Pathogenesis Of Hepatitis B Virus Infection Research Articles

Generation and Utilization of a Monoclonal Antibody against Hepatitis B Virus Core Protein for a Comprehensive Interactome Analysis.

Hepatitis B virus (HBV) core antigen (HBc) is a structural protein that forms the viral nucleocapsid and is involved in various steps of the viral replication cycle, but its role in the pathogenesis of HBV infection is still elusive. In this study, we generated a mouse monoclonal antibody (mAb) against HBc and used it in antibody-based in situ biotinylation analysis in order to identify host proteins that interact with HBc. HBc antigen was produced with a wheat germ cell-free protein synthesis system and used to immunize mice. Among the established hybridoma clones, a single clone (mAb #7) was selected and further characterized for its ability in the antibody-based in situ biotinylation analysis to collect host proteins that are in the vicinity of HBc. Using mass spectrometry, we identified 215 HBc-interacting host proteins, three of which bind HBc most significantly under hypoxic conditions. Our results indicate that mAb #7 can be used to systematically identify host proteins that interact with HBc under pathophysiological conditions, and thus may be useful to explore the molecular pathways involved in HBV-induced cytopathogenesis.

OP-2 IN VITRO CHARACTERIZATION OF HEPATITIS B VIRUS REPLICATION AND VIRAL ANTIGEN EXPRESSION AMONG GENOTYPES

Hepatitis B virus (HBV) has been classified into 10 genotypes (A-I) and numerous subgenotypes (SGTs). There is growing evidence that HBV genotypes (GTs) influence clinical outcomes, HBeAg seroconversion rates, severity of liver disease, and response to interferon therapy. However, there is a paucity of data regarding their distinctive biological characteristics, in particular for GTF, the most prevalent in Latin America. To investigate the impact of HBV genotypes on HBV-DNA levels and viral antigen expression. Full-length HBV genomes representing GTs A-D and SGTs F1b and F4 were transfected in Huh7 cell line. Secreted HBeAg and intra and extracellular HBsAg were quantified by EIA. HBV-DNA was analyzed by real-time PCR. Marked differences were observed in HBV replicative capacity as well as HBeAg and HBsAg antigen expression across genotypes (Table 1). GTC secreted significantly higher levels of HBeAg in relation to the other GTs. GTD showed lower HBsAg extracellular levels than all other GTs, while GTA showed the highest HBsAg intracellular levels. Finally, SGTs F1b and F4 showed significantly lower HBV-DNA levels. Regarding the ratio of extra and intracellular HBsAg, GTs A and D showed the lower ratios compared to SGTs F1b and F4, while SGTs F1b and F4 showed the highest HBsAg/HBV-DNA ratio. This study provides new insights into the impact of HBV genotypes on HBV antigen expression and HBV-DNA levels. The uneven expression of antigens, as well as their intracellular accumulation, could be associated with the role of genotypes in pathogenesis. Likewise, the extracellular levels of HBsAg and the replication rate might have implications in immunopathogenesis as well as in the exhaustion of the host's immune system. The virus-cell interaction in different genotypes deserves further study to understand its role in the pathogenesis of HBV infection.

Immunobiology and pathogenesis of hepatitis B virus infection.

Hepatitis B virus (HBV) is a non-cytopathic, hepatotropic virus with the potential to cause a persistent infection, ultimately leading to cirrhosis and hepatocellular carcinoma. Over the past four decades, the basic principles of HBV gene expression and replication as well as the viral and host determinants governing infection outcome have been largely uncovered. Whereas HBV appears to induce little or no innate immune activation, the adaptive immune response mediates both viral clearance as well as liver disease. Here, we review our current knowledge on the immunobiology and pathogenesis of HBV infection, focusing in particular on the role of CD8+ T cells and on several recent breakthroughs that challenge current dogmas. For example, we now trust that HBV integration into the host genome often serves as a relevant source of hepatitis B surface antigen (HBsAg) expression during chronic infection, possibly triggering dysfunctional T cell responses and favouring detrimental immunopathology. Further, the unique haemodynamics and anatomy of the liver - and the changes they frequently endure during disease progression to liver fibrosis and cirrhosis - profoundly influence T cell priming, differentiation and function. We also discuss why therapeutic approaches that limit the intrahepatic inflammatory processes triggered by HBV-specific T cells might be surprisingly beneficial for patients with chronic infection.

