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Background and Aims: Little is known about the mechanisms of IL-17 secreting T cells accumulation in HBV-transfected livers. Here, we investigated the role of the chemokines CCL17, CCL20 and CCL22 in this process., Methods: Peripheral blood and liver tissues were obtained from 30 chronic hepatitis B (CHB) patients and 15 healthy volunteers and were evaluated by flow cytometric analysis and immunohistochemistry. Chemokine production by monocyte-derived dendritic cells (MoDCs) cocultured with HBV-transfected or untransfected Huh7 cells was measured by quantitative real-time PCR and enzyme-linked immunosorbent assay. The chemotactic activity of the culture supernatants was also tested., Results: The proportions of IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells were both increased in liver and peripheral blood mononuclear cells of CHB patients compared to those in HVs. CHB patients showed higher intrahepatic levels of CCL17 mRNA, CCL22 mRNA, CCR6 mRNA and CCR4 mRNA than HVs. The expression of CCR6 and CCR4 on the surface of Th17 and Tc17 cells in CHB patients was also significantly higher than that in HVs. Significant correlations existed between the CCR4/CCR6 levels and both the alanine transaminase levels and HBV DNA loads. Contact between MoDCs and pBlue-HBV-transfected Huh7 cells induced the expression of CCL17 and CCL22 dependent on the dose of HBV DNA. However, CCL20 expression was lower in CHB patients than in HVs. Transwell experiments showed that upregulation of CCL17 and CCL22 enhanced the migration of IL-17 secreting T cells., Conclusions: Contact of HBV-transfected cells with MoDCs induces CCL17 and CCL22 chemokine production, which may favour the recruitment of Th17 and Tc17 cells to liver tissue in CHB. Our results reveal the mechanism of IL-17 secreting T cells recruitment to liver tissue and thus provide new immunotherapy targets for CHB patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Restoring antiviral immunity is a promising immunotherapeutic approach to the treatment of chronic hepatitis B virus (HBV) infection. Dendritic cells play a crucial role in triggering antiviral immunity. In this study, we identified immunodominant epitopes prevalent in CD8 + T cell responses. We characterized the hierarchy of HBV epitopes targeted by CD8 + T cells following autologous monocyte-derived dendritic cell (moDC) expansion in HBV-infected subjects with distinct disease stages: treatment-naïve (TN group, n = 168), treatment with complete virological response (TR group, n = 72), and resolved HBV infection (RS group, n = 28). T cell responses against 32 HBV epitopes were measured upon moDC expansion. Several subdominant epitopes that triggered HBV-specific CD8 + T cell responses were identified. These epitopes' responses varied in individuals with different disease stages. Moreover, the most immunodominant and immunoprevalent epitope included the envelope residues 256-270 (Env 256-270 ), corresponding to amino acid residues 93-107 in the small HBV surface protein (SHBs) across three patient groups. The frequency of Env 256-270 -specific interferon-γ-producing T cells was the highest in the RS group and the lowest in the TN group. In addition, individuals with HLA-A*02:03/02:06/02:07 were capable of responding to Env 256-270 . Env 256-270 -specific CD8 + T cells tolerated amino acid variations within the epitope detected in HBV genotypes B and C. This suggests that Env 256-270 in SHBs is crucial in HBV-specific T cell immunity following autologous moDC expansion. It might be a potential target epitope for dendritic-cell-based immunotherapy for CHB patients with complete viral suppression by long-term NAs treatment.

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.

