Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets

Cristiano, Karen; Bisceglie, Di; Jh, Hoofnagle; Sm, Feinstone

doi:10.1007/978-3-7091-5633-9_36

Cited by 25 publications

(27 citation statements)

References 7 publications

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“…Some of these traps have been described by Willems et al (19) and McGuinness et al (18) as: (a) insufficient inactivation of the RT enzyme after cDNA synthesis; (b) possible RT activity of the Taq DNA polymerase, (c) existence of a thermostable hairpin structure in the 5ЈNCR that leads to self-priming of the HCV RNA template; or (d) mispriming of the incorrect strand. Whereas many studies have reported the detection of positive strand RNA in serum of HCV carriers (9,10,(20)(21)(22)(23)) only a few have actually attempted to demonstrate the presence of negative strand RNA using tightly controlled experiments (9,23). All reported studies have been performed using nontagged primers from the 5ЈNCR in RT-PCR assays.…”

Section: Discussionmentioning

confidence: 99%

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Lerat¹,

Berby²,

Trabaud³

et al. 1996

J. Clin. Invest.

260

188

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The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n ϭ 20) and extrahepatic (sera, n ϭ 32; PBMC, n ϭ 26 and fresh bone marrow cells, n ϭ 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5 Ј noncoding region, with or without a tag sequence, or in the nucleocapsid (CAP). Samples were selected to display different viral loads (10 5 -3 ϫ 10 7 genomic equivalent/ml or gram) and viral genotypes ( n ϭ 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, when either 5 Ј noncoding region primer pair was used, whereas both artifacts were dramatically reduced (

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Section: Discussionmentioning

confidence: 99%

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Lerat¹,

Berby²,

Trabaud³

et al. 1996

J. Clin. Invest.

260

188

View full text Add to dashboard Cite

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“…The discongruent results for the two sets of primers at the beginning of and during therapy could not be the result of a reduced sensitivity of the assay (8). In fact, these results are suggestive of the presence of HCV variants in these patients, and the individual sensitivities of these variants for IFN therapy are under investigation.…”

mentioning

confidence: 88%

Detection of hepatitis C virus RNA in patients with chronic hepatitis C virus infections during and after therapy with alpha interferon

Kleter

Brouwer

Heijtink

et al. 1993

Antimicrob Agents Chemother

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In 24 patients with hepatitic C virus (HCV) infection who participated in a randomized trial with alpha 2B interferon, HCV RNA analysis by the polymerase chain reaction with two separate primer sets was performed at weeks 0, 4, and 24 and during a follow-up period of 6 to 9 months. Prior to therapy all patients were HCV RNA positive. During therapy, HCV RNA decreased to an undetectable level (<1 chimpanzee infectious dose per ml) in nine patients at week 4. After week 4, no additional cases of HCV RNA disappearance (<1 chimpanzee infectious dose per ml) were observed. During follow-up, HCV RNA could not be detected in four of the six patients with a sustained alanine aminotransferase response. These results suggest the probable predictive value of HCV RNA levels for detecting the failure of therapy in an early stage of HCV infection.In recent years, several randomized controlled studies were performed with alpha interferon (IFN) for the treatment of non-A, non-B hepatitis (NANBH) (9,10,13,16,19,20). The effect of IFN was evaluated by measuring the alanine aminotransferase (ALT) level in serum, which reflects the activity of liver disease.Recently, the etiological agent for NANBH has been identified (6, 12). The causative agent is now known as hepatitis C virus (HCV). The HCV genome is a positivestranded RNA molecule of about 9,400 nucleotides. Sequence homology among the known HCV strains is about 80% (3, 7, 14, 17, 21).In the study described here, 24 patients were investigated for the presence of HCV RNA during and after IFN therapy, to determine directly the effect of IFN treatment on viremia. HCV RNA analysis by the polymerase chain reaction was performed by using primer sets from the highly conserved noncoding (NC) region and a conserved sequence from nonstructural region 5 (NS5).Patients between 18 and 70 years of age with elevated ALT levels (22 times the upper limit of normal), a biopsyproven chronic NANBH, antibodies to HCV determined by a second-generation enzyme immunosorbent assay (Abbott, North Chicago, Ill.) and a confirmatory assay RIBA IV (Ortho, Raritan, N.J.), and no recent history of hepatitis B virus, hepatitis A virus, cytomegalovirus, or Epstein-Barr virus infection were included in the study. All patients gave informed consent prior to participation in the study. Patients were treated in a randomized controlled trial with either a standard scheme (12 patients) consisting of 3 MU of recombinant IFN-a2B (Intron A; Schering Plough, Kenilworth, N.J.) three times a week for 24 weeks or an experimental scheme (12 patients). In the experimental scheme, therapy was started with 6 MU of recombinant IFN-ot2B three times a week for at least 8 weeks. Therapy was stopped at week 12 if ALT levels remained elevated. If the ALT level normalized, therapy was continued with 3 MU of recombinant IFN-ot2B three times a week for 8 weeks; this was followed * Corresponding author. 595by treatment with 1 MU of recombinant IFN-ox2B three times a week until a normal ALT level was accompanied by an undetectable HCV RN...

