Genomic and phylogenetic analysis of hepatitis C virus isolates: A survey of 535 strains circulating in southern France

Tamalet, Catherine; Colson, Philippe; Tissot‐Dupont, Hervé; Henry, Mireille; Tourres, Christian; Tivoli, Natacha; Botta, D.; Ravaux, Isabelle; Poizot‐Martin, Isabelle; Yahi, Nouara

doi:10.1002/jmv.10505

Cited by 38 publications

(55 citation statements)

References 51 publications

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“…Since 5ЈNC amplification is regularly performed for HCV molecular diagnosis and viral load quantitation, genotyping methods based on this highly conserved region are convenient. However, discrimination among subtypes (especially 1a versus 1b and 2a versus 2c) and certain genotypes (genotype 6 isolates have been identified as genotype 1) is not always reliable using this region, and this often leads to subtyping errors (6,27,30,32). Nucleotide sequencing followed by phylogenetic analysis of more-variable genomic regions, such as NS5b or core, has been recommended for HCV genotyping in consensus proposals (27).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

Martró

González

Buckton³

et al. 2008

J Clin Microbiol

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We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5 noncoding region (5NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5NC reverse hybridization (method 2; InnoLiPA HCV II) and 5NC sequencing (method 3; Trugene HCV 5NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.Hepatitis C virus (HCV) is the most important cause of chronic liver disease and is the leading indication for liver transplantation (2). It is estimated that HCV infects 3% (170 million people) of the world's population (26). HCV possesses a positive-sense single-stranded RNA genome of approximately 9.5 kb, which is flanked by noncoding (NC) regions. The HCV genome encodes at least 11 proteins, which include both structural and nonstructural (NS) proteins (5, 7).HCV is known to have a high rate of genetic heterogeneity. This has allowed HCV strains to be classified into a number of genetically distinct groups, known as genotypes, subtypes, isolates, and quasispecies (5). Genome sequence heterogeneity arises due to poor fidelity of the viral polymerase during replication. Sequence variability is not evenly distributed throughout the genome. The lowest sequence variability is found in the 5ЈNC region, which is the target of choice for many molecular diagnostics assays, including genotyping tests. Nevertheless, nucleotide sequencing coupled with phylogenetic analysis of more-variable genomic regions has been recommended for HCV genotyping in consensus proposals (27). As patients infected with different genotypes respond differently to antiviral drug therapy, identification of the infecting genotype ha...

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Section: Discussionmentioning

confidence: 99%

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

Martró

González

Buckton³

et al. 2008

J Clin Microbiol

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“…Genotyping is also an essential tool for epidemiological studies, since HCV genotypes vary according to epidemic history in different geographical regions (23,24,30,40). Epidemiologic studies of HCV strains from blood donors (7, 18), drug addicts (1, 12, 13), and hospital patients (22,35) have demonstrated a correlation between some subtypes and risk factors.Since the 5Ј noncoding region (5Ј NCR) is one of the most highly conserved regions of HCV, it has historically been used for virus detection and is now one of the best-characterized regions. For practical reasons, the 5Ј NCR was also chosen as the target for various genotyping methods, including the InnoLipa HCV II test (33, 34), sequencing (8,9,10,26), and the duplex mobility assay (39).…”

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confidence: 99%

Analysis of the 5′ Noncoding Region versus the NS5b Region in Genotyping Hepatitis C Virus Isolates from Blood Donors in France

et al. 2006

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The 5 noncoding region (5 NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5 NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (ϳ22%) initially identified as genotype 1b by 5 NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (ϳ61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (ϳ45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5 NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5 NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.Hepatitis C virus (HCV) is a common human pathogen considered as the major cause of parenterally transmitted hepatitis (6). It is an enveloped virus with a positive-sense RNA genome containing a single large open reading frame composed of over 9,000 nucleotides (6, 11). Sequencing of HCV isolates has identified 6 genotypes and more than 70 subtypes (19,25,28,32). Accurate HCV genotyping is important for predicting response to antiviral therapy, since genotypes 1 and 4 are less likely to respond to interferon than genotypes 2 and 3 (14, 21). Genotyping is also an essential tool for epidemiological studies, since HCV genotypes vary according to epidemic history in different geographical regions (23,24,30,40). Epidemiologic studies of HCV strains from blood donors (7, 18), drug addicts (1, 12, 13), and hospital patients (22,35) have demonstrated a correlation between some subtypes and risk factors.Since the 5Ј noncoding region (5Ј NCR) is one of the most highly conserved regions of HCV, it has historically been used for virus detection and is now one of the best-characterized regions. For practical reasons, the 5Ј NCR was also chosen as the target for various genotyping methods, including the InnoLipa HCV II test (33, 34), sequencing (8,9,10,26), and the duplex mobility assay (39). However, recent studies involving NS5b region analysis (3,29,35) show that 5Ј NCR sequence analysis can lead to genotyping errors (5, 35). In this study, we used NS5b region sequence analysis to determine the HCV subtype distribution of 357 isolates collected from blood donors in France between 2002 and 2003. All samples had already been genotyped based on 5Ј NCR analysis. Results of the two seque...

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“…However, several studies showed that subtyping analysis based on 5'UTR may be limited (18,26,27) due to insufficient sequence variation in these highly conserved regions.…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C virus genotype distribution in Turkey remains unchanged after a decade: Performance of phylogenetic analysis of the NS5B, E1, and 5’UTR regions in genotyping efficiency

Alagöz

Karataylı²,

Karataylı³

et al. 2014

Turk J Gastroenterol

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Background/Aims: Hepatitis C virus (HCV) genotyping has a considerable effect on therapy. The aim was to determine the change in prevalence of HCV genotypes in Turkey during the last decade and to compare the performance of DNA sequencing of different targets in the HCV genome (NS5B, E1, and 5'UTR). Materials and Methods: Five hundred HCV RNA-positive patients (226 males, 274 females) were included in the study. The NS5B, E1, and 5'UTR regions of the HCV genome were amplified by polymerase chain reaction (PCR) in patients where possible. Amplified PCR products were sequenced directly, and phylogenetic analysis was performed. A commonly used database, namely www.hcv.lanl.gov, was also used to determine the genotypes. Results: Phylogenetic analysis of the NS5B, E1, and 5'UTR regions showed that 1b was the most frequent genotype, with percentages of 92.5%, 93.5%, and 87.7%, respectively. Genotype 1a was the second most prevalent genotype, with ratios of 6.7%, 5.6%, and 6.6%, whereas genotype 2a was detected in proportions of 0.4%, 0.2%, and 0.8%, respectively. Genotype 5 or 6 was not detected among patients. The phylogenetic analysis showed discordant results with 18 patients' genotypes for different targets. The phylogenetic analysis showed similar results with the hcv.lanl. gov database for the E1 and NS5B sequences. Conclusion: There has been no change in genotyping profiles of Turkey during the last decade, representing 1b as the most prevalent subtype, followed by 1a. Phylogenetic analysis of HCV indicated high performance compared with the hcv.lanl.gov database when sequences of E1 and NS5B regions were analyzed.

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Genomic and phylogenetic analysis of hepatitis C virus isolates: A survey of 535 strains circulating in southern France

Cited by 38 publications

References 51 publications

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

Analysis of the 5′ Noncoding Region versus the NS5b Region in Genotyping Hepatitis C Virus Isolates from Blood Donors in France

Hepatitis C virus genotype distribution in Turkey remains unchanged after a decade: Performance of phylogenetic analysis of the NS5B, E1, and 5’UTR regions in genotyping efficiency

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