Absent in melanoma 2 (AIM2) is a recently recognized cytoplasmic receptor which could sense cytoplasmic double-stranded DNA (dsDNA). After AIM2 detects the presence of parasitic nucleic acids (dsDNA) derived from invasive bacteria or viral genomes (for example, vaccinia virus and cytomegalovirus) within infected cells, AIM2 inflammasome could be formed. The formed AIM2 inflammasome could induce innate immune response and increase expressions of IL-1β and IL-18. Hepatitis B virus (HBV) is a hepatotropic, non-cytopathic double-stranded DNA virus. The immune response to viral antigens or virus is thought to be responsible for both liver damage and viral clearance in patients with HBV infection. However, there are no reports about whether AIM2 inflammasome exists in hepatocytes. In the present study, we investigated the presence and activity of AIM2 inflammasome in human hepatocytes. We found that AIM2 was expressed in cytoplasm of hepatocytes, and IL-18 expression was increased after AIM2 sensed HBV in hepatocytes in vitro. These results showed that AIM2 inflammasome was active in hepatocytes. We also found that hepatic AIM2 expression of chronic hepatitis B (CHB) patients was higher than that of controls. Hepatic AIM2 expression levels were positively correlated to the severity of liver inflammation. IL-18 is already considered to be associated with hepatic injury during HBV infection. In conclusion, we, therefore, believe that AIM2 inflammasome in hepatocytes might play an important role in the development and maintenance of HBV-related hepatitis.

The cellular mechanisms by which hepatitis B virus (HBV) is assembled and exported are largely undefined. Recently, it has been suggested that these steps require the multivesicular body (MVB) and the autophagic machinery. However, the mechanisms by which HBV might regulate these compartments are unclear. In this study, we have found that by activating Rab7a, HBV alters its own secretion by inducing dramatic changes in the morphology of MVB and autophagic compartments. These changes are characterized by the formation of numerous tubules that are dependent upon the increase in Rab7 activity observed in the HBV-expressing HepG2.2.15 cells compared to HepG2 cells. Interestingly, transfection-based expression of the five individual viral proteins indicated that the precore protein, which is a precursor of HBeAg, was largely responsible for the increased Rab7 activity. Finally, small interfering RNA (siRNA)-mediated depletion of Rab7 significantly increased the secretion of virions, suggesting that reduced delivery of the virus to the lysosome facilitates viral secretion. These findings provide novel evidence indicating that HBV can regulate its own secretion through an activation of the endo-lysosomal and autophagic pathway mediated by Rab7 activation., (© 2015. Published by The Company of Biologists Ltd.)

Objective: To explore relations between the opportunities and effects of internal general treatment added Entecavir on acute-on-chronic liver failure (ACLF) of HBeAg-negative chronic hepatitis B in different score ranges of acute-on-chronic liver failure severity., Methods: A total of 108 ACLF of HBeAg-negative chronic hepatitis B patients with different ACLF severity score were treated with internal general treatment added Entecavir. The liver failure severity scores, HBV-DNA loads during the initiation of therapy, recovery phase and in deathbed phase, courses of Entecavir administration and mortalities were studied., Results: For 19 patients with high ACLF score (> or = 12), the difference in ACLF score between pre and post-treatment was not significant. The difference in HBV-DNA load between pre and post-treatment was not significant and the mortality was 18/19. For 30 patients with higher intermediate ACLF score (8-11), the difference in ACLF score between pre and post-treatment was not significant. The difference in HBV-DNA load between pre and post-treatment was significant, and the mortality was 66.67% (20/30). For 36 patients with lower intermediate ACLF score (5-7), the difference in ACLF score between pre and posttreatment was not significant. The difference in HBV-DNA load between pre and post-treatment was significant, and the mortality was 30.56% (11/36). For 23 patients with low ACLF score (< or = 4), the difference in ACLF score between pre and post-treatment was significant. The difference in HBV-DNA load between pre and post-treatment was significant, and the mortality was 8.70% (2/23)., Conclusions: A novel acute-on-chronic liver failure scoring system can syllabify differentiate the relations between the opportunities and efficacies on the Entecavir treatment for HBeAg-negative ACLF.

