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Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication., (© 2011 Blackwell Publishing Ltd.)

Background/aims: Hepatitis C virus infection is a major worldwide health problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be important. With the aim of inhibiting NS3 protease activity as a means to inhibit HCV replication we used a novel bacterial genetic screen to isolate NS3-inhibiting peptide aptamers., Methods: We have isolated and characterized seven NS3-inhibiting peptide aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic RNA replicons undergo upon intracellular expression of these peptide aptamers, assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected replicon cells., Results and Conclusions: The peptide aptamers inhibited NS3 protease activity in vitro with an IC50 in the low micromolar range. Upon transfection, aptamers inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA replicons. Aptamer-based intracellular immunization may emerge as a promising antiviral approach to interfere with the life cycle and pathogenicity of HCV.

The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence with unknown biological function. In this study we sought to characterize the prevalence of specific anti-F antibodies in patients with chronic HCV infection and to analyze the anti-F antibody profile before, during, and after antiviral treatment in order to gain a better understanding of the role of F protein in HCV pathogenesis. Serum samples were collected from 44 patients with chronic HCV infection and from 19 healthy controls. Consecutive samples from 27 patients taken before, during, and after treatment with antiviral therapy. The F and the core proteins were cloned from the HCV genome. The recombinant proteins were expressed in Escherichia coli and affinity purified. A sensitive and specific enzyme-linked immunosorbent assay was developed to assess the prevalence of anti-F antibodies. Eighty-nine percent of chronic HCV patients had evidence of anti-F antibodies, and 95% of them had anti-core antibodies. No correlation of anti-F antibodies was found with response to treatment, genotype, or seroconversion. We conclude that the F protein elicits specific antibodies in most individuals chronically infected with HCV with no correlation with response to treatment. Our results confirm the expression of F protein during natural HCV infection.

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.

Background: Mycophenolate mofetil is used as an immunosuppressive agent in liver transplant recipients. Its active compound, mycophenolic acid, also inhibits the replication of Epstein-Barr virus and human immunodeficiency virus. Based on a study indicating the effectiveness of mycophenolate mofetil on hepatitis B virus (HBV) replication in infected human hepatocyte cells in culture, we examined the efficacy of mycophenolate mofetil in suppressing HBV replication in lamivudine-resistant liver allograft recipients with recurrent HBV infection., Method: The study population included four liver allograft recipients (three males, one female), median age 51 years (range 41-57 years), with recurrent HBV infection who proved to be resistant to lamivudine. All received standard maintenance immunosuppression therapy. Median pretreatment serum alanine aminotransferase level was 75 mu/L (range 39-182 mu/L) and HBV DNA level (quantitative dot blot), 70 pg/ml (range: 10-5,000 pg/ml). Mycophenolate mofetil, 1.0 g p.o. twice daily, was administered for 8 weeks, concomitant with a reduction in the maintenance corticosteroid and cyclosporine doses., Results: After mycophenolate mofetil was administered, the serum alanine aminotransferase level increased in two patients, did not change in one, and decreased in one. Serum HBV DNA levels increased in three patients and decreased (nonsignficantly) in only one patient. Two patients complained of abdominal pain and nausea., Conclusions: Mycophenolate mofetil at the dosage used is not effective in suppressing HBV replication after liver transplantation.

Objective: Hepatitis B virus (HBV) precore mutant infection is associated with a more severe liver disease and a poorer response to interferon. We evaluated the efficacy and tolerance of lamivudine to induce complete and sustained suppression of viral replication in seven patients infected with HBV precore mutant (HBeAg-/HBeAb+/HBV DNA+) (in three patients mutation at codon 1896 was detected by direct sequencing)., Methods: Of the seven patients, five had decompensated HBV cirrhosis in a replicative phase and were liver transplant candidates (Group A) and two patients underwent orthotopic liver transplantation (OLT) for HBV liver cirrhosis and developed recurrent HBV infection in the grafted liver (Group B). Lamivudine 100 mg daily was administered orally for a period of 6-75 wk., Results: After 6-8 wk lamivudine therapy was well tolerated and successfully suppressed HBV replication to an undetectable serum level of HBV DNA by polymerase chain reaction in six patients. In Group A, two patients underwent successful OLT with no evidence of HBV reinfection 2-14 months later. Lamivudine was continued after OLT with no episodes of rejection. Three patients died before a suitable liver could be found (one remained serum HBV DNA+ after 6 wk of lamivudine therapy). In Group B, 9-14 months after lamivudine therapy both patients developed lamivudine resistance (increased liver enzymes, reappearance of serum HBsAg and HBV DNA [by hybridization]). In both patients liver histology had progressed and in both, mutation at codon 552 of the HBV polymerase gene was detected., Conclusions: Lamivudine is well tolerated in patients with decompensated liver cirrhosis due to HBV precore mutant infection who are liver transplant candidates. In four patients (80%) potent suppression of viral replication was detected, allowing OLT to be performed. However, post-OLT, a resistant mutant developed under lamivudine therapy. Combination therapy with other antiviral agents should be evaluated to discourage the emergence of lamivudine-resistant mutants.