AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface...AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.展开更多
Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus （HBV） DNA replication, the covalently closed circular HBV DNA （cccDNA） can be completely cleared. To date, ...Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus （HBV） DNA replication, the covalently closed circular HBV DNA （cccDNA） can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction （PCR） assay for detecting HBV cccDNA in peripheral blood mononuclear cells （PBMCs） and bone marrow mononuclear cells （MMNCs）. Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA （rcDNA）, two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard. Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0×10^2 copies/ml to 3.9×10^8 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative. Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.展开更多
Approximately 350 million people are estimated to be persistently infected with hepatitis B virus(HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA(ccc DNA), a template for...Approximately 350 million people are estimated to be persistently infected with hepatitis B virus(HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA(ccc DNA), a template for all HBV RNAs. Chronic hepatitis B(CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit ccc DNA transcription and inhibit only a late stage in the HBV life cycle(the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating ccc DNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors(HNFs) play the most important roles in ccc DNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB.展开更多
基金Supported by The National Natural Scientifi c Foundation of China,No. 30070958The National Key Technologies Research and Development Program of China during the 11th Five-year Plan Period,No. 2008zx1002-006
文摘AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.
文摘Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus （HBV） DNA replication, the covalently closed circular HBV DNA （cccDNA） can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction （PCR） assay for detecting HBV cccDNA in peripheral blood mononuclear cells （PBMCs） and bone marrow mononuclear cells （MMNCs）. Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA （rcDNA）, two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard. Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0×10^2 copies/ml to 3.9×10^8 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative. Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.
文摘Approximately 350 million people are estimated to be persistently infected with hepatitis B virus(HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA(ccc DNA), a template for all HBV RNAs. Chronic hepatitis B(CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit ccc DNA transcription and inhibit only a late stage in the HBV life cycle(the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating ccc DNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors(HNFs) play the most important roles in ccc DNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB.
