Hepatitis B Virus Pathogenesis Research Articles

Three-dimensional liver organoids as a novel platform for studying hepatitis B virus infection

Hepatitis B virus (HBV) infection is a major global health problem that contributes to chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. Despite advances in antiviral therapy and the availability of preventive vaccines, current treatments often do not completely eradicate the virus, particularly in chronic HBV carriers, and the incidence of liver cancer due to chronic HBV infection remains high. A major obstacle to HBV research is the lack of infection models that can accurately recapitulate the complex interactions between HBV and the host. Existing models, such as hepatoma cell lines and animal models, are limited by species-specific barriers and the lack of support for the complete viral life cycle. These limitations pose significant challenges to the study of HBV pathogenesis and the development of therapies aimed at complete eradication of HBV. In recent years, 3-dimensional (3D) liver organoids have emerged as promising in vitro model systems to study HBV infection and HBV-mediated liver disease. These organoids serve as a suitable physiologically relevant platform for investigating HBV pathogenesis, including the ability of HBV to promote liver tumorigenesis. Furthermore, liver organoids can be genetically modified, patient-derived, expanded, and biobanked, providing a powerful tool for studying HBV pathogenesis and personalized medicine, drug discovery, and screening. In this review, we explore the utilization of 3D liver organoids as a research model for studying HBV infection and HBV-associated liver cancer, highlighting their advantages over conventional models.

Small hepatitis B virus surface antigen (SHBs) induces dyslipidemia by suppressing apolipoprotein-AII expression through ER stress-mediated modulation of HNF4α and C/EBPγ.

Persistent infection with hepatitis B virus (HBV) often leads to disruptions in lipid metabolism. Apolipoprotein AII (apoAII) plays a crucial role in lipid metabolism and is implicated in various metabolic disorders. However, whether HBV could regulate apoAII and contribute to HBV-related dyslipidemia and the underlying mechanism remain unclear. This study revealed significant reductions in apoAII expression in HBV-expressing cell lines, the serum, and liver tissues of HBV-transgenic mice. The impact of HBV on apoAII is related to small hepatitis B virus surface antigen (SHBs). Overexpression of SHBs decreased apoAII levels in SHBs-expressing hepatoma cells, transgenic mice, and the serum of HBV-infected patients, whereas suppression of SHBs increased apoAII expression. Mechanistic investigations demonstrated that SHBs repressed the apoAII promoter activity through a HNF4α- and C/EBPγ-dependent manner; SHBs simultaneously upregulated C/EBPγ and downregulated HNF4α by inhibiting the PI3K/AKT signaling pathway through activating endoplasmic reticulum (ER) stress. Serum lipid profile assessments revealed notable decreases in high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG) in SHBs-transgenic mice compared to control mice. However, concurrent overexpression of apoAII in these mice effectively counteracted these reductions in lipid levels. In HBV patients, SHBs levels were negatively correlated with serum levels of HDL-C, LDL-C, TC, and TG, whereas apoAII levels positively correlated with lipid content. This study underscores that SHBs contributes to dyslipidemia by suppressing the PI3K/AKT pathway via inducing ER stress, leading to altered expression of HNF4α and C/EBPγ, and subsequently reducing apoAII expression.IMPORTANCEThe significance of this study lies in its comprehensive examination of how the hepatitis B virus (HBV), specifically through its small hepatitis B virus surface antigen (SHBs), impacts lipid metabolism-a key aspect often disrupted by chronic HBV infection. By elucidating the role of SHBs in regulating apolipoprotein AII (apoAII), a critical player in lipid processes and associated metabolic disorders, this research provides insights into the molecular pathways contributing to HBV-related dyslipidemia. Discovering that SHBs downregulates apoAII through mechanisms involving the repression of the apoAII promoter via HNF4α and C/EBPγ, and the modulation of the PI3K/AKT signaling pathway via endoplasmic reticulum (ER) stress, adds critical knowledge to HBV pathogenesis. The research also shows an inverse correlation between SHBs expression and key lipid markers in HBV-infected individuals, suggesting that apoAII overexpression could counteract the lipid-altering effects of SHBs, offering new avenues for understanding and managing the metabolic implications of HBV infection.

Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells.

Hepatitis B virus (HBV), particularly through the HBx protein, induces oxidative stress during liver infections. This study reveals that HBx increases reactive oxygen species (ROS) via two distinct mechanisms. The first mechanism is p53-independent, likely involving mitochondrial dysfunction, as demonstrated by elevated ROS levels in p53-deficient Hep3B cells and p53-knocked-down HepG2 cells after HBx expression or HBV infection. The increase in ROS persisted even when p53 transcriptional activity was inhibited by pifithrin-α (PFT-α), a p53 inhibitor. The second mechanism is p53-dependent, wherein HBx activates p53, which then amplifies ROS production through a feedback loop involving ROS and p53. The ability of HBx to elevate ROS levels was higher in HepG2 than in Hep3B cells. Knocking down p53 in HepG2 cells lowered ROS levels, while ectopic p53 expression in Hep3B cells raised ROS. HBx-activated p53 downregulated catalase and upregulated manganese-dependent superoxide dismutase, contributing to ROS amplification. The transcriptional activity of p53 was crucial for these effects, as cells with a p53 R175H mutation or those treated with PFT-α generated less ROS. Additionally, HBx variants with Ser-101 increased p53 and ROS levels, whereas variants with Pro-101 did not. These dual mechanisms of HBx-induced ROS generation are likely significant in the pathogenesis of HBV and may contribute to liver diseases, including hepatocellular carcinoma.

Hepatitis B

Hepatitis B is a viral infection caused by the Hepatitis B virus (HBV), primarily affecting the liver and leading to both acute and chronic disease. With over 250 million individuals suffering from chronic Hepatitis B, it poses a significant global health challenge, being a leading cause of liver cirrhosis, liver failure, and hepatocellular carcinoma. This study aims to elucidate the transmission pathways, treatment options, and necessary public health strategies for Hepatitis B. We analyze the modes of transmission, which include contact with infectious body fluids—primarily through perinatal transmission, unprotected sexual contact, and sharing contaminated needles. Despite the availability of an effective vaccine since the 1980s, Hepatitis B remains endemic in many parts of Asia and Africa due to limited immunization coverage. The pathogenesis of HBV involves complex interactions between the virus and the host immune system, potentially leading to liver inflammation and fibrosis. Clinical presentations vary widely, ranging from asymptomatic infections to severe liver disease. Current treatment options for chronic Hepatitis B include antiviral medications such as tenofovir and entecavir, which suppress viral replication but do not cure the infection. Recent advancements in therapeutic strategies, including novel antivirals and immune modulators, show promise for more effective management and potential cures in the future. Comprehensive public health strategies—such as vaccination, screening, and education—are crucial for controlling the spread of HBV and reducing the global burden of Hepatitis B-related complications.

Tackling hepatitis B Virus with CRISPR/Cas9: advances, challenges, and delivery strategies.

Hepatitis B virus (HBV) infection remains a significant global health challenge, with chronic HBV leading to severe liver diseases, including cirrhosis and hepatocellular carcinoma. Current treatments often fail to eradicate the virus, highlighting the need for innovative therapeutic strategies. The CRISPR/Cas9 system has emerged as a dynamic tool for precise genome editing and presents a promising approach to targeting and eliminating HBV infection. This review provides a comprehensive overview of the advances, challenges, and delivery strategies associated with CRISPR/Cas9-based therapies for HBV. We begin by elucidating the mechanism of the CRISPR/Cas9 system and then explore HBV pathogenesis, focusing on the role of covalently closed circular DNA (cccDNA) and integrated HBV DNA in maintaining chronic infection. CRISPR/Cas9 can disrupt these key viral reservoirs, which are critical for persistent HBV replication and associated liver damage. The application of CRISPR/Cas9 in HBV treatment faces significant challenges, such as off-target effects, delivery efficiency, and immune responses. These challenges are addressed by examining current approaches to enhance the specificity, safety, and efficacy of CRISPR/Cas9. A future perspective on the development and clinical translation of CRISPR/Cas9 therapies for HBV is provided, emphasizing the requirement for further research to improve delivery methods and ensure durable safety and effectiveness. This review underscores the transformative potential of CRISPR/Cas9 in combating HBV and sets the stage for future breakthroughs in the field.

