SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background  Most reports suggest that mutations in the interferon sensitivity-determining region (ISDR) correlate with response to conventional interferon-based therapies in hepatitis C virus-1b (HCV-1b) patients. However, the correlation between ISDR region mutations and response to pegylated interferon plus ribavirin therapy in HCV-1b patients remains unclear.

Aim  To assess whether ISDR mutations correlate with response to Peg interferon plus ribavirin therapy in HCV-1b patients.

Patients and methods  Sixty HCV-1b naive patients who had undergone 6 months of Peg interferon α-2b plus ribavirin and a 6-month follow-up were enrolled. The amino acid sequences of the nonstructural 5A-interferon-induced RNA-dependent protein kinase (NS5A–PKR)-binding domain were determined by polymerase chain reaction and sequencing.

Results  Thirty (50%) patients achieved sustained virological response (SVR). Univariate analysis showed that the proportion of patients with ISDR mutations ≥4 and rapid virological response rate was higher in the sustained virological response group than in the non-SVR group. Viral load was lower in the SVR group than in the non-SVR group. Multivariate analysis revealed that ISDR mutations ≥4 and ribavirin ≥14 mg/kg/day were independent predictors of SVR.

Conclusion  Mutations of the ISDR correlate with SVR to Peg interferon α-2b plus ribavirin therapy in HCV-1b patients.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Hepatitis C virus (HCV) infection frequently causes chronic liver disease leading to cirrhosis and hepatocellular carcinoma over a 20- to 30-year period.1–3 Interferon (IFN) therapy alone or in combination with ribavirin is currently the standard treatment for chronic HCV infection. However, this approach is not effective in all patients. Parameters reportedly predictive of IFN response including age, pre-treatment viral load, HCV-RNA at week 12, fibrosis stage, body mass index, IFN receptor and HCV genotype.4–10 Most patients with HCV genotypes 2 and 3 infection can achieve sustained virological response (SVR) by current combination therapy of pegylated IFN (PegIFN) plus ribavirin. Conversely, HCV-1b infection, the prevalent genotype in Taiwan, Japan as well as Southern and Eastern Europe,11–14 is predictive of poor response. SVR is achieved in <10% of cases after conventional IFN monotherapy and in approximately 40–50% of cases after combination therapy using PegIFN plus ribavirin in HCV-1b infection.15–18 The significant adverse effects and expense19 of PegIFN and ribavirin can be minimized by accurately predicting which HCV-1b patients are IFN-sensitive and which harbour resistant isolates.

Recent studies have attempted to explain the resistance of HCV-1b infection to IFN therapy. An IFN sensitivity-determining region (ISDR) between codon 2209 and 2248 of the nonstructural 5A (NS5A) domain of the HCV-1b genome has been identified.20 The NS5A with a wild-type ISDR sequence has been reported to inhibit IFN-induced RNA-dependent protein kinase (PKR), an early effector of the IFN-mediated anti-viral response. The PKR mediates various aspects of the anti-viral effect of IFN with the blockade of viral protein synthesis and viral replication within the infected cells.21 Previous Japanese studies suggest that patients infected with the wild-type ISDR strain respond poorly to IFN, while patients exhibiting more than one amino acid mutation have an increased incidence of SVR. Although the correlation has not been confirmed in a Caucasian population,22, 23 major differences in IFN treatment protocols and study designs as well as the lower prevalence of the mutant ISDR type observed in Western countries (0–17%) compared with Japan (16–42%) may at least partially explain this discrepancy.23–31 A recent meta-analysis of ISDR sequences suggests a correlation with response to conventional IFN, which was not evident in earlier studies because of inadequate sample size.32

The PegIFN is produced by the binding of the inert polyethylene glycol moiety to IFN molecules, thus decreasing renal clearance, altering metabolism and increasing the half-life of the PegIFN molecule.33 Large randomized-controlled trials have achieved higher SVR rates using PegIFN combined with ribavirin than conventional IFN combined with ribavirin or PegIFN used alone.17, 18

Although the correlation between ISDR mutations and response to conventional IFN-based therapy in HCV-1 patients is well documented, the correlation between ISDR mutations and response to PegIFN plus ribavirin therapy in HCV-1 patients is rarely reported.34 This study assessed whether ISDR mutations correlated with the response to PegIFN plus ribavirin therapy in HCV-1b patients.

