丙型肝炎病毒高变区基因变异特点初探

对两例ＨＣＶＲＮＡ持续阳性者分三个时间点随访五年,测定了ＨＣＶ的高变区基因序列,每个时间点平均测定２８个克隆,总共测定了１６８个克隆。研究发现ＨＣＶ高变区基因变异有五个明显特点：（１）变异程度大,高变区至少有８９％的核苷酸位点都可能发生变异;（２）变异形式多样,除常见的替代突变外,缺失突变（缺失１、２或３个核苷酸）的克隆数占总克隆数的２２．５％;（３）优势克隆明显,即有若干个克隆高变区的核苷酸和氨基酸序列相同;（４）类似株现象严重;（５）变异幅度大。该研究说明ＨＣＶ高变区基因变异具有高度复杂多样性。     

丙型肝炎病毒基因型及基因组多样性与临床的关系

本文阐述丙型肝炎病毒（ＨＣＶ）基因的全部结构,已知的基因型及超变区克隆间的多样性与肝炎临床病况及治疗的关系。     

中国河南两株庚型肝炎病毒（HGV）NS5区部分cDNA的克隆与序列分析

通过逆转录－聚合酶链反应从我国河南省２例重叠感染ＨＣＶ或ＨＢＶ／ＨＤＶ的献血啼中,分离到ＨＢＶＮＳ５区的部分ｃＤＮＡ,对其进行序列分析比较,结果表明,河南株ＨＧＶＮＳ５工核苷酸与两中国ＨＧＶ主同源性高于国外代表株（８８．５－９０．６％）,但由核苷酸推导的氨基酸的同源性都无明显的地区性区别。ＨＧＶＮＳ５区氨基酸序列较保守,缺乏明显高变区,中国４株ＨＧＶ在７３８４位发生了由Ｃ→Ｔ的变异,从而导致一个人     

Ⅲ型中国株HCVE2/NS1基因与已知分离株的同源性比较

利用逆转录套式ＰＣＲ扩增Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段,将其克隆到ｐｃＤＮＡ３载体上．采用双脱氧链终止法测定插入片段的核苷酸序列．并与已知分离株的相应区域进行同源性比较．首次克隆出Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因（ＨＣ－Ｗ１４）,其核苷酸序列与Ⅲ型日本株ＨＣＶ（ＨＣ－Ｊ６）该区域同源性为８８．３７％,其推定的氨基酸同源性为８９．２９％．而与已知的非Ⅲ型株ＨＣＶ该区域相比,核苷酸及氨基酸的同源性均相对较低．Ⅲ型中国株ＨＣＶ与Ⅱ型中国株ＨＣＶ在Ｅ２／ＮＳ１区域有较大的变异,揭示研制我国的ＨＣＶ疫苗应该考虑这种基因型之间的变异性．     

丙型肝炎病毒抗原决定簇与霍乱毒素B亚基的融合表达以及融合蛋白的抗原性

通过固相化学合成法,合成了编码丙型肝炎病毒（ＨＣＶ）结构区和非结构区的４个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串朕后与ｃｔｘＢ基因融合,构建了１２种表达不同嵌合蛋白的重组质粒。各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量随所融合的抗原决定簇不同在１０～５０μｇ／ｍｌ之间,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化达到了电泳纯。对１１种融合蛋白中ＨＣＶ抗原决定簇的反应原性的研究表明,多数融合蛋白中ＨＣＶ抗原决定簇均能与对应的ＨＣＶ抗体结合。其中以融合蛋白９５０８２为抗原研制的抗－ＨＣＶＥＬＩＳＡ试剂具有良好的灵敏度和特异性。     

我国丙型肝炎病毒囊膜蛋白E2高变区1的序列特征

对２３例国内献血员、血透析及肝炎病人血清用反转录巢式ＰＣＲ技术扩增了ＨＣＶＲＮＡ囊膜蛋白２基因的ｃＤＮＡ片段,并进行了序列测定。结果表明２３例病人ＨＣＶＥ２／ＮＳ１Ｎ末端的核苷酸及氨基酸序列呈现多样性,高变区１（ＨＶＲ１）位于核苷酸第１４５９～１５５９位,氨基酸第３８４～４１０位;我国ＨＣＶ株ＨＶＲ１２７个氨基酸中有１５个位置氨基酸相对稳定,氨基酸组成与分布均与Ｓｅｋｉｙａ报道的１６６个ＨＣＶ株的不同。结果提示,研究我国ＨＣＶ株ＨＶＲ１的序列特征有助于ＨＣＶ的流行病学研究,对研制适用于我国的抗体诊断试剂盒及进行疫苗研究均有重要的意义。     

