Hepatitis B Virus Pathogenesis Research Articles

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

Circulating and Hepatic BDCA1+, BDCA2+, and BDCA3+ Dendritic Cells Are Differentially Subverted in Patients With Chronic HBV Infection.

Background and aims: Chronic hepatitis B virus (HBV) infection is a major health burden potentially evolving toward cirrhosis and hepatocellular carcinoma. HBV physiopathology is strongly related to the host immunity, yet the mechanisms of viral evasion from immune-surveillance are still misunderstood. The immune response elicited at early stages of viral infection is believed to be important for subsequent disease outcome. Dendritic cells (DCs) are crucial immune sentinels which orchestrate antiviral immunity, which offer opportunity to pathogens to subvert them to escape immunity. Despite the pivotal role of DCs in orientating antiviral responses and determining the outcome of infection, their precise involvement in HBV pathogenesis is not fully explored.Methods: One hundred thirty chronically HBV infected patients and 85 healthy donors were enrolled in the study for blood collection, together with 29 chronically HBV infected patients and 33 non-viral infected patients that were included for liver biopsy collection. In a pioneer way, we investigated the phenotypic and functional features of both circulating and intrahepatic BDCA1+ cDC2, BDCA2+ pDCs, and BDCA3+ cDC1 simultaneously in patients with chronic HBV infection by designing a unique multi-parametric flow cytometry approach.Results: We showed modulations of the frequencies and basal activation status of blood and liver DCs associated with impaired expressions of specific immune checkpoints and TLR molecules on circulating DC subsets. Furthermore, we highlighted an impaired maturation of circulating and hepatic pDCs and cDCs following stimulation with specific TLR agonists in chronic HBV patients, associated with drastic dysfunctions in the capacity of circulating DC subsets to produce IL-12p70, TNFα, IFNα, IFNλ1, and IFNλ2 while intrahepatic DCs remained fully functional. Most of these modulations correlated with HBsAg and HBV DNA levels.Conclusion: We highlight potent alterations in the distribution, phenotype and function of all DC subsets in blood together with modulations of intrahepatic DCs, revealing that HBV may hijack the immune system by subverting DCs. Our findings provide innovative insights into the immuno-pathogenesis of HBV and the mechanisms of virus escape from immune control. Such understanding is promising for developing new therapeutic strategies restoring an efficient immune control of the virus.

In Vivo Model Systems for Hepatitis B Virus Research.

Hepatitis B virus (HBV) affects more than 257 million people globally, resulting in progressively worsening liver disease, manifesting as fibrosis, cirrhosis, and hepatocellular carcinoma. The exceptionally narrow species tropism of HBV restricts its natural hosts to humans and non-human primates, including chimpanzees, gorillas, gibbons, and orangutans. The unavailability of completely immunocompetent small-animal models has contributed to the lack of curative therapeutic interventions. Even though surrogates allow the study of closely related viruses, their host genetic backgrounds, immune responses, and molecular virology differ from those of HBV. Various different models, based on either pure murine or xenotransplantation systems, have been introduced over the past years, often making the choice of the optimal model for any given question challenging. Here, we offer a concise review of in vivo model systems employed to study HBV infection and steps in the HBV life cycle or pathogenesis.

Management of Hepatitis B Virus Infection: 2018 Guidelines from the Canadian Association for the Study of Liver Disease and Association of Medical Microbiology and Infectious Disease Canada.

Hepatitis B virus (HBV) infection is an important public health problem in Canada. In keeping with evolving evidence and understanding of HBV pathogenesis, the Canadian Association for the Study of Liver Disease periodically publishes HBV management guidelines. The goals of the 2018 guidelines are to (1) highlight the public health impact of HBV infection in Canada and the need to improve diagnosis and linkage to care, (2) recommend current best-practice guidelines for treatment of HBV, (3) summarize the key HBV laboratory diagnostic tests, and (4) review evidence on HBV management in special patient populations and include more detail on management of HBV in pediatric populations. An overview of novel HBV tests and therapies for HBV in development is provided to highlight the recent advances in HBV clinical research. The aim and scope of these guidelines are to serve as an up-to-date, comprehensive resource for Canadian health care providers in the management of HBV infection.

