HBV基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系

 目的：探讨乙型肝炎病毒（HBV）基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系。
方法：选择血清HBsAg阳性且HBV DNA定量≥1×1010 IU/L的孕妇及其新生儿,以新生儿是否发生免疫预防失败将孕妇分为免疫预防失败组（15例）和免疫预防成功组(45例),采用PCR扩增直接测序法对2组孕妇血中HBV进行基因分型和前S/S基因突变的检测,对比分析2组的不同。
结果：（1）基因型：2组孕妇血中HBV的基因型均为B和C型,均以B型为主,2组基因型分布相比,差异无统计学意义（P>0.05）。（2）突变率：HBV 前S/S基因2个片段的突变率在免疫预防失败组与免疫预防成功组间相比,差异均无统计学意义（P>0.05）;而B基因型与C基因型间相比,差异均有统计学意义（P<0.05）;但在同一基因型内,2个片段的突变率在免疫预防失败组与免疫预防成功组相比,差异均无统计学意义（P>0.05）。对HBV 前S/S基因2个片段的遗传树分析也显示不同基因型的基因突变率不同,但同一基因型的突变率在2组间差异无统计学意义（P>0.05）。（3）突变热点：在免疫失败组的4个病例中发现4个突变热点：529G-A、530A-G、826A-G1和166het-dupC各1例;在免疫成功组的6个病例中发现3个突变热点：A530T 1例,A530G 2例, T531C 3例。
结论：（1）不同HBV基因型间前S/S基因突变率不同。（2）HBV前S/S基因突变普遍存在,但并不是每个突变都与母婴传播免疫失败有关,仅分析基因突变率对研究免疫失败并无意义,寻找与免疫失败有关的特异性突变位点可能更有意义。     

乙肝疫苗母-婴阻断失败者病毒全基因变异分析

目的 探讨乙肝疫苗母-婴阻断失败者中乙肝病毒(HBV)全基因变异状况.方法 用PCR方法对启东地区11对乙肝疫苗免疫失败儿童-母亲配对血清及6例疫苗接种成功儿童的"大三阳"母亲血清扩增HBV全长基因,克隆测序后以Clustal X软件进行序列比对.病毒载量采用实时荧光定量PCR方法测定.结果 疫苗阻断失败和成功儿童的母亲HBV平均滴度分别为(1.2×107±3.1×1010)copies/ml和(1.6×107±8.8×1010)copies/ml,两组之间差异无统计学意义(P>0.05).在11例HBV DNA阳性的儿童中,4例(36.4%)有HBsAg"a"决定簇的氨基酸改变,表现为T125A、I126T、Q129H、M133V、D144V和G145A等6种不同突变形式,但母亲中HBsAg"a"决定簇均为野生型.疫苗接种失败的儿童中HBV preS1、preS2、S、X、preC/C和P基因的平均突变率分别为0.38%、0.22%、0.27%、0.17%、0.11%和0.11%.preS1区nt2999-3157、S区nt529-677、C区nt1955-2016和P区nt923-1001、nt2489-2602为病毒基因组中最易发生突变的区域.结论 经主动和被动免疫后,儿童体内HBV突变可发生于所有开放阅读框架内.除s基因外,preS和P基因的突变可能也与免疫逃逸有关.     

乙型肝炎病毒e系统血清转换中HBV前C、BCP基因检测及临床意义

目的：探讨乙型肝炎患者HBeAg和HBeAb双阳性状态下HBV前C区基因及BCP区基因变异情况,探讨前C区A1896及BCP区T1762与A1764变异与HBe转换的关系。方法：采用时间分辨荧光免疫分析方法定量检测乙肝“二对半”,对HBeAg／HBeAb双阳性标本采用基于膜显色的DNA芯片检测HBV nt1896、nt1762、nt1764位基因。结果：35例HBeAg、HBeAb、HBVDNA阳性的患者均存在nt1762和nt1764的突变,有14例患者出现了nt1896的突变。结论：在乙型肝炎HBe转换过程中均伴有BCP区T1762和A1764的突变,部分存在A1896位点的突变,T1762和A1764的突变早于A1896的突变,A1896的突变主要在e抗体产生过程中或产生以后。     