同型HCV不同分离株NS5区基因片段的比较分析

对同一地区两例输血后丙型肝炎进行ＰＣＲ分型,结果为Ⅱ型。分别将其ＮＳ５区基因部分片段克隆到ｐＵＣ１８和Ｍ１３ｍｐ１８中,序列分析结果表明在ＮＳ５区基因相同区段,二者的核苷酸同源性为８９．０４％,氨基酸同源性为９０．７５％。而且在分离株Ｖ中第７０～７２位发现阅读框内终止密码子ＴＧＡ。与Ⅱ型代表株ＨＣＶＢＫ序列相比,相同区段的核苷酸和氨基酸同源性分别为：９１．５％,９１．９２％和９１．９１％,９１．９１％。对比分析表明,在同一地区基因型相同的不同分离株间ＮＳ５区基因仍存在较大变异,分离株Ｖ可能为缺陷性病毒     

乙型肝炎病毒X基因准种特点的研究

以乙型肝炎病毒（ＨＢＶ）Ｘ基因序列的异质性表现来探讨ＨＢＶ准种在　慢性感染者中的存在状态。以中国株ＨＢＶ基因序列为依据,设计特异性多聚酶链反应引物,　自９例慢性ＨＢＶ感染患者血清中扩增ＨＢＶ　Ｘ基因,克隆入ｐＧＥＭ　Ｔｅａｓｙ质粒,随机挑选克隆进行Ｄ　ＮＡ测序以确定病毒的变异程度。３７例测序结果提示来源于不同患者ＨＢＶ　Ｘ基因序列高度保守　,但每个序列均不一致。Ｘ区除了存在广泛的碱基点替换突变外,序列的缺失突变占测序克　隆总数的５１．４％（１９／３７）;氨基酸缺失及移框突变多发生于１２３位氨基酸残基之后,可导致Ｘ蛋　白多种羧基端形式。结果提示ＨＢＶ长期携带者体内有ＨＢＶ准种共存,Ｘ区内存在热点缺失突变区,该区突变结果可能影响Ｘ蛋白反式激活功能。     

从中国病人克隆丙型肝炎病毒基因组C区基因及其在大肠杆菌中的表达

本文通过逆转录（ＲＴ）－聚合酶链式反应（ＰＣＲ）,从两份分别来自湖南省娄底地区丙型肝炎病人和河北省秦皇岛市职业献血员丙型肝炎病毒（ＨＣＶ）ＲＮＡ打点杂交阳性的血清中,扩增并克隆到１段５６３ｂｐ的ＨＣＶ基因组Ｃ区抗原基因Ｃ２６９／８３１,并通过ＰＣＲ得到了３个表达片段Ｃ８３１、Ｃ８０１和Ｃ５８７。测定Ｃ２６９／８３１基因的全序列后发现,中国人ＨＣＶ湖南分离株与ＨＣＶ－Ⅰ型株ＨＣＶ－ＵＳ和Ⅱ型株ＨＣＶ－ＢＫ在该基因区段的核苷酸／氨基酸序列的同源性,分别为９０．３％／９４．６％和９５．２％／９４．６％。利用原核高效表达载体ｐＢＶ２２０在大肠杆菌中有效地表达了非融合的Ｃ区抗原基因重组蛋白ＣＬ、ＣＭ和ＣＳ。通过免疫筛选法及Ｗｅｓｔｅｍ印迹法对约占菌体可溶性蛋白１１％的表达产物进行了鉴定。采用ＴｒｉｔｏｎＸ－１００和盐析处理表达产物,再进行离子交换层析纯化,得到可用于检测ＨＣＶ血清抗体的核壳蛋白（Ｃ）抗原。通过不同分子量抗原的表达,发现由Ｃ区Ｎ端８９个氨基酸组成的多肽ＣＳ其抗原性与由１５８或１６８个氨基酸组成的多肽ＣＭ或ＣＬ相同,但抗原的稳定性和表达量显著优于后两种抗原。本研究为研制ＨＣＶ抗体诊断试剂盒奠定了重要基础。     

