慢性乙型肝炎患者体内乙型肝炎病毒准种特点的初步研究

目的 探讨乙型肝炎病毒（HBV）准种在慢性乙型肝炎患者中的存在状态。方法 以已知中国株HBV基因序列为依据。设计特异性聚合酶链反应（PCR）引物,自3例慢性乙型肝炎患者体内扩增HBV全S基因片段,克隆人pGEMTeasy质粒,用限制性片段长度多态性（RFLP）法确定HBV的变异现象,DNA测序确定病毒的变异程度。结果 分别自3例患者血清中克隆出29、28和8个阳性克隆,以EcoRⅠ酶切患者1和2的RFLP结果提示各有5种不同的长度带型,PCR产物XhoI酶切RFLP结果表现为高度保守,测序结果发现HBV体内毒株DNA序列高度一致,同一患者两株全S区序列测序结果表明DNA序列的同源性大于97%。结论 慢性乙型肝炎患者体内有HBV准种共存,且呈现出一定的优势克隆现象。可能与患者预后有关。     

慢性乙型肝炎患者体内乙型肝炎病毒准种特点的初步研究

探讨乙型肝炎病毒（HBV）在慢性患者中存在状态，以HBV基因序列为依据。设计特异性多聚酶链反应（PCR）引物，自2例慢性患者体内扩增HBV全S基因片段，克隆入T载体。限制片段长度多态性（RFLP）法确定HBV的变异现象，DNA测序确定病毒的变异程度，质粒EcoRI酶切RFLP结果提示自2例患者血清中克隆出的29和28个阳性克隆中各有5种不同的长度带型，PCR产物XhoI酶切RFLP结果则表现为高度保守，测序结果发现HBV体内毒株碱基序高度一致，同一患者两株全S区序列测序结果表明DNA序列的同源性大于97%。本结果提示HBV慢性患者体内有HBV准种共存，且呈现出一定的优势克隆现象。     

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。     

乙型肝炎病毒大S蛋白基因纵向研究方法及应用

目的:建立乙型肝炎病毒(HBV)大S蛋白基因纵向研究方法,并将其应用于疾病进程中HBV的准种动态研究.方法:以2例HBV感染者(对象A,男,38岁;对象B,女,22岁.对象A研究期间未经任何抗病毒治疗,B则1年持续接受ADV抗病毒药物治疗)体内不同时期的血清样本核酸提取物为模板,通过特异性扩增获取病毒大S蛋白基因序列并克隆测序,依据生物信息学方法对发生于序列结构不同区域的变异进行分析,利用比对数据对不同样本HBV准种予以描述与评价.结果:所得24条HBV大S蛋白基因序列中2条源于对象B样本的基因序列PreS2编码区存在30 nt的缺失,其余22条序列大小均为1203bp;24条序列的HBV基因型均为c型.对象A大S蛋白基因序列分散值DA、DB、Dc分别为:0.29、0.16、0.23,对象B则为0.80、2.30、1.82.来自对象A的全部10条HBV大S蛋白基因序列内部的"a"决定簇与参考基因完全一致;对象B的14条序列期初有2条(29%)期终有6条(86%)存在G145R突变,期终序列中有1条序列"a"决定簇内发现4个位点的突变.结论:人体内感染HBV以准种形式存在且可以用分散值DA、DB、Dc对其进行描述,并应用于HBV准种动态的纵向研究.而抗病毒药物ADV的1年期治疗可使本例HBV准种分散性增高,可能增加本例耐药性突变出现的风险,同时增加本例"a"决定簇的变异出现.     

HePG2.2.15细胞株中乙型肝炎病毒基因异质性的研究

目的以乙型肝炎病毒(HBV)前C/C基因异质性来探讨在HepG2.2.15细胞体内是否存在HBV准种。方法依据Genebank中HBVayw亚型全基因序列,选择保守区域设计出特异性多聚酶链反应引物,自HepG2.2.15细胞培养上清中扩增HBV前C/C基因序列,克隆入pGEM-TEasy载体,挑选15个克隆测序以比较病毒的变异程度。结果测序结果发现HepG2.2.15细胞HBV前C/C基因存在异质性,碱基序列之间的同源性大于93%,突变类型全部为碱基替换。结论结果提示,长期培养HepG2.2.15细胞内有HBV准种共存,这种细胞模型内HBV异质性现象可能影响其在基因治疗等方面的应用。     

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。     

乙型肝炎病毒核心启动子缺失突变的初步观察

目的研究乙型肝炎病毒(HBV)e抗原阴性患者HBV核心基因启动子变异方式。方法自5例慢性HBV感染者血清中分别提取HBVDNA,应用多重PCR法鉴定其基因型。PCR扩增X基因序列,克隆入pMD19T载体,共挑选23个克隆测序,与已知HBV基因相应序列比较该患者体内HBV基因变异程度。结果 5例患者中3例基因型分别为B、C和B/C混合型,2例患者通过现行多引物方法无法分型。16个(16/23,69.6%)克隆在X基因下游出现大段缺失突变,其中15例缺失长度为234bp,1例为245bp。缺失突变区域包括C基因启动子区,并导致前-C区起始密码子ATG编码缺失,这种缺失突变在5例患者中均被检出。结论 CP/HBeAg起始密码子缺失突变可能是HBeAg阴性慢性乙型肝炎的一种变异模式。     

