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Hepatitis C virus (HCV) genotype 3a is considered a significant risk factor for the development of liver diseases and hepatocellular carcinoma for most of the cases in Pakistan. Because of the limited efficiency of the current therapy, RNA interference (RNAi), which results in sequence-specific degradation of HCV RNA, has potential as a powerful alternative molecular therapeutic approach. The envelope genes (E1 and E2) of HCV come in immediate contact with cells during infection and therefore might be a relevant target for new drug development. In the present study, the expression of E1 and E2 genes of HCV genotype 3a was dramatically reduced at both the mRNA and protein level using gene-specific small interfering RNAs (siRNA) when compared to mock-transfected and cells treated with control siRNAs. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was also demonstrated by combined treatment of siRNAs against the E1 and E2 genes, which resulted in a significant decrease in HCV viral copy number. This clearly demonstrates that the RNAi-mediated silencing of HCV E1 and E2 is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a. [ABSTRACT FROM AUTHOR]

Background: Several factors have been proposed to assess the clinical outcome of HCV infection. The correlation of HCV genotypes to possible serum markers in clinical prediction is still controversial. The main objective of this study was to determine the existence of any correlation between HCV genotypes to viral load and different clinical serum markers. Methods: We performed a prospective cross-sectional and observational study. About 3160 serum HCV RNA positive patients were chosen from 4020 randomly selected anti-HCV positive patients. Statistical analysis was performed using the SPSS 16 software package. ROC (receiver operating characteristics) curves were used to compare diagnostic values of serum markers to predict genotypes. Results: The most prevalent genotype was 3a (73.9%) followed by 1a (10.7%), 4a (6.4%) and 3b (6.1%) in Pakistani population. No correlation was found between viral load and serum markers for genotype 3a in a large no. of sample (n = 2336). While significant correlation was observed between viral load and AST in genotype 3b, ALP with viral load and ALT for genotype 1a. Patients with genotype 4a showed a significant inverse correlation with viral load and Hb level and AST with ALP. For genotype 4a, AUC (area under the curve) of ALT, ALP, AST, bilirubin, Hb level and viral load was 0.790, 0.763, 0.454, 0.664, 0.458 and 0.872 respectively. Conclusions: In conclusion, there was a significant variable response of HCV genotypes with serum markers. Severity of disease is independent of serum marker level in genotype 3a, while the liver damage in genotype 4a may associate with viral cytopathic effect as well as the immune-mediated process. An index using six serum markers may correctly predict genotype 4a in patients with ≥75% accuracy. [ABSTRACT FROM AUTHOR]

HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of tight junction's proteins that are responsible for establishing barriers for controlling the flow of molecules around cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-terminal tail. In cellular proteins, interplay between phosphorylation and O-β-GlcNAc modification is believed to be functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo. Netphos 2.0 and Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction; and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may have potential for O-β-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O-β-GlcNAc modification on Ser 192, Ser 205, Ser 206; and Thr 191 may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV viral entry complex. [ABSTRACT FROM AUTHOR]

Background: Chronic HCV is one of the major causes of morbidity and mortality in the present day world. The assessment of disease progression not only provides useful information for diagnosis and therapeutic supervision judgment but also for monitoring disease. Different invasive and non invasive methods are applied to diagnose the disease from initial to end stage (mild fibrosis to cirrhosis). Although, liver biopsy is still considered as gold standard to identify liver histological stages, an assessment of the disease development based on non-invasive clinical findings is also emerging and this may replace the need of biopsy in near future. This review gives brief insight on non-invasive methods currently available for predicting liver fibrosis in HCV with their current pros and cons to make easier for a clinician to choose better marker to assess liver fibrosis in HCV infected patients. Methods: More than 200 studies regarding invasive and noninvasive markers available for HCV liver disease diagnosis were thoroughly reviewed. We examined year wise results of these markers based on their sensitivity, specificity, PPV, NPV and AUROCs. Results: We found that in all non-invasive serum markers for HCV, FibroTest, Forn's Index, Fibrometer and HepaScore have high five-year predictive value but with low AUROCs (0.60∼0.85) and are not comparable to liver biopsy (AUROC = 0.97). Even though from its beginning, Fibroscan is proved to be best with high AUROCs (> 0.90) in all studies, no single noninvasive marker is able to differentiate all fibrosis stages from end stage cirrhosis. Meanwhile, specific genetic markers may not only discriminate fibrotic and cirrhotic liver but also differentiate individual fibrosis stages. Conclusions: There is a need of marker which accurately determines the stage based on simplest routine laboratory test. Genetic marker in combination of imaging technique may be the better non invasive diagnostic method in future. [ABSTRACT FROM AUTHOR]