母婴传播感染HBV儿童及其母亲体内HBV preS/S基因准种序列以及变异特点研究

目的：对经母婴传播获得乙型肝炎病毒（HBV）感染子女及其母亲无症状携带者（AsC)体内HBV preS/S基因进行研究，了解来源相同的HBV毒株在不同程度病毒血症情况下preS/S有无准种存在及其特点。方法：应用T－A克隆技术构建重组质粒pGEM-preS/S、双酶切进行鉴定，每个病人选6个酶切鉴定正确的克隆测序并进行分析。结果：3对AsC母子呈不同程度病毒血症，HBV均为基因型B／血清型adw2。36个克隆序列建立的进化树显示3对母子的preS/S为同一分支HBV preS/S进化而来，每例病人6个序列呈准种分布。低病毒血症HBV preS/S的核苷酸变异率明显高于高病毒血症，2个低病毒血症病人的变异位点绝大多数相同，其中错义变异多位于T－细胞表位和B－细胞表位内或／和附近。变异率和变异位点均与年龄无关。结论：经母婴传播而获得HBV感染儿童及其母亲无症状携带者，无论病毒血症高低，体内HBV preS/S序列均呈准种分布。来源相同的HBV preS/S在不同程度病毒血症病人体内差异较大，而与年龄无关。高病毒血症病人变异率低，而在低病毒血症变异率较高，但变异是有规律的，可能与免疫逃逸有关。     

乙型肝炎病毒C区基因多态性的检测分析

目的 探讨乙型肝炎患者和健康携带者乙型肝炎病毒（hepatitis B virus,HBV）C区基因变异规律及其临床意义。方法采用实时荧光定量PCR检测乙肝患者和HBV健康携带者外周血HBV DNA载量;PCR结合DNA测序对HBV C区基因进行多态性检测,并应用DNAstar软件进行比对和分析。结果①与标准株相比,所有标本C区基因序列均出现变异。在≥2例标本中出现的突变31处,包括PreC基因3处,C基因28处;其中9处为错义突变,1处为终止突变,其余21处均为同义突变。nt 1827 c→a和nt 2221 c→t出现在所有标本,有6处同义突变出现于较多标本中。4例乙肝患者标本在nt1896位发生g→a突变,另有4例乙肝患者在HBcAgCTL识别表位84～101处发生2处突变即S87G和I97F或197L。②DNA扩增及测序的成功与否与HBV的DNA拷贝数密切相关,本次研究DNA扩增及测序成功的标本其DNA拷贝数多大于40 193/ml,DNA拷贝数较低的标本不易获得成功。结论HBV基因组极易发生突变,C区基因有2个位点的突变见于所有扩增成功标本,为此研究标本所特有,提示这些突变热点可能具有地域特异性。C区基因变异可引起HBeAg和HBcAg的结构及功能改变,进而导致病毒逃逸机体对病毒的免疫清除作用,或影响HBeAg的检出。     

HBV前C基因突变及分型与HBV母婴传播关系的研究

目的探讨HBV(乙型肝炎病毒)前C基因突变、血清型及基因型分型与HBV母婴传播的关系。方法100份HBsAg阳性孕产妇及其新生儿的外周血,发生母婴传播血清为病例组,其他为对照组。ELISA法检测乙肝两对半,根据S基因判断HBV血清型和基因型。对HBV前C基因进行扩增及序列分析。结果99份HBV血清型全为adw型;39例发生母婴传播HBV基因C型25例;B型13例;E型1例。60例未发生母婴传播,HBV基因B型50例;C型10例。两组HBV基因分型结果差异有统计学意义(P0.05)。39例发生母婴传播中27例发生了点突变,12例无任何突变,突变发生率为69%。结论HBV母亲C基因型可能更易发生母婴传播。HBV母婴传播中大多存在HBV前C基因变异并导致氨基酸取代,其中主要涉及丝氨酸、苏氨酸、酪氨酸等的磷酸化位点,这些重要位点氨基酸的取代可能在HBV母婴传播的发病中起作用。     

