丙型肝炎病毒准种多样性与病毒血症水平、疾病活动度及干扰素疗效的关系

目的　了解丙型肝炎病毒 (HCV)准种变异与病毒血症水平、疾病活动度及干扰素疗效的关系。方法　采用针对HCVE2高变区 1 (HVR1 )的单链构象多态性分析法 (SSCP)对 68例慢性丙型肝炎患者进行HCV准种检测 ,分析准种数目与HCVRNA、ALT、AST水平及肝组织活动指数 (HAI)的相关性。对其中 48例给予干扰素治疗 ,分析准种数目对干扰素应答效果的影响。结果　 61例HVR1SSCP阳性 ,HCV准种数目为 (6 2± 2 4)条。准种数量与HCVRNA水平显著相关 (P <0 0 1 ) ,与ALT、AST及HAI无明显相关 (P >0 0 5)。干扰素治疗患者中 ,43例HVR1阳性 ,持续应答者治疗前HCV准种数量 (3 3± 1 2 ,n =1 1 )显著少于获得治疗终点应答 (ETR)伴复发者 (6 3± 2 2 ,n =1 2 ,P <0 0 5)或无应答者 (8 0± 3 3 ,n=2 0 ,P <0 0 1 )。治疗结束时 ,干扰素组仍有 1 6例检测出HCV准种 ,但准种条数降为 (3 4± 1 2 )条 ,与未接受干扰素治疗病例的准种数目 (6 8± 2 5)相比差异有显著性 ,P <0 0 1 ) ,且其中 1 0例准种模式发生了改变。结论　HCV准种多样性可引起较高的病毒血症水平 ,但与疾病活动度无关 ;准种数目可作为预测慢性丙型肝炎干扰素疗效的指标。     

HCV准种多样性及其与临床关系的研究

目的     

Dynamics of serum hepatitis C virus load and quasispecies complexity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) infection is a dynamic process during which viral genetic variants continuously develop as a result of the virus adaptation to the host's immune system. The level of viremia and the complexity of the hypervariable region 1 (HVR 1) quasispecies of hepatitis C virus during antiviral therapy reflect the dynamic balance between the viral and host components in response to therapy. OBJECTIVE: The aim of the study was to evaluate the dynamics of HCV viremia and the complexity of the HVR 1 quasispecies during the induction phase of a triple combination therapy regimen in nonresponders to earlier anti-HCV treatment. STUDY DESIGN: Ten patients with chronic hepatitis C undergoing antiviral combination therapy with interferon-alpha, ribavirin, and amantadine were studied. The serum HCV RNA level was monitored by a quantitative RT-PCR assay up to 3 months after start of treatment. The HVR 1 quasispecies complexity was analysed by an "in house" nested RT-PCR mediated single-strand conformation polymorphism (SSCP) assay. RESULTS: Baseline serum HCV RNA levels ranged from 1.94x10(6) to 5.53x10(6) copies/ml. In all patients, HCV subtype 1b was found. At the start of therapy, the SSCP assay revealed a high complexity pattern (at least six SSCP bands) in all patients. None of the patients responded within 4 weeks of treatment, however, the serum HCV RNA level decreased by one to two logs in eight patients. At week 4 after start of treatment, there was a decrease of SSCP bands in five patients. In four patients, SSCP bands remained unchanged and in one patient SSCP bands increased. At month 3 after start of treatment, serum HCV RNA was not detectable in one patient. CONCLUSION: Because of the low number of patients involved in this study, prediction of therapeutical success based on the quasispecies complexity was not possible. Larger studies are urgently needed.     

