Located within 5' untranslated region of HCV RNA is internal ribosome entry site (IRES) which directs cap-independent translation of viral polyprotein. Mutations in IRES sequence have been shown to cause changes in efficiency of protein translation in vitro in few instances. No study has been done to investigate association between frequency of nucleotide sequence variations in IRES region of HCV-3 RNA and response to pegylated interferon-α plus ribavirin therapy. Hence, this study was planned to analyze relationship between frequency of nucleotide sequence variations of HCV-3 IRES region and response to therapy. Twenty-seven HCV-3 patients were studied, of whom 19 responded to therapy and 8 did not. Alanine aminotransferase and aspartate aminotransferase levels were significantly lower in responders compared to non-responders. HCV RNA detection and genotyping was performed by nested-PCR and RFLP respectively. Viral load quantification in pre and post therapy samples was done by real time PCR. The viral load was significantly lower in the patients after treatment as compared to before treatment. HCV IRES region from pre-treatment sera of 27 HCV-3 infected patients was amplified by nested PCR and sequenced. Secondary structure of IRES region of HCV-3 was predicted using the M fold Web Server. Mutational analysis revealed hot spot of mutations in HCV-3 IRES region from 40-80 and 210-280 nucleotides. Though more mutations were found in non-responders as compared to responders, this difference was statistically insignificant. Therefore, in addition to IRES region of HCV-3, some other host and viral factors may contribute to therapy outcome.

The clinical exploitation of type I interferon (IFN) as an antiviral and antineoplastic agent is based on the properties originally attributed to this cytokine family, with schedules reflecting only their antiviral and antiproliferative activities. Nevertheless, type I IFN has emerged as a central activator of the innate immunity. As current schedules of treatment for chronic hepatitis C and for hematological and solid tumors, based on the continuous administration of recombinant type I IFN or pegylated formulations, disregard viral resistance, host genetic variants predicting treatment outcome and mechanisms of refractoriness, new administration schedules, the combination of type I IFN with new drugs and the increased monitoring of patients' susceptibility to type I IFN are expected to provide a new life to this valuable cytokine.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV is able to evade host defense mechanisms, including both innate and acquired immune responses, to establish persistent infection, which results in a broad spectrum of pathogenicity, such as lipid and glucose metabolism disorders and hepatocellular carcinoma development. The HCV genome is characterized by a high degree of genetic diversity, which can be associated with viral sensitivity or resistance (reflected by different virological responses) to interferon (IFN)-based therapy. In this regard, it is of importance to note that polymorphisms in certain HCV genomic regions have shown a close correlation with treatment outcome. In particular, among the HCV proteins, the core and nonstructural proteins (NS) 5A have been extensively studied for their correlation with responses to IFN-based treatment. This review aims to cover updated information on the impact of major HCV genetic factors, including HCV genotype, mutations in amino acids 70 and 91 of the core protein and sequence heterogeneity in the IFN sensitivity-determining region and IFN/ribavirin resistance-determining region of NS5A, on virological responses to IFN-based therapy.

Hepatitis C virus (HCV) is considered a significant risk factor in HCV-induced liver diseases and development of hepatocellular carcinoma (HCC). Nucleotide substitutions in the viral genome result in its diversification into quasispecies, subtypes and distinct genotypes. Different genotypes vary in their infectivity and immune response due to these nucleotide/amino acid variations. The current combination treatment for HCV infection is pegylated interferon α (PEG-IFN-α) with ribavirin, with a highly variable response rate mainly depending upon the HCV genotype. Genotypes 2 and 3 are found to respond better than genotypes 1 and 4, which are more resistant to IFN-based therapies. Different studies have been conducted worldwide to explore the basis of this difference in therapy response, which identified some putative regions in the HCV genome, especially in Core and NS5a, and to some extent in the E2 region, containing specific sequences in different genotypes that act differently with respect to the IFN response. In the review, we try to summarize the role of HCV proteins and their nucleotide sequences in association with treatment outcome in IFN-based therapy.

Chronic hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In order for HCV to persist, the virus must escape immune recognition or inhibit the host immune response. The NS5A protein contains the interferon sensitivity-determining region (ISDR) and is able to repress dsRNA-dependent protein kinase (PKR) thus influencing the response to interferon (IFN) therapy. Patients who respond to IFN therapy have stronger antibody reactivity against the NS5A compared to IFN non-responders. Therefore, given the possible role for the ISDR in IFN resistance and differential antibody reactivity, it is possible that variation in ISDR may be involved in viral immune escape and development of persistent HCV infection employing aspects of host mimicry. In this study, pre-treatment samples obtained from HCV infected patients were used to investigate the effect of different NS5A ISDR variants on the IFN antiviral response and their involvement in immune evasion. The NS5A was identified as a homologue of the variable region of immunoglobulins (Ig). The IFN resistant genotypes had higher levels of similarity to Ig compared to IFN sensitive genotypes. Expression of NS5A-6003 (HCV genotype 1b) and NS5A-6074 (HCV genotype 2a) was able to rescue vesicular stomatitis virus (VSV) from IFN inhibition and restore luciferase activity. A correlation between Ig-like NS5A structure and also antibody response with the outcome of IFN treatment was observed.