Association between host TNF-α, TGF-β1, p53 polymorphisms, HBV X gene mutation, HBV viral load and the progression of HBV-associated chronic liver disease in Indonesian patients.

In developing countries, including Indonesia, there is a high mortality rate associated with the progression of hepatitis B virus (HBV)-associated chronic liver disease (CLD). The pathogenesis of HBV infection is influenced by viral and host factors. To determine potential associations between these factors, host single nucleotide polymorphisms (SNPs) on TNF-α, TGF-β1 and p53, HBV X gene mutation and HBV viral load were investigated in patients with HBV-associated CLD in Surabaya, Indonesia. Sera were collected from 87 CLD patients with HBV infection. TNF-α, TGF-β1 and p53 SNPs were genotyped by PCR restriction fragment length polymorphism. The HBV X gene was sequenced and compared with reference strains to determine mutations and the viral load was measured using reverse transcription-quantitative PCR. In Indonesian patients, no association between TNF-α, TGF-β1 and p53 SNPs and CLD or X gene mutation were identified. A total of 23% (20/87) of samples had HBV X gene mutations, including ten substitution types, one deletion and one insertion. Multinomial regression analysis revealed that the K130M/V131I mutations were correlated with CLD progression (OR, 7.629; 95% CI, 1.578-36.884). Significant differences in viral load were found in HBV-infected patients who had X gene mutations, such as R87W/G, I127L/T/N/S and K130M/V131I mutations (P<0.05). The presence of K130M and V131I mutations may be predictive for the progression of HBV-associated CLD in Indonesia.

Development of Direct-acting Antiviral and Host-targeting Agents for Treatment of Hepatitis B Virus Infection

Hepatitis B virus (HBV) infection affects approximately 300 million people worldwide. Although antiviral therapies have improved the long-term outcomes, patients often require life-long treatment and there is no cure for HBV infection. New technologies can help us learn more about the pathogenesis of HBV infection and develop therapeutic agents to reduce its burden. We review recent advances in development of direct-acting antiviral and host-targeting agents, some of which have entered clinical trials. We also discuss strategies for unbiased high-throughput screens to identify compounds that inhibit HBV and for repurposing existing drugs.

MicroRNA-125b-5p mediates post-transcriptional regulation of hepatitis B virus replication via the LIN28B/let-7 axis

ABSTRACTMicroRNAs (miRNAs) are able to modulate hepatitis B virus (HBV) replication and play an important role in the pathogenesis of HBV infection. Recently, we have identified that serum miR-125b-5p levels correlated with HBV DNA levels and liver necroinflammation. In the present study, we addressed how miR-125b-5p regulated HBV replication at the different steps, inclduing viral transcription, assembly, and virion production and investigated the underlying mechanisms. We found that miR-125b-5p overexpression increased HBV replication without altering HBV transcription. This is the first demonstration of post-transcriptional miRNA regulation of HBV replication. In contrast, transfection of miR-125b-5p inhibitor resulted in downregulation of HBV replication in hepatoma cells. Further, miR-125b-5p inhibited the phosphorylation of retinoblastoma protein and blocked cell cycle progression at the G1/S phase in hepatoma cell lines. Our results indicate that certain miRNAs are able to arrest the cell cycle at G1 phase and may increase HBV replication. RNA sequencing revealed several liver-specific metabolic pathways regulated by miR-125b-5p, which was also found to suppress LIN28B and induce let-7 family members. Additional data demonstrated that miR-125b-5p targeted the LIN28B/let-7 axis to stimulate HBV replication at a post-transcriptional step.

Characteristics of CpG Islands and their quasispecies of full-length hepatitis B virus genomes from patients at different phases of infection

BackgroundCpG islands in hepatitis B virus (HBV) genome are potential targets for methylation mediated gene silencing, and may be involved in the pathogenesis of HBV infection. To date, their characteristics in HBV quasispecies (QS) remain largely unknown. The purpose of this study was to investigate the characteristics of CpG islands in HBV QS. MethodsForty patients diagnosed as acute hepatitis B (AHB, n = 10), immune-tolerant HBV carriers (IT, n = 9), chronic hepatitis B (CHB, n = 11), or acute on chronic liver failure (ACLF, n = 10), were enrolled in this case–control study. A total of 599 clones were isolated, and full-length HBV genomes were sequenced.ResultsCpG island II (CGII) in AHB group was shorter in length and its QS heterogeneity was lower than that in the chronic infection group. Among the chronic infection subgroups, CGII and CpG island III (CGIII) in IT group were longer and their heterogeneity was lower compared to CHB and ACLF groups. Length of CGII correlated with HBV DNA levels positively while the complexity and diversity of CGII correlated with HBV DNA levels negatively. Moreover, CGII and CGIII were shorter in genotype B than those in genotype C, while QS complexity and diversity of either CGII or CGIII had no significant difference between genotype B and C.ConclusionsOverall, our results suggest that the distribution, length and QS heterogeneity of CpG islands in full-length HBV genome differ across clinical phases of infection, of which the mechanism warrants further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3192-3) contains supplementary material, which is available to authorized users.

