A conceptual framework for dynamics of cccDNA in hepatitis B virus / 中华肝脏病杂志

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.

Advances in research on small molecule regulators targeting HBV cccDNA generation and transcription / 药学学报

Hepatitis B virus (HBV) represents a significant global public health challenge. Despite the availability of several approved drugs for hepatitis B treatment, the persistence of covalently closed circular DNA (cccDNA) renders HBV eradication elusive, thereby leading to disease relapse after drug withdrawal. This paper reviews the regulatory mechanisms of cccDNA formation, transcription and replication, and summarizes the research progress of related small molecule regulators from the perspective of medicinal chemistry.

A short half-life of cccDNA offer or ignite hope for hepatitis B cure under nucleos(t)ide analogues treatment / 中华肝脏病杂志

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the template for HBV replication. Currently, there is a lack of therapeutic drugs that directly target cccDNA. Therefore, blocking cccDNA supplements as fast as possible and reducing the existing cccDNA is the key to achieving a complete cure of chronic hepatitis B. Previous studies have suggested that cccDNA had a long half-life, but a recent study showed that it only took a few months to update cycle of cccDNA pool, and its number was much less than previously predicted. In the future, with the advent of new antiviral drugs that can completely inhibit HBV replication, it is expected that the cccDNA pool will be completely cleared due to its supplement complete blockade, so as to achieve virological cure of chronic hepatitis B.

Research advances in anti-hepatitis B virus therapy targeting covalently closed circular DNA / 临床肝胆病杂志

The incurable chronic infection caused by hepatitis B virus (HBV) is the major health burden worldwide. Covalently closed circular DNA (cccDNA) exists in the nucleus of infected cells as a stable minichromosome, and when a new therapy realizes inactivation or eliminates persistent cccDNA in infected hepatocytes, the natural process of chronic infection and long-term antiviral therapy will no longer exist. This article introduces the methods targeting cccDNA, such as gene editing and epigenetic modification, so as to achieve the complete cure of HBV infection.

A new method for simultaneous quantitative determination of hepatitis B virus pregenomic RNA and DNA / 第二军医大学学报

Objective To design and establish a new method for simultaneous determination of hepatitis B virus (HBV) pregenomic RNA (pgRNA) and DNA from nucleic acid extracts. Methods We established a duplex fluorescence quantitative PCR system to determine HBV pgRNA and DNA. DNA gel electrophoresis and quantitative PCR were used to test the specificity and sensitivity. We tested the feasibility and accuracy by determining the HBV pgRNA and DNA in HepG2.2.15 cells and the culture supernatants. Results The established duplex fluorescence quantitative PCR system has a good specificity and sensitivity. When it was used to determine cell culture supernatants with different dilution ratios, the dilution ratios and results were well correlated. However, this method was more suitable for the determination of HBV pgRNA and DNA in cell culture supernatants, rather than cell samples. Conclusion Our method can avoid inaccuracy of HBV RNA determination caused by HBV DNA contaminant in nucleic acid extracts, and realize simultaneous detection of HBV pgRNA and DNA in one PCR reaction, which greatly improves the determination efficiency and has potential clinical application value..

Research progress on molecular mechanism of hepatitis B virus reactivation after liver transplantation / 器官移植

Liver transplantation is the most effective method for hepatitis B-related liver failure, liver cirrhosis and hepatocellular carcinoma. However, the reactivation of hepatitis B virus (HBV) after liver transplantation is not conducive to the recovery of liver function and leads to poor clinical prognosis. The prevention and treatment of HBV reactivation is currently the focus of research by physicians and surgeons. The current viral suppression strategies can not completely eradicate HBV nor completely prevent the recurrence of HBV infection in the future. This article aims to explore the molecular mechanism of HBV reactivation after liver transplantation, in order to more effectively prevent the recurrence of hepatitis B after liver transplantation.

