ISDR变异对聚乙二醇干扰素联合利巴韦林治疗1b型慢性丙型病毒性肝炎疗效的影响
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摘要
研究背景
丙型肝炎病毒(Hepatitis C virus, HCV)感染常引起慢性肝病,感染HCV25～30年后肝硬化发生率为5%-25%,HCV相关肝硬化患者10年后肝功能失代偿发生率为30%,肝细胞癌年发生率为1%-3%。目前尚无有效的疫苗预防。有效的抗病毒治疗可改善患者长期生存率与生活质量。因此,抗病毒治疗的长期目标为降低HCV相关肝硬化、肝衰竭与肝细胞癌的发生率,降低HCV相关病死率,改善患者生活质量。HCV感染一般病情缓慢进展,抗病毒疗效评价多采用短期的临床指标,包括病毒学应答、生化学应答与肝组织学应答等指标。其中病毒学应答指标中的持续病毒学应答(sustained virological response, SVR)是当前评判疗效的最主要指标。SVR是指治疗结束24周后应用敏感PCR方法检测血清HCV RNA仍为阴性,被视为疾病的“病毒学治愈”。
干扰素(interferon, IFN)联合利巴韦林(ribavirin, RBV)是目前慢性丙型肝炎(chronic hepatitis C, CHC)抗病毒治疗的标准方案。多项大型临床研究均表明,聚乙二醇干扰素(Pegylated interferon, Peg-IFN)联合RBV的方案SVR可达65%,疗效明显优于普通IFN联合RBV方案。
HCV基因型是影响CHC患者抗病毒治疗应答的重要因素,不同基因型患者治疗疗程与方案不同,AASLD(?)临床指南推荐HCV 1型患者Peg-IFN联合足量RBV治疗疗程48周,而2型和3型患者应用Peg-IFN及低剂量的RBV治疗疗程24周,在患者开始抗病毒治疗前,应检测HCV基因型。HCV基因型分布存在明显地区和人群差异；HCV不同基因型和亚型感染者的临床表现、肝病严重程度存在差异。由于目前国内尚无市售HCV分型试剂盒可供使用,建立一种可靠、灵敏、简洁、实用的HCV基因分型技术,对丙型肝炎的临床研究和相关的分子流行病学研究均具有重要意义。
影响抗病毒疗效的基线指标很多,其中患者基因型与基线HCV RNA载量是目前最重要的两个预测指标。其他指标还包括性别、年龄、体重、有无胰岛素抵抗以及肝脂肪病变与纤维化程度、是否酗酒以及是否使用静脉毒品等。这些预测指标有助于临床医生与患者对于可能的治疗结局有充分的认知,也可作为个体化治疗方案的依据。
在中国、日本以及东南欧流行的HCV 1b基因亚型感染抗病毒治疗应答较差。对于HCV 1b型慢性丙型病毒性肝炎患者,单用普通干扰素治疗SVR率只有10%-40%,标准抗病毒治疗方案Peg-IFN联合RBV治疗也仅达到约40%-50%。而Peg-IFN联合RBV治疗费用昂贵、副作用大。因此,病毒相关因素如基因型的检测及lb型疗效的影响因素预测将有助于临床合理地选择病例进行干扰素的治疗,从而提高干扰素疗效。
日本学者Enomoto等研究发现HCV lb型NS5A 2209～2248氨基酸序列氨基酸突变数目可作为干扰素抗病毒疗效的独立预测因素,并把该区命名为干扰素敏感性决定区(Interferon sensitivity determining region, ISDR)。他们认为野生型株(HCV J)为耐干扰素株,其他病毒株的ISDR序列如与之相同则为野生株,会对IFN无应答；如与野生型株ISDR序列相差4个氨基酸以上为突变株,对IFN持续应答；差别在1-3个氨基酸之间为中间型,以无应答为主。随后日本学者的系列研究证实了这个发现。M Pascu等进行Meta分析认为：ISDR氨基酸残基突变在HCV lb型患者中可以做为一个亚型用于预测IFN治疗的应答反应,可用于预测IFN治疗疗效。
然而有关ISDR国内是否可做为HCV治疗的预测因素仍存在争议。唐振亚等对武汉地区12例IFN治疗患者进行研究,认为中国分离株ISDR高度保守,中国的野生株对干扰素治疗产生SVR；胡芸文等发现上海丙型肝炎患者血清HCV的ISDR序列相对保守,干扰素治疗前24份血清HCV病毒株均为ISDR野生型株和中间型株,未发现突变株,认为国内少见ISDR氨基酸残基突变,ISDR不能做为疗效预测因素。然而,他们的研究对象为普通干扰素治疗患者,样本量过小,未进行统计分析；而Shen等对江苏地区应用IFN治疗的20例患者进行回顾性分析,认为ISDR氨基酸残基突变影响干扰素治疗疗效,Yen等对及台湾地区应用Peg-IFN联合RBV治疗的60例患者ISDR氨基酸残基突变与SVR显著相关。国内有关Peg-IFN联合RBV治疗的HCV 1b慢性丙型病毒性肝炎ISDR氨基酸残基突变的研究尚未见报道。
本研究主要从影响疗效的HCV病毒学因素方面进行研究,包括以下三部分组成：1、采用RDH法建立HCV基因分型技术；2、建立一种以HCV 1b型ISDR特异性引物为基础的RT-PCR法,对广东地区HCV ISDR进行序列测定,了解广东地区ISDR氨基酸残基突变的分布情况；3、回顾性分析ISDR及治疗前、随访后等临床资料,分析ISDR氨基酸残基突变对Peg-IFN联合RBV治疗lb亚型CHC的影响因素,探讨影响SVR的主要预测因素及ISDR的临床意义。具体内容如下：
一、反向点杂交技术检测丙型肝炎病毒基因型的方法建立与应用
目的：建立HCV基因分型技术PCR-反向点杂交法(PCR-RDH)方法：应用生物信息学软件针对HCV 5’端非编码区(5’UTR)和核心蛋白区(C)设计特异性捕获探针及生物素标记引物,建立HCV基因分型的PCR-反向点杂交技术。应用本技术对115份丙肝血清标本进行基因型和基因亚型检测,同时对其中38份标本中的HCV进行RT-PCR基因扩增、测序、系统进化树分析确实HCV基因型和亚型,以评价反向点杂交法的准确性及临床应用价值。结果：115份血清标本中,反向点杂交法基因型及基因亚型检出率为96.5%(111/115),15份阴性对照全部为阴性。111例基因型明确标本中lb型63例(56.8%)；2a型9例(8.1%)；3a型4例(3.6%),3b型6例(5.4%)；6a型28例(25.2%),1b/2a混合型1例(0.9%)。经测序分型确定此法检测准确度为100%,特异性为100%。HCV基因型反向点杂交法检测准确可靠、简单经济、高效,适用于临床检测。与10余年前研究相比,目前广东地区的HCV基因型分布仍以lb型为主,但呈现出1b型比例下降,3a、6a型比例升高的趋势。
二、HCV 1b型ISDR的检测方法的建立
目的：建立一种以HCV 1b型ISDR特异性引物为基础的RT-PCR方法,对广东地区HCV ISDR进行序列测定,了解广东地区ISDR氨基酸残基突变的分布情况。方法：根据已知GenBank中的HCV 1b基因型D90208的全长序列及参考文献设设计HCV 1b NS5A ISDR引物序列,选择南方医院感染内科门诊就诊和住院的55例HCV 1b型慢性丙型肝炎患者的血清提取HCV RNA,采用巢式PT-PCR,扩增HCV 1b NS5A的ISDR,并与HCV J株进行序列比对确定ISDR区氨基酸残基突变数目,分析广东地区HCV 1b型患者ISDR分布情况。结果：HCV ISDR区扩增产物全长249bp,电泳显示条带清晰,大小符合预期分子量,无非特异性条带扩增,测序结果经BLAST分析为目的片段。广东地区HCV 1b型ISDR分布为：ISDR野生株(无突变)占25.5%(14/55),中间型(突变数目1一3个)占72.7%(40/55),突变株(突变数目超过3个者)占1.8%(1/55)。
变异对ISDR联合RBV治疗1b亚型CHC疗效的影响Peg-IFN目的：探讨广东地区
患者HCV 1b变异对ISDR联合RBV抗病毒Peg-IFN治疗的影响因素及临床意义。方法：选择经检测并接受ISDR联合RBVPeg-IFN治疗的型CHC44例患者进行回顾性分析。结果：SVR率为43.2%(19/44),HCV 1b组与Peg-IFNα-2a组SVR相比,两者无统计学差异(P=0.797)；Peg-IFNα-2b氨基酸残基突变数目>2个与早期病毒学应答及SVR显著相关(P=0.018ISDR及氨基酸残基突变与EVR及SVR有统计学差异,而与P=0.005), ISDRETVR无统计学差异(P=0.056)：在年龄组(年龄≤40岁)中,ISDR与年龄相关(P=0.012),年龄小则ISDR突变数目多；ISDR与输血及非输血传播途径无关(P=0.117)。;ISDR 2218位氨基酸突变最多,在SVR组中突变中有14例(14/18,占77.8%),NR组中11例(11/15,占73.3%),无统计学差异(P=0.767)。多因素Logistic回归分析显示：影响SVR的最主要因素是ISDR氨基酸残基突变(OR=17.038,95%可信区间为：1.923-150.944,P=0.011),其次是年龄≤40岁(OR=12.259,95%可信区间为：0.012～0.559,P=0.011),AST影响SVR (OR=1.021,95%可信区间为：1.003～1.039,P=0.020)。
结论：
1、本研究建立的PCR-反向点杂交法对我国常见的HCV基因亚型lb、2a、3a、3b和6a检测的准确性、特异性及灵敏度较好,适宜我国临床推广应用；
2、广东地区的HCV基因型分布仍然以1b型多见,呈现出lb型比例下降,3a、6a型比例升高的趋势；
3. HCV ISDR区扩增顺利,无非特异性条带扩增,测序及BLAST分析为目的片段,方法可靠。广东地区HCV 1b型ISDR分布以野生株及中间株为主,多突变株比例较低(1.8%)4、聚乙二醇干扰素联合利巴韦林治疗广东地区HCV 1b型CHC患者SVR率为43.2%(19/44), Peg-IFNα-2a组与Peg-IFNα-2b组SVR相比无统计学差异；
5、ISDR氨基酸残基突变数目≥2个与早期病毒学应答及SVR相关；ISDR氨基酸残基突变与输血及非输血传播途径无关；
6、广东地区ISDR氨基酸残基突变以2218位氨基酸突变居多,但对SVR的影响未见统计学差异,需扩大样本深入研究；
7、ISDR氨基酸残基突变数目、年龄≥40岁和AST是Peg-IFN联合RBV治疗CHC 1b型患者获得SVR的独立预测因素,其中ISDR氨基酸残基突变数目和年龄≥40岁对SVR的影响最大(OR=17.038, OR=12.259);
8、ISDR氨基酸残基突变对SVR的影响可能与HCV病毒进化相关。
BACKGROUD
Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver diseases. It was reported that 5% to 25% HCV infection would develop to cirrhosis during a period of 20 to 30 years. Patients with HCV-related cirrhosis are at risk for developing to the end-stage liver disease (a risk of approximately 30% over 10 years) as well as hepatocellular carcinoma (HCC) (a risk of approximately 1% to 3% per year). There is still no effective vaccine for prevention. Effective antiviral therapy can improve long-term survival and quality-of-life of the patients. Therefore, the goal of treatment is to prevent complications of HCV infection; this is principally achieved by eradication of the infection. Because of the slow evolution of chronic HCV infection over several decades, it has been difficult to demonstrate that therapy prevents complications of liver disease. Accordingly, treatment responses are defined by a surrogate virological parameter rather than a clinical endpoint. Short-term outcomes can be evaluated biochemically, virologically, and histologically. The most important is the sustained virological response (SVR), defined as the absence of HCV RNA from serum by a sensitive PCR assay 24 weeks of following up after discontinuation of therapy. This is generally regarded as a "virological cure".
The currently recommended therapy of chronic HCV infection is the combination of a pegylated interferon alfa and ribavirin (RBV). Interferon plus RBV is the chronic hepatitis C (CHC) standard regimen of antiviral therapy. Several large clinical studies showed that pegylated interferon combined with RBV can approaches the SVRs of 65%, significantly better than non-prgylated.
HCV genotyping is useful in epidemiological studies and clinical management. The optimal duration of treatment should be based on HCV genotypes. AASLD guidelines recommend that patients with genotype 1 should be treated for 48 weeks with peg-interferon alfa-2a plus standard weight-based RBV, whereas those with genotype 2 and 3 could be treated with peg-interferon alfa-2a plus low dose RBV for 24 weeks. It is highly recommended that HCV genotyping should be performed in all HCV-infected persons prior to interferon-based treatment. HCV genotypes have a geographic and population distribution. Different genotypes have different clinical manifestations and correlations with the severity of liver disease. However, there is still no commercially available HCV genotyping kit in China. Therefore, It is greatly significant to develop a simple, sensitive, reliable and practical genotyping method for the clinical and molecularly epidemiological studies.
There are many parameters for estimating the response to interferon-based therapies. Of which, genotype and pretreatment viral load are two major predictors. Other indicators include gender, age, body weight, with or without insulin resistance and fatty liver disease and fibrosis, alcohol abuse and intravenous drugs or not. These predictors not only can help clinicians and patients evaluate the possible outcomes, but also can be considered as the basis of individualized treatment.
HCV-1b, the prevalent genotype in China, Japan, Southern and Eastern Europe, commonly indicates the poor response to antiviral therapy. SVR is achieved in 10 to 40 percent of cases after conventional IFN monotherapy and in approximately 40 to 50 percent of cases after combination therapy using Peg-IFN plus RBV in patients with HCV-lb infection.Since Peg-IFN-based therapy is expensive and may lead to serious adverse effects, it would be useful for clinicians to choose the optimal case by the detection of virus-related factors such as genotypes and predictors for HCV-lb infection with the aim of improving the effect of IFN-based therapies.
Enomoto et al found a significant correlation between the response to interferon in patients with HCV-1b infection and the number of amino acid (aa) substitutions that were present before therapy in a small region of NS5A2209-2248. The NS5A2209-2248 region was therefore named the interferon sensitivity determining region (ISDR). Patients infected with genotype-1b HCV harbouring more than three mutated sites in this region are more likely to become sustained responders (SR) than those whose HCV quasispecies harbour few ISDR mutations. There were 3 groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). These initial findings have been further confirmed by other Japanese studies. M Pascu et al found, by a meta-analysis of all available studies, the number and patterns of mutations within the ISDR can be considered as an independent factor to assess the response to IFN in patients with HCV genotype lb infection.
However, controversial data were reported from mainland China. Tang Zhenya et al preformed a study including 12 patients in Wuhan who received IFN treatment and found that the ISDRs of isolates highly conserved, and the wild strains is sensitive to IFN. Hu Yunwen et al found none of them was mutant type in Shanghai.3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. ISDR sequence alone could not predict outcome of IFN treatment. However, their study sample size was too small and not for statistical analysis. And Shen et al analyzed retrospectively 20 cases and supported the position that the mutations within the ISDR of NS5A gene affected the effect of interferon therapy. Yen et al found the mutations of the ISDR significantly correlated with SVR to Peg-IFNa-2b plus RBV in patients with HCV-lb in Taiwan. However, the correlation between ISDR mutations and the response to Peg-IFN-based therapies in HCV-1 patients is rarely reported in China.