Patients and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

A total of 826 naive patients with chronic HCV infection received IFN-based therapy at this hospital between April 2002 and August 2006. Chronic hepatitis C was diagnosed by elevated serum aminotransferase levels, histological examination and the detection of serum HCV-RNA. Each patient was confirmed positive for anti-HCV antibody by third-generation enzyme-linked immunosorbent assay. Patients were excluded, if they tested positive for serum hepatitis B surface antigen, anti-HIV antibody or exhibited other causes of hepatocellular injury (e.g. any history of alcoholism, autoimmune hepatitis, primary biliary cirrhosis or treatment with hepatotoxic drugs). Of 826 patients, 363 had genotype 1b infection (excluding co-infection with other HCV genotypes). Of 363 genotype 1b patients, 93 patients received PegIFNα-2b plus ribavirin therapy (excluding patients who initially received PegIFNα-2b but were subsequently shifted to conventional IFN or PegIFNα-2a during treatment). The PegIFNα-2b (Peg-Intron, Schering-Plough Corporation, Kenilworth, NJ, USA) was given at 1.5 μg/kg subcutaneously once weekly. Ribavirin (Rebetol, Schering-Plough, Auxerre, France) was given at 1000–1200 mg orally daily. Sixty HCV-1b patients who had completed a 6-month treatment course and a 6-month follow-up, were enrolled in this study. Thirty-four patients were male, and 26 patients were female [median age: 56 (34–75) years]. Cases of SVR were defined by absence of serum HCV-RNA 24 weeks after treatment. Rapid virological response (RVR) was defined by absence of serum HCV-RNA at week 4 of treatment.

Qualitative and quantitative assay of HCV-RNA and HCV genotyping

Serum was prepared in a laminar flow bench and frozen at −80 °C until use. The RNA was extracted from serum. Qualitative HCV-RNA was assessed by polymerase chain reaction (PCR)-based assay (cobas amplicor hepatitis c virus test, version 2.0, Roche Molecular Systems, Branchburg, NJ, USA) with an 50 IU/mL lower detection limit. Quantitative HCV-RNA were determined by branched DNA assay (versant hcv rna, version 3.0, Bayer Diagnostics, Tarrytown, NY, USA) with a detections range of 615–700 000 IU/L (1 IU/L could be converted to 5.2 copies/mL).35 Genotyping of HCV was performed by line-probe assay (Inno LiPA HCV II, Innogenetics, Ghent, Belgium) based on reverse hybridization of PCR amplicons on a nitrocellulose strip coated with genotype-specific oligonucleotide probes.36

Amplification of HCV NS5A–PKR-binding domain (aa 2209–2274) by RT-PCR and sequence analysis

The RNA was extracted from 140 μL of serum using QIAamp Viral RNA Mini Kits (Qiagene, Inc., Hilden, Germany). One-tenth of extracted RNA was used for nested RT-PCR. The NS5A fragments were amplified using the external 5′-outer primer (5′-TGGATGGAGTGCGGTTGCACAGGTA; nt 6703–6727); 3′-outer primer (5′-TCTTTCTCCCTGGAGGTGGTATTGG; nt 7296–7320); 5′-inner primer (5′-CAGGTACGCTCCGGCGTGCA; nt 6722–6741) and 3′-inner primer (5′-GGGGCCTTGGTAGGTGGCAA; nt 7275–7294).

All amplifications entailed 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. Nested-PCR products were sequenced directly using an ABI Prism 377 automated DNA sequencer and DNA dye terminator cycle sequencing kit (Perkin-Elmer, Oak Brook, IL, USA). The sequencing assay was validated, and sequence analyses were performed in duplicate.

