Lysosomotropic agents as HCV entry inhibitors

HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH4Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH4Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH4Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH4Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH4Cl showed more than 50% reduction of virus infectivity at 50 μM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV

In-vitro model systems to study Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design

Liver Stiffness Measurement and Biochemical Markers in Senegalese Chronic Hepatitis B Patients with Normal ALT and High Viral Load

Despite the high prevalence of chronic hepatitis B (CHB) in Africa, few studies have been performed among African patients. We sought to evaluate liver stiffness measurement by FibroScan® (LSM) and two biochemical scores (FibroTest®, Fibrometer®) to diagnose liver fibrosis in Senegalese CHB patients with HBV plasma DNA load ≥3.2 log(10) IU/mL and normal alanine aminotransferase (ALT) values.LSM and liver fibrosis biochemical markers were performed on 225 consecutive HBV infected Senegalese patients with high viral load. Patients with an LSM range between 7 and 13 kPa underwent liver biopsy (LB). Two experienced liver pathologists performed histological grading using Metavir and Ishak scoring.225 patients were evaluated (84% male) and LB was performed in 69 patients, showing F2 and F3 fibrosis in 17% and 10% respectively. In these patients with a 7-13 kPa range of LSM, accuracy for diagnosis of significant fibrosis according to LB was unsatisfactory for all non-invasive markers with AUROCs below 0.70. For patients with LSM values below 7 kPa, FibroTest® (FT), and Fibrometer® (FM) using the cut-offs recommended by the test promoters suggested a fibrosis in 18% of cases for FT (8% severe fibrosis) and 8% for FM. For patients with LSM values greater than 13 kPa, FT, FM suggested a possible fibrosis in 73% and 70%, respectively.In highly replicative HBV-infected African patients with normal ALT and LSM value below 13 kPa, FibroScan®, FibroTest® or Fibrometer® were unsuitable to predict the histological liver status of fibrosis

siRNAs: Potential therapeutic agents against Hepatitis C Virus

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. 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. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HC

Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Curation of viral genomes: challenges, applications and the way forward

BACKGROUND: Whole genome sequence data is a step towards generating the 'parts list' of life to understand the underlying principles of Biocomplexity. Genome sequencing initiatives of human and model organisms are targeted efforts towards understanding principles of evolution with an application envisaged to improve human health. These efforts culminated in the development of dedicated resources. Whereas a large number of viral genomes have been sequenced by groups or individuals with an interest to study antigenic variation amongst strains and species. These independent efforts enabled viruses to attain the status of 'best-represented taxa' with the highest number of genomes. However, due to lack of concerted efforts, viral genomic sequences merely remained as entries in the public repositories until recently. RESULTS: VirGen is a curated resource of viral genomes and their analyses. Since its first release, it has grown both in terms of coverage of viral families and development of new modules for annotation and analysis. The current release (2.0) includes data for twenty-five families with broad host range as against eight in the first release. The taxonomic description of viruses in VirGen is in accordance with the ICTV nomenclature. A well-characterised strain is identified as a 'representative entry' for every viral species. This non-redundant dataset is used for subsequent annotation and analyses using sequenced-based Bioinformatics approaches. VirGen archives precomputed data on genome and proteome comparisons. A new data module that provides structures of viral proteins available in PDB has been incorporated recently. One of the unique features of VirGen is predicted conformational and sequential epitopes of known antigenic proteins using in-house developed algorithms, a step towards reverse vaccinology. CONCLUSION: Structured organization of genomic data facilitates use of data mining tools, which provides opportunities for knowledge discovery. One of the approaches to achieve this goal is to carry out functional annotations using comparative genomics. VirGen, a comprehensive viral genome resource that serves as an annotation and analysis pipeline has been developed for the curation of public domain viral genome data . Various steps in the curation and annotation of the genomic data and applications of the value-added derived data are substantiated with case studies