【作者】 赵耀；

【导师】 黄爱龙；

【作者基本信息】 重庆医科大学 ， 生物制药与生物医用材料， 2011， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus, HBV)感染是全球性公共卫生问题,影响超过2亿人。慢性乙肝病毒感染大大增加了肝硬化和肝细胞癌(HCC)的风险,80-90%的肝癌患者由乙肝病毒感染引起。虽然自1982年有高效的乙肝病毒疫苗问世,但仍有超过4.0亿慢性携带者,且75%的携带者居住在亚洲太平洋地区。根据HBV本身的变异情况被分为不同的基因型。越来越多的临床和流行病学观察表明,不同的基因分型存在不同的生物学和临床的表现。与同为肝炎病毒的丙型肝炎病毒(HCV)相同,HBV持续存在和发展会演变为慢性肝病。但HCV感染的基因分型已成为治疗的重要提示,而HBV的基因型在抗病毒治疗中起到的作用仍然知之甚少。对于HBV基因型与药物反应性的问题,似乎还更加复杂,目前基因型与干扰素的联系是较为清楚,而核苷(酸)类似物与基因型看起来确没有什么联系。但越来越多的研究表明HBV基因型对于慢性乙肝病毒的感染的治疗和预后有影响,而在中国大约有95%的人群感染的基因型为B、C型。因此我们需要建立一种迅速、高效和高灵敏度,并能对B和C基因型分型和定量的手段,动态地监测基因型和HBV-DNA水平变化来寻找核苷(酸)类似物与HBV基因型之间的联系。目的:乙型肝炎病毒(HBV)是人类慢性肝脏疾病的重要原因,是一个重大的公共卫生问题。病毒载量和HBV基因型在确定明确临床疗效,反应及乙肝患者抗病毒治疗上扮演关键角色。目前对于病毒基因型检测和病毒DNA的定量检测不能同时完成。我们想建立一种新的迅速、高效和高灵敏度的方法来同时对HBV的基因型B和C分型并定量。并用该方法监测慢乙肝患者在ADV用药48周过程中HBV B和C基因型的变化,以明确核苷(酸)类似物与HBV基因型之间的联系。方法:在本研究中,同时定量和基因分型的方法建立在实时定量PCR技术的基础上,能够一次反应同时对HBV B和C基因型进行分型并定量。对基因型B和C的RT区型,根据型间特异、型内保守的原则,设计了特异性的引物和探针。最终获得能够对基因型B和C准确定量分型,并且没有交叉反应的方法。该方法对于混合感染的检出率要明显高于直接测序法。我们用该方法纵向研究了98位阿德福韦(ADV)治疗48周的慢乙肝患者基因型及病毒载量的变化。患者中53位慢乙肝患者有(0, 4, 8, 12, 24, 36和48周)7个时点的样本,其余45位有((0, 8, 12, 24和48周)5个时点的样本。结果:在ADV治疗起始时,基因型的分布为:68.37% (67/98)的B型,10.20% (10/98)的C型和21.43% (21/98)的B/C混合型。经过48周的治疗后,除一位患者的HBV-DNA低于检测下限外,其余患者基因型的分布为:80.41% (78/97)的B型,15.59% (19/97)的B、C混合型。所有的感染单一基因型C的患者,基因型(100% [10/10])均发生了改变,而仅有10.61% (7/66)的单一B型感染患者发生改变。14个B/C型混合感染的样本中,在48周的时其中有10个样本变成了单一的基因型B。另外,感染单一基因型C与单一基因型B和混合基因型B/C的患者相比,有更高的e抗原血清学转化率(70.00% [7/10]),并且80.00%(8/10)的患者HBV-DNA拷贝数下降大于10的2次方。在整个48周的治疗过程中,有32为患者发生了基因型的改变(32.99%, 32/97),而在整个治疗过程中曾出现过的基因型的患者共有38位,占38.78%。结论:本研究提供了一个经济、快速、可靠的基因分型和定量的新方法,能够提供病毒载量和基因型的连续检测,适合应用于亚洲人群病程监控,且对于基因型混合的检出率高。借助已经建立的准确、可靠的HBV定量和分型的方法,探讨了基因型对抗病毒药物的敏感性。发现基因型C比基因型B对ADV的治疗更加敏感,表现在基因型的改变,血清学转化及HBV-DNA水平下降等方面。还发现在抗病毒治疗过程中基因型的改变是普遍存在的。并且基因型混合的比例也比想象的更高。提示在将来临床抗病毒治疗中,慢乙肝患者乙肝病毒基因型的鉴定也是相当重要的。更多还原