We studied the HCV virological factors that estimating the response to interferon-based therapies, and this thesis consisted of three studies. The first study aimed to develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. The second study was to establish a reverse transcription polymerase chain reaction (RT-PCR) method to amplify the HCV lb ISDR in Guangdong area, then the amino acid sequence of NS5A 2209-2248 was determined by direct sequencing, and to investigate the mutation number of amino acid in ISDR compared with HCV J strain. The last study was to perform a retrospective analysis on 44 patients who had been tested by ISDR and treated with Peg-IFN and RBV in order to discuss the primarily predictive factor that affected the therapeutic effect and the clinical significance of ISDR by analyzing clinical characters before treatment. Details are as follows:
1. A novel hepatitis C virus genotyping method using RT-PCR reverse dot hybridization technique and its application
Objective:To develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. Methods:The primers and the probes targeting the 5'untranslated region (5'UTR) and Core region of HCV genotypes 1b,2a,3a,3b and 6a were specially designed. And then the RT-PCR Reverse Dot Hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C from Nanfang Hospital were genotyped by the method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method. Results:Of 115 patients,111 (96.5%) were successfully genotyped to be 1b,2a, 3a,3b,6a and mix-infection of lb/2a at frequencies of 56.8%,8.1%,3.6%,5.4%, 25.2% and 0.9% respectively.15 healthy control samples were all negative. The accuracy and reliability of genotyping method of PCR-RDH was confirmed by amplification of HCV Core and NS5B regions from 38 cases followed by DNA sequencing and phylogenetic analysis. The newly developed HCV genotyping method was proved to be reliable and specific, and may be suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased when compared with 10 years ago.
2. Establishment of test method on ISDR of HCV genotype lb
Objective:To establish a establish a reverse transcription polymerase chain reaction (RT-PCR) method based on the special primers of the interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV), and to detect the sequence of the HCV ISDR and to investigate the mutation number of amino acid in ISDR compared with HCV J strain in Guangdong area, China. Methods: According to the total length sequences of HCV genotype lb D90208 in GenBank and the ISDR primer sequences of HCV lb NS5A in references, we selected 55 blood serum samples from patients with HCV-lb infection from out-patients and in-patients in the department of infectious diseases, Nanfang Hospital. We adopted nested PT-PCR to enlarge ISDR of HCV lb NS5A and compared sequences with HCV J strain in order to define the mutation numbers of amino acid in ISDR and to analyze the distribution of ISDR patterns among patients with HCV genotype lb in Guangdong area. Result:The size of ISDR-amplified products was 249bp, which was determined clearly in the electrophoretic pattern and consistent with the expected molecular size. There were no any non-specific bands. The sequencing results were in accord with the target fragments confirmed by BLAST. We could find out that,25.5%(14/55) of HCV genotype lb were wild-type (no mutation), while 72.7% (40/55) were intermediate-type (1 to 3 mutation numbers) and 1.8%(1/55) was mutant-type isolates (more than 3 mutation numbers) in Guangdong area.
3. Impact of Mutation in HCV ISDR on the Efficacy of pegylated interferon/ribavirin Therapy in Patients with Chronic Hepatitis C Virus Genotype lb Infection
Objective:To assess whether ISDR mutations correlate with the response to Peg interferon plus RBV therapy among patients with HCV genotype lb in Guangdong area. Methods:We retrospectively analyzed 44 patients with HCV genotype lb infection who had received Peg-IFN/RBV for 24 weeks to 48 weeks. Pretreatment serum samples were collected. The amino acid sequence of ISDR was determined by direct sequencing of the HCV genome amplified by the RT-PCR and was compared with the established sequence for HCV-J. Result: The sustained virological response (SVR) rate was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group (P =0.797). The statistical analysis indicated significant correlations between the mutation number (over 2) in ISDR of HCV-lb and SVR/EVR (P=0.005/P=0.018). However, no significant difference could be found between mutate number and EVTR (P=0.056). In young group (age≤40), we could find out that the younger, the more mutate numbers in ISDR (P=0.012). But ISDR was not significantly related with blood transfusion and non-blood transmission (P=0.117). The mutation of ISDR amino acid 2218 was prevalent in mutate group, and there was no statistical difference between SVR group (14/18,77.8%) and Nonresponse group (11/15,73.3%) (P=0.767). Multivariate regression analysis indicated that the independent variables related to SVR were the mutation number in ISDR (odds ratio,17.038; 95% confidence interval,1.923～150.944; P=0.011), age group (≤40 years) (odds ratio,12.259; 95% confidence interval,0.012～0.559; P=0.011) and AST (odds ratio,1.021; 95% confidence interval,1.003～1.039; P=0.020).
Conclusion:
1. The newly developed HCV genotyping method was proved to be reliable and specific, and it might be suitable for clinical and epidemiological investigations.
2. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased in comparison with 10 years ago.
3. Amplification of ISDR of HCV was carried out successfully without any non-specific bands. The sequencing results were in accord with the target fragments. It shown that, the distribution of ISDR of HCV genotype lb in Guangdong area was mainly wild strains and intermediate strains, while mutant strains accounted for a small proportion (1.8%).
4. The SVR rate of Guangdong patients with HCV genotype lb who was treated by Peg-IFN/RBV was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group.
5. The mutation number of ISDR (over 2) was significantly associated with EVR and SVR. ISDR was not significantly related with blood transfusion and non-blood transmission.
6. The mutation of ISDR amino acid 2218 was the prevalent in Guangdong area; but there was no statistical difference for SVR. Further studies with an expanded sample size are needed.
7. The mutations number in ISDR, age less 40 years and AST are independent predictors of the response to antiviral therapy in chronic hepatitis C lb patients treated with peg-interferon plus RBV.
