Nucleotide sequence diversity of hypervariable region 1 of hepatitis C virus in Japanese hemophiliacs with chronic hepatitis C and patients with chronic posttransfusion hepatitis C

Hemophiliac patients with chronic hepatitis C might be exposed to and become infected with multiple hepatitis C virus (HCV) strains by means of frequent use of blood products, even if they are infected with a single subtype of HCV. To test this hypothesis, we analyzed the genetic diversity of hypervariable region 1 (HVR1) of HCV in chronically infected hemophiliacs and in patients with chronic posttransfusion hepatitis with a single HCV inoculation. The diversity of nucleotide sequences in HVR1 of serum HCV RNA was compared between 21 hemophiliacs infected with a single HCV subtype and 16 patients with posttransfusion HCV infection. The number of HCV quasispecies was determined by fluorescence single-strand conformation polymorphism (SSCP) analysis. Direct sequencing was performed to determine the diversity in HVR1. The number of HCV quasispecies in the blood was 5.2 +/- 2.0 clones in hemophiliacs and 4.0 +/- 2.3 clones in posttransfusion patients, a nonsignificant difference (P = .0943). The number of sites at which the nucleotide was not homogenous in all quasispecies was significantly higher in hemophiliacs (13.0% +/- 7.4%) than in posttransfusion hepatitis patients (2.7% +/- 2.8%; P < .0001). In conclusion, there was a high degree of genetic variation in HVR1 of HCV specimens isolated from hemophiliacs compared with posttransfusion patients. These findings indicate the possibility that multiple infections of a single HCV subtype may occur among patients frequently exposed to blood products; single HCV subtypes may therefore derive from multiple origins.     

Genetic changes in hypervariable region 1 as a possible mechanism for biochemical responders with interferon therapy

AIM: Approximately 10% of chronic hepatitis C patients who were treated with interferon have a sustained normalization of aminotransferase levels after interferon therapy without HCV RNA clearance. We investigated genetic change in hypervariable region 1 (HVR 1) to see if the mutation in this region is responsible for their sustained biochemical response and relapse thereafter. A total of 250 patients with chronic hepatitis C who were treated with interferon were studied for 8 to 11 years post therapy. Of these, 26 were biochemical responders (BR) whose ALT remained normal without viral clearance for more than 6 months after IFN therapy. HVR 1 was examined by direct sequencing of the PCR products, and found that no significant HVR 1 differences were observed in samples obtained pretreatment, posttreatment or during flares from these patients, suggesting that mutations in this region were not responsible for either the persistence of the HCV RNA or for the ALT relapses thereafter.     

Hepatitis C virus quasispecies and response to interferon therapy in patients with chronic hepatitis C: a prospective study

We prospectively examined whether the complexity of hepatitis C virus (HCV) quasispecies is related to the response to interferon (IFN) therapy. Among 64 patients who had histologically proven chronic hepatitis and were treated with natural IFN-α, 53 patients were analysed. The other 11 patients discontinued therapy because of adverse effects of IFN. The complexity of the hypervariable region 1 (HVR 1) in quasispecies was determined using both clone number determined by fluorescence single-strand conformation polymorphism (SSCP) and nucleotide diversity determined by direct sequencing. These parameters were measured not only before treatment but also at completion and 6 months after therapy, if serum HCV RNA was detectable. This population of patients was different from the general Japanese population with regard to the high prevalence of patients infected with genotype 2a or 2b (49%), who had a higher viral load than those with genotype 1b ( P  = 0.021). Twenty-two patients (41.5%) were sustained responders. Genotype non-1b ( P  = 0.0009) and a smaller clone number ( P  = 0.008) were significantly associated with a sustained response. In multivariate analysis, these variables were independently associated with a sustained response (i.e. genotype: odds ratio 6.84, 95% CI 1.84–30.12; and clone number: odds ratio 1.26, 95% CI 0.99–1.68). The clone number and nucleotide diversity did not change significantly between pretreatment and at completion or 6 months after therapy. These results suggest that lower complexity of HVR 1 quasispecies predicts a preferable response to IFN therapy that is independent of viral load, especially in the population of the relatively high prevalence of patients infected with genotype 2.     