【Abstract】 Hepatitis B virus is one of the most serious and prevalent health problems, affecting more than 2 billion people worldwide. Chronic HBV infection greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV infection is associated with up to 80–90% of HCC patients in China, India, Korea, Singapore and Vietnam. Although highly effective vaccines against hepatitis B virus have been available since 1982, there are still more than 400 million chronic carriers, 75% of whom reside in Asia Pacific region. The evolutionary history of major hepatitis viruses in different human populations includes the origin of phylogenetic variants named genotypes. Several clinical and epidemiological observations suggest that genetic differences in viral genotypes may underlie differences in biological and clinical behaviours. Similarly to hepatitis C virus (HCV), HBV persistence and progression to chronic liver disease are thought to result from a combination of viral and host factors. More importantly, although for HCV infections genotyping has become an essential part of therapeutic algorithms, the evidence that HBV genotypes play any role in response to antiviral treatment is much less clear. The problem is further compounded by the fact that drugs which have exclusive antiviral activity against HBV, such as nucleos(t)ide analogues (NUC’s), seem not to be influenced by genotypes, whereas interferon-a does.Several studies indicate that HBV genotypes may affect the disease profiles and clinical outcome in chronic HBV infection. Genotypes B and C are the two most common HBV genotypes in China accounting for approximately 95% of patients. Here we wanted to develop a rapid and sensitive method for concurrent quantification and identification of HBV genotypes B and C in a single-step reaction. And we used this novel method to evaluate the relationship with HBV genotype and drug sensitivity.Background/Aims. Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantitation assays are currently in use with a different effectiveness. In this study, a real time genotyping and quantitative PCR (RT-GQ-PCR)-based method was wanted to develop and to provide simultaneous identification and quantification of genotypes B and C in a single reaction. And we investigated the genotype changes of hepatitis B virus (HBV) genotypes B and C in chronic hepatitis B (CHB) patients during adefovir dipivoxil treatment and try to evaluate the relationship with HBV genotype and drug sensitivity.Methods. The genotype-specific PCR primers and hybridization probes were selected to genotype and quantify viral loads simultaneously for HBV genotypes B and C from infected patients. Our RT-GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes was observed. The RT-GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Genotyping 127 samples from HBV infected Chinese patients with RT-GO-PCR revealed 56.7% HBV as genotype B, 13.4% as genotype C and 29.8% as mixed-genotypes B and C. Longitudinal study of HBV genotypes was performed in 98 patients with adefovir dipivoxil treatment. Serum samples from 53 CHB patients were included in seven time points (0, 4, 8, 12, 24, 36 and 48 week) and 45 in five time points (0, 8, 12, 24 and 48 week).Results. At the baseline of adefovir dipivoxil treatment, genotypes were B 68.37% (67/98) of cases, C 10.20% (10/98) and mixed B/C 21.43% (21/98). After 48 weeks’treatment, there are B 80.41% (78/97) of cases, mixed genotype B/C 15.59% (19/97) and genotype C 0% (0/97), with one negative patient. All genotype C (100%, 10/10) were changed, while only 10.61% (7/66) of genotype B changed. In the mixed genotype B/C, 14 samples had genotype changes, of which 10 samples (71.43% [10/14]) converted into a single genotype B at 48 weeks. Genotype C has the higher HBeAg seroconverison rate (70.00% [7/10]) and the higher rate (80.00% [8/10]) of HBV-DNA levels reduction compared to genotype B (42.84% [29/67]; 49.25% [33/67]) and mixed genotype B/C (57.14% [12/21]; 47.62% [10/21]). Genotype changes were observed in 32 patients (32.99%, 32/97). In the 48 weeks of ADV treatment, a total of 38 patients (38.78%, 38/98) there have been mixed genotypes.Conclusions: This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections and is suitable for diagnosis of HBV, sequential monitoring of viral load levels in response to antiviral therapy, and determining the relationship between the genotype, viral load and stage of disease in Asians. We found that hepatitis genotype C is more sensitive and has better virologic response than genotype B to adefovir dipivoxil treatment. Our data suggest that the existence of genotype switch and mixed genotypes is ubiquitous in the antiviral therapy. Identification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B.更多还原