Statistical analysis

Chi-squared test, Fisher’s exact test and the Mann–Whitney U-test were used for statistical analysis. Receiver operating characteristics curves were constructed to identify the best cut-off values for mean daily ribavirin dose in SVR patients. Independent factors possibly affecting response to combination therapy were determined by stepwise multiple logistic regression analysis. A P-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Comparison of baseline features in patients with and without SVR

The clinical features of patients in relation to SVR are shown in Table 1. Thirty (50%) patients achieved SVR, and 30 (50%) did not. The HCV-RNA at week 4 was available in 38 patients. Twenty-two (58%) patients achieved RVR, and 16 did not. In patients who achieved RVR, the positive predictive value of SVR was 86%, and negative predictive value was 88%. The SVR and non-SVR patients did not significantly differ in terms of age, gender, total IFN dose, weekly IFN dose per kilogram, total ribavirin dose, median number of amino acid mutations in the NS5A–PKR-binding domain, proportion of patients with ribavirin ≥14 mg/kg/day or incidence of liver cirrhosis. However, the proportion of patients with ISDR mutations ≥4 and RVR rate was higher in the SVR group than in the non-SVR group. Viral load was lower in the SVR group than in the non-SVR group. The baseline NS5A–PKR-binding domain was analysed by direct sequencing, and the deduced amino acid sequences of 60 HCV-1b patients were aligned and compared with that of the HCV-1b prototype (HCV-J; Figure 1).

Table 1.   Comparison of baseline features in patients with sustained virological response (SVR) and non-SVR
 SVR (n = 30)Non-SVR (n = 30)P-value
  1. RVR, rapid virological response; ISDR, interferon sensitivity-determining region; NS5A, nonstructural 5A; PKR, interferon-induced RNA-dependent protein kinase.

  2. Data were presented as the median and the interquartile range [IQR; range from the 25th (Q1) to the 75th (Q3) percentile].

Age (years)54.5 (50.5–65.0)59.5 (47–65.3)0.564
RVR vs. non-RVR (N)19:23:14<0.001
Gender (male/female)19:1115:150.297
HCV-RNA (log copies/mL) 6.1 (5.4–6.6)6.6 (5.9–6.9)0.044
Cirrhosis, N (%)7 (23)13 (43)0.10
Amino acid mutation in:
 ISDR ≥ 4, N (%)9 (30)2 (7)0.02
 PKR-binding domain 6 (4–9)5 (4–6)0.094
 PegIFNα-2b total dose (μg)2330 (1920–2400)2400 (2000–2400)0.210
 PegIFNα-2b (μg/kg/week)1.4 (1.3–1.6)1.4 (1.3–1.6)0.620
 Ribavirin total dose (mg)162 400 (129 050–171 150)136 200 (117 600–168 000)0.113
 Ribavirin daily dose (mg/kg/day):  ≥14 vs. <14 (N)21:914:160.067
image

Figure 1.  Alignment of amino acid of NS5A–PKR-binding domain (aa 2209–2274) before combination therapy of pegylated interferon and ribavirin in 60 hepatitis C virus-1b patients. ‘R’ represents sustained virological response (SVR); ‘N’ represents non-SVR.

Download figure to PowerPoint

Comparison of baseline features in subgroups classified by ISDR mutations

After dividing these patients into three categories (Table 2), as described by Enomoto et al.,20 12 (20%) patients had wild-type sequence with no amino acid changes; 11 (18%) had mutant type with four or more amino acid changes; 37 (62%) had an intermediate type with one to three amino acid changes. The three patient groups did not significantly differ in age, gender, cirrhosis rate, IFN dose, ribavirin dose or RVR rate. However, the SVR rate was higher in patients with mutant-type ISDR sequence (nine of 11, 82%) than in those with intermediate-type ISDR sequence (16 of 37, 43%) (P = 0.025). Viral load was lower in those with mutant type ISDR sequence than in those with intermediate type ISDR sequence (P = 0.008).

Table 2.   Comparisons of baseline features in subgroup patients as divided by the types of ISDR mutations defined by Enomoto et al.
ISDR typeWild (n = 12)Intermediate (n = 37)Mutant (n = 11)P-value
  1. HCV, hepatitis C virus; ISDR, interferon sensitivity-determining region; SVR, sustained virological response; RVR, rapid virological response.

  2. * Significant differences between intermediate type vs. mutant type.

  3. † Significant differences between intermediate type and mutant type.

  4. Data were presented as the median and the interquartile range [IQR; range from the 25th (Q1) to the 75th (Q3) percentile].

