母婴传播后母子体内乙型肝炎病毒前S/S基因变异研究

目的 通过经母婴传播获得乙型肝炎病毒(HBV)感染的慢性无症状乙型肝炎表面抗原携带者(ASC)母子体内HBV PreS/S基因序列研究,了解来源相同的HBV在不同程度病毒血症情况下PreS/S基因变异的特点。方法 选择15对母孕前为HBV感染、未接种过乙型肝炎疫苗且均未使用过抗HBV药物的ASC母子。应用T-A克隆技术构建重组质粒pGEM-PreS/S、双酶切进行鉴定,每个患者选2个酶切鉴定正确的克隆测序并进行分析。结果 选择15对ASC母子,根据HBV病毒血症高低分为3组,每组5对,A组母子均为高病毒血症,B组子女为高病毒血症、母亲为低病毒血症,C组子女为低病毒血症、母亲为高病毒血症。高病毒血症患者均为乙型肝炎e抗原( ),低病毒血症均为抗-HBe( )。母子HBV亚型相同,各组中有4/5对母子为B/adw2、1/5对母子为C/adrq 亚型。对每组中B/adw2亚型HBV患者的PreS/S基因进行分析显示:高病毒血症组间或低病毒血症组间HBV PreS/S基因变异数目及位点差异均无显著性,变异与年龄无关,低病毒血症患者变异数目及位点明显高于高病毒血症患者。两个低病毒血症组PreS/S基因绝大多数变异位点相同(113个),其中变异热点85个、可引起37个氨基酸变异,这些变异的氨基酸大多位于免疫表位内或(和)其附近。结论 HBV变异可能与感染的时间长短无关;在发生乙型肝     

乙型肝炎病毒基因组nt531位突变特异性聚合酶链反应

目的:为调查国内乙型肝炎病毒(HBV)nt531位变异株的流行情况提供方法学。方法:根据已知的HBV基因组序列并结合国内主要流行adr和adw亚型的特点,合成在nt531位分别为C、G和T的3种不同引物,建立了突变特异性聚合酶链反应(msPCR)方法。结果:利用msPCR对10个乙型肝炎疫苗免疫失败儿童体内扩增的HBV S基因片段进行了初步检测,表明该法可分别特异性扩增nt531为C、G或T的HBV DNA,并可推测nt531为A的HBV DNA。结论:该法是鉴定HBV基因组nt531位变异株的有效方法之一。     

暴露后预防失败的HBeAg阳性母亲的儿童HBsAgα决定簇的分子分析

本文目的是为了调查研究乙型肝炎病毒(HBV)S基因中α决定簇的突变而引起的免疫失败。方法:HBV S基因中α决定簇通过巢式多聚酶链反应的方法在血清中扩增,此血清来自11位无症状HBeAg阳性母亲所生的HBV阳性的婴儿和儿童。所获得的基因序列被翻译成相对应的氨基酸并与HBV adr亚型的氨基酸序列相比较。所有被调查研究的婴儿均在出生后24小时之内接种重组乙型肝炎疫苗并完成所推荐的免疫过程,在限定的时间间隔内完成3至4次免疫接种。结果:在11个被检测的样本中鉴定基因型和亚型的通常的偏离。其中只有两个在a决定簇中存在     

拉米夫定耐药HBV毒株的RT区序列分析

目的分析拉米夫定耐药HBV毒株部分RT区序列，以了解RT区YMDD基序之外HBV的变异情况。方法采用PCR方法对拉米夫定临床耐药患者治疗前及耐药后血清HBV部分RT区序列进行扩增，扩增的核苷酸片段覆盖HBV RT的B区～E区及S基因“a”决定簇，对PCR产物进行直接测序，将HBV RT区核苷酸和推导的氨基酸序列-与治疗前50株HBV野毒株序列和Genebank中77株不同基因型野毒株序列进行比较，同时对HBV进行血清分型。结果60例患者有YMDD变异，其中26例为adw亚型，占43．33％；34例为adr亚型，占56．67％。另外12例患者(16．67％)未发现YMDD变异。16例患者出现11个RT保守区氨基酸变异(不包括550和526位氨基酸变异)；25例患者出现19个HBVRT非保守区氨基酸变异。结论拉米夫定耐药株的氨基酸变异不仅见于RT B区的526和C区550两个位点，B、C区的其他位点、保守区以及RT非保守区也可发生多个变异。     

