Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication-inducible cell line

AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregenomic RNA(pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction(PCR)assay.The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay.HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.RESULTS:When HepG2.117 cells were grown in the presence of EGCG,the expression of HBeAg was suppressed,however,the expression of HBsAg was not affected.HBV precore mRNA level was also downregulated by EGCG,while the transcription of precore mRNA was not impaired.The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent,however,HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment,indicating that EGCG targets only replicative intermediates of DNA synthesis.CONCLUSION:In HepG2.117 cells,EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

Extrahepatic and Intrahepatic Replication and Expression of Hepatitis C Virus

In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minusstrand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.

Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

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.