Effect of PBMC HBV cccDNA in HBsAg-positive mothers on neonatal Th1/Th2 cytokines / 中华疾病控制杂志

Objective To explore the effect of PBMC HBV cccDNA in HBsAg-positive mothers on neonatal Th1, Th2 cytokines and the ratio of Th1/Th2. Methods HBsAg-positive mothers and their neonates delivered in the Third People’s Hospital of Taiyuan between June 2011 and July 2013 were recruited. Questionnaires on general information were collected by an in-person interview. Electrochemiluminescence immunoassay (ECLIA) were utilized to detect HBV serological markers.HBV cccDNA in PBMC was detected with real-time PCR-TaqMan Probe method, Th1 cytokines (interleukin 2, interferon-γ and tumor necrosis factor-α) and Th2 cytokines (interleukin 4, interleukin 6 and interleukin 10) were detected with Procarta Plex Multiplex Immunoassays. Results Univariate analysis showed that the levels of IL-2, IL-6 and IL-10 in the positive group were significantly higher than those in the negative group, while the ratio of Th1/Th2 was lower than that in the negative group (P=0.034, P=0.007, P=0.048, P=0.029). The levels of IL-6 and IL-10 in neonates delivered by vagina were significantly higher than those by cesarean section, while the ratio of Th1/Th2 was lower than that by cesarean section (P<0.001). The level of IL-10 in positive group of neonatal HBsAg was significantly higher than that in negative group, while TNF-α and Th1/Th2 ratio were lower than negative group (P=0.011, P<0.001, P=0.027). The degree of Th2 predominant response was reflected by ratio of Th1/Th2. After adjusting potential confounding factors in non-conditional logistic regression analysis, compared to those born to mothers with PBMC HBV cccDNA negative, neonates whose mother with PBMC HBV cccDNA positive had an increased risk of having a strong Th2 predominant response (OR=2.42,95% CI:1.16-5.04, P=0.018). The risk of a strong Th2 predominant response in neonates delivered by vagina was 5.49 times higher than those by cesarean section (OR=5.06, 95% CI: 2.95-8.67, P<0.001). Conclusion HBsAg-positive mothers’ PBMC HBV replication and vaginal delivery may increase the risk of having a Th2 predominant response in neonates. It is suggested that we should pay attention to the effect of maternal PBMC HBV replication and the mode of delivery on neonatal Th1/Th2 cytokines.

Influence of interferon and nucleos(t)ide analogues on HBV cccDNA and functional cure of chronic hepatitis B / 临床肝胆病杂志

At present, interferon (IFN) and nucleos(t)ide analogues (NAs) remain the most important methods for the treatment of chronic hepatitis B in clinical practice, but neither of them can effectively eliminate the virus and cure hepatitis B. As the template for HBV transcription and replication, HBV covalently closed circular DNA (cccDNA) persistently exists in the nucleus in the form of minichromosome and is considered the most important reason for chronic and refractory HBV infection. Since it is hard to completely eliminate cccDNA, functional cure of chronic hepatitis B through sustained silencing of cccDNA has become a major goal of clinical and basic research in recent years. This article reviews the influence of current treatment methods on cccDNA, the factors regulating the amount and activity of cccDNA, and the key obstacles to eradication of cccDNA pool, with perspectives of cccDNA research towards a functional cure of chronic hepatitis B.

Detection of HBV-cccDNA level in children with hepatitis B virus associated glomerulonephritis and its clinical significance / 吉林大学学报(医学版)

