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Objective:To evaluate the risk of HBV recurrence after liver transplantation in patients with end-stage hepatitis B related liver disease, and to explore the indications for antiviral therapy withdrawal.Methods:The data of HBV DNA, cccDNA in liver puncture tissues and peripheral blood in 31 patients after liver transplantation was retrospectively analyzed.Results:Among the 31 patients, 15 (48%) had detectable and quantified HBV DNA in liver biopsy tissue, while their HBV related serological indicators were negative, suggesting an occult HBV infection in some patients. The study found 15 out of 19 cases who were taking Entecavir were cccDNA negative (78.9%), compared to 5 out of 12 cases (41.6%) in Lamivudine regiment ( P=0.03). Conclusions:Hidden HBV infection can be detected by amplifying cccDNA and HBV DNA in liver puncture tissue by using ddPCR. Entecavir is superior to lamivudine in the clearance of cccDNA.
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Chronic hepatitis B virus (HBV) infection is a primary cause for liver cancer. And the main challenge of curing hepatitis B is the elimination of the stable covalently closed circular DNA (cccDNA) of the viral genome. The formation of HBV cccDNA requires the filling of single-stranded region and the ligation of nicks in relaxed circular DNA (rcDNA) strands. Previously, our group reported that proliferating cell nuclear antigen (PCNA) was involved in the formation of HBV cccDNA. However, the underlying mechanism of the conversion of HBV rcDNA to cccDNA is poorly understood. In the present study, we aim to explore the mechanism by which PCNA contributes to the conversion of HBV rcDNA to cccDNA. Our data showed that PCNA was involved in the process of HBV rcDNA repair. The knockout of PCNA by the CRISPR/Cas9 system remarkably blocked the conversion of HBV rcDNA to cccDNA, while the ectopic expression of PCNA could effectively rescue the event (P<0. 001). Knockout of PCNA significantly slowed down the conversion kinetics of HBV rcDNA to cccDNA (P<0. 01). Mechanically, the DNA binding domain of PCNA was required for the process of HBV rcDNA repair to cccDNA (P<0. 01). Thus, we conclude that PCNA confers the conversion of HBV rcDNA to cccDNA by its DNA binding domain. Clinically, PCNA might serve as a novel target for antiviral therapy.
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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.
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Chronic hepatitis B virus (HBV) infection remains a major public health threat worldwide. Current antiviral drugs can effectively inhibit the replication of HBV, but they fail to achieve a complete cure. As the original template for HBV replication, covalently closed circular DNA (cccDNA) is intrinsically stable in vivo and is regarded as the molecular basis for persistent HBV infection and the key target for the cure of HBV infection. Studies on biosynthesis, metabolism, and regulation of HBV cccDNA have been limited by the low copy number of cccDNA within cells and the lack of sensitive and reliable detection methods. Establishment of appropriate research models of cccDNA helps to understand related biological processes. This article reviews the latest research advances in cell models and mouse models of HBV cccDNA, in order to facilitate future studies on HBV virology and development of antiviral drugs against HBV.
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Elimination of HBV cccDNA from hepatocytes infected with chronic HBV virus is considered to be the key to eradicating HBV. Monitoring HBV cccDNA before, during, and after viral treatment is essential for routine treatment of patients with chronic hepatitis B. With the introduction of new anti-HBV treatment technologies and new drugs targeting HBV cccDNA, Accurate and sensitive HBV cccDNA assays are urgently needed to evaluate efficacy. In recent years, HBV cccDNA detection methods have achieved gratifying results in both traditional PCR methods and digital PCR methods popular in recent years. In this paper, the advances in HBV cccDNA quantitative detection by qPCR, Magnetic bead capture hybridization, rolling circle amplification combined with in situ PCR, digital PCR and digital PCR assay in single cells were reviewed.
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In situ hybridization (ISH) is a new technique which combines molecular biology, histochemistry, and cytology. It can quantify and locate specific nucleic acids at the cellular and chromosomal levels and is widely used in virological research. ISH is of great significance for the detection of hepatitis B virus (HBV) nucleic acids (RNA and replicative intermediate DNA) and covalently closed circular DNA. This article reviews the development of ISH and its application in HBV research.
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Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is stably maintained in hepatocytes in the form of minichromosome and is considered the most important cause of chronicity of HBV infection, presence of HBV after antiviral therapy, and recurrence of hepatitis after drug withdrawal. However, due to a lack of antiviral regimens targeting cccDNA itself or the formation and transcription of cccDNA, there is an urgent need for treatment strategies targeting cccDNA. With the gradual understanding of epigenetic modification of histones of the cccDNA minichromosome, epigenetic therapy is expected to become a potential therapy for HBV. This article reviews the current status and future directions of HBV DNA methylation and cccDNA-bound histone modification, in order to provide new thoughts for epigenetic therapy for HBV.