Hypervariable region 1 quasispecies in hepatitis C virus genotypes 1b and 3 infected patients with normal and abnormal alanine aminotransferase levels

The role of hepatitis C virus (HCV) heterogeneity in the severity of chronic hepatitis C infectionremains unclear. Our aim was to study the hypervariable region 1 (HVR1) heterogeneity in patients with chronic hepatitis C infected with genotype 1b or 3 and with normal or abnormal alanine aminotransferase (ALT). HVR1 quasispecies were assessed by single strand conformational polymorphism (SSCP) in 67 patients with chronic hepatitis C, including 35 with persistently normal ALT and 32 with abnormal ALT. Sixty-two patients underwent a liver biopsy. Among the 67 patients, 40 were infected with genotype 1b and 27 with genotype 3. In univariate analysis, low heterogeneity (≤ 3 bands at SSCP) was significantly associated with normal ALT ( P  < 0.001), milder histological lesions (activity, P =0.02; fibrosis, P =0.04), and at the limit of significance for genotype 1b ( P =0.07). In multivariate analysis, low heterogeneity was significantly and independently associated with normal ALT ( P =0.09) and genotype 1b ( P =0.03). In patients with chronic hepatitis C, a low viral heterogeneity is significantly and independently associated with normal ALT and genotype 1b. These results are consistent with the view that patients with normal ALT have a different immune response against HCV resulting in a low HCV heterogeneity.     

Modulation of hepatitis C virus quasispecies heterogeneity by interferon-α and ribavirin therapy

SUMMARY. To determine the effects of interferon-α (IFN-α) and ribavirin therapy on hepatitis C virus (HCV) quasispecies heterogeneity, 29 patients with chronic HCV infection treated with either IFN-α (n = 15), ribavirin (n = 7) or placebo (n = 7) were studied. HCV quasispecies heterogeneity was determined by single-strand conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1). For patients receiving IFN-α, HVR1 was amplified in 14 of 15 patients before, and in six of seven patients after therapy. After controlling the amount of amplicon loaded, a reduction in the number of SSCP bands was observed with IFN-α therapy (median number of SSCP bands per patient was eight before therapy and two after therapy). In the seven patients within each of the ribavirin- and placebo-treated groups, there was no significant difference in the viraemia level, number of SSCP bands per patient or the SSCP band pattern, before and after therapy. These findings suggest that at the doses given, IFN-α, but not ribavirin, exerts a selective pressure on HCV quasispecies heterogeneity.     

Combined Therapy with Interferon and Ribavirin in Chronic Hepatitis C Does Not Affect Serum Quasispecies Diversity

Our aim was to investigate if interferon plus ribavirin has any effect on serum HCV quasispecies distribution and the relationship between diversity of HCV quasispecies and treatment response. In all, 21 patients were treated with interferon plus ribavirin for 48 weeks. The presence of HCV quasispecies was determined in serum samples at baseline and at the fourth week of treatment by SSCP analysis of the hypervariable region. SSCP pattern was defined as single or multiple band. A single band was found in six patients and multiple bands in nine. No significant difference was found between SSCP pattern in pretreatment samples and response to the therapy. In none of the patients were observed changes in number of SSCP bands between samples taken at baseline and in the fourth week of the therapy. In conclusion, the complexity of HCV quasispecies before the therapy was not related to treatment response; combined therapy did not affect serum HCV quasispecies.     

Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion

BACKGROUND & AIMS: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. METHODS: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2-3 weeks for 6 months, at 10 months, and at 11-16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5' untranslated region (5'UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). RESULTS: Seven patients cleared infection within 7-24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8-22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5'UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. CONCLUSIONS: Changes in E2/HVR1 quasispecies 8-22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host.     

Significance of serum soluble Fas antigen level in chronic hepatitis C patients treated with interferon: Relationship to the therapeutic response

BACKGROUND AND AIM: Fas system-mediated cytotoxicity is thought to be involved in the development of liver injury in hepatitis C virus (HCV) infection. In this study, we investigated serum soluble Fas antigen levels in chronic hepatitis C patients treated with interferon and their correlation with the therapeutic response. METHODS: The subjects were 67 chronic hepatitis C patients who underwent a 24-week course of alpha-interferon therapy. Patients were categorized into three groups; sustained responders (n = 22), transient responders (n = 24), and non-responders (n = 21), according to changes in the serum alanine aminotransferase level during and after therapy. The viral genotype, viremic level and diversity in the hypervariable region were examined before therapy. Serum soluble Fas antigen levels were assayed by using serum samples taken at the beginning and the end of therapy. RESULTS: In the univariate analysis, serum soluble Fas antigen levels tended to be higher in non-responders (10.0 +/- 3.4 ng/mL) than in sustained responders (8.5 +/- 3.0 ng/mL) and transient responders (8.2 +/- 2.1 ng/mL; P = 0.13 and P < 0.05). The non-response to therapy was observed in eight of the 15 (53%) patients with serum soluble Fas antigen > or = 11 ng/mL, compared with 13 of the 52 (25%) patients with serum soluble Fas antigen < 11 ng/mL (P < 0.05). As for the multivariate analysis, the only significant factor contributing to the sustained response was a low HCV viremic level (P = 0.0046). Significant factors contributing to the non-response were a high serum alanine aminotransferase (P = 0.0407) and a high serum soluble Fas antigen level (P = 0.0483). CONCLUSIONS: High production levels of soluble Fas antigen may be associated with a poor response to interferon therapy in chronic hepatitis C patients.     

