• PBMCs
• genotype
• hepatitis C virus
• patients
• sequence analysis

• challenge model
• controlled human infection model
• hepatitis C virus
• trial design
• vaccine

• Humans
• Hepacivirus
• Sample Size
• Hepatitis C/prevention & control
• Hepatitis C/drug therapy
• Vaccines
• Hepatitis C, Chronic/prevention & control
• Hepatitis C, Chronic/drug therapy
• Antiviral Agents/therapeutic use

• K24 DA034621 NIDA NIH HHS
• R01 DA048063 NIDA NIH HHS
• US National Institutes of Health

• CpG methylation
• HCV
• SHP-1
• next-generation sequencing

• Humans
• CpG Islands
• Epigenesis, Genetic
• DNA Methylation
• Genes, Tumor Suppressor
• Carcinoma, Hepatocellular/genetics
• Carcinoma, Hepatocellular/metabolism
• High-Throughput Nucleotide Sequencing
• Liver Neoplasms/genetics
• Liver Neoplasms/metabolism
• Hepatitis C/genetics
• Cell Line

Occult hepatitis C virus infection (OCI) is the absence of HCV RNA in serum and the presence of actively replicating HCV RNA in hepatocytes and peripheral blood mononuclear cells (PBMCs), as evidenced by the presence of antigenomic negative sense single-stranded RNA. This study aimed to determine the prevalence of OCI in Egyptian lymphoma patients and assess changes in biochemical parameters in patients with confirmed OCI. The current study was conducted on 100 apparently healthy subjects as control group and 100 patients with lymphoma as a case group. HCV RNA was extracted and detected in both plasma and PBMCs using qRT-PCR. Total protein, albumin, ALT, AST, and total and direct bilirubin were measured in serum. OCI was detected in 6% of the patient group. OCI patients had lower levels of total protein and serum albumin and higher ALT and AST compared with lymphoma patients without OCI. Our study revealed that six out of 100 patients with lymphoma disorders had occult HCV infection (6%). Therefore, the possibility of this infection should be considered in patients with lymphoma.

The sustained virologic response and elimination of HCV is widely viewed as a true cure of chronic hepatitis C as it associates with amelioration of histological liver damage and improved clinical outcomes. Therefore, the existence and clinical burden of occult HCV infection (OCI) has been a controversial issue for many years. In this review, we summarize recently published data that adds new information on the molecular and clinical background of OCI and its epidemiological significance. We also identify and discuss the most important methodological pitfalls, which can be a source of inconsistency between studies. Data that have accumulated so far, strongly support the existence of extrahepatic HCV replication in individuals negative for serum HCV-RNA by conventional clinical tests. OCI emerges as a condition where the immune system is unable to fully resolve infection but it is continuously stimulated by low levels of HCV antigens, leading to progression of liver pathology and extrahepatic HCV-related complications. Moreover, the development of monitoring strategies or management guidelines for OCI is still hampered by the lack of clear definition and the confusion regarding its clinical significance. Careful study design and the introduction of uniform protocols for the detection of low-level HCV-RNA are crucial for obtaining reliable data on OCI.

• Clinical Trial Protocols as Topic
• Government Regulation
• Hepacivirus
• Hepatitis C/drug therapy
• Human Experimentation/ethics
• Human Experimentation/legislation & jurisprudence
• Humans
• Viral Hepatitis Vaccines

• Z99 DK999999 Intramural NIH HHS
• ZIA DK054504 Intramural NIH HHS
• ZIA DK054505 Intramural NIH HHS

Despite the link between HCV and malignant lymphoproliferative disorders has been established, the association between occult hepatitis C virus infection and malignant lymphoproliferative disorders remains obscure. The present study intended to identify the possible association between occult HCV infection and malignant lymphoproliferative disorders. Newly diagnosed patients with LPDs were screened for the presence of HCV-RNA in both plasma and PBMCs. PBMCs of the subjects were also, examined by transmission and immuno-electron microscopy. LPD patients showed a high percentage of HCV infection (71.9%): OCI-HCV (37.5%) and HCV (34.38%). Meanwhile, 28.13% of LPD patients did not show any evidence of HCV infection. Ultrastructural examination of PBMCs revealed the presence of intracytoplasmic vacuoles enclosing viral like particles, which were less prominent in occult HCV patients. The possibility of occult HCV should be considered in patients with LPDs which can be helpful in the management of the treatment protocol in order to set up a balance between the control of the tumor progression and minimizing post chemotherapy complications related to HCV infection.

