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。     

慢性乙型肝炎患者体内乙型肝炎病毒准种特点的初步研究

目的 探讨乙型肝炎病毒（HBV）准种在慢性乙型肝炎患者中的存在状态。方法 以已知中国株HBV基因序列为依据。设计特异性聚合酶链反应（PCR）引物,自3例慢性乙型肝炎患者体内扩增HBV全S基因片段,克隆人pGEMTeasy质粒,用限制性片段长度多态性（RFLP）法确定HBV的变异现象,DNA测序确定病毒的变异程度。结果 分别自3例患者血清中克隆出29、28和8个阳性克隆,以EcoRⅠ酶切患者1和2的RFLP结果提示各有5种不同的长度带型,PCR产物XhoI酶切RFLP结果表现为高度保守,测序结果发现HBV体内毒株DNA序列高度一致,同一患者两株全S区序列测序结果表明DNA序列的同源性大于97%。结论 慢性乙型肝炎患者体内有HBV准种共存,且呈现出一定的优势克隆现象。可能与患者预后有关。     

重型乙型肝炎患者肝组织和血清中乙型肝炎病毒前C区准种的比较

乙型肝炎病毒存在准种现象。为研究重型乙型肝炎患者血清和肝组织中乙型肝炎病毒（HBV）DNA前C区准种组成特点，本研究从3例重型乙型肝炎患者的血清和肝组织中提取HBV DNA，经巢式聚合酶链反应（PCR）扩增出HBV前C区，产物克隆后经单链构象多态性／异源双链分析（SSCP／HDA）筛选不同的克隆并测序，比较血清和肝组织中HBV前C区准种组成的异同。     

人类免疫缺陷病毒-1的核酸遗传学特点

目的研究人类免疫缺陷病毒（HIV）-1膜蛋白基因（env）的准种与变异特点.方法采用套式聚合酶链反应（PCR）对5例艾滋病患者血浆HIV-1 env基因C2～V3区进行PCR扩增,产物经纯化后克隆至pMD-18T载体中,每例患者分别挑取5～8个克隆,共34株,完成阳性克隆的鉴定、抽提纯化和测序,将获得的DNA序列及核苷酸序列进行分析,并计算其核苷酸同源替换率（ds）与非同源替换率（dn）的比值.结果同一患者体内HIV准种株平均为83.5%,各克隆株之间的核苷酸同源性在88%～99%之间;不同患者之间的核苷酸同源性在75%～91%;34株序列的V3区平均变异频率明显高于C2～C3区（P＜0.05）;而V3区和C2～C3区的ds/dn比值均＞1.结论艾滋病患者体内有大量HIV准种存在,基因的变异在V3区发生较频繁,免疫选择压力在该区变异中不起主要作用.     

山东省丙型肝炎病毒分离株NS5区核苷酸序列分析

目的：了解山东省丙型肝炎病毒（HCV）分离株的基因型及其基因的变异情况，方法：应用德国UBI HCV EIA 4．0诊断试剂盒筛选山东省部分地区64例临床检验为抗－HCV阳性的血清标本，有54例阳性，随意抽取其中28例，应用逆转录套式－聚合酶链反应（RT－nested-PCR)扩增319bp的HCV NS5区基因片段，结果12例出现特异性条带，随后将这12个NS5区片段直接进行T载体克隆，并用Sanger法对克隆成功的10个NS5区基因片段进行序列测定，将所得到的10个序列与GenBank中所有的HCV分离株进行同源性比较。结果：10例山东省部分地区HCV分离株的基因型均属于HCV－1b型，对获得的10个NS5区片段进行是性分析发现，所有的核苷酸变化都是由于替代作用引起的，没有碱基的插入和缺失；大部分的突变属于同义突变，占突变总数的74％。RNA－依赖性RNA聚合酶的G－D－D基序和所有的半胱氨酸都完全保守，结论：本研究证明了HCV－1b型是山东省部分地区主要的基因型。     

丙型肝炎病毒高变区准种异质性与干扰素疗效的关系

丙型肝炎病毒(HCV)基因组具有高度的变异性，在机体内以准种的形式存在，尤其以E2／NS1区384～410和474～480位氨基酸变异程度最高，分别称为HVR1和HVR2。近年的研究发现，HCV准种感染除与引起HCV感染慢性化有关外，还与HCV对干扰素(IFN)治疗的抵抗作用关系密切。本研究采用克隆测序法对10例应用IFN治疗的1b型慢性丙型肝炎患者进行了HVR1和HVR2准种异质性检测，以探讨HCV准种感染与IFN疗效的关系。     

慢性乙型肝炎患者体内乙型肝炎病毒准种特点的初步研究

探讨乙型肝炎病毒（HBV）在慢性患者中存在状态，以HBV基因序列为依据。设计特异性多聚酶链反应（PCR）引物，自2例慢性患者体内扩增HBV全S基因片段，克隆入T载体。限制片段长度多态性（RFLP）法确定HBV的变异现象，DNA测序确定病毒的变异程度，质粒EcoRI酶切RFLP结果提示自2例患者血清中克隆出的29和28个阳性克隆中各有5种不同的长度带型，PCR产物XhoI酶切RFLP结果则表现为高度保守，测序结果发现HBV体内毒株碱基序高度一致，同一患者两株全S区序列测序结果表明DNA序列的同源性大于97%。本结果提示HBV慢性患者体内有HBV准种共存，且呈现出一定的优势克隆现象。     

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。     

慢性乙型肝炎患者病毒准种特性的初步研究

目的 探讨慢性乙型肝炎(CHB)患者血清中乙型肝炎病毒(HBV)是否存在准种特性,并初步了解HBV准种的复杂性和遗传差异性。 方法用多聚酶链反应(PCR)技术从1例CHB患者血清中扩增HBV整个PreC/C基因区,然后用T载体克隆PCR产物,从转化阳性的克隆中随机选出34个克隆进行核酸序列分析。结果在34个测序克隆中发现存在28种不同的序列,序列间差异性介于0.2％～2.1％。变异位点分布于整个区域。所有序列nt1896位均无变异。 结论 在CHB患者体内HBV存在复杂的准种特性。     

利用非同位素的单链构型多态性分析检测丙型肝炎病毒的准种

在感染丙型肝炎病毒(HCV)的患者体内,存在着被称为准种(quasispecies)的病毒异源性群体,在第二个膜基因(HCV-E2)中的高变区(HVR)显示出特别高的型内变异并被认为是中和抗体的目标,本研究的目的是优化扩增HVR-1的、不依赖于基因型的引物系统,建立了一个敏感的SSCP分析方法,以快速、非同位素检测血清中占优势的HCV准种。利用优化的SSCP分析的方法,调查了5个HCV慢性感染的患者在α-干扰素治疗前后准种组成的改变,HCV基因型是通过DNA序列测定和多基因分析来确定,另外,还检测了血清病毒血症和血清转氨酶(ALT)水平,SSCP分析在干扰素治疗前后的两个时间点分别进行,在IFN治疗的前3个月中有     

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.     

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.     

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     

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.     

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。     

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.     

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.