乙型肝炎

抗乙型肝炎病毒新药特必夫定；拉米夫定治疗慢性乙型肝炎发生HBV YMDD变异的研究；乙型肝炎病毒PreS1基因的原核表达及免疫学性质鉴定；尿酸对HBV表位肽S28-39负载DC诱导免疫效应的影响；慢性乙型肝炎患者血清瘦素与脂代谢的相关性研究-附150例检测分析；     

隐匿性乙型肝炎:免疫组织化学和S基因序列分析

目的　了解不明原因慢性肝病中隐匿性乙型肝炎所占比例 ,隐匿性乙型肝炎的发病机制及临床、病理特点。方法　给予 2 0例不明原因慢性肝病患者肝穿刺病理检查 ,应用免疫组织化学方法检测肝组织内的乙型肝炎病毒表面抗原 (HBsAg)、核心抗原 (HBcAg) ,丙型肝炎病毒NS3、NS4抗原。采用荧光定量PCR方法对血清HBVDNA进行定量 ,用套式PCR方法扩增HBVS基因 ,对PCR产物进行直接测序 ,比较S基因的核苷酸和推导出的氨基酸序列的差异。结果　 5例患者经肝脏病理检查表现为慢性炎症 ,3例在肝组织内HBsAg、HBcAg同时阳性 ,2例仅HBcAg阳性。S基因的氨基酸序列分析显示 ,1例患者S基因的 74位密码子发生终止变异 ,另 1例在HBsAg的“a”决定簇内有 2个氨基酸发生变异 (T13 1N ,M 13 3S) ,其他 3例患者HBsAg的“a”决定簇内未发现变异。 结论　在我国隐匿性乙型肝炎是不明原因肝病的主要原因之一 ,低水平的血清HBVDNA可以引起慢性肝炎。部分隐匿性乙型肝炎HBsAg阴性的原因是S基因变异引起的 ,而有些患者则可能因为血清HBsAg水平过低 ,导致HBsAg检测阴性。     

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。     

乙型肝炎疫苗母婴阻断失败与乙型肝炎病毒S基因变异

目的　探讨母亲感染HBV基因变异在乙型肝炎 (乙肝 )疫苗母婴阻断失败过程中所起的作用。方法　PCR直接测定母婴HBVS基因序列 ,对婴儿存在HBVS基因变异而母亲未发现者 ,进一步通过克隆筛选法对母亲所感染的HBV进行S基因亚群分析。结果　 11例乙肝疫苗母婴阻断失败者中有 3例幼儿存在S基因“a”决定簇的氨基酸变异 ,其中 1例幼儿母亲也存在相同的变异。对另 2例母亲所感染的HBV进行S基因亚群分析发现 ,1例母亲 9个克隆所测序列与其原序列完全一致 ;另 1例母亲 8个克隆中 ,有 6个克隆与其原序列一致 ,另 2个克隆与其幼儿所感染的HBV序列一致。结论　乙肝疫苗母婴阻断失败者感染的HBVS基因变异株可能早已存在于母亲体内 ,通过免疫选择传染给患儿。     

