Active Hepatitis B Virus Replication Research Articles

Sirtuin 1 Dynamic Interplay of IFI16 and STING Interferon-Stimulated Gene Regulate Hepatitis B Virus Replication

Background: Hepatitis B virus (HBV) infection remains a significant global health concern, affecting millions worldwide and often leading to severe liver diseases. Chronic conditions associated with HBV contribute to the development of liver fibrosis, a crucial prognostic factor necessitating urgent therapeutic strategies. Objectives: This study aims to elucidate the interplay between interferon (IFN)-inducible protein 16 (IFI16), Sirtuin 1 (Sirt1) (NAD-dependent deacetylase), and STING (Stimulator of Interferon Genes) in the context of HBV infection, focusing on understanding their roles in viral replication and innate immune responses. Methods: This study investigates the interaction between Sirt1 and IFI16 during active HBV replication using siRNA-mediated knockdown and co-transfection techniques. HBV replication is assessed following IFI16 silencing, and the synergistic inhibition of IFI16 and Sirt1 is evaluated. Western blotting, electrophoresis, and immunoprecipitation methods are employed to explore STING's role in DNA-mediated innate immunity and interferon-stimulated gene activation during viral infection. Results: While individual knockdown of IFI16 has minimal impact on HBV replication, with a reduction of less than 10%, dual inhibition of IFI16 and Sirt1 resulted in a significant reduction in viral replication by approximately 70%. This underscores the synergistic role of these proteins in the context of HBV infection. Furthermore, the study implicates STING as a promising therapeutic target for viral infections, shedding light on its regulatory role in innate immune responses and interferon-stimulated genes (ISGs). Conclusions: Our study reveals the complex interplay between IFI16, Sirt1, and STING in HBV infection, highlighting potential therapeutic targets. While in vitro findings offer valuable insights, in vivo validation and further exploration of broader pathway interactions are essential. Future efforts should prioritize translating these findings into clinical applications for HBV treatment.

Liver mechanosignaling as a natural anti-hepatitis B virus mechanism

The mechanisms underlying the natural control of hepatitis B virus (HBV) infection have long been an intriguing question. Given the wide physiological range of liver stiffness and the growing attention to the role of mechanical microenvironment in homeostasis and diseases, we investigated how physical matrix cues impact HBV replication. High matrix stiffness significantly inhibited HBV replication and activated YAP in primary hepatocyte culture system, a key molecule in mechanosignaling. YAP activation notably suppressed HBV transcription and antigen expression. Several YAP-induced genes exhibited strong anti-HBV effects. Single-cell analysis of liver tissue from male individuals with active HBV replication revealed a strong significant negative correlation between YAP signature activation and HBV transcript levels. Intraperitoneal administration of YAP small molecule agonist potently controls HBV in male mouse models. These findings unveil a mechanism that involves the mechanical environment of hepatocytes and YAP to clear hepatotropic viral infection in the liver, providing new perspectives for HBV cure studies and antiviral development.

Measuring HBV pregenomic RNA may be a potential biomarker to determine HBV functional cure in HIV/HBV-co-infected patients with HBsAg loss.

Functional cure of hepatitis B virus (HBV) is an optimal treatment goal for chronic hepatitis B, with the loss of hepatitis B surface antigen (HBsAg) being a crucial indicator. However, the adequacy of HBsAg loss for evaluating functional cure of HBV in patients co-infected with HBV/human immunodeficiency virus (HIV) remains controversial. In this study, we measured HBV pregenomic RNA (pgRNA), a potential biomarker that correlates with covalently closed circular DNA, in the frozen plasma of 98 patients with HBsAg loss from a large HIV/HBV co-infection cohort in Guangzhou, China. HBV pgRNA was still detected in 43.9% (44/98) of the patients, suggesting active HBV replication in individuals with HBsAg loss. Our observations imply that HBsAg loss may not be a reliable predictor of HBV functional cure in cases of HIV/HBV co-infection.

Inhibitory effect of small-molecule compound AM679 targeting elongation-factor binding protein 2 on hepatitis B virus in vitro

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation

BackgroundPreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive. MethodsWe investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP. ResultsOur study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation. ConclusionsThese findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

FibroScan Predicts Liver Fibrosis Progression in Chronic HBV Infection Patients with No Clear Indication for Antiviral Therapy: A Retrospective Cohort Study.

