Viruses and Hematopoiesis

病毒和造血作用

基本信息

批准号：
8344868
负责人：
NEAL S YOUNG
金额：
$ 56.65万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8344868
关键词：
Achievement Acute Acute Hepatitis Adult Anemia Animals Annual Reports Antibodies Antibody Formation Antigens Aplastic Anemia Apoptosis Asians Baculoviruses Bilirubin Biochemical Biological Assay Blood Bone Marrow CD34 gene Capsid Capsid Proteins Cell Culture System Cell Culture Techniques Cell Cycle Arrest Cell Death Cell Line Cells Childhood China Chronic Clinic Clinical Codon Nucleotides Communicable Diseases Congestive Heart Failure Development Disease E2F1 gene Enzyme-Linked Immunosorbent Assay Epitopes Erythema Infectiosum Erythroid Erythroid Progenitor Cells Etiology Exanthema Family Female Fetus Functional disorder G2 Phase Gel Generations Genetic Genetic Transcription Globosides Goals Hematology Hematopoiesis Hematopoietic Hepatitis Hepatitis C Hepatitis Viruses Hospitals Human Human Parvovirus B19 Human Volunteers Hydrops Fetalis Immune Immune system Immunoglobulin G Immunoglobulin M In Vitro Infection Infectious Agent Institution Laboratories Liver Mammalian Cell Maps Mediating Methods Minority Modification Molecular Mothers Nonstructural Protein Nucleotides PTPN11 gene Pancytopenia Parvoviridae Parvovirus Parvovirus Infections Pathogenesis Patients Pattern Peptide Elongation Factor 2 Phase Phosphorylation Physiological Play Prevention Production Proteins Publishing Pure Red-Cell Aplasia Recombinant Vaccines Recording of previous events Research Research Personnel Risk Factors Role Sampling Second Pregnancy Trimester Serologic tests Serological Serum Sickle Cell Anemia Single Stranded DNA Virus Specimen Structure-Activity Relationship Surface Syndrome System Transaminases United States United States National Institutes of Health Vaccines Viral Viral Proteins Viral hepatitis Virus Virus Diseases Work arthropathies base calmodulin-dependent protein kinase III cost effective erythroid differentiation in utero in vivo inhibitor/antagonist interest liver transplantation male member neutralizing antibody novel pathogen progenitor protein expression receptor research study self assembly stillbirth tissue culture tool transcription factor two-dimensional vaccine candidate

