【作者】 孙晓风；

【导师】 郑玉建； 张跃新； 侯金林；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 HBV是环境中存在的一种对人致病性较强的病毒。HBV感染后常可引起急性和慢性肝炎,肝硬化甚至肝癌,是中国现阶段最为突出的公共卫生问题之一。慢性肝炎病毒(HBV)感染呈世界性流行。我国属HBV感染高流行区。HBV具有易变性及准种的特性。目前,根据HBV全基因组序列差异≥8%或S基因序列≥4%,将HBV分为A、B、C、D、E、F、G、H等8个基因型。HBV基因型有明确的地理和人种的分布特征。HBV在慢性感染过程中,变异可自发产生,也可在体内因宿主免疫压力或药物压力下而产生。如慢性乙型肝炎可因前C区变异和/或C区基因变异,不表达HBeAg而分为HBeAg阳性和HBeAg阴性两大类。HBV C基因基本核心基因启动子变异(BCP)和前C区变异(PC),可能与肝病严重程度有关。也具有一定的地域分布差别。HBV聚合酶有四个功能区,其中RT区有7个催化功能的基序,依次称为G、F、A、B、C、D、E区,其中,B、C、D区是P基因耐药变异最常见的部位。不同的核苷类似物耐药株的变异位点不同。新疆是个多民族居聚区,HBV感染及疾病谱有其特殊性。研究表明,新疆维吾尔族HBV基因型以D基因型为多,与我国内地以B、C基因型为主的流行情况有所不同,为进一步证实新疆地区的不同民族HBV感染者的基因型分布特征,了解新疆不同民族的基因亚型的分布,前C区变异和C区变异,耐药变异等情况,为今后防治HBV奠定了理论的基础。目的:进一步证实新疆慢性HBV感染者的HBV基因型分布特征,了解新疆不同民族的基因亚型,PC、BCP变异及耐药变异的特征,为进一步研究防治HBV提供科学依据。尤其是研究新疆HBV感染者基因型、亚型分布有无特征。新疆HBV感染者的HBV PC变异和BCP变异与HBV感染病情严重性和HBeAg阴性有无关联性。了解P基因区变异与HBV不同基因型是否有联系以及抗病毒药物效果与P基因区变异之间的关系,旨在为今后防治HBV感染提供科学依据。方法:收集331例新疆慢性HBV感染者的血清,其中慢性乙型肝炎(CHB)240例,乙肝肝硬化(LC)64例,肝癌(HCC)27例。1)采用聚合酶链反应限制性片段长度多态性(PCR-RFLP)方法,进行基因型、基因亚型、PC变异、BCP变异的检测,并对部分标本用直接测序法进行验证,所得的测序结果采用生物信息学技术进行分析比较。2)对随机选择的14例标本进行全基因测序,选择14例用核苷类似物治疗的CHB及4例未用核苷类似物的标本进行P基因区测序,然后用生物信息学技术进行分析并与基因库进行比对分析。结果:1)用PCR-RFLP技术对新疆331例HBV感染者的HBV基因型、基因亚型进行检测,随机选取部分标本用直接测序法验证。结果显示331例慢性HBV感染者的HBV基因型为:B基因型12.7%,C基因型64%,C/D重组体16%,D基因型7.3%。进一步检测基因亚型显示B基因亚型均为Ba亚型,未检测到Bj亚型。在C基因型中,C1亚型42.9%,C2亚型57.1%。不同民族基因型分析显示,在汉族HBV感染者中,B基因型14.9%,C基因型72.9%,C/D重组体9.5%,D基因型2.7%,而维吾尔族HBV感染者中B基因型8.3%,C基因型45.9%,C/D重组体29.4%,D基因型16.5%。维吾尔族与汉族比较,基因型分布有差别(P<0.05)。进一步基因亚型结果显示Ba亚型中,汉族78.6%,维吾尔族21.4%。C基因亚型汉族76.4%,其中,C1亚型39.5%,C2亚型60.5%;而维吾尔族23.6%,C1亚型54%,C2亚型46%。在不同临床类型中,慢性乙型肝炎中,C1亚型26.7%,C2亚型33.3%。在乙肝肝硬化中,C1亚型30%,C2亚型45.3%。在肝癌中,C1亚型30.8%;C2亚型46.2%。2)用PCR-RFLP技术检测HBV PC变异和BCP变异结果显示,PC变异30.2%,BCP变异34.4%。进一步比较PC变异和BCP变异,在不同亚型分布显示,Ba亚型中PC变异35.7%,BCP变异28.6%;C1亚型中PC变异37.4%,BCP变异41.8%;C2亚型PC变异25.6%,BCP变异31.4%;C/D重组体PC变异20.8%,BCP变异35.8%。C1亚型BCP变异明显高于Ba亚型,有统计学意义(P<0.05),PC变异在各亚型中无明显差别(P>0.05)。维吾尔族HBV感染者中,Ba亚型PC变异1.8%,BCP变异5.5%;C1亚型PC变异3.7%,BCP变异11%;C2亚型PC变异5.5%,BCP变异7.3%;C/D重组体PC变异6.4%,BCP变异12.8%,C/D重组体的PC变异、BCP变异明显高于Ba亚型,差异有统计学意义(P<0.01)。C1亚型、C2亚型介于两者之间。3)使用PCR-RFLP技术对HBV PCR-测序方法,对14例病人进行全基因的测序。全基因测序证实2例C/D重组体,12例D基因型与RFLP方法保持一致。测序结果显示2例C/D重组体,12例D基因型与GenBank现有的序列有差异。全部在GenBank上收录。4)用PCR-测序方法,对新疆18例HBV感染者的HBV P基因区测序,采用生物信息学技术进行分析,在P基因区变异结果显示,14例服用阿德福韦治疗,CHB标本中有13例出现P基因区变异。进一步分析显示,主要在聚合酶B区附近的变异(16个)和D区(6个)附近。5例C1亚型在聚合酶B区附近的变异有9个;聚合酶D区附近的变异有1个。5例C2亚型在聚合酶B区附近的变异有3个;聚合酶D区附近的变异有3个。6例Ba亚型在聚合酶B区附近的变异有4个;聚合酶D区附近的变异有8个。1例D基因型在聚合酶B区附近的变异有3个;聚合酶D区附近的变异有1个。1例C/D1亚型在聚合酶B区附近的变异2个。34例YMDD变异中,B基因型11.8%,C基因型64.7%,C/D基因型20.6%例,D基因型2.9%,提示C基因型易出现YMDD耐药变异。在基因亚型中,10例C1亚型45.4%,12例C2亚型54.5%,YMDD变异在C2亚型比C1亚型的多。结论:1)新疆地区331例HBV感染者的HBV采用PCR-RFLP方法,部分标本测序验证,采用生物信息学技术进行分析,结果显示新疆HBV基因型以C基因型为主,且以C2亚型为主。