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Objective To evaluate the association of genetic variation in interleukin 28 B ( IL-28B) gene and viral factors with treatment outcome in chronic hepatitis C virus (HCV)-infected patients in Pakistan. Methods Patients with chronic HCV infection who received interferon plus ribavirin combination therapy were enrolled in the study. The viral loads were checked at the third and sixth month of treatment and 6 months after the completion of the treatment. Genotyping of single-nucleotide polymorphism ( SNP) rs12979860 of IL-28B gene was performed by the restriction fragment length polymorphism method and direct sequence analysis. Results A total of 111 patients successfully completed antiviral therapy and the follow-up period. Early virological response ( EVR) and a sustained virological response ( SVR) were achieved in 92 and 91 patients, respectively. Patients infected with HCV genotype 3a had a higher SVR rate compared with other HCV genotypes. A higher C allele frequency (81.9%) was observed in patients with SVR than those with non- SVR ( P = 0.00001). The odds ratio of achieving SVR was more than 10-fold greater for the CC genotype than the CT/ TT genotypes of SNP rs12979860 ( OR 10.351, 95% CI 1.925-55.653, P = 0.006). Other positive predictors observed for SVR were female gender and EVR. Conclusion The CC genotype of IL-28B SNP rs12979860 is an independent predictive factor for SVR in chronic HCV-infected patients in Pakistan. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which progresses to hepatocellular carcinoma (HCC) afflicting > 170 million people worldwide. HCV 3a is the most common genotype (about 70% of all genotypes) circulating in Pakistan. Expression of HCV individual gene of 3a would facilitate therapeutic and vaccines strategies against chronic HCV and liver Cirrhosis. The aim of the present study was the establishment of stable Huh-7 cell lines expressing structural and non structural proteins of HCV Genotype 3a Pakistani isolate obtained from chronic HCV patients. Methods: Blood samples were obtained from chronic HCV-3a positive patients. HCV individual genes were amplified using PCR with gene specific primers having restriction sites. These gene amplicons were cloned in mammalian expression vector PcDNA3.1+. Huh-7 cell lines were transfected with these constructed plasmids having structural or non-structural HCV genes in confluent cells with lipofectamine. Positive clones were selected with G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated genes were demonstrated by RT-PCR, sequencing and Western blot analysis. Results: We successfully cloned and express five HCV-3a genes in PcDNA3.1+ mammalian expression vector. Results of western blot and sequencing PCR confirmed the stable expression of these five genes. Conclusion: The stable cell-lines expressing HCV-3a individual genes would be a useful tool to investigate the role of various HCV proteins on HCV disease outcome and testing of new therapeutic strategies against HCV. [ABSTRACT FROM AUTHOR]

Background: Current standard therapy commonly followed for chronic Hepatitis C Virus (HCV) in Pakistan is interferon alpha plus ribavirin combination therapy (IFN α/ribavirin) and pegylated interferon plus ribavirin (PegIFN/ribavirin). PegIFN/ribavirin has increased rate of sustained virological response than standard IFN α/ribavirin therapy. Objective of current study was to analyze rate of early and delayed response to antiviral treatment as well as rate of relapse response in patients following standard treatment IFN α/ribavirin and in patients following pegylated interferon treatment. Methods: Baseline serum samples of 153 patients enrolled for IFN α/ribavirin and 50 patients for PegIFN/ribavirin were collected. After total RNA extraction, genotyping was and HCV RNA viral load was done. Subsequently HCV RNA viral load was estimated at 4 weeks of treatment, at 12 weeks, at 24 or 48 weeks and finally after 6 months follow up period. All the data was statistically analyzed using fisher's exact test. Results: Total 86 patients out of 153 patients following conventional IFN α/ribavirin therapy completed treatment and 69% of them showed Rapid Virological Response (RVR). Whereas 50 patients following PegIFN/ribavirin treatment completed treatment and 80% of them achieved RVR. Total 64 out of 86 patients following IFN α/ribavirin therapy completed follow up period and 53.5% of them achieved Sustainded Virologcal Response (SVR). Forty-five out of total 50 patients who received PegIFN/ribavirin treatment completed 6 months follow up period and among these 70% achieved SVR. SVR rates were significantly associated with RVR (p < 0.001), age (p < 0.001) and gender (p < 0.01) Conclusions: Rate of sustained virological response can be determined by factors like rapid virological response and age since they share significant association with one another. More over rate of SVR was more prominent in males than in females. [ABSTRACT FROM AUTHOR]