项目摘要

B19 Parvovirus: B19 parvovirus is a small, nonenveloped, single-stranded DNA virus, the only member of the Parvoviridae family that is known to be pathogenic in humans. B19 parvovirus infection is common in childhood, and most adults have been exposed to the virus as determined by serologic assays for anti-viral IgG. B19 parvovirus is the etiologic agent in fifth disease, a childhood exanthem; fifth disease manifests in adulthood as chronic arthropathy. Hematologically, B19 parvovirus causes several diseases: transient aplastic crisis of hemolytic syndromes, leading to severe and sometimes fatal acute anemia, as in patients with sickle cell disease; hydrops fetalis, in which infection of the mother in the second trimester is transmitted in-utero to the developing fetus, leading to severe anemia, congestive heart failure and stillbirth; chronic pure red cell aplasia due to a persistent infection, the result of inability of the host to mount an adequate neutralizing antibody response. The Hematology Branchs notable achievements in B19 parvovirus research include its first propagation in cell culture; elucidation of a detailed transcription map that led to the virus reclassification into a new genus; identification of the cellular receptor, globoside or P antigen, and determination that genetic absence of the receptor leads to insusceptibility in vitro and in vivo; description of the neutralizing epitopes present on the unique region of VP1, which are external to the capsid surface; and production of a recombinant vaccine candidate, based on expression of B19 capsid proteins in a baculovirus system and subsequent self assembly of the proteins into empty capsids, with adjustment of VP1 content to maximize neutralizing antibody responses in animals and humans. In recent years, investigators in the Branch have also developed powerful tools for the study of B19 parvovirus in tissue culture: both an infectious clone, which allows modification of viral proteins at the nucleotide level and therefore detailed molecular mapping of structure-function relationships, and utilization of CD34 cells driven to erythroid differentiation obtained from normal human volunteers as a basis for a productive cell culture system, permitting propagation of the virus under physiologic conditions. In the last year, we have built on our previous, now published, observation that the E2F master family of transcription factors plays a role in B19 pathophysiology. The major nonstructural protein (NS1), expressed soon after parvovirus entry into the target cell, alters E2F1-E2F5 expression, leading to stable arrest of the cell cycle in G2 phase. In current work, we have focused on the B19 11-KD protein B19 which induces apoptosis in erythroid progenitor cells. Utilizing two-dimensional gel analyses in order to profile protein expression, we found that expression of the 11-KD protein led to phosphorylation of elongation factor 2 (EF2). Both 11-KD induced EF2 phosporylation and apoptosis are specifically down-regulated by an inhibitor of EF2 kinase. These findings provide a new mechanism underlying cell death in erythroid progenitors after B19 parvovirus infection. In work towards development of a B19 parvovirus vaccine, we found that alteration of codon usage allows higher expression of B19 parvovirus capsid proteins in mammalian cell lines. High level expression can be obtained for clones of NIH 3T cells, and these capsid proteins should generate empty capsids and be translationally modified as in native target cells. Production of empty B19 parvovirus capsids in mammalian cells would be much more cost effective than in the more cumbersome baculovirus system. Virus Infection and Aplastic Anemia: There is a long history of failed attempts to isolate a virus for seronegative hepatitis (non-A, non-B, non-C serologies). While the proportion of acute hepatitis in the United States without a viral etiology is tiny, as many as 20% of hepatitis cases in Asian clinics are seronegative. Seronegative acute hepatitis differs from known viral hepatitis in its demographic features and clinical consequences. In particular, there is a higher rate of severe late complications of fulminant hepatitis and of post-hepatitis aplastic anemia following seronegative acute hepatitis. For bone marrow failure, the pattern is stereotypical: patients are more often male than female, usually young, and without known risk factors for hepatitis virus exposure; the hepatitis is transient but severe, with marked elevations in bilirubin and serum transaminases; pancytopenia is profound and historically almost always fatal. Due to inability to isolate a putative infectious agent, using a wide variety of molecular, immunological and biochemical methods, from either bone marrow or blood of patients with post-hepatitis aplastic anemia or in liver samples obtained from patients undergoing liver transplantation for fulminant hepatitis, we have collaborated with other institutions to obtain blood from patients in the acute phase of seronegative hepatitis. These samples also may be more likely to contain infectious material than are those obtained months following the onset of the hepatitis and its likely clearance by the immune system. In early experiments, we utilized 454 deep sequencing. However, the number of sequences obtained was beyond the computational abilities of our collaborating core laboratory, and the number of sequences present in both control normal sera and in patient material was large and not easily distinguished. We have now undertaken high through-put deep sequencing by Solexa. Samples have been obtained from patients with acute seronegative hepatitis from collaborators at a large infectious disease hospital in China. Preliminary results indicate the presence in a significant minority of these cases, but not in control specimens, of novel viral sequence. We thus may have a putative final hepatitis viral pathogen. Current efforts include expression of the capsid proteins and generation of an ELISA assay for IgG and IgM antibodies, as well as development of a quantitative PCR assay for viral sequence in clinical specimens.

