Near-full genome characterisation of two natural intergenotypic 2k/1b recombinant hepatitis C virus isolates

Abstract

Few natural intergenotypic hepatitis C virus (HCV) recombinants have been characterised, and only RF1_2k/1b has demonstrated widespread transmission. The near-full length genome sequences for two cases of 2k/1b recombinants (CYHCV037 and CYHCV093) sampled in Cyprus were obtained using strain-specific RT-PCR amplification and sequencing protocols. Sequence analysis confirmed their similarity with the original RF1_2k/1b strain from St. Petersburg, N687. These two isolates significantly contribute to the sequence data available on this recombinant and confirm its increasing spread among individuals from Eastern Europe, and its association with transmission through intravenous drug use. Phylogenetic analyses reveal clustering of the sequence 3' to the recombination point, not seen in the topology of the 5' sequences, implying a more complicated evolutionary history than that held to date. The increasing cases of HCV recombinant strains underline the requirement of their contribution to the standardised rules of HCV classification and nomenclature, molecular epidemiology, diagnosis, and treatment.

Figure 1

Simplot analyses of the two 2k/1b recombinant candidate strains, showing percentage sequence similarity over nucleotide position. (a, c) are for strain CYHCV037, and (b, d) for strain CYHCV093. The plots on the top are the results of a genotype-level analysis, with genotypes corresponding to line colours annotated below the plots. The bottom plots are the results of a subtype-level analysis, once the genetically closest parental genotypes had been determined using the top plots. The subtypes corresponding to the coloured lines are annotated below the plots.

Figure 2

Bootscan plot of percentage permuted trees over nucleotide position across the full length of the 2k/1b recombinant strains. (a, b) are for the strains described in this study and (c, d) for the only near-full 2k/1b genomes submitted to GenBank to date. Strain identifiers are indicated at the top left corner of each plot. In brackets are the GenBank accession numbers of the sequences. The annotation below shows the HCV subtype of the consensus sequences corresponding to each line colour in the plots.

Figure 3

Neighbour-Joining trees for (a) the region 5′ of the recombination point and (b) the region 3′ of the recombination point, as seen by the indications on the genome maps above each tree, for the 2k/1b recombinant strains identified in this study (highlighted in black), other 2k/1b sequences obtained from GenBank, and reference stains of the corresponding parental genotypes. The trees are rooted with 1b and 2k strains, respectively. Grouping can be seen within the corresponding parental subtypes and is highlighted with coloured boxes. Numbers at branch nodes indicate the percentage bootstrap support for 1,000 replicates. The scale at the bottom left of each tree indicates the divergence between any two sequences obtained by summing the branch lengths between them.