Development and evaluation of an automated hepatitis C virus NS5B sequence-based subtyping assay

Virco BVBA, Mechelen, Belgium.
Clinical Chemistry and Laboratory Medicine (Impact Factor: 2.71). 08/2010; 48(8):1095-102. DOI: 10.1515/CCLM.2010.236
Source: PubMed


Hepatitis C virus (HCV) genotyping and accurate subtype determination is becoming increasingly important to better understand viral evolution, the development of resistance to STAT-C, and possibly even for the treatment and management of chronic HCV-infected patients.
A subtyping assay based on a 329-bp sequence of the NS5B region, together with an automated subtype interpretation tool was developed. Clinical samples of the six major genotypes were used to assess assay performance characteristics.
The NS5B BLAST-based subtyping assay showed clinical sensitivity for amplification of 89% (n=603 random samples). Assessment of analytical sensitivity of amplification for genotypes 1, 2, 3 and 4 revealed a suitable performance for high viral load samples and decreased only with low viral loads. The results were 100% and 99% accurate at the genotype and subtype level, respectively. A concordance of 97% on genotype level and 62% on subtype level was observed by comparison with subtype results from 5' non-coding-based assays with a panel of 276 isolates.
The HCV NS5B subtyping assay has been validated for research use. Based on its performance, it is the method of choice in cases where subtype rather than genotype information is needed.

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    • "However, current HCV NS5B-based genotyping uses only partial short nucleic acid sequences of NS5B. Koletzki et al. [32] used a 329-bp NS5B gene fragment (nucleotides 8280–8610 in HCV sequence) to classify HCV and showed an 89% clinical sensitivity for amplification. Cantaloube et al. [30] used 339-bp nucleotide segments (nucleotides 8370–8708) in the NS5B region to classify HCV and achieved a 98.32% of sensitivity for TABLE 2. The theoretical amplification ability of the potential primers for all NS5B from hepatitis C virus (HCV) database "
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    ABSTRACT: Nucleotide sequencing of the phylogenetically informative region of NS5B remains the gold standard for HCV genotyping. Here we developed a new methodology for sequencing new NS5B regions to increase the accuracy and sensitivity of HCV genotyping and subtyping. The 8 new primers were identified by scanning the full-length NS5B regions from 1127 HCV genomic sequences found in HCV databases. The ability of each pair of primers to amplify HCV subtypes were scored, and the new primers were able to amplify the NS5B region better than the previously used primers, therefore more accurately subtyping HCV strains. Sequencing the DNA amplified by the new primer pairs can specifically and correctly detect the 5 standard HCV subtypes (1a, 2a, 3b, 6a, and 1b). We further examined patient samples and found that the new primers were able to identify HCV subtypes in clinical sample with high sensitivity. This method was able to detect all subtypes of HCV in 567 clinical samples. Importantly, 3 novel HCV subtypes (1b-2a, 1b-2k, and 6d-6k) were identified in the samples, which have not been previous reported in China. In conclusion, sequencing the NS5B region amplified by the new NS5B primers is a more reliable method of HCV genotyping and a more sensitive diagnostic tool then sequencing using the previously described primers, and could identify new HCV subtypes. Our research is useful for clinical diagnosis, guidance of clinical treatment, management of clinical patients, and studies on the epidemiology of HCV. Copyright © 2015. Published by Elsevier Ltd.
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    ABSTRACT: Abstract The present study describes the strains of hepatitis C virus (HCV) isolated from Tunisian hemodialysis patients. Thirty-three HCV strains isolated from different dialysis centers in Tunis City were amplified by RT-PCR in a region of the NS5b gene, genotyped by sequencing, and compared to international sequences by phylogenetic analysis. The phylogenetic tree showed that 16 HCV isolates have been identified as subtype 4k (48.5%), 7 as unspecified HCV-4 subtype (21.2%), 5 as subtype 4a et 1b (each 15.2%). The analysis of this tree revealed that the HCV-1b strains were closely related to Anglo-Saxon and European isolates, while the HCV-4 isolates are genetically similar to Egyptian and African strains. Phylogenic analysis of 33 Tunisian isolates with international HCV strains on a region of the NS5b gene demonstrated that the subtype 4k submerged the Tunis city and a new subtype of HCV4 seems to be suspect in this area.
    Viral immunology 02/2013; 26(1). DOI:10.1089/vim.2012.0043 · 1.45 Impact Factor
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    ABSTRACT: Introduction:Accurate genotyping of hepatitis C virus (HCV) is important in determining the optimal regimen, dose and duration of antiviral therapy of chronic HCV, as well as estimating the response rate. The 5-untranslated region (5'UTR) of HCV RNA is used in commercial genotyping, but probes and lengths of amplicons are proprietary and vary across assays.Objective:Evaluation of the HCV 5'UTR for factors involved in reliable determination of HCV genotypes.Study Design:Serum from four subjects with chronic HCV with disparate results on commercial genotyping and four controls were analyzed. HCV RNA was extracted from serum samples and the 5'UTR and NS5B region were sequenced. Ten clones from each region were compared to prototype sequences and analyzed for genotype assignment using five programs. Results were compared to commercial assays. 5'UTR sequences were sequentially shortened from either the 5' end or 3' end, or both, with genotyping of resultant fragments.Results:Sequences were obtained for the 5'UTR in all eight subjects, but for the NS5B in five. Genotype assignment was identical between the two regions in those 5 with complete sequencing. Genotyping by sequencing gave different results from the commercial assays in the four experimental samples but agreed in the four controls. Shortening of sequences affected results and sequences of less than 200 bases were inaccurate. Neither Hamming distance nor quasispecies affected results.Conclusion:Sequencing of the HCV 5'UTR provided reliable genotyping results and resolved discrepancies identified in commercial assays, but genotyping by sequencing was highly dependent upon sequence length.
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