Chronic hepatitis B virus (HBV) infection patients who do not fulfill the typical treatment indications should be followed up. This study aimed to evaluate the risk of liver fibrosis progression (LFP) and assess the role of noninvasive tests (NITs) of liver fibrosis in monitoring LFP in these patients. A total of 116 patients with active HBV replication, persistently normal or minimally elevated alanine aminotransferase (ALT) levels, and no or mild hepatic necroinflammation or fibrosis based on liver biopsy tests at baseline and followed by a repeated liver biopsy assessment during follow-up. LFP was defined as increase in METAVIR fibrosis score by 1 score or more. Among 116 patients, 40 (34.5%) progressed by at least one fibrosis stage, 16 (13.8%) progressed by at least two fibrosis stages at a median follow-up interval of 27 months (IQR: 12-36). Multivariate analysis confirmed the significant association of an increase in liver stiffness measurement (LSM) value with LFP on histology (p =0.005). The AUROC of LSM value increase rate is significantly higher than that of serum-based NITs of liver fibrosis for the prediction of LFP (p < 0.05). An increase in LSM by 20% is the optimal cutoff for the prediction of LFP. LFP is non-negligible in patients with active HBV replication, persistently normal or minimally elevated ALT, and initially no or minimal hepatic necroinflammation or fibrosis. Serial LSM tests would be more reliable in identifying LFP than serum-based NITs, and easier to obtain than serial liver biopsy tests.

Combining Preoperative Clinical and Imaging Characteristics to Predict MVI in Hepatitis B Virus-Related Combined Hepatocellular Carcinoma and Cholangiocarcinoma

Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA) is a rare form of primary liver malignancy. Microvascular invasion (MVI) indicates poor postsurgical prognosis in cHCC-CCA. The objective of this study was to investigate preoperative predictors of MVI in hepatitis B virus (HBV) -related cHCC-CCA patients. A total of 69 HBV-infected patients with pathologically confirmed cHCC-CCA who underwent hepatectomy were included. Univariate and multivariate analyses were conducted to determine independent risk factors that were then incorporated into the predictive model associated with MVI. Receiver operating characteristic analysis was used to assess the predictive performance of the new model. For the multivariate analysis, γ-glutamyl transpeptidase (OR, 3.69; p = 0.034), multiple nodules (OR, 4.41; p = 0.042) and peritumoral enhancement (OR, 6.16; p = 0.004) were independently associated with MVI. Active replication of HBV indicated by positive HBeAg showed no differences between MVI-positive and MVI-negative patients. The prediction score using the independent predictors achieved an area under the curve of 0.813 (95% CI 0.717-0.908). A significantly lower recurrence-free survival was observed in the high-risk group with a score of ≥1 (p < 0.001). γ-glutamyl transpeptidase, peritumoral enhancement and multiple nodules were independent preoperative predictors of MVI in HBV-related cHCC-CCA patients. The established prediction score demonstrated satisfactory performance in predicting MVI pre-operatively and may facilitate prognostic stratification.

High Level of Hepatitis B Core–Related Antigen Associated With Increased Risk of Hepatocellular Carcinoma in Patients With Chronic HBV Infection of Intermediate Viral Load

Chronic hepatitis B virus (HBV) infection is a risk factor for hepatocellular carcinoma (HCC). Serum levels of HB core-related antigen (HBcrAg) have been associated with active replication of HBV. We investigated whether HBcrAg levels are associated with development of HCC, especially in patients who do not require antiviral treatment. We collected data from 2666 adults positive for hepatitis B surface antigen (HBsAg), infected with HBV genotypes B or C, and without liver cirrhosis, who had long-term follow-up at the National Taiwan University Hospital from 1985 through 2000. None of the patients received antiviral treatment during the follow-up. Baseline levels of HBV DNA, HBsAg, and HBcrAg were determined retrospectively and participants were followed for a mean of 15.95 years. The primary end point was an association between serum level of HBcrAg and HCC development. HCC developed in 209 patients in the cohort (incidence rate, 4.91 cases/1000 person-years). We found a positive association between baseline level of HBcrAg and HCC development; HBcrAg level was an independent risk factor in multivariable analysis. In the subgroup of hepatitis B e antigen-negative patients with HBV DNA levels from 2000 to 19,999 IU/mL (intermediate viral load [IVL]) and normal levels of alanine aminotransferase, HBcrAg levels of 10 KU/mL or more identified patients at increased risk of HCC (hazard ratio, 6.29; confidence interval, 2.27-17.48). Patients with an IVL and a high level of HBcrAg had a risk for HCC that did not differ significantly from that of patients with a high viral load (≥20,000 IU/mL). Patients with an IVL but a low level ofHBcrAg had a low risk of HCC, with an annual incidence rate of0.10% (95% confidence interval, 0.04%-0.24%). In a long-term follow-up study of 2666 patients with chronic HBV infection (genotypes B or C), level of HBcrAg is an independent risk factor of HCC. Moreover, HBcrAg level of 10 KU/mL identifies patients with an IVL who are at high risk for HCC.