Objective: To investigate whether hepatitis B virus (HBV) can induce the expression of the host-encoded cytokine interleukin-32 (IL-32) and its effects on host signaling mechanisms related to HBV pathogenesis., Methods: A eukaryotic expression vector harboring an enhanced green fluorescent protein was constructed with HBV genomic sequences (pIRES2-HBV-EGFP) and transfected into HepG2 cells. In addition, the nuclear factor-kappa B (NF-kB) subunits, p50 and p65, were transfected respectively into HepG2 cells. In both cases, 48 hrs after transfection, IL-32 expression was determined at the mRNA and protein levels using real-time PCR and ELISA and western blot, respectively. The HepG2 cells transfected with pIRES2-HBV-EGFP were also treated with the NF-kB inhibitor SN50 at various concentrations, and the effects on IL-32 protein expression 48 hrs later were evaluated by western blot. Significance of between-group differences was assessed by the Student's t-test., Results: Transfection with pIRES2-HBV-EGFP led to significantly higher IL-23 expression than transfection with empty vector (mRNA: 2.8-fold higher and protein: 4.5-fold higher; both P less than 0.05). Transfection of p50 and p65 proteins led to significantly higher IL-32 expression (both P less than 0.05), and NF-kB activation was found to be required for HBV-induced IL-32 expression., Conclusion: IL-32 expression is induced by HBV in HepG2 cells. This host-encoded cytokine, and its downstream activation of NF-kB, may be involved in the pathogenesis of HBV, especially in the subsequent liver inflammation that accompanies HBV infection.

Objectives: The role of interleukin-32 (IL-32) in chronic hepatitis B (CHB) remains unclear. In order to identify the role of IL-32 in CHB, we detected the expression levels of IL-32 in liver samples of CHB patients and analyzed the correlation between IL-32 and liver inflammation/fibrosis., Methods: Real-time PCR and immunohistochemistry were used to detect the expression levels of IL-32 in liver tissues of patients with CHB. The correlation between hepatic IL-32 expression and the severity of liver inflammation/fibrosis was assessed using Spearman's correlation., Results: Hepatic IL-32 expression was increased in CHB patients and increased with the severity of liver inflammation/fibrosis. Moreover, hepatic IL-32 expression was significantly positively correlated with serum ALT level and negatively related with serum ALB level., Conclusions: Our results suggest that IL-32 could be implicated in HBV-related liver inflammation/fibrosis. We believe that IL-32 might play important roles in the pathogenesis of CHB., (Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study was to compare the epidemiological, biochemical and virological characteristics among patients co-infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) according to the mode of HCV contamination., Methods: The study included 133 patients with chronic HBV/HCV co-infection. They were studied and subdivided into two groups (drug addicts group and Blood transfusion group) according to the mode of HCV contaminnation. The epidemiological, biochemical and virological characteristics were collected. Univariate analysis was performed with the SPSS 16.0., Results: 78 patients were infected by the mode of drug addicts (IDU), whereas 55 were infected by the mode of blood transfusion( PTCH). Patients in drug addicts group had yonger age, shorter HBV and HCV infection history, and lower cirrhosis percentage than those of patients in PTCH group (P <0.05). However,serum levels of ALT (t =4.760, P =0.000), AST (t = 3.798, P = 0.000), TBil (t = 4.274, P = 0.000) of IDU patients were higher than those of PTCH patients. There was difference of sex composition between two groups (chi2 = 18.706, P = 0.000)., Conclusions: The clinical characteristics of patients with HBV/HCV coinfection were significantly different among different HCV contamination mode. PTCH patients have the characteristics of older age, more cirrhosis and mild degree of liver injury; IDU patients have the characteristics of yonger age,fewer cirrhosis and severe liver injury.

HBV replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes. Moreover, liver damage in patients with HBV infection is immune-mediated and cytokines play important roles in immune-mediated liver damage after HBV infection. Interleukin-32 (IL-32) is a proinflammatory cytokine and plays a critical role in inflammation. However, the role of HBV in IL-32 expression remains unclear. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IL-32 expression through the promoter of IL-32 at positions from -746 to +25 and in a dose-dependent manner. Furthermore, we demonstrate that increase of NF-κB subunits p65 and p50 in Huh7 cells also augments IL-32 expression, and the NF-κB inhibitor blocks the effect of HBx on IL-32 induction. These results indicate that NF-κB activation is required for HBx-induced IL-32 expression. In conclusion, IL-32 is induced by HBx in Huh7 cells. Our results suggest that IL-32 might play an important role in inflammatory response after HBV infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To identify the HBV genotype-specific tag sequence., Methods: The large S region sequences from 930 HBV genomes were aligned to identify the genotype-specific tag sequences. PCR was used to check the genotyping effect of these tags., Results: Two tag sequences, sequence between 149-169 and sequence between 461-483, were identified in the large S region. Using primers specific to these tag sequences, the genotype of HBV can be specifically identified., Conclusion: These tag sequences can be used for HBV genotyping.