Clinical implications of viral quasispecies heterogeneity in chronic hepatitis C

To determine the clinical significance of viral quasispecies heterogeneity, 59 patients with chronic hepatitis C were studied using single-stranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1); of these, 48 were subsequently treated with interferon-α. The SSCP method was validated using clones of known nucleotide sequence. HVR1 was amplified in 54 of 59 (92%) patients. The median number of SSCP bands per sample was 6 (range: 2–12). Increased quasispecies heterogeneity correlated with the estimated duration of HCV infection (P < 0.05), parenteral-acquired HCV infection (vs. sporadic, P < 0.05), serum HCV RNA levels (P < 0.05), and HCV genotype 1 infection (P < 0.05), but not with age, serum AST, ALT, or Knodell score. Patients who had complete and sustained response to interferon-α (n = 11) had lower pre-treatment quasispecies heterogeneity compared to patients who had complete response with relapse (n = 18, P < 0.05) or no complete response (n = 16, P < 0.01). However, multivariate analysis revealed that HCV viremia was a stronger predictor of response to interferon-α. These findings indicate that the estimated duration of HCV carriage, serum HCV RNA levels, and HCV type 1 are important determinants for the evolution of HCV quasispecies heterogeneity; and that increased HCV quasispecies heterogeneity is another marker associated with a poor subsequent response to interferon-α. © 1996 Wiley-Liss, Inc.     

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): Influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 ± 2.3 vs. 4.7 ± 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 ± 1.7 vs. 5.4 ± 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis C. J. Med. Virol. 54:256–264, 1998. © 1998 Wiley-Liss, Inc.     

Influence of three successive antiviral treatments on viral heterogeneity in nonresponder chronic hepatitis C patients.

The purpose of the present study was to assess the viral diversity of hepatitis C virus (HCV) in six nonresponder patients during three unsuccessful treatments. These patients were treated successively with IFN-alpha2a (IFN-alpha) at a posology of 3.10(6) units (MIU) three times a week, 10 MIU three times a week, and a combination of IFN-alpha (3 MIU) plus ribavirin (1,000 mg/day). However, only two chronically infected patients could be included in the study due to the persistence of HCV RNA during the three successive treatments. The viral diversity was analysed by cloning and sequencing the HVR-1 region. The treatment of the two nonresponder patients was associated with the persistence of a wide diversity in the viral population and with the emergence of new or minor variants. Under the influence of standard doses of IFN-alpha, a rearrangement of the quasispecies present was observed at this time point. No significant change in viral load or in the complexity of the quasispecies was observed. A second treatment with a high dose of IFN-alpha induced a significant decrease in the associated viral load and, in one case, resulted in a radical change of the viral diversity. Administration of a combination of IFN-alpha and ribavirin did not affect the evolution of the variants but was followed by the emergence of various multiple variants. These results reinforce the hypothesis of the presence of preexisting quasispecies best adapted to the host environment, and therefore resistant to any current therapy.     

Rapid changes in hepatitis C virus quasispecies produced by a single dose of IFN-alpha in chronically infected patients.

The effects of a single dose of 3 international megaunits of interferon-alpha2b (IFN-alpha2b) on hepatitis C virus (HCV) load and quasispecies were examined 24 h after administration in 12 previously untreated, chronically infected patients. All patients had viremia loads appreciably reduced relative to baseline values, thus confirming that the viral load is rapidly affected by IFN-alpha2b. Five patients also exhibited changes in plasma HCV quasispecies composition that were clearly evident by single-strand conformation polymorphism, analysis, thus indicating that one dose of IFN-alpha2b may suffice to produce rapid perturbations in the genetic heterogeneity of circulating HCV. Prior to IFN-alpha2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. The latter finding raises the possibility that in these patients, the differences in quasispecies composition between pre-IFN and post-IFN plasma resulted from increased representation of lymphoid tissue-originated variants in the latter sample, possibly because of poor sensitivity to IFN-alpha2b of HCV replication in the lymphoid compartment.     

Floating density of hepatitis C virus particles and response to interferon treatment

Hepatitis C virus (HCV) particles can be classified into two major fractions according to their floating density in serum. However, the genomic heterogeneity of each fraction and the relationship between this viral characteristic and interferon (IFN) response in patients with chronic hepatitis are not known. In this study, floating density and single strand conformation polymorphism (SSCP) of the hypervariable region 1 (HVR1) of HCV were examined in 16 patients with chronic hepatitis prior to IFN treatment. The ratio of HCV RNA titers in the top (T) and bottom (B) fractions, or T:B ratio, was 10:1 in 4 patients, 1:1 in 7, and 1:10 in 5. Three of the 4 patients with a 10:1 ratio showed a sustained response to IFN, while none of the 5 patients with a 1:10 ratio demonstrated a sustained response (P < 0.05). All 4 patients with a 10:1 ratio had 1 or 2 SSCP bands, and 4 of the 5 patients with a 1:10 ratio had 4 or 5 bands (P < 0.01). Furthermore, the number of SSCP bands in the top fraction from 6 sustained responders (1.8 ± 0.3) was significantly smaller than from 10 non sustained responders (4.1 ± 0.8) (P < 0.05). Thus, patients with a high T:B ratio and low heterogeneity in HVR1 demonstrated sustained responses to IFN, while those with low T:B ratios and high heterogeneity did not. J. Med. Virol. 55:12–17, 1998. © 1998 Wiley-Liss, Inc.     

