乙型肝炎病毒基因组剪接变异体结构分析

目的 了解乙型肝炎病毒(HtBV)基因组剪接变异体的基因结构及特点。方法 从慢性乙型肝炎患者血清中扩增并克隆HBV基因组剪接变异体DNA，测序并比较基因结构特点。结果 共获得10种HBH基因组剪接变异体，基因组大小介于765～2039bp之间。导致剪接变异体产生的5′端供体位点和3′端受体位点各6个。HBVH基因组剪接变异体在C、前-Sl、前-S2及S编码区存在不同程度的缺失，但均保留与致病密切相关的X基因以及病毒复制、包装所必须的DNA序列。结论 HBV基因组剪接变异体在慢性乙型肝炎患者血清中普遍存在。     

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。     

HBsAb阳性隐匿性HBV感染者血清、肝组织HBV全基因序列分析

目的 了解HBsAb阳性隐匿性HBV感染者血清和肝组织中的HBV基因序列,并比较其差异性.方法 以1例长期随访HBsAb阳性隐匿性HBV感染者作为研究对象,用多种试剂盒检测其血清HBsAg、HBsAb,提取外周血血清和肝组织HBV DNA进行全基因组分段扩增,行序列测定及同源性比较.结果 多种试剂盒检测均提示该例患者HBsAg阴性、HBsAb阳性;血清HBV DNA为103～ 105拷贝/mL;血清和肝组织来源的HBV DNA全基因测序完全相同,均为3 215个碱基、B基因型,与参照序列核苷酸同源性为98.82％,各编码区均没有缺失或移码突变,不同编码区的核苷酸序列同源性为98.37％～ 100％,氨基酸序列同源性为98.18％～ 100％,在S区存在几种变异如PreS1的Q80H、S的C64Y、E164G、L175S,但前S区、“a”决定簇、1 762/1 764、1 896位点均未见变异.结论 HBsAb阳性隐匿性HBV感染者血清和肝组织来源的HBV基因序列无明显差异.     

重型乙型肝炎患者血清乙型肝炎病毒全基因克隆及测序

目的建立重型乙型肝炎患者血清乙型肝炎病毒（HBV）DNA克隆并测序，从全基因水平分析HBV基因变异与重型乙型肝炎发病的关系。方法10例重型乙型肝炎患者血清提取HBV DNA，聚合酶链反应（PCR）扩增HBV全基因。PCR产物构建到PUCm-T载体上，转化至大肠杆菌感受态DH-5α细胞，经酶切鉴定，获得含3．2Kb HBVDNA的重组克隆菌，全基因测序，分析各读码框核苷酸和氨基酸变化。结果4例成功构建HBV DNA克隆，并完成全基因测序。其中3例在前C区发生G1896A变异，产生一个终止密码子，导致HBeAg缺失；1例在C启动子区1762、1764双位点出现突变；有多处点突变及缺失变异分布于PreS2区及C区已知细胞毒T淋巴细胞、B淋巴细胞和T淋巴细胞的细胞表位。结论该法可用于临床研究HBV病毒基因结构与重型乙型肝炎发病的关系，并为进一步研究其HBV基因功能奠定基础。     

全基因组分析乙型肝炎病毒缺失变异及其与干扰素疗效的关系

干扰素是治疗慢性乙型肝炎(CHB)的主要药物之一,但其有效率仅为20%～40%[1-3].研究显示,HBV一些特定位点的变异具有抵抗干扰素的作用,这些位点的变异虽然可以抵抗干扰素的作用,但不足以说明干扰素抵抗的机制.目前大多数研究是以HBV的部分片段或特定位点作为研究对象,并不能反映患者体内病毒的真实状态,具有一定的局限性.为进一步了解HBV的生物学特性对干扰素疗效的影响,本研究采用全基因克隆的方法[4]从CHB患者血清中扩增HBV全基因组DNA,分离检测HBV基因组缺失突变体,分析HBV变异与干扰素疗效之间的关系.     

