抗—HBe阳性慢性乙型肝炎病毒感染：病毒前C变异与病变活动

抗－ＨＢｅ（＋）的慢性活动性肝炎５１例，以聚合酶链反应检出ＨＢＶ ＤＮＡ４８例（９４．１％）。对６例以反应产物直接序列分析，发现前Ｃｎｔ８３发生Ｇ→Ａ（Ａ８３）点突变，使密码２８由于ＴＧＧ变异为终止密码ＴＡＧ而不能编码ＨＢｅＡｇ。以抗－ＨＢｅ（＋）的慢性无症状ＨＢＶ携带者３０例为对照，仅１２例（４０％）检出弱ＨＢＶ，ＤＮＡ带。５例序列分析未发现Ａ８３变异。结果提示：变异病毒逃避免疫清除而继续活跃复     

聚合酶链反应检测HBV－DNA的临床意义

本文采用聚合酶链反应（ＰＣＲ）和ＥＬＩＳＡ法对２７３７例乙肝患者血清中ＨＢＶ－ＤＮＡ和乙肝病毒标志物进行检测，结果发现各组ＨＢＶ－ＤＮＡ的检出率：①ＨＢｓＡｇ（＋）、ＨＢｅＡｇ（＋）和抗ＨＢｃ（＋）组为９９．２７％；②ＨＢｓＡｇ（＋）、抗ＨＢｅ（＋）和抗ＨＢｃ（＋）组为４４．７７％；③ＨＢｓＡｇ（＋）和抗ＨＢｃ（＋）组为４２．８６％；④ＨＢＶ标志物均阴性为１５．９２％。结果表明ＨＢＶ－ＤＮＡＰＣＲ检出先于其它乙肝病毒标志物，可早期发现乙肝。ＰＣＲ法直接检测ＨＢＶ－ＤＮＡ更有利于临床对乙肝的诊断和治疗。     

以聚合酶链反应法研究乙型肝炎HBV－DNA与e系统的关系

本文采用聚合酶链反应（ＰＣＲ）法检测了１２２例乙型肝炎患者血清ＨＢＶＤＮＡ。对照患者ｅ系统的模式，发现ＨＢｅＡｇ（＋），抗－ＨＢｅ（－）者ＨＢＶＤＮＡ阳性率为９０．９％，抗－ＨＢｅ（＋）、ＨＢｅＡｇ（－）者ＨＢＶＤＮＡ阳性率为５０．１％，ＨＢｅＡ（－）、抗－ＨＢｅ（－）者ＨＢＶＤＮＡ阳性率为３５．７％。结果表明，ＰＣＲ法较ｅ系统更能准确反映ＨＢＶ的复制情况。     

以聚合酶链反应法研究乙型肝炎HBV—DNA与e系统的关系

本文采用聚合酶链反应（ＰＣＲ）法检测了１２２例乙型肝炎患者血清ＨＢＶＤＮＡ。对照患者ｅ系统的模式，发现ＨＢｅＡｇ（＋），抗－ＨＢｅ（－）者ＨＢＶＤＮＡ阳性率为９０．９％，抗－ＨＢｅ（＋），ＨＢｅＡｇ（－）者ＨＢＶＤＮＡ阳性率为５０．１％，ＨＢｅＡｇ（－），抗－ＨＢｅ（－）者的ＨＢＶＤＮＡ阳性率为３５．７％。结果表明，ＰＣＲ法较ｅ系统更能准确反映了ＨＢＶ的复制情况。     

抗HBe(+)慢性乙型肝炎的重组干扰素治疗应答及其影响因素

目的探讨抗ＨＢｅ／ＨＢＶＤＮＡ（＋）慢性乙型肝炎（ＣＨＢ）的干扰素治疗效果及其影响因素。方法对１５例ＨＢｅ（＋）和２５例ＨＢｅＡｇ（＋）ＣＨＢ患者用重组干扰素α－１ｂ治疗，治疗前、后和随访半年后检测ＨＢＶ及前Ｃ区Ａ１８９６终止变异。结果抗ＨＢｅ（＋）慢性乙型肝炎干扰素治疗近期应答率为７３％（１１／１５），与ＨＢｅＡｇ（＋）组比较差异无显著性（Ｐ＞００５）；治疗前抗ＨＢｅ（＋）组ＨＢＶＤＮＡ含量明显低于ＨＢｅＡｇ（＋）组（Ｐ＜０００５）；但前Ｃ区Ａ１８９６变异检出率４７％（７／１５）显著高于ＨＢｅＡｇ（＋）组的１６％（４／２５），Ｐ＜００５；４例复发者都是Ａ１８９６变异感染。结论血清ＨＢＶＤＮＡ水平可能是影响其应答的重要因素，前Ｃ区Ａ１８９６变异并不影响干扰素的近期应答率，但可能是其复发的一个重要因素。     