汉族HBV基因型以C基因型为主,且以C2亚型为主;维吾尔族HBV基因型以C基因型、C/D重组体为多,且以C1基因亚型为多。汉族C2基因型明显高于维吾尔族,而维吾尔族C/D重组体、D基因型明显高于汉族。在新疆地区,HBV基因型中依次为C基因型>C/D重组体>B基因型>D基因型;汉族基因型依次为C基因型>B基因型>C/D重组体>D基因型;维吾尔族基因型依次为C基因型>C/D重组体>D基因型>B基因型。C基因亚型汉族中C2亚型>C1亚型,维吾尔族中C1亚型>C2亚型。在乙肝肝硬化、肝癌中,C2所占亚型比例明显高于慢性乙型肝炎C2亚型的比例,有统计学意义(P<0.05),在肝癌中C2亚型明显高于C1亚型。2)对新疆331份HBV感染的血清进行HBV PC、BCP变异研究结果显示,C1亚型BCP变异明显高于Ba亚型,PC变异在各亚型中无明显差别。Ba亚型BCP变异发生率最低,C/D重组体PC变异发生率最低。说明HBV不同的亚型之间存在PC、BCP变异的差异。维吾尔族HBV感染者C/D重组体的PC变异、BCP变异明显高于Ba亚型,差异有统计学意义(P<0.01)。C1亚型、C2亚型介于两者之间。在慢性乙型肝炎、乙肝肝硬化、肝癌中,HCC组的PC变异和BCP变异明显高于CH、LC组。3)对新疆14例HBV感染的HBV全序测序,在新疆首次经过以上方法,首次报道出一种新的基因型,命名为C/D重组体。维吾尔族中的C/D重组体所占比例高于汉族C/D重组体的比例。,说明C/D重组体存在族别的差异。已在GenBank上注册。4)对新疆18例P基因区变异位点进行分析。14例用阿德福韦治疗,13例出现变异,主要集中在聚合酶B区和D区附近。而且口服核苷类似物,随着时间的延长,出现HBV P基因变异的可能性增加;C1亚型变异主要集中在聚合酶B区附近,Ba亚型变异主要集中在聚合酶D区附近。YMDD变异的C2亚型比C1亚型的比例高,这方面的研究还需要更多实验证实。C1亚型变异主要集中在聚合酶B区附近,Ba亚型变异主要集中在聚合酶D区附近。更多还原

【Abstract】 Hepatitis B Virus has a strong human pathogenicity in environment. It causes acute and chronic hepatitis, liver cirrhosis, and liver cancer.At the present stage, HBV infection is one of public health problem in China. HBV infection assumes cosmopolitism prevalence. It has distinct geographic distribution. There is highest prevalence of HBsAg (9.09%)in China. Incident rate and fatality of chronic hepatitis B, liver cirrhosis, liver cancer will be increased by it.HBV has mutability and quasispecies characteristics. Now, HBV can be classified into at least eight genotypes (A-H) based on a divergence in the entire nucleotide sequence greater than 8% or S gene nucleotide sequence greater than4%, based on a divergence greater than 4% but less than 8% in the complete nucleotide sequence, HBV genotype has been divided into subgenotypes. HBV genotypes and subgenotypes have a distinct geographical distribution and a close relationship with ethnicity, as well as the relationship with clinical outcomes or responses to antiviral treatment. In China, genotype B and C are the most prevalent HBV strains.HBV showed different geographical distribution in our country, genotype C was predominant in northern China, genotype B was more prevalent in southern provinces. HBV mutation may appear with spontaneous, immunity and drug pressure. According to HBeAg, CHB was divided into HBeAg positive type and HBeAg negative type.PC and BCP mutations lead to HBeAg negative type on clinical manifestations.Long-term nucleoside analogue leads to HBV P genovariation.Recently, HBV genotype has been studied in Xinjiang.There is HBV genotypes, subgenotype distinction between hans and Uygurs. The aim of this article is to study HBV molecular virology and clinical character deeply between hans and uighurs. Object:Xinjiang has its specificity, so it is necessary to study hans and Uygurs