厦门市乙型肝炎病毒亚基因型分布的初步研究

目的探讨厦门地区乙型肝炎患者血清乙型肝炎病毒（HBV）基因型的分布情况。方法应用混合性型特异性引物聚合酶链反应法结合琼脂糖电泳，根据PCR产物片段大小直接判定HBV基因型；应用PCR扩增全S基因序列-DNA测序-生物信息学分析方法确定部分样本的亚基因型，并探讨前前S区编码的流行情况。结果在217例可以通过混合性型特异性引物聚合酶链反应法确定基因型的感染者中，B型83例（38．2％）、C型71例（32．7％），B／C混合型63例（29．0％）。HBeAg阳性患者中感染B基因型占50％，B／C型混合感染占22％；抗-HBe阳性患者中B／C型混合感染36％，B型38％；在慢性乙型肝炎患者中，以B基因型多见（46％），在肝硬化和肝癌患者中，以C（41％和42％）或B／C混合基因型（38％和33％）感染为主；20例患者血清经过DNA测序-生物信息学分析方法确定了亚基因型，其中B2亚型2例，C2亚型18例，其中混合性型特异性引物聚合酶链反应判断为B型的4例经过测序分析为C2亚型。B2亚型不编码前前S区，18例C2亚型均编码前前S区。结论本研究提示厦门地区乙型肝炎患者群的HBV基因型B、C型的感染率大致相同，亚基因型分析提示C2亚基因型占90％，可能是B／C基因型重组的结果。C2亚基因型病毒株均编码前前S区，提示前前S区的编码可能具有亚基因型特异性。     

儿童乙型肝炎病毒感染者血清中病毒基因与e系统的关系及预后

目的 了解儿童乙型肝炎病毒(HBV)感染者血清中，乙型肝炎病毒基因(HBV DNA)与e系统的关系及病情发展趋势。方法采用聚合酶链反应方法(PCR)，酶联免疫吸附检测法(ELISA)。结果73例乙型肝炎病毒携带者(ASC)中e系统阳性68例，HBV DNA阳性61例，29例乙型肝炎患儿中e抗原阳性28例，HBV DNA阳性28例，两组数据都远远高于成人感染组。结论儿童HBV感染中，ASC与肝炎患儿的HBV DNA与e系统阳性率有非常显著的差异。     

Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan.

Mutants of a determinant of hepatitis B surface antigen (HBsAg) identified in vaccinated children pose a potential threat to long-term success of vaccination programs. We examined the mutants of a determinant (residues 110-160) of HBsAg in hepatitis B virus (HBV) DNA-positive children identified during previous serosurveys in Taipei undertaken just before (1984), 5 years after (1989), and 10 years after (1994) universal vaccination began. In HBV DNA-positive children from 3 surveys, the prevalence of a determinant mutants increased from 8 of 103 (7.8%) in 1984 to 10 of 51(19.6%) in 1989 and 9 of 32 (28.1%) in 1994 and was higher in those fully-vaccinated than unvaccinated (12/33 vs. 15/153, P =. 0003). Most amino acid changes of the variants clustered in residues 125-129 and 140-149. In all 27 children with detectable mutants, the mean age of those vaccinated was younger than those unvaccinated (4. 8 +/- 3.8 vs. 7.9 +/- 2.3 yrs, P <.05); and mutations occurred in a region with greatest local hydrophilicity (residues 140-149) more frequently in those vaccinated than in those unvaccinated (10/12 vs. 6/15, P =.0253). More mutated residues and more mutations at neutralizing epitopes, such as N146, C147, T148, and C149, were found in the 1994 survey. Vaccinated children may contract variant infections through vertical or horizontal transmission. Universal vaccination has accelerated an accumulation of HBsAg a determinant mutants with amino acid changes critical for immune escape in vaccinated children who became carriers, suggesting that new vaccination strategies should be considered.     

