Genetic Heterogeneity, Scroepidemiology and Evolutionary Dynamics of Hepatitis C Virus (HCV) in Association with Antiviral Therapy

Abstract

Hepatitis C virus (HCV) is a global public health problem currently circulating in the human blood for centuries however gets the recognition in 1989. Prevalence and incidence rates vary from country to country. So far, it has affected millions of people from all ethnic origins, all walks of life and all socioeconomic status. Most of the patients develop the persistent infection due to failure in clearance of the virus from their blood and finally leads to cirrhosis and hepato-cellular carcinoma (HCC). Currently. the available conventional antiviral therapy against HCV is interferon (IFN) in combination with Ribavirin (RBV). To attain the sustained virological response (SVR) is the main objective in treating HCV. There are several mechanisms evolved by the HCV that facilitate the persistence of virus and further lead the patient's status as non-responder. Emergence of quasispecies as a result of high genomic variability and immune escape variants is supposed to drive viral resistance to therapy. However, the precise mechanism underlying viral non-response to treatment is still not clear. This provoked us to explore the factors in order to further unravel the mechanism of viral resistance to therapy. This study was basically conducted to explore role of seroepidemiolgy, genetic heterogeneity and evolution in viral resistance to therapy. In order to accomplish the task, initially 500 HCV ELISA patients were selected who met the selection criteria. Among these, 451 patients were constantly followed throughout the study period and were divided into two groups on the basis of their treatment response. Group 1 constitutes the 376 patients who became HCV RNA negative in response to therapy while group 2 comprised the 75 patients who did not respond to therapy at all and remained HCV RNA positive even till the end of therapy. Patients were diagnosed as non-responders or responders on the basis of viral load determined by Real-Time PCR. From analysis of serum markers both the groups 1 and 2 were compared with positive and negative control groups. Moreover both the treatment groups were followed from the early diagnosis till the 6 months after the end of treatment. Furthermore, to determine the genetic heterogeneity and tracking the evolutionary changes comparative sequence analysis was performed of 75 nonGenetic Heterogeneity, Seroepidemiology and Evolutionary Dynamics of Hepatitis C Virus (HCV) in XVll Association with Antiviral Therapy Abstract responders with 75 responders (randomly picked from 376). To proceed further reverse transcriptase peR was performed by targeting three genomic regions, 5'UTR, core and NS5B. The amplified products were sequenced directly and obtained sequences were cleaned, aligned and submitted to GenBank. Maximum Parsimony (MP) method was used for phylogenetic analysis and dendrograms were dragged to study the evolutionary behavior of different viral types and subtypes of the present study. All the serum markers related with liver diseases showed significant variation in non-responders as compared to those who become HeV RNA negative in response to therapy. Moreover the patients in group 1 exhibited the presence of genotype 3 which is most prevalent genotype of Pakistan and more susceptible to respond the antiviral therapy whereas the patients who were non-responders possess the Hev isolates with rare genotype in Pakistan i.e 4 and 5 while the most important finding in the present study was the presence of genotype 6q and 6v which is being reported first time from Pakistan. Further, at nucleotide and amino acid level the genetic distance and mutation was high in non-responders in comparison to responders. Difference in percentage composition of individual amino acids was observed between the two groups while amino acid heterogeneity analysis exhibited the different pattern of amino acid substitution and also variation in conserved and variable sites of both the proteins in the two groups. Number of predicted N-phosphorylation and N-glycosylation sites was high in group 2 (nonresponders) as compared to group 1 (with SVR). To evaluate the stereo-chemical structure of the proteins, predicted 3D models were selected on the basis of C-score and referred as best for further analysis. According to Ramachandran plot, satisfactory atomic models considered useful for further studies, i.e. to calculate HeV genotypes conservation at structural level and to find out critical binding sites for drug designing. Genetic heterogeneity was found at both nucleotide and amino acid level using different bioinformatics tools. In addition a computational approach was used to design siRNA that could block the synthesis of Hev RNA and can be used against more than one genotypes if become practice in future. Therefore, here it can be assumed that host and viral factors both play their role equally in viral resistance to therapy. According to present results host serum markers i.e. Alanine amino transferase (AL T), Aspartate Genetic Heterogeneity, Seroepidemiology and Evolutionary Dynamics of Hepatitis C Virus (HCV) in xviii Association with Antiviral Therapy Abstract amino transferases (AST) , Total Bilirubin (TBL), total protein, albumin, low density lipoprotein (LDL), high density lipoprotein (HDL), total cholesterol (TCOL), triglycerides (TGs), hemoglobin (Hb), prothrombin time (PTT) and platelet count (PL T) act as indicator of viral response to therapy. Moreover, viral heterogeneity both at nucleotide and amino acid level contributed equally in resistance to therapy. Besides this, appearance of rare genotypes is also one of the important factors that hurdle the way to positive response of conventional treatment. Computational analysis showed that genetic diversity, any change or mutation in core region, NS5B and 5'UTR might be the cause of HCV strains to resist IFN therapy. Furthermore, prediction of phosphorylation and glycosylation sites could help in targeting the proper sites for drug designing. A single designed siRNA can be used as an alternative for current therapy against more than one resistant HCV genotypes and can be more economical and affective against HCV if become common practice in future However functional analysis are mandatory to confirm the computational analysis.