Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.     

乙型肝炎病毒感染不同状态下血清病毒反转录酶区准种特点及其临床意义

目的 研究HBV感染不同状态下血清病毒反转录酶(RT)区准种特点和临床意义.方法 50例未接受抗病毒治疗的HBV感染者分为慢性HBV携带者(ASC)组10例、慢性乙型肝炎(CHB)组30例和乙型肝炎肝硬化(LC)组10例.收集患者外周血血清,抽提HBV DNA,PCR扩增RT区基因组后克隆、测序.每例患者获得15～30个序列,进行HBV RT区的准种异质性分析及相关基因突变统计等生物信息学分析.多均数比较采用方差分析,中位数比较采用非参数检验分析,非计量资料比较采用x2检验.结果 总共测序1221个克隆,其中ASC组152个,CHB组780个,LC组289个.3组间基因型构成差异无统计学意义.准种复杂度为LC组＞CHB组＞ASC组,3组间差异有统计学意义(F=33.400,P＜0.05).准种离散度为LC组＞CHB组＞ASC组,LC组与CHB组及ASC组均差异有统计学意义(F=18.070,P＜0.05),CHB组与ASC组间差异无统计学意义.结论 慢性HBV感染过程中,免疫清除期比免疫耐受期具有更宽的HBV变异谱系,随着病程的延长和病情加重,HBV的准种趋向复杂.     

Hepatitis B and D viruses replication interference: Influence of hepatitis B genotype

AIM: To study the hepatitis B virus(HBV) and hepatitis D virus(HDV) replication interferences in patients with chronic hepatitis delta infected with different HBV genotypes.METHODS: We conducted a transversal study including 68 chronic hepatitis delta(CHD)(37 HIVpositive) patients and a control group of 49 chronic hepatitis B(CHB)(22 HIV-positive) patients. In addition, a dynamic follow-up was performed in 16 CHD patients. In all the samples, the surface antigen of hepatitis B(HBs Ag) serum titers were analyzed with the Monolisa HBs Ag Ultra system(Bio-Rad), using as quantification standard a serial dilution curve of an international HBs Ag standard. Serum HBV-DNA titers were analyzed using the Roche Cobas Taq Man(Roche, Barcelona, Spain), and the serum HDV-RNA using an in-house real-time q RT-PCR method, with Taq Man probes. HBV genotype was determined with the line immunoassay Li PA HBV genotyping system(Innogenetics, Ghent, Belgium). In those patients negative for Li PA assay, a nested PCR method of complete HBs Ag coding region, followed by sequence analysis was applied.RESULTS: No differences in the HBV-DNA levels were found in CHB patients infected with different HBV genotypes. However, in CHD patients the HBV-DNA levels were lower in those infected with HBV-A than in those with HBV-D, both in HIV negative [median(IQR): 1.25(1.00-1.35) vs 2.95(2.07-3.93) log10(copies/m L), P = 0.013] and HIV positive patients [2.63(1.24-2.69) vs 7.25(4.61-7.55) log10(copies/m L), P 0.001]. This was confirmed in the dynamic study of the HBV/HDV patients. These differences induce an under-estimation of HBV-A incidence in patients with CHD analyzed with Li PA assay. Finally, the HBs Ag titers reflected no significant differences in CHD patients infected with HBV-A or D.CONCLUSION: Viral replication interference between HBV and HDV is HBV-genotype dependent, and more evident in patients infected with HBV-genotype A, than with HBV-D or E.     

熔点曲线法研究乙型肝炎病毒准种和临床表现的关系

目的研究乙型肝炎病毒（HBV）准种与临床的关系，从HBV准种这个角度阐述相同基因型HBV导致不同病情的机制。方法选取B基因型HBV感染的慢性携带者血清32份和慢性乙型肝炎重度患者血清28份作为检测对象，采用熔点曲线法比较两组血清波峰数量的差异。结果慢性乙型肝炎重度患者血清的波峰数量明显多于慢性携带者血清（P〈0．05），提示前者所携带HBV的准种数量多于后者。结论HBV准种与乙型肝炎临床表现存在一定关系，在HBV基因型相同时，病情较重的乙型肝炎与HBV准种数量多有关，可在一定程度上解释同一基因型的不同患者出现不同临床表现的现象。     