Mutant p53 protein is prevalent in Aflatoxin B1 than in Hepatitis B Core antigen associated chronic liver diseases in Southern Nigeria

Background: Aflatoxin B1 (AFB1), hepatitis B virus (HBV) and p53 mutation are major risk factors for liver cancer. However, the presence of AFB1 and expression of mutant p53 protein during active HBV replication (nuclear expression of Hepatitis B Core Antigen; HBcAg) and HBV clearance (nuclear expression of HBcAg) is still under-investigated in Africa. Objectives: This study assessed the prevalence, extent of expression, interaction, and risk associated with single and binary expression of AFB1, HBcAg and mutant p53 (mtp53) in liver diseases in relation to age and sex. Materials and Methods: This retrospective study included 14 and 74 cases of liver cancer (LC) and chronic hepatitis, respectively. Tissues were histochemically and immunohistochemically stained, scored and documented as positive (+) or negative (-) for antigen. Result: In this study, the prevalence of AFB1, HBcAg, and mtp53 were 76.1%, 40.9%, and 61.4%, respectively. Higher prevalence and expression of AFB1 was observed in chronic hepatitis than in LC (p= 0.035 and 0.028, respectively). Higher prevalence of AFB1 and mtp53 and lower prevalence of HBcAg were observed in females than in males (p= 0.186, 0.0003 and 0.062, respectively). Cytoplasmic expression of HBcAg was higher in males (58 than in females were 58.6% (17/29) and 14.3% (1/7), respectively (p= 0.088). AFB1+ and HBcAg+ females were more and less likely to develop LC than males (OR: 3.00 and 0.39, 95%CI: 0.10-4.18 and 0.44-12.20, p= 0.012 and 0.003), respectively. AFB1+mtp53- (23.9%), AFB1+mtp53+ (52.3%), HBcAg+mtp53- (13.6%), HBcAg+mtp53+ (27.3%) and AFB1+HBcAg+ (34.1%) increased liver cancer risk (OR: 6.45, 21.50, 2.80, 0.78, and 5.11; 95%CI: 0.89-29.29, 0.95-43.86, 2.96-156.13, 0.60-13.01, and 0.14-4.25; p= 0.057, 0.002, 0.189, 0.772, and 0.067, respectively). Conclusion: This study suggests that women are at a higher risk of AFB1 exposure and p53 mutation, but are at a lower risk of viral replication than men.

Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B

Treatment with nucleos(t)ide analogues (NA) leads to hepatitis B virus (HBV) DNA suppression in most patients with chronic hepatitis B (CHB), but HBV surface antigen (HBsAg) loss rates are low. Upon NA discontinuation, HBV DNA can return rapidly with ensuing alanine aminotransferase flares and induction of cytokines. Several studies reported higher HBsAg loss rates after stopping therapy, but at present it is unclear if cell-mediated immune responses are altered after treatment discontinuation. The aim of this study was to characterise T cell responses during the early phase of virological relapse, following discontinuation of NA therapy in HBeAg-negative patients. A total of 15 HBeAg-negative patients with CHB on long-term NA treatment were included in a prospective study and subjected to structured NA discontinuation. T cell responses were studied at the end of NA therapy and 4, 8 and 12 weeks thereafter. The T cell phenotype of patients with CHB on long-term NA therapy was markedly different compared to healthy individuals, but was only slightly altered after discontinuation of therapy. T cells from patients with HBsAg loss expressed low levels of KLRG1 and PD-1 at all time-points and high levels of Ki-67 and CD38 at week 12 after treatment cessation. In vitro peptide stimulated HBV-specific T cell responses were increased in several patients after NA cessation. Blocking of PD-L1 further enhanced HBV-specific T cell responses, especially after discontinuation of therapy. Relapse of active HBV replication after stopping therapy may trigger an immunological environment that enhances the responsiveness of HBV-specific T cells in vitro. Together with other immune interventions, this approach might be of interest for the development of novel therapeutic options to induce HBsAg loss in CHB. Relapse of hepatitis B virus replication after discontinuation of nucleos(t)ide analogue therapy in certain patients with chronic hepatitis B may alter the phenotype of T cells and enhance the responsiveness of hepatitis B virus-specific T cells to in vitro peptide stimulation. Blocking PD-L1 can further augment these hepatitis B virus-specific T cell responses. Interestingly, T cells of patients that subsequently achieve hepatitis B surface antigen loss are less exhausted at all time-points after stopping treatment and display a higher proliferative capacity 12-weeks after treatment discontinuation. These findings contribute to the understanding of the immunological events that occur during discontinuation of nucleos(t)ide analogue therapy.