Molecular characterization of hepatitis B virus surface antigen mutants in Singapore patients with hepatocellular carcinoma and hepatitis B virus carriers negative for HBsAg but positive for anti‐HBs and anti‐HBc

Background and Aims : Mutations on the a‐determinant of hepatitis B virus surface antigen (HBsAg), capable of escaping detection and vaccination, are identified in HBsAg‐positive/anti‐HBs‐positive vaccinated infants. We studied the prevalence of these mutants in HBsAg‐negative/anti‐HBc‐positive chronic HBV carriers and patients with hepatocellular carcinoma (HCC). Methods : DNA sequence coding for the antigenic a‐determinant of HBsAg was amplified from either HCC genomic DNA or serum samples of the selected patients and sequenced. The replicative mutant genomes were reconstituted in vitro and their reactivity to commercial kits measured. Results : Mutations within and/or outside the a‐determinant were identified in patients seronegative for HBsAg. They were then reconstituted in vitro and transiently transfected into HepG2 cells. Culture medium containing secreted HBV viral particles was collected and assayed for their binding to commercial kits. Drastic decrease of reactivity to these kits was seen with most of the identified mutations, including those located outside the a‐determinant. Conclusion : The existence of a more complex antigenic structure of HBsAg is indicated by the decreased reactivity to detection of mutations, some of which are outside the a‐determinant, escape vaccination and may persist in seronegative patients. The high proportion of HBsAg mutants that are integrated in HCC genomes suggests a role of these mutants in hepatocarcinogenesis, possibly leading to mutant HBV‐related HCC. © 2002 Blackwell Publishing Asia Pty Ltd     

Current aspects of hepatitis B surface antigen mutants in Singapore

Mutations occurring on the major antigenic 'a' determinant of hepatitis B surface antigen (HBsAg) have been detected in immunized Singapore children, despite immunoprophylaxis with hepatitis B immune globulin and HBV vaccine. These vaccine-escape HBsAg mutants display a predominance of the Gly₁₄₅-to-Arg₁₄₅ mutation in the antigenic 'a' determinant. Our latest follow-up studies indicate the stability of this as well as other vaccine-escape HBsAg mutants over time. We have also identified HBsAg mutants in immunized children with amino acid substitutions outside the 'a' determinant. Transmission of various vaccine-escape HBsAg mutants have been shown in our epidemiological studies. Detection of HBsAg mutants, carrying amino acid changes at various position of the 'a' determinant in random population, points to their emergence in Singapore before the implementation of the vaccination programme. Of significant interest is the recent identification of vaccine-escape HBsAg mutants in hepatocellular carcinoma.     

检测乙型肝炎病毒"a"决定簇热点突变基因芯片的制备和初步应用

目的为快速、高通量检测HBV"a"决定簇热点突变,制备基因芯片,并进行了初步应用。方法应用HBV基因保守序列设计PCR扩增引物,针对"a"决定簇突变设计简并探针,制备了检测126A、126S、144A、145R、145E、144A 145R和144A 145E突变的基因芯片。通过对质粒参考品或样本进行测定,以评价芯片的特异性、灵敏度、重复性和检测劣势株的能力,并对45例乙型肝炎患者的血清样本同时应用基因芯片和PCR产物直接测序法进行检测比较。结果所制备的基因芯片能特异性检测出质粒参考品,灵敏度为5×103拷贝/μl。同一样本检测10次,芯片内和芯片间变异系数均小于15%。当劣势株占HBV毒株10%以上时,基因芯片即可检出。芯片法测得45例样本中126A、126S-1和126S-2的阳性率(分别为46.67%、35.56%和24.44%)显著高于PCR产物直接测序法(分别为9.00%、4.44%和2.22%;P值分别为0.000、0.000和0.002),克隆测序验证了基因芯片法的特异性。结论基因芯片法可快速、有效地检测HBV特定位点突变株,为HBV突变的大规模筛查提供了方法。     

Licensed recombinant hepatitis B vaccines protect chimpanzees against infection with the prototype surface gene mutant of hepatitis B virus.

