重型乙型肝炎患者肝组织和血清中乙型肝炎病毒前C区准种的比较

乙型肝炎病毒存在准种现象。为研究重型乙型肝炎患者血清和肝组织中乙型肝炎病毒（HBV）DNA前C区准种组成特点，本研究从3例重型乙型肝炎患者的血清和肝组织中提取HBV DNA，经巢式聚合酶链反应（PCR）扩增出HBV前C区，产物克隆后经单链构象多态性／异源双链分析（SSCP／HDA）筛选不同的克隆并测序，比较血清和肝组织中HBV前C区准种组成的异同。     

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。     

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。     

乙肝病毒C基因启动子变异与乙肝病情的关系

研究乙型肝炎病毒（HBV)C基因启动子（CP）变异与无症状慢性HBV携带者（AsC)肝炎发作及与慢性乙肝病情的关系。．通过PCR及其产物直接测序，检测4例AsC、27例慢性乙肝和3例慢性重型乙肝患者血清的HBV CP序列，并定量测定血清的HBV DNA。（1）CP主要变异为nt1726-1730聚集（1726A→C、1727A →T、1730C→G）和nt1762 1764双变异（1762A→T和1764G→A）。（2）CP聚集变与AsC首次肝炎 急性发作有关。11例中，8例出现CP聚集变异。且1例在AsC状态时无CP变异，肝炎发作时出现CP聚集变异。（3）CP聚集变异合并CP双突变的乙肝患者,表现为重型肝炎或迅速进展为肝硬化，HBV DNA高水平；HBeAg／抗HBe转移。CP聚集变异与AsC肝炎急性发作有关；CP聚集变异 与CP双变异同时存在，使慢性乙肝病情加重。     

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.     

重型乙型肝炎e抗原阴性患者前C变异株及其体外翻译

目的：探讨HBeAg阴性的慢性重型乙型肝炎患者HBV前C区变异及前C区T1862突变体对e抗原合成和分泌影响,方法：选取9例重型乙型重病毒性肝炎患者,用PCR方法扩增血清中HBV前C／C基因区,并克隆后测序和序列分析,构建HBV前C区T1862位点突变体表达质粒pGEMT,经体外翻译比较野毒株和变异株的表达产硪,结果：HBV前C区有3个位点异使氨其酸序列改变,A1896,A1899和T1862,A1899并不单独出现,前C区T1862突变并不阻止HBVe前体外合成,同时,有2例并未检测到前C区的变异,结论：部分重型肝炎HBeAg阳性原因降了T1896变异外,还存在T1862变异,前C1862突变对HBVe抗原前体蛋白合成并无影响,可能e抗原前在分泌过程中受阻。     

乙型肝炎病毒X区核苷酸序列变异的检测

目的：了解乙型肝炎患者血清中乙型肝炎病毒（HBV）DNA X区核苷酸序列的变异情况。方法：采用聚合酶链反应（PCR）扩增24例乙型肝炎患者血清HBV DNA X区产物，并直接测序进行分析。结果：24例患者血清中HBV DNA X区都有程度不等的点突变（2－15），11例同时具有nt1762(A→T),nt1764(G→A）发生变异，8例患者同时在nt1636-nt1741几处位点发生变异。结论：HBV DNA X区核苷酸位于nt1762,nt1764双位变异与HBeAg阴性表型有关。     

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。     

无血清免疫学标志的乙型肝炎病毒株分子生物学某些特性的初步探讨

目的：了解无血清免疫学标志的HBV株分子生物学的改变。方法：收集104例不明原因性肝炎患者的血清，套式PCR扩增HBVS、X、C基因，HBVS、X区扩增产物测序和变异位点分析。结果：检测出隐匿性HBV感染13例。S基因区未发现有义突变，X基因区及重叠核心启动子区、增强子Ⅱ区发现多位点变异。结论：X基因区及重叠核心启动子区、增强子Ⅱ区多位点变异可能是无血清免疫学标志HBV株的分子基础。为深人研究隐匿性HBV感染的临床检测及防治奠定基础。     

小分子乙型肝炎病毒基因组缺失突变体结构分析

目的 了解小分子HBV基因组缺失突变体的基因结构及特点.方法 采用一步法PCR从慢性乙型肝炎患者血清中扩增全基因组HBV DNA,回收并克隆＜1 kh的小分子HBV DNA,测序并以BioEdit及VectorNTI 6.0软件分析基因结构特点.结果 获得基因长度介于174～986 bp之间的64种、共124个小分子HBV缺失突变体DNA,按基因结构特点分为3类,即3种GT-AG剪接变异体、29种"常规"缺失突变体及32种基因组内部含polyA的缺失突变体.所有小分子基因组缺失突变体在HBV各编码区及基因调控区均存在不同程度缺失,其中66%(42/64种)保留与HBV复制(包装)相关的所有顺式调控序列,48%(31/64种)保留X编码区.结论 乙型肝炎患者血清中普遍存在小分子HBV基因组缺失突变体,深入了解这些变异体的结构和功能,有助于进一步探索HBV的致病机制.     

