The HBV E genotype discover in Dai nationality in Xishuangbanna, Yunnan Province

To investigate the distribution of Hepatitis B virus (HBV) genotypes among the population of Dai nationality in Xishuangbanna, Yurman Province HBV genotypes of the Serum samples were tested by PCR-RFLP. This is the first time to discover the B+E genotypes in China. This finding provides new information for understanding the distribution of HBV genotype in China and a provides a basis for establishing a Chinese gene bank.     

黑龙江省乙型肝炎血清流行病学调查分析

为了掌握黑龙江省乙型肝炎(乙肝)病毒(HBV)流行现状和人群免疫水平,评价我省儿童乙肝疫苗预防接种效果,为制订乙肝预防控制策略提供依据。采用横断面调查方法,选择全省7个国家级疾病监测点,在每个监测点随机抽取2个乡级单位,1～59岁作为目标人群共调查3337名人群。黑龙江省1～59岁人群乙肝病毒表面抗原(HBsAg)阳性率、乙肝病毒表面抗体(抗-HBs)阳性率和乙肝病毒核心抗体(抗-HBc)阳性率经标化后为5.65%、55.45%、30.50%,1～3岁人群乙肝表面抗原阳性率明显低于15～59岁人群。1～3岁、4～10岁和11～14岁人群乙肝疫苗全程接种率为99.43%、89.40%和64.81%;首针及时接种率分别为86.64%、75.57%和49.87。城市和农村HbsAg阳性率分别为3.26%和5.04%。医院出生儿童乙肝疫苗首针及时接种率高于在家出生儿童。结果显示接种乙肝疫苗可明显提高抗-HBs阳性率,提高人群对HBV的免疫保护能力,降低HBsAg携带率。     

反义前C／C基因转移表达抗乙型肝炎病毒作用的研究

为了观察逆转录病毒载体包装细胞系统介导反义基因转移表达的抗乙型肝炎病毒（HBV）作用,将HBVayw前C／C基因（preC／C）片段反向插入逆转录病毒载体质粒。将重组体转染逆转录病毒包装细胞PA317后,获得重组逆转录病毒。用重组逆转录病毒感染2．2．15细胞后发现,感染后第3天,细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量即明显减少,抑制作用于感染后第5天达到高峰,其中对HBsAg表达的抑制率为27．0％,对HBeAg表达的抑制率为59.5％。反义基因重组逆转录病毒感染2.2．15细胞对HBV抗原表达的抑制作用至少可以持续到转导后的第11天。空载及正义基因重组逆转录病毒感染对2．215细胞HBV抗原表达无明显抑制作用。此外,反义基因重组逆转录病毒感染对2.2.15细胞HBVDNA复制也有抑制作用．无细胞毒性。     

湖北咸宁地区乙型肝炎病毒基因型的调查

乙型肝炎病毒(Hepatitis B virus,HBV)是引起急性和慢性肝炎的最主要的病原[1]。目前根据HBsAg的共同抗原决定簇“α”和两对相互排斥的抗原决定簇将HBV分为ayw1, ayw2, ayw3, ayw4,ayr, adw2, adw2, adw4, adrq 和adrq-9种不同的血清学亚型,1988年Okamoto[2]等根据HBV基因组核苷酸的差异又提出了HBV基因型的概念,并以全基因组核苷酸差异≥8%,定为基因型分型标准。目前从世界不同地区分离的乙型肝炎病毒分离株已被分为A、B、C、D、E、F、G、H等8种不同的基因型[3~5]。包括中国、日本和东南亚在内的亚洲地区主要流行B、C两种基因…     

Enhanced host immune responses in presence of HCV facilitate HBV clearance in coinfection