SVR, N (%) 5 (42)16 (43)*9 (82)*0.025*
RVR, N (%)4 (67)13 (50)5 (83)0.294
Age (years)60.5 (54.3–66.8)56 (46.0–64.5)55.0 (49.0–70.0)0.254
Gender (male/female)6:622:156:50.837
HCV-RNA (log copies/mL)6.1 (5.7–6.5)6.5 (5.9–6.8)†5.6 (4.9–6.1)†0.008†
Cirrhosis, N (%)4 (33)12 (32)4 (36)0.971
PegIFNα-2b total dose (μg)2400 (2075–2460)2400 (1950–2400)2240 (1920–2400)0.274
PegIFNα-2b (μg/kg/week)1.5 (1.3–1.6)1.5 ± 1.91.4 (1.3–1.6)0.729
Ribavirin total dose (mg)155 500 (119 950–168 000)149 800 (128 300–168 900)123 600 (84 400–168 000)0.116
Ribavirin (mg/kg/day): ≥14 vs. <147:524:134:70.242

Multivariate analysis of pre-treatment factors associated with SVR

Stepwise multiple logistic regression analysis of the factors including age, gender, viral load, IFN dose, ribavirin dose, cirrhosis rate and amino acid mutations in ISDR revealed that ISDR mutations ≥4 and ribavirin ≥14 mg/kg/day were the independent predictors of SVR (Table 3). For predicting SVR, ISDR mutations ≥4 had a positive predictive value of 82%, a negative predictive value of 57%, a sensitivity of 30% and a specificity of 93%.

Table 3.   Stepwise multiple logistic regression analysis of factors associated with sustained viral response
 OR95% CIP-value
  1. ISDR, interferon sensitivity-determining region.

Ribavirin (mg/kg/day): ≥14 vs. <144.81.33–17.240.017
Amino acid mutations in ISDR: ≥4 vs. <4 11.51.84–72.040.009

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In Taiwan, the Bureau of National Health Insurance provides reimbursement for 6 months of PegIFN plus ribavirin therapy for all chronic hepatitis C patients, irrespective of genotypes. Therefore, most HCV-1b patients receive a fixed duration of 6 months treatment, irrespective of early virological response (defined at week 12 as a minimum 2-log decline from baseline HCV-RNA level). Previous studies report a 41–42% SVR rate in HCV-1 patients for 24 weeks of conventional IFN-α plus ribavirin therapy and a 42–66% SVR rate for an equivalent period of PegIFN-α plus ribavirin.37–39 In this study, the SVR rate was 50% in HCV-1b patients, which is consistent with previous reports.37–39

The resistance to anti-viral therapy such as IFN in chronic HCV infection remains an important and controversial issue. Patients infected with genotypes 2 or 3 exhibit a much better response to anti-viral treatment than patients infected with genotype 1.17, 18 Therefore, HCV-1 isolates are presumably not susceptible to the anti-viral effects of IFN-α. Results of clinical and in vitro studies show that the NS5A protein of HCV-1 isolates may be involved in HCV resistance to IFN-α.22, 23, 40–42 At least two possible mechanisms for inhibiting the anti-viral actions of IFN-α by NS5A have been reported. First, NS5A is known to inhibit kinase activity by interacting with PKR.21, 43 Second, NS5A also induces expression of the proinflammatory chemokine interleukin-8, which is associated with inhibition of IFN-α action.44

Enomoto et al., in a comparative study of the full-length HCV-1b genome of IFN-α sensitive and resistant strains, demonstrated an association between ISDR, a 40-amino-acid sequence in the NS5A region (NS5A 2209–2248) and IFN sensitivity.20, 24 Enomoto et al., found that patients infected with the wild-type ISDR sequence were unresponsive to IFN monotherapy, whereas all patients infected with the ‘mutant type’ exhibited SVR.20, 24

In this study, according to the Enomoto classification, the proportion of patients with mutant type ISDR in the SVR patient group was significantly higher than that in the non-SVR patient group (30% vs. 7%, P = 0.02). The SVR rate in the mutant-type patients was significantly higher than that of intermediate-type patients (82% vs. 43%, P = 0.025). Further, after adjusting ribavirin dose, multivariate analysis indicated that ISDR mutations ≥4 were still highly predictive of SVR (odds ratio: 11.5, 95% confidence interval: 1.84–72.04, P = 0.009). This finding suggests that inhibiting PKR activity by NS5A may play an important role in the response to combined PegIFNα-2b and ribavirin therapy in HCV-1b patients.