阿德福韦耐药乙型肝炎病毒毒株生物学特性的研究

目的 体外研究阿德福韦(ADV)耐药相关变异对于HBsAg产生及HBV复制等生物学特性的影响.方法 收集12例在ADV治疗过程中出现病毒学突破的慢性乙型肝炎患者血清,对其HBV反转录(RT)区进行PCR扩增和测序分析.对其中4例患者的HBV进行全基因扩增、测序、序列分析及克隆.将优势株的HBV全基因插入PHY106载体,构建成1.1拷贝HBV基因组表达载体,进而转染Huh7细胞,ELISA法检测细胞培养上清液中HBsAg及HBeAg水平,了解不同ADV耐药相关变异类型对HBsAg分泌的影响.抽提转染细胞内病毒核心颗粒HBV DNA,实时荧光定量PCR方法 检测HBV DNA水平.将rtA181T/sW172*变异株与rtA181非变异株质粒按不同比例混合,共转染Huh7细胞,检测上清液中HBsAg及细胞内病毒核心颗粒HBV DNA水平.结果在12例出现病毒学突破的患者中,10例出现ADV耐药相关位点变异,以rtA181变异为主,其中5例有rtA181T变异,4例为rtA181T/S+rtN236T变异.将含rtA181T/sW172*变异的质粒转染细胞后,上清液中不能检测到HBsAg,而含其他变异类型的质粒转染细胞后,上清液中HBsAg和细胞内病毒核心颗粒HBV DNA水平与非变异株相似;含不同变异的HBV临床分离株转染后的细胞内核心病毒颗粒HBV DNA水平未见明显差异.将rtA181T/sw172*变异株及rtA181非变异株的质粒共转染后.随着rtA181非变异株比例的增加,上清液中HBsAg水平也逐渐增高,但细胞内核心病毒颗粒HBV DNA水平无明显差异.结论 rtA181变异是ADV耐药相关性变异的主要类型,rtA181T变异较多见.rtA181T/sW172*变异株可引起HBsAg分泌障碍,但rtA181非变异株可纠正rtA181T/sW172*变异株的HBsAg分泌障碍.不同类型的ADV耐药相关变异对HBV的复制能力无显著影响.     

突变乙型肝炎病毒核心蛋白的克隆及表达

目的 克隆并表达发生长片段缺失的HBV核心蛋白(HBV-C)基因,并对其进行DNA序列、蛋白质结构和抗原性分析.方法 采用PCR方法从1株野生型HBV基因组中扩增得到发生长片段缺失的HBV-C基因,克隆至pUCm-T质粒,进行测序、同源性比较和蛋白质结构分析;再将基因编码区克隆至原核表达载体pET-28a,构建含HBV-C基因的重组表达质粒,转化至大肠埃希菌BL21中进行诱导表达并检测其抗原性.结果 PCR扩增出的HBV-C基因长度经序列分析表明,其核苷酸序列缺失了220 bp至317 bp之间的98个碱基,造成从第74个氨基酸起发生移码突变并失去了抗原性.结论 成功克隆和表达了发生长片段缺失的HBV-C基因.     

Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood

AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.     

HBV前S缺失突变对病毒复制力及表面抗原启动子Ⅱ转录活性的下调作用

目的：分析HBV前S缺失突变病毒株体外复制力及其对表面抗原启动子（surface antigen promoter, SP）Ⅱ转录活性的影响。方法研究对象为119例解放军第三〇二医院的住院患者,包括38例慢性乙型肝炎（慢乙肝）轻中度、40例慢乙肝重度和41例慢加急性肝衰竭。从患者血清中提取HBV DNA,PCR扩增HBV全长基因组,统计前S缺失突变的发生率。挑选代表前S缺失突变株及其相应对照的HBV全长序列克隆至pGEM-Teasy载体中。用BspQⅠ/ScaⅠ双酶切1.0倍HBV基因组,转染HepG2细胞,72 h后检测病毒复制力;用PCR分别扩增含前S缺失突变型和野生型的HBV SPⅡ启动子片段,构建pGL3-SPⅡ双荧光素酶真核报告表达载体,转染HepG2细胞48 h后检测相对荧光素酶活性,分析前S1缺失突变对重叠的SPⅡ荧光素酶表达的影响。结果①HBV基因组前S缺失突变检出率在慢乙肝轻中度、慢乙肝重度和慢加急性肝衰竭3组中逐渐递增,分别为5.3%、12.5%和24.4%,差异有统计学意义（P〈0.05）;②前S缺失突变病毒株的复制力较相应野生株降低69%;③与野生型相比,前S1缺失突变使重叠的SPⅡ转录活性降低了36%。结论 HBV前S缺失突变发生率随乙肝进展而升高,前S缺失突变株复制力降低,导致重叠的SPⅡ转录活性降低。     

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM： To establish a cell model harboring replicative clinical hepatitis B virus （HBV） isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B （CHB） patients.
METHODS： The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction （PCR）. All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel.
RESULTS： A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent.
CONCLUSION： The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.     

In vitro resistance to interferon of hepatitis B virus with precore mutation

AIM: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-α), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS: HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS: We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind lll and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result: showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION: These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.     

乙型肝炎病毒表达载体pHY106在筛选抗病毒药物中的意义

目的应用新的HBV表达载体,建立体外筛选临床抗HBV药物的方法。方法克隆对拉米夫定耐药的CHB患者体内HBV全基因组,然后将其亚克隆到HBV真核表达载体pHY106,体外转染Huh7细胞,检测转染后不同时间HBsAg、HBeAg、HBV DNA及HBV复制中间体水平。分析拉米夫定和阿德福韦对HBV基因表达和复制的抑制作用,指导临床用药。结果成功构建全部8个HBV临床分离株全基因组真核表达质粒,HBV聚合酶基因YMDD有5个产生YVDD突变,3个产生YIDD突变。该质粒上HBV基因能在Huh7细胞中进行复制和表达,拉米夫定不能体外抑制HBV复制,阿德福韦抑制了HBV在Huh7细胞中的复制,抑制程度与药物浓度相关。阿德福韦在患者体内也抑制了HBV的复制。结论新建立的体外筛选抗HBV药物的方法能用于临床快速筛选抗HBV药物,对CHB的治疗用药具有指导意义。