Serum Interleukin (IL)-9 and IL-10, but not T-Helper 9 (Th9) Cells, are Associated With Survival of Patients With Acute-on-Chronic Hepatitis B Liver Failure.

CD4+ T helper (Th) cells are reported to be essential for initiating and maintaining an effective immune response to hepatitis B virus (HBV) infection. Th9 cells are a new subset of CD4+ Th cells that produce interleukin (IL)-9 and IL-10. The present study aimed to investigate the percentage of Th9 cells relative to the number of CD4+ cells in peripheral blood.We also measured serum IL-9 and IL-10 levels in different stages of HBV infection and their relationship with progress and prognosis of liver disease. Whole blood samples from 111 patients with HBV infection, including 39 chronic hepatitis B (CHB), 25 HBV-liver cirrhosis (HBV-LC), 21 acute-on-chronic liver failure (ACLF) patients, and 26 healthy controls were collected.The percentage of Th9 cells and serum IL-9 and IL-10 levels were determined. There was no significant difference in the percentage of Th9 cells and serum IL-9 and IL-10 levels among different groups, nor were these related to hepatitis B e antigen status, complications of cirrhosis, inflammation index, or prognosis indexes. There was no change in the percentage of Th9 cells before and after antiviral treatment in CHB patients. There was no correlation of Th9 cells with survival of ACLF patients. However, IL-9 and IL-10 levels were significantly higher in the nonsurvived ACLF patients compared to survived ACLF patients. Furthermore, baseline IL-9 level predicted the prognosis of ACLF patients with 87.5% sensitivity and 61.5% specificity.Thus, our data indicate that Th9 cells were unlikely involved in the pathogenesis of HBV infection, but elevation in IL-9 and IL-10 may signal poor prognosis for ACLF.

Genetic variation of hepatitis B virus and its significance for pathogenesis.

Hepatitis B virus (HBV) has a worldwide distribution and is endemic in many populations. Due to its unique life cycle which requires an error-prone reverse transcriptase for replication, it constantly evolves, resulting in tremendous genetic variation in the form of genotypes, sub-genotypes, and mutations. In recent years, there has been considerable research on the relationship between HBV genetic variation and HBV-related pathogenesis, which has profound implications in the natural history of HBV infection, viral detection, immune prevention, drug treatment and prognosis. In this review, we attempted to provide a brief account of the influence of HBV genotype on the pathogenesis of HBV infection and summarize our current knowledge on the effects of HBV mutations in different regions on HBV-associated pathogenesis, with an emphasis on mutations in the preS/S proteins in immune evasion, occult HBV infection and hepatocellular carcinoma (HCC), mutations in polymerase in relation to drug resistance, mutations in HBV core and e antigen in immune evasion, chronicalization of infection and hepatitis B-related acute-on-chronic liver failure, and finally mutations in HBV x proteins in HCC.

Gut microbiota modulate the immune effect against hepatitis B virus infection.

The immunological mechanisms by which hepatitis B virus (HBV) initiates and maintains acute or chronic infection, even the formation of cirrhosis and hepatocellular carcinoma, are still undefined. An increasing number of studies have shown that intestinal flora regulate immune homeostasis, and, thus, protect the immunologic function against hepatitis virus infection. In this article, we discuss gut microbiota and its potential immune effects against HBV infection. It may provide a novel insight into the pathogenesis of HBV infection, as well as a potential therapeutic target to HBV-related disease.

Enhanced hepatitis B virus (HBV) pre-genomic RNA levels and higher transcription efficiency of defective HBV genomes.