Evolution of hepatitis C virus genome in chronically infected patients receiving ribavirin monotherapy

Recent results of clinical trials suggest that combination of interferon and ribavirin exhibits an enhanced antiviral effect in the treatment of chronic hepatitis C. To investigate the effect of ribavirin on hepatitis C virus (HCV) infection, we analysed the evolution of the genetic heterogeneity of HCV in relation to the anti-HCV humoral response in patients treated by ribavirin alone. The study population included 35 patients with liver biopsy proven chronic hepatitis C infected with HCV genotype 1. Among them, 26 were treated with ribavirin for at least 12 months and nine untreated patients served as a control group. Serum samples were analysed before and at 6 and 12 months of therapy. Three regions of the HCV genome, i.e. HVR1, a domain of NS5A including part of the interferon sensitivity determining region (ISDR), and a segment of NS5B, were amplified by RT-PCR using specific primers. The PCR products were then studied using single-strand conformation polymorphism (SSCP) analysis followed by either direct sequencing, or cloning and sequencing. In parallel, the humoral anti-E1 response was studied using an ELISA (Innotest HCV E1Ab, Innogenetics). The results of HCV genome analysis showed no significant effect on the amino acid sequence evolution of the HVR1, NS5A and NS5B regions of HCV. Analysis of a phylogenetic tree from the major quasispecies variants showed the absence of correlation with ribavirin response, and the absence of selection of viral strains during ribavirin treatment. A trend towards a decrease in the anti-E1 Ab response was also observed. Altogether these results suggest that ribavirin may not exhibit a direct antiviral effect, but may trigger a favourable response to interferon by modulating the immune response against HCV.     

Multigene tracking of quasispecies in viral persistence and clearance of hepatitis C virus

AIM: To investigate the evaluation of hepatitis C virus (HCV) quasispecies in the envelope region and its relationship with the outcome of acute hepatitis C. METHODS: HCV quasispecies were characterized in specimens collected every 2-6 mo from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, 33 cloned cDNAs representing each specimen were assessed by a combined method of analysis of a single-stranded conformational polymorphism and heteroduplex analysis. The rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were analyzed. RESULTS: Serum samples collected from chronic phase of infection had higher quasispecies complexity than those collected from acute phase of infection in all individuals examined. The genetic diversity (genetic distance) within HVR1 was consistently higher than that in the complete E1(0.0322±0.0068 vs-0.0020±0.0014, P<0.05) and E2 regions outside HVR1 (0.0322±0.0068 vs 0.0017±0.0011, P<0.05) in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region (2.76×10-3±1.51×10-3) predominated and gradually increased, as compared with that in the E1 and E2 regions outside HVR1 (0.23×10-3±0.15×10-3, 0.50×10-3±0.10×10-3). By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions including HVR1 were consistently lower in individuals with clearance of viremia. CONCLUSION: HCV persistence is associated with a complexity quasispecies and positive selection of HVR1 by the host immune system.     

丙型肝炎病毒高变区1抗体的交叉反应性分析

目的探索检测丙型肝炎病毒（HCVJ高变区1（HVR1）抗体交叉反应性的意义。方法采用16种重组的HVRI抗原包被,酶联免疫吸附检测不同病程及不同干扰素治疗效果HCV感染者血清中HVR1抗体的情况,分析其高变区抗体交叉反应性。结果在慢性丙型肝炎、肝硬化和肝细胞癌这三组患者血清中HVR1抗体交叉反应阳性数目分别为10．93±4．98、12±5．57和10．64±4．83,三组间差异无统计学意义（P〉0．05）;对干扰素治疗效果好的患者HVR1抗体交叉反应阳性数为13．85±2．85,治疗效果差的患者为7±5．27,前者显著高于后者（P〈0．05）。但两者治疗前HCV病毒载量差异无统计学意义（P〉0．05）。结论HVRI抗体的交叉反应性与HCV感染的严重程度无明显关系,但可能成为一项干扰素疗效的辅助预测指标。     