Hepatitis C virus genetic variability in 52 human immunodeficiency virus-coinfected patients

The aim of this study was to examine whether hepatitis C virus (HCV) pretreatment quasispecies complexity was linked to virological response or other clinical and biological parameters, in human immunodeficiency virus (HIV)-coinfected patients undergoing anti-HCV treatment. In addition, HCV quasispecies composition is described longitudinally in these patients before, during, and after treatment. The 52 HIV-coinfected patients were included in a randomized therapeutic trial. At inclusion, they had CD4(+) counts of >250/micro l, HIV plasma load of <10,000 copies/ml, and chronic HCV infection with genotype 1 (n = 27), 2 (n = 2) or 3 (n = 23). These values were compared at baseline with 32 HCV-only-infected, interferon-naive patients who were infected with genotype 1, 2, or 3 (n = 16, 1, or 15, respectively). HCV complexity was studied by single-strand conformation polymorphism (SSCP) in E2 hypervariable region 1 (HVR1), and diversity was evaluated at inclusion in 20 coinfected patients by sequencing four major SSCP bands. The baseline number of SSCP bands was identical in HIV-infected and control patients. In HIV-infected patients, HCV complexity was not predictive of sustained virological response to anti-HCV treatment and was unrelated to epidemiological factors, immunological parameters linked to HIV infection (CD4(+) counts, T CD4(+) proliferative responses to HIV-1 p24), protease inhibitor treatment, HCV plasma load, or genotype. HCV diversity was lower in genotype 2- and 3-infected patients. Six months after completion of the anti-HCV treatment, in comparison with baseline, SSCP profiles were modified in 13 of the 21 nonresponding coinfected patients with analyzable samples. In conclusion, in HIV-infected patients, HCV variability had no significant influence on virological response to anti-HCV treatment.     

Quasispecies as predictive factor of rapid, early and sustained virological responses in chronic hepatitis C, genotype 1, treated with peginterferon-ribavirin.

BACKGROUND: The relationship between the complexity of the hypervariable region 1 quasispecies of HCV and responsiveness to therapy is not completely clear. OBJECTIVE: To investigate the importance of quasispecies as a predictive factor of rapid (RVR), early (EVR) and sustained (SVR) virologic response. METHODS: Prospective analysis of 82 patients with chronic hepatitis C, genotype 1, treated with peginterferon alfa-2a and ribavirin. Quasispecies (SSCP), HCV-RNA and viral load were determined at baseline and 2, 4, 8 and 12 weeks after beginning treatment. RESULTS: Less fibrosis and lower serum GGT activity were the only predictive factors for EVR. SVR predictive factors were age < or =40 years, viral load < or =600,000IU/mL, and quasispecies < or =5 bands. By logistic regression analysis, the independent factors determining SVR were age (P=0.011), viral load (P=0.027), and quasispecies (P=0.010). Quasispecies and viral load were not predictive factors of RVR. During treatment, quasispecies decreased rapidly in the SVR group. In non-EVR patients, quasispecies were slightly lower up to 8 weeks and then increased. CONCLUSIONS: Quasispecies are an important predictive factor for SVR, but are no better predictors than viral load. Quasispecies are not predictive factors for RVR or EVR.     