血清HBeAg阴性与HBeAg/IC及A1896变异的关系

目的探讨血清ＨＢｅＡｇ阴性（双抗体夹心法）与ＨＢｅＡｇ／ＩＣ形成及ＨＢＶ变异株Ａ１８９６的关系，评价ＨＢｅＡｇ／ＩＣ检测的临床意义．方法单克隆抗ＨＢｅ固相ＥＬＩＳＡ检测血清中ＨＢｅＡｇ／ＩＣ；套式多聚酶链反应检测ＨＢＶＤＮＡ；３＇碱基特异多聚酶链反应判断Ａ１８９６变异；ＥＬＩＳＡ检测ＨＢｅＡｇ、抗ＨＢｅ，研究对象为１１７例慢性ＨＢＶ感染者，２０例健康对照统计处理采用卡方检验．结果ＨＢｅＡｇ／ＩＣ阳性血清中ＨＢＶＤＮＡ检出率明显高于ＨＢｅＡｇ／ＩＣ阴性血清，Ｐ＜０００１（９１３％ｖｓ３６２％）；２９份ＨＢｅＡｇ阴性、ＨＢＶＤＮＡ阳性血清中仅５例（１７２％）检出Ａ１８９６，而且其中２例与野毒株（Ｇ１８９６）混合感染并伴ＨＢｅＡｇ／ＩＣ阳性．２９份中１７份（５８７％）为ＨＢｅＡｇ／ＩＣ阳性的Ｇ１８９６感染；血清抗ＨＢｅ阳性组Ａ１８９６检出率高于抗ＨＢｅ阴性组，Ｐ＜００５（２５％ｖｓ３２％）．结论ＨＢｅＡｇ／ＩＣ为ＨＢＶ活跃复制指标；临床ＨＢｅＡｇ阴性、ＨＢＶＤＮＡ阳性患者仍多数为Ｇ１８９６感染，ＨＢｅＡｇ／ＩＣ形致双抗体夹心法不能检出ＨＢｅＡｇ；抗ＨＢｅ应答可能为促使前Ｃ变异的重要因素     

聚合酶链反应检测HBV DNA的临床意义

应用ＰＣＲ对１７１例肝病血清检测结果，ＨＢＶＤＮＡ阳性率５９．１％，ＥｌｉＳＡ检测ＨＢｓＡｇ阳性率４３．２７％，二者比较有显著性差异（Ｐ〈０．０１），ＨＢｓＡｇ（＋）／ＨＢｅＡｇ（＋）组ＨＢＶＤＮＡ阳性率７９．５％，而ＨＢｓＡｇ（＋）抗－ＨＢｅ（＋）组主５７．９％，ＨＢｓＡｇ（－）／抗－ＨＢｓ（＋）组，ＨＢＶＤＮＡ阳性率３６．８％。     

乙型肝炎病毒前C区A83变异株的临床检测及分析

目的 了解ＨＢＶ前Ｃ区Ａ８３变异的临床意义。方法 用错配ＰＣＲ－ＲＦＬＰ检测ＨＢＶ感染者血清ＨＢＶ前Ｃ区Ａ８３变异株。结果 该变异株在急性肝炎未检出;在慢性肝炎、肝硬化和重型肝炎检出率分别为６６．７％、４８．９％和４５．５％;慢性无症状ＨＢＶ携带者为３．３％;各型肝炎均以血清抗ＨＢｅ（＋）组的变异检出率最高;肝硬化血清变异株阳性者的腹水发生率和肝功功能异常率较阴性者无显著差别。结论 机体对ＨＢｅＡ     