The emergence in vaccinated individuals of hepatitis B virus (HBV) mutants with amino acid substitutions within the a determinant of the surface protein has raised the possibility that such variants represent neutralization escape mutants. We previously demonstrated that one such mutant HBV, strain AS, with an arginine substituted for glycine at surface gene codon 145, was infectious and pathogenic in seronegative chimpanzees. In the present study, the protective efficacy of licensed hepatitis B vaccines was evaluated against challenge with this mutant virus. Four chimpanzees were immunized with 1 of 2 licensed recombinant hepatitis B vaccines. Shortly after the chimpanzees developed antibodies to hepatitis B surface antigen (anti-HBs), they were challenged intravenously with mutant HBV strain AS. Two unvaccinated chimpanzees served as positive controls. The 4 vaccinated chimpanzees did not develop evidence of HBV infection or hepatitis during 2 years following virus challenge. In contrast, the 2 unvaccinated chimpanzees developed HBV infection and hepatitis. Serum anti-HBs in the vaccinated chimpanzees reacted not only with wild-type surface antigen, but also with mutant surface antigen by competition enzyme-linked immunosorbent assay (ELISA). Thus, immunization of chimpanzees with licensed recombinant hepatitis B vaccines stimulates anti-HBs that is broadly reactive and affords protection against infection with a surface gene mutant of HBV, suggesting that properly immunized individuals are not at significant risk of infection with this prototype variant strain of HBV.     

Chronologic changes in serum hepatitis B virus DNA,genotypes, surface antigen mutants and reverse transcriptase mutants during 25‐year nationwide immunization in Taiwan

We investigated breakthrough infection and hepatitis B virus (HBV) genetic changes in immunized subjects after 25 years of a universal infant immunization. Specifically, serum HBV DNA, genotypes, surface antigen mutants and nucleoside analog‐resistant (NAr) mutants were assessed in 2853 subjects (<25 years old) surveyed in 2009, and these data were compared with the data from previous serosurveys. A comparison across different age‐stratified groups using the 2009 data revealed a significant increase in the seropositive rate of anti‐HBc (5.51% vs 12.38%, P=.001) and HBV DNA (1.13% vs 3.96%, P=.007) between those 17‐22 and 23‐24 years of age, possibly due to selective infant immunization in 1984‐1986. Well‐characterized NAr mutants, potential NAr mutants and surface “a” determinant mutants were detected in none, 15 (45.5%) and nine (27.3%) of 33 HBV DNA‐positive subjects, respectively. Of 15 immunized, HBV DNA‐positive young adults (18‐24 years), three (20%) carried “a” determinant mutants. Amongst 1176 HBsAg‐negative subjects evaluated for occult HBV infection, those seropositive for anti‐HBc had a higher seropositive rate for HBV DNA (10/110, 9.1% vs 7/1066, 0.66%; P<.001) and “a” determinant mutants (4/110, 3.6% vs 0/1066; P<.001) than those seronegative for anti‐HBc. Overall, the HBsAg‐positive subjects in six serosurveys showed no significant increase in genotype C frequency in the comparison between the vaccinated and unvaccinated cohorts (25/98, 25.5% versus 14/79, 17.7%, P=.188). Over the 25‐year programme, there was no increase in the prevalence of genotype C in HBsAg carriers and no increase in breakthrough HBV infection or surface mutant prevalence beyond adolescence. Nucleic acid amplification should still be considered the primary screening method for occult hepatitis B detection in high‐risk recipients.     

Hepatitis B virus genotypes, phylogeny and occult infection in a region with a high incidence of hepatocellular carcinoma in China