Antiviral Compounds from Myxobacteria.

Viral infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis C virus (HCV) pose an ongoing threat to human health due to the lack of effective therapeutic agents. The re-emergence of old viral diseases such as the recent Ebola outbreaks in West Africa represents a global public health issue. Drug resistance and toxicity to target cells are the major challenges for the current antiviral agents. Therefore, there is a need for identifying agents with novel modes of action and improved efficacy. Viral-based illnesses are further aggravated by co-infections, such as an HIV patient co-infected with HBV or HCV. The drugs used to treat or manage HIV tend to increase the pathogenesis of HBV and HCV. Hence, novel antiviral drug candidates should ideally have broad-spectrum activity and no negative drug-drug interactions. Myxobacteria are in the focus of this review since they produce numerous structurally and functionally unique bioactive compounds, which have only recently been screened for antiviral effects. This research has already led to some interesting findings, including the discovery of several candidate compounds with broad-spectrum antiviral activity. The present review looks at myxobacteria-derived antiviral secondary metabolites.

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.

Hepatitis B virus (HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host miRNAs and may deregulate miRNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viral-derived miRNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBV-derived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBV-induced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.

Nature of Host Immunity during Hepatitis B Virus Infection and designing Immune Therapy.

Hepatitis B virus (HBV) infections represent one of the major public health problems in global context. More than 2 billion people in the world have been infected with this virus at some point of time in their life and millions are chronically infected, indicating that chronic HBV-infected subjects remain as a living source of HBV transmission. The public health impact of this is tremendous. Considerable numbers of chronic HBV-infected individuals would eventually develop progressive liver diseases and their complications like hepatic failure, liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Epidemiological studies have suggested that about 0.6 to 1.2 million people die annually from HBV-related liver diseases. These figures about death due to HBV and sufferings from HBV-related diseases indicate a notion of medical emergencies about HBV. In addition to these, the impact of HBV on health care delivery system moves beyond these numbers of HBV-related patients and HB-related deaths. This is because significant insights have already been developed about epidemiology, virology, and pathogenesis of HBV. Also, an effective and widely used preventive vaccine is available against HBV. In addition to these, antiviral drugs against HBV have been developed from early 1980s and several such drugs are now available commercially in the open market around the worldwide. Unfortunately, the ongoing therapeutic regimens could not stand the test of time and new insights about HBV pathogenesis are required for the development of new, novel, and evidence-based therapies for chronic HBV infections.How to cite this article: Akbar SMF, Al-Mahtab M, Khan SI. Nature of Host Immunity during Hepatitis B Virus Infection and designing Immune Therapy. Euroasian J Hepato-Gastroenterol 2018;8(1):42-46.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection.

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs.Highlights: (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver. (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle. (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo.Research in Context To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

Chronic hepatitis B virus and liver fibrosis: A mathematical model.

Hepatitis B virus (HBV) infection is a liver disorder that can result in cirrhosis, liver failure and hepatocellular carcinoma. HBV infection remains a major global health problem, as it affects more 350 million people chronically and kills roughly 600,000 people annually. Drugs currently used against HBV include IFN-α that decreases viremia, inflammation and the growth of liver fibrosis, and adefovir that decreases the viral load. Each of these drugs can have severe side-effects. In the present paper, we consider the treatment of chronic HBV by a combination of IFN-α and adefovir, and raise the following question: What should be the optimal ratio between IFN-α and adefovir in order to achieve the best ‘efficacy’ under constraints on the total amount of the drugs; here the efficacy is measured by the reduction of the levels of inflammation and of fibrosis? We develop a mathematical model of HBV pathogenesis by a system of partial differential equations (PDEs) and use the model to simulate a ‘synergy map’ which addresses the above question.

Hepatitis B virus inhibits the expression of complement C3 and C4, in vitro and in vivo.

