Viruses and Hematopoiesis

病毒和造血作用

• 批准号: 8558015
• 负责人: NEAL S YOUNG
• 金额: $ 52.89万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/8558015
• 关键词: Achievement; Acute; Acute Hepatitis; Adult; Amino Acid Sequence; Anemia; Animals; Annual Reports; Antibodies; Antibody Formation; Antigens; Aplastic Anemia; Apoptosis; Apoptotic; Asians; Baculoviruses; Base Pairing; Bilirubin; Biochemical; Biological Assay; Blood; Blood Banks; Bone Marrow; CD34 gene; CD36 gene; Capsid; Capsid Proteins; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Childhood; China; Chinese Hamster Ovary Cell; Chronic; Circoviridae; Circovirus; Clinic; Clinical; Codon Nucleotides; Communicable Diseases; Congestive Heart Failure; Development; Disease; Distal; E2F transcription factors; Epitopes; Erythema Infectiosum; Erythroid; Etiology; Exanthema; Failure; Family; Fetus; Functional disorder; Gene Amplification; Genes; Genetic; Genetic Transcription; Genome; Globosides; Goals; Hemagglutinin; Hematology; Hematopoiesis; Hematopoietic; Hepatitis; Hepatitis C; Hepatitis Viruses; Hospitals; Human; Human Parvovirus B19; Human Volunteers; Hydrops Fetalis; Immune; Immune system; Immunization; Immunoblotting; Immunoglobulin G; Immunoglobulin M; In Vitro; Infection; Infectious Agent; Inflammation; Influenza; Influenza A virus; Institution; Laboratories; Lentivirus Vector; Liver; M2 protein; Mammalian Cell; Maps; Mediating; Methods; Modification; Molecular; Mothers; Mutation; Nonstructural Protein; Nucleotides; Pancytopenia; Parvoviridae; Parvovirus; Parvovirus Infections; Pathogenesis; Patients; Pattern; Phase; Phospholipase A2; Physiological; Prevention; Production; Proline; Proteins; Publishing; Pure Red-Cell Aplasia; Recombinant Vaccines; Recombinants; Research; Research Personnel; Risk Factors; Role; SH3 Domains; SH3-Binding Motif; Sampling; Second Pregnancy Trimester; Sequence Analysis; Serological; Serum; Sickle Cell Anemia; Single Stranded DNA Virus; Staining method; Stains; Structural Protein; Structure-Activity Relationship; Subfamily lentivirinae; Sucrose; Surface; Syndrome; System; Tertiary Protein Structure; Testing; Transaminases; United States; United States National Institutes of Health; Vaccines; Viral; Viral Proteins; Viral hepatitis; Virus; Virus Diseases; Work; arthropathies; base; density; erythroid differentiation; flu; in utero; in vivo; influenza virus vaccine; interest; knock-down; liver transplantation; male; member; mutant; neutralizing antibody; novel virus; overexpression; particle; pathogen; prevent; progenitor; promoter; receptor; self assembly; stillbirth; tissue culture; tool; vaccine candidate; vaccine development

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. We have pursued our previously published observation of the role of the E2F transcription factors focusing on the 11-KD non structural protein which induces apoptosis in CD36 erythroid progenitor target cells. We constructed recombinant lentiviruses with siRNAs to target EF2K; these siRNAs knock down the levels of EF2K. We are currently testing whether EF2K deficiency can prevent apoptosis in CD36 cells. In addition, sequence analysis shows that the 11-kD protein is proline-rich and contains four SH3 binding motifs (PPXP); we created serial 11-kD mutants with altered PPXP domains. Substitution mutations in two of the distal SH3 binding motifs abolished 11-KD apoptotic activity. We hypothesized that the interaction between B19 11-kD protein and host SH3 domain containing factors results in activation of EF2K and apoptosis of B19 target cells; this hypothesis is being tested functionally. We previously found that codon optimization of B19 capsid genes increased viral capsid protein production in non-permissive cells. To produce large amounts of B19 empty viral capsids for vaccine development, codon optimized VP1 and VP2 genes were cloned into an SP lentivirus vector, in which VP1 and VP2 expression is under the control of a qmate-inducible promoter. To test whether the phosphalipase motif of VP1 unique region contributed to viral induced inflammation, we abolished enzymatic activity by constructing a mutant VP1 gene substituted D175A. We have established two mammalian cell lines that stably overexpress native or PLA2 negative mutant VP1 or VP2 capsid proteins. Immunoblot shows a VP1/VP2 rratio of 1:5 in these cells lines. B19 empty capsids have been purified by Optiprep density grading centrification, and direct electromicroscopy shows typical parvovirus-like particles. In order to produce a chimeric vaccine agains other major viral pathogens, we modified constructs by insertion of a hemagglutinin region or M2 protein of influenza A virus into the VP1 unique region. These flu proteins elicit broadly neutralizing antibodies. Chimeric empty capsids may be useful in the development of a universal influenza vaccine, as well as for vaccines to elicit antibodies against both influenza and parvovirus. Early results indicate that purified empty capsids from transfected cells contain particles with antigens of both viruses present. We are currently working to establish stable CHO cell lines able to produce large amounts of empty capsids for immunization of animals. Virus infection and aplastic anemia: There have been repeated failures to identify a viral etiology for seronegative hepatitis (non-A, non-B, and non-C). While seronegative hepatitis is rare in the United States, 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, 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 entering 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. Using Solexa deep sequencing, we have isolated viral sequences from samples obtained from a large infectious disease hospital in Chonching in the west of China. Contigs have been assembled to a complete viral sequence of approximately 3500 base pairs. Sequence analyses indicates a nonstructural protein and capsid protein domain, similar to the parvoviruses but with relatively low homology either in nucleotide or amino acid sequence. The noncapsid protein has features of a circovirus while the capsid protein more closely resembles a parvovirus. Virus sequence is present at an average of about 1,000 to 10,000 genome copies in a majority of patients with seronegative hepatitis. Sequence is not detectable by gene amplification in contemporary healthy blood bank donors. Immunoblot shows the presence of IgM antibody to capsid protein in the majority of hepatitis patients, and IgG antibody in both patients and controls. Particles have been visualized by electromicroscopy after purification on a sucrose density cushion. Our results suggest a novel virus, genaeologically between Circoviridae and Parvoviridae, and possibly disease-associated. Current efforts are directed to immunogold EM staining and expression of the viral proteins in a variety of cells, including construction of an infectious clone. Sero-epedemiologic studies are required, with samples from China and elsewhere, to determine more certainly the role of this virus, termed NIH-CQ, in human hepatitis and other diseases.