Hepatitis B virus infection and active replication promote the formation of vascular invasion in hepatocellular carcinoma

BackgroundVascular invasion, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), is associated with the postoperative recurrence of hepatocellular carcinoma (HCC). We aimed to investigate the potential impact of hepatitis B virus (HBV) activity on the development of vascular invasion.MethodsPatients with HBV and tumor-related factors of HCC who had undergone hepatectomy were retrospectively enrolled and analyzed to identify the risk factors for developing vascular invasion.ResultsA total of 486 patients were included in this study. The overall proportion of patients with vascular invasion, including MVI and PVTT, was 60.3% (293/486). The incidence of MVI was 58.2% (283/486) whereas PVTT was 22.2% (108/486). Univariate analysis revealed that positive Hepatitis B virus surface Antigen (HBsAg) was significantly associated with the presence of vascular invasion. In a multivariate regression analysis carried out in patients with HBV-related HCC, positive Hepatitis B virus e Antigen (HBeAg)(OR = 1.83, P = 0.019) and a detectable seral HBV DNA load (OR = 1.68, P = 0.027) were independent risk factors of vascular invasion. The patients in the severe MVI group had a significantly higher rate of positive seral HBsAg (P = 0.005), positive seral HBeAg (P = 0.016), a detectable seral HBV DNA load (> 50 IU/ml) (P < 0.001) and a lower rate of anti-viral treatment (P = 0.002) compared with those in the mild MVI group and MVI-negative group. Whereas, HCC with PVTT invading the main trunk showed a significantly higher rate of positive HBsAg (P = 0.007), positive HBeAg (P = 0.04), cirrhosis (P = 0.005) and a lower rate of receiving antiviral treatment (P = 0.009) compared with patients with no PVTT or PVTT invading the ipsilateral portal vein. Patients with vascular invasion also had a significantly higher level of seral HBV DNA load than patients without vascular invasion (P = 0.008).ConclusionsIn HCC patients, HBV infection and active HBV replication were associated with the development of vascular invasion.

Expression of microRNA let-7a positively correlates with hepatitis B virus replication in hepatocellular carcinoma tissues.

Let-7a miRNA is downregulated in various cancers. However, in hepatocellular carcinoma (HCC) patients infected with hepatitis B virus (HBV), the relationship between let-7a and HBV replication has not been fully elucidated. Liver specimens were collected from 23 HCC patients with chronically active HBV. The serum levels of the HBV antigens hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the HBV antibodies, anti-HBs, anti-HBe and anti-hepatitis B core antigen (anti-HBc) were measured using the microparticle enzyme immunoassay. Let-7a levels and HBV DNA copy numbers were measured by quantitative real-time PCR (qRT-PCR) and analyzed statistically. A let-7a specific antisense oligonucleotide was introduced to the HBV-producing cell line HepG2.2.15 and a change in HBV DNA copy numbers was assessed by qRT-PCR. HCC patients with highly active HBV replication (>106 DNA copies/mL) showed higher levels of serum HBsAg and anti-HBc than patients with less active HBV replication (<103 DNA copies/mL). The level of let-7a was lower in malignant tissues than in adjacent normal tissues. However, patients with highly active HBV replication demonstrated a significantly higher level of let-7a in hepatocarcinoma tissue than patients with less active HBV replication ( P < 0.05). A higher level of let-7a was observed in the HBV-producing cell line HepG2.2.15 than in HepG2 cells ( P < 0.05), and let-7a down-regulation by antisense oligonucleotides led to a reduction in HBV DNA copy numbers ( P < 0.05), indicating a correlation between the let-7a level and HBV replication. Down-regulation of let-7a reduces HBV replication and could prevent the development of HCC, suggesting it could be an effective therapeutic treatment for HBV infection. Impact statement Although interferon and nucleic acid analogues effectively suppress HBV replication in HBV patients, there is no treatment which eradicates the virus. Moreover, the therapeutic effect can be reduced by virus mutations or drug resistance. Let-7a is a miRNA initially found in the nematode as a master regulator of developmental processes, but also exists in humans. It has been reported that the transcription of let-7a was much lower in HCC than in normal liver tissues and specific miRNA could directly promote virus replication. Therefore we hypothesized that transcription of let-7a promotes HBV replication, which might compromise the therapeutic effects of antivirus treatments. In our present study, we demonstrated a correlation between let-7a transcription and HBV replication in surgical specimens obtained from patients with HCC, as well as in HCC cell lines. Our finding might be the base for a new approach to improve HBV infection treatments in the future.