The in vitro replication phenotype of hepatitis B virus (HBV) splice variants Sp3 and Sp9 and their impact on wild-type HBV replication.

The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.

Management of autoimmune and viral hepatitis in immunotherapy: a narrative review.

Cancer immunotherapy has firmly established itself as a pillar of cancer care due to its advantages over traditional anti-tumor therapy but also carries limitations due to potential for severe adverse reactions. This review highlights the current understanding and management of patients with autoimmune and viral hepatitis immune in the setting of immune checkpoint inhibitor (ICI) therapy. A literature search was conducted on PubMed, Scopus, Google Scholar SEER*Stat databases (from inception to December 2022) using search terms: "immune checkpoint inhibitor", "autoimmune hepatitis", "viral hepatitis", "HBV pathogenesis", "HCV pathogenesis", "HBV reactivation", "HCV reactivation", "cancer immunotherapy", "immune related adverse events", "immune related hepatitis". Pre-existing autoimmune disease (AD), whether active or inactive, can predispose patients receiving ICI therapy to develop autoimmune disease flares or immune-related adverse events (irAEs). Thus, patients with AD have routinely been excluded from clinical trials and data on safety of ICI therapy are limited. Hepatic irAE can be seen in ICI therapy and is a distinct entity from autoimmune hepatitis (AIH). ICI therapy alters the immune environment in patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Patients who had prior exposure to HBV are at risk for viral reactivation. However, the prevalence of viral hepatitis in patients receiving immunotherapy is underrecognized and can lead to increases in liver biochemical tests as well as deterioration of liver function ultimately limiting treatment. The high morbidity and mortality associated with immune-related hepatitis emphasizes the need for screening of underlying diseases, including autoimmune and viral hepatitis, prior to initiation of ICI. Presence of AIH or chronic viral hepatitis is the most important risk factor for hepatic adverse events in ICI therapy. Screening for AIH, HBV and HCV is paramount in patients who will undergo ICI therapy.

Conserved use of the sodium/bile acid cotransporter (NTCP) as an entry receptor by hepatitis B virus and domestic cat hepadnavirus

The Orthohepadnavirus genus includes hepatitis B virus (HBV) that can cause chronic hepatitis and hepatocarcinoma in humans. Recently, a novel hepadnavirus in cats, domestic cat hepadnavirus (DCH), was identified that is genetically close to HBV. DCH infection is associated with chronic hepatitis in cats, suggesting a similarity with HBV pathogenesis and the potential to use DCH as a novel animal model for HBV research. HBV is shown to use the sodium/bile acid cotransporter (NTCP) as a major cell entry receptor, but the equivalent receptor for DCH remains unknown. Here we sought to identify the entry receptor for DCH. HBV- and DCH-derived preS1 peptides efficiently bound to both human and cat NTCPs, and residue 158 of NTCP proteins determined the species-specific binding of the DCH preS1 peptide. Myrcludex B, an HBV entry inhibitor, blocked the binding of the DCH preS1 peptide. Thus, DCH and HBV may share cell entry molecules, suggesting a possibility of inter-species transmission. Furthermore, our study suggests that DCH can be useful as a novel model for HBV research.

Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

Association between the Expression Patterns of Hepatitis B Surface Antigen and Hepatitis B Core Antigen with Clinicopathological Parameters and Antiviral Therapy in Liver Biopsies Obtained from Chronically Infected Hepatitis B Positive Omani Patients

Background: Hepatitis B virus (HBV) infection is considered a major global health problem. The main objectives of the current study is to determine the patterns of expression of the hepatitis B surface antigen (HBsAg) and HBV core antigen (HBcAg) in liver tissue samples obtained from hepatitis B virus-infected Omani patients and to associate between the pattern of the expression of HBsAg and HBcAg with the other clinical parameters and anti-viral therapy. Methods : The expression patterns of HBsAg and HBV core antigen HBcAg were determined by immunohistochemistry (IHC), in 58 formalin-fixed paraffin-embedded liver tissue biopsies obtained from chronic hepatitis B Omani patients. The association between positivity for HBV antigens with gender, age group, histological appearances, and antiviral therapy was determined. Results: IHC-positive staining of HBsAg was demonstrated in 28 patients (48.3%) patients, of whom 4 (6.9%) also showed HBcAg expression. The expression pattern of HBsAg was predominantly cytoplasmic and was seen in 25 (89.3%) of patients, whereas expression of HBcAg was nuclear in 3 (75%) of patients. HBsAg and HBcAg IHC positivity were more common among males than females and among those aged 39–58 years (P = 0.130 and 0.569, respectively). The presence of lymphocytic infiltration in the majority of liver biopsies examined in the present study indicates that the liver damage could be attributed to immunologically mediated events, especially because HBV is a non-cytopathogenic virus. No significant statistical association was found between positivity for HBsAg/HBcAg by IHC and antiviral treatment. Conclusions: Determination of the expression patterns of HBV antigens in liver biopsies obtained from chronic hepatitis B Omani patients and the association between these expression patterns with other clinical histopathological parameters and anti-viral therapy, will contribute greatly to a better understanding of the pathogenesis of HBV in this unique cohort group of infected individuals.

A spatiotemporally controlled recombinant cccDNA mouse model for studying HBV and developing drugs against the virus

Covalently closed circular (ccc) DNA is the template for hepatitis B virus (HBV) replication. The lack of small animal models for characterizing chronic HBV infection has hampered research progress in HBV pathogenesis and drug development. Here, we generated a spatiotemporally controlled recombinant cccDNA (rcccDNA) mouse model by combining Cre/loxP-mediated DNA recombination with the liver-specific “Tet-on/Cre” system. The mouse model harbors three transgenes: a single copy of the HBV genome (integrated at the Rosa26 locus, RHBV), H11-albumin-rtTA (spatiotemporal conditional module), and (tetO)7-Cre (tetracycline response element), and is named as RHTC mouse. By supplying the RHTC mice with doxycycline (DOX)-containing drinking water for two days, the animals generate rcccDNA in hepatocytes, and the rcccDNA supports active HBV gene expression and can maintain HBV viremia persistence for over 60 weeks. Persistent HBV gene expression induces intrahepatic inflammation, fibrosis, and dysplastic pathology, which closely mirrors the disease progression in clinical patients. Bepirovirsen, an antisense oligonucleotide (ASO) targeting all HBV RNA species, showed dose-dependent antiviral effects in the RHTC mouse model. The spatiotemporally controlled rcccDNA mouse is convenient and reliable, providing versatile small animal model for studying cccDNA-centric HBV biology as well as evaluating antiviral therapeutics.

Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16

More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.

The Multiple Facets and Disorders of B Cell Functions in Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infection continues to be a global public health burden. B cells play a pivotal role in mediating HBV clearance and can participate in the development of anti-HBV adaptive immune responses through multiple mechanisms, such as antibody production, antigen presentation, and immune regulation. However, B cell phenotypic and functional disorders are frequently observed during chronic HBV infection, suggesting the necessity of targeting the disordered anti-HBV B cell responses to design and test new immune therapeutic strategies for the treatment of chronic HBV infection. In this review, we provide a comprehensive summary of the multiple roles of B cells in mediating HBV clearance and pathogenesis as well as the latest developments in understanding the immune dysfunction of B cells in chronic HBV infection. Additionally, we discuss novel immune therapeutic strategies that aim to enhance anti-HBV B cell responses for curing chronic HBV infection.

