Genotyping hepatitis C viruses from Southeast Asia by a novel line probe assay that simultaneously detects core and 5' untranslated regions

Abstract

Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently divided into six genotypes. A line probe assay (LiPA), which targets the 5' untranslated region (5'UTR) of the HCV genome, is widely used for genotyping. However, this assay cannot distinguish many genotype 6 subtypes from genotype 1 due to high sequence similarity in the 5'UTR. We investigated the accuracy of a new generation LiPA (VERSANT HCV genotype 2.0 assay), in which genotyping is based on 5'UTR and core sequences, by testing 75 selected HCV RNA-positive sera from Southeast Asia (Vietnam and Thailand). For comparison, sera were tested on the 5'UTR based VERSANT HCV genotype assay and processed for sequence analysis of the 5'UTR-to-core and NS5b regions as well. Phylogenetic analysis of both regions revealed the presence of genotype 1, 2, 3, and 6 viruses. Using the new LiPA assay, genotypes 6c to 6l and 1a/b samples were more accurately genotyped than with the previous test only targeting the 5'UTR (96% versus 71%, respectively). These results indicate that the VERSANT HCV genotype 2.0 assay is able to discriminate genotypes 6c to 6l from genotype 1 and allows a more accurate identification of genotype 1a from 1b by using the genotype-specific core information.

FIG. 1.

Midpoint rooted neighbor-joining tree of the 5′UTR-to-core region among selected HCV field viruses from SEA and prototype strains. The tree was constructed for nucleotides 47 to 695 of the HCV genome (numbering is according to reference strain HCV-H GenBank accession number M67463) by using the Tajima-Nei model with rate heterogeneity parameter. Confidence values (>70%) calculated by bootstrap analysis (1,000 replicates) are indicated at the major branching points. Branch lengths are drawn to scale. The prototype HCV strains obtained from GenBank are indicated in boldface.

FIG. 2.

(A and B) Comparison of INNO-LiPA strip patterns obtained from VERSANT HCV genotype assay (5′UTR lines) (A) and the new VERSANT HCV genotype 2.0 assay (5′UTR/core lines) (B). Both assays were tested with HCV genotype 1a, 1b, 2a, 3a, 6a, and 6c to 6l samples. Type-specific line numbers and interpreted genotypes of 5′UTR and core regions are indicated on the left side of each panel. (C) Patterns of the VERSANT HCV genotype 2.0 assay obtained from subtype-indeterminate samples (from Th41, D89 [genotype 1a], Th48 [genotype 3b]) and from nontypeable samples (from Th6, Th44 [genotype 1b], and Th52 [genotype 6f]). Sample codes and specific probe numbers are indicated. MKR, marker line; CONJ CTRL, conjugate control; AMPL CTRL, amplification control; AC NCR, amplification control of noncoding region; AC CORE, amplification control of core.

FIG. 3.

Alignment of the 5′UTR and core sequences of a subset of samples obtained from SEA. Sample codes and genotypes are indicated in the left-hand column. Residues identical to the major sequence are indicated by dots. The positions of specific probes of the VERSANT HCV genotype assay (5′UTR) are boxed. The first nucleotide position of each box is indicated in the bottom left corner. The LiPA line number outside the box indicates the predicted reactivity of each isolate in the sequence alignment. The core sequence alignment starts with the ATG initiation codon (positions 342 to 345 of the HCV genome; the numbering system is as described for Fig. 1). Slashes indicate discontinuous sequences within the 5′UTR.