifications would probably involve a longer extension step during PCR and generously degenerate primers for a highly variable, first-read sequence, in order to ensure primer–cDNA hybridization.
Phylogenetic analysis of this new complete genome along with representatives of the six HCV genotypes described previously was restricted to the coding portion of the genome. Maximum-likelihood phylogenetic reconstructions (Fig. 1) revealed that the new sequence grouped clearly with HCV genotype 1 sequences, but grouped separately from any of the subtypes (1a, 1b and 1c) described for this genotype. According to this reconstruction, genotype 1 would consist of two sister clusters, one with subtypes 1a and 1c, with subtype 1b and the new sequence constituting a different cluster. Therefore, subtype 1b would be the closest to this new sequence (Fig. 1). Moreover, all of the nodes within genotype 1, including the one constituted by subtype 1b and the new sequence, were well-supported by three different measurements of node support (Fig. 1).
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). In agreement with the phylogenetic analysis, the shortest distances were found between the new sequence and the group of sequences belonging to subtype 1b.
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), with subtype 1b sequences always appearing closest to the new sequence and subtypes 1a and 1c grouping together in a different cluster. In the discordant topology obtained with the NS4A gene, the new sequence occupied a basal position with respect to the other type 1 subtypes, although with low bootstrap support. This could be reflecting a low phylogenetic signal as a consequence of the short length of this gene (162 nt). Therefore, this new subtype 1 sequence did not correspond to any variant coming from a rare recombination event between genotypes (Colina et al., 2004; Kalinina et al., 2002) or genotype 1 subtypes.
A partial fragment (267 nt) of the NS5B gene was subjected to further phylogenetic analysis in which 34 additional sequences of genotype 1 were included, mostly from isolates of different African countries (Candotti et al., 2003; Jeannel et al., 1998; Ndjomou et al., 2003) (Fig. 2). In general, sequences from the same country tended to group in well-supported clusters. This could reflect relatively recent epidemiological events, as indicated by the short genetic distances connecting these isolates. In the case of Cameroon sequences, many distant clusters were found to be dispersed along the tree, which is probably related to extensive sampling in an area with extremely high heterogeneity among HCV-1 isolates (Ndjomou et al., 2003). The new sequence did not resemble closely any partial or complete sequence described previously, nor did it present any significant node support that could relate it to any group of sequences within the genotype 1 cluster. However, a poorly supported node connected the new sequence to a group of Cameroon sequences. It is worth noting that Equatorial Guinea is a small country located to the south of Cameroon. The new sequence was related more closely to subtype 1b than to subtypes 1a or 1c, coinciding with the results from the complete-genome analysis (Fig. 1). However, this relationship was less evident in the NS5B gene analysis, due to the almost-continuous branching of isolates along the phylogenetic tree.
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; Simmonds, 2004). However, this variability apparently co-exists with clear discontinuities between genotypes, subtypes and even isolates. In HCV, these discontinuities allow virus populations to be classified into six genotypes and many different subtypes. To date, only three confirmed subtypes (1a–1c) and nine provisionally assigned subtypes (1d–1l) have been described for HCV genotype 1 (Simmonds et al., 2005). As for other genotypes, confirmed subtypes of genotype 1 were established early on, both on the basis of genetic distances and on phylogenetic analysis of full-length sequences. Studies based on age-stratified seroprevalence data (Alter et al., 1999) and phylogenetic analyses (Pybus et al., 2001) indicate that HCV has recently spread rapidly in industrialized nations where subtypes 1a and 1b are considered prevalent. Clinical studies on treatment response or disease progression of such infections account for most of the genome information deposited in sequence databases, mainly as partial genome sequences. Like other highly prevalent subtypes, subtypes 1a and 1b have possibly been distributed widely through unsafe practices between intravenous drug users and through medical practices (i.e. blood transfusion, surgery, intravenous medication and haemodialysis). Moreover, transmission patterns and dated infection rates differ among countries and between viral subtypes (Nakano et al., 2004).
In this study, we have revealed the existence of a new and distinct variant of HCV from Equatorial Guinea by sequencing and analysing its complete genome. This genome is as divergent from representative variants of confirmed genotype 1 subtypes as these are from each other. In consequence, we have provided evidence that this could be a truly new subtype. However, the consensus proposal for a unified system of naming HCV genotypes (Simmonds et al., 2005) states that new subtype designation should be provided only in those cases where there is evidence of their spreading in identified transmission networks. According to this consensus, such evidence will qualify when the new subtype is found in ‘several independently infected individuals’. In such cases, if distinctness is proved, then a provisional designation of a new subtype could be established through partial sequences (core/E1 and NS5B regions) or confirmed designation if at least one HCV isolate from the provisionally designated subtype is sequenced completely. However, as shown by the NS5B phylogenetic analysis of highly diverse isolates, many new sequences from this African area could potentially be designated provisional subtypes without being epidemiologically relevant. Therefore, we propose the generic name ‘subtype 1’ for the new variant reported here and, in order to avoid an increasing profusion of labels for provisional subtypes, we also suggest the same procedure for provisionally assigned subtypes (1d–1l), only considering them to be newly confirmed subtypes if they become epidemiologically or clinically relevant (Ndjomou et al., 2003; Simmonds et al., 2005).
Our current view of HCV variants distributed in separate subtypes within some of the major genotypes is probably the result of the recent epidemiological history of this virus. The most common subtypes come about through the extremely successful spread of a few variants that enter a ‘fast track’, spreading into a susceptible population whose new behavioural patterns help the virus to spread further (Simmonds, 2004). A clear example of this dynamic is the current spread of HCV subtype 1a in Western countries, where its prevalence is rising steadily (Pybus et al., 2005; Thomson & Finch, 2005) thanks to having entered the transmission route of intravenous drug users at the same time as a significant proportion of the general population has begun to engage in this risky behaviour.
If the genetic differences revealed between the new sequence reported here and the previously characterized subtypes are assumed to be of the same order in the whole genome as in the portion of the NS5B gene that we have analysed, including African samples from three different studies (Candotti et al., 2003; Jeannel et al., 1998; Ndjomou et al., 2003), then it is likely that the current clear-cut separations between subtypes 1a, 1b and 1c will become diluted. This will come about because many intermediate forms will appear and a new range of subtypes will be defined following the new nomenclature rules (Simmonds et al., 2005). The relevant question then will be: is such a division of any use? We believe that recognizing the intrinsically high variability of the genome sequence of RNA viruses should not necessarily be reflected in their endless subdivision into discrete categories, especially if these do not correspond to clear evolutionary splits or are not particularly relevant from a clinical or epidemiological point of view. In addition, conflicting reports on response, course, likelihood of becoming a chronic infection, etc. (Simmonds, 2004) for different subtypes/genotypes may be reconciled more easily if a more accurate reconstruction of the evolutionary history of HCV is obtained and such observations are freed from a biologically untenable view and are interpreted in an appropriate context.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 November 2005;
accepted 7 February 2006.
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) of 1.16 for the 
distribution of substitution rates at variable sites. The number of sequences included in each subtype group for the analysis is indicated in parentheses next to the subtype name.