【作者】 张立新；

【导师】 秦成勇；

【作者基本信息】 山东大学 ， 内科学， 2011， 博士

【摘要】 研究背景和目的：丙型肝炎(Hepatitis C)是威胁人类身体健康的重要传染病,其病原体是丙型肝炎病毒(Hepatitis C virus, HCV),全球范围内大约有1.7亿人感染了HCV,我国的感染率约为3%。HCV感染容易慢性化,慢性丙型肝炎约有20%-30%会在20年内缓慢进展为肝硬化和肝癌,HCV感染成为导致肝癌及终末期肝病的重要原因。HCV为单股正链RNA病毒,整个基因组只有一个开放读码框(Open reading frame, ORF),位于基因组中央,在基因组的5’和3’末端各含有一段非编码序列(Non-cording Region, NCR)。基因组从5’NCR依次为C、E1、E2、P7、NS2、NS3、NS4A、NS4B、NS5A、NS5B及3’NCR, C区编码核壳蛋白,E1、E2区编码包膜糖蛋白,P7及NS2到NS5分别编码不同功能的非结构蛋白。HCV具有高度变异性,但不同区段变异率不同,有些区段变异性较大,而另有些区段则遗传保守性很高,其中5’NCR和C区最保守,E区最容易变异。HCV 5’NCR是整个基因组最为保守的区域,长度和序列非常稳定,这个区段核苷酸突变少,并且其变异可以代表基因型。HCV可以分为6个基因型及不同亚型,HCV基因型分布有明显的地域差别,我国大陆主要是1b、2a型及少量的la、2b、3b、4a及6a型等。研究表明,HCV基因型与临床有密切的关系,基因1型HCV感染者往往病毒载量较高,疾病进展快,对干扰素的应答较差,但是基因型对临床的影响仍然存在一定的争议。HCV 5’NCR是病毒复制的起始部分,在病毒的复制及病毒蛋白的翻译方面起到很重要的作用,5’NCR某些核苷酸的特异性变异可能会影响病毒的复制水平及对干扰素的治疗应答。NS5A是HCV编码的非结构蛋白,在HCV多蛋白的成熟和RNA的复制过程中具有十分重要的作用,具有抗肝细胞凋亡作用,并下调干扰素a(IFNa)刺激的抗病毒效应。HCV NS5A某些区域的基因变异可能导致其抗干扰素作用的减弱,研究最多的区域是ISDR,日本学者研究发现NS5A羧基末端第2209到2248氨基酸的40个氨基酸区域的序列与干扰素治疗的敏感性有关,将这个区域称为干扰素敏感决定区(ISDR),认为ISDR变异株(4个以上氨基酸突变)对干扰素的应答较好,但是欧洲和美国的一些研究并未证实ISDR的突变与治疗转归的关系,考虑ISDR外还存在与干扰素疗效相关的序列,如V3区、干扰素及利巴韦林耐药区(IFN/RBV resistance determining region, IRRDR)等。目前对ISDR变异与干扰素应答关系的研究较多,但是对NS5A区基因全序列的变异对干扰素应答影响的研究还很少,国内尚未见报道。为了明确HCV 5’NCR及NS5A的变异情况以及变异对临床的影响,本研究应用基因芯片法及基因测序法检测山东地区HCV感染者5’NCR区,对进行抗病毒治疗的基因1b型慢性丙型肝炎病人进行NS5A序列测定,观察HCV 5’NCR及NS5A的变异情况及其临床意义。材料与方法：1.2005年1月至2008年10月期间济南市传染病医院门诊及住院的慢性丙型肝炎及肝硬化病人170例,抗HCV及HCV RNA均阳性,并排除了HAV、HBV、HEV、HIV感染及酒精性肝炎和药物性肝炎,空腹抽静脉血5m1。应用聚乙二醇干扰素a-2a 180ug或a-2b 1.0-1.5ug/kg治疗,基因1型疗程12个月联合利巴韦林800-1200mg,非基因1型疗程6个月联合利巴韦林800-1000mg。应用基因芯片法检测HCV基因型,观察基因型与疾病严重程度、感染途径、HCV RNA定量及干扰素应答的关系。2.2008年1月至2009年12月期间济南市传染病医院住院的慢性丙型肝炎病人118例,进行HCV 5’NCR的序列测定,应用分子生物学软件ClustalX2.0及Mega4.0进行分析,以Mega4.0软件构建遗传进化树,以此了解不同病毒株的亲源性；与国内外HCV流行株相比较,观察不同病毒株5’NCR区核苷酸序列的变异,了解山东地区HCV 5’NCR的基因变异特点；观察特征性的基因变异与HCVRNA定量水平及干扰素应答的关系。3.对35例进行抗病毒治疗的基因1b型慢性丙型肝炎病人治疗前的血清进行NS5A序列测定,全长NS5A序列设计5对引物,分5段进行测序,测序结果应用ClustalX2.0及Mega4.0软件进行核苷酸及氨基酸序列的比对,与HCV 1b型标准株HCV-J比较,观察NS5A全基因的核苷酸及氨基酸变异率及特异性的区域ISDR、V3区、IRRDR等的变异情况,以及以上变异与干扰素应答的关系。结果：1.基因芯片法检测HCV基因型结果为：基因1b型63.1%,2a型28.6%,1a及3b型均为1.19%,1b+2a及1a+1b混合型6%；肝硬化和慢性肝炎两组病人基因型分布无差别；有输血史者占67.9%,有输血史者及无输血史者基因型分布无差别；基因1b型与基因2a型血清HCV RNA定量log值分别为：6.42±1.0及5.69±1.15,t=3.89,p=0.0001；基因1b型与基因2a型抗病毒治疗SVR率分别为63.6%、90%,0.01<p<0.05。2.慢性丙型肝炎病人的5’NCR区基因序列分析结果表明：基因型分别为1b型65例、基因2a型45例、1a型2例、3a型1例、3b型2例及6a型3例。3.42例1b型慢性丙型肝炎病人5’NCR区序列跟国内外多数参考株序列相同,23例病人发生1-2碱基变异,存在120位C-T(9例)和204位C-T(8例)两个位点的特征性变异。2例1a病人5’NCR区基因序列与中国株序列相同,与2株美国株同源性为99.5%-100%。2a型病人5’NCR序列同源性为97.8%-100%,存在与参考株完全相同的病毒株,共有11个位点存在碱基变异,有222位及247位两个位点的特征性变异,根据这两个位点碱基的不同可以将2a型病人分为3组：均为T组、均为C组及分别为C、T组。3a型5’NCR和标准株同源性为98.1%,有4个碱基不同。2例3b型病人的5’NCR与国内外流行株同源性分别为99.1%-100%。6a病人都具有特征性的第-145位的CA插入,病人及参考株之间仅存在3个位点的碱基的不同,其中2例病人序列与中国株之一相同,与另一中国株及香港株同源性均为99.5%,另外1例病人与参考株的同源性为98.5%-99.5%。4.1b型5’NCR区120位C-T变异株和野毒株HCV RNA定量分别为5.16+1.40 log和6.14+1.01 log(p=0.041),差别有显著性；而1b型5’NCR区204位C-T变异株和野毒株HCV RNA定量分别为5.95±0.95 log和6.14±1.01log(p=0.23)。2a型病人根据5’NCR第222位及247位的碱基分为3组,3组病人HCV RNA定量没有明显差别。1b型及2a型5’NCR区变异株与野毒株比较干扰素的应答率没有差异。5.35例病人中有20例得到全序列HCV NS5A测序结果,其中11例实现持续病毒学应答(SVR),9个病人无应答或停药后复发(NR)。与HCV标准株J株进行比较,NS5A变异率较高,核苷酸变异数目为136+9.2,氨基酸变异数目为31.3士4.2,但病人之间同源性较高。SVR及NR两组病人NS5A全长序列的氨基酸变异数目没有差别(32.4+4.4及30.4士3.7,p=0.302), ISDR为突变型、中间型及野生型人数分别为：1、6、2及0、4、7(p>0.05), IRRDR氨基酸变异数目在SVR组及NR组分别为：6+1.33及4.4+1.14(p=0.014), SVR组及NR组氨基酸变异数≥6的比例分别为6/9及2/11(p=0.04),差异有显著性。结论：1.山东地区HCV主要流行株是基因1b型,其次为2a型,存在少量的1a、3a、3b、6a型及混合型感染。其中基因3a和6a型感染以前在山东地区未见报道。2.HCV感染者大部分有输血史,基因型与疾病的进展无关,1b型感染者HCVRNA定量(病毒载量)较高,且持续病毒学应答较差。3.HCV 5’NCR区序列高度保守,与国内外流行株同源性高,变异率低。基因1b型及2a型5’NCR区均有特征性的核苷酸变异,1b型HCV感染者5’NCR第120位C-T的变异减弱了病毒的复制水平,其余位点的变异不影响病毒的复制水平,5’NCR的变异对干扰素的应答无明显的影响。4.与HCV J株比较,山东地区慢性丙型肝炎病人HCV NS5A区核苷酸及氨基酸变异率较高,但病人之间NS5A序列的同源性较高。ISDR的氨基酸变异率较高者抗病毒治疗应答者较好,但差异没有统计学意义,IRRDR氨基酸变异数≥6预示丙型肝炎抗病毒治疗疗效较好。更多还原

