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Background and Aims: Hepatitis B virus (HBV) infection causes oxidative stress (OS) and alters mitochondria in experimental models. Our goal was to investigate whether HBV might alter liver mitochondria also in humans, and the resulting mitochondrial stress might account for the progression of fibrosis in chronic hepatitis B (CHB)., Approach and Results: The study included 146 treatment-naïve CHB mono-infected patients. Patients with CHB and advanced fibrosis (AF) or cirrhosis (F3-F4) were compared to patients with no/mild-moderate fibrosis (F0-F2). Patients with CHB were further compared to patients with chronic hepatitis C (CHC; n = 33), nonalcoholic steatohepatatis (NASH; n = 12), and healthy controls ( n = 24). We detected oxidative damage to mitochondrial DNA (mtDNA), including mtDNA strand beaks, and identified multiple mtDNA deletions in patients with F3-F4 as compared to patients with F0-F2. Alterations in mitochondrial function, mitochondrial unfolded protein response, biogenesis, mitophagy, and liver inflammation were observed in patients with AF or cirrhosis associated with CHB, CHC, and NASH. In vitro , significant increases of the mitochondrial formation of superoxide and peroxynitrite as well as mtDNA damage, nitration of the mitochondrial respiratory chain complexes, and impairment of complex I occurred in HepG2 cells replicating HBV or transiently expressing hepatitits B virus X protein. mtDNA damage and complex I impairment were prevented with the superoxide-scavenging Mito-Tempo or with inducible nitric oxide synthase (iNOS)-specific inhibitor 1400 W., Conclusions: Our results emphasized the importance of mitochondrial OS, mtDNA damage, and associated alterations in mitochondrial function and dynamics in AF or cirrhosis in CHB and NASH. Mitochondria might be a target in drug development to stop fibrosis progression., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle., Methods: Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBV naf ). In vitro infection assays were performed to address the impact of HBV naf ., Results: Hierarchical clustering individualized two clusters of HBV naf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 ± 6.4 vs 6.6 ± 6.2 Log 10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBV naf , pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1% ± 2.5% vs 4.1% ± 1.8% of HBV naf , p < 0.0001) but remained highly prevalent in C2 patients, whatever the level of replication. C2 patients samples used in infection assays showed that: (1) HBV naf secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBV naf in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication., Conclusion: Besides the critical role of HBV replication in circulating HBV naf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle., Clinical Trial Registration: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458)., (© 2022. Asian Pacific Association for the Study of the Liver.)

Hepatitis B splicing-regulated protein (HBSP) of the hepatitis B virus (HBV) was uncovered a few years ago but its function remains unknown. HBSP expression occurs from a spliced viral transcript that increases during the course of liver disease. This study aimed at characterizing the impact of HBSP on cellular signaling pathways in vitro and on liver pathogenesis in transgenic (Tg) mice. By RT-qPCR array, NF-κB-inducible genes appeared modulated in HepG2 cells transduced with a HBSP-encoding lentivirus. Using luciferase and Western blot assays, we observed a decreased activation of the NF-κB pathway in HBSP-expressing cells following TNF-α treatment, as illustrated by lower levels of phosphorylated IκB-α. Meanwhile, the level of phosphorylated JNK increased together with the sensitivity to apoptosis. The contrasting effects on JNK and IκB-α activation upon TNF-α stimulation matched with a modulated maturation of TGF-β-activated kinase 1 (TAK1) kinase, assessed by 2-dimensional SDS-PAGE. Inhibition of the NF-κB pathway by HBSP was confirmed in the liver of HBSP Tg mice and associated with a significant decrease of chemically induced chronic liver inflammation, as assessed by immunohistochemistry. In conclusion, HBSP contributes to limit hepatic inflammation during chronic liver disease and may favor HBV persistence by evading immune response., (© FASEB.)

Current treatments for HBV chronic carriers using interferon alpha or nucleoside analogues are not effective in all patients and may induce the emergence of HBV resistant strains. Bay 41-4109, a member of the heteroaryldihydropyrimidine family, inhibits HBV replication by destabilizing capsid assembly. The aim of this study was to determine the antiviral effect of Bay 41-4109 in a mouse model with humanized liver and the spread of active HBV. Antiviral assays of Bay 41-4109 on HepG2.2.15 cells constitutively expressing HBV, displayed an IC(50) of about 202 nM with no cell toxicity. Alb-uPA/SCID mice were transplanted with human hepatocytes and infected with HBV. Ten days post-infection, the mice were treated with Bay 41-4109 for five days. During the 30 days of follow-up, the HBV load was evaluated by quantitative PCR. At the end of treatment, decreased HBV viremia of about 1 log(10) copies/ml was observed. By contrast, increased HBV viremia of about 0.5 log(10) copies/ml was measured in the control group. Five days after the end of treatment, a rebound of HBV viremia occurred in the treated group. Furthermore, 15 days after treatment discontinuation, a similar expression of the viral capsid was evidenced in liver biopsies. Our findings demonstrate that Bay 41-4109 displayed antiviral properties against HBV in humanized Alb-uPA/SCID mice and confirm the usefulness of Alb-uPA/SCID mice for the evaluation of pharmaceutical compounds. The administration of Bay 41-4109 may constitute a new strategy for the treatment of patients in escape from standard antiviral therapy.