AIM: To determine the genotypes and phylogeny of hepatitis B viruses (HBVs) in asymptomatic HBV carriers, and the prevalence of occult HBV infection in Long An County, Guangxi Zhuang Autonomous Region, an area with a high incidence of hepatocellular carcinoma. METHODS. A nested polymerase chain reaction (nPCR) was used for detection of HBV DNA in serum samples from 36 blood donors with asympmatic HBV infection, and in serum samples from 52 HBsAg negative family members of the children who did not receive hepatitis B vaccination in Long An County. PCR products were sequenced, and the genotype of each HBV sequence was determined by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases using the BLAST programme. Phylogenetic trees were constructed by the quartet maximum likelihood analysis using the TreePuzzle software. RESULTS: Twenty (55.56%) of 36 HBV asymptomatic carriers were positive for HBV DNA. They were all genotype C by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases. The full-length HBV DNA sequence isolated from the sample No. 624 contained 3215 bases. No interesting mutations were found in this isolate. The homology analysis showed that this strain was closer to the Vietnamese HBV genotype C strain, with a homology of 97%, compared its relation to the same genotype of HBV isolated in Shanghai. Six (11.5%) of the 52 HBsAg negative family members were positive for HBV DNA. A point mutation was found in the sample No. 37, resulting in the substitution of amino acid glycine to arginine in the “a” determinant. Other samples with positive HBV DNA did not have any unusual amino acid substitutions in or around the “a” determinant, and were attributed to the wild-type HBV. CONCLUSION: The HBVs isolated from asymptomatic carriers of Long An County were all identified as genotype C, and the prevalence of occult HBV infection in the population of the county is as high as 11.5%. It is suggested that genotype C and persistent occult HBV infection may play an important role in the development of HCC in the county.     

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

INTRODUCTIONIn spite of the progress made in vaccine development, hepatitis B virus (HBV) infection remains a major health care problem worldwide. Upon exposure to HBV, 5%-10% of healthy adults fail to mount a protective antibody response and become chron…     

Seroepidemiology of Hepatitis B virus infection in Saudi children 8 years after a mass hepatitis B vaccination programme

OBJECTIVES: On October 1 1989, a programme was begun in Saudi Arabia in which the HBV vaccine was added as the 'seventh' primary immunogen of the Extended Programme of Immunization (EPI). In 1990, another programme was launched by the Ministry of Health to vaccinate all school children. Eight years after this mass vaccination programme, the efficacy of HBV vaccine was evaluated in a community-based study. METHODS: A community-based study was carried out in Saudi children in urban and rural areas, covering all the regions of Saudi Arabia. After informed consent, blood samples were obtained and tested for HBV markers. RESULTS: Among 4791 vaccinated Saudi children aged 1-12 years, only 15 were found to be HbsAg-positive (0.31%). HbsAg-positivity was 0.16% in children vaccinated at birth compared with 0.7% in those vaccinated at school entry. The overall HbsAg carrier rate dropped from 6.7% in 1989 to 0.3% in 1997 (P<0.00001). Similarly, there was a significant reduction in the prevalence of anti-HBc from 4.2% in 1989 to 0.46% in 1997 (P<0.00001). The overall seroconversion rate to HB vaccine among 4087 Saudi children up to 12 years of age was about 77%. Seroconversion rate in those vaccinated at birth was 77% compared with 71% in those vaccinated at school entry. After 8 years of receiving the third vaccine dose, close to 65% of the children had an anti-HBs titre of more than 10 IU/l compared with about 28% who had an anti-HBs titre of more than 100 IU/l after the same period. CONCLUSION: The result of this study demonstrates the tremendous impact of the mass HB vaccination programme on the seroepidemiology of HBV infection in Saudi Arabia. The ultimate goal of preventing HBV-related chronic liver disease and hepatocellular carcinoma in Saudi Arabia is foreseeable in the near future.     

Hepatitis B surface antigen variants in vaccinees, blood donors and an interferon-treated patient

Variants of hepatitis B virus (HBV), with amino acid substitutions in the major antigenic 'a' determinant of hepatitis B surface antigen (HBsAg), have been described mainly in vaccinated children. In the present study in addition to vaccinated children, we have investigated Chinese blood donors positive for anti-HBc alone, and a patient with continuing liver disease after interferon-induced seroconversion to anti-HBs. Variants were detected in two of four children with break-through infections. One child had a double mutation (P142S and G145R) and the other a G145A substitution. Three of seven anti-HBc positive Chinese blood donors had a T131I substitution, whilst the interferon-treated patient had a treble amino acid substitution (P142S, G145R and N146D). The present results indicate that HBsAg variants may exist in individuals other than vaccinated children.