The biological role of ISDR remains unclear. Studies revealed that the NS5A protein binds and represses PKR. The PKR-binding domain of NS5A, spanning amino acid positions 2209–2274, comprises the ISDR, and the NS5A–PKR interaction is disrupted by ISDR mutation, which corresponds to IFN sensitivity.21

Enomoto et al. found that HCV-1b patients with wild-type ISDR sequence were unresponsive to conventional IFN monotherapy.24 However, a previous report by the authors indicated 23% of HCV-1b patients with wild-type ISDR sequence had SVR after conventional IFN plus ribavirin therapy,45 whereas the figure in this study was 42%. In vitro studies have also shown that NS5A regions involved in the inhibition of PKR activity may not be required to inhibit the anti-viral actions of IFN.42, 46 Indeed, according to western reports, most patients with HCV-1 infection and SVR exhibit only a small number of mutations (one to three amino acid mutations) detectable within the ISDR.32, 41, 47–49 Thus, the sensitivity to IFN α-based therapy in these patients is not convincingly explained by the ISDR hypothesis. Therefore, elucidating the role of NS5A/PKR interaction requires investigation of other mechanisms whereby NS5A protein antagonizes IFN-α activity (e.g. interleukin-844). This may explain why 42% of wild-type ISDR patients have SVR and that PegIFN/ribavirin may be able to overcome the wild type in one-third of cases. However, further study is needed to clarify this hypothesis.

Recent studies show that a virological response at week 4 (RVR) is highly predictive of SVR.37–39 However, the association between ISDR mutations and RVR is rarely reported. In this study, the mutant type ISDR patients had a higher RVR rate than nonmutant type ISDR patients, although the difference did not reach significant difference. The lack of correlation between ISDR mutations and RVR may have been because of inadequate sample size. Further large-scale studies are needed to clarify this issue.

Recent studies have focused on the PKR-binding domain in the NS5A.21 Whether the mutations in the PKR-binding domain are associated with IFN response is still controversial.49, 50 The above data showed that the median number of amino acid mutations in the PKR-binding domain did not significantly differ between the SVR and the non-SVR group patients.

