Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences

Abstract

Nucleotide sequence analysis of the NS5B region was performed to identify genotypes of 8,479 hepatitis C virus (HCV) RNA-positive patient samples collected in the Canadian province of Quebec. Genotypes could be determined for 97.3% of patients. Genotypes 1 to 6 were found in 59.4, 9.0, 25.7, 3.6, 0.6, and 1.8% of patients, respectively. Two isolates did not classify within the six genotypes. The subtype 1 distribution was 76.7% 1a, 22.6% 1b, and 0.7% others, while the subtype 2 distribution was 31.8% 2a, 47.6% 2b, 10.9% 2c, 4.1% 2i, and 5.6% others. Subtype 3a accounted for 99.1% of genotype 3 strains, while all genotype 5 samples were of subtype 5a. The subtype 4 distribution was 39.2% 4a, 15.4% 4k, 11.6% 4d, 10.2% 4r, and 23.6% others. The subtype 6 distribution was 40.4% 6e, 20.5% 6a, and 39.1% others. The 5' untranslated region (5'UTR) sequences of subtype 6e were indistinguishable from those of genotype 1. All samples that did not classify within the established subtypes were also sequenced in C/E1 and 5'UTR. C/E1 phylogenetic reconstructions were analogous to those of NS5B. The sequences identified in this study allowed the provisional assignments of subtypes 1j, 1k, 2m, 2r, 3i, 4q, 6q, 6r, and 6s. Sixty-four (0.8%) isolates classifying within genotypes 1 to 6 could not be assigned to one of the recognized subtypes. Our results show that genotyping of HCV by nucleotide sequence analysis of NS5B is efficient, allows the accurate discrimination of subtypes, and is an effective tool for studying the molecular epidemiology of HCV.

FIG. 1.

Neighbor-joining trees for NS5B sequences (340 bp) (positions 8276 to 8615) and C/E1 sequences (424 bp) (positions 869 to 1292). Phylogenies for genotype 1 NS5B (a), genotype 2 NS5B (b), genotype 3 C/E1 (c), genotype 4 C/E1 (d), and genotype 6 NS5B (e) sequences are shown. The phylogenetic tree for genotype 1 sequences was rooted using genotype 4a sequence ED43 as the outgroup, while the phylogenetic trees for sequences of genotypes 2, 3, 4, and 6 were rooted using genotype 1a sequence HCV-1 as the outgroup. The numbers at the nodes represent the percent bootstrap support for 1,000 replicates. Only values over 75% are shown. Bars at the base of the trees show the genetic divergence. Letters following isolate names indicate countries of origin of patients: BI, Burundi; CA, Canada; CD, Democratic Republic of Congo; CG, Congo; CI, Côte d'Ivoire; CM, Cameroon; DO, Dominican Republic; HT, Haiti; IN, India; IS, Indian subcontinent; IT, Italy; KH, Cambodia; LA, Laos; LB, Lebanon; MA, Morocco; PA, Pakistan; RW, Rwanda; VN, Vietnam.

FIG. 1.

Neighbor-joining trees for NS5B sequences (340 bp) (positions 8276 to 8615) and C/E1 sequences (424 bp) (positions 869 to 1292). Phylogenies for genotype 1 NS5B (a), genotype 2 NS5B (b), genotype 3 C/E1 (c), genotype 4 C/E1 (d), and genotype 6 NS5B (e) sequences are shown. The phylogenetic tree for genotype 1 sequences was rooted using genotype 4a sequence ED43 as the outgroup, while the phylogenetic trees for sequences of genotypes 2, 3, 4, and 6 were rooted using genotype 1a sequence HCV-1 as the outgroup. The numbers at the nodes represent the percent bootstrap support for 1,000 replicates. Only values over 75% are shown. Bars at the base of the trees show the genetic divergence. Letters following isolate names indicate countries of origin of patients: BI, Burundi; CA, Canada; CD, Democratic Republic of Congo; CG, Congo; CI, Côte d'Ivoire; CM, Cameroon; DO, Dominican Republic; HT, Haiti; IN, India; IS, Indian subcontinent; IT, Italy; KH, Cambodia; LA, Laos; LB, Lebanon; MA, Morocco; PA, Pakistan; RW, Rwanda; VN, Vietnam.

FIG. 2.

Unrooted neighbor-joining tree of NS5B sequences (340 bp) (positions 8276 to 8615) showing the relationship of QC69 and QC272 to representatives of genotypes 1 to 6, CS101285, and CS101300 sequences. The bar at the base of the tree shows the genetic divergence.

FIG. 3.

Comparison of sequences in the 5′UTR region (196 bp) (positions 96 to 291). (a) Comparison of QC69, QC272, genotype 6a QC169 and QC242, and genotype 6 QC148 sequences to HCJ6 (genotype 2a) and EUHK2 (genotype 6a) sequences. Nucleotides different from those of genotype 1a HCV-1 are indicated. (b) Thirty-seven genotype 6 sequence profiles obtained for 100 non-6a, non-6b variants. Nucleotides different from those of genotype 1b HCV-BK are indicated.