1 Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, Uruguay
2 Populations, Génétique et Evolution, CNRS, 91198 Gif-sur-Yvette, France
3 Laboratorio de Biología Molecular, Centro Nuclear RACSO, Instituto Peruano de Energía Nuclear IPEN, Av. Canadá 1470, San Borja, Apartado 1687, Lima 41, Peru
4 Servicio de Inmunología, Hospital Nacional Edgardo Rebagliati Martins HNERN, Domingo Cueto s/n, Jesús María, Lima 11, Peru
5 Laboratorio de Organización y Evolución del Genoma. Instituto de Biología. Facultad de Ciencias. Iguá 4225, 11400 Montevideo, Uruguay
6 Division of Human Health, International Atomic Energy Agency, Wagramerstrasse 5, 1400 Vienna, Austria
Correspondence
Juan Cristina
cristina{at}cin1.cin.edu.uy
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ABSTRACT |
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The GenBank/EMBL accession numbers of the sequences reported in this work are AJ438618, AJ438622 and AJ582126AJ582135.
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INTRODUCTION |
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RNA viruses exploit all known mechanisms of genetic variation to ensure their survival (Domingo & Holland, 1997). Their high rate of mutation and replication allow them to move through sequence space at a pace that often makes their DNA-based host's evolution look glacial in comparison (Worobey & Holmes, 1999
). Over the last two decades it has become increasingly clear that many RNA viruses add the capacity to exchange genetic material with one another. Thus, in addition to producing large amounts of the raw material of evolution (mutations), these viruses also possess mechanisms (recombination) that, in principle, allow them both to purge their genomes of accumulated deleterious changes (Muller, 1964
) and to create or spread beneficial combinations of mutations in an efficient manner.
Until 1999, there was no evidence for recombination in flaviviruses, although the possibility had been considered (Blok et al., 1992; Kuno, 1997
; Monath, 1994
). Accordingly, the vast majority of work on flaviviruses, including vaccine studies and phylogenetic analyses in which genotypes were identified and sometimes correlated with disease severity (Chen et al., 1990
; Leitmeyer et al., 1999
; Rico-Hesse, 1990
), has rested on the implicit assumption that evolution in the family Flaviviridae is clonal, with diversity generated through the accumulation of mutational changes.
Recent studies have shown this assumption to be invalid, as homologous recombination has now been demonstrated in pestiviruses (bovine viral diarrhoea virus) (Becher et al., 2001), flaviviruses (all four serotypes of dengue virus) (Holmes et al., 1999
; Tolou et al., 2001
; Uzcategui et al., 2001
; Worobey & Holmes, 1999
), hepaciviruses (GB virus C/hepatitis G virus) (Worobey & Holmes, 2001
) and Japanese encephalitis or St Louis encephalitis virus (Twiddy & Holmes, 2003
). There have been few reports on recombination between HCV strains of different genotypes (Kalinina et al., 2002
; Yun et al., 1996
) and it has been suggested that these events are rare in vivo and that the resultant recombinants are usually not viable (Simmonds et al., 1994
; Smith & Simmonds, 1997
).
Selected HCV genome regions within the 5'UTR, core, E1 or NS5, which have been shown to be conserved within a given HCV genotype, are used for the classification of HCV strains (Simmonds et al., 1994; Simmonds, 1999
). Most methods for direct HCV genotyping include amplification of different genome regions, such as the 5'UTR, core, E1 or NS4, by PCR with type-specific primers or by restriction fragment length polymorphism analysis of PCR products (Ohno et al., 1997
; Okamoto et al., 1993
; Stuyver et al., 1993
, 1995
). Indirect HCV genotyping may be achieved by demonstration of type-specific antibodies by ELISA (Dixit et al., 1995
; Simmonds et al., 1993
). Thus, present methods of HCV genotype identification do not take recombination into account.
Given the implications of recombination for virus evolution (Worobey & Holmes, 1999) and the development of vaccines, virus control programmes, patient management and antiviral therapies, it is clearly important to determine the extent to which recombination plays a role in HCV evolution. Recombination plays a significant role in the evolution of RNA viruses by creating genetic variation. For example, the frequent recovery of recombinant isolates of poliovirus (Georgescu et al., 1994
; Kew & Nottay, 1984
) that result from recombination involving vaccine strains shows that recombination has the potential to produce escape mutants' in nature as well as in experiments. Recently, recombination has also been detected in other RNA viruses for which multivalent vaccines are in use or in trials (Holmes et al., 1999
; Suzuki et al., 1998
; Worobey et al., 1999
). We think the potential for recombination to produce new pathogenic hybrid strains needs to be carefully considered whenever vaccines are used or planned to control RNA viruses. Assumptions that recombination either does not happen or is unimportant in RNA viruses have a history of being proved wrong (Worobey & Holmes, 1999
).