Hepatitis B virus (HBV)/Hepatitis C virus (HCV) coinfection is frequently observed because of the common infection routine. Despite the reciprocal inhibition exerted by HBV and HCV genomes, the coinfection of HBV and HCV is associated with more severe forms of liver diseases. However, the complexity of viral interference and underlying pathological mechanism is still unclarified. With the demonstration of absence of direct viral interplay, some in vitro studies suggest the indirect effects of viral-host interaction on viral dominance outcome. Here, we comprehensively investigated the viral replication and host immune responses which might mediate the interference between viruses in HBV/HCV coinfected Huh7-NTCP cells and immunocompetent HCV human receptors transgenic ICR mice. We found that presence of HCV significantly inhibited HBV replication in vitro and in vivo irrespective of the coinfection order, while HBV did not affect HCV replication. Pathological alteration was coincidently reproduced in coinfected mice. In addition to the participation of innate immune response, an involvement of HCV in up-regulating HBV-specific immune responses was described to facilitate HBV clearance. Our systems partially recapitulate HBV/HCV coinfection and unveil the uncharacterized adaptive anti-viral immune responses during coinfection, which renews the knowledge on the nature of indirect viral interaction during HBV/HCV coinfection.     

HCV/HBV复合抗原基因的克隆、表达及免疫应答研究

为探讨ＨＣＶ／ＨＢＶ 复合疫苗的可行性,将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ与ＨＢｓＡｇ 基因连接成ＰＣＸＳ基因,与β－半乳糖苷酶（ＧＺ）基因融合后在大肠杆菌及减毒鼠伤寒沙门氏菌中获得表达．目的蛋白ＧＺ－ＰＣＸＳ可被抗－ＨＢｓ 及抗－ＨＣＶ 抗体所特异识别．ＧＺ－ＰＣＸＳ抗原皮下注射免疫ＩＣＲ小鼠后,诱发了较高水平的抗－ＧＺ－ＰＣＸＳＩｇＧ反应．构建的重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲ／ＰＣＸＳ）口服免疫小鼠后,诱发了高水平的ＣＤ８＋ Ｔ细胞增殖反应及抗ＧＺ－ＰＣＸＳＩｇＧ反应．所有免疫小鼠均未见明显的毒副作用．该研究揭示,ＨＣＶ／ＨＢＶ 复合抗原可诱发特异性体液免疫及细胞免疫应答,而活菌苗口服可能是理想的免疫途径,为ＨＣＶ／ＨＢＶ 双价疫苗研究提供了一定的理论及实验依据．     

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.     

乙型肝炎病毒S-preS1-C融合基因在酵母中的表达及抗原性分析

以HBV全基因组质粒为模板,扩增出目的区段:S(1-222AA),preS1(10-50AA)和C(2-30AA),以酶切-PCR的方法进行连接,克隆入表达载体pPIC3.5k,电穿孔转化后利用G418加压筛选出多拷贝整合菌株,诱导表达并对表达产物进行检测。结果显示融合后的基因为ss1c,长度约为906bp。在酵母中成功地进行了表达,表达产物(SSIC)的大小约为31kDa,并形成直径约为22nm的病毒样颗粒。表达产物与HBs抗体、preS1抗体和HBc抗体均有特异反应。为进一步研究治疗性疫苗提供了重要的基础。     

反义前S／S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒（HBV）的作用,将HBVayw前S／S基因（PreS／S）片段反向插入逆转录病毒载体质粒,构建preS／S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒。用重组病毒转导2．2．15细胞后,第3天即可见细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71％,HBeAg抑制率为27％,抑制作用至少可持续到转导后第11天。空载及正向插入基因的重组逆转录病毒转导对2,2．15细胞内HBV抗原表达无抑制作用。     

Development of HBsAg-binding aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial 115 mer library of ~1.1×10 15 random-sequence RNA molecules using the SELEX procedure.The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells.This is the first reported RNA aptamer which could bind to a HBV specific antigen.This newly isolated aptamer could be modified to deliver imaging,diagnostic,and therapeutic agents targeted at HBV-infected cells.     

