慢性丙型肝炎患者HCV准种的变化及其意义

目的　探讨HCV准种与丙型肝炎慢性化的关系。方法　以 3 0例HCVRNA(HVR1区PCR )阳性的丙型肝炎患者为研究对象 ,采用单链构象多态性分析 (SSCP)进行HCV准种检测。结果　 8例急性丙型肝炎、15例慢性丙型肝炎和 7例肝硬化患者的SSCP条带数分别为 ( 3 .13± 1.0 7)、( 5 .0 7± 1.48)和 ( 5 .5 7± 2 .15 ) ,慢性肝炎组、肝硬化组与急性肝炎组SSCP条带数比较有显著差异 (P <0 .0 5 )。通过输血或使用血制品感染HCV者共 17例 ,SSCP条带数为 ( 5 .3± 1.8) ,散发感染组 11例 ,SSCP条带数为 ( 3 .9± 1.6) ,两组比较有显著差异 (P <0 .0 5 )。 2 2例慢性丙型肝炎和肝硬化患者的SSCP条带数与病程相关系数分别为 0 .5 72 (Pearson’s相关 )和 0 .5 5 6(Spearman’s相关 ) ,与性别、年龄、ALT水平无相关性。结论　HCV准种数量与丙型肝炎慢性化密切相关 ;HCV准种数量与丙型肝炎感染途径相关 ,输血或血制品感染者HCV准种数量增多 ;HCV准种数量与病程呈正相关 ,感染时间长 ,HCV准种数量多。     

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。     

慢性丙型肝炎患者病毒因素及宿主细胞免疫对干扰素治疗应答的影响

目的：探讨丙型肝炎病毒（HCV）因素及机体细胞免疫因素对干扰素疗效的影响。方法：对40例慢性丙型肝炎患者进行干扰素治疗，分析HCV基因型，准种多样性，血清HCV RNA水平及肝组织HCV特异性细胞毒T淋巴细胞（CTL）活性与干扰素应答的关系。结果：经过6个月的干扰素治疗，21例获得治疗终点应答，其中10例呈持续应答，19例无应答，HCV1型患者应答率（43．3％）明显低于非1型患者（80％，P＜0．05），应答患者中治疗前血清HCV准种数目及HCVRNA水平明显低于无应答患者（P＜0．05，P＜0．01），而其肝组织HCV特异性CTL活性阳性率则显著高于无应答者（P＜0．05），结论：病毒因素和宿主因素均是影响慢性丙型肝炎干扰素治疗效果的重要因素，非1型感染，低准种数目，低病毒血症水平及肝组织HCV特异性CTL活性阳性者预示对干扰素应答良好。     

丙型肝炎病毒高变区1融合蛋白的表达、纯化及其初步应用

目的　克隆并表达中国不同地区不同基因型的丙型肝炎病毒高变区 1(HCVHVR1)蛋白 ,并用表达纯化产物检测慢性丙型肝炎患者血清中的相应抗体 ,分析其临床意义。方法　根据 31株HCVHVR1序列分析及免疫原性预测结果 ,选择 4株克隆 (1b型 3株 ,2a型 1株 ) ,从 pGEMT E2克隆中扩增得到 4个HVR1片段 ,将其分别克隆到原核表达载体 pQE4 0中 ,表达产物经纯化后用以检测慢性丙型肝炎患者血清中的HVR1抗体。结果　构建的HVR1原核表达载体在大肠埃希菌中成功表达了 4种相对分子质量约为 2 80 0 0的二氢叶酸还原酶 (DHFR) HVR1融合蛋白 ,纯化蛋白的获得率约为 32 0～ 80 0 μg/ 10 0ml培养液。这 4种融合蛋白 (SH1b、BJ1b、SD1b、SD2a)与慢性丙型肝炎患者的血清结合率分别为 72 .8% (5 1/ 70 )、6 0 % (4 2 / 70 )、4 8.6 % (34/ 70 )和 5 8.6 % (4 1/ 70 )。在 2 0例用干扰素治疗的丙型肝炎患者中 ,5 7% (4 / 7)的干扰素治疗无应答者治疗前血清能与之反应 ,而应答者血清中只有 15 .3% (2 / 13)能与之反应。干扰素治疗应答者血清与 4种融合蛋白反应的A值高于治疗无应答者 (P <0 .0 5 )。结论　选择的 4株HCVHVR1片段在大肠埃希菌中获得成功表达 ,所得 4种HVR1融合蛋白能与HCV感染者血清发生较广泛的反应     

