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.     

Pretransplantation hepatitis C virus quasispecies may be predictive of outcome after liver transplantation

The evolution of hepatitis C virus (HCV) envelope variation was studied using a liver-transplant model to evaluate the role of HCV quasispecies for hepatocyte infection. Twelve HCV polymerase chain reaction (PCR)-positive liver-transplant recipients (6 with posttransplantation biochemical hepatitis and 6 without hepatitis [controls]) were prospectively evaluated and underwent detailed quasispecies analysis of pre- and postoperative serum samples. HCV amino acid sequence diversity and complexity at the first hypervariable region (HVR1) of the second envelope protein (E2) was correlated with outcome. Recurrence of HCV-induced allograft injury was defined by persistently elevated serum alanine transaminase (ALT) levels. The major variant (sequences >10% of all clones) of recipients with hepatitis accounted for a significantly smaller percent of all preoperative clones compared with controls (41% +/- 6% vs. 69% +/- 8%; P <.015). Recipients with hepatitis had an increased number of pretransplantation major variants (2.5 +/- 0.3 vs. 1.1 +/- 0.2; P <.006). Eighty-three percent of controls had a predominant variant (accounting for >50% of clones) compared with 17% of those with recurrence (P <.05). These differences did not persist postoperatively. In addition, recipients without a pretransplantation predominant variant demonstrated an increased allograft fibrosis score (2.3 +/- 0.3 vs. 0.5 +/- 0.3; P <.015) at 181 to 360 days posttransplantation compared with those in whom a predominant variant was present. Increased HCV envelope complexity may act as a stronger antigenic stimulus or improve hepatocyte receptor binding and lead to allograft hepatitis and fibrosis. Although pretransplantation differences in HCV quasispecies did not persist postoperatively, pretransplantation quasispecies may be a predictor of HCV-induced hepatitis and graft fibrosis after liver transplantation.     

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.     

Evolution of hepatitis C viral quasispecies after liver transplantation

BACKGROUND & AIMS: To determine whether HCV quasispecies diversity correlated positively with liver disease progression after orthotopic liver transplantation (OLT). METHODS: We studied 11 patients undergoing OLT for HCV-related cirrhosis with recurrent hepatitis C in 2 groups according to the stage of hepatic fibrosis on follow-up. The mild group had stage 1 or 2 fibrosis; the severe group, stage 3 or 4 fibrosis. HCV quasispecies diversity was assessed by cloning and sequencing in pretransplantation and posttransplantation serum samples. RESULTS: In the mild fibrosis group, intrasample hypervariable region 1 (HVR1) genetic distance and nonsynonymous substitutions increased after OLT, whereas in the severe fibrosis group, these parameters decreased in follow-up. In contrast, intrasample diversity progressed similarly in both groups in the adjacent sequences flanking HVR1. There was an inverse correlation between the stage of hepatic fibrosis and amino acid complexity after OLT. Among all patients, the estimated rate of amino acid change was greater initially and became more constant after 36 months. CONCLUSIONS: After OLT, a more complex HCV HVR1 quasispecies population was associated with mild disease recurrence. Among those patients with severe recurrent hepatitis C, HCV appeared to be under greater immune pressure. The greatest change in viral amino acid sequences occurred in the first 36 months after OLT.     

Post-transplant quasispecies pattern remains stable over time in patients with recurrent cholestatic hepatitis due to hepatitis C virus

BACKGROUND/AIMS: Several studies have shown that cholestatic recurrent hepatitis is associated with very high HCV RNA loads in liver transplant recipients. The aim of this study was to investigate whether a correlation exists between cholestatic hepatitis post-transplant and the population of viral quasispecies. METHODS: One hundred and nine serial sera samples were tested from 15 recurrent HCV patients. Four of these patients showed severe cholestatic recurrent hepatitis, 11 patients demonstrated non-cholestatic recurrent hepatitis post-transplant. Quasispecies were detected by RT-PCR amplification of the HVR1 followed by single-stranded conformational polymorphism analysis. RESULTS: Forty-one samples from four cholestatic patients were tested. All four patients showed very stable quasispecies patterns post-transplant. One cholestatic patient also showed a stable quasispecies band pattern following retransplantation, again associated with severe cholestatic hepatitis. Sixty-eight samples were tested from the 11 non-cholestatic patients. In contrast, these patients showed significantly more quasispecies bands than the cholestatic patients. The noncholestatic patients also displayed fluctuating band patterns post-transplant. Serial samples were tested after retransplantation in one non-cholestatic patient, with a fluctuating pattern again seen. There was a negative correlation between the HCV RNA load in serum and the number of quasispecies bands. CONCLUSIONS: Stable hepatitis C viral quasispecies associated with persistently high viral load in post-transplant cholestatic hepatitis suggest that viral escape from immune pressures may play a role in the pathogenesis of this condition.     