Occult HBV infection in the oncohematological setting.

Occult hepatitis B infection (OBI), a virological condition characterized by a low release of Hepatitis B Virus (HBV) from liver cells and low HBV-DNA levels in serum and/or liver tissue of HBsAg-negative subjects, may reactivate in oncohematological patients undergoing immunosuppression by aggressive chemotherapy or hematopoietic stem cell transplantation. The entity of OBI reactivation varies from an increase in HBV replication without liver damage to an active HBV replication followed by liver cell necrosis, frequently severe and in some cases life threatening. Because of a possible severe outcome associated with OBI reactivation (hepatic failure or death due to the discontinuation of chemotherapy), prophylaxis with anti-HBV nucleot(s)ide analogues is recommended in relation to the foreseeable degree of immunosuppression. This review article focuses on the clinical impact of OBI in the oncohematological setting and is addressed to all health care workers having in care oncohematological patients or involved in the treatment of HBV infection and OBI prophylaxis. International guidelines have indicated lamivudine prophylaxis in hematopoietic stem cell transplantation and when high-dose corticosteroids or anti-CD20 or anti-CD52 monoclonal antibodies are used. Entecavir or tenofovir should replace lamivudine for patients with advanced liver diseases for whom reactivation of OBI may be life threatening. When anti-CD20 or anti-CD52 sparing schedules or other non-aggressive chemotherapies are used, monitoring may be indicated, but very early treatment with highly effective antiviral drugs (entecavir or tenofovir) should be administered once a reactivation of OBI has occurred.

Hepatitis B Virus (HBV) Core-Related Antigen During Nucleos(t)ide Analog Therapy Is Related to Intra-hepatic HBV Replication and Development of Hepatocellular Carcinoma.

Although nucleos(t)ide analog (NA) therapy effectively reduces the hepatitis B virus (HBV) DNA load in the serum of patients with chronic hepatitis B, it does not completely reduce the incidence of hepatocellular carcinoma (HCC). A total of 109 patients who had chronic hepatitis B and were receiving NA therapy were analyzed. Multivariate Cox regression analysis showed that age (>60 years had a hazard ratio [HR] of 2.66), FIB-4 index (an index of >2.1 had a HR of 2.57), and the presence of HBV core-related antigen (HBcrAg; HR, 3.53) during treatment were significantly associated with the development of HCC. The amount of HBV DNA and pregenomic RNA in liver were significantly higher in 16 HBcrAg-positive patients, compared with 12 HBcrAg-negative patients, suggesting active HBV replication in HBcrAg-positive livers. Hepatic gene expression profiling showed that HBV-promoting transcriptional factors, including HNF4α, PPARα, and LRH1, were upregulated in HBcrAg-positive livers. HepAD38 cells overexpressing LRH1 increased HBV replication, characterized by higher HBV DNA and pregenomic RNA levels, during long-term exposure to entecavir. Conversely, overexpression of precore/core in HepG2 cells increased levels of these transcriptional factors. Metformin efficiently repressed HBV replication in primary human hepatocytes. Modulating HBV transcriptional factors by metformin in combination with NA therapy would potentiate anti-HBV activity and reduce the incidence of HCC in HBcrAg-positive patients.