Defective hepatitis B virus (dHBV) particles contain genomes corresponding to singly spliced HBV RNA. A limited number of studies show that dHBV is present in all chronically HBV-infected patients. Clinical studies have linked dHBV and dHBV gene products to high virus loads and liver damage. The replication characteristics of dHBV genomes remain poorly understood. We found that the splice donor/acceptor sites critical for the formation of dHBV genomes are conserved across HBV genotypes. We report a novel method to create dHBV constructs from corresponding wild-type (WT) HBV constructs. We assessed the transcriptional characteristics of the dHBV constructs with those of the corresponding WT construct using a cell culture model. Interestingly, dHBV constructs had higher pre-genomic RNA levels, transcription efficiency, HBV e antigen levels and intracellular HBV core antigen levels compared with the corresponding WT HBV constructs. Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their potential role in the pathogenesis of HBV infection.

Changes in glucose-6-phosphate dehydrogenase expression results in altered behavior of HBV-associated liver cancer cells.

Hepatocellular carcinoma (HCC) is regarded as a major global health care issue, and chronic hepatitis B virus (HBV) infection is considered to be involved in pathogenesis of HCC. To increase knowledge of HCC pathogenesis, as well as discover potential novel molecules for anti-cancer therapy, mass spectrometry and isobaric tag for relative and absolute quantitation (iTARQ) were employed. The differences between nine HBV-related HCC and adjacent non-HCC tissue specimens were studied. In total, 222 proteins were analyzed for differential expression in the two types of samples. Among these proteins, several were further confirmed by immunohistochemical, immunoblotting, and real-time RT-PCR analysis. RNA interference induced downregulation of glucose-6-phosphate dehydrogenase (G6PD) and decreased HBV replication by fivefold by the IFN pathway. Decreased G6PD expression resulted in decreased hepatoma cell migration and invasion in cell culture. In summary, the investigation provides new information on pathogenesis of HBV infection and suggests G6PD as a novel anti-HCC target. G6PD suppression may contribute to treatment strategies for inhibiting tumor progression.

Interleukin 18 gene promoter polymorphisms and susceptibility to chronic hepatitis B infection: a review study.

Context:The variation in clinical outcome of hepatitis B virus (HBV) infection is determined by virological, immunological and host genetic factors. Genes encoding cytokines are one of the candidates among host genetic factors. Polymorphisms in gene promoter can lead to different levels of cytokine expression and unique immune response. Being involved in the inflammatory cytokine network, interleukin-18 (IL-18) plays an important role in pathogenesis of HBV infection. The aim of this review is considering available literature on the association between IL-18 gene promoter single nucleotide polymorphisms (-137 C/G and -607 A/C) and susceptibility to chronic HBV infection.Evidence Acquisition:Published literature from PubMed, EMBASE, and other databases were retrieved. All studies investigating the association of IL-18 gene promoter SNPs, -137 C/G and -607 A/C, with susceptibility to chronic HBV infection were included.Results:Findings showed that the genotype -607A/A is associated with the susceptibility to chronic hepatitis B. Furthermore, allele C at position -137 is suggested to play a protective role against development of chronic HBV infection.Conclusions:Host genetic factors play an important role in determining the outcome of HBV infection. It is suggested that IL-18 genotype -607 A/A is associated with susceptibility to chronic hepatitis B. Furthermore, the carriage of allele C at position -137 may play a protective role in the development of chronic HBV infection.

Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular PreS1/2

Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because excess viral surface protein (HBsAg) is secreted in large amounts as soluble particles containing preS proteins, the role of soluble preS1/2 in hepatocyte (HepG2) death modulation is an important issue to be explored. An increase of cell death induced by preS1/2 was observed. Also, cell death was associated with the down-regulation of FLIP and activation of caspase 8, caspase 9, and BID. Additionally, hepatocytes exhibited a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) in the pathogenesis of HBV infection.

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host–virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110–154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419–525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.

Programmed hepatocytes cell death associated with Bid activation and FLIP dowregulation in response to extracellular PreS1/2

Event Abstract Back to Event Programmed hepatocytes cell death associated with Bid activation and FLIP dowregulation in response to extracellular PreS1/2 Siham Salmen Halabi1*, Masyelley Rojas1, Lisbeth Berrueta1, Darrell L. Peterson1 and Luisa Barboza1 1 Instituto de Inmunologia clinica, Universidad de Los Andes, Cellular Immunology, Venezuela Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because the viral surface protein is extensively secreted and it is found in considerable amount in soluble particles, we explored the possible role of soluble preS1/2 in hepatocyte (HepG2) death modulation. We observed an increase of cell death induced by preS1/2, which was associated with down-regulation of FLIP and activation of caspase 8, caspase 9 and BID. Additionally, hepatocytes exhibit a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) on pathogenesis of HBV infection