HBV molecular virology and clinical character. HBV genotype, subgenotype data will be analysed.PC and BCP regions of HBV genome mutation data and HBV P regions mutation data will be supplied. All of them will help to improve the level of prevention and treatment.Contents:1)To investigate the virological characteristics of these genotype, subgenotypes and their clinical characteristics in Xinjiang hans and Uygurs;2) To investigate the virological features of the precore (PC) and basal core promoter (BCP) regions of the HBV genome and their clinical characteristics in Xinjiang Han and Uygurs;3) To investigate the virological characteristics of P regions mutation of the HBV genome caused by nucleoside analogue antiviral treatment.Method:1) 331 HBV patients collected were from Xinjiang Province.A total of chronic hepatitis B patients with detectable HBV genotypes, subgenotype, PC and BCP regions were detected by PCR-restriction fragment length polymorphism (PCR-RFLP), a part of samples were validated by direct sequencing, nucleotide sequence analysis of the phylogenetic tree, bioinfomatic.2)Randomized 14 samples has been dealed with by whole gene sequencing, 14 samples HBV P regions mutation caused by nucleoside analogue was assayed, 4 samples HBV P regions was assayed, sequence analysis of the phylogenetic tree, bioinfomatic.Results:Collect 331 HBV patients were from Xinjiang Province.A total of chronic hepatitis B patients were detected by PCR-RFLP, a part of samples validate by direct sequencing, nucleotide sequence analysis of the phylogenetic tree, bioinfomatic. 222 hans and 109 Uygurs, mean age±standard deviation, 38.7±12.7 years.1) Amongst 331 HBV patients studied, HBV genotypes showed B genotype 12.7%, C genotype 64%, C/D recombinant 16%, D genotype 7.3%.Compare with Genotype B, Genotype C, recombinant C/ D and Genotype D, they showed the distinctly distribution in Han patients and Uighur patients.Further analysis showed that all genotype B strains belong to Ba subgenotype, Bj subgenotype was not found. Genotype C had two subgenotypes, C1 42.9%, C2 57.1% in Xinjiang. Han HBV genotype showed B 14.9%, C 72.9%, recombinant C/D 9.5%, D 2.7%, Uighur HBV genotype showed B 8.3%, C 45.9%, recombinant C/D 29.4%, D 16.5%. Han HBV subgenotype showed Ba 78.6%, C1 39.5%, C2 60.5% ; Uygurs Ba 21.4%, C1 54%, C2 46%?2) Collect 331 HBV patients were from Xinjiang Province. HBV PC and BCP mutation of total chronic hepatitis B patients were detected by PCR-RFLP, a part of samples were validated by direct sequencing, nucleotide sequence analysis of the phylogenetic tree, bioinfomatic. HBV PC mutation showed PC mutation 30.2%, BCP mutation 34.4%.Further analysis showed C1 was associated with the