Objective: To investigate the prevalence of fatty liver in Guangzhou and its relation to hepatitis B virus (HBV) infection, hyperlipidemia and abnormal alanine aminotransferase (ALT)., Methods: A retrospective analysis was made on clinical data of 4365 participants who received health check-up in our hospital., Results: The prevalence of fatty liver was 18.2%, 19.2% in males and 17.1% in females, respectively. There was no significant difference between males and females (P = 0.07). Among 793 subjects with fatty liver, 440 were males and 353 were females. The prevalence of fatty liver was 16.7% in HBV infection group and 18.3% in the group without HBV infection, no significant difference was seen between these two groups (P = 0.45). The prevalence of fatty liver was 42.1% in hyperlipidemia group, and 11.6% in the group with normal serum triglyceride (TG) and total cholesterol (TC), respectively, there was a significant difference between these two groups (P < 0.05). The abnormal ALT was seen in 32.5% of the fattty liver group, which was significantly higher than that (8.6%) in the group without fatty liver (P < 0.05)., Conclusions: The prevalence of fatty liver was not significantly different between males and females in Guangzhou. Fatty liver was not related with HBV infection but closely related with age and hyperlipidemia. Fatty liver was a common cause of abnormal ALT.

Hepatitis B virus has been linked to the pathogenesis and carcinogenesis of hepatocellular carcinoma. Components of viruses have been identified within pathological specimens of hepatocellular carcinoma tissue. We characterized the in vitro response of human normal liver cells (L-02 cells) to components of infectious agents related to toll-like receptors. Immortalized human normal liver cells (L-02 cells) exhibited increased proliferation in response to exposure to CpG DNA. This molecule is a well-characterized surrogate for DNA viruses, which are common in the liver. Our experiments show that L-02 cells and some hepatoma cell lines such as HepG2, HuH7, Hep3B, express TLR 9 (CpG-specific). CpG DNA, HBV DNA, DNA of HBV middle envelope protein (MP) containing a number of CpG, supernatant of HepG2.2.15 (HepG2 cells transfected HBV) excreting HBV DNA and extraction of nucleic acids from HepG2.2.15 supernatant can all activate NF-kappaB. In addition, L-02 cells were less susceptible to TNF-alpha-induced apoptosis as measured by Annexin V-FITC staining when stimulated with CpG. mRNA of DNA methyltransferase 1 (DNMT-1) and BCL-2 was increased when L-02 cells were stimulated with CpG DNA. Our study has identified a possible novel mechanism that indicates how CpG DNA of HBV DNA may contribute to the malignant transformation of benign liver cells.

Objective: To obtain specific small interfering RNAs (siRNA) for hepatitis B virus (HBV) and evaluate their interfering effect., Methods: Three siRNAs were transfected into HepG2.2.15 cells, and the amount of HBV mRNA in the cell culture medium was quantified with real-time fluorescence quantitative RT-PCR. HBsAg in the culture media was assayed with Western blotting at different time points after transfection., Results: The cells transfected with specific siRNAs showed decreased levels of HBV mRNA and HBsAg (P<0.05), but those with nonspecific siRNA transfection as the negative control did not show such changes (P>0.05)., Conclusion: Specific siRNA can significantly inhibit protein expression and mRNA synthesis of HBV in HepG2.2.15 cells in vitro.