Recent Drug Development in the Woodchuck Model of Chronic Hepatitis B.

Infection with hepatitis B virus (HBV) is responsible for the increasing global hepatitis burden, with an estimated 296 million people being carriers and living with the risk of developing chronic liver disease and cancer. While the current treatment options for chronic hepatitis B (CHB), including oral nucleos(t)ide analogs and systemic interferon-alpha, are deemed suboptimal, the path to finding an ultimate cure for this viral disease is rather challenging. The lack of suitable laboratory animal models that support HBV infection and associated liver disease progression is one of the major hurdles in antiviral drug development. For more than four decades, experimental infection of the Eastern woodchuck with woodchuck hepatitis virus has been applied for studying the immunopathogenesis of HBV and developing new antiviral therapeutics against CHB. There are several advantages to this animal model that are beneficial for performing both basic and translational HBV research. Previous review articles have focused on the value of this animal model in regard to HBV replication, pathogenesis, and immune response. In this article, we review studies of drug development and preclinical evaluation of direct-acting antivirals, immunomodulators, therapeutic vaccines, and inhibitors of viral entry, gene expression, and antigen release in the woodchuck model of CHB since 2014 until today and discuss their significance for clinical trials in patients.

Identification of a Novel HBV Encoded miRNA Using Next Generation Sequencing.

Hepatitis B Virus (HBV) encoded miRNAs were previously described and suggested to play a role in HBV replication and pathogenesis. In this study we aim to identify novel HBV encoded miRNAs in plasma and liver tissue samples from chronic hepatitis B (CHB) patients and determine their role in CHB pathogenesis and HBV replication. RNA next generation sequencing was performed on plasma and liver tissue samples from ten CHB patients and uninfected controls. The interaction of the potential miRNA-like structures with the RNA-induced silencing complex (RISC) was determined using RNA immunoprecipitation. Expression levels of the HBV encoded miRNAs were measured in liver tissue samples derived from a conformation cohort. The effect of HBV encoded miRNAs overexpression on HBV replication, expression of predicted target genes, and induction of interferon stimulated genes in cell lines were assessed. Three potential miRNA-like structures transcribed by HBV were identified in liver tissue, of which one miRNA, HBV-miR-6, was recognized using RISC. HBV-miR-6 expression was demonstrated in liver tissue samples from 52 of the 87 CHB patients. HBV-miR-6 levels correlated with hepatic HBV-DNA and plasma HBsAg levels. Overexpression of HBV-miR-6 in vitro did not affect HBV replication, and predicted both target genes expression and interferon stimulated genes expression after stimulation. A potential novel HBV encoded miRNA was identified and validated in liver tissue from CHB patients. It is suggested that HBV-miR-6 may play a role in the process of viral excretion or particle formation in vivo.

Multiomics Analysis of Endocytosis upon HBV Infection and Identification of SCAMP1 as a Novel Host Restriction Factor against HBV Replication.

Hepatitis B virus (HBV) infection remains a major global health problem and the primary cause of cirrhosis and hepatocellular carcinoma (HCC). HBV intrusion into host cells is prompted by virus–receptor interactions in clathrin-mediated endocytosis. Here, we report a comprehensive view of the cellular endocytosis-associated transcriptome, proteome and ubiquitylome upon HBV infection. In this study, we quantified 273 genes in the transcriptome and 190 endocytosis-associated proteins in the proteome by performing multi-omics analysis. We further identified 221 Lys sites in 77 endocytosis-associated ubiquitinated proteins. A weak negative correlation was observed among endocytosis-associated transcriptome, proteome and ubiquitylome. We found 33 common differentially expressed genes (DEGs), differentially expressed proteins (DEPs), and Kub-sites. Notably, we reported the HBV-induced ubiquitination change of secretory carrier membrane protein (SCAMP1) for the first time, differentially expressed across all three omics data sets. Overexpression of SCAMP1 efficiently inhibited HBV RNAs/pgRNA and secreted viral proteins, whereas knockdown of SCAMP1 significantly increased viral production. Mechanistically, the EnhI/XP, SP1, and SP2 promoters were inhibited by SCAMP1, which accounts for HBV X and S mRNA inhibition. Overall, our study unveils the previously unknown role of SCAMP1 in viral replication and HBV pathogenesis and provides cumulative and novel information for a better understanding of endocytosis in response to HBV infection.