上海地区HIV/HCV重叠感染者HCV准种的分子流行病学研究

目的 观察HIV/HCV重叠感染和高效抗反转录病毒治疗(HAART)对HCV准种的影响.方法 通过PCR、测序及单链构象多态性分析建立HCV高变1区(HVR1)准种变异率检测方法,运用该方法对我国上海地区48例HIV/HCV重叠感染者HCV准种变异的分子流行病学进行研究.结果 与单独HCV感染组和HIV/HCV重叠感染...     

丙型肝炎病毒NS5A基因在自然感染过程中的准种动态

干扰素敏感决定区（ISDR）的准种特性与干扰素的疗效密切相关。体外研究发现，野生型的丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）能与干扰素（内源或外源的）诱导的双链RNA依赖的蛋白激酶（PKR）结合并相互作用，从而抑制PKR的抗病毒活性。部分揭示了NS5A与HCV持续感染的关系。为了进一步调查ISDR，PKR-BD，V3和NS5A其他功能区的变异情况，本研究从7例慢性丙型肝炎患者10年前与10年后血清中扩增并克隆NS5A基因，进行序列分析发现，野生型干扰素敏感决定区毒株在所有感染者中具有选择优势。     

1例早期HIV感染者体内病毒准种的各基因片段分析

目的比较1例早期艾滋病病毒（HIV）感染者体内病毒不同基因片段准种复杂度。方法使用套式聚合酶链反应（Nested-PCR）,扩增HIV感染者外周血淋巴细胞中病毒的gag、pol、vif-vpr和env区的部分基因,通过克隆、测序方法观察该感染者体内病毒准种分布情况,比较不同基因片段检测病毒准种的可靠性。结果不同基因片段检测病毒准种结果显示,该早期感染者体内的病毒不同基因片段显示的准种基因离散率不同,其中gag区显示出的病毒准种复杂度最高,反映为该区段病毒准种的基因离散率最高,为0．008;其他各区基因（pol、vif-vpr、env）的离散率依次为0．006、0．001、0．000。结论该HIV感染者体内病毒准种的基因中,gag区基因的突变率较高,提示Gag在机体感染HIV早期承受的免疫压力较高,因此在病毒准种研究中,gag区应该作为重要的基因区域。     

广东省单纯丙型肝炎病毒及其与人类免疫缺陷病毒共感染者丙型肝炎病毒的基因亚型分析

目的 了解广东省人类免疫缺陷病毒(HW) /HCV共感染者与单纯HCV感染者的感染途径、HCV的基因亚型分布及其遗传特征,为丙型肝炎病毒感染的治疗与预防提供依据. 方法采用巢式逆转录-聚合酶链反应扩增广东省95例HIV/HCV共感染者及99例单纯HCV感染者HCVNS5B基因区域,扩增产物测序后进行HCV基因亚型分析,遗传分析利用MEGA4软件.不同种基因亚型型内基因距离以碱基置换率表示.结果 HIV/HCV共感染者HCV有5种基因亚型,其中6a型占53.7％ (51/95)、3a型占17.9％ (17/95)、1b型占15.8％ (15/95)、3b型占11.6％ (11/95)、1a型占1.0％(1/95);1b亚型内基因距离为6.30％±1.27％,高于其他基因亚型.HIV/HCV共感染者主要通过静脉注射毒品感染(75/95,78.9％),其基因亚型主要为6a型,占60.0％ (45/75).单纯HCV感染者HCV有7种基因亚型,其中1b型占67.7％ (67/99)、6a型占17.2％(17/99)、3a型占6.1％ (6/99)、2a型占5.％(5/99)、3b型占2.0％(2/99)、4a型占1.0％ (1/99)、5a型占1.0％(1/99);1b亚型内基因距离为5.17％±1.03％,高于其他基因亚型.单纯HCV感染者主要通过输血或血液制品感染(80/99,80.8％),其HCV基因亚型主要为1b型,占76.2％ (61/80).结论 广东地区HIV/HCV共感染者及单纯HCV感染者HCV基因亚型呈现多样性,HIV/HCV共感染者与单纯HCV感染者主要感染途径不同,HCV主要基因亚型也不同.     