A possible role of hypervariable region 1 quasispecies in escape of hepatitis C virus particles from neutralization

We examined serial changes in the hypervariable region 1(HVR1) quasispecies both in immune and nonimmune complexed hepatitis C virus (HCV) particles from 12 patients with chronic hepatitis C to elucidate the mechanism by which genetic diversification of HCV during the course of infection allows escape of virus from the humoural immune response. Immune and nonimmune complexes were separated by differential flotation centrifugation and immunoprecipitation, and their HVR1 quasispecies were determined by subcloning and sequencing. The presence of a specific antibody against a specific viral clone in serum was examined in two patients by Western blotting of the corresponding recombinant HVR1 protein. The distribution of HVR1 quasispecies in both immune and nonimmune complexes conspicuously changed over time in most of the patients studied. In seven patients, various HCV clones serially shifted from nonimmune complexes to immune complexes. In four of them, a group of clones with similar HVR1 sequences to each other remained predominant in nonimmune complexes, whereas minor clones with sequences considerably divergent from the predominant clones shifted from nonimmune complexes to immune complexes. These results suggest a mechanism for persistent infection of HCV, in which major HCV clones escape from neutralization by anti-HVR1 antibodies by generating considerably divergent minor 'decoy' clones which may be preferentially neutralized.     

Retreatment with interferon for chronic hepatitis C after transient response

Approximately half of all patients with chronic hepatitis C show an initial biochemical response to interferon, but only 15% to 20% of patients achieve a sustained response. We studied the efficacy of retreatment with interferon for patients with chronic hepatitis C who showed transient biochemical responses to initial treatment. Thirty patients who relapsed were retreated 1 to 52 months (median 14) after the end of initial treatment, according to the previously used regimens. The responses were correlated with the pre-retreatment patient data. The liver histologic grades, compared with those found before the initial treatment, were better in eight (27%) patients but worse in six (20%), whereas the fibrosis stage was improved in five (17%) but worsened in eight (27%). All patients displayed end-of-retreatment biochemical responses. Of the 30 patients, 10 (33%) achieved sustained aminotransferase normalization and serum hepatitis C virus (HCV) RNA clearance, but the remaining 20 patients showed relapse within 1 year after cessation of retreatment. Univariate analysis associated the sustained response with low pre-retreatment viral loads (0.8 +/- 0.7 MEq/mL vs. 9.1 +/- 6.5 MEq/mL; p = 0.006), short treatment intervals (13 +/- 13 months vs. 22 +/- 14 months; p = 0.031), and low histologic grades (1.3 +/- 0.7 vs. 1.9 +/- 0.7; p = 0.039). However, multivariate analysis indicated that only the pre-retreatment viral load was predictive of the sustained response (p = 0.049). These findings suggest that transient responders to interferon are likely to respond to retreatment but the achievement of a sustained response depends on the HCV viral load before retreatment.     

HCV quasispecies evolution: association with progression to end-stage liver disease in hemophiliacs infected with HCV or HCV/HIV

Patients with inherited bleeding disorders who received clotting factor concentrates before 1987 have high rates of hepatitis C virus (HCV) or HCV/HIV infection. We evaluated HCV quasispecies evolution in longitudinally collected specimens comparing those from patients with progression to end-stage liver disease (ESLD; cases) to those with compensated chronic hepatitis (controls). Plasma samples were obtained from the National Cancer Institute Multicenter Hemophilia Cohort Study. Controls were matched for age, sex, infection duration, and presence/absence of HIV. Samples from early infection were compared to those obtained after onset of ESLD in the cases. The first hypervariable (HVR1) and core proteincoding regions were amplified, subcloned, and sequenced. Complexity and diversity were determined. More than 700 sub-clones from 10 pairs of patients (8 with HIV) followed over approximately 9.3 years were evaluated. HVR1 complexity narrowed over time in the cases, whereas it increased in controls (P = .01). Similar trends were observed for diversity within HVR1 and the core region (P = .04). HCV-infected patients with inherited bleeding disorders undergo quasispecies evolution over time. Evolution patterns differ for progressors and nonprogressors. Further understanding of these mechanisms may help identify factors related to progression rate and treatment response.     

Hepatitis cRNA quasispecies complexity in patients with alcoholic liver disease.

