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Hepatitis C virus genotyping using an oligonucleotide microarray based on the NS5B sequence.

ABSTRACT: The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV genome. The biochip contains 120 oligonucleotides that identify genotypes 1 to 6 and 36 (1a, 1b, 1c, 1d, 1e, 2a, 2b, 2c, 2d, 2i, 2j, 2k, 2l, 2m, 3a, 3b, 3k, 4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r, 4t, 5a, 6a, 6b, 6d, 6g, 6h, and 6k) subtypes. The procedure included amplification of a 380-nucleotide (nt) fragment of NS5B and its hybridization on the biochip. Tests on 345 HCV-positive samples showed that the assay agreed with NS5B sequencing 100% for the genotype and 99.7% for the subtype. The hybridization on the microarray and the NS5B sequence were in 100% agreement for identifying the most common subtypes, 1a, 1b, 4a, 4d, and 3a. This approach is a promising tool for HCV genotyping, especially for implementing the new anti-HCV drugs that require accurate identification of clinically relevant subtypes.

SUBMITTER: Gryadunov D

PROVIDER: S-EPMC3020838 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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Hepatitis C virus genotyping using an oligonucleotide microarray based on the NS5B sequence.

Gryadunov Dimitry D Nicot Florence F Dubois Martine M Mikhailovich Vladimir V Zasedatelev Alexander A Izopet Jacques J

Journal of clinical microbiology 20100915 11

The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV ...[more]

PMID: 20844214

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Project description:BACKGROUND:Current HCV treatments are genotype specific although potential pan-genotype treatments have recently been described. Therefore, genotyping is an essential tool for the therapeutic management of HCV infection and a variety of technologies have been developed for HCV genotypes determination. Sequences analysis of HCV sub-genomic regions is considered as gold standard and is widely used for HCV genotyping. Here, we compared HCV genotyping using core and NS5B regions in routine practice in HCV-positive Cameroonian patients. METHODS:All plasma samples received at Centre Pasteur of Cameroon (CPC) in 2016 for HCV genotyping were included. Viral loads were determined using the Abbott Real Time assay. Further, genotyping was based on the amplification and sequencing of core and NS5B regions following by phylogenetic analysis of corresponding sequences. RESULTS:A total of 369 samples were received during the study period with high viral load values (median: 930,952?IU/ml; IQR: 281,833-2,861,179). Positive amplification was obtained in at least one genomic region (core or NS5B) for all the samples with similar amplification rate in the two genomic regions (p?=?0.34). Phylogenetic analysis showed that among the 369 samples, 146 (39.6%) were classified as genotype 4, 132 (35.8%) as genotype 1, 89 (24.1%) as genotype 2, in both core and NS5B regions. Interestingly, for two samples (0.54%) discordant genotypes were obtained in both regions with the core region classified as genotype 4 while the NS5B was identified as genotype 1 indicating the presence of putative HCV recombinant virus or multiple infections in these samples. Discrimination of HCV subtypes was most likely possible with NS5B compared to core region. CONCLUSIONS:We found high amplification rates of HCV in both core and NS5B regions, and a good concordance was obtained at genotype level using both regions except for two samples where putative 1-4 recombinants/multiple infections were detected. Therefore, HCV genotyping based on at least two genomic regions could help to identify putative recombinants and improve therapeutic management of HCV infection.

Dataset Information

Hepatitis C virus genotyping using an oligonucleotide microarray based on the NS5B sequence.

Publications

Hepatitis C virus genotyping using an oligonucleotide microarray based on the NS5B sequence.

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