Detection of Extrahepatic Hepatitis C Virus Replication by a Novel, Highly Sensitive, Single-Tube Nested Polymerase Chain Reaction

Guilfoyle, Dennis E.; Hu, Yuan; Park, Soon; Shahidi, Azra; Hirshfield, Irvin N.

doi:10.1309/33ta-jlb7-48kl-mxvg

Cited by 10 publications

(9 citation statements)

References 28 publications

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“…Strand-specific intracellular HCV RNA was detected using a recently established procedure that combined previously published methods [27], [28] with minor modifications [4], [13]. Positive- and negative-strand-specific HCV RNAs were detected by a nested polymerase chain reaction (PCR) method.…”

Section: Methodsmentioning

confidence: 99%

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HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

et al. 2014

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BackgroundVarious kinds of autoimmune diseases have been reported to have a significant relationship with persistent hepatitis c virus (HCV) infection and Th17 cells. Previously, our group reported that the existence of HCV in T lymphocytes could affect the development of CD4+ helper T cells and their proliferation, in addition to the induction of immunoglobulin hyper-mutation.MethodsTherefore, we analyzed the relationship between persistent infection of HCV and the mechanism of Th17 cell induction ex vivo and in vitro.ResultsThe prevalence of autoimmune-related diseases in chronic hepatitis c patients (CH-C) was significantly higher than in other types of chronic hepatitis (hepatitis B and NASH). A significantly higher frequency of IL6 and TGF-β double-high patients was detected in CH-C than in other liver diseases. Moreover, these double-high patients had significantly higher positivity of anti-nuclear antibody, cryoglobulinemia, and lymphotropic HCV and higher amounts of IL1-β, IL21, IL23. In addition to the previously reported lymphotropic SB-HCV strain, we found a novel, genotype 1b lymphotropic HCV (Ly-HCV), by deep sequencing analysis. Lymphotropic-HCV replication could be detected in the lymphoid cells with various kinds of cytokine-conditions including IL1β, IL23, IL6 and TGF-β in vitro. Infection by HCV could significantly enhance the development of Th17 cells. The HCV protein responsible for inducing the Th17 cells was HCV-Core protein, which could enhance the STAT-3 signaling and up-regulate the expression of RORγt as a Th17 master gene.ConclusionInfection by lymphotropic HCV might enhance the Th17 development and contribute to understanding the pathogenesis of autoimmune-related diseases.

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Section: Methodsmentioning

confidence: 99%

“…Other groups have also reported the existence of HCV in T lymphocytes[14], [15]. HCV replication in T lymphocytes could suppress Interferon-γ (IFN-γ)/signal transducers and activators of transcription factor 1 (STAT-1) signaling that might affect signal transducers and activators of transcription factor 3 (STAT-3) signaling[4], [13].…”

Section: Introductionmentioning

confidence: 99%

HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

et al. 2014

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“…Strand-specific intracellular HCV RNA was detected by using a recently established procedure that combined previously published methods [9, 18] with minor modifications [13]. Positive- and negative-strand-specific HCV RNAs were detected by a nested polymerase chain reaction (PCR) method.…”

Section: Methodsmentioning

confidence: 99%

Lymphotropic HCV strain can infect human primary naïve CD4+ cells and affect their proliferation and IFN-γ secretion activity

et al. 2010

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Background Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear. Aim The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment. Methods SB strain cell culture supernatant (2 × 104 copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4+CD45RA+CD45RO− naïve CD4+ cells. Interferon (IFN)-γ and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out. Results Negative strand HCV RNA was detected in CD4+, CD14+, and CD19+ cells. Among CD4+ cells, CD4+CD45RA+RO− cells (naïve CD4+ cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected naïve CD4+ cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected naïve CD4+ cells. Moreover, the production of IFN-γ was significantly suppressed in SB-infected naïve CD4+ cells. Conclusions Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary naïve CD4+ cells.

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“…Strand-specific intracellular SB-HCV RNA was detected by using a recently established procedure that combined methods published elsewhere [31, 32], with minor modifications [5]. Positive strand–specific and negative strand–specific SB-HCV RNA were detected by use of a nested polymerase chain reaction (PCR) method.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C Virus Infection of T Cells Inhibits Proliferation and Enhances Fas-Mediated Apoptosis by Down-Regulating the Expression of CD44 Splicing Variant 6

et al. 2009

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Background A lymphotropic hepatitis C virus strain (HCV, SB strain, hereafter “SB-HCV”) has been shown to infect established T cell lines (Molt-4 and Jurkat) and primary human naive CD4+ T cells. During T cell development and activation, transient expression of CD44 splicing variant 6 (CD44v6) plays a significant role. Methods SB-HCV was used to infect Molt-4 cells, and their cellular proliferation and CD44 expression was examined. Results SB-HCV–infected Molt-4 cells expressed a significantly lower level of the CD44v6 isoform. The infected cells could be divided into 2 carboxyfluorescein succinimidyl ester (CFSE) groups, CFSE–high (indicating low proliferation activity; 34.2% of the cells) and CFSE-low (indicating high proliferation activity; 62.5% of the cells), whereas uninfected cells consisted of only a CFSE-low population. Of the CFSE-high cells, 82.4% were positive for the HCV protein NS5A, whereas only 1.2% of the CFSE-low cells were positive for this protein. Among the HCV proteins, NS5A alone caused the down-regulation of CD44v6 expression. After cells were stimulated with phorbol myristate acetate, the amount of phosphorylated mitogen-activated protein (MAP) kinase was significantly reduced in CFSE-high, SB-HCV–infected Molt-4 cells. After Fas ligand stimulation, SB-HCV–infected Molt-4 cells had increased cleavage of caspase 8 and 3 and enhanced apoptosis, compared with the rates of cleavage and apoptosis in control groups, indicating that SB-HCV infection increased Fas-mediated apoptosis. Conclusion HCV replication in T cells suppresses cellular proliferation and enhances susceptibility to Fas signaling by inhibiting CD44v6 signaling and expression.

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Detection of Extrahepatic Hepatitis C Virus Replication by a Novel, Highly Sensitive, Single-Tube Nested Polymerase Chain Reaction

Cited by 10 publications

References 28 publications

HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

Lymphotropic HCV strain can infect human primary naïve CD4+ cells and affect their proliferation and IFN-γ secretion activity

Hepatitis C Virus Infection of T Cells Inhibits Proliferation and Enhances Fas-Mediated Apoptosis by Down-Regulating the Expression of CD44 Splicing Variant 6

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