Alcohol abuse is described as a major cofactor in the development of hepatitis C (HCV) associated liver disease and may play a role in the outcome of interferon-based treatment interventions. The association between HCV viral heterogeneity and alcohol has not been previously described. This study was designed to evaluate the quasispecies nature of the HCV population in patients with compensated and decompensated alcoholic liver disease, to test the hypothesis that alcoholics have greater complexity than matched nonalcoholic controls. A nonisotopic heteroduplex complexity assay (HCA) was first validated by comparison with results of quasispecies complexity determined by subcloning and sequencing of amplicon products from the E2/NS1 hypervariable coding region (HVR). Subsequently, this methodology was applied to comparison of 20 compensated (Child's-Pugh A) and decompensated (Child's-Pugh B/C) alcoholic and 20 nonalcoholic controls. The complexity of the HVR, as well as the 5' Untranslated (5'UT) and the NS5b coding domains were evaluated. The HCA methodology provided a reasonable semiquantitative measure of HCV RNA quasispecies variability compared with subclone sequence homology comparison. Overall, alcoholic patients had greater quasispecies complexity (2.65 bands) than nonalcoholic controls (1.6 bands; P =.01). Subset analysis revealed that compensated alcoholic patients had a mean of 3.1 homo/heteroduplex bands per sample whereas Child's-Pugh B/C alcoholics showed intermediate complexity. A similar quasispecies complexity difference was seen in the 5'UTR, but not in the NS5b coding domain. Quasispecies complexity was not associated with viral titer, complementary DNA concentration, or genotype. The differences in quasispecies complexity may help explain reports of poor interferon responsiveness in alcoholic patients.     

Effect of ribavirin and amantadine on early hepatitis C virus RNA rebound and clearance in serum during daily high-dose interferon

The early rebound in serum HCV RNA during HCV dynamic studies with high-dose interferon may be due to de novo infection with interferon escape quasispecies. We simultaneously measured serum alanine aminotransferase (ALT) and HCV RNA at rapid intervals in chronic HCV liver disease patients during interferon therapy alone or in combination with ribavirin and amantadine. HCV RNA declined rapidly between 0 and 48 hr in all patients (phase 1). Ribavirin and amantadine significantly increased this phase 1 decline. In all four monotherapy patients with viral rebound, the increasing levels of HCV RNA were associated with a parallel increase in serum ALT, consistent with a hepatitis flare or de novo infection. By contrast, in the four monotherapy patients without viral rebound, and all eight patients receiving combination therapy, the slow progressive phase two decay was associated with declining serum ALT levels. Ribavirin or ribavirin and amantadine significantly and incrementally increased the phase two HCV RNA clearance. Dynamic sequencing in the HVR1 region in one rebound patient confirmed the potential for rapid evolutionary changes during interferon therapy. These preliminary data suggest that early viral rebound might be associated with de novo infection with interferon escape HCV variants, which in turn are attenuated by ribavirin and amantadine.     

丙型肝炎病毒感染自然过程中的准种变化

目的观察丙型肝炎病毒(HCV)持续感染者与自然阴转者外周血HCV准种构成的变化规律。方法应用基因扩增、分子克隆和测序的方法,对未接受过治疗的4例HCV持续感染者与4例自然阴转者前后间隔10年血清中HCV高变区1(HVR1)基因片段进行了序列分析及遗传进化关系比较。结果与持续感染者相比,自然阴转者外周血HCVHVR1区准种群体组内平均遗传距离、熵值较小。4例持续感染者中有3例10年前后血清HCVHVR1准种群体组内与组间遗传距离有明显差异。8例感染者中有7例血清HCV准种KA/KS值大于1。结论在丙型肝炎的自然病程中HCV准种遗传复杂度、变异度大小可能与丙型肝炎的转归相关;HCV准种构成可能发生改变。     

Expression of hepatitis C virus hypervariable region 1 and its clinical significance

AIM, To explore the properties of hypervariable region 1(HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders.METHODS: Gene fragments of HVR1 of four HCV strains(three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1. The purified DHFR-HVR1 proteins were then used to detect the anti-HVR1 antibodies in 70 serum samples of patients with chronic hepatitis C.RESULTS: Four DHFR-HVR1 fusion proteins were successfully expressed in E.coli (320-800ug fusion proteinsper 100ml culture). Each fusion protein (SH1b, BJ1b,SD1b and SD2a) reacted with 72.8% (51/70), 60% (42/70), 48.6% (34/70), and 58.6% (41/70) of the anti-HCV positive patients‘ sera respectively by ELISA. 57.1% (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3% (2/13) of responders reacted with all of them. The O.D. values of sera from IFN therapy responders were significantly higher than those of nonresponders (P&lt;0.05).CONCLUSION: The selected HVR1 fusion proteins expressed in E. coil can broadly react with HCV-infected patients‘ sera. The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment. With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies. A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.