Prediction of promiscuous epitopes in NSP2 of Chikungunya virus: An in-silico approach.

This study is focused towards developing a global consensus sequence of nonstructural protein 2 (NSP2), a protease of Chikungunya Virus (CHIKV) and predict immunogenic promiscuous T-cell epitopes based on various bioinformatics tools. To date, no epitope data is available for the Chikungunya virus in the IEDB database. In this study, 100 available nucleotide sequences of NSP2-CHIKV belonging to different strains were downloaded from the National Centre for Biotechnology Information (NCBI) database. The nucleotide sequences were subjected to translated sequencing using the EXPASY tool followed by protein alignment using the CLC workbench and a global consensus sequence for the respective protein was developed. IEDB tool was used to predict HLA-I and HLA-II binding promiscuous epitopes from the consensus sequence of NSP2-CHIKV. Thirty-four B-cell based epitopes are predicted and the promiscuous epitope is VVDTTGSTKPDPGD at position 341-354. Twenty-six MHC-I short peptide epitopes are predicted to bind with HLA-A. The promiscuous epitopes predicted to bind with HLA-A*01:01 are VTAIVSSLHY, SLSESATMVY, FSKPLVYY, QPTDHVVGEY at positions 317-326, 84-93, 535-544 and 15-24 with percentile ranks 0.17, 0.39, 0.51 and 0.81, respectively. Twenty-four MHC-II short peptide epitopes are predicted for HLA-DRB. The promiscuous epitope predicted to bind with HLA-DRB*01:01 is VVGEYLVLSPQTVLRS from 20-35 with a lowest percentile rank of 0.01. These predicted epitopes can be effective targets towards development of vaccine against CHIKV. Epitopes predicted in this study displayed good binding affinity, antigenicity and promiscuity for the HLA classes. These predicted epitopes can prove to be translationally important towards the development of CHIKV.

Prediction of promiscuous epitopes in NSP2 of Chikungunya virus: An in-silico approach

@# This study is focused towards developing a global consensus sequence of nonstructural protein 2 (NSP2), a protease of Chikungunya Virus (CHIKV) and predict immunogenic promiscuous T-cell epitopes based on various bioinformatics tools. To date, no epitope data is available for the Chikungunya virus in the IEDB database. In this study, 100 available nucleotide sequences of NSP2-CHIKV belonging to different strains were downloaded from the National Centre for Biotechnology Information (NCBI) database. The nucleotide sequences were subjected to translated sequencing using the EXPASY tool followed by protein alignment using the CLC workbench and a global consensus sequence for the respective protein was developed. IEDB tool was used to predict HLA-I and HLA-II binding promiscuous epitopes from the consensus sequence of NSP2-CHIKV. Thirty-four B-cell based epitopes are predicted and the promiscuous epitope is VVDTTGSTKPDPGD at position 341-354. Twenty-six MHC-I short peptide epitopes are predicted to bind with HLA-A. The promiscuous epitopes predicted to bind with HLA-A*01:01 are VTAIVSSLHY, SLSESATMVY, FSKPLVYY, QPTDHVVGEY at positions 317-326, 84-93, 535-544 and 15-24 with percentile ranks 0.17, 0.39, 0.51 and 0.81, respectively. Twenty-four MHC-II short peptide epitopes are predicted for HLA-DRB. The promiscuous epitope predicted to bind with HLA-DRB*01:01 is VVGEYLVLSPQTVLRS from 20-35 with a lowest percentile rank of 0.01. These predicted epitopes can be effective targets towards development of vaccine against CHIKV. Epitopes predicted in this study displayed good binding affinity, antigenicity and promiscuity for the HLA classes. These predicted epitopes can prove to be translationally important towards the development of CHIKV.

Quantitative evaluation of insulin resistance markers in Pakistani patients suffering from HCV-associated type 2 diabetes mellitus.

The current study was designed to determine the role of the host genes involved in the development of chronic hepatitis C-associated type 2 diabetes mellitus. This study was carried out in patients in four different stages of chronic hepatitis C virus (HCV) infection, including treatment-naïve HCV patients, HCV-positive patients with type 2 diabetes mellitus (T2DM), non-responders and responders. The mRNA expression level of host genes, such as glucose-6-phosphatase (G6Pase), tumor necrosis factor α (TNF-α) and different adipokines including adiponectin, leptin and resistin, was quantified and compared to healthy controls. HCV infection was found to be associated with insulin resistance, a step towards type 2 diabetes mellitus (T2DM). The results also suggest the potential role of adipokines in chronic HCV (CHC)-associated T2DM. The upregulation of gluconeogenic genes, such as G6Pase and resistin, and a decreased mRNA expression level of adiponectin suggest the potential role of selected markers in the CHC-associated T2DM in Pakistani population. Based on these results, it is concluded that upregulation of TNF-α, G6Pase and resistin in chronic HCV patients leads to gluconeogenesis, eventually favoring T2DM. Collectively, these findings suggest that CHC patients are more prone to T2DM.

Molecular characterization and fluorescence analysis of HCV non-structural proteins NS3, NS3-4A and NS4A of genotype 3a.

Hepatitis C virus (HCV) chronically infects almost 2% of world's population. Chronic infection can lead to liver failure and hepatocellular carcinoma (HCC). Approximately 10% of the Pakistani population is infected with HCV and type 3 is the most prevalent genotype with 75-90% prevalence. In this study we have developed transiently expressing cell culture based system for the expression of HCV non-structural NS3, NS3-4A and NS4A proteins of genotype 3a. HCV non-structural genes NS3, NS3-4A and NS4A were cloned in to pFLAG-CMV2 and pEGFP-C1vectors. All vectors were transfected separately to Huh-7 cells and their protein expression was analyzed by Western blot and immunofluorescence. All proteins were expressed correctly and in the transfection we have obtained 42-70% efficiency for all clones. This system can be used for the development of novel antiviral strategies to inhibit the viral replication, to study apoptosis pathways induced by HCV, for the evaluation of vaccine candidates and also to study the role of HCV different signaling pathways.

Hepatitis C virus infection in vitro triggers endoplasmic reticulum stress and downregulates insulin receptor substrates 1 and 2 through upregulation of cytokine signaling suppressor 3.

Hepatitis C virus (HCV) infection is highly prevalent worldwide and most of HCV infections enter into chronic phase subsequently leading to insulin resistance (IR) and clinical complications. Although the clinics of chronic HCV infection is well described, there is need to better understand the molecular mechanisms of HCV-induced IR. Therefore this study was aimed to unveil the role of host genes involved in the development of HCV-induced IR. For this purpose the expression of selected genes in HCV-infected and non-infected Huh-7 cells at various time post infection (p.i.) was assayed by real-time PCR. HCV infection was found to trigger endoplasmic reticulum (ER) stress response as demonstrated by an increase in the expression of calreticulin (Cal) gene but no change in the expression of Gadd153 gene. The infection also enhanced the expression of suppressor of cytokine signaling 3 (SOCS-3), responsible for the degradation of insulin receptor substrates (IRS). Moreover, it led to a decreased expression of key signaling molecules IRS-1 and IRS-2, unchanged expression of SOCS-7 and increased expression of downstream signaling molecule Akt. Altogether these findings indicate that the HCV infection induces ER stress and IR in Huh-7 cells in vitro.