8. The impct of ISDR amino acids mutation on SVR might be correlated with evolution of HCV.
丙型肝炎病毒(Hepatitis C virus, HCV)感染常引起慢性肝病,感染HCV25～30年后肝硬化发生率为5%-25%,HCV相关肝硬化患者10年后肝功能失代偿发生率为30%,肝细胞癌年发生率为1%-3%。目前尚无有效的疫苗预防。有效的抗病毒治疗可改善患者长期生存率与生活质量。因此,抗病毒治疗的长期目标为降低HCV相关肝硬化、肝衰竭与肝细胞癌的发生率,降低HCV相关病死率,改善患者生活质量。HCV感染一般病情缓慢进展,抗病毒疗效评价多采用短期的临床指标,包括病毒学应答、生化学应答与肝组织学应答等指标。其中病毒学应答指标中的持续病毒学应答(sustained virological response, SVR)是当前评判疗效的最主要指标。SVR是指治疗结束24周后应用敏感PCR方法检测血清HCV RNA仍为阴性,被视为疾病的“病毒学治愈”。
干扰素(interferon, IFN)联合利巴韦林(ribavirin, RBV)是目前慢性丙型肝炎(chronic hepatitis C, CHC)抗病毒治疗的标准方案。多项大型临床研究均表明,聚乙二醇干扰素(Pegylated interferon, Peg-IFN)联合RBV的方案SVR可达65%,疗效明显优于普通IFN联合RBV方案。
HCV基因型是影响CHC患者抗病毒治疗应答的重要因素,不同基因型患者治疗疗程与方案不同,AASLD(?)临床指南推荐HCV 1型患者Peg-IFN联合足量RBV治疗疗程48周,而2型和3型患者应用Peg-IFN及低剂量的RBV治疗疗程24周,在患者开始抗病毒治疗前,应检测HCV基因型。HCV基因型分布存在明显地区和人群差异；HCV不同基因型和亚型感染者的临床表现、肝病严重程度存在差异。由于目前国内尚无市售HCV分型试剂盒可供使用,建立一种可靠、灵敏、简洁、实用的HCV基因分型技术,对丙型肝炎的临床研究和相关的分子流行病学研究均具有重要意义。
影响抗病毒疗效的基线指标很多,其中患者基因型与基线HCV RNA载量是目前最重要的两个预测指标。其他指标还包括性别、年龄、体重、有无胰岛素抵抗以及肝脂肪病变与纤维化程度、是否酗酒以及是否使用静脉毒品等。这些预测指标有助于临床医生与患者对于可能的治疗结局有充分的认知,也可作为个体化治疗方案的依据。
在中国、日本以及东南欧流行的HCV 1b基因亚型感染抗病毒治疗应答较差。对于HCV 1b型慢性丙型病毒性肝炎患者,单用普通干扰素治疗SVR率只有10%-40%,标准抗病毒治疗方案Peg-IFN联合RBV治疗也仅达到约40%-50%。而Peg-IFN联合RBV治疗费用昂贵、副作用大。因此,病毒相关因素如基因型的检测及lb型疗效的影响因素预测将有助于临床合理地选择病例进行干扰素的治疗,从而提高干扰素疗效。
日本学者Enomoto等研究发现HCV lb型NS5A 2209～2248氨基酸序列氨基酸突变数目可作为干扰素抗病毒疗效的独立预测因素,并把该区命名为干扰素敏感性决定区(Interferon sensitivity determining region, ISDR)。他们认为野生型株(HCV J)为耐干扰素株,其他病毒株的ISDR序列如与之相同则为野生株,会对IFN无应答；如与野生型株ISDR序列相差4个氨基酸以上为突变株,对IFN持续应答；差别在1-3个氨基酸之间为中间型,以无应答为主。随后日本学者的系列研究证实了这个发现。M Pascu等进行Meta分析认为：ISDR氨基酸残基突变在HCV lb型患者中可以做为一个亚型用于预测IFN治疗的应答反应,可用于预测IFN治疗疗效。
然而有关ISDR国内是否可做为HCV治疗的预测因素仍存在争议。唐振亚等对武汉地区12例IFN治疗患者进行研究,认为中国分离株ISDR高度保守,中国的野生株对干扰素治疗产生SVR；胡芸文等发现上海丙型肝炎患者血清HCV的ISDR序列相对保守,干扰素治疗前24份血清HCV病毒株均为ISDR野生型株和中间型株,未发现突变株,认为国内少见ISDR氨基酸残基突变,ISDR不能做为疗效预测因素。然而,他们的研究对象为普通干扰素治疗患者,样本量过小,未进行统计分析；而Shen等对江苏地区应用IFN治疗的20例患者进行回顾性分析,认为ISDR氨基酸残基突变影响干扰素治疗疗效,Yen等对及台湾地区应用Peg-IFN联合RBV治疗的60例患者ISDR氨基酸残基突变与SVR显著相关。国内有关Peg-IFN联合RBV治疗的HCV 1b慢性丙型病毒性肝炎ISDR氨基酸残基突变的研究尚未见报道。
本研究主要从影响疗效的HCV病毒学因素方面进行研究,包括以下三部分组成：1、采用RDH法建立HCV基因分型技术；2、建立一种以HCV 1b型ISDR特异性引物为基础的RT-PCR法,对广东地区HCV ISDR进行序列测定,了解广东地区ISDR氨基酸残基突变的分布情况；3、回顾性分析ISDR及治疗前、随访后等临床资料,分析ISDR氨基酸残基突变对Peg-IFN联合RBV治疗lb亚型CHC的影响因素,探讨影响SVR的主要预测因素及ISDR的临床意义。具体内容如下：
一、反向点杂交技术检测丙型肝炎病毒基因型的方法建立与应用
目的：建立HCV基因分型技术PCR-反向点杂交法(PCR-RDH)方法：应用生物信息学软件针对HCV 5’端非编码区(5’UTR)和核心蛋白区(C)设计特异性捕获探针及生物素标记引物,建立HCV基因分型的PCR-反向点杂交技术。应用本技术对115份丙肝血清标本进行基因型和基因亚型检测,同时对其中38份标本中的HCV进行RT-PCR基因扩增、测序、系统进化树分析确实HCV基因型和亚型,以评价反向点杂交法的准确性及临床应用价值。结果：115份血清标本中,反向点杂交法基因型及基因亚型检出率为96.5%(111/115),15份阴性对照全部为阴性。111例基因型明确标本中lb型63例(56.8%)；2a型9例(8.1%)；3a型4例(3.6%),3b型6例(5.4%)；6a型28例(25.2%),1b/2a混合型1例(0.9%)。经测序分型确定此法检测准确度为100%,特异性为100%。HCV基因型反向点杂交法检测准确可靠、简单经济、高效,适用于临床检测。与10余年前研究相比,目前广东地区的HCV基因型分布仍以lb型为主,但呈现出1b型比例下降,3a、6a型比例升高的趋势。
二、HCV 1b型ISDR的检测方法的建立
目的：建立一种以HCV 1b型ISDR特异性引物为基础的RT-PCR方法,对广东地区HCV ISDR进行序列测定,了解广东地区ISDR氨基酸残基突变的分布情况。方法：根据已知GenBank中的HCV 1b基因型D90208的全长序列及参考文献设设计HCV 1b NS5A ISDR引物序列,选择南方医院感染内科门诊就诊和住院的55例HCV 1b型慢性丙型肝炎患者的血清提取HCV RNA,采用巢式PT-PCR,扩增HCV 1b NS5A的ISDR,并与HCV J株进行序列比对确定ISDR区氨基酸残基突变数目,分析广东地区HCV 1b型患者ISDR分布情况。结果：HCV ISDR区扩增产物全长249bp,电泳显示条带清晰,大小符合预期分子量,无非特异性条带扩增,测序结果经BLAST分析为目的片段。广东地区HCV 1b型ISDR分布为：ISDR野生株(无突变)占25.5%(14/55),中间型(突变数目1一3个)占72.7%(40/55),突变株(突变数目超过3个者)占1.8%(1/55)。
变异对ISDR联合RBV治疗1b亚型CHC疗效的影响Peg-IFN目的：探讨广东地区
患者HCV 1b变异对ISDR联合RBV抗病毒Peg-IFN治疗的影响因素及临床意义。方法：选择经检测并接受ISDR联合RBVPeg-IFN治疗的型CHC44例患者进行回顾性分析。结果：SVR率为43.2%(19/44),HCV 1b组与Peg-IFNα-2a组SVR相比,两者无统计学差异(P=0.797)；Peg-IFNα-2b氨基酸残基突变数目>2个与早期病毒学应答及SVR显著相关(P=0.018ISDR及氨基酸残基突变与EVR及SVR有统计学差异,而与P=0.005), ISDRETVR无统计学差异(P=0.056)：在年龄组(年龄≤40岁)中,ISDR与年龄相关(P=0.012),年龄小则ISDR突变数目多；ISDR与输血及非输血传播途径无关(P=0.117)。;ISDR 2218位氨基酸突变最多,在SVR组中突变中有14例(14/18,占77.8%),NR组中11例(11/15,占73.3%),无统计学差异(P=0.767)。多因素Logistic回归分析显示：影响SVR的最主要因素是ISDR氨基酸残基突变(OR=17.038,95%可信区间为：1.923-150.944,P=0.011),其次是年龄≤40岁(OR=12.259,95%可信区间为：0.012～0.559,P=0.011),AST影响SVR (OR=1.021,95%可信区间为：1.003～1.039,P=0.020)。
结论：
1、本研究建立的PCR-反向点杂交法对我国常见的HCV基因亚型lb、2a、3a、3b和6a检测的准确性、特异性及灵敏度较好,适宜我国临床推广应用；
2、广东地区的HCV基因型分布仍然以1b型多见,呈现出lb型比例下降,3a、6a型比例升高的趋势；
3. HCV ISDR区扩增顺利,无非特异性条带扩增,测序及BLAST分析为目的片段,方法可靠。广东地区HCV 1b型ISDR分布以野生株及中间株为主,多突变株比例较低(1.8%)4、聚乙二醇干扰素联合利巴韦林治疗广东地区HCV 1b型CHC患者SVR率为43.2%(19/44), Peg-IFNα-2a组与Peg-IFNα-2b组SVR相比无统计学差异；
5、ISDR氨基酸残基突变数目≥2个与早期病毒学应答及SVR相关；ISDR氨基酸残基突变与输血及非输血传播途径无关；
6、广东地区ISDR氨基酸残基突变以2218位氨基酸突变居多,但对SVR的影响未见统计学差异,需扩大样本深入研究；
7、ISDR氨基酸残基突变数目、年龄≥40岁和AST是Peg-IFN联合RBV治疗CHC 1b型患者获得SVR的独立预测因素,其中ISDR氨基酸残基突变数目和年龄≥40岁对SVR的影响最大(OR=17.038, OR=12.259);
8、ISDR氨基酸残基突变对SVR的影响可能与HCV病毒进化相关。
BACKGROUD
Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver diseases. It was reported that 5% to 25% HCV infection would develop to cirrhosis during a period of 20 to 30 years. Patients with HCV-related cirrhosis are at risk for developing to the end-stage liver disease (a risk of approximately 30% over 10 years) as well as hepatocellular carcinoma (HCC) (a risk of approximately 1% to 3% per year). There is still no effective vaccine for prevention. Effective antiviral therapy can improve long-term survival and quality-of-life of the patients. Therefore, the goal of treatment is to prevent complications of HCV infection; this is principally achieved by eradication of the infection. Because of the slow evolution of chronic HCV infection over several decades, it has been difficult to demonstrate that therapy prevents complications of liver disease. Accordingly, treatment responses are defined by a surrogate virological parameter rather than a clinical endpoint. Short-term outcomes can be evaluated biochemically, virologically, and histologically. The most important is the sustained virological response (SVR), defined as the absence of HCV RNA from serum by a sensitive PCR assay 24 weeks of following up after discontinuation of therapy. This is generally regarded as a "virological cure".