In conclusion, ISDR mutations correlated with SVR to PegIFNα-2b plus ribavirin therapy in HCV-1b patients in the examined population of Taiwan patients. Thus, ISDR mutations are apparently predictive of SVR before treatment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Declaration of personal interests: None. Declaration of funding interests: This study was supported by grant NMRPD 140841 (NSC 94-2314-B-182-067) and NMRPD 160571 (NSC 96-2628-B-182-020-MY2) from Chang Gung Memorial Hospital and the National Council of Science, Taiwan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997; 349: 82532.
  • 2
    Kiyosawa K, Sodeyama T, Tanaka E, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990; 12: 6715.
  • 3
    Lee CM, Lu SN, Changchien CS, et al. Age, gender, and local geographic variations of viral etiology of hepatocellular carcinoma in a hyperendemic area for hepatitis B virus infection. Cancer 1999; 86: 114350.
  • 4
    Lin R, Liddle C, Byth K, Farrell GC. Virus and host factors are both important determinants of response to interferon treatment among patients with chronic hepatitis C. J Viral Hepat 1996; 3: 8596.
  • 5
    Pagliaro L, Craxi A, Cammaa C, et al. Interferon-alpha for chronic hepatitis C: an analysis of pretreatment clinical predictors of response. Hepatology 1994; 19: 8208.
  • 6
    Hung CH, Lee CM, Lu SN, et al. Is delayed normalization of alanine aminotransferase a poor prognostic predictor in chronic hepatitis C patients treated with a combined interferon and ribavirin therapy? J Gastroenterol Hepatol 2002; 17: 130711.
  • 7
    Shiratori Y, Kato N, Yokosuka O, et al. Predictors of the efficacy of interferon therapy in chronic hepatitis C virus infection. Tokyo-Chiba Hepatitis Research Group. Gastroenterology 1997; 113: 55866.
  • 8
    Hung CH, Lee CM, Lu SN, et al. Long-term effect of interferon alpha-2b plus ribavirin therapy on incidence of hepatocellular carcinoma in patients with hepatitis C virus-related cirrhosis. J Viral Hepat 2006; 13: 40914.
  • 9
    Poynard T, McHutchison J, Goodman Z, Ling MH, Albrecht J. Is an “a 1a carte” combination interferon alfa-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis C? Hepatology 2000; 31: 2118.
  • 10
    Lee CM, Kee KM, Hung CH, et al. Hepatic interferon receptor mRNA expression: clinical relevance and its relationship with effectiveness of interferon plus ribavirin therapy in patients with genotype 1b hepatitis C virus infection. Antivir Ther 2006; 11: 1723.
  • 11
    Nousbaum JB, Pol S, Nalpas B, Landais P, Berthelot P, Brechot C. Hepatitis C virus type 1b (II) infection in France and Italy. Collaborative Study Group. Ann Intern Med 1995; 122: 1618.
  • 12
    McOmish F, Yap PL, Dow BC, et al. Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative survey. J Clin Microbiol 1994; 32: 88492.
  • 13
    Kao JH, Chen PJ, Lai MY, et al. Genotypes of hepatitis C virus in Taiwan and the progression of liver disease. J Clin Gastroenterol 1995; 21: 2337.
  • 14
    Lee CM, Lu SN, Hung CH, et al. Hepatitis C virus genotypes in southern Taiwan: prevalence and clinical implications. Trans R Soc Trop Med Hyg 2006; 100: 76774.
  • 15
    Martinot-Peignoux M, Marcellin P, Pouteau M, et al. Pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype are the main and independent prognostic factors of sustained response to interferon alfa therapy in chronic hepatitis C. Hepatology 1995; 22: 10506.
  • 16
    Poynard T, Leroy V, Cohard M, et al. Meta-analysis of interferon randomized trials in the treatment of viral hepatitis C: effects of dose and duration. Hepatology 1996; 24: 77889.
  • 17
    Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001; 358: 95865.
  • 18
    Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 97582.
  • 19
    Cornberg M, Wedemeyer H, Manns MP. Treatment of chronic hepatitis C with pegylated interferon and ribavirin. Curr Gastroenterol Rep 2002; 4: 2330.
  • 20
    Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon sensitive and resistant hepatitis C virus 1b: sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 1995; 96: 22430.
  • 21
    Gale M Jr, Blakely CM, Kwieciszewski B, et al. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 1998; 18: 520818.
  • 22
    Duverlie G, Khorsi H, Castelain S, et al. Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity. J Gen Virol 1998; 79: 137381.
  • 23
    Zeuzem S, Lee JH, Roth WK. Mutations in the nonstructural 5A gene of European hepatitis C virus and response to interferon alfa. Hepatology 1997; 25: 7404.
  • 24
    Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996; 334: 7781.
  • 25
    Khorsi H, Castelain S, Wyseur A, et al. Mutations of hepatitis C virus 1b NS5A 2209-2248 amino acid sequence do not predict the response to recombinant interferon-alfa therapy in French patients. J Hepatol 1997; 27: 727.
