Jinyu Wang

Transcription of Hepatitis B Virus Covalently Closed Circular DNA Is Regulated by CpG Methylation during Chronic Infection

The persistence of hepatitis B virus (HBV) infection is maintained by the nuclear viral covalently closed circular DNA (cccDNA), which serves as transcription template for viral mRNAs. Previous studies suggested that cccDNA contains methylation-prone CpG islands, and that the minichromosome structure of cccDNA is epigenetically regulated by DNA methylation. However, the regulatory effect of each CpG island methylation on cccDNA activity remains elusive. In the present study, we analyzed the distribution of CpG methylation within cccDNA in patient samples and investigated the impact of CpG island methylation on cccDNA-driven virus replication. Our study revealed the following observations: 1) Bisulfite sequencing of cccDNA from chronic hepatitis B patients indicated that CpG island I was seldom methylated, 2) CpG island II methylation was correlated to the low level of serum HBV DNA in patients, and in vitro methylation studies confirmed that CpG island II methylation markedly reduced cccDNA transcription and subsequent viral core DNA replication, 3) CpG island III methylation was associated with low serum HBsAg titers, and 4) Furthermore, we found that HBV genotype, HBeAg positivity, and patient age and liver fibrosis stage were also relevant to cccDNA CpG methylation status. Therefore, we clearly demonstrated that the status of cccDNA methylation is connected to the biological behavior of HBV. Taken together, our study provides a complete profile of CpG island methylation within HBV cccDNA and new insights for the function of CpG methylation in regulating HBV cccDNA transcription.

Comparative Analysis of CpG Islands among HBV Genotypes

DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.

Impact of Nucleos(t)ide Analogue Combination Therapy on the Estimated Glomerular Filtration Rate in Patients With Chronic Hepatitis B

AbstractMonotherapy with telbivudine or adefovir can affect estimated the glomerular filtration rate (eGFR). However, only a few studies have assessed changes in eGFR in patients who have chronic hepatitis B (CHB) and are receiving nucleos(t)ide analogue (NA) combination therapy. In our study, we aimed to evaluate the effects of long-term NA combination therapy on eGFR in Chinese CHB patients.This retrospective study included 195 CHB patients. Patient subgroups included those treated with lamivudine plus adefovir (n = 73), telbivudine plus adefovir (n = 51), and entecavir plus adefovir (n = 35); untreated patients (n = 36) served as a control group.After an average follow-up duration of 24 months with combination therapy, analysis of changes in eGFR from baseline values, calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and Modification of Diet in Renal Disease (MDRD) formulas, showed decrease by11.08 and 18.34 mL/min (P < .001), respectively, in the lamivudine plus adefovir group; decrease by 3.73 and 10.04 mL/min (P = .012), respectively, in the entecavir plus adefovir group; and increase by 0.91 and 2.12 mL/min (P = .46), respectively, in the telbivudine plus adefovir group. The eGFR in the telbivudine plus adefovir group was similar to that for the untreated group. The eGFR decreases due to adefovir therapy could be rescued by adding telbivudine, and the eGFR increase due to telbivudine could be compromised by adding adefovir.Adefovir in combination with lamivudine or entecavir therapy was significantly associated with decreased eGFR, but telbivudine could rescue the eGFR decrease that results from adefovir treatment.

Low Serum Hepatitis B Surface Antigen Level Predicts Compensated Cirrhosis Caused by Chronic Hepatitis B in HBeAg Positive Patients in East China

Backgrounds: Serum hepatitis B surface antigen (HBsAg) levels are associated with fibrosis in patients with chronic hepatitis B (CHB) infection. Objectives: The aim of our study was to evaluate serum HBsAg level as a biomarker for compensated cirrhosis in hepatitis B e antigen (HBeAg) positive CHB patients. Patients and Methods: Two-hundred and one HBeAg-positive Chinese CHB patients with or without cirrhosis were enrolled in this retrospective study. Cirrhosis was diagnosed based on liver biopsy. Furthermore, patients with decompensated cirrhosis were excluded. A statistical analysis was performed regarding the association between serum HBsAg level and compensated cirrhosis. Results: Patients with compensated cirrhosis had a significantly lower mean serum HBsAg level compared to those without cirrhosis (3.27 Log10 IU/mL VS 4.17 Log10 IU/mL, P < 0.001). Furthermore, examining the correlation with compensated cirrhosis revealed that lower level of serum HBsAg was a significant factor in multivariate analysis. The area under the receiver operating characteristics curve of serum HBsAg was 0.856 for compensated cirrhosis. A positive predictive value of 66.2% and negative predictive value of 90.7% were obtained with a cut-off value of < 3.60 Log10 IU/mL (4000 IU/mL) of serum HBsAg. Moreover, the rate of compensated cirrhosis increased to 75.0% after combining with APRI > 2. Conclusions: In HBeAg positive CHB patients, low serum HBsAg level is a useful predictor of compensated cirrhosis.