乙型肝炎病毒X基因准种与变异特点的研究

以乙型肝炎病毒 (ＨＢＶ)Ｘ基因序列的异质性表现来探讨ＨＢＶ准种在慢性感染者中的存在状态。用中国株ＨＢＶ基因序列为依据 ,设计特异性聚合酶链反应 (ＰＣＲ)引物 ,自 3例慢性ＨＢＶ感染患者血清中扩增ＨＢＶＸ基因 ,克隆入 ｐＧＥＭＴｅａｓｙ质粒 ,随机挑选克隆进行ＤＮＡ测序以确定病毒的变异程度。测序结果提示 ,来源于不同患者ＨＢＶＸ基因序列高度保守 ,序列同源性为 98.9%～ 99.8% ,但每个序列均不一致 ,符合准种判断标准。Ｘ区存在广泛的点替换突变 ;缺失突变占测序克隆总数的 33.3% ,变异区域集中于 12 3位氨基酸编码核苷酸之后…     

HBsAg与抗-HBs同时阳性慢性乙肝患者HBV基因全序列分析

目的 报告HBsAg与抗-HBs同时阳性慢性乙肝患者基因序列变化特点.方法 对三例HBsAg与抗-HBs同时阳性慢性乙肝患者血清中HBV DNA基因全序列进行PCR扩增、测序,并对测序结果及S区氨基酸进行分析.结果 测序结果发现三例患者血清中HBV S区氨基酸均发生替换突变,并且,三例患者中HBV DNA发生碱基替换的位点大部分相同,且替换后碱基基本相同.结论 HBsAg与抗-HBs同时阳性慢性乙型肝炎患者常发生S区氨基酸序列突变,双阳现象的出现亦可能与S区外其他位点碱基突变有关.     

一种新的HBeAg阴性慢性乙型肝炎病毒变异机制

目的 检测HBV核心启动子区(CP)基因变异方式.方法 自HBV慢性感染患者血清中提取HBV DNA,扩增CP区域序列,克隆入pMD19 T载体,选择阳性克隆进行DNA测序,与已知HBV基因相应序列比较患者体内HBV基因变异位点以及变异形式.结果 自21例患者中共挑选74个克隆测序,54个克隆中病毒基因序列CP区发生大段缺失突变,长度达234个核苷酸,另有1个克隆发生245个核苷酸缺失突变.缺失突变区域包括CP区、HBeAg起始密码子和直接重复序列(DR)Ⅰ区,命名为CP缺失突变,发生CP缺失突变的病毒株同时存在A1585T替换突变,这两个部位的突变具有联动特征.结论 观察到一种导致HBeAg阴性慢性乙型肝炎的新方式,即CP、HBeAg起始密码子缺失突变,并提出一种简明的CP缺失检测方式.     

乙型肝炎病毒表面抗原一级结构多态性的初步研究

目的 研究慢性乙型肝炎患者体内乙型肝炎病毒（HBV）表面抗原一级结构的多态性。方法 设计特异性引物，自7例慢性乙型肝炎患者血清中扩增S基因全长或全基因组片段，TA克隆法克隆到T载体中，随机选择克隆测序。结果 共20个克隆被测序。20个克隆全S蛋白的总一致率仅为32．0％，13株全长为401氨基酸残基的克隆氨基酸一致效率为82．5％。患者血清中发现2株克隆编码截短型表面抗原中蛋白，在前S1或前S2的免疫决定区或可能的肝细胞结合部位均发现缺失突变。结论 慢性乙肝患者体内存在HBV准种群，病毒编码的截短型表面抗原中蛋白可为HBV诱导原发性肝癌提供一种途径，应加强对HBsAg一级结构多态性的研究。     

HBV“相似株”与“优势株”的混杂可能为慢性重型乙型肝炎的一个特点

目的了解慢性重型乙型病毒性肝炎患者体内HBV的状况。方法选3例慢性重型乙型病毒性肝炎患者,以HBV前S基因为研究区域,采用半巢式PCR法,从此3例患者中血清中扩增出HBV前S基因,并分别将其克隆于噬菌体M13mp18、M13mp19载体中,每例患者随机筛选10个阳性克隆,进行序列分析及遗传距离分析。结果此3例病人存在着“相似株”与“优势株”的混杂及HBVadr与HPBadrwl两种亚型的混合感染。优势株内可能存在引起机体免疫性增高的区域。结论 HBV“相似株”与“优势株”的混杂可能为慢性重型乙型肝炎的一个特点。     

Hepatitis B Virus Genomes of Chronic Hepatitis Patients Do Not Contain Specific Mutations Related to Acute Exacerbation