乙肝疫苗联合乙肝免疫球蛋白对阻断母婴垂直传播乙型肝炎病毒的临床分析

目的探讨孕妇产前用乙肝免疫球蛋白（HBIG）与乙型肝炎疫苗联合免疫阻断母婴传播的效果。方法将504例HBsAg（＋）孕妇分为A（预防组）,B（对照组）两组。A组：246名HBsAg阳性孕妇孕晚期每月分别注射基因重组型乙肝疫苗10μg、HBIG200IU（200IU/ml）,新生儿出生后采股静脉血,同时在出生后24h内注射HBIG200IU,然后在0、1、6月龄接种基因重组型乙肝疫苗,每次10μg。B组：258例产前未注射HBIG和基因重组型乙肝疫苗的HBsAg阳性孕妇,其所生新生儿在0、1、6（30μg、30μg、30μg）月龄只用基因重组型乙肝疫苗免疫。A、B两组婴儿都分别在0、3、6、9、12、24月龄静脉采血,用酶联免疫吸附试验（ELISA）检测HBV标志物,同时随访。结果A组的宫内感染率为3.25%,B组为4.16%,差异无统计学意义（χ^2=1.43,P〉0.05）。A组没有发生慢性HBV感染的婴儿,而B组中有7例婴儿发生慢性HBV感染,B组婴儿发生慢性HBV的感染率显著高于A组（χ^2=4.41,P〈0.05）。结论产前用HBIG和新生儿HBIG联合免疫可降低慢性HBV感染率,阻断宫内感染的慢性化,提高产程感染的阻断效果。     

2623例新生儿听力筛查和GJB2基因检测结果分析

目的探讨常规听力筛查的同时进行GJB2基因检测的可行性。方法采取知情同意、自愿选择的原则,对2623例新生儿出生后2-3天采集足跟血,利用飞行时间质谱技术对GJB2耳聋基因进行检测,包括5个热点突变位点235delC、299-300delAT、35delG、l76-191dell6、167delT突变,并采用GSI耳声发射仪（DPOAE）进行新生儿听力筛查。结果2623例新生儿中GJB2基因检测阳性率3．20％,其中听力初筛通过婴儿中基因阳性率2．50％,听力初筛未通过婴儿中基因阳性率5．21％,听力初筛未通过GJB2耳聋基因阳性率高于听力初筛通过婴儿,差异性显著（P〈0．01）。l例GJB2235del纯合突变经ABR检查确诊为双耳中重度听力损失。结论 将JB2基因筛查和常规听力筛查联合对早期发现新生儿语前听力损失或迟发的听力损失,及婚育指导具有重要意义。     

山东省人群乙型肝炎病毒＂a＂抗原决定簇突变分析

目的 了解目前山东省社区人群乙型肝炎病毒（HBV）＂a＂抗原决定簇突变率和突变形式,探讨乙肝疫苗（HepB）接种对＂a＂抗原决定簇突变的影响.方法 在全省1～59岁社区人群中通过多阶段随机抽样确定调查对象,通过询问（15岁以上）或查阅接种记录（14岁以下）了解调查对象HepB免疫史;采集血标本,酶联免疫吸附方法 检测血清乙肝表面抗原（HBsAg）,阳性者提取血清DNA,采用巢式PCR方法 扩增HBV S基因,测序后与标准序列进行比较.结果 共对7601人进行调查和血标本采集,得到HBsAg阳性标本239份（3.14%）,可用于HBV DNA提取206份,扩增HBV S基因并成功测序102份.15份血清标本检测到13种HBV＂a＂抗原决定簇突变,突变率为14.70%（15/102）.新生儿普种HepB前、后出生调查对象间,以及有、无HepB免疫史调查对象间＂a＂抗原决定簇突变率差异均无统计学意义.结论 目前山东社区人群中＂a＂抗原决定簇突变率较低且突变位点比较分散;未发现HepB接种对人群＂a＂抗原决定簇突变产生影响.     