The currently recommended therapy of chronic HCV infection is the combination of a pegylated interferon alfa and ribavirin (RBV). Interferon plus RBV is the chronic hepatitis C (CHC) standard regimen of antiviral therapy. Several large clinical studies showed that pegylated interferon combined with RBV can approaches the SVRs of 65%, significantly better than non-prgylated.
HCV genotyping is useful in epidemiological studies and clinical management. The optimal duration of treatment should be based on HCV genotypes. AASLD guidelines recommend that patients with genotype 1 should be treated for 48 weeks with peg-interferon alfa-2a plus standard weight-based RBV, whereas those with genotype 2 and 3 could be treated with peg-interferon alfa-2a plus low dose RBV for 24 weeks. It is highly recommended that HCV genotyping should be performed in all HCV-infected persons prior to interferon-based treatment. HCV genotypes have a geographic and population distribution. Different genotypes have different clinical manifestations and correlations with the severity of liver disease. However, there is still no commercially available HCV genotyping kit in China. Therefore, It is greatly significant to develop a simple, sensitive, reliable and practical genotyping method for the clinical and molecularly epidemiological studies.
There are many parameters for estimating the response to interferon-based therapies. Of which, genotype and pretreatment viral load are two major predictors. Other indicators include gender, age, body weight, with or without insulin resistance and fatty liver disease and fibrosis, alcohol abuse and intravenous drugs or not. These predictors not only can help clinicians and patients evaluate the possible outcomes, but also can be considered as the basis of individualized treatment.
HCV-1b, the prevalent genotype in China, Japan, Southern and Eastern Europe, commonly indicates the poor response to antiviral therapy. SVR is achieved in 10 to 40 percent of cases after conventional IFN monotherapy and in approximately 40 to 50 percent of cases after combination therapy using Peg-IFN plus RBV in patients with HCV-lb infection.Since Peg-IFN-based therapy is expensive and may lead to serious adverse effects, it would be useful for clinicians to choose the optimal case by the detection of virus-related factors such as genotypes and predictors for HCV-lb infection with the aim of improving the effect of IFN-based therapies.
Enomoto et al found a significant correlation between the response to interferon in patients with HCV-1b infection and the number of amino acid (aa) substitutions that were present before therapy in a small region of NS5A2209-2248. The NS5A2209-2248 region was therefore named the interferon sensitivity determining region (ISDR). Patients infected with genotype-1b HCV harbouring more than three mutated sites in this region are more likely to become sustained responders (SR) than those whose HCV quasispecies harbour few ISDR mutations. There were 3 groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). These initial findings have been further confirmed by other Japanese studies. M Pascu et al found, by a meta-analysis of all available studies, the number and patterns of mutations within the ISDR can be considered as an independent factor to assess the response to IFN in patients with HCV genotype lb infection.
However, controversial data were reported from mainland China. Tang Zhenya et al preformed a study including 12 patients in Wuhan who received IFN treatment and found that the ISDRs of isolates highly conserved, and the wild strains is sensitive to IFN. Hu Yunwen et al found none of them was mutant type in Shanghai.3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. ISDR sequence alone could not predict outcome of IFN treatment. However, their study sample size was too small and not for statistical analysis. And Shen et al analyzed retrospectively 20 cases and supported the position that the mutations within the ISDR of NS5A gene affected the effect of interferon therapy. Yen et al found the mutations of the ISDR significantly correlated with SVR to Peg-IFNa-2b plus RBV in patients with HCV-lb in Taiwan. However, the correlation between ISDR mutations and the response to Peg-IFN-based therapies in HCV-1 patients is rarely reported in China.
We studied the HCV virological factors that estimating the response to interferon-based therapies, and this thesis consisted of three studies. The first study aimed to develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. The second study was to establish a reverse transcription polymerase chain reaction (RT-PCR) method to amplify the HCV lb ISDR in Guangdong area, then the amino acid sequence of NS5A 2209-2248 was determined by direct sequencing, and to investigate the mutation number of amino acid in ISDR compared with HCV J strain. The last study was to perform a retrospective analysis on 44 patients who had been tested by ISDR and treated with Peg-IFN and RBV in order to discuss the primarily predictive factor that affected the therapeutic effect and the clinical significance of ISDR by analyzing clinical characters before treatment. Details are as follows:
1. A novel hepatitis C virus genotyping method using RT-PCR reverse dot hybridization technique and its application
Objective:To develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. Methods:The primers and the probes targeting the 5'untranslated region (5'UTR) and Core region of HCV genotypes 1b,2a,3a,3b and 6a were specially designed. And then the RT-PCR Reverse Dot Hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C from Nanfang Hospital were genotyped by the method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method. Results:Of 115 patients,111 (96.5%) were successfully genotyped to be 1b,2a, 3a,3b,6a and mix-infection of lb/2a at frequencies of 56.8%,8.1%,3.6%,5.4%, 25.2% and 0.9% respectively.15 healthy control samples were all negative. The accuracy and reliability of genotyping method of PCR-RDH was confirmed by amplification of HCV Core and NS5B regions from 38 cases followed by DNA sequencing and phylogenetic analysis. The newly developed HCV genotyping method was proved to be reliable and specific, and may be suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased when compared with 10 years ago.