慢性乙型肝炎拉米夫定耐药患者HBV基因型及ALT变化的观察

观察慢性乙型肝炎患者用拉米夫定治疗后HBVP基因变异与不同HBV基因型感染及HBV DNA复升水平和转氨酶变化.收集51例慢性乙型肝炎患者用拉米夫定治疗52-78周后发生YMDD变异的血清标本,对照组128例未用拉米夫定治疗的慢性乙型肝炎患者血清标本,应用聚合酶链反应方法,测定HBV DNA基因型;用限制性片段长度多态性分析方法(PCR RELP)测定HBV DNA YMDD变异;同时进行HBV DNA定量分析.结果显示51例拉米夫定治疗后HBV DNA基因变异患者以B型和C型为主,分别为10例(19.6%)和39例(76.47%),B C混和型2例(3.92%),未见其它基因型.拉米夫定治疗引起HBVDNAYMDD变异可以发生在不同HBV基因型感染的慢性乙型肝炎患者中,与对照组比较二者没有显著性差异.     

乙型肝炎病毒全长基因的扩增及克隆

目的 建立血清标本经聚合酶链反应(PCR)扩增乙型肝炎病毒(HBV)全长基因的新方法,并初步进行克隆分析,从而为HBV分子生物学及致病机理研究奠定基础.方法 针对位于整个HBV基因序列中的两个缺口区设计含酶切位点的引物,扩增3.2 kb HBV全长DNA,经酶切及连接后克隆人PUC18载体.结果 用PCR方法成功获得了3.2 kb HBV DNA,经Sal Ⅰ酶切克隆入PUC18载体,获得重组质粒PUC18/HBV3.2,酶切鉴定和PCR扩增证实重组质粒中含有3.2 kb HBV全长DNA,成功构建了HBV全基因克隆.结论 成功建立了从患者血清中克隆HBV全基因组的方法,为从全基因水平深入研究乙型肝炎病毒变异与致病机理的关系奠定了基础.     

HBeAg阴性乙型肝炎与前C/BCP区变异的相关性

通过对HBV基因的研究表明,HBeAg阴性慢性乙型肝炎(CHB)与HBV DNA中前C(PC)、基本核心启动子(BCP)区的基因突变相关,并指出PC/BCP区的突变不仅影响血清HBeAg的表达,也与机体内HBV DNA的低复制、疾病的进展和抗病毒治疗的应答反应密切相关,使得CHB的治疗变得更为复杂。就HBeAg阴性CHB目前治疗现状、HBV PC/BCP区基因的结构与变异、变异对HBeAg阴性CHB疾病进展以及对乙型肝炎抗病毒治疗的影响进行综述。     

A～D基因型乙型肝炎病毒全长逆转录酶区PCR方法的建立及应用

目的 建立适用于扩增我国常见的乙型肝炎病毒(HBV)A～D基因型全长逆转录酶(RT)区(包含全长HBsAg编码区)的聚合酶链反应(PCR)法,并确定其在分析临床标本中的应用价值.方法 建立我国HBV基因序列库,设计适用于扩增A～D基因型HBV全长RT区引物,以A～D基因型HBV重组质粒为模板建立半巢式PCR(snPCR)法,并确定该法灵敏度.用所建立的snPCR对44份HBV DNA定量阳性(>5.0×102 拷贝/ml)慢性乙型肝炎患者血清标本进行扩增及PCR产物直接测序.结果 琼脂糖凝胶电泳及测序证实,snPCR可扩增A～D基因型HBV全长RT区,对A、B、C和D基因型HBV质粒扩增的灵敏度分别为1.2×103、7.0×102、6.0×102和6.0×102 拷贝/ml.snPCR检测HBV DNA >5×102 拷贝/ml血清标本的阳性率为88.64% (39/44),扩增阴性血清标本HBV DNA滴度均处于103 拷贝/ml水平.扩增目的 产物经直接测序确证无误.生物信息学分析显示,样品中B和C基因型分别占35.90%(14/39)和64.10%(25/39);其中15.38%(6/39)发生RT区变异,包括4例为已知耐药变异;7例(17.95%)存在HBsAg编码区变异,其中2例为已知免疫逃逸变异;6例(15.38%)发生RT区和HBsAg编码区"镜像改变".结论 建立了一种新的扩增全长RT区(涵盖全长HBsAg编码区)的snPCR.该法结合直接测序法,可同时分析我国HBV A～D基因型已知和潜在的耐药变异位点.     