HFRSV内影像型抗独特型人单抗重链可变区基因克隆

利用反转录－ＰＣＲ方法,从分泌肾综合症出血热病毒（ＨＦＲＳＶ）内影像型抗独特型人单抗杂交瘤细胞系（Ｃ８）中,成功地克隆了抗ＨＦＲＳＶ１ｄ人单抗重链可变区基因,并将此基因重组入Ｍ１３噬菌体ＤＮＡ中,测定了此重链可变区基因全序列。经计算机分析,基因全长共３５１ｂｐ、编码１１７个氨基酸,氨基酸序列同源性分析发现所推得的该单抗ＣＤＲ２区与ＨＦＲＳＶＧ２蛋白Ｃ端有同源区,此区可能具有较强的抗原性。     

Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients.

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.     

四份中国HCV阳性血清中包膜蛋白E1/E2准种基因的序列分析及新型插入突变的发现

为分析四份中国丙型肝炎病毒（HCV）阳性血清中包膜蛋白E1/E2基因的准种特征。本研究对从4份中国HCV阳性血清（1b亚型：274、366、383;2a亚型：283）中提取的HCV核酸,采用逆转录-聚合酶反应扩增编码全长E1/E2蛋白（191～764aa）的基因片段,随机挑取多个克隆测序。根据E1/E2基因核苷酸的序列与其他相关序列（来自于GenBank）构建亲缘性关系进化树,进行核苷酸与氨基酸同源性分析并对重要的基因位点进行分析。共获得阳性克隆序列43个（274株10个,283株12个,366株13个,383株8个）,发现高变区HVR1、HVR2的基因异质性高,而其他抗体中和表位及跨膜区I、II及N末端糖基化位点相对保守。并首次发现在HCV 2a亚型（283血清）中多个准种序列存在1279nt（E1区,313aa）处单碱基插入优势基因突变,导致HCV包膜蛋白编码突变与中断（E2区,398aa）。本研究对中国HCV代表株包膜蛋白E1/E2编码基因的准种多样性及一种新型插入突变进行了描述,可为进一步研究HCV免疫逃避与慢性化机制提供重要信息。关键词：丙型肝炎病毒;包膜蛋白;序列分析;准种;插入突变     

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.     

Effect of Retreatment with Interferon Alone or Interferon plus Ribavirin on Hepatitis C Virus Quasispecies Diversification in Nonresponder Patients with Chronic Hepatitis C

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.     

Hypervariable region 1 sequence stability during hepatitis C virus replication in chimpanzees

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.     

Two optimal mutation rates in obligate pathogens subject to deleterious mutation

Pathogen species with high mutation rates are likely to accumulate deleterious mutations that reduce their reproductive potential within the host. By altering the within-host growth rate of the pathogen, the deleterious mutation load has the potential to affect epidemiological properties such as prevalence, mean pathogen load, and the mean duration of infections. Here, I examine an epidemiological model that allows for multiple segregating mutations that affect within-host replication efficiency. The model demonstrates a complex range of outcomes depending on pathogen mutation rate, including two distinct, widely separated mutation rates associated with high pathogen prevalence. The low mutation rate prevalence peak is associated with small amounts of genetic diversity within the pathogen population, relatively stable prevalence and infection dynamics, and genetic variation partitioned between hosts. The high mutation rate peak is characterized by considerable genetic diversity both within and between hosts, relatively frequent invasions by more virulent types, and is qualitatively similar to an RNA virus quasispecies. The two prevalence peaks are separated by a valley where natural selection favors evolution toward the optimal within-host state, which is associated with high virulence and relatively rapid host mortality. Both chronic and acute infections are examined using stochastic forward simulations.     

Evolution of Hepatitis C Virus Quasispecies in Hypervariable Region 1 and the Putative Interferon Sensitivity-Determining Region during Interferon Therapy and Natural Infection

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.     

Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus.

We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.     

HCV Genome-Wide Genetic Analyses in Context of Disease Progression and Hepatocellular Carcinoma

Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV’s sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.