Clinical pathology of recurrent hepatitis B after liver transplantation

To investigate the clinical pathology of recurrent hepatitis B after orthotopic liver transplantation (OLT). The clinical manifestation and hepatic pathological characteristics of 12 patients with recurrent hepatitis B after OLT were examined in this study by using hematoxylin and eosin staining,immunochemical staining of hepatitis B surface antigen and hepatitis B core antigen,tissue in situ hybridization of hepatitis B virus (HBV) DNA, and Mallory's trichrome staining.The survival rate of these OLT patients was assessed by Kaplan-Meier analysis. The early stage of recurrent HBV infection in patients with OLT was characterized by active HBV replication and mild-to-moderate inflammation in the liver. Three of the 12 patients who were treated with combination therapy group were carriers of YMDD mutants and all three showed improvement in liver function and hepatic histology after receiving adefovir dipivoxil,instead of lamivudine,in the early stage of recurrent hepatitis B after OLT. Among the patients treated with lamivudine monotherapy, four did not achieve improvement at the early stage of recurrent hepatitis B and developed fibrosing cholestatic hepatitis (FCH). Recurrent hepatitis B in patients who underwent OLT was characterized by mild-to-moderate viral hepatitis at the early stage and FCH at the later stage. Effective antiviral intervention at the early stage may reverse recurrent hepatitis B and prevent the disease progression to fatal FCH.

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

High prevalence of asymptomatic HBV chronic carriage in HIV infected long term survivors

Hepatitis B virus (HBV) infection is common in individuals infected with human immunodeficiency virus, and coinfection is associated with higher rates of HBV replication and more rapid liver disease progression than HBV monoinfection. This study evaluates the prevalence and virological profiles of hepatitis B infection in a cohort of long term survivors, with multiple antiretroviral treatments. 164 HIV-infected subjects (median age: 24 years) on combined antiretroviral therapy (cART) (median duration: 13 years), were evaluated for serologic markers of HBV infection (HBsAg, total anti-HBc and anti-HBsAg antibodies). Markers of HBV infectivity (HBeAg and HBV DNA) were evaluated in all HBsAg carriers; HBV genotype and lamivudine resistance mutations were analyzed in the cases with HBV DNA >103 IU/mL. 65.9% of the patients (108/164) had markers of past or present HBV infection (antiHBc positives), out of which 51.8% (56/108) were chronic HBV carriers and 30.5% had resolved HBV infection. All subjects were equally exposed to HBV infection, irrespective of their current immune status. Out of 21 patients with isolated anti-HBc antibodies, only 4 had detectable HBV DNA, presumably having occult hepatitis B. HBV chronic carriage rate was not influenced by the immune status. Overall, only 17.8% of the chronic carriers had active HBV replication; severely immune-depressed patients tend to maintain active viral replication more frequently than those with moderate or absent immunosuppression. The majority of the coinfected individuals (68.3%) showed no sign of liver fibrosis (APRI score 1.5); HBV DNA was directly correlated with APRI score. HBV genotype A was present in all but one of the tested patients. 98.8% of the coinfected subjects have been treated with a cART regimen that includes a drug dually active against HIV and HBV (in 98% of the cases lamivudine (3TC), for a mean time of 6.9 years and in 29.7% of the cases the current dually active drug was tenofovir). 3TC-resistance mutations were present in only 4 coinfected subjects. We found a strikingly low percentage of long term HIV/HBV coinfected patients from our group with active liver disease. A high prevalence of asymptomatic HBV chronic carriage was associated with a good immune status, suggesting that dually active antiretrovirals have an important role in delaying progression of liver disease in HIV/HBV coinfected patients.

Coinfection of hepatitis B and hepatitis delta virus in Belgium: A multicenter BASL study. Prospective epidemiology and comparison with HBV mono‐infection

