The quasispecies nature of hepatitis C virus (HCV) may have important implications concerning resistance to antiviral agents. To determine whether HCV NS5A quasispecies composition and dynamics are related to responsiveness to combined interferon (IFN) and ribavirin therapy, extensive sequence analyses of cloned RT-PCR amplification products of HCV-1b NS5A quasispecies of sequential isolates from 15 treated (nine sustained responders and six non-responders) and three untreated patients were performed. Accumulation of mutations in NS5A during therapy was relatively frequent in the V3 domain, but unusual elsewhere. Amino acid changes were the result of the imposition of minor variants that were already present before treatment and always occurred within the first week of therapy. Before treatment, the complexity and diversity of quasispecies were lower in isolates from responders than in those from non-responders, particularly in the V3 domain, where differences in nucleotide entropy (0?35 vs 0?64, P=0?003), genetic distance (0?0145 vs 0?0302, P=0?05) and non-synonymous substitutions (0?0102 vs 0?0203, P=0?036) were statistically significant. These differences became more apparent during treatment, because complexity and diversity remained stable or tended to increase in non-responders, whereas they tended to decrease in responders. These observations suggest that the composition and dynamics of HCV NS5A quasispecies, particularly in the V3 domain, may play a role in the response to combined IFN/ribavirin therapy.

The quasispecies nature of hepatitis C virus (HCV) is thought to play a central role in modulating viral functions. Recent work has linked NS5A protein with viral replication, resistance to interferon (IFN), and control of cellular growth, probably through the interaction of its protein kinase R (double stranded RNA-activated protein kinase, PKR) binding domain (PKR-bd) with cellular PKR, but knowledge of how PKR-bd viral population evolves during disease progression is limited. Since we have previously described an association between amino acid composition of the PKR-bd and the presence of HCC, in this report we further investigated the dynamic behavior of viral population parameters by sequencing an average of 20 clones per sample in 27 samples from 19 untreated patients with different degrees of liver disease, 8 of whom were followed over time. Viral population parameters varied widely from patient to patient, but no differences were observed in the complexity, diversity, types of nucleotide changes, or evolutionary pattern of the quasispecies according to the stage of liver disease. In five samples, we detected "quasispecies-tails"; that is, clones whose minimum genetic distance to the remaining clones of their own quasispecies were higher than the maximum genetic distance found between any other two clones of the same sample. In summary, independent of the degree of liver disease, or the mutations detected within the consensus sequence of the PKR-bd, the NS5A of HCV presents a flexible and variable quasispecies structure that remains largely stable during the natural course of an HCV infection, highlighting the central role of NS5A protein in viral life cycle.

The features of Hepatitis C virus (HCV) quasispecies within an envelope segment including the hypervariable region 1 were analysed at an early time point post-infection in seven patients that acquired HCV from a single common donor during a nosocomial outbreak. The grouping of patients according to viral load was reflected in the structure of the quasispecies. A higher viral load correlated with the presence of a predominant HCV genome and a corresponding lower quasispecies complexity. The quasispecies complexity itself was not correlated with HCV clearance or persistence. Thus, the relationship between an intrapatient HCV quasispecies and the clinical outcome of an HCV infection is more complex than previously anticipated. INTRODUCTIONHepatitis C virus (HCV) is an enveloped positive-strand RNA virus of the family Flaviviridae. Only about 15-30 % of HCV infections are spontaneously cleared, the remaining result in virus persistence with subsequent development of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Alter et al., 1999). Because worldwide over 170 million people are infected carriers, HCV is a major health problem and a key issue in antiviral research.The mechanisms responsible for the high rate of viral persistence are thought to be the result of a complex host-virus interaction early after infection (Racanelli & Rehermann, 2003). However, little is known about these early virus and host determinants because the acute phase of infection is often asymptomatic and thus most diagnoses are made during the chronic stage, i.e. months or years after the events that determined the clinical course of the infection. While HCV induces strong humoral and cellular immune responses, their roles in virus clearance or persistence have not been fully elucidated. Studies in humans and chimpanzees indicate that a robust intrahepatic CD4 + and CD8 + T-cell response during the first weeks after infection is associated with viral clearance (Cooper et al., 1999;Major et al., 2004;Thimme et al., 2002). Also, antibodies may play a role here because an early antibody recognition of the hypervariable region 1 (HVR1) in the envelope E2 protein was correlated with virus clearance (Allander et al., 1997), and infection of chimpanzees could be inhibited by a human hyperimmune serum against HVR1 (Farci et al., 1996). In line with this are studies on the rate of HVR1 evolution that have suggested that the HVR1 region is under immune pressure exerted by neutralizing antibodies (Booth et al., 1998;Kato et al., 1993; Shimizu et al., 1994;Weiner et al., 1992). Nevertheless, by using infectious retroviral pseudotypes, a recent study has shown that neutralizing antibody responses early after infection do not seem to play a role in the resolution of an acute infection (Logvinoff et al., 2004 aspect is that HCV seems to have a wide cell tropism and can infect not only hepatocytes but also cells of the immune system (Bain et al., 2001;Sung et al., 2003). Another important feature is that HCV behaves in infected patients as a c...