• hepatitis C virus
• lymphoproliferative disorders
• occult HCV

The hepatitis C virus (HCV) is known as a main etiological cause of chronic hepatitis. HCV infection disturbs cholesterol metabolism of the host, which is frequently observed in patients suffering from chronic hepatitis C (CHC). The course of viral infections remains under strict control of microRNA (miRNA). In the case of HCV, miR-122 exerts a positive effect on HCV replication in vitro. The purpose of this study was to investigate the impact of peginterferon alpha (pegIFN-α) and ribavirin treatments on the expression of miR-122 and the cholesterol level in the peripheral blood mononuclear cells (PBMCs) of CHC patients. We report here that the level of miR-122 expression in the PBMCs decreased after the antiviral treatment in comparison to the pretreated state. Simultaneously, the level of cholesterol in the PBMCs of CHC patients was higher six months following the treatment than it was pretreatment. Consequently, it seems that the decrease of miR-122 expression in the PBMCs of CHC patients is one of the antiviral effects connected with the pegIFN-alpha/ribavirin treatments.

• HCV
• PBMCs
• cholesterol
• miR-122
• pegIFN-alpha
• ribavirin

• Direct-acting antiviral agents
• Hepatic pathology
• Liver fibrosis
• Occult hepatitis C
• RNAscope assay

• T cells
• hepatitis C virus
• lymphocytes
• lymphotropism
• virus variants

• Hepatocellular carcinoma (HCC)
• chronic hepatitis B
• chronic hepatitis C
• cirrhosis
• sustained virologic response (SVR)

• hcv
• complement
• b cell
• cd21
• raji

• Antigens, CD19
• B-Lymphocytes/immunology
• B-Lymphocytes/virology
• Cells, Cultured
• Hepacivirus/physiology
• Hepatitis C, Chronic/immunology
• Hepatitis C, Chronic/virology
• Humans
• Leukocytes, Mononuclear/immunology
• Leukocytes, Mononuclear/virology
• Receptors, Complement/immunology

• Z01 CL002068-17 Intramural NIH HHS

Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states.

IMPORTANCE Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Hepatitis C virus (HCV) frequently causes chronic hepatitis, while spontaneous recovery from infection is infrequent. Persistence of HCV after self-limited (spontaneous) resolution of hepatitis C was rarely investigated. The current study aimed to assess incidence and robustness of HCV persistence after self-resolved hepatitis C in individuals with normal liver enzymes and undetectable virus by conventional tests. Applying high sensitivity HCV RNA detection approaches, we analyzed plasma and peripheral blood mononuclear cells (PBMC) from individuals with previous hepatitis C infection. Parallel plasma and PBMC from 24 such non-viraemic individuals followed for 0.3–14.4 (mean 6.4) years were examined. Additional samples from 9 of them were obtained 4.5–7.2 (mean 5.9) years later. RNA was extracted from 250 μl plasma and, if HCV negative, from ~5 ml after ultracentrifugation, and from ex vivo stimulated PBMC. PBMC with evidence of HCV replication from 4 individuals were treated with HCV protease inhibitor, telaprevir. HCV RNA was detected in 14/24 (58.3%) plasma and 11/23 (47.8%) PBMC obtained during the first collection. HCV RNA replicative strand was evident in 7/11 (63.6%) PBMC. Overall, 17/24 (70.8%) individuals carried HCV RNA at mean follow-up of 5.9 years. Samples collected 4.5–7.2 years later revealed HCV in 4/9 (44.4%) plasma and 5/9 (55.5%) PBMC, while 4 (80%) of these 5 PBMC demonstrated virus replicative strand. Overall, 6/9 (66.7%) individuals remained viraemic for up to 20.7 (mean 12.7) years. Telaprevir entirely eliminated HCV replication in the PBMC examined. In conclusion, our results indicate that HCV can persist long after spontaneous resolution of hepatitis C at levels undetectable by current testing. An apparently effective host immune response curtailing hepatitis appears insufficient to completely eliminate the virus. The long-term morbidity of asymptomatic HCV carriage should be examined even in individuals who achieve undetectable HCV by standard testing and their need for treatment should be assessed.