拉米夫定与乙型肝炎病毒准种及变异特点的关系

目的 研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒聚合酶(HBV P)基因序列的准种组成及变异特点。 方法 目的片段经聚合酶链反应扩增后克隆,每份标本选择3 3个克隆,用单链构象多态性/异源双链分析法对准种复杂性及变异特点进行分析。 结果 拉米夫定治疗前患者体内HBV P基因序列准种数为7～14(平均9.8),高于治疗后准种数4～8(平均5.7),t=3.98,P<0.05。6例患者治疗前的准种分布中有一种或两种优势准种,但比例较低(33.3％～81.8％);治疗后显著升高(78.8％～90.9％),t=3.42,P<0.05。选择优势克隆测序,6例患者经拉米夫定治疗后2例出现M550V/L526M变异,3例为M550I变异,1例无YMDD变异。此外为个体化的点突变,无明显趋势。 结论在拉米夫定的药物筛选作用下HBV准种的组成改变,同时出现YMDD序列变异。     

23株HBeAg阳性慢性乙型肝炎患者病毒的全基因组分析

目的研究HBeAg阳性慢性乙型肝炎患者HBV变异特点。方法PCR扩增并克隆HBeAg阳性慢性乙型肝炎患者血清中HBV全基因组DNA,测序并进行基因结构分析。结果获得23株HBV全基因组DNA,它们均属于c或B基因型。与中国HBVB、C基因型参照序列相比,HBeAg阳性慢性乙型肝炎患者来源的HBV在表面抗原、P蛋白、X蛋白的反式激活区及增强子II／核心启动子区发生了一些有意义的共有变异。结论HBV变异可能与HBeAg阳性慢性乙型肝炎的发生、发展有关。     

新疆维吾尔族慢性乙型肝炎患者HBV基因型分布及其特点

目的探讨新疆维吾尔族慢性乙型肝炎患者HBV基因型分布及其特点。方法采用型特异性引物巢式PCR法对127例维吾尔族慢性乙型肝炎患者进行基因分型,并测序验证。结果基因D型占39．4%（50/127）,基因B型占22．0%（28/127）,基因C型占16．5%（21/127）,基因BD混合型占9．4%（12/127）,基因CD混合型占8．7%（11/127）,基因BCD混合型占3．9%（5/127）; HBeAg阳性与HBeAg阴性的维吾尔族慢性乙型肝炎患者基因型分布,差异无统计学意义（x^2= 6．033,P＞0．05）;不同年龄维吾尔族慢性乙型肝炎患者HBV基因型分布差异无统计学意义（x^2= 3．137,P＞0．05）;不同性别维吾尔族慢性乙型肝炎患者HBV基因型分布差异亦无统计学意义（x^2= 8．058,P＞0．05）。结论新疆维吾尔族慢性乙型肝炎患者HBV基因型以D型占优势,其次可见B、C型及BD、CD、BCD混合型。同一疾病谱的慢性HBV感染者基因型分布可能与宿主HBeAg状态、年龄、性别无明显关系。     

舟山群岛慢性乙型肝炎病毒感染者基因型与临床病理的关系

目的观察中国舟山群岛慢性HBV感染者的基因型与临床病理变化的关系。方法180例两代以上生活在舟山群岛的当地居民,血清HBV标志物和HBV DNA均阳性的各类慢性肝病患者,男147例,女33例,年龄(39.0±11.3)岁,HBV携带者(ASC)17例,CHB轻度57例,中度48例,重度9例,重型肝炎(SHB)6例,肝炎后肝硬化(LC)39例,HCC 4例。采用PCR结合Taqman MGB探针技术,对血清中HBV基因型进行分型检测,同时检测HBV血清标志物、HBV DNA、肝功能。对其中的129例患者做肝组织活检。结果舟山群岛慢性HBV感染者HBV基因型C型135例,占75.0%,B型40例,占22.2%;B、C混合型5例,占2.8%,未见有A、D型。随着病情加重,C型所占比例逐渐增高,在ASC、CHB、LC和SHB中C型分别占41.2%(7/17例)、75.4%(86/114例)、87.2%(34/39例)、100%(6/6例);在HCC中,B、C基因型各占2例。肝组织病理学检查中,99例C基因型患者,中、重度炎症(G3～4)有84例,占84.8%,30例B基因型患者G3～4有7例,占23.3%,z=6.47,P<0.01。C基因型在中、重度纤维化(S3～4)的分布与B基因型比较,差异有统计学意义。在中、重度肝脏病理损伤中C型病理改变与临床诊断符合率较好;而在轻度肝脏病理损伤中,B型病理改变与临床诊断符合率较好,C型符合率较差。结论中国舟山群岛HBV基因型为C、B及BC混合型,以C基因型占优势,C基因型的病理损伤程度较B型严重。     