HBV rtA181位点突变的病毒学特点及临床耐药研究

目的 回顾性研究HBV rtA181位点突变模式与临床核苷（酸）类似物耐药的关系及挽救治疗效果.方法 核苷（酸）类似物治疗过程中发现HBV rtA181位点突变的45例慢性乙型肝炎或肝硬化患者,检测其血清HBV DNA水平,PCR产物直接测序检测HBV变异模式及基因型,收集突变前用药史及突变后的挽救治疗方案及疗效相关资料.结果 HBV rtA181位点共发现三种突变形式,其突变率分别为rtA181T 66.67％ （30/45）,rtA181V 31.11％ （14/45）,rtA181S 2.22％ （1/45）,rtA181T多见于HBV C基因型患者（P＜0.05）;涉及rtA181的HBV突变模式共发现13种,多位点联合突变率为57.78％ （26/45）,其中rtA181与rt236位点联合突变率为40.00％ （18/45）,与rt204位点联合突变率为22.2％ （10/45）,多位点突变患者较单位点突变患者有病毒载量分布更高的趋势,但差异无统计学意义（P＞0.05）,多位点突变与基因型及突变形式无关（P＞0.05）;发生rtA181突变的患者97.78％ （44/45）有拉米夫定（LAM）和（或）阿德福韦酯（ADV）治疗史,另外1例有替比夫定（LdT）治疗史.其后的挽救治疗采用加用或换用恩替卡韦（ETV）、LAM联合ADV以及LdT联合ADV均能在一个月内将HBV DNA降低2个lg值,继续随访效果最显著的方法为加用或换用ETV.结论 HBVrtA181位点突变模式多样,与LAM、ADV或LdT长期单药或序贯治疗有关,加用或换用ETV可取得显著的挽救治疗效果.     

Management of mother-to-child transmission of hepatitis B virus: Propositions and challenges

Chronic hepatitis B virus (HBV) infection due to mother-to-child transmission (MTCT) during perinatal period remains an important global health problem. Despite standard passive–active immunoprophylaxis with hepatitis B immunoglobulin (HBIG) and hepatitis B vaccine in neonates, up to 9% of newborns still acquire HBV infection, especially these from hepatitis B e antigen (HBeAg) positive mothers. Management of HBV infection in pregnancy still need to draw careful attention because of some controversial aspects, including the failure of passive-active immunoprophylaxis in a fraction of newborns, the effect and necessity of periodical hepatitis B immunoglobulin (HBIG) injection to the mothers, the safety of antiviral prophylaxis with nucleoside/nucleotide analogs, the benefit of different delivery ways, and the safety of breastfeeding. In this review, we highlight these unsettled issues of preventive strategies in perinatal period, and we further aim to provide an optimal approach to the management of preventing MTCT of HBV infection.     

Reduced doses of hepatitis B immune globulin in the prevention of perinatal transmission of hepatitis B

From October 1982 to May 1983, newborn infants of 79 hepatitis B surface antigen (HBsAg)-positive women were enrolled in a study of the efficacy of hepatitis B immune globulin (HBIG) in the prophylaxis of perinatal transmission of hepatitis B virus (HBV) infection. HBIG 0.5 ml or 0.25 ml was given to the newborn within 15 minutes of birth and at 3 and 6 months. The mother-infant pairs were followed-up every 3 months for at least 9 months. Similar observations of untreated infants were used for comparison. Among infants of hepatitis B e antigen (HBeAg)-positive carrier mothers, the HBsAg carrier rates at 3 months were similar in the 0.5-ml and 0.25-ml HBIG dose groups. At 12 months the difference--17.7% of 17, 40% of 15--did not reach statistical significance, but the differences between these rates and that of the untreated control-85.7% of 35--did. Among infants of HBeAg-negative carrier mothers, HBV infection rates in both dose groups were similar to those of untreated infants. In the treated groups at 12 months about 45% of infants of HBeAg-positive mothers and 90% of infants of HBeAg-negative mothers were still negative for HBsAg and anti-HBs. Vaccination to induce active antibody is necessary to prevent postnatal infection and chronic carriage of HBV. To reduce the cost of combined passive and active hepatitis B immunoprophylaxis in children born to HBeAg-positive carrier mothers, 0.25 ml of HBIG could be used instead of the usually recommended 0.5 ml.     