• Adult
• Aged
• Base Sequence
• Female
• Follow-Up Studies
• Hepacivirus/metabolism
• Hepatitis C, Chronic/blood
• Hepatitis C, Chronic/drug therapy
• Humans
• Leukocytes, Mononuclear/metabolism
• Leukocytes, Mononuclear/virology
• Male
• Middle Aged
• Molecular Sequence Data
• Oligopeptides/administration & dosage
• RNA, Viral/blood
• Remission, Spontaneous
• Time Factors

• MOP-126056 Canadian Institutes of Health Research

• Hepatitis C Virus
• Mononuclear Leukocytes
• Real-Time Polymerase Chain Reaction

Hepatitis C virus (HCV) can replicate in cells of the immune system and productively propagate in primary T lymphocytes in vitro. We aimed to determine whether exposure to authentic, patient-derived HCV can modify the proliferation capacity, susceptibility to apoptosis and phenotype of T cells.

Primary total T cells from a healthy donor were used as targets and plasma-derived HCV from patients with chronic hepatitis C served as inocula. T cell phenotype was determined prior to and at different time points after exposure to HCV. T cell proliferation and apoptosis were measured by flow cytometry-based assays.

The HCV inocula that induced the highest intracellular expression of HCV also caused a greatest shift in the T cell phenotype from predominantly CD4-positive to CD8-positive. This shift was associated with inhibition of CD4+ but not CD8+ T cell proliferation and did not coincide with altered apoptotic death of either cell subset.

The data obtained imply that exposure to native HCV can have an impact on the relative frequencies of CD4+ and CD8+ T cells by selectively suppressing CD4⁺ T lymphocyte proliferation and this may occur in both the presence and the absence of measurable HCV replication in these cells. If the virus exerts a similar effect in vivo, it may contribute to the impairment of virus-specific T cell response by altering cooperation between immune cell subsets.

The online version of this article (doi:10.1186/s12985-015-0322-4) contains supplementary material, which is available to authorized users.

• Chronic hepatitis C virus infection
• Interferon alpha therapy
• Interferon alpha-induced gene expression
• Peripheral blood mononuclear cells

• Cognitive impairment
• anxiety
• depression
• hepatitis C
• virological response

• HCV-RNA
• Liver
• Occult HCV
• PBMC

• T-cell response
• flow cytometry
• occult HCV infection
• peripheral blood mononuclear cells
• real-time PCR
• sustained virological responders

• HCV RNA
• HCV antibodies
• liver cells
• occult hepatitis C
• peripheral blood mononuclear cells

• Chemotherapy
• Hepatitis C Virus
• Interferon
• Ribavirin
• SVR

It has been reported that the direct binding of hepatitis C virus (HCV) and/or the replication of HCV in the extrahepatic organs and, especially, lymphoid cells, might affect the pathogenesis of extrahepatic diseases with HCV infection. More than one decade ago, several reports described the existence of HCV-RNA in peripheral blood mononuclear cells. Moreover, many reports describing the existence of HCV in B lymphocytes and B cell lymphoma have been published. In addition to B lymphocytes, it was reported that HCV replication could be detected in T lymphocytes and T cell lines. Among the extrahepatic diseases with HCV infection, mixed cryoglobulinemia-related diseases and autoimmune-related diseases are important for understanding the immunopathogensis of HCV persistent infection. Moreover, HCV persistent infection can cause malignant lymphoma. The biological significance of lymphotropic HCV has not yet become clear. However, several candidates have been considered for a long time. One is that lymphotropic HCV is an HCV reservoir that might contribute to the recurrence of HCV infection and difficult-to-treat disease status. The other important issue is the carcinogenesis of the lymphoid cells and disturbances of the immune responses. Therefore, the extrahepatic diseases might be induced by direct interaction between HCV and lymphoid cells. In this article, we summarize various studies showing the direct effect of HCV on lymphoid cells and discuss the biological significance of lymphotropic HCV.