As an RNA virus, hepatitis C virus (HCV) shows a characteristically high level of nucleotide diversity. Accumulation of nucleotide substitutions in the virus has resulted in diversification into quasispecies, subtypes and distinct genotypes. Pathobiological studies linking nucleotide and amino acid sequences with clinical findings have identified relationships between certain genotypes and characteristic biological properties. Genotype 3 HCV infection was found to be associated with a high level of liver steatosis. Genotypes 1 and 4 were found to be more resistant to interferon (IFN) based therapies than genotypes 2 and 3. Studies of genotype 1 sequences obtained from patients treated with IFN have identified a relationship between favorable response to interferon therapy and amino acid substitutions in the NS5A region (interferon response determining region; ISDR). Further studies have identified a relationship between the effect of IFN therapy and other regions of the NS5A protein. More recently, a relationship has been found between poor response to peg-IFN plus ribavirin combination therapy and substitutions at amino acid 70 and 91 in the core protein. Furthermore, a correlation between human genetic variation in the IL28B (IFN-lamda 3) locus and core amino acid substitutions has been characterized. In this review we briefly summarize the discovery, classification and nomenclature of HCV genotypes and subtypes. We also discuss amino acid substitutions within specific regions that have been reported to be associated with outcome of IFN and peg-IFN plus ribavirin combination therapy.

  Several host and viral factors have been reported to influence the effectiveness of pegylated interferon plus ribavirin combination therapy for chronic hepatitis C. In Japan, where the age of treated patients is comparatively high, recent studies have reported poor response to treatment in older female patients, but little is known about the relationship between advanced age in women and previously reported factors.

  Using a database of 1167 patients chronically infected with hepatitis C virus (HCV) genotype 1b, we analyzed the amino acid sequences of the HCV core protein and interferon sensitivity determining region (ISDR) and examined the relationships among predictive factors.

  The proportion of patients with substitutions at core 70, which is associated with poor response to pegylated interferon plus ribavirin therapy, increased with age only in female patients. A similar trend was observed for ISDR wild type (wt). We also found that core 70 wt is associated with core 91 wt (P = 5.4 × 10(-9) ) as well as ISDR wt (P = 0.025). HCV RNA levels were higher in patients with core and ISDR wt (P < 0.001). Furthermore, core amino acid mutations were associated with advanced fibrosis and higher inflammatory activity (P = 0.028 and 0.048, respectively) as well as higher gamma-glutamyltranspeptidase, alanine aminotransferase and low-density lipoprotein cholesterol levels (P < 0.001, 0.006 and 0.001, respectively).

  A combination of factors account for poor response rate in older female patients in Japan. Elucidating the relationship between amino acid substitutions and metabolic alteration is an important step in understanding the mechanism of HCV interferon resistance.

In the non-structural protein 5A (NS5A) of hepatitis C virus (HCV), mutations within the interferon sensitivity-determining region (ISDR), the PKR-binding domain (PKR-BD), the variable region 3 (V3), and the interferon/ribavirin resistance-determining region (IRRDR) have been correlated with the IFN-based therapy response. In Tunisia, where a high prevalence of HCV-1b has been found, no data regarding the implication of NS5A in treatment response were available. The current study examined the relationship between the pre-treatment mutation number within ISDR, PKR-BD, V3, IRRDR, as well as in the entire ISDR-V3 region of NS5A (aa 2209-2379) and the response to the 48-week course of combined IFN plus ribavirin therapy in 15 HCV-1b-infected Tunisian patients. Referring to HCV-J sequence, a significant high genetic variability was observed within PKR-BD in the sustained virological responder patients compared to non-responders (P = 0.040). More importantly, when considering the entire region from ISDR to V3, referred to as NS5A(ISDR-V3), a clear difference in the mutation number was observed between sustained virological responders (19.6 +/- 3.16) and non-responders (15.0 +/- 1.41) (P = 0.002). Additionally, a more detailed analysis of NS5A(ISDR-V3) region revealed an elevated degree of mutation rate within the region located between amino acids 2282 and 2308 (P = 0.0006). Interestingly, an analysis of specific amino acid variations defined proline and serine at position 2300 as signature patterns for sensitive and resistant strains, respectively. The genetic variability within the NS5A region of HCV-1b strains was associated with the response to the combined IFN plus ribavirin therapy in our Tunisian cohort.

