1 Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 4to piso, Buenos Aires 1113, Argentina
2 Unidad de Hepatología, Hospital Argerich, Buenos Aires, Argentina
Correspondence
Rodolfo Héctor Campos
rcampos{at}ffyb.uba.ar
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences determined in this work are AY876391AY876493.
Supplementary material is available in JGV Online.
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MAIN TEXT |
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In this work, we investigated the composition and molecular evolution of the HCV E2 quasispecies in two patients during the course and follow-up of two successive treatments with alpha interferon (IFN-) and IFN-
/ribavirin. The analysed fragment (nt 14251972) was composed of HVR1 (nt 14911571) and 467 nt flanking this region. This genomic fragment thus contained both variable and conserved regions and was amplified by RT-PCR using Pfu DNA polymerase (Promega) to diminish the introduction of artefactual mutations (primer sequences are available in Supplementary Table S1, available in JGV Online). PCR products were cloned and independent clones were bidirectionally sequenced (ALFexpress II; Amersham Biosciences). Sequences were aligned using the CLUSTAL X program (Thompson et al., 1997
). Nucleotide analyses were performed with PAUP* version 4.0b10 (Swofford, 2002
). The best-fitting nucleotide substitution models for the different data analysed in each patient were chosen with MODELTEST version 3.06 (Posada & Crandall, 1998
) (see Supplementary Table S2, available in JGV Online). Phylogenetic trees were constructed according to the maximum-likelihood method. To assess diversity, genetic distances were calculated for all pairs of sequences. Phylogenetic trees constructed on the basis of amino acid sequences were obtained using the neighbour-joining method in the MEGA2 program (Kumar et al., 2001
). The reliability of the phylogenetic trees was assessed by bootstrap resampling (1000 datasets). Patient A, infected with a genotype 1b strain, was treated for 6 months with IFN-
2b and with IFN-
2b plus ribavirin for another 6 months. He did not respond to either therapy. Serum samples were taken: (I) just before the IFN-
treatment; (II) 6 months after the end of the monotherapy; (III) 4 months later, at the beginning of the combined treatment; and (IV) 3 months and (V) 6 months after the end of this therapy. Patient B, infected with HCV genotype 1a, was treated for 6 months with IFN-
2b but did not respond. He received a second treatment for 12 months of IFN-
2b plus ribavirin. This patient initially responded to the combined therapy, but relapsed at the end of the treatment. Serum samples were taken: (I) just before the first treatment; (II) 7 months after the end of this IFN-
treatment; (III) 3 months later, before the beginning of the IFN-
/ribavirin treatment; (IV) at the end of the combined treatment; and (V) 6 months later.
Phylogenetic analysis of the nucleotide sequences from all clones from patient A showed a shift pattern of evolution, with selection of clearly distinct lineages of sequences at different times of the infectious process, supported by high bootstrap values (Fig. 1a). In the basal sample, we found two different clusters of sequences: Ia (seven clones) and Ib (three clones). In sample II, the viral sequences composed a single group that was different from both lineages found in sample I. Three months later, there was a single lineage of sequences, different from the previous ones. We found a single clone in sample II that grouped with lineage III and a single clone in sample III that was associated with lineage II. Clone sequences from samples IV and V formed a new group genetically distant from the previous lineages. In this patient, the divergence between lineage sequences was remarkably higher than within them (see Supplementary Table S3, available in JGV Online). This inter-lineage evolution has been previously observed (Pawlotsky et al., 1999
; Alfonso et al., 2004
). In contrast, analysis of all clones sequenced for patient B revealed a phylogenetic structure that reflected the continual selection of genomes within lineages, a process that could be defined as intra-lineage evolution (Fig. 1b
). Clones from sample I grouped into four lineages of sequences (Fig. 1b: a
, six clones; b, one clone; c, two clones; d, one clone). At the end of the therapy (samples II and III), there were only two lineages of sequences: lineage a, where populations IIa and IIIa appeared phylogenetically close to Ia clones, and lineage b composed of populations IIb and IIIb that clustered with clone Ib. At the end of the combined therapy, all the viral sequences detected were included in or derived from lineage a. The sequences designated IVa were intermingled with those from lineage a, and the sequences designated IVa2 formed a different lineage. Sample V showed a group of viral sequences that clustered intermingled with sequences from population IVa2. Both populations constituted the so-called lineage a2. This lineage was considered to be derived from lineage a, since they shared a monophyletic origin in the tree. The viral sequences found at any one time seemed to derive from variants of the previous sample. This temporal evolutionary pattern has been described as the most possible representative for HCV intra-host dynamics (Grenfell et al., 2004
). The genetic distances (see Supplementary Table S4, available in JGV Online) coincided with the clustering of the above-mentioned lineages.