抗HCV阳性慢性肝病患者HBV感染及前C／C区基因变异

探讨ＨＢＶ基因变异在ＨＢｓＡｇ阴性抗ＨＣＶ阳性慢性肝病中的意义。方法用巢式聚合酶链反应（ＰＣＲ）与限制片段长度多态性相结合，对５６例慢性肝病患者６例ＨＢｓＡｇ阴性抗ＨＣＶ阳性（Ａ组）．１９例ＨＢｓＡｇ阳性抗ＨＣＶ阳性（Ｂ组）及３１例单独ＨＢＶ成染（Ｃ组）进行前Ｃ区密码２８终止变异（Ａ８３）和Ｃ区密码９７异亮氨酸亮氨酸变异（Ｌ９７）分析。结果Ａ组和Ｂ组Ａ８３和Ｌ９７变异检出率分别为３３％和４２％显著低于Ｃ组８１％（Ｐ值＜０．００１）：而Ａ组和Ｂ组间无统计学差异（Ｐ值＞０．０５）。结论ＨＢＶ和ＨＣＶ双重感染ＨＢｓＡｇ阴性与Ａ８３和Ｌ９７变异无相关。     

慢性乙型肝炎干扰素疗法的适应证选择

目前用于慢性乙型肝炎抗病毒治疗的干扰素主要是ＩＦＮ α，其适应证基本只有一个，即ＨＢＶ复制活跃的活动性慢性乙型肝炎。 慢性乙型肝炎有经典型（ｃｌａｓｓｉｃａｌ）和异型（ａｔｙｐｉｃａｌ）之分。经典型的血清病毒学标志特征是血清Ｈ ＢｓＡ ｇ ＋、ＨＢｅＡｇ＋和抗－ＨＢｃ＋、血清ＨＢＶＤＮＡ十，由野生株ＨＢＶ感染所致，也称ＨＢｅＡｇ阳性慢性乙型肝炎；异型的血清病毒学标志特征是血清ＨＢＳＡｇ＋、抗ＨＢｅ＋和抗ＨＢｃ＋，血清ＨＢＶ ＤＮＡ＋，主要由前Ｃ基因变异株或Ｘ区Ｃ基因启动子变异株ＨＢＶ感染所致，也称抗－…     

The frequency of pre-core gene mutations in chronic hepatitis B infection: a study of Malaysian subjects

A retrospective study was carried out to determine the frequency of the pre-core stop codon mutant virus in a group of chronic hepatitis B carriers: 81 cases were considered [33 hepatits B e antigen (HBe) positive and 48 HBe negative]. All of the HBe positive cases had detectable viral DNA by hybridization analysis; in the case of the HBe negative cases, one third had detectable viral DNA by hybridization analysis and two thirds had HBV DNA detectable by polymerase chain reaction (PCR) amplification. Pre-core stop codon mutant detection was carried out on all specimens using allele-specific oligonucleotide hybridization following PCR amplification of the target sequence. The pre-core mutant was detected in 13/33 (39.4%) of HBe positive cases and in 32/48 (66.7%) of HBe negative cases. Sequence analysis was carried out on 8 of the 16 HBe negative specimens that did not carry the pre-core mutant virus to determine the molecular basis for the HBe minus phenotype in these cases: the 1762/1764 TA paired mutation in the second AT rich region of the core promoter was detected in five cases; a start codon mutation was detected in one case. The predominant mutation resulting in the HBe minus phenotype in our isolates was the 1896A pre-core ("pre-core stop codon") mutation; other mutations responsible for the phenotype included the core promoter paired mutation and pre-core start codon mutation. In view of the high frequency of the pre-core mutant virus, sequence analysis was performed to determine the virus genotype on the basis of the nucleotide sequence of codon 15. The sequences of 21 wild type virus (14 HBe positive and 7 HBe negative cases) were examined: 15 were found to be codon 15 CCT variants (71.4%); the frequency in the HBe positive group was 12/14 (85.7%), while that in the HBe negative group was 3/7 (42.9%). The high frequency of the codon 15 CCT variant in association with the frequent occurrence of the pre-core mutant in our isolates concurs with the results of other studies.     

Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

AIM: To investigate the role of pre-core and basal core promoter(BCP) mutations before and after hepatitis Be antigen(HBe Ag) seroconversion.METHODS: The proportion of pre-core(G1896A) and basal core promoter(A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus(HBV) DNA, hepatitis B surface antigen(HBs Ag), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBe Ag seroconversion(n = 25), in those who were persistently HBe Ag positive(n = 18), and in those who were persistently anti-HBe positive(n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years.RESULTS: Although the pre-core mutant became predominant(24% to 65%, P = 0.022) in the HBe Ag seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBe Ag positive status(HBV DNA: P = 0.003; HBs Ag: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA(P = 0.012) and HBs Ag(P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status.CONCLUSION: There is an opposite association of the pre-core mutation with viral load before and after HBe Ag seroconversion in patients with HBV infection.     

Prevalence of hepatitis B virus precore stop codon mutations in chronically infected children

AIM: To find out whether there is a significant difference in the prevalence of the precore stop codon mutation between HBeAg positive and anti-HBe positive children. METHODS: We investigated a large pediatric population of 155 European children (mean age 10.9 years) with chronic hepatitis B by PCR and direct sequencing. Ninety were HBeAg positive and 65 had seroconversion to anti-HBe. Additionally genotyping was performed. RESULTS: Seventy-four (48%) of the sequenced HBV strains were attributed to genotype D and 81 (52%) to genotype A. In the group of 90 HBeAg positive patients, 2 (2.2%) 1896-G-to-A transitions leading to precore stop codon mutation were found, and in the group of 65 anti-HBe positive children, 5 (7.7%) were identified harbouring HBeAg-minus mutants. The difference was not statistically significant (P=0.13). CONCLUSIONS: HBeAg minus variants as predominant viral HB strains play a minor role in the course of chronic hepatitis B in European children.     

88例乙型肝炎病毒携带者前C区1 896位变异分析

目的 研究乙型肝炎病毒(HBV)携带者前C区1 896位变异情况.方法 采用聚合酶链反应(PCR)和酶联定量检测法分析,检测88例HBV携带者(其中HBeAg阳性50例,HBeAg阴性38例,HBV DNA均阳性)前C区1 896位变异.结果 50例HBeAg阳性患者中前C区1 896位变异率为8%,38例HBeAg阴性患者中,其前C区1 896位变异率为31.5%,88例HBV携带者总变异检出率为18.2%.HBV携带者HBeAg阴性组变异率明显高于HBeAg阳性组(P＜0.05).结论 HBV携带者普遍存在前C区1 896变异.     

乙型肝炎病毒前S2序列异质性的初步研究

目的 通过对乙型肝炎病毒（HBV）前S2基因序列的研究,证实HBV在慢性感染者中以准种状态存在,并对前S2序列异质性进行初步的阐明。方法 以中国株HBV基因序列为依据,设计特异性PCR引物,自51例慢性HBV感染患者血清中扩增HBV前S2基因序列,采用聚丙烯酰胺凝胶电泳技术展示缺失突变,随机选择克隆测序,确定病毒的变异程度。结果 聚丙烯酰胺凝胶电泳结果发现,52.9%（27／51）患者血清中可获得2或3条扩增条带;测序结果发现前S2基因序列存在缺失突变高发区,氨基酸序列分析示缺失突变主要位于前S2编码蛋白的氨基端。结论 HBV前S2序列内有一缺失高变区,并在患者体内表现为多种变异形式;氨基端附近的变异可能影响中和抗体的识别作用。结果提示HBV慢性携带者体内有HBV准种共存。     

Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity

BACKGROUND/AIMS: We aimed to clarify the clinical relevance of hepatitis B virus pre-core mutant detection in patients with chronic hepatitis B using a newly developed assay. METHODS: Viral genotypes and pre-core mutations were studied in relation to viral persistence and liver disease severity using INNO-LiPA methodology. The study group included 151 patients with chronic hepatitis B, 85 positive for HBeAg (group I) and 66 positive for anti-HBe (group II). RESULTS: The prevalence of viral genotypes in group I was: 64% A, 1% B, 15% C, 19% D, 0% E, 0% F and in group II: 39% A, 0% B, 2% C, 56% D, 2% E, 2% F (p<0.001). The prevalence of mutations at pre-core codon 28 (M2) was lower in group I (5%) than in group II (64%) (p<0.001). The prevalence of pre-core promoter mutations was also lower in group I (21%) than in group II (61%) (p<0.001). M2 mutations were more frequently detected in genotype D than in genotype A (p<0.001), while the other mutations were not influenced by viral genotype. Serum HBV DNA levels were significantly lower in group II versus group I (p<0.001), and in patients with any of the pre-core mutations versus wild-type sequence (p<0.01). Although cirrhosis was more frequent in group II (37%) versus group I (22%) and in patients with either one of the pre-core mutation (31%) versus wild-type sequence (25%), there was no statistical difference in liver severity assessed by ALT levels and Knodell score. CONCLUSION: Pre-core mutants, whose molecular pattern is strongly dependent on viral genotypes, are associated with viral persistence in anti-HBe positive patients with ongoing chronic hepatitis B. The availability of this rapid assay should allow a precise monitoring of viral pre-core mutants during the course of chronic hepatitis B.     

