Diversifications in the HCV genome are the results of viral RNA polymerase infidelity, immune-driven selection and exertion of therapy pressure or a combination of these processes. PCR-SSCP analysis is a well established technique to study genetic variability, including the presence of mutations and quasispecies[10–13
]. Millions of viruses are produced via
replication and eliminated each day due to exertion of immune pressure and interferon therapy[16
]. Ribavirin does not have any effect on the viremia level of HCV-infected patients, but it reduces the ALT level, increases the likelihood of sustained virological response and reduces the probability of relapse[16
The HVR and NS5B genes were selected in the present study because the persistence of infection may be associated with positive selection of HVR quasispecies by the host immune system and because of the major role of NS5B in the replication of hepatitis C. Factors like age, sex and duration of infection may be possible causes of the occurrence of quasispecies; in this study, we studied virological responses in patients infected with quasispecies before the initiation of therapy and looked for alterations in the SSCP patterns during therapy. Several conflicting reports about a possible correlation of therapy response and disease severity with the presence of quasispecies have been published[15,20,24–27
]. The genes encoding HVR protein exhibit a high degree of variability, giving rise to differing phenotypic traits, including alterations in receptor-binding affinity and escape from immune recognition[28
]. Substitutions in the NS5B gene of HCV may correlate with the virological response to interferon-based therapy[29–31
The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in CAH patients, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in hepatitis C patients in this study. The genetic diversity at baseline can be predictive of distinct virological behavior during the course of combination therapy. Among the 13 patients infected with complex quasispecies with variations in the HVR gene, 10 were non-responders at baseline. This correlates significantly with the persistent of infection as the response rate against interferon may be due to two mutually compatible approaches; the infecting virus might have become naturally resistant to interferon or the virus could have evolved into quasispecies. The non-responders had normal signal transduction pathways required for the intracellular activity of interferon[32
]. The recognition of the HVR epitope (envelope) by immune cells and the exertion of interferon-based therapy play a major role in the evolution of quasispecies.
Several studies have shown an association of epidemiological factors and treatment regime with the presence of quasispecies[13,14,19,33
]. Complex quasispecies with variations in the HVR and NS5B genes of HCV were significantly associated with virological response during wk 48 of therapy. Virus quasispecies evolution within patients increases the pathogenicity of HCV. The resulting viral quasispecies are heterogeneous and sensitive to selection pressures. Complex variants escape from immune pressure and the antiviral spectrum of the combination therapy[34
]. No mutant strains have the capacity for replication, but only selective mutants can replicate, which may be responsible for persistence of infection. The circulating viremia in blood and liver cells is significantly associated with disease prognosis[35
]. There was no statistically significance difference in the HAI scores among patients with different degrees of HVR quasispecies at baseline. However, there was a significant difference in the HAI scores at baseline between patients infected with different genetic variants (CQS vs
NQS) of the NS5B gene. Selective adaptive mutations in the resistant strains occurring during replication influence the RDRP activity and lead to increases in viremia level and HAI scoring[7–9
]. These results suggest the significance of evolution of quasispecies on virological responses and disease severity.
The amino acid variability in HVR and NS5B was evaluated from cDNA-derived sequences. The occurrence of HVR quasispecies influences the therapeutic outcome; because no strains isolated from non-responder patients showed close pairing, it is possible that some other factors may be responsible for the persistence of infection. However, the possibility of resistant strains can not be denied. The NS5B sequences of non-responder strains were on the same branch, showing the possibility of resistant strain infection in these patients. However, adaptive mutations in NS5B lead to persistence of infection and a severe course of liver disease in patients. These findings reveal that the occurrence of HVR quasispecies, including the presence of resistant strains, is a major factor influencing the therapeutic outcome and disease severity in chronic hepatitis C patients. Genetic variability was not uniformly distributed throughout the HCV genome.
In conclusion, viral quasispecies heterogeneity can be an important marker to predict the response of combination therapy in patients with chronic hepatitis C. Quasispecies complexity on the basis of SSCP pattern could be helpful in differentiation of SVR and NR patients. Complex quasispecies heterogeneity causes high HCV viremia, which was significantly correlated with the severity of liver disease and prognosis during the course of combination therapy. However, there was no significant alteration in SSCP profile during therapy. The occurrence of resistant strains may be another factor contributing to the persistence of HCV infection. Quasispecies in both HVR and NS5B genes may have a major impact on treatment and be a good predictor of virological response. Complex quasispecies are resistant to combination therapy and influence disease prognosis during therapy. Further evaluation of viral quasispecies complexity in the HVR and NS5B genes, as well as other HCV genes (whole genome), is required for a better understanding of the influence of quasispecies on the persistence of infection and disease prognosis during the course of therapy in HCV-infected patients.