【Abstract】 Background and Aims:Hepatitis C is an important infectious disease which threatens people’s health. Approximately 17 million people are infected with the hepatitis C virus (HCV) worldwide. The overall prevalence of HCV in China is 3.2%. HCV infections progress easily to chronic hepatitis. Twenty to thirty percent of HCV infections lead to liver cirrhosis and hepatocellular carcinoma within two decades. HCV infection has become a main cause of hepatocellular carcinoma and end stage liver disease.HCV is an enveloped virus with a positive-sense RNA genome. The genome contains one centrally located open reading frame (ORF) that encodes structural and nonstructural proteins. Short non-coding regions at each end of the genome (5’NCR and 3’NCR) are required for replication of the genome. Regions of the genome include 5’NCR, C, El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, NS5B and 3’NCR from the 5’terminus to the 3’terminus. The core (C) region encodes a nucleocapsid protein and the E1 and E2 regions encode two envelope proteins. The P7 region and genes from NS2 to NS5 encode nonstructural proteins with various functions.HCV is highly mutable with mutation rates varying among different regions of the viral genome. The 5’NCR and the core region are relatively conserved while the E regions show the highest variation. HCV 5’NCR is the most conserved region in terms of both length and sequence. Rare mutations in this region constitute an important basis for genotyping. Six major genotypes have been identified, each of which further divided into several subtypes. Distribution of HCV genotypes and subtypes varies geographically. The predominant HCV genotype in China’s mainland is lb, followed by 2a and low frequencies of la,2b,3b,4a, and 6a. Because sequences in the 5’NCR determine the genotype, it is speculated that variations in this region relates to clinical features, especially the response to antiviral treatment. It is reported that HCV genotypes may account for some of the variance in the clinical course of infection. Patients infected with genotype 1 often demonstrate higher viral loads and lower response rates to antiviral treatment than those with other genotypes. However, the correlation between genotypes and the clinical course is still debated. HCV 5’NCR contains the origin of replication and plays an important role in genome replication and protein translation. Consequently, some mutations in this region might affect the efficiency of viral replication and response to interferon (IFN) treatment.NS5A is a nonstructural protein that functions in RNA replication and maturation of the viral polyprotein. It inhibits apoptosis of the hepatocyte and downregulates the antiviral response stimulated by IFN a. Mutations in certain NS5A sequences may weaken its anti-IFN effect. Researchers from Japan discovered that the sequence from amino acid 2209 to 2248 was related to sensitivity to IFN, and named it the interferon sensitivity-determining region (ISDR). They found that mutations of four or more amino acids in this region resulted in improved response to IFN. However, studies from Europe and America did not confirm the relationship between mutations in ISDR and response to treatment, leading to speculations that there are other sequences in NS5A related to IFN sensitivity. To date, there have been many