未经治疗的慢性乙型肝炎患者乙型肝炎病毒耐药变异、基因型和血清型研究

目的 研究未经核苷(酸)类似物(NA)治疗的慢性乙型肝炎患者HBV耐药变异、基因型、基因亚型和血清型特点.方法 从北京大学附属医院收集97例未经NA治疗的慢性乙型肝炎患者血清,用半巢式聚合酶链反应-直接测序法获得HBV全长逆转录酶区序列,用生物信息学技术筛查该区内11个经典耐药变异位点并鉴定基因型、基因亚型和血清型.用统计分析软件SPSS11.0进行t检验和χ~2检验. 结果 HBV在11个经典耐药变异位点上均为野生型氨基酸;B基因型和C基因型分别占36.1%(35/97)和63.9%(62/97),前者均属B2亚型,后者C2亚型占91.9%(57/62),C1亚型占6.5%(4/62),1例未能分出亚型.已知出生地的患者中,71.9%(23/32) B基因型感染者出生于我国南方地区,81.6%(40/49) C基因型感染者出生于北方地区,基因型地域分布特点明显,χ~2=23.19,P<0.01.血清型为adr者占60.8%(59/97),与C基因型相关;为adw者占38.1%(37/97),与B基因型相关,χ~2=87.83,P<0.01.结论 未经NA治疗的慢性乙型肝炎患者体内野毒株为优势株,其基因型、基因亚型和血清型与患者出生地有关.     

慢性乙型肝炎患者病毒准种特性的初步研究

目的 探讨慢性乙型肝炎(CHB)患者血清中乙型肝炎病毒(HBV)是否存在准种特性,并初步了解HBV准种的复杂性和遗传差异性。 方法用多聚酶链反应(PCR)技术从1例CHB患者血清中扩增HBV整个PreC/C基因区,然后用T载体克隆PCR产物,从转化阳性的克隆中随机选出34个克隆进行核酸序列分析。结果在34个测序克隆中发现存在28种不同的序列,序列间差异性介于0.2％～2.1％。变异位点分布于整个区域。所有序列nt1896位均无变异。 结论 在CHB患者体内HBV存在复杂的准种特性。     

乙型肝炎病毒基因型与病情轻重的关系

目的研究乙型肝炎病毒(HBV)基因型与肝脏损伤程度的相关性。方法随机选取87例慢性无症状乙型肝炎表面抗原携带者(ASC)、157例慢性乙型肝炎(CHB)、22例肝硬化(LC)和18例肝细胞癌(HCC)患者外周血测定HBV S基因序列以明确其基因型。结果基因型以B型和C型为主,分别为26.1％和73.2％。在B型与C型中,ASC、CHB、LC和HCC的构成比差异有非常显著性(X2=15.09,P<0.001),C型与B型相比,C型发展致CHB和HCC所占百分比显著高于B型,分别为59.6％比43.2％,X2=10.87,P<0.001;和7.7％比1.4％,X2=7.41,P<0.001。而LC所占百分比差异无显著性。结论基因型C型与B型相比,C型HBV感染易引起较重肝脏损伤。     

慢性乙型肝炎急性发作患者血清病毒准种动力学研究

目的揭示慢性乙型肝炎急性发作及重型化过程中血清乙型肝炎病毒(HBV)基因组前C／C 及基本核心启动子(BCP)区准种的动态变化规律。方法选取1例慢性乙型肝炎两次急性发作伴重型化患者病程中5份血清样本,采用聚合酶链反应-TA克隆-构象敏感凝胶电泳-测序的方法,研究慢性乙型肝炎重型化过程中血清HBV准种的动态变化。结果血清HBV准种复杂性在慢性乙型肝炎急性发作时为10～ 12,在好转时为14～17(t=3．133,P<0．05),且随时间推移有逐渐升高的趋势;在此过程中优势准种组成及比例亦发生了变化(t=3．295,P相似文献   

Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B

AIM： To study the relationship between hepatitis B virus （HBV） DNA levels and liver histology in patients with chronic hepatitis B （CHB） and to determine the prevalence and characteristics of hepatitis B e antigen （HBeAg） negative patients.
METHODS： A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status.The correlation between serum HBV DNA levels and liver damage （liver histology and biochemistry） was explored.
RESULTS： Of the 213 patients with serum HBV DNA levels higher than 10^5 copies/mL, 178 （83.6%） were HBeAg positive, 35 （16.4%） were HBeAg negative. The serum HBV DNA levels were not correlated to the age,history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse （ALT）,aspartate aminotrans-ferase （AST） in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST,while serum DNA levels correlated with ALT （r = 0.351, P = 0.042）. The grade （G） of liver disease correlated with ALT and AST （P 〈 0.05, r = 0.205, 0.327 respectively）in HBeAg positive patients. In HBeAg negative patients,correlations were shown between ALT, AST and the G （P 〈 0.01, and r = 0.862, 0.802 respectively）. HBeAg negative patients were older （35 ± 9 years vs 30 ±9 years, P 〈 0.05 ） and had a longer history of HBV infection （8 ± 4 years vs 6 ± 4 years, P 〈 0.05） and a lower HBV DNA level than HBeAg positive patients （8.4± 1.7 Log HBV DNA vs 9.8 ± 1.3 Log HBV DNA, P 〈0.001）. There were no significant differences in sex ratio,ALT and AST levels and liver histology between the two groups.
CONCLUSION： Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA mor     

拉米夫定耐药后序贯治疗中HBV准种的演变

目的:探讨拉米夫定(LAM)耐药后序贯治疗中HBV准种的演变特点.方法:收集7例拉米夫定耐药患者序贯治疗中的血清,对HBV聚合酶基因逆转录酶区进行PCR扩增克隆并测序,结合临床用药分析序列突变,探讨其演变特征.结果:患者1、4、5在序贯治疗中LAM耐药变异株一直存在,主要以M204I+L80I、M204I+L80I+L180M、M204V+L180M+G173L、M204V+L180M为主,所占比例在不断发生变化,患者2、3在序贯治疗中检测出野生株,但患者2在后续治疗中又选择出LAM耐药变异株.患者6在ADv联合ETV治疗时、患者7在单用ADV治疗时测时分别测出双重耐药株M204v+L180M+G173L+T184A和M204V+180M+G173L+236D.结论:拉米夫定耐药后序贯治疗中原耐药突变不易消失,且容易在此基础上筛选出交叉耐药株或多重耐药株,他的出现影响序贯治疗的应答甚至导致序贯治疗的失败.     

e抗原阳性慢性乙型肝炎患者感染病毒的基因型与抗病毒治疗疗效的关系

目的 探讨HBV基因型与抗病毒治疗疗效的关系。方法 应用PCR-微板核酸分子杂交ELISA法检测90例HBeAg阳性CHB患者的HBV基因型,对其中41例患者给予拉米夫定（100 mg／d）抗病毒治疗48周,49例患者给子干扰素α（3 MU／次,隔日1次）抗病毒治疗48周,治疗前、治疗过程中和治疗结束时分别检测血清生化指标（ALT）、病毒学血清标志物（HBeAg和抗-HBe）和HBV DNA水平。结果 90例CHB患者中HBV B基因型者16例,C基因型者74例。41例患者应用拉米夫定治疗,感染B和C基因型患者48周时对拉米夫定治疗应答率分别为33.3％和20%,差异无统计学意义;49例患者应用干扰素α治疗,48周时感染基因B型患者的ALT复常率、HBeAg消失率和HBV DNA阴转率均高于感染基因C型患者（分别为60.0％和20．5％,50．0％和17．9％, 50．0％和17．9％）,两组比较差异有统计学意义,但HBeAg血清转换率差异无统计学意义。结论 基因B和C型对拉米夫定抗病毒治疗的疗效无影响,基因B型对干扰素α治疗的疗效优于C型。     

