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, Adrian M. Di; Hoofnagle, Jay H.

doi:10.1002/hep.1840140109

Cited by 160 publications

(35 citation statements)

References 13 publications

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“…This method can detect a single point mutation and is widely used to screen mutations in various oncogenes (Cottrell et at., 1992). In addition, the nested PCR used in the present study to generate the ssDNA is known to have the same level of specificity as Southern blot hybridization for the detection of a target gene (Cristiano et al, 1991).…”

Section: Discussionmentioning

confidence: 94%

Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis

Enomoto¹,

Kurosaki²,

Tanaka³

et al. 1994

Journal of General Virology

184

100

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Hepatitis C virus (HCV) populations in vivo consist of heterogeneous mixtures of genetically different but closely related variants defined as a 'quasispecies'. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5'-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymoi]~h-ism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.

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Section: Discussionmentioning

confidence: 94%

Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis

Enomoto¹,

Kurosaki²,

Tanaka³

et al. 1994

Journal of General Virology

184

100

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“…Unfortunately, in the absence of an efficient in vitro replication system or convenient animal model, the causal relationship between CTL and variant viral peptide sequence in HCV infection cannot be tested directly (34)(35)(36). Furthermore, our stimulation method does not allow us to distinguish between immunodominant versus subdominant epitopes nor could we study the CTL response and virus sequence from the onset of their infection.…”

Section: Discussionmentioning

confidence: 99%

“…The viral cDNA was amplified by PCR with appropriate precautions to prevent PCR contamination as described by Kwok and Higuchi (27), and the PCR products were analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. The primer sets and PCR conditions to amplify the ten epitopes are described as follows: PCR products containing the HCV fragment were sequenced with the corresponding internal HCV primers, Sequenase 2.0 (Amersham Corp., Arlington Heights, IL), 35 S dATP (Amersham Corp.) using dideoxynucleotide chain-termination method according to the manufacturer's instructions, analyzed on a 6% denaturing acrylamide gel, and autoradiographed. Ambiguous or variant (i.e., non-HCV-1) sequences were confirmed by sequencing of both sense and antisense strands, and/or by sequencing of second independently amplified PCR product.…”

Section: Methodsmentioning

confidence: 99%

Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus.

Chang¹,

Rehermann²,

McHutchison³

et al. 1997

J. Clin. Invest.

313

251

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This study was performed to test the hypothesis that cytotoxic T lymphocyte (CTL) selection of hepatitis C virus (HCV) escape variants plays a role in HCV persistence. The peripheral blood CTL responsiveness of patients with wellestablished chronic hepatitis C to a panel of 10 prototype HCV peptides (genotype 1a) was compared with the corresponding sequences encoded by the infecting viruses in each patient. Variant viral peptide sequences were threefold more frequent in the presence of a CTL response than in its absence, and CTL responses were detected nearly twice as often in association with variant rather than with prototype viral peptide sequences. Furthermore, over half of the patients were infected with potential CTL escape variants that contained nonimmunogenic and noncross-reactive variant peptides many of which displayed reduced HLA-binding affinity. Surprisingly, follow up analysis over a period of up to 46 mo revealed that, in contrast to the relatively high frequency of escape variants initially observed, the subsequent emergence rate of CTL escape variants was very low. Interestingly, the one escape variant that was detected proved to be a CTL antagonist. Collectively, these observations suggest that CTL selection of epitope variants may have occurred in these patients before their entrance into the study and that it may have played a role in HCV persistence. The low apparent rate of ongoing CTL selection in chronically infected patients, however, suggests that if CTL escape occurs during HCV infection it is probably an early event. ( J.

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“…Although the time required for the development of HCV-specific antibodies after acute infection is reduced, these tests remain unsatisfactory for the diagnosis of acute infection. HCV RNA PCR, performed in individual research laboratories, has rapidly become the diagnostic standard for HCV infection (19)(20)(21)(24)(25)(26)(27)(28)(29). Most investigators prefer primers from the 5' UTR since HCV nt analyses have indicated that the 5' UTR has a very high homology (2 97%) among different HCV strains (8,13,14).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, multiple weeks are required for development of anti-HCV after HCV infection (18), which precludes the use of anti-HCV for the diagnosis of acute infection. The second technique detects HCV RNA through a reverse transcription and polymerase chain reaction (HCV RNA PCR) (19)(20)(21). Although HCV RNA PCR is a sensitive technique, extensive application is limited by cost, labor intensity, risk of false-positive resultsthrough contamination (22,23), and disparate results among laboratories using different primers (21,24 (25)(26)(27)(28).…”

Section: Introductionmentioning

confidence: 99%

Direct detection of circulating hepatitis C virus RNA using probes from the 5' untranslated region.

Hu¹,

Yu²,

Vierling³

1992

J. Clin. Invest.

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Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 983%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10' chimpanzee infectious doses5o/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards. (J. Clin.

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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 160 publications

References 13 publications

Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis

Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis

Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus.

Direct detection of circulating hepatitis C virus RNA using probes from the 5' untranslated region.

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