Hepatitis C Virus persistence, virus and host related factors
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Hepatitis C is a global health problem caused by infection with hepatitis C virus (HCV). Most patients fail to clear the virus and develop persistent infection that frequently leads to cirrhosis and hepato-cellular carcinoma (HCC). The only available antiviral therapy is interferon (lFN) in combination with Ribavirin (RBV). High genornic variability that results in quasi-species and immune escape variants is supposed to lead to viral persistence. However, the exact mechanism underlying viral persistence is still not clear. This incited us to investigate the virus and host related factors in order to further unravel the mechanisrn of viral persistence. HCV genotype 3 is prevalent in Pakistan as shown by RFLP analysis of 5' UTR, but some isolates remain unresolved. Sequencing and phylogenetic analysis showed that the presence of additional restriction sites of HaeIII and RsaI (that cause failure of RFLP) resulted in clustering of unresolved samples in a separate branch of un-rooted Neighbor-Joining (NJ) tree (bootstrap value of 71%). Another, cluster I (having additional stretches of nucleotides) was found at a bootstrap value of 8l%. Secondary structure analysis showed a major distortion in the stem loop III d of 5'- UTR region. This study showed the presence of unique sequence variability in the 5'- UTR of HCV type 3 isolates from Pakistan. Mutations found in these sequences lead to conformational changes in stem loop III that may alter the translation initiation of the virus. The translation initiation of HCV, regulated by internal ribosomal entry site (IRES), includes whole 5'-UTR and a portion of core region. HCV genotype 3, the most prevalent genotype in Pakistan, is reported as the best responding genotype to combination therapy of IFN and/or RBV. Despite this, a high proportion (30%) of patients infected with genotype 3 remains as non-responders (NR) when treated with lFN plus RBV. The HCV IRES translation efficiency (TE) was observed both in SR (sustained responders) and NR patients. Low TE was observed for IRES derived from SR patients, whereas IRES of NR patients showed significantly greater TE. TE of different IRES constructs were further analysed in a cell culture system in the presence or absence of IFN and/ RBV. A dose dependent inhibition of relative TE by INF-α and RBV was observed. A greater inhibitory effect was observed after INF-α addition, on relative TE of IRES constructs isolated from SR patients as compared to IRES of the NR group, however, no difference between the SR and the NR group was found when cells were exposed to RBV. IFN and RBV when given in combination, also had a negative effect on translation, but this effect was not synergistic. These results are suggestive of a positive correlation between the effect of antiviral agents in cells and clinical response of the patients. Secondary structure analysis of HCV RNA (IRES) showed that in case of NR the nucleotide substitutions and insertions were only few as compared to SR sequences relative to a reference genotype 3a strain. In the SR samples, substitutions, deletions and insertions were mainly clustered in stem loop I, II and III of the 5'-UTR. A possibility of differential binding of host cellular factors to the IRES regions of different sequences, resulting in varied TE of the viral polyprotein also seems to be an obvious affect. Host genetic factors also account for the variability in the rate of disease progression seen in patients with chronic hepatitis C. The capacity for cytokine production in individuals has a major genetic component. It has been suggested that polymorphism in pro-inflammatory cytokine genes may have a role in determining the progression of viral persistence in chronic HCV.IFN-γ polimorphism i.e. G to T transition at -I79 position has been focused in the present study. It was found that in our population G allele is present and in addition to this a new polymorphic site was also found at -35 position. Both G allele and new polymorphic sites are equally distributed in controls and HCV infected patients. Moreover, these polymorphic sites were not associated with the viral clearance or persistence. Host cellular immune defense, including CD4+ and CD8+ T-cell response is activated in patients with HCV infection. The intensity of intra-hepatic CDS+ T-cell response during the early stages of infection may be a critical determinant of disease resolution and control of viral replication. Present study showed that in intra-hepatic CD8+ T cell assay HCV epitopes of core and NS3 regions were more reactive in patients with viral clearance as compared to those having persistent infection at pre-treatment level. Since CTL escape variants seem to occur in persistent chronic HCV infection, a multi-specific CTL response must be induced to avoid selecting for escape variants. The findings suggested that high translation efficiency and generation of immune escape variants cads to viral persistence. In contrast, viral clearance is associated with effective and strong immune system as well as low translation efficiency of the virus. These findings would be beneficial as pre-diagnostic marker as well as helpful to predetermine the treatment strategies for the NR; further more adjuvant therapy could also be suggested. The findings would be valuable for consideration of more aggressive therapy towards those patients having an increased risk of developing persistent chronic HCV.
Yasmeen, A. (2004). Hepatitis C Virus persistence, virus and host related factors (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.