Hepatitis C Virus Genotyping: Interrogation of the 5′ Untranslated Region Cannot Accurately Distinguish Genotypes 1a and 1b

Abstract

Although the 5 untranslated region (5 UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5 UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5 UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position ؊99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position ؊99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5 UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.Hepatitis C virus (HCV) is an enveloped positive singlestranded RNA virus which is the major cause of chronic hepatitis worldwide (2, 12). Chronic hepatitis resulting from HCV infection may lead to severe sequelae, including hepatic cirrhosis and hepatocellular carcinoma (3). HCV demonstrates a high degree of sequence variation throughout its genome and exists in vivo as a group of heterogeneous but closely related quasispecies (8,20). However, the levels of heterogeneity differ considerably among the various regions of the virus, ranging from as little as 10% in the 5Ј untranslated region (5Ј UTR) to 50% or more within the E1 region (8,10,32). Based on the analysis of variable coding regions in the viral genome, distinct genotypes as well as subtypes have been defined (10,16,34). The distribution of HCV genotypes differs geographically, with subtype 1a being most common in the United States, subtype 1b being most common in Europe and Japan, and other genotypes being prevalent in other parts of the world (for a review, see reference 41). Several studies have shown that HCV genotype influences the response to therapy with alpha interferon alone or in combination with ribavirin (1,14,23,43). In general, HCV type 2 and 3 isolates have higher rates of response to therapy than type 1 isolates. In addition, some studies suggest that g...

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

“…Also, in accordance with previous studies 6,8,12,14,17,18,21 , discrepant results at the subtype level, mainly subtypes 1a and 1b, were found here (two samples identified as 1a by NS5B sequencing were subtyped as 1b by 5' NC sequencing). Although both regions provide similar results at the genotype level, which is adequate for clinical practice, especially in the management of antiviral therapy, the NS5B sequencing appears to be more useful for epidemiological investigations.…”

“…As observed by other authors 6,8,12,18 , no difference at the genotype level was found for the 42 samples that could be successfully amplified and sequenced in the the 5' NC and NS5B regions. Also, in accordance with previous studies 6,8,12,14,17,18,21 , discrepant results at the subtype level, mainly subtypes 1a and 1b, were found here (two samples identified as 1a by NS5B sequencing were subtyped as 1b by 5' NC sequencing).…”

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

“…However, the genotyping methods employed in those studies, which were based on 5' noncoding (5'NC) region analyses, did not permit the correct identification of HCV subtypes (10)(11)(12)(13)(14)(15)(16)(17). To our knowledge, the present study is the first to analyze Brazilian HCV samples using two genotyping methods: line probe assay (LiPA, based on the 5'NC region) and the nucleotide sequencing analysis of the nonstructural 5B (NS5B) region, which has been used as a reference method for accurate genotyping (10)(11)(12)(13)(14)(15)(16)(17). Thus, our objective was to determine which HCV genotypes and subtypes occur in hemodialysis patients in Central Brazil and also to compare the genotyping efficiency of LiPA and direct sequencing of the NS5B region.…”

“…On the other hand, this region does not contain sufficient information for the recognition of all different types and subtypes. Thus, in the present study we chose the NS5B region for sequence analysis since this region appears to be much more accurate for identifying variations in the nucleotide sequences (10)(11)(12)(13)(14)(15)(16)(17).…”

Phylogenetic Analysis of Isolated HCV Strains from Tunisian Hemodialysis Patients

“…These results corroborate those of Scott et al (41) who reported that, while most assays target the highly conserved 5' noncoding region of the HCV genome, mutations can occur in this region leading to misclassification of HCV genotypes in 5%-8% of cases. Similarly, the study of Chen et al (8) showed that the reverse line probe assay LiPA was not heterogeneous enough for use in determination of HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.…”