The immune system serves an important function in Hepatitis B virus (HBV) infection, and the complement system is a major component of innate immunity. However, the regulatory effect of HBV on complement proteins has not yet been fully elucidated. The present study focused on investigating the impact of HBV on the expression of complement proteins C3 and C4. A total of 226 patients with a clinical diagnosis of HBV infection were enrolled in the study, including 153 with chronic hepatitis B (CHB) and 73 with hepatocellular carcinoma (HCC), whereas 116 healthy individuals were included as a control group. Immunoturbidimetric detection was performed to determine the levels of complement C3 and C4 in the serum of the patients with HBV and the control group. The results revealed that the mean ± standard deviation C3 and C4 content was 1.223±0.237 and 0.226±0.052 g/l for the control group, 0.687±0.150 and 0.145±0.070 g/l for the patients with CHB, and 0.829±0.332 and 0.174±0.088 g/l for the patients with HCC, respectively. The levels of complement C3 and C4 in the patients with CHB or HCC were significantly lower than the control group (P<0.05). The HBV infectious clone pHBV1.3 was used to transfect Huh7 cells; Huh7 cells transfected with the pBlue-ks empty vector were used as the blank control. The changes in mRNA and protein expression of complements C3 and C4 were detected by RT-PCR and western blotting. When compared with the control cells, the Huh7 cells transfected with pHBV1.3 exhibited reduced C3 and C4 mRNA and protein expression levels. It was concluded that HBV can inhibit the expression of complement C3 and C4 in vitro and in vivo, which may lay the foundation for revealing the pathogenesis of HBV.

Hepatitis B virus lymphotropism: emerging details and challenges

The hepatitis B virus (HBV) is predominantly a hepatotropic virus but also infects cells of the lymphatic system. HBV genomes (DNA, messenger (m)RNA, covalently closed circular (ccc) DNA) and proteins have been found in extrahepatic sites such as peripheral blood mononuclear cells (PBMC), lymph nodes, spleen, bone marrow and cerebrospinal fluid. HBV entry into hepatocytes occurs by binding of the HBV preS1 surface protein to its specific receptor, the bile acid transporter, sodium taurocholate co-transporting polypeptide (NTCP). Although the mechanism of HBV entry into lymphatic cells is unknown, the pre S1 encoded surface protein is thought to be involved. Extrahepatic HBV infection has been studied in both chronic HBV (CHB) and in occult HBV infection (OBI). Studies have shown that HBV genomes are present in different PBMC subsets from chronically infected carriers. Unique HBV variants have been found in PBMC compared to plasma or liver in both nucleos(t)ide analogue (NA) treated and untreated CHB carriers, suggesting replication and compartment specific evolution of HBV. In HBV coinfection, HBV genomes were found in PBMC from hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) co-infected individuals. Moreover, during pregnancy, the trans placental passage of HBV infected PBMC from highly viremic mothers to infants is one of the postulated means of vertical transmission of HBV. Taken together, HBV infection in extrahepatic sites (i.e., PBMC) is implicated in multiple facets of HBV pathogenesis such as persistence, viral evolution and vertical transmission.

Proteomics Based Identification of Autotaxin As An Anti-Hepatitis B Virus Factor and a Promoter of Hepatoma Cell Invasion and Migration

Background/Aims: Hepatitis B virus (HBV) infection is a major cause of cirrhosis and hepatocellular carcinoma. Therefore, we aimed to obtain further information on HBV pathogenesis, and to search for novel putative molecules for anti-HBV therapy. Methods: We utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify the secretory proteins that are differentially expressed in the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line. Immunohistochemistry (IHC) was employed to assess the clinical relevance of the observations. Small interfering (si)RNA-based silencing transfection methods were carried out to study the function of ENPP2. Results: Totally, 133 unique proteins were identified as differentially expressed in HepG2.2.15 cell line compared with HepG2 cell line. Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 precursor (ENPP2) is one of the most significantly up-regulated secretory proteins associated with HBV replication. This differential expression of ENPP2 was further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. To study the function of ENPP2, we knockdown ENPP2 expression in HepG2.2.15 cell line by RNA interference. ENPP2 silencing increased HBV replication approximately 2.3-fold by enhancing, via the type I IFN signaling pathway, HBV cccDNA (covalently closed circular DNA) translation into viral RNA. Moreover, attenuation of ENPP2 expression inhibited both the invasion and migration ability of hepatoma cells in vitro via interacting with the molecules in the tumor microenvironment. Conclusion: Our study demonstrates that ENPP2 may be a novel anti-HBV target and indicate that suppression of its expression may inhibit the invasion and migration ability of hepatoma cells.

Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians

Tumor necrosis factor alpha (TNF-α) is one of the important cytokine in generating an immune response against hepatitis B virus (HBV). Genetic polymorphisms might influence gene transcription, leading to disturbance in cytokine production. We hypothesized that single nucleotide polymorphism (SNPs) in TNF-α gene could affect the pathogenesis of HBV. To test this hypothesis, we investigated the role of TNF-α polymorphism [−863C/A (rs1800630), −308G/A (rs1800629), −376G/A (rs1800750), −857C/T (rs1799724) and +489G/A (rs1800610)] in the susceptibility to chronic hepatitis B (CHB) infection. Polymorphisms of the TNF-α (−863C/A (rs1800630), −308G/A) were analyzed by Polymerase chain reaction sequence specific primer (PCR-SSP) while TNF-α (−376G/A, −857C/T and +489G/A) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 104 patients with CHB and 104 healthy controls. The plasma level of TNF-α was measured using Enzyme-linked immunosorbent assay (ELISA). The study showed a significant increase in the frequency of −863CC, −376GA, −857CC, −857TT and +489GA genotypes and −863C, −376A, −857C, and +489A alleles in CHB patients compared to controls. In addition, CAGCG haplotype had a highest frequency in CHB patients. A strong Linkage Disequilibrium (LD) between TNF-α −863C/A (rs1800630) and −376G/A (D′ = 0.7888, r2 = 0.0200); −308G/A and −857C/T (D′ = 0.9213, r2 = 0.1770); −308G/A and +489G/A (D′ = 0.9088, r2 = 0.1576) was demonstrated. CHB patients had significantly lower levels of TNF-α compared to controls. In conclusion, our preliminary results suggest that −863C/A (rs1800630), −308G/A, −376G/A, and +489G/A of the TNF-α gene may play a role in HBV susceptibility in Egyptians. The significant reduction in TNF-α in CHB patient was independent of any particular genotype/haplotype in TNF-α.

Aberrant production of soluble inducible T cell co‑stimulator and soluble programmed cell death protein 1 in patients with chronic hepatitis B.

Previous studies demonstrated that immune dysregulation is an important cause of hepatitis B virus (HBV)‑mediated liver damage. Co‑stimulators including programmed cell death protein 1 (PD‑1) and inducible T cell co‑stimulator (ICOS) are involved in the pathogenesis of HBV. In the present study, the serum levels of soluble (s)PD‑1 and sICOS in patients with chronic HBV infections, were investigated, and the association between sPD‑1 and sICOS levels and liver injury degree was investigated. Serum sPD‑1 and sICOS levels were increased in the HBV‑patient group particularly in the HBV external core antigen positive group. In the immune clearance group, sPD‑1 and sICOS were increased compared with the tolerance group. Furthermore, the relative mRNA expression levels were also increased in patients with HBV. However there was no correlation between sPD‑1 and sICOS levels and HBV antibodies or PD‑1/ICOS mRNA copies. The altered sPD‑1 and sICOS serum levels in the different HBV groups may reflect the dysregulation of T cell activation, and may be associated with the HBV pathological process.

Cytochrome P450 Genes (CYP2E1 and CYP1A1) Variants and Susceptibility to Chronic Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infection is a worldwide health concern which is associated with significant morbidity and mortality. Both viral and host factors have a significant effect on infection, replication and pathogenesis of HBV. The aim of this study was to investigate the effect of CYP2E1 and CYP1A1 genetic variants on susceptibility to HBV. 143 individuals including 54 chronic HBV patients and 89 healthy controls were enrolled in the genotyping procedure. rs2031920 and rs3813867 at CYP2E1 as well as rs4646421 and rs2198843 at CYP1A1 loci were studied in all subjects using PCR-RFLP (restriction fragment length polymorphism) analysis. Both variants at CYP2E1 locus were monomorphic in all studied subjects. Genotype frequency of rs4646421 was significantly different between chronic HBV patients and healthy blood donors (P=0.04, OR 4.31; 95% CI 1.04-17.7). Furthermore, individuals carrying at least one C allele (CC or CT genotypes) for rs4646421 seemed to have a decrease risk of hepatitis in comparison with TT genotype (P=0.039). Our results showed a relationship between rs4646421 TT genotype (rare genotype) and the risk for developing chronic HBV infection (four times higher). Further studies are needed to examine the role of CYP1A1 polymorphism in susceptibility to chronic HBV infection.