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“…If HCV peptides are presented to the immune system, why does the virus persist? One potential explanation for this phenomenon is that HCV seems to accumulate mutations in both its structural and NS proteins [36,70] , and escape mutants may emerge under the presence of immune selection pressure exerted by CTLs against wild-type virus. Despite the importance of the CTL epitope viral mutation for immune evasion, in HCV infection many highly targeted epitopes have a low mutation frequency.…”

Section: Discussionmentioning

confidence: 99%

Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

Wang

Buchli

Schiller

et al. 2010

WJG

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AIM:To understand how interactions between hepatitis C virus (HCV) and the host's immune system might lead to viral persistence or effective elimination of HCV. METHODS:Nucleotides 3519-3935 of the non-structural 3 (NS3) region were amplified by using reverse transcription polymerase chain reaction (PCR). PCR products of the HCV NS3 regions were integrated into a PCR ® T7TOPO ® TA vector and then sequenced in both directions using an automated DNA sequencer. Relative major histocompatibility complex binding levels of wild-type and variant peptides were performed by fluorescence polarization-based peptide competition assays. Peptides with wild type and variant sequences of NS3 were synthesized locally using F-moc chemistry and purified by high-performance liquid chromatography. Specific cytotoxic T lymphocytes (CTLs) clones toward HCV NS3 wild-type peptides were generated through limiting dilution cloning. The CTL clones specifically recognizing HCV NS3 wild-type peptides were tested by tetramer staining and flow cytometry. Cytolytic activity of CTL clones was measured using target cells labeled with the fluorescence enhancing ligand, DELFIA EuTDA. RESULTS:The pattern of natural variants within three human leukocyte antigen (HLA)-A2-restricted NS3 epitopes has been examined in one patient with chronic HCV infection at 12, 28 and 63 mo post-infection. Results obtained may provide convincing evidence of immune selection pressure for all epitopes investigated. Statistical analysis of the extensive sequence variation found within these NS3 epitopes favors a Darwinian selection model of variant viruses. Mutations within the epitopes coincided with the decline of CTL responses, and peptide-binding studies suggested a significant impact of the mutation on T cell recognition rather than peptide presentation by HLA molecules. While most variants were either not recognized or elicited low responses, such could antagonize CTL responses to target cells pulsed with wild-type peptides. CONCLUSION:Cross-recognition of CTL epitopes from wild-type and naturally-occurring HCV variants may lead to impaired immune responses and ultimately contribute to viral persistence.

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Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets

Cited by 25 publications

References 7 publications

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Detection of hepatitis C virus RNA in patients with chronic hepatitis C virus infections during and after therapy with alpha interferon

Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

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