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Aim: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA-dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear., Methods: HCV quasispecies were studied by cloning and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-alpha2b for 3 mo followed by IFN-alpha2b alone or combined IFN-R therapy for 9 additional months. Patients were categorized into two groups based on their response to the treatments: 7 with sustained virological response (SVR) (quasispecies = 150) and 3 non-responders (NR) to IFN-R (quasispecies = 106)., Results: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispecies during therapy. Analysis of amino acids substitutions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate the two groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients., Conclusion: These results suggest that detailed molecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.

We investigated whether an HCV NS3 protease quasispecies heterogeneity was associated with progression from viral cirrhosis to hepatocellular carcinoma (HCC). The NS3 protease quasispecies structure of 10 HCV-1b cirrhotic patients (controls) was compared with that of 10 paired HCV-1b cirrhotic patients who displayed progression to HCC (cases). NS3 protease genetic complexity and diversity did not differ significantly between cases and controls. Amino acid substitutions were detected at 20 (11%) and 25 (14%) sites in at least two variants of the NS3 protease in cases and controls, respectively. Significant differences in the percentage of substituted clones were observed for 10 NS3 sites. Mutations Y56F, I71V, T72I, Q86P, P89S, S101G/D, R117H, S122G/T/N, V132I and V170I were more frequently observed in the NS3 protease sequences of controls than in those of cases. Residue V107 was substituted in NS3 cases but not in controls. However, these differences did not allow the definition of a specific NS3 profile related to HCC occurrence. The NS3 secondary structure B1-1 previously identified as potentially predictive of HCC was identified with a higher frequency in cases quasispecies (84.2%) than in controls (55.9%; P < 0.05). Our results suggest that there may be a relationship to fibrosis progression when diversity parameters are considered together with secondary structure profiles. Further investigations are required to determine the cellular interactions of HCV NS3 protease in the context of carcinogenesis.

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5' noncoding (5' NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5' NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5' NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Aim of the Study: The hepatitis C virus (HCV) non-structural NS5A protein has been controversially implicated in the resistance of HCV to interferon therapy in clinical studies. In Japan, mutations in the interferon sensitivity-determining region (ISDR) in the NS5A gene were associated with response to interferon therapy in patients infected with genotype 1b. In contrast, studies from Europe did not confirm such association. More recently, it has been suggested that the V3 domain outside the putative ISDR might also have amino acids changes that may be involved in the resistance to IFN. In this study, the relationship between NS5A mutations in ISDR and V3 domains and virological response to therapy were investigated., Materials and Methods: The NS5A gene was sequenced from 35 HCV genotype 1b infected patients at D0 of a prospective clinical trial of interferon therapy and interferon plus Ribavirin combination therapy., Results: In the ISDR domain, we did not observe any significant differences in amino acids changes between responders (1.7 +/- 1.8, n = 19, range 0-6) and non-responders (1.1 +/- 0.8, n = 14, range: 0-3), (P = 0.483), to therapy before the beginning of treatment. In the V3 domain, we found more mutations in responders (6.5 +/- 1.9, range: 2-11) than in non-responders (4.7 +/- 1.2, range: 3-8) (P = 0.0013), before the beginning of treatment., Conclusion: Our results confirm that, in Europe, the ISDR domain is not predictive for treatment success but suggest that the V3 domain have greater variability in responders than non-responders.