Hepatitis C virus genotypes in French haemophiliacs: Kinetics and reappraisal of mixed infections

Tuveri, Rosella; Rothschild, Chantal; Pol, Stanislas; Reijasse, Didier; Persico, T.; Gazengel, C.; Bréchot, Christian; Thiers, Valérie

doi:10.1002/(sici)1096-9071(199701)51:1<36::aid-jmv6>3.0.co;2-t

Cited by 32 publications

(33 citation statements)

References 30 publications

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“…Among the discordant results at the genotype level (eight specimens [2.8%]), five were confirmed as mixed-genotype infections by cloning and pyrosequencing and/or QCMD. Mixed-genotype infections have been widely reported, particularly in those multiply exposed to HCV infection (1,24,31). Detection rates vary depending on the patient population studied and the HCV genotyping method used (9,12,15).…”

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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“…To be further spread, the recombinant not only must be viable but also must have been selected for. Superinfection of patients carrying another HCV strain or with mixed infections has been repeatedly reported (11,24,33). However, the frequency of such infections is not well known.…”

Section: Discussionmentioning

confidence: 99%

A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg

Kalinina

Nordér

Мукомолов

et al. 2002

J Virol

283

263

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Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5 untranslated region (5UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1_2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.Hepatitis C virus (HCV) is the major agent of parenterally transmitted non-A, non-B hepatitis worldwide (1). HCV is an enveloped virus classified in the family Flaviviridae. It has a single-stranded, positive-sense, nonsegmented RNA genome of approximately 9,500 nucleotides (nt) with a single, long open reading frame encoding a polyprotein of ϳ3,030 amino acids with the gene order C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The structural proteins are C (core) and E1 and E2 (envelope glycoproteins). The function of the p7 gene product is presently unknown. The NS2 through NS5 regions encode putative nonstructural proteins (2).HCV is characterized by a high degree of genetic heterogeneity. Its variability is, however, distributed unequally within the genome, and some regions, such as NS3 and NS5B, are considerably conserved. HCV is classified into six genotypes and an increasing number of subtypes based on degree of genomic sequence divergence (26,27). Evolution of HCV is believed to be mediated by point, insertion, or deletion mutations. Another mechanism for viral evolution is through recombination. There has so far been little evidence of recombination between HCV strains of different genotypes (38), and it has been suggested that these events are rare in vivo and that the resultant recombinants are usually not viable (26,28,34).Relatively short subgenomic region...

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“…Multitypic HCV infection has been reported in individuals at risk of multiple or repeated HCV exposures, e.g., patients with bleeding disorders given inadequately sterilized clotting factor concentrates (11,16,21) and injection drug users (IDUs) (1). Infection of a host with multiple HCV genotypes potentially complicates pre-antiviral therapy assessments.…”

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confidence: 99%

“…Thus, studies using these approaches to evaluate the prevalence of multitypic HCV infection likely lead to underestimates. Other approaches have been adopted to study multitypic HCV infection specifically (5,9,10,18,(20)(21)(22). Those involving PCR cloning and sequencing are potentially the most effective to identify minority populations, but the impracticality and expense of sequencing large numbers of clones restrict their wider applicability.…”

mentioning

confidence: 99%

Multitypic Hepatitis C Virus Infection Identified by Real-Time Nucleotide Sequencing of Minority Genotypes

Buckton¹,

Ngui²,

Arnold³

et al. 2006

J Clin Microbiol

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The prevalence of concurrent multitypic hepatitis C virus (HCV) infection is uncertain. A sensitive and specific approach to identifying minority HCV genotypes in blood is presented. Following serum extraction and reverse transcription PCR to amplify cDNA originating from the viral 5 noncoding region, the amplified product mixture was treated with genotype-specific restriction endonuclease to digest the dominant genotype. Residual amplicons were subjected to PCR cloning and then to real-time DNA sequencing using a Pyrosequencer to identify the remaining genotypes. Dilution experiments showed that minority genotypes may be detected when they represent 1:10,000 of the total population and in serum specimens with viral loads as low as 1,000 IU/ml. Of 37 patients with bleeding disorders and 44 injecting drug users, infection by more than one HCV genotype was found in 7 (19%) and 4 (9%) patients, respectively. The low rate of detection in people at high risk of repeated HCV infection suggests that multitypic HCV carriage is uncommon.

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Hepatitis C virus genotypes in French haemophiliacs: Kinetics and reappraisal of mixed infections

Cited by 32 publications

References 30 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

A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg

Multitypic Hepatitis C Virus Infection Identified by Real-Time Nucleotide Sequencing of Minority Genotypes

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