Sequence variation among different hepatitis C virus (HCV) isolates has adaptive significance and reflects the modes and intensities of selection mechanisms operating on the virus. In this work, we sought to investigate using classical population genetics parameters, the genetic variability of HCV genotype 1 using the 5' UTR and NS5A regions from treatment non-responding and responding groups of patients. Both regions showed low genetic variability and the 5' UTR showed neutral deviation. No differences were observed in the nonsynonymous/synonymous nucleotide substitution ratio among groups for NS5A. The analysis of molecular variance test of the 5' UTR region showed an 11.94% variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Mutations in several subgenomic regions of hepatitis C virus (HCV) have been implicated in influencing the response to interferon (IFN) therapy. Sequences within HCV NS5A (PKR binding domain [PKRBD], IFN sensitivity-determining region [ISDR], and variable region 3 [V3]) were analyzed for the pretreatment serum samples of 60 HCV genotype 1-infected patients treated with pegylated IFN plus ribavirin (1b, n = 47; 1a, n = 13) but with different treatment outcomes, those with sustained virologic responses (SVR; n = 36) or nonresponders (NR; n = 24). Additionally, the sequence of the PKR/eIF-2alpha phosphorylation homology domain (E2-PePHD) region was determined for 23 patients (11 SVR and 12 NR). The presence of > 4 mutations in the PKRBD region was associated with SVR (P = 0.001) and early virologic responses (EVR; 12 weeks) (P = 0.037) but not rapid virologic responses (4 weeks). In the ISDR, the difference was almost statistically significant (68% of SVR patients with mutations versus 45% without mutations; P = 0.07). The V3 region had a very high genetic variability, but this was not related to SVR. Finally, the E2-PePHD (n = 23) region was well conserved. The presence of > 4 mutations in the PKRBD region (odds ratio [OR] = 9.9; P = 0.006) and an age of < or = 40 years (OR = 3.2; P = 0.056) were selected in a multivariate analysis as predictive factors of SVR. NS5A sequences from serum samples taken after 1 month of treatment and posttreatment were examined for 3 SVR and 15 NR patients to select treatment-resistant viral subpopulations, and it was found that in the V3 and flanking regions, the mutations increased significantly in posttreatment sera (P = 0.05). The genetic variability in the PKRBD (> 4 mutations) is a predictive factor of SVR and EVR in HCV genotype 1 patients treated with pegylated IFN and ribavirin.

The interferon sensitivity determining region (ISDR) in the non-structural 5A protein (NS5A) gene of hepatitis C virus genotype 1b (HCV-1b) has been demonstrated in Japanese patients; however, contradictory data have been reported from other parts of the world, particularly from Europe and the USA. This study investigated whether mutations in the ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic HCV-1b infection.

Forty-five Chinese patients with chronic HCV infection were enrolled in a retrospective study. Of these patients, 20 with HCV-1b infection completed an interferon monotherapy course and 6-month follow-up. The pretreatment mutations in the nucleic acid sequence of NS5A 2209-2248 (ISDR) of HCV-1b were identified by sequencing RT-PCR products followed by comparison with the prototype sequence (HCV-J). Correlation with the clinical efficacy of interferon alpha therapy was then analyzed retrospectively.

The statistical analysis indicated a significant correlation between the mutations in ISDR of HCV-1b and sustained virological response to interferon alpha treatment (P < 0.05), and an association of the mutations in ISDR with the pretreatment serum alanine aminotransferase levels (P < 0.01) in Chinese patients with chronic HCV-1b infection.

The results support the concept that the response of HCV-1b infected patients towards interferon therapy is influenced by the mutations within the ISDR of NS5A gene.

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon alpha resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon alpha-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon alpha/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194-2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients.

Etiologic variations of hepatocellular carcinoma (HCC) exist in different geographic areas of the world. Hepatitis B virus infection is associated with HCC. However, hepatitis C virus (HCV) infection plays an increasingly more important role in the development of HCC and is associated with more than 30% of HCC in Taiwan. The prevalence of HCV infection and HCV genotypes vary in different geographic areas. The prevalence of HCV genotype 1b (HCV-1b) was around 50-70% in Taiwan and even varied in different townships. In addition to host factors, HCV genotypes may be associated with the development of HCC. In our study, the prevalence of HCV-1b in patients with HCC was significantly higher than in those with liver cirrhosis and chronic hepatitis; multivariate analysis revealed that the disease severity was significantly correlated with age and HCV-1b. Furthermore, HCV-1b was associated with a lower response rate to interferon (IFN) therapy than HCV-2. Our study has demonstrated that mutations in the IFN sensitivity-determining region, spanning nucleotides 2,209-2,248 in the NS5A region, correlate with the sustained virological response to combination therapy with IFN and ribavirin in patients with chronic HCV-1b infection in Taiwan. A third-generation enzyme immunoassay for antibody to HCV can be used to predict viremia and monitor the virological response.