2. Establishment of test method on ISDR of HCV genotype lb
Objective:To establish a establish a reverse transcription polymerase chain reaction (RT-PCR) method based on the special primers of the interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV), and to detect the sequence of the HCV ISDR and to investigate the mutation number of amino acid in ISDR compared with HCV J strain in Guangdong area, China. Methods: According to the total length sequences of HCV genotype lb D90208 in GenBank and the ISDR primer sequences of HCV lb NS5A in references, we selected 55 blood serum samples from patients with HCV-lb infection from out-patients and in-patients in the department of infectious diseases, Nanfang Hospital. We adopted nested PT-PCR to enlarge ISDR of HCV lb NS5A and compared sequences with HCV J strain in order to define the mutation numbers of amino acid in ISDR and to analyze the distribution of ISDR patterns among patients with HCV genotype lb in Guangdong area. Result:The size of ISDR-amplified products was 249bp, which was determined clearly in the electrophoretic pattern and consistent with the expected molecular size. There were no any non-specific bands. The sequencing results were in accord with the target fragments confirmed by BLAST. We could find out that,25.5%(14/55) of HCV genotype lb were wild-type (no mutation), while 72.7% (40/55) were intermediate-type (1 to 3 mutation numbers) and 1.8%(1/55) was mutant-type isolates (more than 3 mutation numbers) in Guangdong area.
3. Impact of Mutation in HCV ISDR on the Efficacy of pegylated interferon/ribavirin Therapy in Patients with Chronic Hepatitis C Virus Genotype lb Infection
Objective:To assess whether ISDR mutations correlate with the response to Peg interferon plus RBV therapy among patients with HCV genotype lb in Guangdong area. Methods:We retrospectively analyzed 44 patients with HCV genotype lb infection who had received Peg-IFN/RBV for 24 weeks to 48 weeks. Pretreatment serum samples were collected. The amino acid sequence of ISDR was determined by direct sequencing of the HCV genome amplified by the RT-PCR and was compared with the established sequence for HCV-J. Result: The sustained virological response (SVR) rate was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group (P =0.797). The statistical analysis indicated significant correlations between the mutation number (over 2) in ISDR of HCV-lb and SVR/EVR (P=0.005/P=0.018). However, no significant difference could be found between mutate number and EVTR (P=0.056). In young group (age≤40), we could find out that the younger, the more mutate numbers in ISDR (P=0.012). But ISDR was not significantly related with blood transfusion and non-blood transmission (P=0.117). The mutation of ISDR amino acid 2218 was prevalent in mutate group, and there was no statistical difference between SVR group (14/18,77.8%) and Nonresponse group (11/15,73.3%) (P=0.767). Multivariate regression analysis indicated that the independent variables related to SVR were the mutation number in ISDR (odds ratio,17.038; 95% confidence interval,1.923～150.944; P=0.011), age group (≤40 years) (odds ratio,12.259; 95% confidence interval,0.012～0.559; P=0.011) and AST (odds ratio,1.021; 95% confidence interval,1.003～1.039; P=0.020).
Conclusion:
1. The newly developed HCV genotyping method was proved to be reliable and specific, and it might be suitable for clinical and epidemiological investigations.
2. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased in comparison with 10 years ago.
3. Amplification of ISDR of HCV was carried out successfully without any non-specific bands. The sequencing results were in accord with the target fragments. It shown that, the distribution of ISDR of HCV genotype lb in Guangdong area was mainly wild strains and intermediate strains, while mutant strains accounted for a small proportion (1.8%).
4. The SVR rate of Guangdong patients with HCV genotype lb who was treated by Peg-IFN/RBV was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group.
5. The mutation number of ISDR (over 2) was significantly associated with EVR and SVR. ISDR was not significantly related with blood transfusion and non-blood transmission.
6. The mutation of ISDR amino acid 2218 was the prevalent in Guangdong area; but there was no statistical difference for SVR. Further studies with an expanded sample size are needed.
7. The mutations number in ISDR, age less 40 years and AST are independent predictors of the response to antiviral therapy in chronic hepatitis C lb patients treated with peg-interferon plus RBV.
8. The impct of ISDR amino acids mutation on SVR might be correlated with evolution of HCV.
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[8]http://www.chinacdc.cn/n272442/n272530/n272757/index.html[Z].
[9]Ghany M G, Strader D B, Thomas D L, et al. Diagnosis, management, and treatment of hepatitis C:an update[J]. Hepatology.2009,49(4):1335-1374.
[10]Manns M P, Mchutchison J G, Gordon S C, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C:a randomised trial[J]. Lancet.2001,358(9286):958-965.
[11]Fried M W, Shiffman M L, Reddy K R, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection[J]. N Engl J Med.2002,347(13):975-982.
[12]Hadziyannis S J, Sette H J, Morgan T R, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C:a randomized study of treatment duration and ribavirin dose[J]. Ann Intern Med.2004,140(5): 346-355.
[13]Weck K. Molecular methods of hepatitis C genotyping[J]. Expert Rev Mol Diagn.2005,5(4):507-520.
[14]Romero-Gomez M, Del M V M, Andrade R J, et al. Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients[J]. Gastroenterology.2005,128(3):636-641.
[15]黄辉红.HCV基因型和亚型：分型技术,临床意义及其流行病学研究[D].南方医科大学,2010.
[16]Zhou Y, Li W, Zeng Y, et al. The New Epidemiological Features of HCV Infection in Southern China[J]. Hepatol Int.2011,5(3):236.
[17]慢性丙型肝炎抗病毒治疗专家委员会.慢性丙型肝炎抗病毒治疗专家共识[J/CD].中华实验和临床感染病杂志(电子版).2009,3(3)：343-352.
[18]Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[J]. Hepatology(Baltimore, Md.). 2005,42(4):962-973.
[19]Okamoto H, Kurai K, Okada S I, et al. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates:comparative study of four distinct genotypes[J]. Virology.1992,188(1):331-341.
[20]Nakao T, Enomoto N, Takada N, et al. Typing of hepatitis C virus genomes by restriction fragment length polymorphism[J]. J Gen Virol.1991,72 (Pt 9): 2105-2112.
[21]Buoro S, Pizzighella S, Boschetto R, et al. Typing of hepatitis C virus by a new method based on restriction fragment length polymorphism[J]. Intervirology. 1999,42(1):1-8.
[22]王静,王露楠.丙型肝炎基因型定量检测及分型检测方法的研究进展[J].世界华人消化杂志.2007,15(27)：2897-2902.
[23]Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay[J]. J Gen Virol.1993, 74(Pt 6):1093-1102.
[24]Bouchardeau F, Cantaloube J F, Chevaliez S, et al. Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay[J]. J Clin Microbiol.2007,45(4):1140-1145.
[25]陈连英,王跃国,施英娟,等.HCV基因分型检测芯片的临床应用及意义[J].现代检验医学杂志.2005,20(1)：8-10.
[26]Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region[J]. J Clin Invest.1995, 96(1):224-230.
[27]Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection[J]. N Engl J Med.1996,334(2):77-81.
[28]Schinkel J, Spaan W J, Kroes A C. Meta-analysis of mutations in the NS5A gene and hepatitis C virus resistance to interferon therapy, uniting discordant conclusions[J]. Antivir Ther.2004,9(2):275-286.
[29]Pascu M, Martus P, Hohne M, et al. Sustained virological response in hepatitis C virus type lb infected patients is predicted by the number of mutations within the NS5A-ISDR:a meta-analysis focused on geographical differences[J]. Gut. 2004,53(9):1345-1351.
[30]Polyak S J, Paschal D M, Mcardle S, et al. Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication[J]. Hepatology.1999,29(4):1262-1271.
[31]Song J, Fujii M, Wang F, et al. The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon[J]. J Gen Virol.1999,80 (Pt 4): 879-886.
[32]唐振亚,Kock J, Moradpour D,等.慢性丙型肝炎患者干扰素敏感决定区突变的意义[J].中华肝脏病杂志.1999,7(1)：57.
[33]胡芸文,唐美芳.慢性丙型肝炎患者丙型肝炎病毒NS5A区序列与干扰素疗效的关系[J].中华实验和临床病毒学杂志.2002,16(002)：114-118.