Hepatitis B Virus-Induced Calreticulin Protein Is Involved in IFN Resistance

IFN-α is a widely used treatment for hepatitis B virus (HBV) infection, and IFN resistance caused by viral and/or host factors is currently a challenging clinical problem. A better understanding of the molecular mechanisms underlying IFN immunotherapy in the treatment of viral infection would be very beneficial clinically and is of immense clinical importance. Calreticulin (CRT) is an endoplasmic reticulum luminal calcium-binding chaperone that is involved in the regulation of calcium homoeostasis, the folding of newly synthesized proteins, and many other cellular functions. However, little is known about the role of CRT in HBV infection. In this study, we observed high levels of CRT expression in the sera and PBMCs of patients with HBV relative to those of healthy individuals. HBV upregulated the expression of CRT at the transcriptional level. Further investigation showed that HBV-induced CRT enhanced HBV replication by antagonizing the IFN pathway. CRT suppressed the production of endogenous IFN-α by reducing the nuclear translocation of IFN regulatory factor-7 but not IFN regulatory factor-3. Furthermore, CRT also suppressed the antiviral activity of IFN-α by inhibiting the phosphorylation of STAT1 and decreasing the expression of two IFN-α downstream effectors, protein kinase R and 2',5'-oligoadenylate synthetase. Our results offer new insights into the pathogenesis of HBV infection and may provide potential targets for anti-HBV therapy.

Proteomic analysis of the interleukin‐4 (IL‐4) response in hepatitis B virus‐positive human hepatocelluar carcinoma cell line HepG2.2.15

Hepatitis B virus (HBV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. In recent decades, significant progress toward understanding the molecular virology and pathogenesis of HBV infection has been made. In addition, multiple treatment modalities have been developed for persons with HBV infection. In the present study, we demonstrated that IL-4 inhibits the expression of hepatitis B surface antigen and hepatitis B e antigen in a HBV stably transfected hepatocellular carcinoma cell line (HepG2.2.15). To reveal the anti-HBV mechanism of IL-4 by proteomics, 2-DE and MS technology were utilized to profile global changes in protein expression in HepG2.2.15 cells after IL-4 treatment. A total of 56 differentially expressed proteins were identified in IL-4-treated HepG2.2.15 cells. To find out the interaction of these changed proteins by bioinformatics, signaling network analysis with the STRING tool showed that the identified proteins are primarily involved in transcription and proteolysis. Taken together, these results offer valuable clues for understanding the molecular mechanisms of the IL-4-mediated anti-HBV response.

Toll-Like Receptors Signaling Contributes to Immunopathogenesis of HBV Infection

Innate and adaptive immune systems have important role in the pathogenesis of acute and chronic infection with hepatitis B virus (HBV). These immune responses are mediated through complex interactions between the innate immune response and adaptive immune response. Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize the molecular patterns associated with microbial pathogens. So far, TLR1 to 13 were found in human or mice and investigated to detect the target molecules and the downstream mechanisms of these unique systems. Stimulation by their ligands initiates the activation of complex networks of intracellular signaling transduction and innate and adaptive immune-related cells (NK, NK-T, monocytes, dendritic cells, T cells, B cells, and Tregs, etc.). However, reports on such relationships between HBV and TLRs have been relatively rare in comparison to those on HCV and TLRs, but have recently been increasing. Thus, a review of TLRs involved in the pathogenesis of HBV infection may be needed toward better understanding of the immunopathogenesis of HBV infection.

Hepatitis B virus infection and the risk of hepatocellular carcinoma

Epidemiological studies have provided overwhelming evidence for a causal role of chronic hepatitis B virus (HBV) infection in the development of hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and carcinogenesis of HBV-associated HCC are still elusive. This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis. The role of HBV in tumor formation appears to be complex, and may involve both direct and indirect mechanisms. Integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion, and it has been shown to enhance the host chromosomal instability, leading to large inverted duplications, deletions and chromosomal translocations. It has been shown that the rate of chromosomal alterations is increased significantly in HBV-related tumors. Prolonged expression of the viral regulatory HBV x protein may contribute to regulating cellular transcription, protein degradation, proliferation, and apoptotic signaling pathways, and it plays a critical role in the development of hepatocellular carcinoma.