Epidemiological data on hepatitis delta virus (HDV) infection in Belgium are lacking. A multicenter questionnaire-based registry on HDV infection was collated between March 1, 2008 and February 28, 2009. It consisted of patients coinfected with hepatitis B virus (HBV) and HDV. The data samples were compared to those of a concurrent registry on HBV infection. Prospective data of patients with HBV-HDV coinfection were collected. Active HBV replication is defined as HBeAg positivity or HBV DNA > 2,000 IU/ml. Forty-four patients from 15 centers were registered. A comparison of 29 patients infected with HDV (registered in the concurrent HBV registry) was made against 785 HBV mono-infected patients. The seroprevalence of patients coinfected with HBV and HDV in Belgium is reported to be 3.7% (29/785), consisting solely of the HBV-HDV coinfected patients in the HBV registry. This rises to 5.5% (44/800) if all patients infected with HDV from the two registries combined are included. The patients coinfected with HBV and HDV had higher (P < 0.05) ALT values and more advanced liver disease (Metavir score ≥F2), but had less active HBV replication and lower HBV DNA titers when compared with the patients infected only with HBV. Additionally, the majority of HBV-HDV coinfected patient was male, and 13.6% (6/44) of the patients that were coinfected HBV and HDV were also infected with HCV. In conclusion, this study provided much needed epidemiological data on the current state of HDV infection in Belgium.

Prevalence of hepatitis B virus and hepatitis C virus in patients with human immunodeficiency virus infection in central China

Co-infection of hepatitis B virus (HBV) and/or hepatitis C virus (HCV) with human immunodeficiency virus (HIV) has an adverse effect on liver disease progression. This study investigated the prevalence of HBV and/or HCV co-infection in HIV-infected patients in Central China. A total of 978 HIV-infected patients from Hunan Province were enrolled. HBV serum markers, anti-hepatitis-C-virus antibody (anti-HCV), HBV DNA, and HBV genotypes were analyzed. The prevalence of hepatitis B surface antigen (HBsAg) and anti-HCV in HIV-infected patients was 19.4% and 62.4%, respectively. The prevalence of anti-HCV in HIV-positive intravenous drug users was 93.6%. Among HBsAg-positive patients, 88.1% were found to have at least one HBV serum marker. The rates of HIV mono-infection, HBV/HIV dual infection, HCV/HIV dual infection, and HBV/HCV/HIV triple infection were 30.4%, 7.2%, 50.2%, and 12.2%, respectively. Antibody to HBsAg (Anti-HBs) was more common in anti-HCV-positive than anti-HCV-negative patients (53.3% vs 40.2%, P=0.000), but isolated hepatitis B core antibody (anti-HBc) was more common in anti-HCV-negative than anti-HCV-positive patients (24.2% vs 12.3%, P=0.000). Hepatitis B e antigen (HBeAg) and sexual transmission were independent risk factors for active HBV replication. Intravenous drug use and male sex were independent risk factors, but old age and presence of HBeAg were independent protective factors for anti-HCV. Co-infection of HBV and/or HCV with HIV infection is common in central China. HCV status is associated with anti-HBs and isolated anti-HBc in co-infected patients.

Variability in the Precore and Core Promoter Regions of HBV Strains in Morocco: Characterization and Impact on Liver Disease Progression

BackgroundHepatitis B virus (HBV) is one of the most common human pathogens that cause aggressive hepatitis and advanced liver disease (AdLD), including liver cirrhosis and Hepatocellular Carcinoma. The persistence of active HBV replication and liver damage after the loss of hepatitis B e antigen (HBeAg) has been frequently associated with mutations in the pre-core (pre-C) and core promoter (CP) regions of HBV genome that abolish or reduce HBeAg expression. The purpose of this study was to assess the prevalence of pre-C and CP mutations and their impact on the subsequent course of liver disease in Morocco.Methods/Principal FindingsA cohort of 186 patients with HBeAg-negative chronic HBV infection was studied (81 inactive carriers, 69 with active chronic hepatitis, 36 with AdLD). Pre-C and CP mutations were analyzed by PCR-direct sequencing method. The pre-C stop codon G1896A mutation was the most frequent (83.9%) and was associated with a lower risk of AdLD development (OR, 0.4; 95% CI, 0.15–1.04; p = 0.04). HBV-DNA levels in patients with G1896A were not significantly different from the other patients carrying wild-type strains (p = 0.84). CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were associated with higher HBV-DNA level and increased liver disease severity. Multiple logistic regression analysis showed that older age (≥40 years), male sex, high viral load (>4.3 log10 IU/mL) and CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were independent risk factors for AdLD development. Combination of these mutations was significantly associated with AdLD (OR, 7.52; 95% CI, 4.8–8; p<0.0001).ConclusionsThis study shows for the first time the association of HBV viral load and CP mutations with the severity of liver disease in Moroccan HBV chronic carriers. The examination of CP mutations alone or in combination could be helpful for prediction of the clinical outcome.