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Hepatitis B virus (HBV) infection is the main cause of chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is the template for HBV replication and exists stably in hepatocytes, which is the main factor for persistent infection. However, existing antiviral drugs including nucleos(t)ide analogues and interferons do not directly target cccDNA, and thus it is difficult to achieve the clinical cure of CHB. With the progress in biotechnology and in-depth understanding of the features of HBV cccDNA, more and more studies have been conducted on antiviral therapies targeting cccDNA, but there is still a long way before clinical application. This article briefly reviews related research advances.
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Current antiviral treatment regimens seldom clear hepatitis B virus (HBV) infection and achieve complete cure, and therefore, many patients need long-term or even life-long antiviral therapy with nucleos(t)ide analogues. The root cause is the presence of covalently closed circular DNA (cccDNA) with transcriptional activity within hepatocytes. In order to help readers understand the research advances in HBV cccDNA, this issue invites leading experts in China to introduce the research advances from the following six aspects: anti-HBV treatment and functional cure of chronic hepatitis B, drugs and biotechniques targeting HBV cccDNA, transcriptional regulation of HBV cccDNA and prospects of anti-HBV treatment, in vitro cell models and experimental mouse models of HBV cccDNA, application of in situ hybridization in detection of HBV nucleic acid and cccDNA, and quantification of HBV cccDNA.
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Chronic hepatitis B (CHB) is a worldwide public health problem and current therapeutic drugs cannot completely eliminate the covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in the liver cell nucleus. The presence of cccDNA leads to the persistent HBV infection, which is hard to be completely eliminated. At present, the research on cccDNA is getting increasing attention. Only by comprehensively understanding it can we find a better way to cure HBV infection. This article reviewed the structure, regulation, detection and elimination of HBV cccDNA.
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Functional cure of chronic hepatitis B ( CHB) through antiviral therapy is the goal and main focus of research worldwide.The criteria of functional cure of CHB are undetectable serum HBV DNA, persistent loss of HBsAg, with or without anti-HBs seroconversion, persistence of cccDNA in a transcriptionally inactive status and the absence of spontaneous relapse after treatment cessation .Current antiviral therapy can effectively control viral replication , but is still quite far from achieving functional cure. From the virological point of view , HBV cccDNA reservoir and its transcription activity can hardly be inhibited by the existing antiviral therapy , and the integration of viral genomic DNA leads to the continuous expression of HBsAg, which are the two main causes of the low functional cure rate in CHB patients. Furthermore, the high frequency of viral mutation can also result in a reduction of HBsAg loss .This article reviews the new strategies and advances about functional cure of CHB based on the virology research .
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Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in infected hepatocytes is the main cause of off-therapy viral rebound. The half-life of cccDNA is only 33-50 days, so the conversion of newly synthesized rcDNA to cccDNA in the nucleus is essential for the maintenance of cccDNA pool in infected hepatocytes. Though not directly targeting the existing cccDNA, current nucleos(t)ide analogues (NAs) may exhaust the cccDNA reservoir by blocking the rcDNA formation. Indeed, a prolonged consolidation therapy post loss of serum HBV DNA can achieve sustained remission and thus safe drug discontinuation in a small proportion of chronic hepatitis B (CHB) patients. In recent studies, we and others have demonstrated that it is the serum HBV RNA that reflects the cccDNA activity in infected hepatocytes, particularly among the patients on NAs. Here we suggest that instead of measuring serum HBV DNA only, simultaneous measurement of both viral DNA and RNA would improve the accuracy to reflect the cccDNA activity; therefore, the virological response should be redefined as consistent loss (less than the lower limit of detection) of both serum HBV DNA and RNA, which indicates the safety of drug discontinuation. Accumulating evidence has suggested that for the CHB patients with lower serum HBsAg, switch-to or add-on pegylated interferon (Peg-IFN) treatment would result in loss of serum HBsAg in a relatively large proportion of CHB patients. Since serum HBV RNA is an ideal biomarker to reflect the intrahepatic cccDNA activity, for the patients with a serum HBsAg level lower than 1 500 IU/ml after long-term NAs treatment, the serum HBV RNA should be measured. If serum HBV RNA is detected, peg-IFN should be added on; if serum HBV RNA is not detected, NAs treatment should be switched to peg-IFN treatment. We believe the therapy based on serum HBV RNA would make the functional cure of CHB (serum HBsAg loss or even conversion to anti-HBs) more efficient.
