scholarly journals Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Kazuya Shiogama ◽  
Ken-ichi Inada ◽  
Michinori Kohara ◽  
Hidemi Teramoto ◽  
Yasuyoshi Mizutani ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleic Acid ◽  
Oligonucleotide Probe ◽  
High Sensitivity ◽  
Locked Nucleic Acid ◽  
Hcv Rna ◽  
Chimeric Mice ◽  
Rt Pcr ◽  
Modified Oligonucleotide

Background.In situhybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

scholarly journals Therapeutic Silencing of MicroRNA-122 in Primates with Chronic Hepatitis C Virus Infection

Science ◽  
2009 ◽  
Vol 327 (5962) ◽  
pp. 198-201 ◽  
Author(s):  
Robert E. Lanford ◽  
Elisabeth S. Hildebrandt-Eriksen ◽  
Andreas Petri ◽  
Robert Persson ◽  
Morten Lindow ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Locked Nucleic Acid ◽  
Hcv Rna ◽  
Hepatitis C Virus Infection ◽  
Chronic Hepatitis C Virus ◽  
Cultured Liver Cells ◽  
Liver Biopsies ◽  
Rna Accumulation ◽  
Modified Oligonucleotide

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)–modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

scholarly journals Nucleic Acid Testing for Diagnosis of Perinatally Acquired Hepatitis C Virus Infection in Early Infancy

2020 ◽  
Author(s):  
Charitha Gowda ◽  
Stephanie Smith ◽  
Linda Crim ◽  
Katherine Moyer ◽  
Pablo J Sánchez ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antibody Test ◽  
Case Definition ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Case Definitions ◽  
Loss To Follow Up ◽  
Perinatally Acquired

Abstract Background Most US children with perinatal hepatitis C virus (HCV) exposure fail to receive the recommended anti-HCV antibody test at age ≥18 months. Earlier testing for viral RNA might facilitate increased screening, but sensitivity of this approach has not been established. We hypothesized that modern HCV-RNA RT-PCR platforms would adequately detect infected infants. Methods Nationwide Children’s Hospital electronic health records from 1/1/2008 to 30/6/2018 were reviewed to identify perinatally exposed infants tested by HCV-RNA RT-PCR at age 2–6 months. Diagnostic performance was determined using a composite case definition: (1) infected children had positive repeat HCV-RNA testing or positive anti-HCV at age ≥24 months; (2) uninfected children lacked these criteria and had negative anti-HCV at age ≥18 months. Results 770 perinatally exposed infants underwent HCV-RNA testing at age 2–6 months. Of these, 28 (3.6%) tested positive; viremia was confirmed in all who underwent repeat testing (n = 27). Among 742 infants with negative HCV-RNA results, 226 received follow-up anti-HCV testing at age ≥18 months, of whom 223 tested negative. Three children had low-positive anti-HCV results at age 18–24 months that were negative upon retesting after age 24 months, possibly indicating waning maternal antibodies. Using the composite case definitions, early HCV-RNA screening demonstrated sensitivity of 100% (87.5–100%, Wilson-Brown 95% CI) and specificity of 100% (98.3–100%). Conclusions Modern HCV-RNA RT-PCR assays have excellent sensitivity for early diagnosis of perinatally acquired infection and could aid HCV surveillance given the substantial loss to follow-up at ≥18 months of age.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3328-3337 ◽  
Author(s):  
Domenico Sansonno ◽  
Claudio Lotesoriere ◽  
Vito Cornacchiulo ◽  
Massimo Fanelli ◽  
Pietro Gatti ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Progenitor Cells ◽  
Hcv Infection ◽  
Hematopoietic Progenitor Cells ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

scholarly journals Hepatitis C Virus Persistence after Spontaneous or Treatment-Induced Resolution of Hepatitis C

2004 ◽  
Vol 78 (11) ◽  
pp. 5867-5874 ◽  
Author(s):  
Tram N. Q. Pham ◽  
Sonya A. MacParland ◽  
Patricia M. Mulrooney ◽  
Helen Cooksley ◽  
Nikolai V. Naoumov ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mononuclear Cells ◽  
Lymphoid Cells ◽  
Nucleic Acid Hybridization ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Virus Eradication ◽  
Peripheral Blood Mononuclear ◽  
Negative Strand