To determine the specific viral variants associated with acute exacerbation of chronic hepatitis from hepatitis B virus (HBV) infection, we analyzed the complete nucleotide sequences of the HBV genome in serial serum samples from two chronic active hepatitis patients who seroconverted from HBeAg to anti-HBe. HBV DNA was amplified by polymerase chain reaction (PCR) and sequenced. A 1896 precore stop codon mutant (G to A at nt 1896) coexisting with the wild sequence was found in both patients prior to seroconversion from HBeAg to anti-HBe. Core promoter mutations at nucleotide positions 1762 (A to T) and 1764 (G to A) were found in both patients throughout the observation period. Mutations were observed in the HBV genome of the two patients at different time points, and there was no correlation between the mutations and liver disease or DNA polymerase levels. The nucleotide divergence rate and the composition of quasispecies in the HBV sequence at the time of acute exacerbation were almost the same as were found at other time points. These results suggest that acute exacerbation does not appear to be caused by a characteristic HBV species. The multiple factors that cause generalized HBV replication activation may contribute to acute exacerbation.     

乙型肝炎病毒不同株的混杂感染可能是慢性重型肝炎的一个特点

目的　了解慢性重型乙型病毒性肝炎患者体内ＨＢＶ的状况。方法　选３例慢性重型乙型病毒性肝炎患者,以ＨＢＶ前Ｓ基因为研究区域,采用半套式ＰＣＲ法,从３例患者血清中扩增出ＨＢＶ前Ｓ基因,并分别将其克隆于噬菌体Ｍ１３ｍｐ１８、Ｍ１３ｍｐ１９载体中,每例患者随机筛选１０个阳性克隆,进行序列分析及遗传距离分析。结果　此３例患者存在着“相似株”与“优势株”的混杂及ＨＢＶ ａｄｒ与ＨＰＢ ａｄｒｗｌ两种亚型的混合感染。优势株内可能存在引起机体免疫性增高的区域。结论ＨＢＶ“相似株”与“优势株”的混杂可能是慢性重型乙型肝炎的一个特点。     

Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases

BACKGROUND/AIMS: PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches. METHODS: We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by NlaIII enzyme that cuts the wild ATG-start-codon but not the mutated one. RESULTS: We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease (P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients (P<0.02). CONCLUSIONS: Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.     

Impact of hepatitis B virus (HBV) preS/S genomic variability on HBV surface antigen and HBV DNA serum levels

To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. CONCLUSION: In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection.     

Hepatitis B virus precore mutations and HBeAg negative reactivation of chronic hepatitis B after interferon therapy

The objective of this study was to look for HBV precore mutations in three patients with chronic active hepatitis B who developed HBV-DNA-positive/HBeAg-negative reactivation after HBe seroconversion induced by interferon therapy. Direct sequencing of polymerase chain reaction products was performed on serum collected before and after HBe seroconversion. In two patients precore sequence showed only wild-type HBV before and after interferon therapy. In one patient, precore sequence showed only wild-type HBV before interferon therapy and a mixed infection by wild-type HBV and precore mutant viruses (1858 and 1896 nucleotide mutations) after treatment. The presence of HBeAg/anti-HBe immune complexes was found after HBe seroconversion in all cases. Our results suggest that: 1) precore mutations are not always found in patients with chronic hepatitis B who develop HBV DNA-positive/HBeAg-negative reactivation; and 2) HBeAg negativity, despite the presence of wild-type HBV, might be due to HBeAg/anti-HBe immune complexes. We speculate that the production of these immune complexes may be favored by interferon therapy.     

Hepatitis B e antigen in sera from individuals infected with hepatitis B virus of genotype G

Hepatitis B virus (HBV) genotype G (HBV/G) was detected in sera from four individuals by polymerase chain reaction with hemi-nested primers deduced from an insertion of 36 nt in the core gene that is specific for this genotype. Despite two stop codons in the precore region characteristic of HBV/G, all patients were positive for hepatitis B e antigen (HBeAg) in serum. When 10 HBV clones were propagated from one patient, and sequenced within precore region and a section of the core gene, 6 clones were HBV/G while 2 were genotype A (HBV/A); a recombination between HBV/G and HBV/A occurred in the remaining 2 clones. Mixed infection of HBV/G and HBV/A, as well as the recombination, was demonstrated in the sequence of preS1 and preS2 regions also. Coinfection with HBV/G and HBV/A was demonstrated in the other three patients, and their recombination in two patients. Ten HBV clones were propagated from one patient at two time points separated by 1 year. Clones of HBV/A, HBV/G and their recombination were found in 9 : 1 : 0 when the patient was positive for HBeAg, while the proportion shifted to 0 : 8 : 2 after the patient seroconverted to anti-HBe. In conclusion, HBV/G is frequently found as a coinfection with HBV/A. This coinfection would explain the presence of HBeAg in individuals infected with HBV/G. Along with seroconversion to anti-HBe, HBV/G would be selected accompanied by the recombination with HBV/A. Further studies should be performed to confirm these findings.