内江地区慢性乙型肝炎患者的基因型及拉米夫定联合阿德福韦酯抗病毒治疗效果

目的 探讨内江地区慢性乙型肝炎患者的基因型及拉米夫定联合阿德福韦酯抗病毒的治疗效果。方法 将201例慢性乙型肝炎患者进行HBV基因型的测定,其中120例HBeAg（+）慢性乙型肝炎患者随机分为三组：A组、B组和C组,每组40例。A组给予拉米夫定(LAM)治疗;B组给予恩替卡韦(ETV)治疗;C组给予LAM联合阿德福韦酯(ADV)治疗,比较治疗情况。结果 201例慢性乙型肝炎患者中B型119例（59.2%）,C型68例（33.8%）,B/C混合型10例（5.0%）,未知型4例（2.0%）,各分型之间的性别、年龄差异无统计学意义（P＞0.05）。C型感染者HBeAg阳性率为86.8%,显著高于B型的51.3%（P＜0.05）。HBeAg（+）的3组患者治疗12、24和48周时,B组和C组患者的ALT复常率、HBV DNA阴转率及48周时的HBeAg血清转换率均显著高于A组（P＜0.05）;C组患者与B组比较差异无统计学意义（P＞0.05）。治疗期间均未见不良反应发生。C组患者（包括B型19例、C型21例）中B型的HBV DNA阴转率及HBeAg血清转换率显著高于C型（P＜0.05）。结论 地处西南方的内江地区慢性乙型肝炎患者主要以B型为主,C型次之,B型和C型共占93.0%,其他型别仅占较少部分。ETV方案或LAM联合ADV方案治疗HBeAg（+）慢性乙肝疗效优于LAM治疗。初始LAM联合ADV治疗基因B型HBeAg（+）慢性乙型肝炎疗效优于C型。     

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial ...     

Novel evidence suggests Hepatitis B virus surface proteins participate in regulation of HBV genome replication

Naturally occurring mutations in surface proteins of Hepatitis B virus (HBV) usually result in altered hepatitis B surface antigen (HBsAg) secretion efficiency. In the present study, we reported two conserved residues, M75 and M103 with respect to HBsAg, mutations of which not only attenuated HBsAg secretion (M75 only), but also suppressed HBV genome replication without compromising the overlapping p-gene product. We also found M75 and M103 can initiate truncated surface protein (TSPs) synthesis upon over-expression of full-length surface proteins, which may possibly contribute to HBV genome replication. However, attempts to rescue replication-defective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful, which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

不同佐剂对含S+PreS1融合抗原的乙型肝炎病毒颗粒疫苗免疫原性的影响

本研究在前期工作基础上,用CHO细胞表达的含PreS1+S融合抗原的新型基因工程HBV颗粒疫苗(HBSS1)与Al(OH)3、CpG及CpG+Al(OH)3等佐剂配伍,在Balb/C小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFNELISpot检测)。结果表明:CpG佐剂结合HBSS1颗粒疫苗可快速诱导(单针免疫)高水平的抗PreS1及S抗体,IgG2a/IgG1比率1,同时可诱导较高抗原特异的细胞免疫应答;Al(OH)3+CpG双佐剂组一次免疫后可诱导产生最高的抗S抗体滴度(1:105),其产生的抗体亚类包括IgG1、IgG2a与IgG2b;在S抗原N端(13~49aa)存在优势CTL表位。结论:CpG佐剂结合HBSS1颗粒疫苗应是发展新型治疗性乙肝疫苗的较佳选项。     

Differing roles of protein kinase C on the signal transduction of tumour necrosis factor-alpha and -beta on PANC-1 cells: In vitro autoradiographic investigation

We investigated alterations in protein kinase C (PKC) activity of PANC-1 cells following treatment with tumour necrosis factor (TNF)-alpha or TNF-beta by an in vitro autoradiographic method. Binding studies performed on whole cells using [³H]phorbol-12,13-dibutyrate (PDBu) as a ligand revealed strong activation of PKC by TNFs within 30 min. The effect was similar to that seen after 30 min treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). After treatment for 24 h, TNF-beta caused a marked down-regulation of PKC similar to that seen after 24 h treatment with TPA; significant activation persisted, however, in the cells treated for 24 h with TNF-alpha. Our data suggest that PKC activation may play a more important role in the TNF-alpha signal transduction pathway than in that of TNF-beta.