白细胞介素-28B基因多态性与丙型肝炎病毒感染自然清除的相关性

目的 探讨IL-28B基因多态性与HCV感染自然清除的相关性.方法 HCV感染者280例,其中慢性丙型肝炎患者200例,HCV感染后复查自发清除者80例,检测IL-28B基因rs8099917位点基因型,并分析IL-28B基因型与HCV感染自然清除的关系.统计学处理采用x2检验.结果 慢性丙型肝炎组与自发清除组IL-28B rs8099917位点TT、非TT(TG和GG)基因型的频率差异有统计学意义(x2=15.874,P＜0.01).TT型在自发清除组中的频率为86.2％,高于慢性丙型肝炎组的62.0％.慢性丙型肝炎患者、自发清除者IL-28B rs8099917 T和G等位基因频率分别为78.0％、22.0％和92.5％、7.5％.自发清除人群中以T等位基因频率为主(92.5％),两组等位基因的差异有统计学意义(P＜0.01).TT型HCV感染者自发清除HCV的可能性是非TT型感染者的2.84倍,而非TT型(TG+GG)HCV感染者慢性化的危险性是TT型的1.36倍.结论 宿主IL-28B rs8099917位点TT基因型与HCV感染自发清除相关,可作为HCV感染自发清除的一个重要预测因素.     

转化生长因子β1信号肽区单核苷酸多态性与慢性丙型肝炎的相关性

目的 探讨HCV感染者转化生长因子β1(TGF β1)信号肽区密码子25基因多态性与HCV感染的相关性. 方法 采用扩增阻碍突变系统方法,分析106名健康对照者和85例慢性HCV感染者的TGF β1信号肽区密码子25基因多态性. 结果 TGF β1信号肽区密码子25的基因型分布与基因频数在HCV感染组和对照组之间,慢性丙型肝炎组和HCV相关肝硬化组之间的差异均无统计学意义.密码子25基因型分布在ALT正常组和ALT升高组之间的差异无统计学意义,但G基因频数在ALT升高组(G=108)较ALT正常组(G=47)高(P-0.040).密码子25基因型分布在尢病毒血症组(GG=18,CG=5,CC=3)和病毒血症组(GG=55,CG=4)之间的差异有统计学意义(P=0.005),且G等位基因更多出现干病毒血症组(P=0.000).结论 TGF β1信号肽区密码子25G→C突变影响HCV感染肝脏炎性反应,G等位基因与HCV感染病毒血症相关,TGF β1信号肽区密码子25G→C突变可能是影响HCV感染结局的因素之一.     

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.     

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.     

Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes

Differences in the composition of the hepatitis C virus (HCV) quasispecies between plasma and blood mononuclear cells (BMC) strongly suggest that BMCs support viral replication. We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin-complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg- and HIV-seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)-bound fraction, and total and sorted BMCs (CD19+, CD8+, CD4+, and CD14+ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (d(N)/d(S) ratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19+, 10/11 CD14+, 4/11 CD8+ and 0/11 CD4+ cell samples. HVR1 sequences were significantly different between plasma and at least one cellular compartment in all nine cases analyzed, and between B cells (CD19+) and monocytes (CD14+) in all five available cases. IgG-bound variants were distinct from cellular variants. D(N)/d(S) ratios were similar (n = 3) or lower (n = 6) in cellular compartments compared with plasma and the IgG-bound fraction. In conclusion, HCV compartmentalization is a common phenomenon. B cells and monocytes harbor HCV variants showing a low rate of nonsynonymous mutations, a feature that might contribute to the persistence of HCV infection.     

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.     

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.     

Clinical relevance of hepatitis C virus quasispecies

Summary. It has been shown that hepatitis C virus (HCV) populations in infected individuals are composed of quasispecies with diverse mutations. The analysis of these variants may reveal mechanisms of the persistence of HCV infection, carcinogenesis and resistance to antiviral therapy. Recently, genetic features of interferon-resistant HCV have been elucidated through the analysis of interferon-resistant quasispecies, making it possible to predict interferon efficacy by detecting interferon-resistant strains.     

Evolution of hepatitis C virus hypervariable region 1 in immunocompetent children born to HCV-infected mothers

Summary.  Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d _N / d _S rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site ( d _N ) and synonymous nucleotide substitutions per synonymous site ( d _S )] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.