Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

Gryadunov, Dmitry; Nicot, Florence; Dubois, Martine; Mikhailovich, Vladimir; Заседателев, А. С.; Izopet, Jacques

doi:10.1128/jcm.01265-10

Cited by 20 publications

(16 citation statements)

References 52 publications

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“…[18][19][20][21]. Sequencing of a genome region divergent enough to discriminate genotypes and subtypes, such as HCV NS5B, core, and envelope regions, is considered to be the most accurate method [22,23].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Accurate genotyping of hepatitis C virus through nucleotide sequencing and identification of new HCV subtypes in China population

Tong

Liu

et al. 2015

Clinical Microbiology and Infection

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Nucleotide sequencing of the phylogenetically informative region of NS5B remains the gold standard for hepatitis C virus (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 eight 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 was 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 five 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 samples with high sensitivity. This method was able to detect all subtypes of HCV in 567 clinical samples. Importantly, three 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 than 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.

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Section: Accepted Manuscriptmentioning

confidence: 99%

Accurate genotyping of hepatitis C virus through nucleotide sequencing and identification of new HCV subtypes in China population

Tong

Liu

et al. 2015

Clinical Microbiology and Infection

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“…Some low-density microarray systems have been developed to identify different HCV genotypes, but samples with mixed infections were not considered during the clinical evaluations of these systems. Moreover, the low sample throughput and cost of these assays limit their breadth of application, especially in clinical use (Gryadunov et al, 2010). Suspension bead arrays can overcome these limitations (Miller and Tang, 2009), thereby reducing labor requirements and sample processing.…”

Section: Discussionmentioning

confidence: 99%

A reliable multiplex genotyping assay for HCV using a suspension bead array

Yang

Wang

Cheng

et al. 2014

Microbial Biotechnology

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The genotyping of the hepatitis C virus (HCV) plays an important role in the treatment of HCV because genotype determination has recently been incorporated into the treatment guidelines for HCV infections. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. We therefore developed a multiplex genotyping assay to determine HCV genotypes using a bead array. Synthetic plasmids, genotype panels and standards were used to verify the target-specific primer (TSP) design in the assay, and the results indicated that discrimination efforts using 10 TSPs in a single reaction were extremely successful. Thirty-five specimens were then tested to evaluate the assay performance, and the results were highly consistent with those of direct sequencing, supporting the reliability of the assay. Moreover, the results from samples with mixed HCV genotypes revealed that the method is capable of detecting two different genotypes within a sample. Furthermore, the specificity evaluation results suggested that the assay could correctly identify HCV in HCV/human immunodeficiency virus (HIV) co-infected patients. This genotyping platform enables the simultaneous detection and identification of more than one genotype in a same sample and is able to test 96 samples simultaneously. It could therefore provide a rapid, efficient and reliable method of determining HCV genotypes in the future.

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“…Genotyping by sequence analysis of the NS5B region has been shown to correlate well with genotyping using the 5= UTR, with the added advantage of superior subtyping (21)(22)(23)(24). To some extent, the NS5B region is considered the gold standard for genotyping and subtyping.…”

Section: Discussionmentioning

confidence: 99%

Important Factors in Reliable Determination of Hepatitis C Virus Genotype by Use of the 5′ Untranslated Region

et al. 2013

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Accurate genotyping of hepatitis C virus (HCV) is important for determining the optimal regimen, dose, and duration of antiviral therapy for chronic HCV infection, as well as for estimating the response rate. The 5= untranslated region (UTR) of HCV RNA is used in commercial genotyping, but the probes and the lengths of the amplicons are proprietary and vary among the assays. In this study, factors involved in the reliable determination of HCV genotypes utilizing the 5= UTR were evaluated. Serum samples from four subjects with chronic HCV infection and 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. The results were compared to those from commercial assays. 5= UTR sequences were sequentially shortened from either the 5= end, the 3= end, or both ends, with genotyping of the resultant fragments. Sequences were obtained for the 5= UTR in all eight subjects and for the NS5B region in five subjects. The genotype assignments were identical between the two regions in the five subjects with complete sequencing. Genotyping by sequencing gave different results than those from the commercial assays in the four experimental samples but agreed in the four controls. Shortening of the sequences affected the results, and the results for sequences of <200 bases were inaccurate. Neither the Hamming distance nor the quasispecies affected the results. 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. H epatitis C virus (HCV), a member of the Flaviviridae family, possesses a single-stranded positive-sense RNA genome approximately 9.6 kb in length. The HCV genome is characterized by 5= and 3= untranslated regions (UTRs) that abut a single large open reading frame. The single polyprotein translated from the HCV genome includes an initial structural region comprising envelope (E1 and E2) and core regions and a nonstructural region that is posttranslationally processed into at least 4 polypeptides (NS2 to NS5). In a manner similar to that of other RNA viruses, HCV circulates in infected individuals as a population of closely related but diverse viral sequences, referred to as quasispecies. Heterogeneity of the HCV genome is a consequence of mutations during viral replication, which is mainly due to the error-prone viral RNA polymerase and the lack of an associated repair mechanism. Different HCV isolates display significant nucleotide sequence variability depending on the genomic region. The E1 and E2 regions are highly variable, whereas the 5= UTR is the most conserved (1, 2).The accumulation of mutations in the viral genome over time has led to the emergence of multiple HCV genotypes. Phylogenetic analysis of full-length or partial sequences of HCV strain...

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Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

Cited by 20 publications

References 52 publications

Accurate genotyping of hepatitis C virus through nucleotide sequencing and identification of new HCV subtypes in China population

Accurate genotyping of hepatitis C virus through nucleotide sequencing and identification of new HCV subtypes in China population

A reliable multiplex genotyping assay for HCV using a suspension bead array

Important Factors in Reliable Determination of Hepatitis C Virus Genotype by Use of the 5′ Untranslated Region

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