Hepatitis G virus/GBV-C persistence: absence of hypervariable E2 region and genetic analysis of viral quasispecies in serum and lymphocytes

Persistent infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is common and may last for years. In addition, the principal site of virus replication remains undefined. Sequencing studies of E2 in four patients showed that a hypervariable region equivalent to that of hepatitis C virus (HCV) was absent and that viral quasispecies were less frequent than in HCV infection, particularly with respect to amino acid variation. Recurrence of viraemia following interferon treatment did not result in the emergence of new quasispecies. Virus persistence therefore does not appear to be related to immune escape by strains bearing a hypervariable E2 region. We also investigated whether virus replication occurred in peripheral blood mononuclear cells. The positive-RNA strand of the virus, but no negative strand, was detected in both serum and lymphocytes. The lymphocytes harbouring the virus were CD4 and CD19 positive. Direct sequencing and cloning of amplicons from the region of the non-structural 3 (NS3) protein showed that the nucleotide sequences in lymphocytes were different from those in serum and did not represent any of the minor serum quasispecies. Although evidence of replication in lymphocytes has not been forthcoming, the differences in sequences between serum and lymphocytes suggest that circulating virus originates from a non-hepatic site, other than lymphocytes.     

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。     

Constrained genomic and conformational variability of the hypervariable region 1 of hepatitis C virus in chronically infected patients

We analysed the genomic and conformational variability of the hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) to evaluate the importance of its biological role. A total of 865 genotype 1b HVR1 subclones were collected from serially sampled sera in 11 patients with chronic hepatitis C, four of whom received interferon therapy. Consequently, 169 distinct sequences were examined for amino acid substitutions as well as hydrophilic or hydrophobic profile at each amino acid position within HVR1. Secondary structure of HVR1 was also predicted by the method of Robson in 90 distinct sequences from eight patients, including three interferon-treated patients. Some positions within the HVR1 were invariable or nearly so as to amino acid substitution. Hydrophilic or hydrophobic residues exclusively predominated at several positions. These constrained amino acid replacement and hydrophilic or hydrophobic profiles were conserved irrespective of interferon therapy, though the frequency of amino acid replacement was greater at almost all amino acid positions within the HVR1 in interferon-treated patients. The quasispecies of HCV showed various secondary structures of HVR1, but many sequences seemed to have common characteristics. β sheet conformations around both the N-terminus and position 20 (numbered from the NH₂ terminus of E2 envelope glycoprotein), and/or coil structures around the C-terminus of HVR1 could be identified. These results suggest that HVR1 amino acid replacements are strongly constrained by a well-ordered structure, in spite of being tolerant to amino acid substitutions, and imply an important biological role of the HVR1 protein in HCV replication.     

The minimum number of clones necessary to sequence in order to obtain the maximum information about hepatitis C virus quasispecies: a comparison of subjects with and without liver cancer

Most studies of hepatitis C virus (HCV) quasispecies have reported the results of sequencing only three to five clones per sample. The possibility that sequencing so few clones might not provide a representative picture of the quasispecies present in a sample has never been evaluated. The present study was conducted to evaluate whether sequencing greater numbers of clones results in better information about the HCV quasispecies number and distribution, and to compare the HCV quasispecies in liver cancer cases and controls. RNA was extracted from serial serum samples from six subjects with HCV-associated liver cancer and 11 age- and sex-matched HCV-infected controls without liver cancer. The hypervariable region 1 (HVR1) of the HCV genome was amplified, cloned, and sequenced. For further studies of 12 serum samples from two liver cancer cases and two matched controls, successive groups of 10 additional clones were sequenced up to a total of 50 clones per serum sample. When only 10 clones were sequenced from each specimen, no consistent differences were seen between the number of HCV quasispecies in the six liver cancer cases and the 11 controls. However, sequencing 40 clones from each of 12 samples from two liver cancer cases and two controls revealed a greater number of quasispecies in liver cancer cases than in controls. Testing an additional 10 clones (50 clones per sample) did not significantly increase the number of quasispecies detected.     

Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment

Summary. Interferon- α (IFN) has been shown to accelerate the evolution of hepatitis C virus (HCV) variants (quasispecies) in nonresponder patients. Different sensitivities of HCV variants to IFN are discussed as a possible mechanism. In the present study, quasispecies were investigated in detail by a newly established and validated direct solid-phase sequencing of the hypervariable region 1 (HVR1), during the initial 3 months of IFN therapy. According to single strand conformation polymorphism (SSCP) analysis, 14 of 26 (54%) virologic nonresponders with quasispecies evolution were identified. Six representative patients with SSCP changes were selected for frequent HVR1 sequencing. Pre-existing variants were identified by cloning and sequencing of the pretreatment serum HCV sample. In one patient the major type was substituted by a minor variant within 3 days of treatment while in the majority of patients the pretreatment major type did not decline before days 26–57 of treatment. Total serum HCV RNA levels remained constant in all patients. In conclusion, although quasispecies evolution during IFN therapy is common, it occurs after a wide range of time intervals after initiation of therapy. Thus, nonresponse to IFN cannot exclusively be explained by changes in the quasispecies.     

Antibody responses to hepatitis C virus hypervariable region 1: evidence for cross-reactivity and immune-mediated sequence variation.

Sequence heterogeneity of hepatitis C virus (HCV) is unevenly distributed along the genome, and maximal variation is confined to a short sequence of the HCV second envelope glycoprotein (E2), designated hypervariable region 1 (HVR1), whose biological function is still undefined. We prospectively studied serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection obtained at baseline and after a defined follow-up period. Extensive serological cross-reactivity for unrelated HVR1 peptides was observed in the majority of the patients. Antibody response was restricted to the IgG1 isotype and was focused on the carboxyterminal end of the HVR1 region. Cross-reactive antibodies could be readily elicited following immunization of mice with multiple antigenic peptides carrying HVR1 sequences derived from our patients. The vigor and heterogeneity of cross-reactive antibody responses were significantly higher in patients with chronic hepatitis compared with those with acute hepatitis and in patients infected with HCV type 2 compared with patients infected with other viral genotypes (predominantly type 1), which suggest that higher time-related HVR1 sequence diversification previously described for type 2 may result from immune selection. The finding of a statistically significant correlation between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses provided stronger evidence in support of the contention that HCV variant selection is driven by the host's immune pressure.     

Genetic complexity and serum reactivity of HVR1 quasispecies of hepatitis C virus in patients with cirrhosis

OBJECTIVE: The RNA genome of hepatitis C virus varies considerably, especially within the hypervariable region 1 (HVR1), a domain located on the 5' end of the E2/NS1 envelope region. Our previous study has suggested there were greater numbers of quasispecies in the liver than in matched serum, independent of the viral load. However, the significance of this finding has not been examined extensively at genetic and serological levels. METHODS: By large scale cloning and sequencing, we studied the genetic complexity of HVR1 quasispecies in two selected patients with cirrhosis. The serum reactivity of peptides representing different HVR1 quasispecies isolated from these cases was also estimated by standard ELISA format. RESULTS: We found the same major (dominant and/or subdominant) viral quasispecies variants in serum and in the cirrhotic liver. Genetic analysis suggested that the evolutionary pressure on HVR1 was higher than on its flanking region in quasispecies derived from the liver, whereas this trend is attenuated in quasispecies from serum. The immunoreactivity to peptides representing different HVR1 quasispecies variants showed considerable cross-reactivity with heterologous sera, whereas the reactivity was strongest against the dominant HVR1 peptide over time in homologous sera. CONCLUSIONS: These findings indicate that the formation and selection of HVR1 quasispecies may not be driven solely by humoral immune pressure, at least in these two cases.     

HCV selection and HVR1 evolution in a chimpanzee chronically infected with HCV-1 over 12 years.

Aim: To study hepatitis C virus (HCV) selection and hypervariable region-1 (HVR1) evolution in a chimpanzee chronically infected with HCV-1 over 12 years after inoculation with a human factor VIII concentrate contaminated with HCV. Methods: From the inoculum, the earliest chimpanzee plasma and 12 annual plasma samples, HCV fragments including HVR1 were amplified followed by cloning and sequencing. Results: Five HCV subtypes - 1a, 1b, 2a, 2b, 3a - and multiple 1a strains were identified in the inoculum. Two 1a strains were found in the earliest chimpanzee sample, while a single HCV-1 strain was detected in the 12 annual samples. None of the chimpanzee sequences were identical to those found in the inoculum. Over 12 years, HVR1 patterns changed irregularly, but a few patterns showed identical nucleotide or amino acid sequences. In the last three years, the variety of HVR1 patterns decreased, while the proportion of major patterns increased. These corresponded to a higher virus load and a lower number of amino acid substitutions. Simultaneously, the HVR1 sequences became more similar to the consensus sequence of the 1a subtype. Conclusion: HCV selection was observed from the inoculum to the inoculated chimpanzee and from the early acute hepatitis to the persistent chronic infection. The selection occurred at three levels: among subtypes after transmission, among isolates during acute hepatitis and among quasispecies in chronic infection.     