Currently, the hepatocellular trafficking pathways that are used by the hepatitis B virus (HBV) during viral infection and shedding are poorly defined. It is known that the HBV uses late endosomal and multivesicular body (MVB) compartments for assembly and release. The intraluminal vesicles (ILVs) generated within MVBs have also been implicated in the late synthesis stages of a variety of pathogenic viruses. We recently observed that the HBV within infected hepatocytes appears to associate with the tetraspanin protein CD63, known to be a prominent and essential component of ILVs. Immunofluorescence microscopy of HBV‐expressing cells showed that CD63 colocalized with HBV proteins (large hepatitis B surface antigens [LHBs] and hepatitis B core) and labeled an exceptionally large number of secreted extracellular vesicles of uniform size. Small interfering RNA (siRNA)–mediated depletion of CD63 induced a substantial accumulation of intracellular LHBs protein but did not alter the levels of either intracellular or extracellular HBV DNA, nor pregenomic RNA. Consistent with these findings, we found that markedly less LHBs protein was associated with the released HBV particles from CD63 siRNA‐treated cells. Importantly, the HBV viral particles that were shed from CD63‐depleted cells were substantially less infective than those collected from control cells with normal CD63 levels. Conclusion: These findings implicate the tetraspanin protein CD63 as a marker and an important component in the formation and release of infectious HBV particles. [ABSTRACT FROM AUTHOR]

Increasing studies have revealed that long noncoding RNAs (lncRNAs) might play vital roles in the development and progression of various diseases including viral infectious diseases. However, the expression and biological functions of lncRNAs in chronic hepatitis B virus (HBV) infection remain largely unknown. Therefore, lncRNA microarray was performed to analyze the lncRNAs' and messenger RNAs' (mRNAs) expression profiles in liver tissues from patients with chronic HBV infection. Subsequently, a comprehensive bioinformatics analysis was conducted to investigate the potential functions of the differentially expressed genes. As a result, a total of 203 differentially expressed lncRNAs and 180 mRNAs were identified in chronic HBV infection. The expressions of five differentially expressed lncRNAs were further validated using quantitative real‐time polymerase chain reaction. Gene ontology, pathway analysis, and gene set enrichment analysis revealed that differentially expressed lncRNAs might be mainly be involved in cytokine‐cytokine receptor interaction and varied biotransformation processes, including fatty acid metabolism, amino acid metabolism, carbon metabolism, and drug metabolism. Additionally, coexpression networks between differentially expressed lncRNAs and mRNAs were constructed to reveal the hub regulator and analyze the functional pathways. This study provided an overview of lncRNA and mRNA expression in liver tissues from patients with chronic HBV infection. These differentially expressed lncRNAs might play crucial roles in the pathogenesis and progression of chronic HBV infection, which deserve further investigation. Highlights: This study provided an overview of lncRNA expression in liver tissues from patients with chronic HBV infection. These lncRNAs might play crucial roles in the pathogenesis and progression of chronic HBV infection through cytokine‐cytokine receptor interaction and multiple biotransformation processes, especially fatty acid degradation. [ABSTRACT FROM AUTHOR]

Background/Aims: The hepatocellular transport pathways and cellular proteins utilized during the packaging and secretion of hepatitis B virus are poorly understood. In this study, we tested if the large GTPase dynamin, a protein involved in vesicle formation and secretion at the trans-Golgi network in hepatocytes, is also used by hepatitis B virus (HBV) in secreting viral proteins.Methods: Using HepG2.2.15 cells expressing the full-length HBV genome, we tested the effects of wild-type and mutant dynamin on the localization and secretion of two hepatitis B antigens, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Distribution of these two antigens was analyzed morphologically in cells transiently transfected with wild-type or mutant dynamin constructs, whereas secretion of the antigens was measured by testing for antigen levels in the media of transfected cells.Results: Mutant dynamin was found to induce a striking redistribution of HBsAg and HBeAg to a perinuclear compartment, as well as a decrease in the levels of HBsAg and HBeAg present in cell culture media indicating a reduction in viral protein secretion. At the electron microscopy level, cells expressing the mutant dynamin showed a marked accumulation of viral particles in dilated cisternae of an uncharacterized cellular compartment.Conclusions: Intact dynamin function is required for secretion of HBV proteins from hepatocytes through an uncharacterized cellular compartment. [Copyright &y& Elsevier]

Highlights: [•] MIF-173 G/C polymorphism was genotyped in patients with CHB or HC in southern China. [•] Frequency of MIF-173 C/C genotype in patients with CHB or HC was increased. [•] Frequency of the C allele at MIF-173 in CHB patients was also increased. [•] Serum levels of MIF were enhanced in CHB or HC patients. [•] So MIF-173 G/C polymorphism could be associated with susceptibility to CHB or HC. [ABSTRACT FROM AUTHOR]