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【Objective】 To analyze the detection characteristics of a novel serum marker, hepatitis B core-associated antigen (HBcrAg), in the HBsAg-/HBV DNA+ blood donors in Wuxi. 【Methods】 A total of 37 previous HBsAg-/HBV DNA+ blood donors were followed up by telephone and their serum was obtained, and the serum of 22 HBsAg-/HBV DNA+ blood donors was detected by electrochemiluminescence and real-time PCR nucleic acid screening as the OBI group for HBcrAg enzyme-linked immunosorbent assay(ELISA). The serum of 20 healthy blood donors who underwent dual ELISA and one nucleic acid testing(NAT) was selected as the healthy control group, and the serum of 20 patients with chronic hepatitis B who were clinically diagnosed by Wuxi Fifth People's Hospital was selected as the experimental CHB group, and HBcrAg ELISA was detected respectively. The correlation analysis between HBcrAg and HBeAb, HBcAb, ALT and HBV DNA in the OBI group was performed. 【Results】 Thirty-seven blood samples were detected by chemiluminescence for HBsAg and NAT, and 22 HBsAg-/HBV DNA+ samples were detected in the OBI group, with a detection rate of 59.46%. The serum HBcrAg expression content (ng/mL) between the OBI group, the healthy control group and the CHB group were (0.92±0.13), (0.47±0.09) and (1.14±0.23), respectively, and the differences were statistically significant (P0.05). 【Conclusion】 The expression of HBcrAg in the OBI group and CHB group was higher than that in the healthy control group, and the serum HBcrAg was not correlated with HBeAb, HBcAb, ALT and HBV DNA to a certain extent. HBcrAg has a good application prospect in screening HBsAg-/HBV DNA+ blood donors.
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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.
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Humanos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Antivirales/uso terapéutico , Replicación Viral , ADN Circular/uso terapéutico , ADN Viral/genética , Hepatitis B/tratamiento farmacológicoRESUMEN
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.
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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.
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Humanos , Antivirales/uso terapéutico , ADN Circular/genética , ADN Viral , Semivida , Hepatitis B/tratamiento farmacológico , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Replicación ViralRESUMEN
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.
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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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Extrachromosomal DNA (ecDNA) is a small segment of circular DNA located outside the chromosome, which has the function of self-replication. Recently, amplification of oncogenes on ecDNA has been proved to be a common phenomenon in tumor cells, and has some characteristics worth studying, such as correlation with patients' poor prognosis. Multiple chromosomal events are involved in the formation of ecDNA, and its amplification can directly increase the number of DNA copies of extra-chromosomal oncogenes and accelerate the generation and development of tumors. Moreover, the segregation pattern of unequal transmission of parental ecDNA cells to offspring not only increases tumor heterogeneity, but also enhances tumor adaptation to environment and response to therapy. This article reviews the current status and potential significance of ecDNA in tumor cells. .
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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..
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Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5′-noncoding region (5′-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5′-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5′-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5′-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5′-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
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OBJECTIVE@#This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA.@*METHODS@#Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed.@*RESULTS@#The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation.@*CONCLUSIONS@#The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
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Humanos , Biología Computacional , Metilación de ADN , ADN Circular/genética , ADN Viral/genética , Genoma Humano , Genómica , Genotipo , Hepacivirus/genética , Hepatitis C/virología , Leucocitos Mononucleares/virología , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Alineación de SecuenciaRESUMEN
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.
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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.
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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.
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BACKGROUND Two novel viruses named circo-like virus-Brazil (CLV-BR) hs1 and hs2 were previously discovered in a Brazilian human fecal sample through metagenomics. CLV-BR hs1 and hs2 possess a small circular DNA genome encoding a replication initiator protein (Rep), and the two genomes exhibit 92% nucleotide identity with each other. Phylogenetic analysis based on the Rep protein showed that CLV-BRs do not cluster with circoviruses, nanoviruses, geminiviruses or cycloviruses. OBJECTIVE The aim of this study was to search for CLV-BR genomes in sewage and reclaimed water samples from the metropolitan area of São Paulo, Brazil, to verify whether the first detection of these viruses was an isolated finding. METHODS Sewage and reclaimed water samples collected concomitantly during the years 2005-2006 were purified and concentrated using methodologies designed for the study of viruses. A total of 177 treated reclaimed water samples were grouped into five pools, as were 177 treated raw sewage samples. Nucleic acid extraction, polymerase chain reaction (PCR) amplification and Sanger sequencing were then performed.e FINDINGS CLV-BR genomes were detected in two pools of sewage samples, p6 and p9. Approximately 28% and 51% of the CLV-BR genome was amplified from p6 and p9, respectively, including 76% of the Rep gene. The detected genomes are most likely related to CLV-BR hs1. Comparative analysis showed several synonymous substitutions within Rep-encoding sequences, suggesting purifying selection for this gene, as has been observed for other eukaryotic circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. MAIN CONCLUSION The results therefore indicated that CLV-BR has continued to circulate in Brazil two and three years after first being detected.
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Humanos , Aguas del Alcantarillado/virología , ADN Viral/genética , Reacción en Cadena de la Polimerasa , Circovirus/aislamiento & purificación , Circovirus/genética , Filogenia , Genoma Viral , Análisis de SecuenciaRESUMEN
The advent of oral antiviral agents has revolutionized hepatitis B treatment. It has led to decreased incidence and mortality related to hepatocellular carcinoma. However, although nucleos(t)ide analogs (NA) are fast and potent in inhibiting hepatitis B virus (HBV) polymerase and reverse transcriptase activity, complete cure of the virus is not possible. The complete eradication of HBV requires the covalently-closed-circular DNA (cccDNA) to be eliminated. Novel gene editing methods, such as zing finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, designed to target specific DNA sequences has great potential for therapeutic application. Among these, the CRISPR/Cas9 system may be the most feasible approach to eradicate HBV cccDNA. Further studies are needed to develop a more efficient and safer method of delivery using the CRISPR/Cas9 system to achieve complete cure of chronic hepatitis B.
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Antivirales , Secuencia de Bases , Carcinoma Hepatocelular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN , ADN Circular , Dedos , Virus de la Hepatitis B , Hepatitis B , Hepatitis B Crónica , Hepatitis , Incidencia , Métodos , Mortalidad , ADN Polimerasa Dirigida por ARNRESUMEN
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.
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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.
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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.
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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.