Hepatitis C virus genetic variability in patients undergoing antiviral therapy

Hepatitis C virus (HCV) has been the subject of intense research and clinical investigations due to its worldwide prevalence and major role in chronic liver disease. Like most RNA viruses, HCV circulates in vivo as a complex population of different but closely related viral variants, commonly referred to as a quasispecies. Recent studies suggest that ribavirin might exert an antiviral effect against HCV through both mutagenic effect and an impairment of RNA replication. The introduction of alpha interferon (IFN-alpha) plus ribavirin combination therapy was an important breakthrough in the treatment of chronic HCV infection. However, the rate of sustained virological response is still unsatisfactory, particularly in patients infected with HCV genotype 1. Viral persistence, a hallmark of HCV, may result from a dynamic control of the host response by the virus. In children with chronic HCV infection, the viral population is initially highly homogeneous, but diversifies during prolonged infection which seems to be a common event during chronic hepatitis C in childhood. Coinfection of human immunodeficiency virus 1 (HIV-1) patients by HCV can complicate the treatment of these patients with highly active antiretroviral therapy (HAART). HIV coinfection is associated with a decrease of HCV quasispecies variability, which appears to be reversed by effective HAART.     

Evolution of viral quasispecies in four dominant HlA-A2 restricted T cell epitopes is not a major reason for viral persistence in interferon-treated patients with chronic hepatitis C

In most patients, chronic hepatitis C virus (HCV) infection persists despite antiviral treatment with interferon-alpha (IFN-alpha) and ribavirin. The aim of the study was to determine whether HCV could evade cellular immune responses through mutations within T cell epitopes. Viral sequences flanking four major CTL epitopes within the HCV core and envelope regions were analyzed by PCR amplification, cloning and sequencing in seven HLA-A2 positive HCV patients before, during and after antiviral therapy. In addition, cytotoxic T lymphocyte precursor (CTLp) frequencies specific to these epitopes were quantitated by ELISPOT. A total of 13 coding mutations were observed among 650 cloned and sequenced PCR products under or post IFN treatment but no clear selection of viral variants. In detail, the diversity of quasispecies in the two core epitopes remained fairly stable over time despite variable CTLp induction in some individuals. The overall mutation rate in the two envelope epitopes was higher but there was no correlation with specific CTLp frequencies. In conclusion, although evolution of the viral quasispecies during and after antiviral therapy was demonstrated, immune evasion by epitope specific mutations seemed to be not common in interferon nonresponders because the viral complexity did not increase.     

Non-isotopic detection of hepatitis C virus quasispecies by single strand conformation polymorphism

In patients infected with the hepatitis C virus (HCV), a heterogeneous population of viruses, so-called quasispecies exists in vivo. The hypervariable regions (HVR) within the second envelope gene (HCV-E2) show particularly highly intratypic variability and are considered to be the target of neutralizing antibodies. The aims of the study were to optimize a genotype-independent primer set for amplification of HVR-1 and to establish a sensitive SSCP analysis for rapid and non-isotopic detection of predominant serum HCV quasispecies. Using the optimized SSCP technique, changes of quasispecies composition were investigated in five chronically infected patients with HCV before and during interferon-α treatment. HCV genotyping was performed by sequence and phylogenetic analysis. In addition, serial viremia and serum alanine aminotransferase (ALT) levels were determined. The SSCP analysis was performed at two time points before and during interferon-α therapy, respectively. Four patients showed an alteration of the SSCP pattern during the first three months of interferon-α therapy, whereas in one patient the SSCP pattern changed before therapy and remained stable during treatment with interferon-α. The present approach for non-isotopic analysis of single strand conformation polymorphism provides a direct, rapid, and sensitive technique for detection of the heterogeneous population of HCV quasispecies of different genotypes. Using this test procedure, investigations of large cohorts of patients with chronic hepatitis C can be undertaken. J. Med. Virol. 53:245–251, 1997. © 1997 Wiley-Liss, Inc.     

Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy

Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.     

