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.     

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.     

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.     

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.     

Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C

AIM： To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.
METHODS： Forty seven patients with hepatitis C [32 with chronic active hepatitis （CAH）, 9 with cirrhosis, and 6 with hepatocellular carcinoma （HCC）] were screened for the presence of quasispecies by single stranded conformational polymorphism （SSCP） analysis in the hypervariable region （HVR） and non-structural 5B （NS5B） viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients （11 non-responders and 12 showing a sustained virological response） was sequenced for the assessment of mutations.
RESULTS： The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association （P 〈 0.05） with the non-responders, and leads to persistence of infection. Significant differences （P 〈 0.05） in viral load （log10 IU/mL） were observed among patients infected with complex quasispecies （CQS）, those infected with simple quasispecies （SQS） and those with no quasispecies （NQS）, after 12 wk （CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4） and 24 wk （CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3） in the HVR region. However, a statistically significant difference （P 〈 0.05） was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk （CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3） and 48 wk （CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3） of therapy. Disease severity was significantly associated with viral load during th     

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.     

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.     

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.     

单链构相多态性分析丙型肝炎病毒1b型准种及变异研究

目的 研究丙型肝炎病毒准种及其变异情况；同时探索准种变异规律，是否干扰素对准种具有选择作用。方法 选取５例慢性丙型肝炎患者阳性血清，ＰＣＲ法分别扩增５′Ｃ非编码区（５′ＮＣＲ）、高变区（ＨＶＲ１）、非结构蛋白５Ａ（ＮＳ５Ａ）部分片断，单链构相多态性（ＳＳＣＰ）法测定其准种数量，同时动态观察２３例丙型肝炎患者１ｂ型ＮＳ５Ａ准种变化情况，探讨是否具有对干扰素敏感性不同的准种及干扰素对准种是否具有选择作     

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.     

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.     

慢性肝炎患者血清中丙型肝炎病毒核心区和包膜1区基因变异研究

目的 探讨丙型肝炎病毒（HCV）核心（C）区和包膜1（E1）区变异与其感染慢性化的关系。方法 10例HCV慢性感染者和2例急性感染者血标本,采用逆转录－聚合酶链反应（RT－PCR）扩增HCV的C区羧基端、E1及E2区氨基端片段（1kb),扩增产物进行克隆,以单链构象多态性（SSCP）和异质性双体（HD）分析对每份血清的30个克隆的C／E1区准种（quasispecies)进行筛选,挑选每例标本HCV的优势株与劣势株序列进行测定,并分析推导的氨基酸序列。结果 各例患者血清中HCV的C／E1区扩增片段形成的SSCP条带间差异明显,而HD与同源双体之间差距不明显。HCV急性感染者和慢性感染者血清病毒C区无发生变异,后者E1区氨基酸替换率为1．32％,但功能性氨基酸无改变。结论 HCV的E1区序列变异以准种形式存在,但与免疫逃避可能无关。     

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.     

Relationship of the genomic complexity of hepatitis C virus with liver disease severity and response to interferon in patients with chronic HCV genotype 1b infection [correction of interferon

In patients with chronic hepatitis C, the influence of the genetic heterogeneity of the hepatitis C virus (HCV) on the progression of liver disease and on the responsiveness to interferon therapy is a matter of controversy. In this study we evaluated the genetic complexity of HCV by single-strand conformation polymorphism (SSCP) analysis of amplicons from the hypervariable region 1 (HVR1) in 168 patients with chronic genotype 1b HCV infection, of whom 122 received a single course of interferon therapy (3 MU, three times weekly for 6 months). No correlation was observed between the degree of genetic complexity of HCV (indicated by the number of bands in the SSCP assay) and patient age, serum alanine aminotransferase activity, or serum HCV-RNA concentration, measured by competitive polymerase chain reaction. HCV genomic complexity was not related to gender nor to the presumed source of infection. The number of SSCP bands detected in serum samples from patients with chronic hepatitis, either mild (8.1 +/- 3.9), moderate (8.0 +/- 3.3), or severe (9.2 +/- 3.3), and in patients with liver cirrhosis, either compensated (8.0 +/- 2.9), decompensated (6.3 +/- 2.9), or with superimposed hepatocellular carcinoma (9.5 +/- 2.9), was similar. The number of SSCP bands detected in patients with sustained response (7.5 +/- 3. 9), transient response (8.3 +/- 2.9), or no response (8.2 +/- 3.6) to interferon administration was similar as well. These observations suggest that the genetic complexity of hypervariable region (HVR1) of HCV, as estimated by SSCP analysis, is not related to the severity of liver injury nor to the type of response to interferon therapy. Thus, information offered by SSCP analysis of HVR1 of HCV in chronic HCV genotype 1b infection does not appear to be useful in the clinical management of these patients. (HEPATOLOGY 1999;29:897-903.)     

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。     

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.     

丙型肝炎病毒感染个体的准种源于感染过程中的病毒核酸突变

目的研究丙型肝炎病毒(HCV)准种特性与感染慢性化的关系,以及个体准种特性的来源形式。方法收集HCVRNA阳性的10例急性丙肝、20例慢性丙型肝炎(丙肝)和11例肝细胞癌(HCC)患者,采用单链构型多态性分析(SSCP)方法进行HCV准种检测。结果急性丙肝、慢性丙肝和HCC患者中,SSCP电泳条带数分别为2．7±1．16、4．8±1．68和5．2±2．85。慢性丙肝和HCC的条带数显著高于急性丙肝(P＜0．05)。进行DNA序列分析研究发现,准种高变区间的变异性显著低于本地株间和异地株间的变异性(P＜0．01)。结论SSCP是检测准种相对简便而有效的方法。HCV准种特性与其感染慢性化相关。HCV感染个体准种的来源主要为感染过程中的核酸突变。     

Nonstructural 5A gene variability of hepatitis C virus (HCV) during a 10-year follow up

Background Seven patients with chronic hepatitis C virus (HCV) infection were followed up for 10 years in order to analyze the molecular evolution of the HCV nonstructural 5A (NS5A) gene.Methods Serum samples were obtained from seven patients with anti-HCV who were HCV-RNA positive. The HCV NS5A region, including the interferon sensitivity-determining region (ISDR), protein kinase R binding domain (PKR-BD), and V3 was amplified and cloned, and then sequenced. The nucleotide sequences of the region at the beginning and at the end of the 10 years were aligned with the CLUSTAL W program, version 1.8, and the corresponding amino-acid sequences were deduced.Results The three serine residues at positions 2197, 2201, and 2204, suggested to be important for hyperphosphorylation of NS5A, were highly conserved in different patients and were within the quasispecies of each patient. The wild-type ISDR or minimally mutated strain was dominant in all the patients and the number of quasispecies decreased over time.Conclusions Our findings showed that the functional domains of NS5A were generally conserved over an interval of 10 years. Quasispecies distribution was variable and distinctly clustered over time during the natural course of HCV infection. These results may contribute to better understanding of HCV natural infection.     

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.     

Evaluation of cross-reactive antibody response to HVR1 in chronic hepatitis C

AIM:To evaluate the presence and cross-reactive anti-bodies against hypervariable region 1(HVR1) in hepatitis C virus(HCV) infected patients and its relationship with the progression of the disease.METHODS:Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients.Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis,18 with chronic hepatitis and 16 with live...