HBV viremia in newborns of HBsAg(+) predominantly Caucasian HBeAg(−) mothers

BackgroundHepatitis B virus infection is an important public health problem worldwide and eliminating mother-to-infant transmission is important to decrease the prevalence of chronic HBV-infection. Although, immunoprophylaxis given at birth largely prevents mother-to-infant transmission, perinatal HBV viremia has been reported in HBsAg(?) newborns born mainly to HBeAg(+) women in endemic areas.ObjectivesTo examine the incidence of perinatal HBV viremia in newborns of HBsAg(+) predominantly HBeAg(?) mothers.Study designPeripheral blood was obtained at birth from 109 HBsAg(+) mothers and their newborns before the administration of active–passive immunoprophylaxis. Infants were prospectively followed and appropriately vaccinated.ResultsAlthough most (92.7%) of the HBsAg(+) mothers were HBeAg(?), 73.4% had detectable HBV viremia. Neonatal viremia was detected in 3/8 (37.5%) and 24/101 (23.8%) newborns of HBeAg(+) and HBeAg(?) mothers, respectively (p = 0.386). However, HBV–DNA levels were significantly higher in newborns of HBeAg(+) mothers (p = 0.025). No child developed chronic HBV infection, but one child had evidence of subclinical hepatitis.ConclusionsAlthough the clinical significance of low viremia levels in almost one in four newborns of HBsAg(+) mothers in a low endemicity area is unclear, it may enhance our understanding of HBV mother-to-infant transmission.     

Prophylactic vaccination against hepatitis B: achievements,challenges and perspectives

Large-scale vaccination against hepatitis B virus (HBV) infection started in 1984 with first-generation vaccines made from plasma of chronic carriers containing HBV surface antigen (HBsAg). Thereafter, it was replaced in most countries by second-generation vaccines manufactured in yeast cells transformed with gene S encoding HBsAg. Both generations of vaccines have been applied for universal neonate and early childhood vaccination worldwide and have led to a 70–90 % decrease in chronic HBV carrier rates. However, 10–30 % of newborns from HBsAg/HBeAg-positive mothers cannot be protected by passive/active vaccination alone and become chronic HBV carriers themselves. Asymptomatic occult HBV infections are frequent even in those who have protective levels of anti-HBs. Suboptimal protection may be due to heterologous HBsAg subtypes that are present in 99 % of HBV carriers worldwide. Second-generation vaccines contain partially misfolded HBsAg and lack preS1 antigen that carries the major HBV attachment site and neutralizing epitopes. Third-generation vaccines produced in mammalian cells contain correctly folded HBsAg and neutralizing epitopes of the preS antigens, induce more rapid protection, overcome nonresponse to second-generation vaccines and, most importantly, may provide better protection for newborns of HBV-positive mothers. PreS/S vaccines expressed in mammalian cells are more expensive to manufacture, but introduction of more potent HBV vaccines should be considered in regions with a high rate of vertical transmission pending assessment of health economics and healthcare priorities. With optimal vaccines and vaccination coverage, eradication of HBV would be possible.     

Perinatal transmission of hepatitis B virus in high-incidence countries

Hepatitis B is a serious public health problem throughout the world. Hepatitis B virus (HBV) induces acute hepatitis with a case-fatality rate of about 1%. Even more important, 5-10% of patients infected with HBV become chronic carries and about 25% of these will die due to cirrhosis and hepatocellular carcinoma. The reservoir of HBV chronic carriers in the world is estimated at more than 200 million people and 80% of them reside in Asia and the western Pacific. In high-incidence areas, such as south-east Asia, perinatal transmission of HBV from carrier mothers to newborns appears to be the most important factor for the high prevalence of HBV infection and 70-90% of infants born to HBsAg/HBeAg-positive mothers become chronic carriers. Three possibilities of transmission of HBV from carrier mothers to newborns are suggested: (a) transplacental transmission in utero - it was estimated that such transmission occurred in 5-15% of newborns; (b) transmission during delivery, which is considered the main mode of perinatal transmission; (c) postnatal transmission from mother to newborn, which is not common. HBeAg is the main maternal factor in determining whether infection of newborns will occur; the expression of this antigen seems to be determined genetically. Recently it has shown that immunoprophylaxis is highly effective in preventing the development of the carrier state in infants born to HBsAg/HBeAg-positive mothers. Only 5-10% of high-risk infants are not protected by vaccination. If it becomes possible to immunize the entire world population including all babies born to carrier mothers at birth, and if our knowledge of the mechanisms of perinatal transmission of HBV is accurate, the carriers and acute cases of HB ought to disappear in two to three generations.     