Diagnosis and management of pre-core mutant chronic hepatitis B

Chronic hepatitis due to pre-core hepatitis B virus (HBV) mutants presents as hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). HBeAg-negative CHB represents a late phase in the natural course of chronic HBV infection that develops after HBeAg loss and seroconversion to anti-HBe. It is usually associated with pre-core stop codon mutation at nucleotide 1896 (mainly selected in non-A HBV genotypes), but also with other pre-core changes or with mutations in the basic core promoter region (mainly in HBV genotype A). In chronic HBV infections, pre-core mutants can be detected both in patients with HBeAg-negative CHB and in inactive hepatitis B surface antigen (HBsAg) carriers. The diagnosis of HBeAg-negative CHB is based on HBsAg positivity, HBeAg negativity, and mainly on increased alanine aminotransferase (ALT) and serum HBV-DNA levels and exclusion of other causes of liver disease. The differential diagnosis between patients with CHB and inactive HBsAg carriers can be made only by close follow-up of aminotransferase activity and viraemia levels, although the cut-off level of serum HBV DNA has not been definitely determined. IgM anti-HBc levels have also been suggested as an index that increases the diagnostic accuracy for transient hepatitis flares, while liver biopsy confirms the diagnosis and evaluates the severity of the liver disease. Interferon-alpha (IFN-alpha) and lamivudine are the two drugs that have been tried, mainly in the management of HBeAg-negative CHB. A 12-month course of IFN-alpha achieves sustained biochemical remission in about 20% of patients, which has been associated with improvement in the long-term outcome of this subset. A 12-month course of lamivudine is rather ineffective, maintaining remission in less than 15% of patients after cessation of therapy. Long-term lamivudine is associated with progressively increasing rate of virological and subsequent biochemical breakthroughs due to YMDD mutants, with approximately 30% of patients remaining in remission in the third year of therapy. Several other antiviral agents are currently being evaluated in this setting with combined regimens being the most reasonable step for the near future.     

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.     

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.     

Pre-core mutant infections in the Canadian Inuit

BACKGROUND/AIMS: Previous cross-sectional data suggested that chronic hepatitis B viral (HBV) infections in the Canadian Inuit were inactive. The aim of this study was to confirm these findings and document the prevalence of the subsequently described "pre-core mutant" variant of HBV in this population. METHODS: We obtained sera from residents of five remote Canadian Inuit communities. Residents were selected if they were known to be hepatitis B surface antigen (HBsAg) positive or had a history of liver disease. HBV serology, HBV-DNA, and pre-core mutant testing were performed by commercially available assays, polymerase chain reaction (PCR) and direct sequencing of the viral genome, respectively. RESULTS: Sera were obtained from 176/266 (66%) of selected individuals. Thirty-eight (22%) were HBsAg positive and 16 (9.1%) anti-HBs positive. Of HBsAg positive carriers 25/38 (66%) were male as compared to 68/138 (49%) of the remaining individuals (p<0.05). Of 37 HBsAg positive carriers, none were HBeAg positive, 36 (97%) anti-HBe positive and one (3%) HBeAg and anti-HBe negative. Liver enzyme and function tests were normal in all cases. 30/37 (81%) HBsAg positive carriers were HBV-DNA positive and 26/30 (87%) were pre-core mutant positive. CONCLUSION: The majority of HBV infections in community-based Canadian Inuit are inactive and the prevalence of pre-core mutant infections is the highest reported to date.