Role of quantitative hepatitis B core antibody levels in predicting significant liver inflammation in chronic hepatitis B patients with normal or near-normal alanine aminotransferase levels

Chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) levels are not free from significant hepatic lesions. Recently, there has been an improved understanding of the clinical significance of quantitative hepatitis B core antibody levels (qAnti‐HBc) during CHB management. In this cross‐sectional study, we evaluated the utility of qAnti‐HBc in identifying significant liver inflammation in CHB patients.

Improved Efficacy of a pegylated interferon-α-2a stepwise optimization treatment strategy in the treatment of hepatitis B e antigen-positive chronic hepatitis B patients.

AbstractCurrent pegylated interferon-&agr; (PEG-IFN) treatment for chronic hepatitis B (CHB) e-antigen (HBeAg)-positive patients are suboptimal, and effective ways of improving PEG-IFN treatment efficacy are needed.This retrospective cohort study compared the efficacy of a PEG-IFN stepwise optimization treatment (PEG-IFN SOT) strategy with that of a 48-week PEG-IFN standard therapy (PEG-IFN ST) in HBeAg-positive CHB patients.A total of 110 patients were included in our study. Of these, 70 received the PEG-IFN SOT and 40 received the PEG-IFN ST (control group). We based the decision whether to add adefovir and/or extend the PEG-IFN–based treatment to 96 weeks on the patients’ 12-week or 24-week early virological response (12W EVR, at least a 2 log10 reduction in HBV DNA copies/mL at week 12; 24W EVR, at least 1 log10 reduction in HBsAg IU/mL or HBsAg <1500 IU/mL at week 24) and their 48-week partial response (48W PR, 1.0 ⩽HBeAg ⩽10.0 S/CO or HBeAg >10.0 S/CO but HBsAg <1000 IU/mL).The HBeAg seroconversion rate 24 weeks post-PEG-IFN treatment was significantly higher in the PEG-IFN SOT than the PEG-IFN ST group (50% vs 22.5%, P = 0.005). The HBsAg clearance rates in the PEG-IFN SOT and ST groups were 10% and 0% (P = 0.04), respectively. Receiving PEG-IFN SOT (OR = 0.26, P = 0.01), ALT × ULN at baseline (OR = 0.74, P = 0.003), and achieving 12 and 24W EVR (OR = 0.29, P = 0.03) were independent factors associated with HBeAg seroconversion.PEG-IFN SOT is a promising strategy for achieving high rates of serological response in HBeAg-positive CHB patients.

Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus

Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection.

Differentially Expressed Intrahepatic Genes Contribute to Control of Hepatitis B Virus Replication in the Inactive Carrier Phase

Background The natural history of chronic hepatitis B virus (HBV) infection was divided into 4 phases. Patients in the inactive carrier (IC) status and immune tolerant (IT) phase had normal alanine aminotransferase levels but huge different viral loads. The mechanism underlying low viral replication status in IC phase is unknown. Methods We determined the intrahepatic transcriptomes of 83 chronic hepatitis B patients by microarray analysis of liver biopsies, and screened the effect of differentially regulated genes on HBV replication using specific small interfering RNAs in vitro. Results The gene profile distinguishing active chronic hepatitis from IT and IC was predominantly composed of immune-related genes. The liver transcriptomes between the IT and IC phase were largely similar, and 109 expressed genes were significantly different. By performing systematic screening, 5 candidate genes including EVA1A, which were expressed at a relative higher level in IC phase than IT, were identified to regulate HBV replication and gene expression in cellular models. Conclusions The immune-related pathways were up-regulated in the active chronic hepatitis phase but not in the IT and IC phase. A number of intrahepatic genes highly expressed in the IC phase may participate in the control of HBV replication and determine the inactive status of HBV infection.

