Genotyping of hepatitis C virus isolates by a modified polymerase chain reaction assay using type specific primers: Epidemiological applications

Widell, Anders; Shev, Steven; Månsson, Siv; Zhang, Yongyuan; Foberg, U; Norkrans, Gunnar; Frydén, Aril; Weiland, Ola; Kurkus, Jan; Nordenfelt, Erik

doi:10.1002/jmv.1890440311

Cited by 101 publications

(68 citation statements)

References 31 publications

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“…4, 1999 prevalence of the patients with HCV-2b infection among chronic hepatitis C in Japan is only 5%, 6,37 whereas HCV-2b or -2c is more prevalent than HCV-2a in other geographic areas. [45][46][47][48] Therefore, detailed information related to the HCV subtypes of the study population is necessary for interpreting the HCV study from the different geographic areas where the distribution of HCV subtypes is dissimilar. 44,49,50 Thus, the virological or clinical differences between HCV-2a and -2b infection should be investigated.…”

Section: Discussionmentioning

confidence: 99%

Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection

et al. 1999

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An association has been reported between mutations in the amino acid residues 2209-2248 of the nonstructural protein 5A (NS5A) gene (interferon-sensitivity determining region [ISDR]) and interferon efficacy in hepatitis C virus (HCV)-1b infection. This relationship was analyzed in chronic HCV-2 infection. Forty patients with HCV-2a and 35 with HCV-2b were treated with interferon alfa for 6 months with a total dose of 468 to 860 million units. Pretreatment NS5A sequences were determined by direct sequencing. A higher complete and sustained response rate was observed in HCV-2a than in HCV-2b (70% vs. 34%; P ‫؍‬ .003). Serum HCV-RNA levels were lower in complete responders than nonresponders in HCV-2a (P ‫؍‬ .049) and HCV-2b (P ‫؍‬ .02). The number of amino acid mutations was greater in complete responders than nonresponders in NS5A2193- The current primary therapy for chronic hepatitis C is parenteral administration of type 1 interferons (interferon alfa and beta), while the rate of sustained and complete responses with virus eradication is obtained in only up to 30% of the patients. 1-3 To date, hepatitis C virus (HCV) is classified into at least 9 major genotypes and more than 30 subtypes. 4,5 In Japan, about 70% of the patients with chronic hepatitis C are infected with HCV genotype 1b (HCV-1b), and the rest are infected with HCV genotype 2 (HCV-2) 6 (25% with HCV-2a and 5% with HCV-2b). While the complete response is as low as 10% to 20% in HCV-1b infection, it is considered more than 60% in HCV-2 infection in Japan. [7][8][9][10][11][12] Such differences in interferon efficacy among various HCV genotypes suggest that a certain kind of genetic change of the virus could affect the sensitivity to interferon.We recently identified a region that is associated with the response to interferon in HCV-1b genomes in Japan. 13 An increase in the number of amino acid changes in the nonstructural protein 5A gene (NS5A) (NS5A2209-2248, interferon sensitivity determining region [ISDR]) makes HCV more sensitive to interferon. 14 However, the mechanism of the different response to interferon therapy among patients with HCV-2 infection is still unclear. Some studies suggested that a lower viral load is associated with the good response to interferon in HCV-1b or non-1b, 12,15 the mechanism of which is still unknown. In previous studies, interferon effect and pretreatment viral load were also associated with the mutations in ISDR in HCV-1b. 13,16 Thus, the relation between NS5A structure and interferon effect or viral load in HCV genotypes other than HCV-1b is of great interest.Several studies that focused on biological functions of NS5A revealed some aspects of its properties. In particular, Gale et al. 17 suggested that the NS5A protein inhibits an RNA-dependent protein kinase (PKR), which plays a major role in the viral protection by inhibiting viral protein translation. The NS5A protein of HCV-1b and -1a forms a complex with the PKR and inhibits the phosphorylation of eukaryotic translation initiation factor-2 (eIF-2). Mor...

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Section: Discussionmentioning

confidence: 99%

Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection

et al. 1999

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“…z 'g'~'n was probably transmitted to Europe through in-.s Z z; _ _ o > travenous drug use. Genotype la is predominant in the US, Canada and Northern Europe [5,6,26]. It has Z \ been associated with transmission by stored blood products [27] and intravenous drug use [7,23].…”

Section: Discussionmentioning

confidence: 99%

“…The major groupings of sequence variants are designated HCV types and the more closely related groups observed within some types are termed subtypes following the classification system proposed by Simmonds and other researchers [4]. The genotyping of HCV isolates has become important for epidemiological purposes [5][6][7]. Several studies have suggested the existence of biological differences between HCV genotypes which affect their capacity for producing liver disease and sensitivity to interferon treatment [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Genotyping of hepatitis C virus isolates from Basque Country, Spain

Cilla¹,

García-Bengoechea²,

Pérez-Trallero³

et al. 1996

Epidemiol. Infect.

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SUMMARYThe genotype of HCV was determined in 161 chronic HCV-infected patients. The patients were classified into three groups on the basis of the origin of the HCV infection: 50 patients had a history of intravenous drug use (IVDU) but no HIV infection; 41 patients had received blood transfusions, and 70 patients had no known exposure. The distribution of HCV genotypes was associated with the origin of infection and age of patients: genotype lb was predominant among patients who had received blood transfusions and those without evidence of parenteral exposure (84 6% and 67-7 %, respectively), whereas genotype 3a was present in 65-3 % of IVDUs. Patients with genotype lb were older than those with genotypes la or 3a: 503 + 12 vs. 34 1 + 9-9 and 31 + 5.4 years, respectively. These findings suggest that the pattern of HCV genotypes in our region is changing and that genotype lb may be substituted by 3a as the dominant genotype in the future.

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“…In our study, a phylogenetic analysis of the 5Ј-UTR sequences was able to identify genotype 1 but it did not distinguish subtype 1a from subtype 1b. In addition, analysis of all genotype 1 isolates for the putative subtype-specific mutation at position Ϫ99 (A/G) expected to distinguish 1a from 1b (18,42) was in fact unable to distinguish 1a from 1b. Within genotype 1, nucleotide A at position Ϫ99 was always subtype 1a whereas nucleotide G on the same position was either subtype 1b (33%) or 1a (66%).…”

Section: Vol 41 2003 Hcv Subtyping Of Danish Isolates 1095mentioning

confidence: 98%

“…Many different typing methods have been used, all of which depend on a few relatively specific nucleotide changes: RT-PCR using type-specific amplification (18,42), hybridization of RT-PCR products with type-specific probes (34), restriction fragment length polymorphism of RT-PCR amplicons (27), or heteroduplex mobility analysis of RT-PCR amplicons (41). However, these methods were often found not to be definitive, as more sequence data became available.…”

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confidence: 99%

Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients

et al. 2003

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A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5-untranslated region (5-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.

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Genotyping of hepatitis C virus isolates by a modified polymerase chain reaction assay using type specific primers: Epidemiological applications

Cited by 101 publications

References 31 publications

Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection

Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection

Genotyping of hepatitis C virus isolates from Basque Country, Spain

Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients

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