Full-length sequences of 11 hepatitis C virus genotype 2 isolates representing five subtypes and six unclassified lineages with unique geographical distributions and genetic variation patterns

Abstract

In this study, we characterized full-length hepatitis C virus (HCV) genome sequences for 11 genotype 2 isolates. They were isolated from the sera of 11 patients residing in Canada, of whom four had an African origin. Full-length genomes, each with 18-25 overlapping fragments, were obtained by PCR amplification. Five isolates represent the first complete genomes of subtypes 2d, 2e, 2j, 2m and 2r, while the other six correspond to variants that do not group within any assigned subtypes. These sequences had lengths of 9508-9825 nt and each contained a single ORF encoding 3012-3106 aa. Predicted amino acids were carefully inspected and unique variation patterns were recognized, especially for a 2e isolate, QC64. Phylogenetic analysis of complete genome sequences provides evidence that there are a total of 16 subtypes, of which 11 have been described here. Co-analysis with 68 partial NS5B sequences also differentiated 18 assigned subtypes, 2a-2r, and eight additional lineages within genotype 2, which is consistent with the analysis of complete genome sequences. The data from this study will now allow 10 assigned subtypes and six additional lineages of HCV genotype 2 to have their full-length genomes defined. Further analysis with 2021 genotype 2 sequences available in the HCV database indicated that the geographical distribution of these subtypes is consistent with an African origin, with particular subtypes having spread to Asia and the Americas.

Fig. 1.

Maximum-likelihood phylogenies estimated from (a) full-length nucleotide sequences and (b) predicted amino acid sequences. Except for the 11 isolates from this study (•), reference sequences from subtypes 1a, 1b, 1c, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4q, 5a, 6a, 6i and 7a were also included. Each branch was led by an isolate named in the format: subtype_isolate_accession number. Isolate names that start with ‘2’ alone, without a letter, indicate that a subtype has not been assigned. Bootstrap supports are shown in italics. Bars, 0.10 nucleotide substitutions per site (a); 0.05 amino acid substitutions per site (b). *QC64 contains an extra p7 gene.

Fig. 2.

Neighbour-joining tree reconstructed with full-length genome sequences from 58 genotype 2 isolates. In addition to the 11 sequences from this study, the tree included 47 reference sequences. The latter contained 19 subtype 2a, 25 subtype 2b and one each of subtypes 2c, 2i and 2k. Subtypes 2a and 2b sequences are identified with their GenBank accession numbers. Sequences from this study are identified (•) and include the isolate name. Bar, 0.05 nucleotide substitutions per site.

Fig. 3.

Maximum-likelihood tree for 79 sequences of the NS5B region corresponding to nt 8276–8615 in the numbering of the H77 genome. Yellow branches mark the 18 assigned subtypes, 2a–2r. Green branches mark the eight unassigned lineages. All tips are named in the format: subtype, original country (for country codes see Table 2), sampling time (or missing), isolate ID, and GenBank accession number, each of which is separated by a dot. Bar, 0.1 nucleotide substitutions per site.

Fig. 4.

Analysis of p7 sequences. (a) Phylogeny. p7 sequences from the 11 isolates in this study are marked (•). The insertion of isolate QC64 is indicated (○). Subtypes 1a, 1b, 1c, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4f, 5a, 6a, 6i and 7a were included as references. Bar, 0.1 nucleotide substitutions per site. (b) Multiple sequence alignment. The consensus sequence and distinct amino acids are shown in standard International Union of Pure and Applied Chemistry (IUPAC) codes. Dashes indicate gaps relative to the 2e_QC64 insertion sequence. The isolate names are listed to the right of the alignment and the QC64 insertion is indicated in grey.