Evolution of naturally occurring 5'non-coding region variants of Hepatitis C virus in human populations of the South American region

Abstract

Background: Hepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. Previous and recent studies have suggested a diversification of type 1 HCV in the South American region. The degree of genetic variation among HCV strains circulating in Bolivia and Colombia is currently unknown. In order to get insight into these matters, we performed a phylogenetic analysis of HCV 5' non-coding region (5'NCR) sequences from strains isolated in Bolivia, Colombia and Uruguay, as well as available comparable sequences of HCV strains isolated in South America.

Methods: Phylogenetic tree analysis was performed using the neighbor-joining method under a matrix of genetic distances established under the Kimura-two parameter model. Signature pattern analysis, which identifies particular sites in nucleic acid alignments of variable sequences that are distinctly representative relative to a background set, was performed using the method of Korber & Myers, as implemented in the VESPA program. Prediction of RNA secondary structures was done by the method of Zuker & Turner, as implemented in the mfold program.

Results: Phylogenetic tree analysis of HCV strains isolated in the South American region revealed the presence of a distinct genetic lineage inside genotype 1. Signature pattern analysis revealed that the presence of this lineage is consistent with the presence of a sequence signature in the 5'NCR of HCV strains isolated in South America. Comparisons of these results with the ones found for Europe or North America revealed that this sequence signature is characteristic of the South American region.

Conclusion: Phylogentic analysis revealed the presence of a sequence signature in the 5'NCR of type 1 HCV strains isolated in South America. This signature is frequent enough in type 1 HCV populations circulating South America to be detected in a phylogenetic tree analysis as a distinct type 1 sub-population. The coexistence of distinct type 1 HCV subpopulations is consistent with quasispecies dynamics, and suggests that multiple coexisting subpopulations may allow the virus to adapt to its human host populations.

Figure 1

Phylogenetic analysis of 5'NCR sequences of HCV strains. Strains in the trees are shown by their accession numbers for strains previously described and their genotypes are indicated at the right side of the figure. Bolivian, Colombian and Uruguayan strains are shown by name. Number at the branches show bootstrap values obtained after 1000 replications of bootstrap sampling. Bar at the bottom of the trees denotes distance. In (A) the phylogenetic tree for HCV strains isolated in South America is shown. Strains assigned to a newly genetic lineage in HCV type 1 cluster are shown in red. Argentinean strains [EMBL:DQ077818] (Schijman et al., unpublished data), [EMBL:DQ313454] and [EMBL:AY376833] (Gismondi et al. [8, 9] previously reported as a new genetic lineage inside type 1 strains are shown in italics and an arrows denote its position in the figure. Phylogeny for HCV strains isolated in North America and Europe are shown in (B), (C), respectively.

Figure 2

Signature pattern analysis of type 1 HCV strains isolated in South America. In (A) the consensus nucleotide sequence in the background set of type 1 HCV strains isolated in South America is shown in black. The consensus nucleotide sequence in the query (signature sequence) set is shown in red. Query sequence signature identified by VESPA is shown in green. Numbers in the figure shows IRES nucleotide positions, relative to strain HCV1b [16]. In (B) an alignment of 5'NCR sequences from strains belonging to the third cluster observed in type 1 HCV strains isolated in the South American region with corresponding consensus sequences of type 1 HCV strains isolated in South America (Background1), Europe (Background 2) or North America (Background3) is shown. Strains are shown by accession number for strains previously described, or by name at the left side of the figure. Identity to consensus sequences is indicated by a dash. Gaps introduced during alignment are indicated by a dot.

Figure 3

HCV IRES mutations found in sequence signature strains isolated in South America. The 5'NCR sequences of strain HCV1b [16] is shown. The locations of the nucleotide mutations found in the sequence signature are shown in bold and a solid arrow indicates each particular substitution. Sequences previously identified to belong to a specific IRES domain [16] are indicated by colours and domain number is indicated bellow the sequence. IRES nucleotide substitutions positions previously reported in the literature [16] or in the HCV Database [14] are indicated in bold italics underlined. Each particular previously reported substitution is indicated by a dotted arrow. Δ means deletion. Numbers in the figure denote nucleotide position in HCV sequence according to strain HCV1b [16].

Figure 4

Prediction of stem-loop II IRES RNA secondary structure. mfold results of IRES stem-loop II are shown. Numbers in the figure denote nucleotide positions, ΔG obtained for the structures are shown on the bottom of the figure. In (A) mfold results for consensus type 1 strains isolated in South America is shown. (B) shows mfold results for signature consensus sequences.

Figure 5

Prediction of stem-loop III IRES RNA secondary structure. Mfold results of IRES stem-loop III are shown. The rest same as Fig. 4.