Objective: To analyze the effects of serum hepatitis B virus covalently closed circular DNA (HBV-cccDNA) level on the liver and kidney functions, detection of HBV antigens in kidney tissue, pathological grading and staging of liver tissue in the children with hepatitis B virus associated glomerulonephritis (HBV-GN), and to evaluate the clinical value of HBV-cccDNA level detection in the diagnosis of the children with HBV-GN. Methods: A total of 39 HBV-GN children (observation group) were selected and all of them underwent the liver and kidney biopsy. A total of 40 HBV-carried children with normal liver function were selected as control group. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the children were detected. Molecular beacons PCR method was used to detect the serum HBV-cccDNA level of the children. The morphology of liver and kidney tissues of the children was detected with HE staining. The detection rates of HBsAg, HBeAg and HBcAg in kidney tissue of the children were detected by immunofluorescence. Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate the value of HBV-cccDNA level detection in the diagnosis of HBV-GN. The Ax fluorescence value of HBV-cccDNA > 21 was determined as positive, and the Ax fluorescence value of HBV-cc cDNA 0. 05). Membranous nephropathy (MN) was the main pathological type of kidney biopsy in the HBV-GN. HBeAg and HBcAg were the main components of HBV antigens. The detection rates of HBeAg and HBcAg of the children in HBV-cccDNA positive group were significantly higher than those in HBV-cccDNA negative group (χ2 =5. 652, P = 0. 027; =12. 523, P=0. 001). The ROC curve analysis results showed that the HBV-cccDNA level detection could effectively distinguish the HBV-GN children in observation group from those in control group, AUC=0. 804 (95% Cl 0. 709-0. 883). The levels of HBV-cccDNA of 10 cases of HBV-GN children underwent standardized treatment with complete follow-up treatment data were decreased significantly at the 2nd week after treatment. At the 12th week after treatment, in 7 cases the HBeAg turned negative, without proteinuria and hematuria symptoms, and the AST and ALT levels were normal. The HBV-cccDNA levels of 3 cases with ineffective treatment were higher than that those of the remaining 7 cases. Conclusion; The high expression of HBV-cccDNA is closely related to the liver function, proteinuria and HBV antigen detection in kindney tissue of the HBV-GN children. The detection of HBV-cccDNA level has potential clinical value for the auxiliaty diagnosis and evaluation on the curative effect of the HBV-GN.

Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir / 解放军医学杂志

Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.

The inhibition effect of GLS4JHS on the transcription activity of covalently closed circular DNA in HepAD38 cells / 中华传染病杂志

Objective The hepatitis B virus (HBV) core protein assembly inhibitors GLS4JHS could destroy HBV capsid assembly and the formation of non-capsid polymer structure.The aim of this study is to explore the mechanisms of GLS4JHS in inhibiting HBV replication.Methods HepAD38 cells was used as the study model.TaqMan real-time polymerase chain reaction (PCR) and quantitative real-time PCR with specific primers were used to measure the change in pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) levels under different concentrations.ChIP assay in HepAD38 cells was used to assess the recruitment of HBV core protein and histone modifications.Results The amount of cccDNA and pgRNA decreased with the increasing GLS4JHS concentrations.After the drug concentrations reached 400 nmol/L, cccDNA and pgRNA declined by 94% and 84%, respectively.Both HBV core protein occupancy on the cccDNA and cccDNA-bound H3 histone acetylation were reduced by GLS4JHS.Conclusions GLS4JHS decreases transcriptional activity of cccDNA and reduces pgRNA production by inhibiting cccDNA minichromosome bound to HBV core protein and acetylated histone H3, which results in HBV DNA formation.

Application of CRISPR/Cas9 technology in hepatitis B virus clearance / 医学研究生学报

Chronic hepatitis B virus (HBV) infections remain a major public health problem worldwide, which puts human at high risks of liver cancer and liver cirrhosis.The stable nuclear covalently closed circular DNA (cccDNA) is an important step of the HBV replication cycle, as well as the key of drastically destroy the HBV.However, α-interferons and oral nucleo(s) tide analogs cannot target and destroy the cccDNA.Nowadays, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology can specifically target the HBV cccDNA, which brings hope to conquer HBV.Researches of CRISPR/Cas9 technology applied to clear HBV have been reviewed in this paper.