丙型肝炎病毒包膜2区准种复杂性及其临床意义初探

目的 研究丙型肝炎病毒（HCV）包膜2（E2）区准种复杂性及其临床意义。方法 以9例不同血清丙氨酸转氨酶水平物急性HCV感染者和4例慢性丙型肝炎患者为对象，通过逆转录PCR扩增HCVE2区氨基端片段（560bp)，扩增产物直接克隆，以单链构象多态性（SSCP）/异质性双体（HD）分析对每份血清标本的30个克隆进行筛选及准种复杂性分析，结果 慢性丙型肝炎患者血清HCV准种复杂性明显增高：HCV感染者血清ALT水平变化与准种复杂性无明显关系。结论 HCVE2区准种复杂性与病毒持续性感染有关。征收肝损害程度无关。     

A single nucleotide polymorphism in IL28B affects viral evolution of hepatitis C quasispecies after pegylated interferon and ribavirin therapy

Interleukin‐28B (IL28B) polymorphisms are associated with viral response to peginterferon and ribavirin (RBV) in chronic hepatitis C (HCV). Their recognition represents a breakthrough in the understanding of the role of the host in viral eradication. How these polymorphisms determine viral eradication is unknown. The IL‐28B variants are hypothesized to have a differential impact on HCV quasispecies evolution during treatment with pegylated interferon (PEG‐IFN) and RBV. In this study, HCV RNA levels were measured at early time points in 33 naïve genotype 1 hepatitis C patients and clonal analysis of the entire NS5A region was performed on sera from baseline and Day 7. Site rs12979860 polymorphisms were determined by direct sequencing of PCR products and classified into CC, CT, and TT and were identified in 13, 11, and 9 patients, respectively. The CC polymorphism more commonly was seen in Whites versus Blacks [12/21 (57%) vs. 1/12 (8%), P = 0.009] and HIV‐infected versus mono‐infected [13/25 (52%) vs. 0/8 (0%), P = 0.009]. Patients with CC and non‐CC had similar baseline viral loads. More patients with the CC polymorphism had amino acid substitutions in NS5A compared to non‐CC patients. Despite similar baseline viral diversity, by Day 7, significantly more patients with CC had higher non‐synonymous substitution values compared to non‐CC (P = 0.02). Chronic hepatitis C patients with the CC IL28B polymorphism have a higher number of amino acid substitutions in the NS5A region and early viral evolution due to greater interferon induced selective pressure during this critical period of treatment. J. Med. Virol. 84:1913–1919, 2012. © 2012 Wiley Periodicals, Inc.     

Genetic heterogeneity of the envelope 2 gene and eradication of hepatitis C virus after a second course of interferon-alpha

The heterogeneity of the envelope 2 (E2) gene of the hepatitis C virus (HCV) was involved in the sensitivity of HCV to interferon-alpha (IFN-alpha). To assess the factors leading to virus eradication by IFN-alpha, patients whose first treatment by IFN-alpha failed and who had virus eradication after a second treatment were studied. These patients were paired with subjects in whom both treatments failed. The phosphorylation homology domain of the E2 gene (E2-PHD) had no sequence variation between the two stages in both groups of patients. Therefore, this region has no clinical predictive value within a specific genotype. The hypervariable region 1 (HVR1) was analyzed by cloning and sequencing 20 clones per sample. Comparison of samples showed that the change in quasispecies induced by the first IFN-alpha therapy could be associated with virus elimination obtained after a second treatment. The greater proportion of nonsynonymous mutations that was noted before the second treatment in responders suggest that pretherapeutic immune response is a major factor determining virus elimination and that the immune status of these patients changed between the first and the second treatment.     

丙型肝炎病毒包膜区变异与感染慢性化关系的初步研究

目的 探讨丙型肝炎病毒(HCV)包膜区变异与其感染慢性化的关系。方法 3份HCV慢性感染者和3份急性感染者血标本，采用逆转录—聚合两链反应(RT—PCR)扩增HCV的E1区C端及E2区N端片段(573bp)，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30个克隆的E1／E2区准种(quasispecies)进行筛选，挑选每份标本HCV的优势株与劣势株序列进行测定，分析非同义替换碱基数与同义替换碱基数比率(dN／dS，间接反映选择压力)和推导的氨基酸序列。结果 HCV慢性感染者病毒准种的复杂性和E2区dN／dS明显高于急性感染者。HCV慢性感染者的E2区氨基酸替换率(8．46％)比急性感染者(1．02％)更高，而两者的E1区氨基酸替换率(分别为2．74％和1．09％)均较低。尽管HVR1变异程度更高，但仍存在高度保守的氨基酸位点。结论 HCV持续性感染与准种复杂性增高和宿主对HVR1的免疫选择有关。