highest tendency to develop BCP mutation. HBV recombinant C/D was associated with the highest tendency to develop BCP and PC mutation in Uygur.3) 14 samples were defined that the complete HBV genome were sequenced and compared with HBV sequences retrieved from GenBank, representing HBV genotype A-G.. Entire nucleotide sequences revealed that 2 samples were recombinant C/D, 12 samples were genotyped D.Moreover, they had difference sequences. Entire nucleotide sequences of 14 samples were registered.4) HBV P gene of 18 samples was sequenced and compared with HBV sequences retrieved from GenBank, representing HBV genotype A-G..Amongst 14 samples took adefovir, P gene mutation, the esults showed mutation nearby polymerase B(16) and D(6) appeared of 13 samples.Amongst C subgenotype, mutation nearby polymerase B(9) and D(1) with C1 was appeared in 5 samples, mutation nearby polymerase B(3) and D(3) with C2 was appeared in 5 samples, mutation nearby polymerase B(4) and D(8) with Ba was appeared in 6 samples, mutation nearby polymerase B(3) and D(1) with D was appeared in 1 samples, mutation nearby polymerase B(2) with C/D1 was appeared in 1 samples. Genotype B 11.8%, C 64.7%, C/D 20.6%, D 2.9% were showed in 34 YMDD mutation samples.Conclusion:1) Collect 331 HBV patients were from Xinjiang Province.A total of chronic hepatitis B patients were detected by PCR-RFLP, a part of samples validate by direct sequencing, nucleotide sequence analysis of the phylogenetic tree, bioinfomatic. The results showed that the most common HBV genotypes were C and subgenotype C2, while recombinant C/D was also found in Xinjiang. In Uygurs, the most common HBV genotypes were C, C/D and subgenotype C1.Genotype C/D and D have significantly different in Uygurs and Hans. HBV subgenotype C2 was a more important risk factor in hepatocarcinogenesis than other subgenotypes.2) Collect 331 HBV patients were from Xinjiang Province. We studied HBV PC and BCP mutations. The result showed that the PC and BCP mutations have related with subgenotypes. Recombinant C/D has significantly different with Ba in Uygurs.3) 14 samples were defined that the complete HBV genome were sequenced and compared with HBV sequences retrieved from GenBank, representing HBV genotype A-G.. Recombinant C/D was found by above method first time in Xinjiang Han and Uighur. They have been registered in GenBank.4) 18 samples were defined that the HBV P gene was sequenced and compared with HBV sequences retrieved from GenBank, representing HBV genotype A-G..HBV patients with nucleoside analog(ue) has different mutation site in different subgenotype, concentrated nearby polymerase B and D.Uighur HBV patients has particularity in HBV P regions mutations.更多还原