Background: Defective hepatitis B virus (HBV) particles, generated from singly spliced HBV RNA, have been detected in chronic carriers of HBV. The present study was designed to quantify the expression of defective HBV (dHBV) and wild-type HBV (wtHBV) genomes in the serum of patients with HBV infection and its relation to the severity of liver disease., Methods: HBV and dHBV loads were determined by quantitative polymerase chain reaction in the serum of 89 untreated HBV-infected patients (31 coinfected with human immunodeficiency virus [HIV] type 1) with liver disease of different stages. The ratio of dHBV DNA to total (wtHBV plus dHBV) HBV DNA (dHBV/HBV ratio) was used to express data independently of the level of viral replication., Results: Despite a global correlation between dHBV and wtHBV load, the dHBV/HBV ratio ranged from 0.001% to 69%. The variation in dHBV/HBV ratio was independent of HIV coinfection, HBV genotype, and precore mutations. The mean dHBV/HBV ratio was higher in patients with severe liver necrosis and fibrosis., Conclusions: Our data indicate that an elevated dHBV/HBV ratio is associated with liver necroinflammation and fibrosis disease, suggesting a regulation of dHBV expression according to the severity of the liver disease. The dHBV/HBV ratio may help to better define liver disease stage during HBV infection.

. A new hepatitis B virus (HBV) protein, hepatitis B splice-generated protein (HBSP), has been detected in liver biopsy specimens from patients with chronic active hepatitis. The aim of this study was to characterize the phenotype and functions of peripheral HBSP-specific T cells and to determine whether these T-cell responses may be implicated in liver damage or viral control. Two groups of patients were studied: HBV-infected patients with chronic active hepatitis and HBV-infected patients who were inactive carriers of hepatitis B surface antigen. HBSP-specific T-cell responses were analysed ex vivo and after in vitro stimulation of peripheral blood mononuclear cells. Soluble cytokines and chemokines were analysed in sera and in cell culture supernatants. Few HBSP- or capsid-specific T-cell responses were detected in patients with chronic active hepatitis whereas frequency of HBV-specific T cells was significantly higher in inactive carrier patients. HBSP activated CD8+ and CD4+ T cells that recognized multiple epitopes and secreted inflammatory cytokines. The IL-12 level was significantly lower in sera from asymptomatic carrier patients compared to patients with chronic active hepatitis. IL-12 and IP-10 levels in the sera were significantly and independently correlated with both alanine amino transferase and HBV DNA levels. Our results show that the HBSP protein activates cellular immune responses in HBV-infected patients but has probably no prominent role in liver damage. The pattern of cytokines and chemokines in sera was linked to HBV viral load and was consistent with the level of inflammation during chronic hepatitis. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hepatitis B reactivation has been observed in HIV-infected patients with isolated anti-HBc. However, the impact of isolated anti-HBc on liver fibrosis is not known in this population. Methods: We investigated liver stiffness values (LSV) in a population of HIV-infected patients with isolated anti-HBc, and attempted to identify risk factors for high values. Results: Fifty-one out of 69 patients (74%) had low LSV (≤7.1kPa). In univariate analysis, high LSV (>7.1kPa) were associated with HCV coinfection, the duration of HIV infection, the duration of antiretroviral therapy and lipodystrophy. In age-adjusted multivariate analysis, HCV coinfection (OR 11.5; 95% CI, 3.0–62.9; P =0.001) and lipodystrophy (OR 4.6; 95% CI, 1.1–20.7; P =0.031) remained associated with high liver stiffness values. Conclusions: Lipodystrophy was the only factor associated with high liver stiffness values in our population of HIV-infected patients with isolated anti-Hbc and extensive exposure to antiretroviral drugs active on HBV, apart from HCV coinfection Our study correlates to recent studies the results of which have shown that lipodystrophy, and more generally mitochondrial toxicity, was associated with advanced liver fibrosis in HIV/HCV co-infected patients. [Copyright &y& Elsevier]