B19细节性病毒：B19细小病毒是一种小的，未开发的单链DNA病毒，是Parvoviridae家族的唯一成员，在人类中已知是致病性的。 B19细小病毒感染在儿童时期很常见，大多数成年人都暴露于抗病毒IgG的血清学测定法确定的病毒中。 B19小扇联病毒是第五疾病的病因，是童年时期；第五疾病在成年后表现为慢性关节炎。在血液学上，B19细胞小扇区引起了几种疾病：溶血综合征的短暂性和性危机，导致严重，有时是致命的急性贫血，如镰状细胞疾病的患者；胎儿的水力胎儿，在妊娠中期的母亲感染在胎儿中传播到发育中的胎儿，导致严重的贫血，充血性心力衰竭和死产。慢性纯红色细胞性发育不全是由于持续感染而引起的，这是宿主无法安装足够的中和抗体反应的结果。 B19细小病毒研究中的血液学分支值得注意的成就包括其在细胞培养中的首次传播。阐明导致病毒重新分类为新属的详细转录图；鉴定细胞受体，球蛋白或P抗原，并确定遗传缺失受体会导致体外和体内不舒服。 VP1独特区域中存在的中和表位的描述，该区域是衣壳表面外部的；基于杆状病毒系统中B19衣壳蛋白的表达以及随后将蛋白质的自组装成空的capsids，并调整了VP1含量以最大化动物和人的中和抗体反应，并将蛋白质的自我组装成蛋白质。 In recent years, investigators in the Branch have also developed powerful tools for the study of B19 parvovirus in tissue culture: both an infectious clone, which allows modification of viral proteins at the nucleotide level and therefore detailed molecular mapping of structure-function relationships, and utilization of CD34 cells driven to erythroid differentiation obtained from normal human volunteers as a basis for a productive cell culture system, permitting propagation of the生理条件下的病毒。在去年，我们建立在以前的（现已发表）的观察结果上，即E2F硕士转录因子家族在B19病理生理学中起作用。主要的非结构蛋白（NS1）在细小病毒进入靶细胞后不久表示，改变了E2F1-E2F5的表达，从而导致在G2相中稳定地阻止细胞周期。在目前的工作中，我们专注于B19 11-kD蛋白B19，该蛋白B19诱导红细胞祖细胞中凋亡。利用二维凝胶分析来谱图蛋白表达，我们发现11 kD蛋白的表达导致伸长因子2（EF2）的磷酸化。 11 kD诱导的EF2荧光素化和凋亡都被EF2激酶的抑制剂特别下调。这些发现提供了B19细小性病毒感染后红细胞祖细胞祖细胞死亡的新机制。在开发B19细扇区疫苗的工作中，我们发现密码子使用的改变允许在哺乳动物细胞系中更高表达B19 phvovovirus capsid蛋白。可以为NIH 3T细胞的克隆获得高水平的表达，并且这些衣壳蛋白应产生空的衣壳，并像天然目标细胞中的翻译经过翻译。在哺乳动物细胞中产生空的B19粉状帽的成本要比在更繁琐的杆状病毒系统中更具成本效益。病毒感染和性贫血：经常尝试分离出血清神经肝炎病毒（非A，非B，非C血清学）的历史。尽管没有病毒病因的美国急性肝炎的比例很小，但在亚洲诊所中，多达20％的肝炎病例具有血清症。血浆急性肝炎在人口特征和临床后果方面与已知的病毒肝炎不同。特别是，在血清症状急性肝炎后，暴发性肝炎的严重晚期并发症和肝炎后炎后疾病的发生率更高。对于骨髓衰竭，该模式是刻板印象的：患者比女性，通常是年轻的男性，并且没有已知的肝炎病毒风险因素；肝炎是短暂但严重的，胆红素和血清转氨酶的升高明显。全年症是深刻的，历史上几乎总是致命的。由于无法分离推定的感染剂，使用多种分子，免疫学和生化方法，是从骨髓或血液的血液或血液中的血液或血液的血液中，在接受肝移植的患者中获得的肝炎患者在肝移植的患者中获得了forminative肝炎，我们与其他患者进行了绩效，从而获得了其他阶段的疗程。这些样品也可能比肝炎发作后几个月及其可能通过免疫系统清除的几个月所获得的样品包含感染材料。在早期实验中，我们使用了454个深度测序。但是，获得的序列数量超出了我们协作核心实验室的计算能力，并且在控制正常血清和患者材料中存在的序列数量很大，并且不容易区分。现在，我们已经通过Solexa进行了高度的深层测序。已经从中国一家大型传染病医院的合作者那里获得了急性血清肝炎患者的样品。初步结果表明，在这些病毒序列中，在很大的少数情况下，但在对照样本中的存在。因此，我们可能具有推定的最终肝炎病毒病原体。当前的努力包括表达衣壳蛋白以及用于IgG和IgM抗体的ELISA测定法，以及开发用于临床标本中病毒序列的定量PCR分析。