Long-Term Hepatitis B Virus Infection Induces Cytopathic Effects in Primary Human Hepatocytes, and Can Be Partially Reversed by Antiviral Therapy.

ABSTRACTChronic infection of hepatitis B virus (HBV) remains a major health burden worldwide. While the immune response has been recognized to play crucial roles in HBV pathogenesis, the direct cytopathic effects of HBV infection and replication on host hepatocytes and the HBV-host interactions are only partially defined due to limited culture systems. Here, based on our recently developed 5 chemical-cultured primary human hepatocytes (5C-PHHs) model that supports long-term HBV infection, we performed multiplexed quantitative analysis of temporal changes of host proteome and transcriptome on PHHs infected by HBV for up to 4 weeks. We showed that metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels during long-term HBV infection, which led to cytopathic effects and could be partially rescued by early, rather than late, nucleot(s)ide analog (NA) administration and could be significantly relieved by blocking viral antigens with RNA interference (RNAi). Overexpression screening of the dysregulated proteins identified a series of host factors that may contribute to pro- or anti-HBV responses of the infected hepatocytes. In conclusion, our results suggest that long-term HBV infection in primary human hepatocytes leads to cytopathic effects through remodeling the proteome and transcriptome and early antiviral treatment may reduce the extent of such effects, indicating a role of virological factors in HBV pathogenesis and a potential benefit of early administration of antiviral treatment.IMPORTANCE Global temporal quantitative proteomic and transcriptomic analysis using long-term hepatitis B virus (HBV)-infected primary human hepatocytes uncovered extensive remodeling of the host proteome and transcriptome and revealed cytopathic effects of long-term viral replication. Metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels, which could be partially rescued by early, rather than late, NA therapy and could be relieved by blocking viral antigens with RNAi. Overexpression screening identified a series of pro- or anti-HBV host factors. These data have deepened the understanding of the mechanisms of viral pathogenesis and HBV-host interactions in hepatocytes, with implications for therapeutic intervention.

Tumor Necrosis Factor-α-863C/A and 1031 T/C Single nucleotide polymorphic sites (SNPs) may be putative markers of HBV disease prognosis among Caucasoids: Evidence from a systematic review with meta-analysis

BackgroundThe pathogenesis and prognosis of hepatitis B virus (HBV) infection have been correlated with genetic polymorphisms in the gene loci within the promoter region of the immune system modulator molecules such as the cytokines including the tumor necrosis factor (TNF)-alpha. Besides, these polymorphisms vary at population levels but it is not conclusive whether races are involved or not. We aimed at testing the hypothesis that the SNPs in the promoter region of the TNF-alpha gene may have different effects in the Caucasoid populations by pooling the odds of association with the clearance/increased risk of the HBV infection from a large sample size obtained from many primary studies. We searched Scopus, PubMed, EMBASE, Cochrane, Willy and Google scholar databases for the published studies between January 1998 to December 2020. Studies that investigated the association between the TNF-α-238G/A, -308G/A, -857C/T, -863C/A and -1031 T/C gene promoter polymorphisms with the resolution/increased risk of HBV infection published in English and in peer reviewed journals were included. The odds ratios were used to evaluate the association of the TNF-α-gene SNPs with the risk/resolution of the disease. This study is registered on PROSPERO, number CRD42021266944. ResultsA significant association was observed between the TNF-α-857 homozygous mutation TT and its allele T with reduced risk of infection or resolution of the disease among both the Caucasoids and the Mongoloids (p < 0.05, OR < 1.0). In contrast, the TNF-α-1031 wild type genotypes TT; p = 0.001, OR = 0.634, 95%CI = [0.489 to 0.822%] and its allele T; p = 0.001, OR = 0.701, 95%CI = [0.571 to 0.860%] were significantly associated with reduced risk of infection or increased chances of resolution of the disease among the Caucasoids only. However, the TNF-α-863 homozygous mutation AA or its allele A and the TNF-α-1031 heterozygous mutation CT or the allele C were significantly associated with unresolved HBV infection among the Caucasoids (p < 0.05, OR > 1.0) but not among the Mongoloids. ConclusionTumor Necrosis Factor-α-863C/A and 1031 T/C polymorphic sites may be putative markers of HBV disease prognosis and pathogenesis among the Caucasoid populations but not among the Mongoloid populations. Future research therefore should focus on the role of these presumed TNF-α-polymorphisms in the clinical profile of the HBV infections targeting an African or Negroid population.