  • 26
    Squadrito G, Orlando ME, Cacciola I, et al. Long-term response to interferon alpha is unrelated to “interferon sensitivity determining region” variability in patients with chronic hepatitis C virus-1b infection. J Hepatol 1999; 30: 10237.
  • 27
    Yoshioka K, Kobayashi M, Orito E, et al. Biochemical response to interferon therapy correlates with interferon sensitivity-determining region in hepatitis C virus genotype 1b infection. J Viral Hepat 2001; 8: 4219.
  • 28
    Watanabe H, Enomoto N, Nagayama K, et al. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype 1b infection. J Infect Dis 2001; 183: 1195203.
  • 29
    Hofgartner WT, Polyak SJ, Sullivan DG, Carithers RL Jr, Gretch DR. Mutations in the NS5A gene of hepatitis C virus in North American patients infected with HCV genotype 1a or 1b. J Med Virol 1997; 53: 11826.
  • 30
    Kurosaki M, Enomoto N, Murakami T, et al. Analysis of genotypes and amino acid residues 2209 to 2248 of the NS5A region of hepatitis C virus in relation to the response to interferon-beta therapy. Hepatology 1997; 25: 7503.
  • 31
    Murashima S, Ide T, Miyajima I, et al. Mutations in the NS5A gene predict response to interferon therapy in Japanese patients with chronic hepatitis C and cirrhosis. Scand J Infect Dis 1999; 31: 2732.
  • 32
    Witherell GW, Beineke P. Statistical analysis of combined substitution in nonstructural 5A region of hepatitis C virus and interferon response. J Med Virol 2001; 62: 816.
  • 33
    Reddy KR, Wright TL, Pockros PJ, et al. Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C. Hepatology 2001; 33: 4338.
  • 34
    Chambers TJ, Fan X, Droll DA, et al. Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection. J Virol 2005; 79: 307183.
  • 35
    Strader DB, Wright T, Thomas DL, Seeff LB. American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004; 39: 114771.
  • 36
    Stuyver L, Wyseur A, Van Arnhem W, Hernandez F, Maertens G. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol 1996; 34: 225966.
  • 37
    Lee SD, Yu ML, Cheng PN, et al. Comparison of 6-month course peginterferon alpha-2b plus ribavirin and interferon alpha-2b plus ribavirin in treating Chinese patients with chronic hepatitis C in Taiwan. J Viral Hepat 2005; 12: 28391.
  • 38
    Yu ML, Chuang WL, Dai CY, et al. Different viral kinetics between hepatitis C virus genotype 1 and 2 as on-treatment predictors of response to a 24-week course of high-dose interferon-alpha plus ribavirin combination therapy. Transl Res 2006; 148: 1207.
  • 39
    Jensen DM, Morgan TR, Marcellin P, et al. Early identification of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006; 43: 95460.
  • 40
    Sarrazin C, Herrmann E, Bruch K, Zeuzem S. Hepatitis C virus nonstructural 5A protein and interferon resistance: a new model for testing the reliability of mutational analyses. J Virol 2002; 76: 1107990.
  • 41
    Sarrazin C, Berg T, Lee JH, et al. Improved correlation between multiple mutations within the NS5A region and virological response in European patients chronically infected with hepatitis C virus type 1b undergoing combination therapy. J Hepatol 1999; 30: 100413.
  • 42
    Polyak SJ, Paschal DM, McArdle S, Gale MJ Jr, Moradpour D, Gretch DR. Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication. Hepatology 1999; 29: 126271.
  • 43
    Gale JM, Korth MJ, Tang NM, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997; 230: 21727.
  • 44
    Polyak SJ, Khabar KS, Paschal DM, et al. Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J Virol 2001; 75: 6095106.
  • 45
    Hung CH, Lee CM, Lu SN, et al. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin. J Viral Hepat 2003; 10: 8794.
  • 46
    Paterson M, Laxton CD, Thomas HC, Ackrill AM, Foster GR. Hepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response. Gastroenterology 1999; 117: 118797.
  • 47
    Pawlotsky JM, Germanidis G, Neumann AU, et al. Interferon resistance of hepatitis C virus genotype 1b: relationship to non-structural 5A gene quasispecies mutations. J Virol 1998; 72: 2795805.
  • 48
    Saiz JC, Lopez-Labrador FX, Ampurdanes S, et al. The prognostic relevance of the nonstructural 5A gene interferon sensitivity determining region is different in infections with genotype 1b and 3a isolates of hepatitis C virus. J Infect Dis 1998; 177: 83947.
  • 49
    Sarrazin C, Berg T, Lee J-H, et al. Mutations in the protein kinase-binding domain of the NS5A protein in patients infected with hepatitis C virus type 1a are associated with treatment response. J Infect Dis 2000; 181: 43241.
  • 50
    Murphy MD, Rosen HR, Marousek GI, Chou S. Analysis of sequence configurations of the ISDR, PKR-binding domain, and V3 region as predictors of response to induction interferon-alpha and ribavirin therapy in chronic hepatitis C infection. Dig Dis Sci 2002; 47: 1195205.