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.

Since the discovery of hepatitis B virus (HBV) over five decades ago, there have been many independent studies showing presence of HBV genomes in cells of the immune system. However, the nature of HBV lymphotropism and its significance with respect to HBV biology, persistence and the pathogenesis of liver and extrahepatic disorders remains underappreciated. This is in contrast to studies of other viral pathogens in which the capability to infect immune cells is an area of active investigation. Indeed, in some viral infections, lymphotropism may be essential, and even a primary mechanism of viral persistence, and a major contributor to disease pathogenesis. Nevertheless, there are advances in understanding of HBV lymphotropism in recent years due to cumulative evidence showing that: (i) lymphoid cells are a reservoir of replicating HBV, (ii) are a site of HBV-host DNA integration and (iii) virus genomic diversification leading to pathogenic variants, and (iv) they play a role in HBV resistance to antiviral therapy and (v) likely contribute to reactivation of hepatitis B. Further support for HBV lymphotropic nature is provided by studies in a model infection with the closely related woodchuck hepatitis virus (WHV) naturally infecting susceptible marmots. This animal model faithfully reproduces many aspects of HBV biology, including its replication scheme, tissue tropism, and induction of both symptomatic and silent infections, immunological processes accompanying infection, and progressing liver disease culminating in hepatocellular carcinoma. The most robust evidence came from the ability of WHV to establish persistent infection of the immune system that may not engage the liver when small quantities of virus are experimentally administered or naturally transmitted into virus-naïve animals. Although the concept of HBV lymphotropism is not new, it remains controversial and not accepted by conventional HBV researchers. This review summarizes research advances on HBV and hepadnaviral lymphotropism including the role of immune cells infection in viral persistence and the pathogenesis of HBV-induced liver and extrahepatic diseases. Finally, we discuss the role of immune cells in HBV diagnosis and assessment of antiviral therapy efficacy.

• hepatitis B virus
• lymphotyropism
• virus genome integration
• virus variants
• woodchuck model of hepatitis B

Hepatitis C virus infection is associated with many extrahepatic manifestations such as mixed cryoglobulinemia (MC). Renal manifestation of HCV infection might present as cryo-positive membranoproliferative glomerulonephritis (MPGN). First-line therapy includes antiviral treatment as the underlying infection leads to formation of immune complexes. After introducing direct-acting antiviral agents (DAAs) cure rates of HCV infection increased. Sustained virologic response (SVR) is defined as the absence of HCV RNA in serum by a sensitive test performed 12 or 24 weeks after the end of antiviral treatment. Although HCV RNA is undetectable in the serum, it may be present in hepatocytes and peripheral blood mononuclear cells (occult HCV infection). However, the impact of DAA treatment on occult HCV infection is not clear. We report a case of recurrence of MC with MPGN and development of lymphoproliferative disorder 2 years after achieving SVR.

• Extrahepatic HCV manifestations
• Membranoproliferative glomerulonephritis
• Mixed cryoglobulinemia

• hemodialysis
• nested reverse transcriptase-polymerase chain reaction
• occult hepatitis C virus infection

• metagenomic analysis
• mosquito
• phylogenetic analysis
• virome
• virus detection

• Animals
• China
• Computational Biology
• Dengue Virus/genetics
• Dengue Virus/isolation & purification
• Encephalitis Virus, Japanese/genetics
• Encephalitis Virus, Japanese/isolation & purification
• Genes, Viral
• Metagenomics/methods
• Mosquito Vectors/virology
• Polymerase Chain Reaction
• Sequence Analysis, DNA
• Zika Virus/genetics
• Zika Virus/isolation & purification

• metagenomic analysis
• mosquito
• phylogenetic analysis
• virome
• virus detection

• Animals
• Biodiversity
• China
• Chlorocebus aethiops
• Computational Biology
• Metagenomics
• Mosquito Vectors/virology
• Phylogeny
• Polymerase Chain Reaction
• Sequence Analysis, DNA
• Viruses/classification
• Viruses/genetics

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

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

B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

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.

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

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

• 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 (HCV) co-infection occurs in ∼30–40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNFα, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNFα. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection.

• Binding Sites
• Blotting, Western
• Cells, Cultured
• Coinfection
• Gene Expression Regulation, Viral
• HIV Infections/metabolism
• HIV Infections/pathology
• HIV Infections/virology
• HIV Long Terminal Repeat/genetics
• HIV-1/physiology
• Hepacivirus/pathogenicity
• Hepatitis C/metabolism
• Hepatitis C/pathology
• Hepatitis C/virology
• Hepatocytes/cytology
• Hepatocytes/metabolism
• Hepatocytes/virology
• Humans
• NF-kappa B/genetics
• NF-kappa B/metabolism
• Promoter Regions, Genetic/genetics
• Tumor Necrosis Factor-alpha/pharmacology
• Viral Core Proteins/genetics
• Viral Core Proteins/metabolism
• Virus Replication
• tat Gene Products, Human Immunodeficiency Virus/genetics
• tat Gene Products, Human Immunodeficiency Virus/metabolism

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

• 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.

Recent discovery of occult hepatitis C virus (HCV) infection persisting after spontaneous or antiviral therapy-induced resolution of hepatitis C was made possible by the introduction of nucleic acid amplification assays capable of detecting HCV RNA at sensitivities superseding those offered by clinical tests. Although individuals with this seemingly silent HCV infection are usually anti-HCV antibody reactive and have normal liver function tests, occult HCV infection has also been reported in anti-HCV-negative individuals with persistently elevated liver enzymes of unknown etiology. Studies have shown that HCV RNA can persist for years in serum, lymphomononuclear cells and liver in the absence of clinical symptoms, although histological evidence of a mild inflammatory liver injury can be occasionally encountered. Furthermore, while HCV RNA can be detected in circulating lymphoid cells in approximately 30% of cases, a short-term culture under stimulatory conditions augments HCV replication in these cells allowing detection of virus in otherwise HCV-negative cases. HCV infects different immune cell subsets, including CD4⁺ and CD8⁺ T lymphocytes, B cells and monocytes. Studies employing clonal sequencing and single-stranded conformational polymorphism analyses have revealed unique HCV variants residing in immune cells, further strengthening the notion of HCV lymphotropism. Overall, the data accumulated suggest that occult HCV infection is a common consequence of resolution of symptomatic hepatitis C and that examination of the cells of the immune system is an effective approach to diagnosis of HCV infection and its long-term persistence. Further work is required to fully realize pathogenic and epidemiological consequences of occult HCV persistence.

• Hepatitis C virus
• Chronic hepatitis C
• Occult viral persistence
• HCV lymphotropism
• Consequences of occult HCV persistence

• hepatitis c virus lymphotropism
• hepatitis c virus persistence
• interferon alpha
• monocytes
• occult hepatitis c virus infection
• t and b lymphocytes

• Adult
• Base Sequence
• DNA, Viral/blood
• Female
• Hepacivirus/genetics
• Hepacivirus/immunology
• Hepacivirus/physiology
• Hepatitis C, Chronic/blood
• Hepatitis C, Chronic/drug therapy
• Hepatitis C, Chronic/immunology
• Hepatitis C, Chronic/virology
• Humans
• Interferon-alpha/biosynthesis
• Interferon-alpha/genetics
• Interferon-alpha/immunology
• Leukocytes, Mononuclear/immunology
• Leukocytes, Mononuclear/virology
• Lymphocyte Activation
• Lymphocytes/immunology
• Lymphocytes/virology
• Male
• Molecular Sequence Data
• RNA, Messenger/biosynthesis
• RNA, Messenger/genetics
• Reverse Transcriptase Polymerase Chain Reaction
• Sequence Alignment
• Viral Load
• Virus Replication/immunology

• Adult
• Aged
• Antiviral Agents/therapeutic use
• Female
• Hepacivirus/genetics
• Hepacivirus/isolation & purification
• Hepacivirus/physiology
• Hepatitis C/drug therapy
• Hepatitis C/virology
• Humans
• Interferon-alpha/genetics
• Interferon-alpha/therapeutic use
• Male
• Middle Aged
• Polyethylene Glycols/therapeutic use
• Polymerase Chain Reaction/methods
• RNA, Viral/blood
• Reagent Kits, Diagnostic
• Ribavirin/therapeutic use
• Treatment Outcome
• Viral Load
• Viremia/virology

• DNA, Viral/blood
• Genome, Viral
• Hepacivirus/physiology
• Hepatitis B/physiopathology
• Hepatitis B virus/physiology
• Hepatitis C, Chronic/physiopathology
• Humans
• Lymphocytes/virology
• Male
• Middle Aged
• RNA, Viral/blood
• Superinfection/physiopathology
• Virus Replication/physiology

• Cells, Cultured
• Hepacivirus/genetics
• Hepacivirus/physiology
• Humans
• Microscopy, Electron, Transmission
• Microscopy, Immunoelectron
• RNA Virus Infections
• RNA, Viral/analysis
• T-Lymphocytes/virology
• Viral Envelope Proteins/analysis
• Viral Nonstructural Proteins/analysis
• Virion/ultrastructure
• Virus Cultivation
• Virus Replication