• Hepatitis C virus
• Lymphotropism
• T cell
• B cell
• Immunology

• Animals
• Autoimmune Diseases/immunology
• Autoimmune Diseases/virology
• Cryoglobulinemia/immunology
• Cryoglobulinemia/virology
• Hepacivirus/immunology
• Hepacivirus/pathogenicity
• Hepatitis C/complications
• Hepatitis C/immunology
• Hepatitis C/virology
• Humans
• Immune Evasion
• Lymphocytes/immunology
• Lymphocytes/virology
• Lymphoma/immunology
• Lymphoma/virology

Resolution of chronic hepatitis C is considered when serum HCV RNA becomes repeatedly undetectable and liver enzymes normalize. However, long-term persistence of HCV following therapy with pegylated interferon-α/ribavirin (PegIFN/R) was reported when more sensitive assays and testing of serial plasma, lymphoid cells (PBMC) and/or liver biopsies was applied. Our aim was to reassess plasma and PBMCs collected during and after standard PegIFN/R therapy from individuals who became HCV RNA nonreactive by clinical testing. Of particular interest was to determine if HCV genome and its replication remain detectable during ongoing treatment with PegIFN/R when evaluated by more sensitive detection approaches. Plasma acquired before (n = 11), during (n = 25) and up to 12–88 weeks post-treatment (n = 20) from 9 patients and PBMC (n = 23) from 3 of them were reanalyzed for HCV RNA with sensitivity <2 IU/mL. Clone sequencing of the HCV 5′-untranslated region from plasma and PBMCs was done in 2 patients. HCV RNA was detected in 17/25 (68%) plasma and 8/10 (80%) PBMC samples collected from 8 of 9 patients during therapy, although only 5.4% plasma samples were positive by clinical assays. Among post-treatment HCV RNA-negative plasma samples, 9 of 20 (45.3%) were HCV reactive for up to 59 weeks post-treatment. Molecularly evident replication was found in 6/12 (50%) among PBMC reactive for virus RNA positive strand collected during or after treatment. Pre-treatment point mutations persisted in plasma and/or PBMC throughout therapy and follow-up. Therefore, HCV is not completely cleared during ongoing administration of PegIFN/R otherwise capable of ceasing progression of CHC and virus commonly persists at levels not detectable by the current clinical testing. The findings suggest the need for continued evaluation even after patients achieve undetectable HCV RNA post-treatment.

• Antiviral Agents/administration & dosage
• Antiviral Agents/therapeutic use
• Female
• Hepacivirus/genetics
• Hepacivirus/isolation & purification
• Hepacivirus/physiology
• Hepatitis C, Chronic/drug therapy
• Hepatitis C, Chronic/virology
• Humans
• Interferon-alpha/administration & dosage
• Interferon-alpha/therapeutic use
• Limit of Detection
• Male
• Polyethylene Glycols/administration & dosage
• Polyethylene Glycols/therapeutic use
• RNA, Viral/blood
• Recombinant Proteins/administration & dosage
• Recombinant Proteins/therapeutic use
• Ribavirin/administration & dosage
• Ribavirin/therapeutic use
• Virus Replication

• MOP-126056 Canadian Institutes of Health Research

• hepatitis C virus
• occult infection
• polymerase chain reaction

• Cholesterol
• Chronic hepatitis C infection
• Mir-122
• PBMCs

Accumulated evidence implies that hepatitis C virus (HCV) infects not only the liver but also the immune system. A lymphocyte-specific CD5 molecule was recently identified as essential for infection of T cells with native, patient-derived HCV. To assess whether the proposed hepatocyte receptors may also contribute to HCV lymphotropism, expression of scavenger receptor-class B type 1 (SR-B1), claudin-1 (CLDN-1), claudin-6 (CLDN-6), occludin (OCLN), CD5 and CD81 was examined by real-time RT-PCR and the respective proteins quantified by immunoblotting in HCV-prone and resistant T cell lines, peripheral blood mononuclear cells (PBMC), primary T cells and their subsets, and compared to hepatoma Huh7.5 and HepG2 cells. SR-B1 protein was found in T and hepatoma cell lines but not in PBMC or primary T lymphocytes, CLDN-1 in HCV-resistant PM1 T cell line and hepatoma cells only, while CLDN-6 equally in the cells investigated. OCLN protein occurred in HCV-susceptible Molt4 and Jurkat T cells and its traces in primary T cells, but not in PBMC. CD5 was displayed by HCV-prone T cell lines, primary T cells and PBMC, but not by non-susceptible T and hepatoma cell lines, while CD81 in all cell types except HepG2. Knocking-down OCLN in virus-prone T cell line inhibited HCV infection, while de novo infection downregulated OCLN and CD81, and upregulated CD5 without modifying SR-B1 expression. Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells. This study narrowed the range of factors potentially utilized by HCV to infect T lymphocytes amongst those uncovered using laboratory HCV and Huh7.5 cells. Together with the demonstrated role for CD5 in HCV lymphotropism, the findings indicate that virus utilizes different molecules to enter hepatocytes and lymphocytes.