[34]Shen C, Hu T, Shen L, et al. Mutations in ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic hepatitis C virus genotype lb infection[J]. J Gastroenterol Hepatol.2007,22(11):1898-1903.
[35]Yen Y H, Hung C H, Hu T H, et al. Mutations in the interferon sensitivity-determining region (nonstructural 5A amino acid 2209-2248) in patients with hepatitis C-lb infection and correlating response to combined therapy of pegylated interferon and ribavirin[J]. Aliment Pharmacol Ther.2008, 27(1):72-79.
[36]中华医学会肝病学分会中华医学会传染病与寄生虫病学分会.丙型肝炎防治指南[J].中华肝脏病杂志.2004(04)：194-198.
[37]Bouchardeau F, Cantaloube J F, Chevaliez S, et al. Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay[J]. Journal of Clinical Microbiology.2007,45(4):1140-1145.
[38]Stuyver L, Wyseur A, Van Arnhem W, et al. Second-generation line probe assay for hepatitis C virus genotyping[J]. Journal of Clinical Microbiology.1996, 34(9):2259-2266.
[39]王敏,王会龙,马丽,等.丙型肝炎病毒基因分型检测试剂盒CN200710029946.0[P].2009-3-4.
[40]黄辉红,周元平,杨洁,等.联合分析HCV种属信息区和高度保守区序列确定广东地区HCV基因型和亚型[J].广东医学.2010,31(7)：825-828.
[41]张瑞,杜绍财.丙型肝炎病毒基因分型的研究进展[J].中华检验医学杂志.2006,29(5)：469-471.
[42]Chen Z, Weck K E. Hepatitis C virus genotyping:interrogation of the 5' untranslated region cannot accurately distinguish genotypes la and lb[J]. J Clin Microbiol.2002,40(9):3127-3134.
[43]Hraber P T, Fischer W, Bruno W J, et al. Comparative analysis of hepatitis C virus phylogenies from coding and non-coding regions:the 5' untranslated region (UTR) fails to classify subtypes[J]. Virol J.2006,3:103.
[44]Saiki R K, Walsh P S, Levenson C H, et al. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes[J]. Proc Natl Acad Sci U S A.1989,86(16):6230-6234.
[45]杨光,陈姝,崔金环,等.丙型肝炎病毒逆向点杂交基因分型方法的建立及初步应用[J].中华流行病学杂志.2005(06)：440-443.
[46]Nakano T, Lu L, He Y, et al. Population genetic history of hepatitis C virus lb infection in China[J]. Journal of General Virology.2006,87(1):73-82.
[47]Garten R J, Lai S, Zhang J, et al. Rapid transmission of hepatitis C virus among young injecting heroin users in Southern China[J]. International journal of epidemiology.2004,33(1):182-188.
[48]孙南雄,范晓峰.HCV—1b型NS5A区准种变异与干扰素疗效关系的研究[J].江苏医药.1999,25(012)：914-915.
[49]Zeuzem S, Lee J H, Roth W K. Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa[J]. Hepatology.1997,25(3):740-744.
[50]张永祥,孙南雄,黄祖瑚,等.丙型肝炎病毒1b型NS5A 2209-2248区基因突变与干扰素疗效的关系[J].中华传染病杂志.2002,20(1)：53-55.
[51]张琳,赵桂珍,石理兰,等.张琳,赵桂珍,石理兰,曹丽.丙型肝炎病毒NS5A基因变异与干扰素疗效的关系[J].世界华人消化杂志.2003,11(8)：1135-1138.
[52]刘斌,王宇明.丙型肝炎病毒1b型NS5A区基因复杂性与干扰素α疗效的关系[J].中华传染病杂志.2002,20(2)：90-93.
[53]Tai A W, Chung R T. Treatment failure in hepatitis C:mechanisms of non-response[J]. J Hepatol.2009,50(2):412-420.
[54]周朝晖,佘为民,钱雪梅,等.丙型肝炎病毒1b型NS5A基因变异与干扰素疗效关系研究[J].中国临床医学.2008,15(6)：802-804.
[55]张琳,白菡,黄芬,等.1b型丙型肝炎病毒基因组不同区域基因变异对干扰素疗效的影响[J].中华实验和临床病毒学杂志.2007,21(1)：83-84.
[56]Watanabe H, Enomoto N, Nagayama K, et al. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype lb infection[J]. J Infect Dis.2001,183(8):1195-1203.
[57]Nakagawa M, Sakamoto N, Ueyama M, et al. Mutations in the interferon sensitivity determining region and virological response to combination therapy with pegylated-interferon alpha 2b plus ribavirin in patients with chronic hepatitis C-lb infection[J]. J Gastroenterol.2010,45(6):656-665.
[58]Gale M J, Korth M J, Tang N M, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein[J]. Virology.1997,230(2):217-227.
[59]Taguchi T, Nagano-Fujii M, Akutsu M, et al. Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner[J]. J Gen Virol.2004,85(Pt 4):959-969.
[60]He Y, Yan W, Coito C, et al. The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins[J]. J Gen Virol.2003,84(Pt 3):535-543.
[61]Gale M J, Korth M J, Katze M G. Repression of the PKR protein kinase by the hepatitis C virus NS5A protein:a potential mechanism of interferon resistance[J]. Clin Diagn Virol.1998,10(2-3):157-162.
[62]Garaigorta U, Chisari F V. Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation[J]. Cell Host Microbe.2009,6(6): 513-522.
[2]http://www.who.int/immunization/topics/hepatitis_c/en[Z].
[3]中华医学会肝病学分会,中华医学会传染病与寄生虫病学分会.丙型肝炎防治指南[J].中华肝脏病杂志.2004,12(4)：194-198.
[4]Alberti A, Benvegnu L. Management of hepatitis C[J]. J Hepatol.2003,38 Suppl 1:S104-S118.
[5]Seeff L B. Natural history of chronic hepatitis C[J]. Hepatology.2002,36(5 Suppl 1):S35-S46.
[6]Fattovich G, Giustina G, Degos F, et al. Morbidity and mortality in compensated cirrhosis type C:a retrospective follow-up study of 384 patients[J]. Gastroenterology.1997,112(2):463-472.
[7]Kim W R. The burden of hepatitis C in the United States[J]. Hepatology.2002, 36(5 Suppl 1):S30-S34.
[8]http://www.chinacdc.cn/n272442/n272530/n272757/index.html[Z].
[9]Ghany M G, Strader D B, Thomas D L, et al. Diagnosis, management, and treatment of hepatitis C:an update[J]. Hepatology.2009,49(4):1335-1374.
[10]Manns M P, Mchutchison J G, Gordon S C, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C:a randomised trial[J]. Lancet.2001,358(9286):958-965.
[11]Fried M W, Shiffman M L, Reddy K R, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection[J]. N Engl J Med.2002,347(13):975-982.
[12]Hadziyannis S J, Sette H J, Morgan T R, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C:a randomized study of treatment duration and ribavirin dose[J]. Ann Intern Med.2004,140(5): 346-355.
[13]Weck K. Molecular methods of hepatitis C genotyping[J]. Expert Rev Mol Diagn.2005,5(4):507-520.
[14]Romero-Gomez M, Del M V M, Andrade R J, et al. Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients[J]. Gastroenterology.2005,128(3):636-641.
[15]黄辉红.HCV基因型和亚型：分型技术,临床意义及其流行病学研究[D].南方医科大学,2010.
[16]Zhou Y, Li W, Zeng Y, et al. The New Epidemiological Features of HCV Infection in Southern China[J]. Hepatol Int.2011,5(3):236.
[17]慢性丙型肝炎抗病毒治疗专家委员会.慢性丙型肝炎抗病毒治疗专家共识[J/CD].中华实验和临床感染病杂志(电子版).2009,3(3)：343-352.
[18]Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[J]. Hepatology(Baltimore, Md.). 2005,42(4):962-973.