Background: Interferon is well thought-out as the key defence against all infections including HCV. The only treatment for HCV infection is pegylated interferon alpha (IFN-a) but unluckily more than half of the infected individuals do not act in response to the cure and become chronic HCV carriers. The mechanism how HCV induce interferon resistance is still elusive. It is recently reported that HCV envelope protein 2 interacts with PKR which is the interferon-inducible protein kinase and which in turn blocks the activity of its target molecule called eukaryotic initiation factor elF2. Sequence analysis of Envelope protein reveals it contains a domain homologous to phosphorylation sites of PKR andthe translation initiation factor eIF2alpha. Envelope protein competes for phosphorylation with PKR. Inhibition of kinase activity of PKR is postulated as a mechanism of to interferon (IFN) resistance. Results: Present study involves the insilico investigation of possible role of potential phosphorylation in envelope 2 protein of 3a genotype in interferon resistance. Envelope protein coding genes were isolated from local HCV isolates, cloned and sequenced. Phylogenetic analysis was done and tertiary structure of envelope gene was predicted. Visualization of phosphorylation in tertiary structure reveals that residue 266 and 267 of envelope gene 2 are surface exposed and their phosphorylation may compete with the phosphorylation of PKR protein and possibly involved in mediating Interferon Resistance. Conclusion: A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural analysis has pointed out serine 266 and 267 of the HCV E2 gene as a hopeful claimant for the serine phosphorylation. Recognition of these nucleotide variations may assist to propose genotype precise therapy to avoid and resolve HCV infections. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a member of Flaviviridae family and one of the major causes of liver disease. There are about 175 million HCV infected patients worldwide that constitute 3% of world's population. The main route of HCV transmission is parental however 90% intravenous drug users are at highest risk. Standard interferon and ribavirin remained a gold standard of chronic HCV treatment having 38-43% sustained virological response rates. Currently the standard therapy for HCV is pegylated interferon (PEG-INF) with ribavirin. This therapy achieves 50% sustained virological response (SVR) for genotype 1 and 80% for genotype 2 & 3. As pegylated interferon is expensive, standard interferon is still the main therapy for HCV treatment in under developed countries. On the other hand, studies showed that pegylated IFN and RBV therapy has severe side effects like hematological complications. Herbal medicines (laccase, proanthocyandin, Rhodiola kirilowii) are also being in use as a natural and alternative way for treatment of HCV but there is not a single significant report documented yet. Best SVR indicators are genotype 3 and 2, < 0.2 million IU/mL pretreatment viral load, rapid virological response (RVR) rate and age <40 years. New therapeutic approaches are under study like interferon related systems, modified forms of ribavirin, internal ribosome entry site (HCV IRES) inhibitors, NS3 and NS5a inhibitors, novel immunomodulators and specifically targeted anti-viral therapy for hepatitis C compounds. More remedial therapies include caspase inhibitors, anti-fibrotic agents, antibody treatment and vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV), a major cause of liver disease throughout the world, is difficult to treat with interferon (IFN) (and various formulations and combinations thereof) being the only approved molecule available. It has been investigated recently that proinflammatory chemokine interleukin-8 (IL-8) induced by HCV partially inhibits the antiviral IFN-α therapy. Therefore, the current study was aimed to prospectively utilize the baseline IL-8 levels in the HCV infected serum and predicts its role in sustained virological response (SVR) to IFN-α+ribavirin therapy, in chronic HCV patients in Pakistan. One hundred and ten hepatitis C patients without any other infections underwent IFN-α+ribavirin combination treatment. Baseline IL-8 levels were determined before starting of the therapy for all these patients. Fifteen normal volunteers negative for HCV were kept as control. The baseline IL-8 levels were found significantly higher in all HCV positive patients as compared to normal healthy volunteers (1083.54±85.72pg/ml versus 6.99±1.05pg/ml [mean±SEM], p <0.01) and were also significantly higher in non-responders than responders (p <0.05). Comparatively higher mean baseline IL-8 levels were observed in non-responders (2442.02±159.92pg/ml), than late (1009.31±45.31) and rapid (540.91±27.06pg/ml) responders. Significant relation was observed between baseline IL-8 level and response to IFN therapy (p <0.01). Results of this study suggest that increased levels of IL-8 in HCV infection might be involved in pathogenesis, persistence and resistance to IFN-α+ribavirin combination therapy. [Copyright &y& Elsevier]