Molecular Characterization of Viral and Host Genetic Factors in Hepatitis C Virus Infection

Abstract

Hepatitis C virus (HCV) infection has become a major public health problem, affecting 170 million people worldwide. Chronic infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). No vaccine has been developed against HCV so far. Recommended treatment regimen for chronic hepatitis C is pegylated interferon α-2a or α-2b and ribavirin combination therapy (PEG-IFN-α/RBV) for 24-48 weeks. In addition recently discovered direct acting agents (DAA) have also been used. Despite all these therapies, a significant number of HCV patients show no response to the treatment. It is well known that genetic diversity of hepatitis C virus and individual hosts genetic makeup plays a significant role in the treatment success or failure of HCV patients, therefore, it is necessary to find out the exact reason behind this non-response. In the current study we have investigated the role of viral and host factors with treatment response in HCV-3a patients because HCV-3a genotype is more common in Pakistan. Viral factors included viral genotype, baseline viral load and NS5A gene mutations. HCV genotype 2 and 3 show better sustained virological response (SVR) (80%-90%) compared to those infected with genotype 1and 4 (SVR=40%-52%). Therefore, genotype determination is clinically important before start of therapy. Similarly, determination of genotypes in selected population would be helpful to understand origin, pattern of spread of infection throughout the population and to design the prevention policy for hepatitis infection. The prevalence pattern of genotypes and subtypes is variable from country to country or within the same country. According to clinical and scientific literature, genotype 3a infection is the most prevalent HCV subtype in Pakistan. This data is based on PCR focused genotyping assays. Ideal approach for accurate genotyping is sequencing. In order to select proper treatment for HCV patients, understand epidemiology and validate the results of genotyping, we sequenced HCV 5′ UTR Core and NS5B regions to determine genotype of HCV. 5′ UTR Core and NS5B were found to be the most conserved regions which reflected the variation of full genome. However, 5′ UTR region is widely used for genotyping in all methods due to efficient amplification. One hundred and sixty samples were genotyped by sequencing 241bp 5′ UTR, 25 samples by 441bp Core gene region and 20 samples ii by 388bp NS5B gene Okamoto region. Nucleotide blast, manual sequence analysis and phylogenetic analysis of these three regions of HCV genome confirmed that the most frequent genotype was 3a followed by 1a, 3b, and 4. Genotype 5 and 6 were not detected in any sample. One complete genome of HCV was also sequenced to confirm the results of above methods by phylogenetic analysis. Although HCV 3a responds better to PEG-IFN-α/RBV therapy, we found a significant number of HCV 3a patients who were non responder to the therapy. Mutations in HCV NS5A gene interferon sensitivity determing region (ISDR) and protein kinase R (PKR) binding domain (PKRBD) are associated with non-response to therapy in HCV-1a infected populations. HCV introduce mutations in this region by immune defense mechanism to escape antiviral therapy. In order to determine the role of mutations to non-response of genotype 3a to PEG-IFN-α/RBV therapy, 416 bp segment of NS5A gene covering ISDR and PKRBD was sequenced from responders and non-responders HCV-3a patients for comparison. No significant difference was found in ISDR and PKRBD region, however downstream to the PKRBD of NS5A two important statistically significant mutations were observed; at positions 2309 (Alanine to Serine) and 2326 (Glycine to Alanine). These mutations were then analyzed for tertiary protein structure and important structural changes were observed which might cause non-response to therapy. Moreover, we investigated the correlation of baseline viral load with PEG-IFN-α/RBV therapy and found association of low viral load with better response to therapy. Host factors included age, gender, alanine aminotransferase (ALT) levels, liver fibrosis stage and IL28B gene polymorphisms of HCV infected patients. Among the host factors we did not find any association of gender ALT levels and liver fibrosis stage with the treatment response. However, younger age was a strong predictor of treatment success. For IL28B, SNP rs12979860 was studied in two hundred and nineteen HCV-3a patients treated with standard IFN-α and ribavirin. We have found an important result showing the involvement of CC genotype in protection against infection rather than clearance. The TT genotype has revealed good response to treatment. It shows that the patients with genotype 3 and Il28B CC genotype are more likely to develop chronic disease and CT/TT genotypes give better response to treatment compared to CC genotype. The CC genotype provides protection against infection whereas TT genotype showed good response to the treatment. Further exploration of iii this chromosomal region might be helpful in finding associations for genotype 3 infected patients. This knowledge could facilitate the development of tests for efficient genotyping of patients thus leading to a better and more personalized treatment. Ultimately, this approach may prevent treatment failures and avoid unnecessary side effects. These findings would be beneficial as pre-diagnostic marker for HCV patients. It will be helpful to avoid receiving ineffective antiviral therapy to non-responder (NR) patients and predetermine the treatment strategies (adjuvant therapy of PEG-IFN-α/RBV and direct acting agents).