[19]Okamoto H, Kurai K, Okada S I, et al. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates:comparative study of four distinct genotypes[J]. Virology.1992,188(1):331-341.
[20]Nakao T, Enomoto N, Takada N, et al. Typing of hepatitis C virus genomes by restriction fragment length polymorphism[J]. J Gen Virol.1991,72 (Pt 9): 2105-2112.
[21]Buoro S, Pizzighella S, Boschetto R, et al. Typing of hepatitis C virus by a new method based on restriction fragment length polymorphism[J]. Intervirology. 1999,42(1):1-8.
[22]王静,王露楠.丙型肝炎基因型定量检测及分型检测方法的研究进展[J].世界华人消化杂志.2007,15(27)：2897-2902.
[23]Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay[J]. J Gen Virol.1993, 74(Pt 6):1093-1102.
[24]Bouchardeau F, Cantaloube J F, Chevaliez S, et al. Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay[J]. J Clin Microbiol.2007,45(4):1140-1145.
[25]陈连英,王跃国,施英娟,等.HCV基因分型检测芯片的临床应用及意义[J].现代检验医学杂志.2005,20(1)：8-10.
[26]Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region[J]. J Clin Invest.1995, 96(1):224-230.
[27]Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection[J]. N Engl J Med.1996,334(2):77-81.
[28]Schinkel J, Spaan W J, Kroes A C. Meta-analysis of mutations in the NS5A gene and hepatitis C virus resistance to interferon therapy, uniting discordant conclusions[J]. Antivir Ther.2004,9(2):275-286.
[29]Pascu M, Martus P, Hohne M, et al. Sustained virological response in hepatitis C virus type lb infected patients is predicted by the number of mutations within the NS5A-ISDR:a meta-analysis focused on geographical differences[J]. Gut. 2004,53(9):1345-1351.
[30]Polyak S J, Paschal D M, Mcardle S, et al. Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication[J]. Hepatology.1999,29(4):1262-1271.
[31]Song J, Fujii M, Wang F, et al. The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon[J]. J Gen Virol.1999,80 (Pt 4): 879-886.
[32]唐振亚,Kock J, Moradpour D,等.慢性丙型肝炎患者干扰素敏感决定区突变的意义[J].中华肝脏病杂志.1999,7(1)：57.
[33]胡芸文,唐美芳.慢性丙型肝炎患者丙型肝炎病毒NS5A区序列与干扰素疗效的关系[J].中华实验和临床病毒学杂志.2002,16(002)：114-118.
[34]Shen C, Hu T, Shen L, et al. Mutations in ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic hepatitis C virus genotype lb infection[J]. J Gastroenterol Hepatol.2007,22(11):1898-1903.
[35]Yen Y H, Hung C H, Hu T H, et al. Mutations in the interferon sensitivity-determining region (nonstructural 5A amino acid 2209-2248) in patients with hepatitis C-lb infection and correlating response to combined therapy of pegylated interferon and ribavirin[J]. Aliment Pharmacol Ther.2008, 27(1):72-79.
[36]中华医学会肝病学分会中华医学会传染病与寄生虫病学分会.丙型肝炎防治指南[J].中华肝脏病杂志.2004(04)：194-198.
[37]Bouchardeau F, Cantaloube J F, Chevaliez S, et al. Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay[J]. Journal of Clinical Microbiology.2007,45(4):1140-1145.
[38]Stuyver L, Wyseur A, Van Arnhem W, et al. Second-generation line probe assay for hepatitis C virus genotyping[J]. Journal of Clinical Microbiology.1996, 34(9):2259-2266.
[39]王敏,王会龙,马丽,等.丙型肝炎病毒基因分型检测试剂盒CN200710029946.0[P].2009-3-4.
[40]黄辉红,周元平,杨洁,等.联合分析HCV种属信息区和高度保守区序列确定广东地区HCV基因型和亚型[J].广东医学.2010,31(7)：825-828.
[41]张瑞,杜绍财.丙型肝炎病毒基因分型的研究进展[J].中华检验医学杂志.2006,29(5)：469-471.
[42]Chen Z, Weck K E. Hepatitis C virus genotyping:interrogation of the 5' untranslated region cannot accurately distinguish genotypes la and lb[J]. J Clin Microbiol.2002,40(9):3127-3134.
[43]Hraber P T, Fischer W, Bruno W J, et al. Comparative analysis of hepatitis C virus phylogenies from coding and non-coding regions:the 5' untranslated region (UTR) fails to classify subtypes[J]. Virol J.2006,3:103.
[44]Saiki R K, Walsh P S, Levenson C H, et al. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes[J]. Proc Natl Acad Sci U S A.1989,86(16):6230-6234.
[45]杨光,陈姝,崔金环,等.丙型肝炎病毒逆向点杂交基因分型方法的建立及初步应用[J].中华流行病学杂志.2005(06)：440-443.
[46]Nakano T, Lu L, He Y, et al. Population genetic history of hepatitis C virus lb infection in China[J]. Journal of General Virology.2006,87(1):73-82.
[47]Garten R J, Lai S, Zhang J, et al. Rapid transmission of hepatitis C virus among young injecting heroin users in Southern China[J]. International journal of epidemiology.2004,33(1):182-188.
[48]孙南雄,范晓峰.HCV—1b型NS5A区准种变异与干扰素疗效关系的研究[J].江苏医药.1999,25(012)：914-915.
[49]Zeuzem S, Lee J H, Roth W K. Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa[J]. Hepatology.1997,25(3):740-744.
[50]张永祥,孙南雄,黄祖瑚,等.丙型肝炎病毒1b型NS5A 2209-2248区基因突变与干扰素疗效的关系[J].中华传染病杂志.2002,20(1)：53-55.
[51]张琳,赵桂珍,石理兰,等.张琳,赵桂珍,石理兰,曹丽.丙型肝炎病毒NS5A基因变异与干扰素疗效的关系[J].世界华人消化杂志.2003,11(8)：1135-1138.
[52]刘斌,王宇明.丙型肝炎病毒1b型NS5A区基因复杂性与干扰素α疗效的关系[J].中华传染病杂志.2002,20(2)：90-93.
[53]Tai A W, Chung R T. Treatment failure in hepatitis C:mechanisms of non-response[J]. J Hepatol.2009,50(2):412-420.
[54]周朝晖,佘为民,钱雪梅,等.丙型肝炎病毒1b型NS5A基因变异与干扰素疗效关系研究[J].中国临床医学.2008,15(6)：802-804.
[55]张琳,白菡,黄芬,等.1b型丙型肝炎病毒基因组不同区域基因变异对干扰素疗效的影响[J].中华实验和临床病毒学杂志.2007,21(1)：83-84.
[56]Watanabe H, Enomoto N, Nagayama K, et al. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype lb infection[J]. J Infect Dis.2001,183(8):1195-1203.
[57]Nakagawa M, Sakamoto N, Ueyama M, et al. Mutations in the interferon sensitivity determining region and virological response to combination therapy with pegylated-interferon alpha 2b plus ribavirin in patients with chronic hepatitis C-lb infection[J]. J Gastroenterol.2010,45(6):656-665.
[58]Gale M J, Korth M J, Tang N M, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein[J]. Virology.1997,230(2):217-227.
[59]Taguchi T, Nagano-Fujii M, Akutsu M, et al. Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner[J]. J Gen Virol.2004,85(Pt 4):959-969.
[60]He Y, Yan W, Coito C, et al. The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins[J]. J Gen Virol.2003,84(Pt 3):535-543.
[61]Gale M J, Korth M J, Katze M G. Repression of the PKR protein kinase by the hepatitis C virus NS5A protein:a potential mechanism of interferon resistance[J]. Clin Diagn Virol.1998,10(2-3):157-162.
[62]Garaigorta U, Chisari F V. Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation[J]. Cell Host Microbe.2009,6(6): 513-522.