In previous studies, we subtyped 72 HCV strains isolated in South America (Colina et al., 1999; Vega et al., 2001
; San Roman et al., 2002
; Cristina et al., 2002
) by limited sequencing of the 5'UTR region. In the present study, this work was extended to include sequencing of the core and NS5B regions in order to investigate the congruence of HCV genotype determinations among the different regions of the genome. We found congruent results in 97 % of cases. However, we also found evidence for recombination between type 1 subtypes of HCV in the Peruvian population.
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METHODS |
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RNA extraction, cDNA synthesis and amplification.
HCV RNA was extracted from 140 µl serum samples with the QIAamp viral RNA kit (Qiagen) according to the manufacturer's instructions. The extracted RNA was eluted from the columns with 50 µl RNase-free water. cDNA synthesis and PCR amplification of the 5'UTR, core and NS5B regions were carried out as previously described (Chan et al., 1992; Norder et al., 1998
). To avoid false positive results, the recommendations of Kwok & Higuchi (1989)
were strictly adhered to. Amplicons were purified using the QIAquick PCR purification kit (Qiagen), according to the manufacturer's instructions.
Sequencing.
The primers used for amplification were used for sequencing the PCR fragments. The sequencing reaction was carried out using the Big Dye DNA sequencing kit (Perkin-Elmer) on a 373 DNA sequencer apparatus (Perkin-Elmer) or by manual sequencing using the Thermo Sequenase radiolabelled terminator cycle kit (Amersham).
Sequence analysis.
The sequences for the 5'UTR plus core and NS5B regions were aligned using the CLUSTAL W program (Thompson et al., 1994). Using the MEGA program (Kumar et al., 1994
), phylogenetic trees were created by the neighbour-joining method applied to the distance matrix obtained under the Kimura two-parameter model (Felsenstein, 1993
). As a measure of the robustness of each node, we utilized the bootstrap method (1000 pseudo-replicas).
Recombination analysis.
Putative recombinant sequences were identified with the SimPlot program (Lole et al., 1999), using concatenated (5'UTR plus core plus NS5B) sequences. This program is based on a sliding window method and constitutes a way of graphically displaying the coherence of the sequence relationships over the entire length of a set of aligned homologous sequences. The window width and the step size were set to 200 bp and 10 bp, respectively. Once the recombinant strain and strains representing possible parents were identified, the likely recombination breakpoint was determined by LARD (Holmes et al., 1999
). Briefly, for every possible breakpoint, the sequence alignment was divided into two independent regions for which the branch lengths of a tree of the putative recombinant and its two parent sequences were optimized. The two results (likelihoods) obtained by using the separate regions were then combined to give a likelihood score for that breakpoint position, and the breakpoint position that yielded the highest likelihood was then compared, using a likelihood ratio test, to the likelihood obtained from the same data under a model that permitted no recombination. To assess whether the recombination model gave a significantly better fit to the data than the null hypothesis of no recombination, the likelihood ratios obtained using the real data were evaluated for significance against a null distribution of likelihood ratios produced by using the Monte Carlo simulation of sequences generated without recombination. Sequences were simulated 1000 times using the maximum-likelihood model parameters and sequence lengths from the real data using Seq-Gen (Rambaut & Grassly, 1997
).
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RESULTS |
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Recombination analysis
To gain insight into a possible recombination event, a phylogenetic profile analysis was carried out for the Peruvian strain PE22 and the putative parental-like strains H77 (subtype 1a) and JK1 (subtype 1b). The results of these studies are shown in Fig. 2. As can be seen in the figure, profile analysis of the putative parental-like (H77, JK1) and recombinant (PE22) strains showed a clearly visible point of recombination at position 677 of the analysed sequences, which corresponds to position 58 of the NS5B sequences included in this study (see Fig. 3
). This position corresponds to position 8321 in the HCV genome of the putative parental strain H77.
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DISCUSSION |
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ACKNOWLEDGEMENTS |
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Received 28 June 2003;
accepted 16 September 2003.