STUDY OF HBV AND/OR VARIANTS IN ALCOHOLICS

酗酒者中乙型肝炎病毒和/或变异体的研究

• 批准号: 3112159
• 负责人: Jack R Wands
• 金额: $ 22.19万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3112159
• 关键词: alcoholism /alcohol abuse; antibody; antibody specificity; endonuclease; epitope mapping; gene expression; genetic markers; genetic polymorphism; genome; hepatitis B antigens; hepatitis B virus group; human population genetics; human subject; laboratory mouse; mathematical model; molecular cloning; molecular genetics; natural gene amplification; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; radionuclides; recombinant DNA; serology /serodiagnosis; southern blotting; surface antigens; tissue /cell culture; transposon /insertion element; virus classification; virus genetics

Hepatitis B virus (HBV) and retrovirus(es) such as human immunodeficiency virus (HIV) have several properties in common including: 1) conserved nucleotide sequences in the GAG of HIV and core of HBV genes, 2) replication by a reverse transcriptase mechanism, 3) two direct repeats within their genomes, 4) lymphotrophism, 5) latent tissue infection, 6) frequent point mutations leading to or contributing to antigenic diversity, 7) integration into the cellular DNA of the host and 8) non-acute type oncogenic potential. We have recently detected by monoclonal antibodies and recombinant DNA techniques HBV and/or varients In alcoholics with and without conventional serologic markers that Includes hepatitis B surface antigen (HBsAg), antibodies to hepatitis B surface antigen (anti-HBs), hepatitis B core antigen, (anti-HBc) and hepatitis B e antigen (anti-HBe) as measured by polyclonal antibodies. These agents have been shown to be present In serum and are infectious since they will produce a long incubation hepatitis infection in chimpanzees. We wish to investigate alcoholic populations further and characterize such hepatitis viral agents In more detail at the molecular and antigenic level. We plan to study alcoholic subjects with and without serologic markers of present, recent or past HBV Infection. In this regard, we will employ a newly developed monoclonal based second generation immunoradiometric assay (M2-IRMA) that detects as low as 10-15 pg/mi of HBsAg-associated epitopes in serum. HBV-DNA sequences will be searched for In serum and liver by dot and Southern blot hybridization using a full genomic length HBV DNA probe. We will ,examine antigenic variability in the surface antigen protein by epitope mapping with 10 different monoclonal ,IRMAs. Comparisons will be made to the 9 known HBV subtypes and other varients already Identified by this !technique in other regions of the world. We will employ the polymerase chain reaction (PCR) to detect and amplify HBV and/or varients DNA sequences in serum, lymphocytes and liver of alcoholics. In this procedure we will: 1) capture on a solid phase support, HBV and/or varients with different high affinity monoclonal anti-HBs antibodies that recognize all known subtypes of HBV and thus bind to different it domain epitopes. 2) amplify defined regions of the captured HBV genome such as those conserved in all known hepadna virus(es) (pre-core and core region) as well as those sequences in the more variable pre-S and S gene domain. 3) determine nucleotide sequence variability of the surface antigen gene region which may be the molecular basis for a different antigenic composition or immunologic reactivity. Since the PCR developed in our laboratory in combination with monoclonal anti-HBs antibodies will amplify and thus detect DNA sequences between 2 and 4 viral particles/assays of serum, we will have the capability to clone amplified sequences directly through primer linkers that have a restriction enzyme site. These studies will provide new information on the presence and !characteristics of HBV and/or related agents in alcoholics using molecular and monoclonal antibody techniques. ,Such studies will assess the frequency of infection and the genomic organization of these agents.

肝炎病毒（HBV）和逆转录病毒（例如人类免疫缺陷） 病毒（HIV）具有多种共同特性，包括：1）保守 HIV和HBV基因核心的核苷酸序列，2） 通过逆转录酶机制复制，3）两个直接重复 在其基因组中，4）淋巴营养，5）潜在组织感染，6） 频繁的点突变导致或导致抗原多样性， 7）整合到宿主的细胞DNA和8）非急性类型 致癌潜力。我们最近通过单克隆抗体检测到 重组DNA技术HBV和/或酗酒中的变体和/或 没有传统的血清学标记物，包括乙型肝炎表面 抗原（HBSAG），丙型肝炎表面抗原（抗HB）的抗体， 乙型肝炎核心抗原（抗HBC）和丙型肝炎抗原（抗HBE） 通过多克隆抗体测量。这些代理已被证明是 血清中存在并且具有感染性，因为它们会产生长时间 在黑猩猩中孵育肝炎感染。我们希望调查 进一步的酒精种群并表征这种肝炎病毒剂 在分子和抗原水平上更详细。我们计划学习 酗酒受试者，有或没有现在的血清学标记，最近或 过去的HBV感染。在这方面，我们将采用新开发的 基于单克隆的第二代免疫放射测定法（M2-imma） 在血清中检测低至10-15 pg/mi的HbSag相关表位。 DOT和 Southern印迹杂交使用全基因组长度HBV DNA探针。我们 将检查表面抗原蛋白中的抗原变异性 具有10种不同单克隆的irmas的表位映射。比较将是 由9个已知的HBV亚型和其他已确定的变体制成 这种！在世界其他地区的技术。我们将采用 聚合酶链反应（PCR）检测和扩增HBV和/或变化 血清中的DNA序列，淋巴细胞和酒精中毒的肝脏。在这个 过程我们将：1）捕获固相支持，HBV和/或变体 具有不同的高亲和力单克隆抗HBS抗体的识别 HBV的所有已知亚型，因此与不同的IT结构域表位结合。 2） 放大捕获的HBV基因组的定义区域，例如保守的区域 在所有已知的Hepadna病毒（ES）（前核和核心区域）中以及 更可变的pre-s和s基因结构域中的序列。 3）确定 表面抗原基因区域的核苷酸序列变异性 可能是不同抗原组成的分子基础或 免疫反应性。由于PCR在我们的实验室中发展 与单克隆抗HBS抗体的结合将放大，从而放大 检测2至4个病毒颗粒/血清测定法之间的DNA序列，我们 将有能力直接通过 具有限制酶位点的底漆。这些研究会 提供有关HBV和/或的存在和！特征的新信息 使用分子和单克隆抗体的酗酒药物相关剂 技术。 ，这样的研究将评估感染的频率和 这些代理的基因组组织。