乙型肝炎病毒基因型对乙型肝炎病毒变异的影响

目的探讨乙型肝炎病毒(HBV)基因型对病毒前核心区(前C区,nt1896)及基本核心启动子(BCP,nt1762/1764)变异的影响.方法416例血清HBsAg阳性、HBV DNA定量大于1.0×104拷贝/ml的患者,采用微流基因芯片检测HBV基因型、前C区及BCP变异.结果416例HBV感染者中406例有基因分型结果:B型20.9%、C型65.9%、BC混合型10.8%,10例患者未分出基因型.302例为HBeAg(-)且HBV DNA( )患者,其中248例(82.12%)有前C区或BCP变异,41.06%为前C区变异,31.12?P变异,2种同时变异为9.94%.B型患者前C区变异率为22.9%(20/87),与C型患者前C区变异率39.4%(108/274)及B、C混合型变异率40.0%相比均有显著差异(P＜0.01).在BCP变异及双变异,C型患者变异率均大于B型患者,但差异无统计学意义(P＞0.05).B、C混合型患者前C区及BCP变异率与C型相似.结论HBV基因型可影响病毒前C区及BCP变异,以C型为著.     

天津地区家族聚集性慢性乙型肝炎病毒感染者病毒载量及组织病变与基因型的关系

目的探讨天津地区家族聚集性HBV感染患者基因型特征与病毒载量及肝组织病理的关系。方法随机采集临床确诊家族聚集性的慢性HBV表面抗原携带者(ASC)35例和轻型CHB患者65例。分别进行HBV基因分型、HBV DNA载量、肝组织病理学检测。结果HBV基因型显示:B型7例,HBV DNA主要为低载量(57.14%),病理损害程度较轻。BC混合型11例,HBV DNA以低、中载量为主(45.45%、36.36%),病理损害多为轻至中度≤G2 10例(90.91%),≤S2 9例(81.82%)。C型82例,其中ASC 29例,HBV DNA以高载量为主(72.41%),均有不同程度的病理损伤(100%);CHB 53例,HBV DNA主要为中、高载量(39.62%,49.06%),病理损害≥G2者38例(71.70%),≥S2者25例(47.17%)。结论天津地区家族聚集性ASC和CHB以基因C型为主、病毒载量高、肝组织病理损伤较严重,为乙型肝炎预后不良的主要因素;家族聚集性ASC不仅存在HBV病毒高载量,而且均有不同程度的肝脏病理损伤,临床可视肝组织病理结果酌情抗病毒治疗;肝组织病理学检查作为ASC和CHB患者治疗前的常规检测手段,为规范CHB的抗病毒治疗提供可靠依据。     

Genomic change in hepatitis B virus associated with development of hepatocellular carcinoma

AIM: To determine the genomic changes in hepatitis B virus (HBV) and evaluate their role in the development of hepatocellular carcinoma (HCC) in patients chronically infected with genotype C HBV.METHODS: Two hundred and forty chronic hepatitis B (CHB) patients were subjected and followed for a median of 105 mo. HCC was diagnosed in accordance with AASLD guidelines. The whole X, S, basal core promoter (BCP), and precore regions of HBV were sequenced using the direct sequencing method.RESULTS: All of the subjects were infected with genotype C HBV. Out of 240 CHB patients, 25 (10%) had C1653T and 33 (14%) had T1753V mutation in X region; 157 (65%) had A1762T/G1764A mutations in BCP region, 50 (21%) had G1896A mutation in precore region and 67 (28%) had pre-S deletions. HCC occurred in 6 patients (3%). The prevalence of T1753V mutation was significantly higher in patients who developed HCC than in those without HCC. The cumulative occurrence rates of HCC were 5% and 19% at 10 and 15 years, respectively, in patients with T1753V mutant, which were significantly higher than 1% and 1% in those with wild type HBV (P < 0.001).CONCLUSION: The presence of T1753V mutation in HBV X-gene significantly increases the risk of HCC development in patients chronically infected with genotype C HBV.