Background/Aim: Hepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-a and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection. Results: In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number. Conclusion: From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) Core protein is thought to trigger activation of multiple signaling pathways and play a significant role in the alteration of cellular gene expression responsible for HCV pathogenesis leading to hepatocellular carcinoma (HCC). However, the exact molecular mechanism of HCV genome specific pathogenesis remains unclear. We examined the in vitro effects of HCV Core protein of HCV genotype 3a and 1a on the cellular genes involved in oxidative stress and angiogenesis. We also studied the ability of HCV Core and Cox-2 siRNA either alone or in combination to inhibit viral replication and cell proliferation in HCV serum infected Huh-7 cells. Results: Over expression of Core gene of HCV 3a genotype showed stronger effect in regulating RNA and protein levels of Cox-2, iNOS, VEGF, p-Akt as compared to HCV-1a Core in hepatocellular carcinoma cell line Huh-7 accompanied by enhanced PGE2 release and cell proliferation. We also observed higher expression levels of above genes in HCV 3a patient's blood and biopsy samples. Interestingly, the Core and Cox-2-specific siRNAs down regulated the Core 3a-enhanced expression of Cox-2, iNOS, VEGF, p-Akt. Furthermore, the combined siRNA treatment also showed a dramatic reduction in viral titer and expression of these genes in HCV serum-infected Huh-7 cells. Taken together, these results demonstrated a differential response by HCV 3a genotype in HCVinduced pathogenesis, which may be due to Core and host factor Cox-2 individually or in combination. Conclusions: Collectively, these studies not only suggest a genotype-specific interaction between key players of HCV pathogenesis but also may represent combined viral and host gene silencing as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) has chronically infected a large number of patients, leading to the development of steatosis, cirrhosis and, ultimately, hepatocellular carcinoma. The pathogenesis of HCV has not been fully explained, although steatosis is considered to contribute greatly to liver fibrosis progression, modulating host-cell lipid metabolism. Suspected underlying molecular mechanisms include interactions between HCV proteins and intracellular lipid metabolic pathways. Recent studies have suggested that the nucleocapsid of HCV (core) acts as a pathogenic factor involved in lipid droplet accumulation, changes in lipogenic gene expression and/or the activity of lipogenic proteins in a genotype-specific manner. In this review, we have tried to summarize the current knowledge regarding HCV-induced steatosis and the regulation of expression of host genes and receptors that aid in the viral life cycle and promote liver diseases. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Results: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. Conclusions: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) infection has infected approximately 3% of the world population. HCV genotype 1a is distributed throughout the world, and along with genotype 1b, is relatively resistant to current standards of therapy compared to other HCV genotypes. The present study was designed to produce stable Huh-7 cell lines expressing non-structural proteins of HCV genotype la, representing an in vitro system to facilitate the development of new antiviral drugs against chronic HCV infection. The non-structural genes of HCV genotype 1a were amplified and cloned in a mammalian expression vector pCR 3.1/FIagTag. Huh-7 cells were transfected with one of two expression plasmids, the first containing the NS2, NS3, and NS4a cassette, and second containing the NS5a and NS5b genes. Stable cell lines were produced under the selection of gentamycin (G418). mRNA and protein expression analysis was performed by RT-PCR and Western blotting. The RT-PCR and Western blot results confirmed the stable expression of each of the gene products. Stable Huh-7 cell lines with HCV la non-structural proteins may be helpful for evaluating the role of HCV proteins in molecular pathogenesis, and could facilitate the development of new therapeutic drugs. [Copyright &y& Elsevier]