ABSTRACT It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of hepatitis C virus (HCV) RNA from serum, as determined by conventional laboratory assays, reflects virus eradication. In this study, we examined the expression of the HCV genome in the sera, peripheral blood mononuclear cells (PBMC), and, on some occasions, monocyte-derived dendritic cells (DC) long after resolution of hepatitis C by using a highly sensitive reverse transcription (RT)-PCR-nucleic acid hybridization (RT-PCR-NAH) assay. The samples obtained from 16 randomly selected patients (5 with spontaneous and 11 with treatment-induced resolution), monitored for up to 5 years, were studied by qualitative and semiquantitative RT-PCR-NAH and by real-time RT-PCR to detect the HCV RNA positive strand. The replicative HCV RNA negative strand was examined in PBMC after culture with a T-cell proliferation stimulating mitogen. The findings show that HCV RNA was carried in the convalescent-phase sera and/or PBMC in all 16 individuals investigated. Also, DC from six of seven patients were reactive for the HCV genome. Importantly, traces of the HCV RNA negative strand, suggesting progressing virus replication, were detected in the majority of mitogen-stimulated PBMC, including four samples collected 5 years after recovery. Sequencing of the HCV 5′ untranslated region fragment revealed genotype 1b in four of nine individuals examined and genotypes 1a and 2a in three and two patients, respectively. These results imply that HCV RNA can persist at very low levels in the serum and peripheral lymphoid cells and that an intermediate replicative form of the HCV genome can persist in PBMC for many years after apparently complete spontaneous or antiviral therapy-induced resolution of chronic hepatitis C.

scholarly journals RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128686 ◽  
Author(s):  
Margit Mutso ◽  
Andrei Nikonov ◽  
Arno Pihlak ◽  
Eva Žusinaite ◽  
Liane Viru ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Interference ◽  
Nucleic Acid ◽  
Virus Replication ◽  
Locked Nucleic Acid

Antibody testing and RT-PCR results in hepatitis C virus (HCV) infection: HCV-RNA detection in PBMC of plasma viremia-negative HCV-seropositive persons

Infection ◽  
1998 ◽  
Vol 26 (3) ◽  
pp. 151-154 ◽  
Author(s):  
C. Caudai ◽  
M. G. Padula ◽  
I. Bastianoni ◽  
P. E. Valensin ◽  
V. Shyamala ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Plasma Viremia ◽  
Rt Pcr ◽  
Antibody Testing ◽  
Rna Detection

scholarly journals Hepatitis C Virus Quantitation: Optimization of Strategies for Detecting Low-Level Viremia

2000 ◽  
Vol 38 (2) ◽  
pp. 888-891 ◽  
Author(s):  
Wolfgang T. Hofgärtner ◽  
Jeffrey A. Kant ◽  
Karen E. Weck
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Medical Center ◽  
Significant Proportion ◽  
Cost Effective ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Economic Implications ◽  
Hcv Testing

A long-term assessment of quantitative hepatitis C virus (HCV) testing was performed at the University of Pittsburgh Medical Center. The Quantiplex HCV RNA 2.0 branched-chain DNA (bDNA) assay (Bayer Diagnostics) for hepatitis C viral load determination was used to test 3,471 specimens. bDNA-negative samples were also tested by an in-house qualitative reverse transcriptase (RT)-PCR assay with a measured sensitivity of fewer than 100 HCV genome equivalents per milliliter. Of 1,239 bDNA-negative specimens, 74.1% were negative and 25.9% were positive by RT-PCR, indicating the presence of viremia in a significant proportion of bDNA-negative samples. We discuss the medical and economic implications of these results and propose two alternatives for clinical laboratories to consider in approaching quantitative HCV testing. For laboratories able to perform a sensitive RT-PCR assay for ≤40% of the bDNA test cost, prescreening bDNA requests by RT-PCR may be the most cost-effective approach.

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