Acute self-limiting hepatitis B after immunoprophylaxis failure in an infant.

The occurrence of acute hepatitis after failure of immunoprophylaxis in cases of mother-to-infant transmission of hepatitis B virus (HBV) is uncommon. Because immunoprophylaxis failure is caused by the emergence of an "a" determinant escape mutant, the infants usually become HBV carriers. To evaluate whether mutations in the S gene coding for the surface protein that contains the "a" determinant are associated with acute hepatitis after immunoprophylaxis failure, HBV DNA of an infant in with acute hepatitis developed with seroconversion to anti-HBs antibodies at 12 months of age despite administration of anti-hepatitis B immunoglobulin and hepatitis B vaccine was analyzed. The S gene from HBV DNA isolated from the serum of the infant at 12, 19, and 27 months of age was cloned and sequenced. Mutations affecting amino acid residues in the first loop within the "a" determinant (codons 124-147) were found at 12 months of age. Moreover, a novel deletion mutant, with a 1-bp deletion at nucleotide 449 of the S gene, was found at 19 and 27 months of age. This deletion resulted in a frame shift and it introduced a stop codon (TAG) at codon 176. Because the open reading frame of the S gene is completely overlapped by the polymerase gene, mutations in the S gene may affect the polymerase gene. Based on this case, this study suggests that the observed frame-shift mutation in the S gene might affect the polymerase protein and induce prompt suppression of viral replication.     

Does Hepatitis B Virus Prenatal Transmission Result in Postnatal Immunoprophylaxis Failure?

The objective of this work was to evaluate whether postnatal hepatitis B immunization failure in children is caused by prenatal infections. A prospective study was conducted from October 2006 to September 2008. Fetal samples from HBsAg-positive mothers were retrieved by either amniocentesis or cordocentesis (percutaneous umbilical blood sampling [PUBS]). Hepatitis B virus (HBV) serologic markers (HBVM) and quantitative HBV DNA assays were performed to assess prenatal infection. All neonates were given combined HBV immunoprophylaxis after delivery. The newborns were followed up with HBV serologic testing at 1 year old. For the 252 pregnant women recruited, 16 fetuses were found to be HBV DNA positive, with all HBV DNA levels under 10(4) copies/ml. HBsAg and HBV DNA detected in the uterus were uncommon and were expressed at low levels. In contract to the case with prenatal statuses, neonatal serologies were more similar to their mothers'. The response rate of vaccination was 95%. Six children for whom immunoprophylaxis failed were born to HBeAg-positive mothers with high HBV DNA levels (>10(8) copies/ml), but only one of them was found to be positive for intrauterine HBV DNA (8.5 × 10(2) copies/ml). The presence of intrauterine hepatitis B antigen and DNA does not indicate postnatal HBV infection and vaccination failure.     

Influence of HBV gene heterogeneity on the failure of immunization with HBV vaccines in eastern China

The influence of hepatitis B virus (HBV) gene heterogeneity on the failure of HBV vaccination in eastern China remains unknown. Here, we assigned 78 hepatitis B surface antigen (HBsAg)-carrier mothers to two groups: 41 mothers from whom transmission of HBV to their children was successfully prevented and 37 mothers whose children were HBsAg positive 1 year after HBV vaccination. The DNA loads in mothers of the failure group (4.17E + 07 copies/ml) were significantly higher than those in the success group (8.40E + 06 copies/ml). However, no difference was found in the S gene mutation rate and genotypes between the groups. Interestingly, Thr123Ala and Gly145Arg were observed only in failure-group mothers, whereas Thr126Asn, Thr126Ser, Thr143Asn, Asp144Gly, and Asp144Ala were seen in the success group. Thus, high viral load is an important risk factor for HBV vaccination failure, which is correlated with the positions of mutations in the S gene, but not with mutant frequencies or genotypes. She-Lan Liu and Ying Dong have contributed equally to this work.