• CD36 Antigens/genetics
• CD36 Antigens/metabolism
• CD5 Antigens/genetics
• CD5 Antigens/metabolism
• Cell Line
• Claudins/genetics
• Claudins/metabolism
• Gene Expression Regulation
• Gene Knockdown Techniques
• Hepacivirus/physiology
• Hepatitis C/genetics
• Hepatitis C/virology
• Humans
• Jurkat Cells
• Leukocytes, Mononuclear/metabolism
• Leukocytes, Mononuclear/virology
• Occludin/genetics
• Occludin/metabolism
• Receptors, Virus/genetics
• Receptors, Virus/metabolism
• T-Lymphocyte Subsets/metabolism
• T-Lymphocyte Subsets/virology
• Tetraspanin 28/genetics
• Tetraspanin 28/metabolism

• 77544-1 CIHR
• 91665-1 CIHR
• MOP-77544 CIHR

• Animals
• Female
• Hepacivirus/genetics
• Hepatitis C, Chronic/virology
• Humans
• Leukocytes, Mononuclear/virology
• Male
• RNA, Viral/blood

• hcv eradication
• hcv recovery
• pbmc
• residual virus
• viral adherence

• Adult
• Aged
• Animals
• B-Lymphocytes/virology
• Carrier State/virology
• Disease Reservoirs/virology
• Female
• Hepacivirus/genetics
• Hepatitis C, Chronic/virology
• Humans
• Leukocytes, Mononuclear/virology
• Male
• Middle Aged
• Pan troglodytes/virology
• RNA, Viral/blood
• Viral Load

• Z01 CL002068-17 Intramural NIH HHS
• Z01 CL002118-02 Intramural NIH HHS

• Carcinogenesis
• HCC
• IFN
• Recurrence
• SVR

• CHC, chronic hepatitis C
• Clinical practice
• HBV, hepatitis B virus
• HCC, hepatocellular carcinoma
• HCV, hepatitis C virus
• IFN, interferon
• IU, international unit
• NAH, nucleic acid hybridization
• OCI, occult HCV infection
• PBMC, peripheral blood mononuclear cells
• PCR, polymerase chain reaction
• RBV, ribavirin
• RNA, ribonucleic acid
• SVR, sustained virological response
• hepatitis C
• identification of OCI
• occult HCV infection

The modulation of the gamma-aminobutyric acid type A (GABA A) receptors activity was observed in several chronic hepatitis failures, including hepatitis C. The expression of GABA A receptor subunits α1 and β3 was detected in peripheral blood mononuclear cells (PBMCs) originated from healthy donors. The aim of the study was to evaluate if GABA A α1 and β3 expression can also be observed in PBMCs from chronic hepatitis C (CHC) patients and to evaluate a possible association between their expression and the course of hepatitis C virus (HCV) infection. GABA A α1- and β3-specific mRNAs presence and a protein expression in PBMCs from healthy donors and CHC patients were screened by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. In patients, HCV RNA was determined in sera and PBMCs. It was shown that GABA A α1 and β3 expression was significantly different in PBMCs from CHC patients and healthy donors. In comparison to healthy donors, CHC patients were found to present an increase in the expression of GABA A α1 subunit and a decrease in the expression of β3 subunit in their PBMCs. The modulation of α1 and β3 GABA A receptors subunits expression in PBMCs may be associated with ongoing or past HCV infection.

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro.