The Initial Hepatitis B Virus-Hepatocyte Genomic Integrations and Their Role in Hepatocellular Oncogenesis.

Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD+, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis.

Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4+ and CD8+ T Lymphocytes In Vitro.

Accumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infect in vitro two closely related but functionally distinct immune cell types, CD4+ and CD8+ T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4+ or CD8+ T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4+ than in CD8+ T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4+ and CD8+ cells at estimated mean levels ± standard errors of the means of 6.7 × 102 ± 3.8 × 102 and 1.2 × 102 ± 0.8 × 102 copies/µg RNA, respectively (P < 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4+ and in 1.2% of CD8+ T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4+ and CD8+ cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.IMPORTANCE Although the liver is the main site of HCV replication, infection of the immune system is an intrinsic characteristic of this virus independent of whether infection is symptomatic or clinically silent. Many fundamental aspects of HCV lymphotropism remain uncertain, including the degree to which different immune cells support infection and contribute to virus diversity. We show that authentic, patient-derived HCV productively replicates in vitro in two closely related but functionally distinct types of T lymphocytes, CD4+ and CD8+ cells. The display of viral proteins and unique variants, the production of virions with biophysical properties distinct from those in plasma serving as inocula, and inhibition of replication by an antiviral agent led us to ascertain that both T cell subtypes supported virus propagation. Infection of CD4+ and CD8+ T cells, which are central to adaptive antiviral immune responses, can directly affect HCV clearance, favor virus persistence, and decisively influence the development and progression of hepatitis C.

Primary seronegative but molecularly evident hepadnaviral infection engages liver and induces hepatocarcinoma in the woodchuck model of hepatitis B.

Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100-200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5-3.5 years post-infection, circulating virus transiently increased to 103 copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host's hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients.

Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

BACKGROUND: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays. RESULTS: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) cells/ml, 97.2%). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles. CONCLUSIONS: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.

Patient-derived hepatitis C virus inhibits CD4⁺ but not CD8⁺ T lymphocyte proliferation in primary T cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Repeated exposure to trace amounts of woodchuck hepadnavirus induces molecularly evident infection and virus-specific T cell response in the absence of serological infection markers and hepatitis.

Exposure to multiple small doses of hepatitis B virus (HBV) is a frequent occurrence in high-risk groups, including close relatives of infected individuals, primary care givers, and intravenous drug users. It remains uncertain whether such repeated contact may culminate in a symptomatic infection coinciding with hepatitis in individuals not immunoprotected. In this study, we evaluated consequences of multiple exposures to small, liver-nonpathogenic amounts of infectious hepadnavirus in the woodchuck model of hepatitis B. Virus-naïve animals were intravenously injected with 6 weekly doses of 110 DNase digestion-protected virions of woodchuck hepatitis virus (WHV), injected again with 6 weekly 110-virion doses after 7.5 months, and then challenged or not with a liver-pathogenic dose of 1.1 × 10(6) virions of the same inoculum. The data revealed that two rounds of such repeated exposure did not result in serologically evident infection or hepatitis. However, a low-level WHV DNA-positive infection accompanied by a WHV-specific T cell response in the absence of antiviral antibody reactivity was established. The kinetics of the virus-specific and mitogen-induced (generalized) T cell responses and the inability to induce immunoprotection against challenge with a large, liver-pathogenic virus dose were closely comparable to those previously reported for occult infection initiated by a single liver-nonpathogenic dose of WHV. Thus, repeated exposures to small quantities of hepadnavirus induce molecularly evident but serologically silent infection that does not culminate in hepatitis or generate immune protection. The findings imply that the HBV-specific T cell response encountered in the absence of serological markers of infection likely reflects ongoing occult infection.

Hepatitis C virus infection of human T lymphocytes is mediated by CD5.

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.

Hepatitis C virus load and expression of a unique subset of cellular genes in circulating lymphoid cells differentiate non-responders from responders to pegylated interferon alpha-ribavirin treatment.

Based on investigations of liver biopsy material, certain cellular genes have been implicated as correlates of success or failure to interferon alpha-ribavirin (IFN/RBV) therapy against hepatitis C. The current study aimed at determining whether expression of host genes thought to be relevant to HCV replication in the liver would be correlated with HCV infection status in peripheral blood mononuclear cells (PBMCs) and also with patient responsiveness to IFN/RBV treatment. Therefore, PBMCs from patients with chronic hepatitis C responding (n = 35) or not (n = 49) to IFN/RBV and from healthy controls (n = 15) were evaluated for HCV RNA load and cellular gene expression. Non-responders had 3- to 10-fold higher basal levels of interleukin (IL)-8, IFN-stimulated gene 15 (ISG15), 2',5'-oligoadenylate synthetase (OAS), and Toll-like receptors (TLR)-4, -5, and -7 compared to responders. Non-responders with similar post-treatment follow-ups as responders persistently expressed 6- to 20-fold greater levels of IL-8, ISG15, and OAS after therapy. Higher expression of IFN-α, IFN-γ, and IFN-λ was found in PBMCs of individuals achieving sustained virological response, either before or after therapy. Pre-treatment HCV RNA loads in PBMCs of non-responders were significantly higher (P = 0.016) than those of responders. In conclusion, the data indicate that immune cells of responders and non-responders to IFN/RBV therapy exhibited significantly different virological and host gene expression profiles. Elevated baseline HCV loads and TLR-4, -5, and -7 levels, and persistently high levels of IL-8, ISG15, and OAS were correlated with IFN non-responsiveness. The results warrant further investigations on the utilization of PBMCs for predicting success or failure to IFN-based therapies.

Patient-derived hepatitis C virus and JFH-1 clones differ in their ability to infect human hepatoma cells and lymphocytes.

Hepatitis C virus (HCV) is a hepatotropic virus that also infects cells of the immune system. HCV clones cultivated in human hepatoma Huh-7.5 cells have significantly advanced our understanding of HCV replication and candidate hepatocyte receptors. However, naturally occurring patient-derived HCV, in contrast to the HCV JFH-1 clone, is unable to infect Huh-7.5 cells, while it can replicate in human primary T-cells and selected T-cell lines. To better understand this incongruity, we examined the susceptibility of primary T-cells, PBMCs and T-cell lines to infection with patient-derived HCV, the classical HCV JFH-1 and a cell culture-adapted JFH1(T) known to be highly infectious to Huh-7.5 cells. We also tested whether Huh-7.5 cells are prone to virus readily infecting T-lymphocytes. The results revealed that while primary T-cells and Molt4 and Jurkat T-cell lines were susceptible to patient-derived HCV, they were resistant to infection with either JFH1(T) or JFH-1. However, the JFH1(T) clone interacted more firmly, although non-productively, with the cells than JFH-1. Further, Huh-7.5 cells robustly supported replication of JFH1(T) but not patient-derived, wild-type virus, despite using highly sensitive detection assays. In conclusion, JFH-1 and JFH1(T) clones were unable to establish productive infection in human primary T-cells, PBMCs and T-cell lines known to be prone to infection by patient-derived HCV, while Huh-7.5 cells were resistant to infection with naturally occurring virus infecting immune cells. The data showed that the ability to infect lymphocytes is a characteristic of native virus but not laboratory HCV clones.

Hepatocyte cytotoxicity is facilitated by asialoglycoprotein receptor.

UNLABELLED: It has been recently identified that hepatocytes can act as cytotoxic effectors and can kill contacted cells by way of CD95 ligand-CD95 and perforin-dependent pathways. However, it remained unknown whether hepatocyte-mediated cell killing is indiscriminant or is directed toward targets with particular cell surface characteristics, as well as whether hepatocytes have the capacity to directly eliminate contacted lymphocytes. In this study, we found that desialylation of surface glycoproteins significantly augments cell susceptibility to hepatocyte-mediated killing. Using asialofetuin as a competitive ligand, and by silencing gene transcription with specific small interfering RNA, we found that the asialoglycoprotein receptor (ASGPR) is involved in hepatocyte recognition of cells predestined for killing, including activated autologous T lymphocytes. CONCLUSION: Hepatocytes are constitutively equipped in the molecular machinery capable of eliminating cells brought into contact with their surface in a manner that is reliant, at least in part, upon the recognition of terminally desialylated glycoproteins by hepatocyte ASGPR. The study adds a new dimension to the physiological role of hepatic ASGPR and provides further evidence that hepatocytes can actively contribute to intrahepatic immune regulation and moderation of the local inflammatory response.

Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection.

Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.

Hepatitis B virus quasispecies in hepatic and extrahepatic viral reservoirs in liver transplant recipients on prophylactic therapy.

The characterization of hepatitis B virus (HBV) quasispecies in different compartments in liver transplant (LT) recipients may be helpful in optimizing prophylaxis regimens. The aims of this study were to evaluate liver, peripheral blood mononuclear cells (PBMC), and plasma samples for HBV and to compare the quasispecies in hepatic and extrahepatic sites in LT recipients on long-term prophylaxis. For 12 patients followed for up to 15 years post-LT, liver, plasma, and PBMC samples [all HBV DNA-negative according to conventional polymerase chain reaction (PCR) assays] were evaluated for HBV DNA by a sensitive nested PCR method [covalently closed circular DNA (cccDNA) for liver and PBMC samples] and by the sequencing and phylogenetic analysis of polymerase quasispecies. For the 10 patients on prophylaxis with no clinical recurrence (median time post-LT = 15.5 months, range = 12-96 months), liver samples were HBV DNA-reactive in 9 of 10 cases, plasma samples were HBV DNA-reactive in 3 of 10 cases, and PBMC samples were HBV DNA-reactive in 2 of 7 cases (including 1 case with HBV cccDNA in PBMCs). The sequence analysis showed that all HBV clones had a wild-type (WT) sequence in the liver and PBMCs. In 2 patients with early HBV recurrence post-LT who were treated with nucleosides only, HBV DNA was detected in serum, PBMC, and liver samples, and HBV cccDNA was found in liver samples. An HBV lamivudine-resistant variant with an M204I mutation was identified in liver (70% and 18% of the clones) and plasma samples (100% of the clones), but a WT sequence was found in 70% and 100% of the PBMC clones. In conclusion, despite prophylaxis and the absence of HBV DNA in serum according to conventional assays, HBV is detectable in the serum, liver, and PBMCs of almost all patients, and this supports the use of continued anti-HBV therapy in this group. Antiviral drug-resistant variants are more frequent in the liver versus PBMCs, but both compartments are potential sources of reinfection.

Hepadnaviral Lymphotropism and Its Relevance to HBV Persistence and Pathogenesis.

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.

Primary occult hepadnavirus infection induces virus-specific T-cell and aberrant cytokine responses in the absence of antiviral antibody reactivity in the Woodchuck model of hepatitis B virus infection.

Although the virological features of serologically silent hepadnaviral primary occult infection (POI) have been relatively well recognized in the woodchuck model of hepatitis B virus infection, the characteristics of accompanying immune responses remain unknown. In this study, the kinetics of woodchuck hepatitis virus (WHV)-specific and generalized (mitogen-induced) T-cell proliferative responses and cytokine expression profiles in circulating lymphoid cells and the liver, along with WHV-specific antibody responses, were investigated during experimentally induced POI and subsequent challenge with a liver-pathogenic dose (>10(3) virions) or liver-nonpathogenic dose (50 virions) of the same virus. The data revealed that POI, which does not prompt WHV surface antigenemia, antiviral antibody response, and hepatitis or protect from challenge with a liver-pathogenic virus dose, was accompanied by the appearance of a strong WHV-specific T-cell response directed against multiple viral epitopes that intermittently persisted at low levels for up to 10-months during follow-up. Furthermore, immediately after exposure to a liver-nonpathogenic dose of WHV, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic stimuli and displayed augmented expression of alpha interferon, interleukin-12 (IL-12), and IL-2, but not tumor necrosis factor alpha. Overall, the kinetics of WHV-specific and mitogen-induced T-cell proliferative and cytokine responses in POI were closely comparable to those seen in infection induced by liver-pathogenic viral doses. The data demonstrated that virus-specific T-cell proliferative reactivity is a very sensitive indicator of exposure to hepadnavirus, even to small amounts inducing serologically mute infection. They also showed that hepadnaviral POI is not only a molecularly but also an immunologically identifiable and distinctive entity.

Hepatitis C virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro.

UNLABELLED: Hepatitis C virus (HCV) can persist in the liver, lymphoid cells, and serum of individuals with apparently complete spontaneous or therapy-induced resolution of hepatitis C and can replicate in vivo and in vitro in human T cells. The current study was aimed at assessing the infectivity of HCV persisting at very low levels using the previously established HCV infection system in human T cells. Naive lymphoid cells were exposed to plasma and/or supernatants from cultured peripheral blood mononuclear cells from nine individuals with apparent sustained virological response after completion of antiviral therapy. Exposed cells were analyzed for HCV RNA-positive and HCV RNA-negative strands and, in selected cases, for HCV nonstructural protein 5a (NS5a), the appearance of HCV variants, and the release of virions by immunoelectron microscopy (IEM). The results showed that 11 of the 12 established cultures became HCV RNA-positive strand-reactive, whereas 4 also expressed the virus replicative strand. NS5a protein was detected in the de novo infected cells, and clonal sequencing revealed HCV variants not found in inocula. IEM demonstrated enveloped HCV particles in plasma used as inocula and in culture supernatant from T cells exposed to that plasma. Overall, HCV carried in three of the nine individuals studied elicited productive infection in vitro. CONCLUSION: HCV persisting at very low levels long after therapy-induced resolution of chronic hepatitis C can remain infectious. The retained biological competence of the virus might have implications with respect to the mechanisms of its persistence and the epidemiology of HCV infection.

Hepadnaviral infection augments hepatocyte cytotoxicity mediated by both CD95 ligand and perforin pathways.

BACKGROUND/AIM: Recently, we documented that hepatocytes can eliminate contacted cells via the CD95 ligand (CD95L)-CD95 pathway and that they are also equipped in perforin and granzyme B and can eradicate other cells via the granule exocytosis mechanism. The aim of this study was to assess whether hepadnaviral infection modifies hepatocyte-mediated cell killing. METHODS: Primary hepatocytes from woodchucks with progressing or resolved hepadnaviral hepatitis and hepatocyte lines transfected with woodchuck hepatitis virus (WHV) genes were examined for cytotoxic effector activity against cell targets susceptible to CD95L and/or perforin-dependent killing. Hepatocytes from healthy animals served as controls. RESULTS: Actively progressing and resolved hepadnaviral hepatitis is associated with a significantly greater capacity of hepatocytes to kill contacted cells. Both hepatocyte CD95L- and perforin-dependent cytotoxicity were augmented. Hepatocytes transfected with WHV X gene, but not those with complete WHV genome or virus envelope or core gene, transcribed significantly more CD95L and perforin and killed cell targets more efficiently. Exposure to interferon-gamma profoundly enhanced hepatocyte cell killing. CONCLUSIONS: Hepatocyte cytotoxic potential is significantly augmented during and following resolution of active hepadnaviral hepatitis. Hepatocyte cytotoxic activity may contribute to both liver physiological functions and the pathogenesis and progression of liver disease, including viral hepatitis.

Occult hepatitis C virus infection: what does it mean?

Occult hepatitis C virus infection (OCI) is a recently identified entity of which the existence became evident when nucleic acid amplification assays of enhanced sensitivity were introduced for the detection of hepatitis C virus (HCV) genome and its replication. This form of HCV infection has been found to persist in the presence of antibodies against HCV and normal levels of liver enzymes for years after spontaneous or antiviral therapy-induced resolution of hepatitis C and, therefore, can be termed as secondary OCI. HCV RNA in OCI circulate at fluctuating levels normally not exceeding 200 genome copies per millilitre of serum or plasma, while low levels of virus genome and its replicative intermediate RNA-negative strand are detectable in the liver and, importantly, immune cells, which provide an opportunity to detect active virus replication without the need for acquiring a liver biopsy. In addition to secondary OCI, a form of OCI accompanied by persistently moderately elevated serum liver enzymes in the absence of antibodies to HCV, which can be termed as cryptogenic OCI, has also been described. The current understanding of the nature and characteristics of OCI, methods and pitfalls of its detection, as well as the documented and expected pathological consequences of OCI will be summarized in this review.

[Chronic hepatitis and occult HCV infection]. / Przewlekle zapalenie watroby i infekcja utajona w przebiegu zakazenia wirusem zapalenia watroby typu C.

Hepatitis C virus (HCV) was discovered in 1989. HCV is a positive single-strand RNA. We all have thought, that HCV can replicate only in liver tissue, but now we know, that HCV can replicate in extrahepatic tissue as well. In about 48-86% of HCV infected patients, chronic hepatitis C (CHC) has been noticed and eventually, after tens of years, liver insufficiency, cirrhosis or hepatocellular carcinoma. The current recommended treatment for CHC is a combination of pegylated-interferon alpha and Ribavirin. Presently it is known, that HCV infection can persist as an occult infection. RNA HCV can be detected in patients after successful treatment for CHC or spontaneous elimination. Persistent HCV replication in hepatocytes or lymphoid cells would likely lead to continuous antigenic stimulation of the immune system. This prolonged replication may contribute to the immune tolerance of HCV, impairment of immune response and even further virus persistence. This occult infection grows more important in transplantation.

Diverse Virus and Host-Dependent Mechanisms Influence the Systemic and Intrahepatic Immune Responses in the Woodchuck Model of Hepatitis B.

Woodchuck infected with woodchuck hepatitis virus (WHV) represents the pathogenically nearest model of hepatitis B and associated hepatocellular carcinoma (HCC). This naturally occurring animal model also is highly valuable for development and preclinical evaluation of new anti-HBV agents and immunotherapies against chronic hepatitis (CH) B and HCC. Studies in this system uncovered a number of molecular and immunological processes which contribute or likely contribute to the immunopathogenesis of liver disease and modulation of the systemic and intrahepatic innate and adaptive immune responses during hepadnaviral infection. Among them, inhibition of presentation of the class I major histocompatibility complex on chronically infected hepatocytes and a role of WHV envelope proteins in this process, as well as augmented hepatocyte cytotoxicity mediated by constitutively expressed components of CD95 (Fas) ligand- and perforin-dependent pathways, capable of eliminating cells brought to contact with hepatocyte surface, including activated T lymphocytes, were uncovered. Other findings pointed to a role of autoimmune response against hepatocyte asialoglycoprotein receptor in augmenting severity of liver damage in hepadnaviral CH. It was also documented that WHV in the first few hours activates intrahepatic innate immunity that transiently decreases hepatic virus load. However, this activation is not translated in a timely manner to induction of virus-specific T cell response which appears to be hindered by defective activation of antigen presenting cells and presentation of viral epitopes to T cells. The early WHV infection also induces generalized polyclonal activation of T cells that precedes emergence of virus-specific T lymphocyte reactivity. The combination of these mechanisms hinder recognition of virus allowing its dissemination in the initial, asymptomatic stages of infection before adaptive cellular response became apparent. This review will highlight a range of diverse mechanisms uncovered in the woodchuck model which affect effectiveness of the anti-viral systemic and intrahepatic immune responses, and modify liver disease outcomes. Further exploration of these and other mechanisms, either already discovered or yet unknown, and their interactions should bring more comprehensive understanding of HBV pathogenesis and help to identify novel targets for therapeutic and preventive interventions. The woodchuck model is uniquely positioned to further contribute to these advances.

Kinetics of DNA damage repair response accompanying initial hepadnavirus-host genomic integration in woodchuck hepatitis virus infection of hepatocyte.

Mechanism of initial hepatitis B virus (HBV) integrations and kinetics of DNA repair immediately after infection remain essentially unknown impairing understanding of hepadnaviral oncogenesis. WCM260 hepatocytes susceptible to HBV-compatible woodchuck hepatitis virus (WHV) were examined from 15 min to 72 h post-infection (p.i.). WHV strongly induced reactive oxygen species (ROS), transiently inducible nitric oxide (iNOS) and DNA damage from 15 min p.i. All initial WHV-host fusions had the head-to-tail format indicating their formation by non-homologous end joining (NHEJ). Transcription of poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1), the PARP1 binding partner, were induced in 30 min p.i. and that of 8-oxyguanine DNA glycosylse (OGG1) responding to oxidative DNA damage at 12 h p.i. Nicotinamide adenine dinucleotide (NAD+), a marker of PARP1 activation, and heme oxygenase-1 (HO1), an indicator of pro-oxidative stress, were significantly augmented from 15-30 min p.i. Additionally, PARP1 cleavage activity was evident from 30 min p.i. confirming that PARP1-mediated DNA repair became operational almost instatly after hepatocyte contact with virus. By applying complementary approaches, the study showed that initial WHV integration was due to virus-induced oxidative DNA damage and implied that the NHEJ PARP1-dependent repair pathway determined format of the first virus-host DNA junctions.