studies on ISDR but few studies correlating mutations in the entire NS5A region to IFN response. It has not been reported in China.In order to further investigate mutations in the 5’NCR and NS5A regions and their effect on the clinical course, genotypes of HCV in Shandong Province of east China were analyzed using gene chip assays and sequencing. Clinical significance of the mutations was further evaluated in patients receiving interferon therapy. Materials and Methods:1. One hundred and seventy patients with chronic hepatitis and/or cirrhosis due to HCV infection seen in Jinan Infectious Disease Hospital from January 2005 to October 2008 were enrolled. All patients were positive for anti-HCV and HCV RNA. None was co-infected with HAV, HBV, HEV, HIV, or complicated with drug or alcohol-induced liver diseases. Selected hepatitis patients were treated with pegylated interferon a (pegylated IFN a-2a 180ug or a-2b 1.0-1.5ug/kg) combined with ribavirin. Patients with genotype lb were treated with interferon and 1000-1200 mg of ribavirin for 12 months while those with genotype 2a were treated with interferon and 800-1000 mg of ribavirin for 6months. HCV genotypes were analyzed by a DNA chip assay. Relationships between genotype and disease severity, route of infection, viral load, and response to IFN were evaluated.2. One hundred and eighteen chronic hepatitis C patients hospitalized in Jinan Infectious Disease Hospital from January 2008 to December 2009 were enrolled. The 5’NCR of HCV was sequenced. Sequence variations were analyzed with Clustal X 2.0 and Mega 4.0. Phylogenetic trees based on 5’NCR were constructed. Correlations between viral genomic variations and serum levels of HCV RNA as well as response to IFN were investigated.3. The NS5A region of type lb HCV genomes from 35 patients was sequenced prior to combined IFN a and ribavirin treatment. Nucleotide and amino acid sequences of NS5A were analyzed using Clustal X 2.0 and Mega 4.0. Mutations in the entire region and in specific areas (ISDR and V3) were determined by comparing with the HCV lb standard strain (HCV-J). Correlation between variations in NS5A and response to IFN was studied prospectively.Results1. Genotypes 1b,2a, 1a,3b, 1b/2a, and 1a/1b were present in 63.10%,28.57%, 1.19%,1.19%,5.36%and 0.60%of patients, respectively, according to the gene chip assay. No significant difference was observed in the distribution of genotypes between patients with chronic hepatitis and patients with cirrhosis (x~2=2.35, p> 0.05). Neither was there a difference in HCV genotypes between patients infected via blood transfusion and patients without a transfusion history (x2=7.63, p> 0.05). Viral loads were significantly higher in patients infected with genotype 1b than those with 2a (6.42±1.0 vs 5.69±1.15,p=0.0001). SVR (sustained viral response) in genotype 2a infections was higher than in 1b infections.2. HCV genotypes were determined in 118 patients by 5’NCR sequencing. The numbers of cases among genotypes 1b,2a, 1a,3a,3b, and 6a were 65,45,2,1,2, and 3, respectively.3. Sequences of 5’NCR in 42 type 1b HCV genomes were identical to most of genotype lb isolates in Genbank. Twenty three of the sequences showed 1-2 base substitutions. The two characteristic mutations were 120 C-T (9 cases) and 204 C-T (8 cases). Two la strains were identical to the Chinese reference strain and homology to the two American reference strains were 99.5%and 100%. Homology of 5’NCR among type 2a genomes was 97.8%-100%with 11 base substitutions. There were type 2a sequences