乙型肝炎病毒基因组高变区界定的初步研究

目的:探讨乙型肝炎病毒(HBV)基因组是否存在高保守区或高变区,并探讨前-前-S基因和前-X基因的存在方式. 方法:自GenBank中按血清型搜索符合要求的HBV全基因组序列,并应用Vector 6.0版软件进行核苷酸序列同源性的分析和比较. 结果:GenBank中搜索出28个adr血清型和22个adw血清型HBV病毒株全基因组,比较后发现2种血清型的核苷酸序列总一致率分别为76.6%和73.9%;adr血清型病毒株存有高度变异区和高度变异区,一致率分别为54.5%和92.1%;adw血清型基因组内部含有高度保守区,一致率为85.0%,不含有高度变异区.前-前-S区的存在是较为普遍的现象,而前-X区的编码具有adr血清型特异性特点,在某些病毒基因组中,因为存在A2608→C/T、和C/A2733→T替换突变,导致其前-X基因编码区起始密码子突变,因此部分HBV基因组无前-X基因结构区. 结论:HBV基因组内部可能存在高度保守区,前-前-S区的存在是一个重要的现象,而前-X编码区具有adr血清型特异性的特点.     

Long-term follow-up study of core gene deletion mutants in children with chronic hepatitis B virus infection

Core gene deletion mutants of hepatitis B virus (HBV) have been identified in adults. Because the acquisition of HBV occurs mainly in infancy and childhood in hyperendemic areas, this study aimed to learn the temporal profile of such mutants in children with chronic HBV infection. We have followed up 365 HBV-infected children for more than 10 years and screened out HBV core gene deletion from their sera. Serial serum samples of positive cases were subjected to HBV-DNA nucleotide sequence analyses and quantification. Deletion mutants were found in 18 of the 365 patients (4.9%). Most cases (15 of 18) with deletion mutants heralded hepatitis B e antigen (HBeAg) seroconversion phase, while the other cases (3 of 18) remained in HBeAg-seropositive phase. Deletion mutants disappeared after HBeAg seroconversion except in 1 child. Decreased HBV-DNA levels accompanied deletion mutants for those who finally underwent HBeAg seroconversion, but the HBV-DNA level did not decline if there was no seroconversion. Deletion mutants were not associated with a particularly high peak liver enzyme. Core gene deletion mutants could appear as early as the age of 5. The duration of their appearance was 0.5 to 5 years. Horizontal rather than perinatal transmission of HBV was a favorable factor for these mutants to develop. Deletion fragments were located in the middle part of core gene. The emergence of the mutants was likely the result of host-viral interaction and mostly signified HBeAg seroconversion within 1 year. Core gene deletion mutants appeared preferably in children acquiring HBV by horizontal transmission.     

Quantitative DNA fragment analysis for detecting low amounts of hepatitis B virus deletion mutants in highly viremic carriers

Many variants of hepatitis B virus (HBV) with deletions in the viral genome have been identified. Some of these variants are indicator or even effector of a more severe course of hepatitis. These deletion mutants contribute a variable and sometimes very low proportion to the viral population. For early detection of small amounts of deletion mutants among a large number of wild-type genomes, we applied a new screening method designated quantitative fragment analysis (QFA). By QFA the whole viral genome can be scanned for the presence of deletions or insertions of >/=3 nucleotides representing more than 2% of the viral population. Using QFA we showed that an often described deletion of 8 nucleotides is packaged in viral capsids and not a polymerase chain reaction (PCR) artifact. QFA was applied to study the emergence of deletion mutants in a group of 18 pediatric patients who had been infected from a common source while being under multidrug cancer chemotherapy. All patients had developed a highly viremic asymptomatic HBV carrier state. In 3 of these patients 3 different kinds of HBV deletion mutants were found by QFA: 8 bp deletions within the core promoter, core gene deletions from 8 to 86 bp, and large deletions of up to 1,989 bp spanning the precore/core and the preS/S reading frames. PCR primers that specifically amplify deletion variants enabled the detection of additional patients harboring the investigated variant.     

慢性乙型肝炎病毒感染S区基因缺失及相关因素研究

目的 测定慢性乙型肝炎病毒(HBV)感染者HBV DNA全序列,分析S区基因缺失模式、频率及相关因素.方法 慢性HBV感染者59例,其中HBV携带7例,慢性肝炎31例,肝硬化10例,重型肝炎6例,原发性肝癌5例.结果 25.4%(15/59)慢性HBV感染者有s区基因缺失,未发现S基因缺失.Pre-S基因缺失均见于C基因型患者.Pre-S基因缺失患者中,20%(3/15)HBsAg、抗HBs共存,与无S区缺失者比较有明显差异(P<0.05).PreS基因缺失与病程(偏相关系数0.28,P=0.049)、抗病毒治疗(偏相关系数-0.451,P=0.036)有密切关系.结论 Pre-S基因缺失在基因C型、严重肝病及活动性HBV复制患者多见,可能与病程长及抗病毒治疗有关.Pre-S基因缺失可导致HBV免疫逃避或免疫治疗失败,可能是肝脏疾病发展的重要原因.     

Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection

Several types of naturally occurring pre-S mutants in sera or liver tissues in patients with chronic hepatitis B virus (HBV) infection have been identified. To clarify the prevalence and significance of emergence of pre-S mutants, 140 sera and 18 resected livers from patients with HBV were studied. Replicative status was designated as high, intermediate, and low based on the HBV-DNA levels in serum or the expression of HBV antigens in liver. In vitro transfection and Western blot analysis were performed to characterize expression and secretion of HBsAg by the mutant constructs. Five major types (I to V) of pre-S deletion mutants in serum and liver and 2 types (VI and VII) in liver were identified. Pre-S mutant was 6.4% at high replicative phase, 13% at intermediate, and 37.5% at low or nonreplicative phases in serum. In livers, the same tendency existed: pre-S2 deletion mutants emerged and prevailed at a low replicative phase in hepatocytes that expressed a novel marginal pattern of HBsAg and usually clustered in groups. The deletion sequence of pre-S2 region coincides with human leukocyte antigen-restricted T- and B-cell epitopes. In vitro HBsAg was retained in the hepatocytes and synthesis and secretion of major surface antigen decreased for most of the pre-S mutants. Pre-S mutants prevailed with evolution of chronic HBV, probably under immune pressure. Emergence of pre-S mutants may account for the life-long persistence and discrepancy of HBsAg in serum and liver in HBV and may confer growth advantage in view of the clustering proliferation of hepatocytes harboring pre-S2 mutant.     

Complex HBV populations with mutations in core promoter, C gene, and pre-S region are associated with development of cirrhosis in long-term renal transplant recipients

Long-term immunosuppressed renal transplant recipients with chronic hepatitis B virus (HBV) infection often develop liver cirrhosis (LC) and end-stage liver disease (ESLD). This study investigated accumulation and persistence of specific HBV mutants in relation to the clinical course in these patients (n = 38; mean follow-up, 3.5 years). HBV was analyzed longitudinally via length polymorphism of polymerase chain reaction (PCR) fragments (median, 6.5 serum samples per patient) as well as by cloning and partial sequencing of 346 full-length HBV genomes. Fourteen patients (group 1) developed LC or died from ESLD, whereas 24 patients (group 2) showed no evidence of LC during follow-up. Development of LC and ESLD was associated with persistence of HBV mutant populations characterized by deletions/insertions in core promoter plus deletions in the C gene and/or deletions in the pre-S region (86% of group 1 vs. 17% of group 2; P <.0001). HBV without these mutations or with core promoter mutations alone were predominantly found in group 2 (14% of group 1 vs. 75% of group 2). In patients infected with core promoter mutants, the additional appearance and persistence of deletions in the C gene and/or the pre-S region were accompanied or followed by development of LC and ESLD. The mutations were distributed on individual genomes in various combinations, leading to a high complexity of the virus population. In conclusion, these data suggest that accumulation and persistence of specific HBV populations characterized by mutations in 3 subgenomic regions play a role in pathogenesis of LC and ESLD in long-term renal transplant recipients.     

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5 terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-α therapy ( n =37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Precore and X region mutants in hepatitis B virus infections among renal dialysis patients

SUMMARY. Hepatitis B virus (HBV) variants containing mutations within the X and the precore regions of the viral genome were demonstrated by polymerase chain reaction (PCR) amplification and DNA sequencing in renal dialysis patients with different serological patterns of HBV infection. Among carriers, X region deletion mutants predominated in patients who lost hepatitis B e antigen (HBeAg), or developed anti-HBe, but not in persistently HBeAg-positive patients. The precore region remained wild type in all carriers whether or not they seroconverted from HBeAg to anti-HBe. The frequency of precore and X region mutants was greatest among non-carrier patients with viral antibodies as the only indication of infection and among patients with non-A, non-B hepatitis (NANBH), suggesting an inverse relationship between the presence of wild type HBV markers and the presence of HBV mutants. Furthermore, the detection of one but not the other mutation in many serum samples suggests that these mutations are independently selected for during infection. Finally, the absence of HBV DNA in 21 'uninfected' dialysis patients with normal transaminases and no viral serology, suggests that replication of these mutants is associated with hepatitis. These results have important implications for HBV screening and treatment, as well as for the pathogenesis of chronic infection.