Generation, Maintenance and HBV Infection of Human Liver Organoids.

Hepatitis B virus (HBV) infection represents a major public health problem infecting approximately 400 million people worldwide. Despite the availability of a preventive vaccine and anti-viral therapies, chronic HBV infection remains a major health issue because it increases the risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). The lack of a relevant in vitro model for the study of the molecular mechanisms that drive HBV replication and latency, as well as HBV-related carcinogenesis, has been one of the major obstacles to the development of curative strategies. Here, we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. Human liver organoids can be seeded from both healthy and cirrhotic liver biopsies. They can be expanded in vitro when culturing in a medium containing a specific set of growth factors. When the culture medium is changed into a new medium containing growth factors that promote differentiation, organoids differentiate into functional hepatocytes, which makes them susceptible to infection with recombinant HBV. The novel in vitro primary model system described in this protocol can be utilized as a platform to study HBV pathogenesis and drug screening. Organoids generated from cirrhotic liver biopsies can be a potential tool for personalized medicine, and for modeling HCC and other liver diseases. Graphic abstract.

Hepatitis B virus genomic nucleic acid in the activation and maturation of bone marrow-derived dendritic cells

Hepatitis B virus (HBV) is the etiological agent that causes a self-limiting or chronic infection in the hepatocytes of about 250 million people worldwide. The role of adaptive immune system during HBV infection has been well studied. However, the innate immune system's responses against HBV during the early stage of infection largely remain unclear. In this study, we found that HBV genomic DNA or Salmon Sperm DNA (SSD) was able to induce the innate immune response in the macrophages cell line RAW264.7 but not the hepatocyte cell line, HepG2, indicating that hepatocytes may lack of a functional DNA-sensing pathway and hence are unable to respond to the presence of foreign DNA in the cytosol with type 1 IFN response. Thus, we hypothesized that non-parenchymal cells like the Antigen Presenting Cells (APC) might be crucial in triggering the initial immune response to suppress the virus replication and link the innate and adaptive responses. Using bone marrow-derived DCs (BMDC) as a model, this study demonstrated that HBV genomic DNA is able to induce cytokines like TNF-alpha, IL-6, and IL-12p40 secretion. We also examined the activation and maturation of BMDCs when exposed to the HBV genomic DNA intracellularly and extracellularly. A significant shift of CD86+ and CD40+ cell populations was observed during extracellular exposure of BMDC to Poly I:C and HBV genomic DNA, indicating that TLRs may be vital in the uptake of the extracellular viral DNA to activate the BMDCs. Moreover, transfection of intracellular nucleic acid stimuli, including HBV genomic DNA as well induced BMDCs maturation. Our findings highlight the critical function of DCs in antiviral response as a potential connection between the innate and adaptive immune systems during HBV pathogenesis. Nevertheless, further study is required to determine the role of cytosol DNA sensing pathway in DCs during HBV infection.