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.     

Relationship between interferon therapy and variability in nonstructural gene 5b of hepatitis C virus

The hepatitis C virus (HCV) quasispecies nature in the hypervariable region (HVR) has been reported and found to relate to the effectiveness of interferon (IFN) treatment. However, the quasispecies nature in the nonstructural (NS) 5b region remains to be addressed. To examine this characterization and relationship with IFN therapy, we sequenced six independent HCV clones from each of eight patients. The eight patients were classified as responders or nonresponders to IFN. In the four responders, we found one to three isolates in each of the six clones. In the nonresponders, the six clones consisted of four, five, six, and six isolates, respectively. Compared the (NS) 5b genes of the isolates obtained from the patients with that of the reported hepatitis C virus HC-C2 or HC-J6 isolate the ratio of nonsynonymous to total substitutions ranged from 17.61% to 30.95% in the responders and from 33.11% to 76.47% in the nonresponders. We also compared posttreatment with pretreatment sequences. The average number of varying amino acids ranged from 5.5 to 9.0 in isolates remaining after IFN treatment and from 4.3 to 5.5 in the isolates that disappeared with IFN treatment. Two changed amino acids (glycine to arginine and valine to isoleucine) (compared with the pretreatment clones) were found in the posttreatment clones of one of the responders and one amino acid change (valine to alanine) was found in another responder. These results suggest that the NS5b quasispecies correlates with IFN treatment effectiveness. These results also implied that the heterogeneity in different hierarchical strata has a common impact on IFN treatment, making infected patients resistant to IFN. Our study also provides evidence that HCV elimination and mutation may occur simultaneously during IFN therapy. Received Sept. 19, 1997; accepted Feb. 27, 1998     

Influence of specific CD4+ T cells and antibodies on evolution of hypervariable region 1 during acute HCV infection

BACKGROUND/AIMS: Several studies suggest that the evolutionary rate of HVR1 sequence in acute HCV hepatitis derives from the action of a continuous immune-driven positive selection. However, these studies have not been performed examining the relationship between HVR1 evolution and the development of specific immunity to autologous HVR1 sequences. METHODS: We performed a longitudinal analysis of HVR1 sequences and specific antibodies and CD4+ T cells in ten HCV acutely infected patients with different clinical outcomes (recovery versus persistence). RESULTS: We showed that although both recovered and chronically evolving individuals developed IFN-gamma+ T cells specific for Core and NS sequences, HVR1-specific CD4+ T cells were detected only in patients clearing the virus. On the contrary, all patients displayed anti-HVR1 antibodies that recognized sequences exclusively carried by autologous viruses. Measurements of genetic diversity and the number of non-synonymous per synonymous substitutions within HVR1 sequences before and after antibody appearance showed an increase of these parameters only in concomitance with the appearance of anti-HVR1 antibodies. CONCLUSIONS: The evidence that anti-HVR1 antibodies favor HVR1 variant selection suggests that viral complexity in chronically infected patients could represent a virus adaptive strategy to escape the continuous selective process mediated by anti-HVR1 antibodies.     

Phylogenetic investigation for the risk of hepatitis C virus transmission to surgical and dental patients

Blood loss during treatment carries a potential risk for the transmission of blood-borne pathogens in hospital patients. To determine whether nosocomial transmission of hepatitis C virus (HCV) occurs in surgical wards and dental hospitals, we tested anti-HCV antibodies and HCV RNA in sera from these patients and analysed the hypervariable region 1 (HVR1) sequence of HCV phylogenetically in the HCV RNA-positive patients. Five of 83 patients from a surgical ward were positive for HCV RNA, and six patients from one dental hospital and nine patients from a second were found to be positive for HCV RNA during the examination period. The HVR1 sequences were amplified from these patients' serum, and after subcloning, multiple clones of the HVR1 sequence from each patient were determined. The phylogenetic analysis of these sequences showed that HVR1 species from each patient could be classified into one to three genetic clusters of HVR1 quasi-species and that these clusters were independent of each other among patients. Thus, there was no evidence of HCV transmission in our study, and unrecognized transmission of HCV may be a rare event in surgical and dental patients at university hospitals.     

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.