Antiviral activities of ISG20 against hepatitis C virus / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the impact of interferon-stimulated exonuclease 20 kDa (ISG20) on replication of genotype 2a hepatitis C virus (HCV) subgenomic replicon RNA and infectivity of the cell culture-derived HCV strain JFH1 to determine the potential of exogenously expressed ISG20 as an anti-viral therapy of chronic hepatitis C.</p><p><b>METHODS</b>Plasma vectors containing wild-type (WT) ISG20 or a catalytically-inactive mutant ISG20m were transiently transfected into Huh7, Huh7.5 and HEK293 cells, and the replication of a monocistronic subgenomic JFH1 RNA replicon, SGRm-JFH1BlaRL, was measured. Huh7.5 cells stably expressing ISG20, ISG20m, or the control vector were established by transducing replication incompetent pCX4-Bsr-myc retroviruses encoding WT ISG20, D94G mutant ISG20, or the empty vector, respectively, and selecting with 5 mug/mL of blasticidin for approximately three weeks. The stable Huh7.5 cells were then transfected with HCV replicon RNA and infected with cell culture-derived HCV to investigate inhibition capacity of ISG20 against HCV.</p><p><b>RESULTS</b>Huh7.5-ISG20, Huh7.5-ISG20m, and Huh7.5-Bsr controls cells stably expressing ISG20, ISG20m, or the control vector, respectively, were constructed successfully; the ectopically expressed ISG20 and ISG20m were distributed in both nucleus and cytoplasm, as detected by immuno uorescence. SGRm-JFH1BlaRL replicated efficiently and with similar kinetics in the Huh7.5-Bsr and Huh7.5-ISG20m cells, with expression levels plateauing at 48-96 h post-transfection. In contrast, at all time points examined, SGRm-JFH1BlaRL replication was 9.1% to 16.7% in the Huh7.5-ISG20 cells. The Huh7, Huh7.5 and HEK293 cells transiently expressing ISG20 also showed 16.7% to 25.0% of HCV replication that the respective controls. In addition, the amount of infectious progeny JFH1 virus released in culture supernatants was 9.1% to 12.5% from the Huh7.5-ISG20 cells than from the Huh7.5-Bsr and Huh7.5-ISG20m cells at 48-72 h post-infection, and the latter two cultures produced similar JFH1 virus yields. Finally, the expression of HCV core protein was also lower in the Huh7.5-ISG20 cells, as detected by immunoblot analysis.</p><p><b>CONCLUSION</b>Exogenous expression of ISG20, either in a transient or stable manner, suppresses not only replication of genotype 2a HCV RNA replicons but also JFH1 virus propagation in cultured hepatocytes. The exonuclease activity of ISG20 is required for its antiviral activities against HCV.</p>

Construction and functional characterization of a monocistronic replicon based on the HCV genotype 2a promotor / 中华肝脏病杂志

To construct a hepatitis C virus (HCV) genotype 2a monocistronic replicon and investigate its replication capabilities in the human hepatocarcinoma cell lines, Huh7.5 and Huh7.1, in order to determine its potential as a molecular tool for future in vitro studies of HCV replication and selection studies for putative anti-HCV drugs. Site-directed mutagenesis was used to delete the Core-E1-E2-p7-NS2 fragment (about 3090 bp) from plasmid pJ6JFH1BlaRL. The resultant trianglepJ6JFH1BlaRL plasmid was digested with AgeI and AvrII to release the cDNA fragment (hereafter, referred to as fragment L) containing partial 5'-untranslated region (UTR), the first 12 amino acid (aa) of HCV Core coding sequence, full-length coding sequences for the blasticidin-resistance gene, Renilla luciferase, foot-and-mouth disease virus (FMDV) 2a antiprotease and ubiquitin, and partial coding sequence for HCV NS3. To generate the monocistronic replicon, pSGRmJFH1BlaRL, fragment L was ligated into the pSGR-JFH1 vector that had been digested with AgeI and AvrII to remove the partial 5'-UTR, the first 19 aa of HCV Core coding sequence, the full-length coding sequence for the neomycin phosphotransferase II gene, the internal ribosomal entry site from encephalomyocarditis virus, and partial HCV NS3 coding sequence. A replication-defective mutant replicon, pSGRmJFH1BlaRL/GND, was constructed by a similar procedure using the pSGR-JFH1/GND vector. Fragment L was confirmed in both constructs by sequencing. Replicon RNAs were prepared from XbaI-linearized plasmid DNA templates with Invitrogen's T7 MEGAscript kit, and were purified by DNase I treatment and LiCl precipitation. RNAs were quanti?ed by optical density, and the quality and concentration were con?rmed by agarose gel electrophoresis. Replicon RNAs were transfected into Huh7.5 and Huh7.1 cells using Invitrogen's DMRIE-C transfection reagent at a ratio of 5 mug of lipid to 1mug of RNA. Time course assay of Renilla luciferase activity indicated the replicon's replication function. The pSGRmJFH1BlaRL monocistronic replicon and pSGRmJFH1BlaRL/GND replication-defective mutant replicon were successfully constructed. The pSGRmJFH1BlaRL replicon was replication-proficient in Huh7.5 and Huh7.1 cells, with replication peaking at 72 hours post-transfection and decreasing after 96 hours. No replication was detected at any time point post-transfection for the defective mutant replicon. A monocistronic replicon of HCV genotype 2a was constructed and shown to be replication-proficient in human hepatocarcinoma cell lines.

Comparison of antiviral responses to adefovir dipivoxil therapy of genotype B and genotype C HBV infected patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of HBV genotypes on their response to adefovir dipivoxil (ADV) antiviral therapy.</p><p><b>METHODS</b>HBV genotypes from 177 HBeAg-positive chronic hepatitis B (CHB) patients were identified and the patients were treated with ADV 10 mg per day for 48 weeks. The clinical data in terms of serum HBV DNA seroclearance, mean HBV DNA reduction (log value), HBeAg loss, anti-HBe seroconversion and serum ALT of those patients were analyzed against their HBV genotypes.</p><p><b>RESULTS</b>Genotype B and genotype C were found in 102 and 65 cases, respectively. The mean HBV DNA reduction in patients with genotype B and genotype C at their treatment times of 12, 24 and 48 weeks was 2.2 log10copies/ml, 2.1 log10copies/ml (P more than 0.05), 2.7 log10copies/ml, 2.4 log10copies/ml (P more than 0.05) and 3.6 log10copies/ml, 3.1 log10copies/ml (P less than 0.05), respectively. At the end of the therapy (48 weeks), 43 (42.2%) patients with genotype B HBV infection and 22 (33.8%) patients with genotype C HBV infection had achieved HBV DNA seroclearance (P less than 0.05).</p><p><b>CONCLUSIONS</b>Our results suggest that genotype B HBV has a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than that of genotype C. Longer terms of ADV treatment are needed to confirm this conclusion.</p>