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To perform a more in-depth analysis, we studied HVR1 and the flanking region (FR) separately. HVR1 has been described as a target for positive selection by immune system-mediated neutralization (Weiner et al., 1992; Van Doorn et al., 1995
; Farci et al., 2002
) and for purifying selection (McAllister et al., 1998
; Penin et al., 2001
), suggesting an important role of this region in driving the evolutionary process. It has been widely demonstrated that HVR1 displays high diversity associated with immune escape when different samples from the same or distinct patients are compared (Hijikata et al., 1991
; Kurosaki et al., 1993
). HVR1 may be a target for neutralizing antibodies and HCV persistence may therefore require continuous virus amino acid changes (Taniguchi et al., 1993
; Kantzanou et al., 2003
). Phylogenetic analysis of HVR1 in patient A showed the same topology as for the entire fragment. This genomic region was highly homogeneous in the intra-lineage analysis, but was very heterogeneous in the inter-lineage analysis (Fig. 2
a and b; see also Supplementary Table S3). The analysis in patient B also showed a similar topology to that presented for the entire fragment; the nucleotide sequences that belonged to lineage a2 were highly homogeneous in this region, as was observed for all lineages from patient A (Fig. 2c and d
).
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Many authors have shown the importance of HVR1 in driving the evolutionary process. However, there have been few longitudinal studies that have evaluated the quasispecies dynamics of the genomic region flanking HVR1 (McAllister et al., 1998; Sheridan et al., 2004
). For patient A, the nucleotide FR sequence analysis [see Supplementary Fig. S1(a), available in JGV Online] showed the same topology as that of the entire fragment, but the heterogeneity within lineages was slightly higher than for HVR1 and the mean genetic distances between the lineages were lower than for HVR1 (see Supplementary Table S3). The FR phylogenetic analysis in patient B showed poor discriminative ability. Only lineages b and a2 presented significant bootstrap supports [see Supplementary Fig. S1(b)]. Six of eight synonymous substitutions that characterized lineage a2 belonged to the FR. The sequence of clone III* clustered with lineage b when we analysed HVR1, but did not group with this lineage in the FR. Although it is feasible that it may have been an artefact produced during the PCR or cloning process, the possibility that a recombination event has taken place could not be discarded, since inter- and intra-genotypic recombination in HCV has been reported by some authors in recent years (Kalinina et al., 2002
, 2004
; Colina et al., 2004
).
The amino acid substitutions outside HVR1 that define the main lineages in both patients were not evenly distributed along the studied fragment and most were concentrated between aa 435 and 475. McAllister et al. (1998) have described this region as hypervariable in subtype 1b isolates and mentioned that some substitutions tended to occur among restricted amino acids. For patient A (genotype 1b), we found amino acid substitutions that agreed with this previous report (A/S in aa 441, E/N/H/Y in aa 446, K/R in aa 447 and A/S in aa 450). In this patient, we also assessed a marked clustering of the substitutions at aa 445, 446 and 447. The substitutions that characterized the main lineages in patient B (genotype 1a) resulted in four modified amino acids at positions 439, 447, 467 and 475. Published data provide some evidence that at early time points after the beginning of the therapy, the non-HVR1 region of E2 evolves under purifying selection (Farci et al., 2002
). In contrast, it has recently been suggested that this region may also be subject to immune pressure (Chambers et al., 2005
). Both observations made in our study from patient A and, despite the different viral subtypes, the convergence of modifications at aa 439, 447 and 467 in both patients indicated the participation of this region during the evolutionary process as a target of selection (Table 1
).
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ACKNOWLEDGEMENTS |
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Received 5 April 2005;
accepted 22 June 2005.
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