Effector CD8+ T cell-derived interleukin-10 enhances acute liver immunopathology

Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8+ T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8+ T cell-derived IL-10 in liver immunopathology. Mouse HBV-specific effector CD8+ T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8+ T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8+ T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8+ T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8+ T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. Effector CD8+ T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8+ T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8+ T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease.

Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

Partial Hepatectomy and Castration of HBV Transgenic Mice.

Hepatitis B virus (HBV) is a hepatotropic virus. Its infection can cause liver injury and regeneration, and its replication is affected by the gender. Transgenic mice that carry a 1.3-mer overlength HBV DNA genome productively replicate HBV in hepatocytes and have been very useful for studying the replication and pathogenesis of HBV in vivo. By using this mouse model, the relationship between HBV and liver injury and regeneration as well as the effect of the gender on HBV gene expression and replication has been studied. In this chapter, we describe the surgical procedures of partial hepatectomy and castration and provide examples to demonstrate how these surgical procedures may be used to study the effect of HBV on liver regeneration and the effect of androgen on HBV replication.

Measuring Changes in Cytosolic Calcium Levels in HBV- and HBx-Expressing Cultured Primary Hepatocytes.

Chronic infection with hepatitis B virus (HBV) remains a major worldwide health concern and is the leading cause of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is the only regulatory protein encoded in the HBV genome; HBx stimulates HBV replication in vivo and in vitro. HBx also regulates cytosolic Ca2+ signaling, and altered Ca2+ signaling is associated with the development of many diseases, including HCC. Importantly, many HBx functions, including HBx modulation of cell proliferation, apoptosis, and transcription pathways, have been linked to changes in cytosolic Ca2+ signaling. Additionally, several stages of HBV replication, including capsid formation and activation of the HBV polymerase, are dependent on intracellular Ca2+. Consequently, defining the molecular mechanism that underlies HBV and HBx modulation of cytosolic Ca2+ levels is important for understanding HBV pathogenesis and the role of HBx in HBV replication. Here, we describe a single-cell Ca2+-imaging protocol that we use to investigate HBV and HBx effects on the level of cytosolic Ca2+. We specifically outline two methods that we use to evaluate HBV and HBx regulation of cytosolic Ca2+ levels in cultured primary hepatocytes. This protocol can also be adapted for use in liver cell lines.

Immunopathogenesis of Hepatitis B Virus Infection and Related Complications

Chronic hepatitis B (CHB) is a serious consequence of hepatitis B virus (HBV), which infects and replicates in the liver. It is characterised by prolonged hepatitis B surface antigen seropositivity; this can lead to both cirrhosis and hepatocellular carcinoma (HCC). The infection begins when HBV binds its only known functional receptor, sodium taurocholate cotransporting polypeptide (NTCP), which was identified recently. The discovery of NTCP was a significant breakthrough in the field of HBV research, and has facilitated the establishment of a susceptible hepatoma cell line model for studying the mechanisms underlying HBV pathogenesis. Following productive HBV infection, both cellular and humoral immune cells and molecules, such as T cells and chemokines, are activated to resolve infection by destroying HBV-infected hepatocytes. However, host immunity to HBV is not always protective, most likely due to immune evasion mechanisms employed by HBV. These mechanisms may result in viral persistence, accumulation of mutations, and aberrant epigenetic alterations that lead to HCC. Here we highlight our current understanding of the HBV replication cycle, immunopathogenesis, and related mechanisms underlying the progression of CHB to advanced liver disease, along with the attendant complications.