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Treatment of chronic hepatitis B virus (HBV) infection with the viral DNA polymerase inhibitors or pegylated alpha-interferon has led to a significant retardation in HBV-related disease progression and reduction in mortality related to chronic hepatitis B associated liver decompensation and hepatocellular carcinoma. However, chronic HBV infection remains not cured. The reasons for the failure to eradicate HBV infection by long-term antiviral therapy are not completely understood. However, clinical studies suggest that the intrinsic stability of the nuclear form of viral genome, the covalently closed circular (ccc) DNA, sustained low level viral replication under antiviral therapy and homeostatic proliferation of hepatocytes are the critical virological and pathophysiological factors that affect the persistence and therapeutic outcomes of HBV infection. More importantly, despite potent suppression of HBV replication in livers of the treated patients, the dysfunction of HBV-specific antiviral immunity persists. The inability of the immune system to recognize cells harboring HBV infection and to cure or eliminate cells actively producing virus is the biggest challenge to finding a cure. Unraveling the complex virus-host interactions that lead to persistent infection should facilitate the rational design of antivirals and immunotherapeutics to cure chronic HBV infection.
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Objective:To establish a new fluorescence-based quantitative PCR assay for detecting hepatitis B virus covalently closed circular DNA(HBV cccDNA).To explore the effect of artificial liver support system(ALSS)on total HBV DNA and HBV cccDNA in sera of patients with fulminant hepatitis B.Methods:The serum specimens from 50 patients of fulminant hepatitis B prior to and after treatment with ALSS were collected.Then HBV cccDNA was quantitatively detected by fluorescent PCR with specific primer set and Taqman probe.And the results were analyzed by SAS8.0 statistics software.Results:We successfully established a new fluorescence-based quantitative PCR method for HBV cccDNA.HBV cccDNA was detectable in sera of the patients with fulminant hepatitis B.The level of total HBV DNA and HBV cccDNA decreased significantly following plasma exchange in ALSS.The levels of total HBV DNA and HBV cccDNA had positive correlation with degree of PT,ALT,andAST.Total HBV DNA also had positive correlation with HBV cccDNA.Conclusion:The level of total HBV DNA and HBV cccDNA has positive correlation with degree of PT,ALT,and AST,which might be indication of hepatocyte damage.The finding that significant decrease in total HBV DNA and HBV cccDNA after plasma exchange in ALSS may provide a clue of further research about the fulminant hepatitis B treated with ALSS.
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Objective Assaying HBV cccDNA in periphral blood lymphocyte and discuss its clinical significance.Methods Using lymphocyte isolating solution to isolate lymphocytes in the peripheral blood, and quantitative florescent realtime PCR was used to detect HBV cccDNA.Results 29 cases of 36 CAH patients was detected positive(80.56%),32 cases of 61 CPH patients was detected positive(52.46%) and 1 case of 59 ASC was detected positive(0.02%) respectively.All 47 healthy people specimen was found negative by the method.CAH group,CPH group and ASC & negative control group show significance statistically(P
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Aim To observe the anti-hepatitis B virus effect of total phenolics acid extracted from Oenanthe Javanica(OJTPA) with in vitro experiments.Methods HePG2 2.2.15 cells were treated with OJTPA,which were diluted into 500 mg?L-1,250 mg?L-1 and 125 mg?L-1 groups.The test set up positive control group(entecavir,3 mg?L-1) and normal control group.The cells of the 4 th,8 th and 4 th day ceasing treatment were collected.The content of HBV-DNA and cccDNA was determined with Quantitative Real-Time PCR method,and the inhibition rates were calculated.Results OJTPA could obviously inhibit HBV-DNA and cccDNA under the dosages used in the experiment,and showed a dose-dependent relationship.The inbhibitory rates reached the peak of 62.3% and 62.7% for HBV-DNA and cccDNA respectively,and a high inhibitory rate was retained within 3 days of OJTPA withdrawal.Conclusion OJTPA has significant inhibition on HBV-DNA and cccDNA and there is no significant rebound phenomenon.
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Objective To identify the association strength of the prevalence of HBeAg, cccDNA with the occurrence of HBV related hepatocellular carcinoma (HCC) in high risk male cohort in Qidong area in China. Methods A cohort of 377 middle aged HBV infected men in Qidong was followed from 1989 for 13. 25 years. HCC cases were registered. A matched case-controlled study was conducted on 32 pairs of inherent HCC cases with non-HCC controls. Serum HBeAg was measured by ELISA. cccDNA was detected by semi-nested PCR and verified by DNA sequencing. Standard statistical comparison between the prevalence of each HBV marker in HCC versus control group provided the odds ratio and P-value was used to evaluate its association strength with HCC occurrence. Results Serum HBeAg prevalence was 53. 1% (17/32) in HCC group versus 15. 6% (5/32) in controls, odds ratio (OR) =6. 12, P