Comparison of two Taq-man Real-time PCR methods for detection of HBV cccDNA / 中华实验和临床病毒学杂志

Objective@#To compare two Taq-man Real-time PCR methods for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in serum or liver tissue.@*Methods@#Two sets of primers and probes (common Taq-Man probe and MGB Taq-Man probe) were synthesized according to the reference papers, and the sensitivity and specificity of the two methods were compared using prepared plasmid as standard curve, and HBV DNA samples were exlracted from serum and liver tissue samples of hepatitis B patients. The samples were tested with both methods separately before or after the digestion with a Plasmid-Safe ATP-dependent Dnase (PSAD).@*Results@#Both of these two kinds of detection methods had a good linear relationship with the prepared plasmid as standard curve (R2 0.989 or 0.976 respectively, CV were within 4% ), and obtained good specificity when the HBV DNA samples were tested before or after digestion with PSAD. The common Taq-Man probe had lower Ct value than MGB probe when the samples in the same concentration.@*Conclusions@#Both methods can be used for HBV cccDNA detection. The common Taq-Man probe has slightly higher sensitivity than MGB probe, while the MGB probe has lower background than the common Taq-Man probe in our test. One can select the appropriate probe according to the need.

High-dose tenofovir is not effective in suppressing hepatitis B virus replication in patients with hepatocellular carcinoma progression: a preliminary result

BACKGROUNDS/AIMS: Nucleos(t)ide analogues (NUCs) effectively suppress hepatitis B virus (HBV) replication, but hepatocellular carcinoma (HCC) recurrence often leads to HBV replication despite NUC therapy. The aim of this study was to determine whether high-dose tenofovir (TNF) therapy can suppresses HCC recurrence-associated HBV replication. METHODS: We performed a single-arm prospective study to assess the clinical feasibility of high-dose TNF (hdTNF). We recruited 10 patients during September 2015 and followed up for 3 months or early drop-out. RESULTS: All 10 patients had HCC of advanced stages due to HCC recurrence and gradual progression. The average age of patients was 51.2+/-4.7 years and 9 were male. Three patients did not tolerate the increased TNF dosage and were dropped out early. The other 7 patients were relatively tolerable to the increased dosage of TNF 5 tablets per day. One patient had mild gastrointestinal symptoms and another patient complained of insomnia. Increased HBV replication and HCC progression was observed despite hdTNF for 4-8 weeks. All 7 patients showed tumor progression during the 3 month follow-up. In these patients, blood HBV DNA before hdTNF was 50-200 copies/ml; and 4-8 weeks after hdTNF, the HBV replication status was not improved with blood HBV DNA of 50-300 copies/ml. This clinical study was terminated early after these negative results were confirmed. CONCLUSIONS: The results of this study indicated that high dose of TNF up to 5-fold the recommended dosage is not tolerated by a considerable proportion of patients and also ineffective in suppressing HCC progression-associated HBV replication.

Detection of HBVDNA and HBVcccDNA in Peripheral Blood Mononuclear Cells by a Multiplex Polymerase Chain Reaction / 中国医师杂志

Objective To establish a multiplex polymerase chain reaction (M-PCR) assay to detect HBV DNA in the peripheral blood mononuclear cells(PBMCs) of chronic HB patients. Methods One pair of primer amplifying HBV genome DNA and another pair of primer amplifying HBV covalently closed circular DNA (cccDNA ) were added to one PCR reaction to detect HBV DNA in PBMCs. Results Various forms of HBVDNA including total DNA and cccDNA could be amplified simultaneously. Among the 30 chronic HB patients, both the HBVDNA and HBVcccDNA in the PBMCs of 23 patients were detected, the positive rate of which was 76.6%. The positive rate of HBV cccDNA accounted for 82.1% of total HBV DNA positive rate. Conclusion HBVDNA in the PBMCs could partially replicate. The M-PCR was successfully set up to amplify HBV genome DNA and HBV cccDNA simultaneously.