identical to that of the reference strain. Characteristic mutations were at sites 222 and 247. Type 2a genomes were thus divided into 3 groups according to these sites in 5’NCR, i.e., both T, both C, and C/T. Homology of 5’NCR between the single type 3a strain and the standard strain was 98.1%with 4 base substitutions. Homology of 5’NCR between the two type 3b strains and reference strains was 99.1%and 100%. All three type 6a sequences showed the distinctive CA insertion at site 145 of 5’NCR, with altogether 3 base substitutions when compared with reference strains. Two of the three sequences were identical to one of Chinese strain and homology with another Chinese strain and Hong Kong strain was 99.5%. Homology between the other type 6a strain and the 3 reference strains ranged within 98.5%-99.5%.4. Quantities of serum HCV RNA in patients infected with type 1b variants with a C-T mutation at site 120 of the 5’NCR and in patients infected with the wild strain were 5.16±1.40 log and 6.14±1.01 log, respectively (p=0.041). Quantities of serum HCV RNA in patients infected with lb variants with a C-T mutation at site 204 of 5’NCR were 5.95±0.95 log (p=0.23 when compared with those of patients infected with the wild strain). Quantities of serum HCV RNA in patients with type 2a variants with a C-T mutation at site 220 or site 247 were not statistically different from those of patients infected with the wild strain. The response to IFN did not differ significantly between variants of genotypes 1b and 2a and wild strains.5. Entire sequences of HCV NS5 A were obtained in 20 of 35 patients. Sustained viral response (SVR) was achieved in 11 of the 20 patients, while the other 9 patients experienced no response or relapse (NR). Comparison with the HCV J strain revealed high mutation rates in the NS5A region with substitutions of 136±9.2 nucleotides and 31.3±4.2 amino acids. However, homology of sequences among patients in Shandong province was high. Amino acid substitution numbers within the entire NS5A region were not significantly different between SVR and NR patients (32.4±4.4 and 30.4±3.7, p=0.302). Numbers of cases with mutant-type, intermediate-type, and wild-type ISDR were 1,6,2 among SVR patients and 0,4,7 among NR patients (p> 0.05). Amino acid substitution numbers in the IFN/RBV resistance-determining region (IRRDR) were 6±1.33 in SVR patients and 4.4±1.1 in NR patients (p= 0.014). Therefore amino acid substitution numbers of≥6 in IRRDR was predictive of SVR (p=0.04).Conclusions1. The predominant genotype in patients with chronic HCV infection is lb, followed by 2a and low frequencies of la,3a,3b,6a and mixed genotypes. Genotypes 3 a and 6a have not been reported previously in Shandong province.2. Genotype lb is associated with higher viral loads but not disease severity. Blood transfusion is the route of infection in most of the patients. Patients infected with genotype 2a have higher SVR, while the rate of relapse is high in genotype 1b-infected patients.3. HCV 5’NCR is highly conserved. Sequences of 5’NCR display characteristic mutation patterns in both genotypes 1b and 2a. C-T transition at site 120 of genotype 1b impedes viral replication but other mutations are not as significant. Point mutations in 5’NCR do not affect viral response to IFN.4. There are significant sequence differences in the NS5A region between the J strain and HCV strains in Shandong province, even though NS5A sequences in the province are highly homologous. Amino acid substitution numbers of≥6 in IRRDR is predictive of SVR.更多还原