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Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an in silico approach.

ABSTRACT: BACKGROUND: HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An in silico approach was applied for the analysis of entire HCV proteome of Pakistani origin, aimed to identify the viral epitopes and their conservancy in HCV genotypes 1, 2 and 3 of diverse origin. RESULTS: Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide). Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100%) conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2. CONCLUSIONS: The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was significantly higher in contrast to its conservancy in HCV genotype 1 and 2. Despite of the lower conservancy in genotype 1 and 2, all the predicted epitopes have important implications in diagnostics as well as CTL-based rational vaccine design, effective for most population of the world and especially the Pakistani population.

SUBMITTER: Shehzadi A

PROVIDER: S-EPMC3042956 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an in silico approach.

Shehzadi Abida A Ur Rehman Shahid S Idrees Muhammad M

Virology journal 20110208

<h4>Background</h4>HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effect ...[more]

PMID: 21303499

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Project description:BACKGROUND: Pakistan is facing a threat from hepatitis C infection which is increasing at an alarming rate throughout the country. More specific and sensitive screening assays are needed to timely and correctly diagnose this infection. METHODS: After RNA extraction from specimen (HCV-3a), cDNA was synthesized that was used to amplify full length core gene of HCV 3a. After verification through PCR, DNA sequencing and BLAST, a properly oriented positive recombinant plasmid for core gene was digested with proper restriction enzymes to release the target gene which was then inserted downstream of GST encoding DNA in the same open reading frame at proper restriction sites in multiple cloning site of pGEX4t2 expression vector. Recombinant expression vector for each gene was transformed in E. coli BL21 (DE3) and induced with IPTG for recombinant fusion protein production that was then purified through affinity chromatography. Western blot and Enzyme Linked Immunosorbant Assay (ELISA) were used to detect immuno-reactivity of the recombinant protein. RESULTS: The HCV core antigen produced in prokaryotic expression system was reactive and used to develop a screening assay. After validating the positivity (100%) and negativity (100%) of in-house anti-HCV screening assay through a standardized panel of 200 HCV positive and 200 HCV negative sera, a group of 120 serum specimens of suspected HCV infection were subjected to comparative analysis of our method with commercially available assay. The comparison confirmed that our method is more specific than the commercially available assays for HCV strains circulating in this specific geographical region of the world and could thus be used for HCV screening in Pakistan. CONCLUSION: In this study, we devised a screening assay after successful PCR amplification, isolation, sequencing, expression and purification of core antigen of HCV genotype 3a. Our developed screening assay is more sensitive, specific and reproducible than the commercially available screening assays in Pakistan.

Project description:Plant species of the Poaceae family are not only used as fodder and forage but also contribute substantially to the treatment of various health disorders, particularly in livestock. Consequently, the present study was aimed to document the therapeutic uses of Poaceae practiced by the inhabitants of the Punjab Province for the treatment of various veterinary health disorders. Semi structured interviews, group discussion and field walks were conducted to collect the data. Quantitative indices including cultural significance index (CSI), relative frequency of citations (RFC), fidelity level (FL), relative popularity level (RPL), and Jaccard Index (JI) were used for the data analysis. Traditional uses of 149 species belonging to 60 genera and 16 tribes of 5 sub families of Poaceae were recorded. Whole plants and leaves were the most consistently used parts with 40.94 and 29.53%. The plants were mainly given orally as fodder (59 reports) without processing followed by decoction (35 reports). Most of the species were employed to treat infectious diseases (25.93%), and digestive disorders (14.10%). Triticum aestivum had the highest CSI, RFC and RPL levels at 8.00, 0.96, 1.00, respectively, followed by Oryza sativa and Poa annua. Likewise, T. aestivum and Saccharum spontaneum had 100% FL and ROP. Jaccard index ranged from 12.25 to 0.37. Twelve plant species namely Chrysopogon zizanioides (anti-inflammatory), Pennisetum lanatum (improve bull fertility), Cymbopogon citratus (glandular secretion), Sorghum saccharatum and Themeda triandra (malaria), Aristida funiculate (anticancer), Koeleria argentia (skin allergies), Tetrapogon villosus (antibacterial), Cynodon radiatus (eyes infection), Sporobolus nervosa (Jaundice), Enneapogon persicus (antifungal), and Panicum repens (dysfunctional cattle organs) were reported for the first time, with novel ethnoveterinary uses. The inhabitants of the study area had a strong association with their surrounding plant diversity and possessed significant knowledge on therapeutic uses of Poaceae to treat various health disorders in animals. Plant species with maximum cultural and medicinal values could be a potential source of novel drugs to cure health disorders in animals and human as well.

Dataset Information

Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an in silico approach.

Publications

Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an in silico approach.

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