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 (5UTR) 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 5UTR. 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.Hepatitis C virus (HCV) was first identified in the late 1980s and is a major cause of liver disease throughout the world (40). It is a single-stranded, positive-polarity RNA virus classified in the genus Hepacivirus of the family Flaviviridae. The HCV genome encodes a single long polyprotein with the following gene order:

Complete genome analysis of hepatitis C virus subtypes 6t and 6u

“…Remarkably, subtypes 6l and 6k were isolated from Vietnamese individuals (Lu et al, 2006(Lu et al, , 2007a. D85 on the other hand had 92.5 % sequence similarity to subtype 6o prototype strain QC227 isolated from an Asian immigrant in Canada (Murphy et al, 2007), confirmed by phylogeny of whole genome and short fragment analysis ( Fig. 1 and Supplementary Fig.…”

“…Currently, genotype 6 viruses are classified into 17 subtypes (6a-6q) based on complete genome analysis (Lu et al, 2006(Lu et al, , 2007aTokita et al, 1996Tokita et al, , 1998. Recently, the existence of subtype 6r and 6s viruses was proposed based on sequence analysis of core/ E1 and NS5B from HCV strains isolated from Cambodian immigrants in Canada (Murphy et al, 2007). In addition, a subtype of some viruses could not be assigned and these were classified as novel candidate HCV genotype 6 (Lu et al, 2006;Shinji et al, 2004).…”

“…Even though a range of HCV subtype 6a-6q whole genomes has been characterized, a number of viruses cannot be classified (Murphy et al, 2007). These unassigned samples may represent novel candidate HCV subtypes.…”

Complete genomes of three subtype 6t isolates and analysis of many novel hepatitis C virus variants within genotype 6

“…The branch leading to the three isolates appeared equivalent to the branches directing the 6a-6q and km41 and gz52557 genomes. Consequently, VT21, TV241 and TV249 were assigned to subtype 6t, following the recently described 6r and 6s subtypes (Murphy et al, 2007). Within genotype 6, four major groups were formed.…”

“…Among them, D49 (GenBank accession nos DQ155472 and DQ155530) was from a blood donor in Vietnam with partial sequences available in the core and NS5B regions (Noppornpanth et al, 2006). QC131 (GenBank accession nos EF115932 and EF116155), QC145 (EF115934 and EF116157), QC240 (EF115948 and EF116171) and QC351 (EU127994 and EU127995) were from Vietnamese immigrants in Quebec, Canada with partial sequences available in the E1 and NS5B regions (Murphy et al, 2007). The finding of five additional isolates grouping with VT21, TV241 and TV249 in patients originating from Vietnam consolidates the designation of this new subtype.…”