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

AIM To investigate precore/basal core promoter (PC/BCP) mutants throughout hepatitis B virus (HBV) infection and to determine their relationship to hepatitis B early antigen (HBeAg) titers. METHODS We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012. None of the patients received antiviral therapy. HBV DNA from serum, was quantified by real-time PCR. The HBV genotype was determined by direct sequencing of the S gene. We used the Simpleprobe ultrasensitive quantitative method to detect PC/BCP mutants in each patient. We compared the strain number, percentage, and the changes in PC/BCP mutants in different phases, and analyzed the relationship between PC/BCP mutants and HBeAg by multiple linear regression and logistic regression. RESULTS Patients with HBV infection (n = 191) were assigned to groups by phase: Immune tolerance (IT) = 55, Immune clearance (IC) = 67, Low-replicative (LR) = 49, and HBeAg-negative hepatitis (ENH) = 20. Of the patients (male, 112; female, 79) enrolled, 122 were HBeAg-positive and 69 were HBeAg-negative. The median age was 33 years (range: 18-78 years). PC and BCP mutation detection rates were 84.82% (162/191) and 96.86% (185/191), respectively. In five HBeAg-negative cases, we detected double mutation G1896A/G1899A. The logarithm value of PC mutant quantities (log10 PC) significantly differed in IT, IC, and LR phases, as well as in the ENH phase (F = 49.350, P < 0.001). The logarithm value of BCP mutant quantities (log10 BCP) also differed during the four phases (F = 25.530, P < 0.001). Log10 PC and log10 BCP values were high in the IT and IC phases, decreased in the LR phase, and increased in the ENH phase, although the absolute value at this point remained lower than that in the IT and IC phases. PC mutant quantity per total viral load (PC%) and BCP mutant quantity per total viral load (BCP%) differed between phases (F = 20.040, P < 0.001; F = 10.830, P < 0.001), with PC% and BCP% gradually increasing in successive phases. HBeAg titers negatively correlated with PC% (Spearmans rho = -0.354, P < 0.001) and BCP% (Spearmans rho = -0.395, P < 0.001). The negative correlation between PC% and HBeAg status was significant (B = -5.281, P = 0.001), but there was no such correlation between BCP% and HBeAg status (B = -0.523, P = 0.552). CONCLUSION PC/BCP mutants become predominant in a dynamic and continuous process. Log10 PC, log10 BCP, PC% and BCP% might be combined to evaluate disease progression. PC% determines HBeAg status.

Amino acid substitutions Q129N and T131N/M133T in hepatitis B surface antigen (HBsAg) interfere with the immunogenicity of the corresponding HBsAg or viral replication ability

Variants of hepatitis B surface antigen (HBsAg) influenced its antigenicity and immunogenicity. In our study, we aim to investigate biological significance of amino acid (aa) substitutions in HBsAg, Q129 N and T131 N/M133 T, for glycosylation, antigenicity and immunogenicity of variant HBsAg (vtHBsAg) and viral replication. Expression plasmids of vtHBsAg with aa substitutions Q129 L, T123 N, Q129 N and T131 N/M133 T were constructed. Immunofluorescence (IF) staining and Western blot were simultaneously utilized to examine expression of vtHBsAg proteins in Huh7 cells transfected with vtHBsAg constructs. vtHBsAg of Q129 N and T131 N/M133 T created new N-glycosylation and displayed perinuclear distribution by IF staining with the anti-HA. Antigenicity of vtHBsAg of Q129 N and T131 N/M133 T was reduced compared with wild type (wt) HBsAg. In addition, we discovered impaired ability to induce anti-HBs responses against wtHBsAg in mice immunized with plasmids pHBsAg- Q129 N and T131 N/M133 T. Even so, efficient protective response toward wild type HBV can be primed by the two vtHBsAgs in mice. Further, we discovered that vtHBsAg with Q129 N distinctly impaired HBV replication capacity, but vtHBsAg with T131 N/M133 T had no impact on viral replication. Thus, we conclude that vtHBsAg with Q129 N or T131 N/M133 T creates new N-glycosylation and interferes with both the antigenicity and immunogenicity of vtHBsAg. And vtHBsAg with Q129 N impaired HBV replication ability.