B19 细小病毒：B19 细小病毒是一种小型、无包膜、单链 DNA 病毒，是细小病毒科中唯一已知对人类致病的成员。 B19 细小病毒感染在儿童时期很常见，根据抗病毒 IgG 血清学检测确定，大多数成年人都曾接触过该病毒。 B19 细小病毒是第五种疾病（儿童时期的皮疹）的病原体；第五种疾病在成年后表现为慢性关节病。 在血液学上，B19细小病毒会引起多种疾病：溶血综合征的短暂再生障碍性危象，导致严重且有时致命的急性贫血，如镰状细胞病患者；胎儿水肿，母亲在妊娠中期的感染在子宫内传播给发育中的胎儿，导致严重贫血、充血性心力衰竭和死产；由于持续感染而导致的慢性纯红细胞再生障碍性贫血，这是宿主无法产生足够的中和抗体反应的结果。 血液学分支在 B19 细小病毒研究方面取得的显着成就包括其首次在细胞培养中繁殖；阐明导致病毒重新分类为新属的详细转录图谱；鉴定细胞受体、球苷或P抗原，并确定受体的遗传缺失导致体外和体内不敏感；存在于衣壳表面外部的 VP1 独特区域上的中和表位的描述；基于 B19 衣壳蛋白在杆状病毒系统中的表达以及随后将蛋白自组装成空衣壳，并调整 VP1 含量以最大限度地提高动物和人类的中和抗体反应，生产重组候选疫苗。 近年来，该部门的研究人员还开发了用于研究组织培养中的 B19 细小病毒的强大工具：既是一种感染性克隆，可以在核苷酸水平上修饰病毒蛋白，从而获得结构-功能关系的详细分子图谱，利用从正常人类志愿者获得的驱动红细胞分化的 CD34 细胞作为高效细胞培养系统的基础，允许病毒在生理条件下繁殖。 我们对之前发表的 E2F 转录因子的作用进行了观察，重点关注 11-KD 非结构蛋白，该蛋白诱导 CD36 红系祖细胞靶细胞凋亡。 我们构建了带有 siRNA 的重组慢病毒来靶向 EF2K；这些 siRNA 降低了 EF2K 的水平。 我们目前正在测试 EF2K 缺陷是否可以阻止 CD36 细胞凋亡。 此外，序列分析表明，11-kD 蛋白富含脯氨酸，并包含四个 SH3 结合基序 (PPXP)； 我们创建了具有改变的 PPXP 结构域的系列 11-kD 突变体。 两个远端 SH3 结合基序的取代突变消除了 11-KD 凋亡活性。 我们假设 B19 11-kD 蛋白与宿主 SH3 结构域因子之间的相互作用导致 EF2K 激活和 B19 靶细胞凋亡； 该假设正在接受功能测试。 我们之前发现 B19 衣壳基因的密码子优化增加了非许可细胞中病毒衣壳蛋白的产生。 为了生产大量用于疫苗开发的 B19 空病毒衣壳，将密码子优化的 VP1 和 VP2 基因克隆到 SP 慢病毒载体中，其中 VP1 和 VP2 的表达受到 qmate 诱导型启动子的控制。 为了测试VP1独特区域的磷脂酶基序是否导致病毒诱导的炎症，我们通过构建突变的VP1基因取代D175A来消除酶活性。 我们已经建立了两种稳定过表达天然或 PLA2 阴性突变体 VP1 或 VP2 衣壳蛋白的哺乳动物细胞系。 免疫印迹显示这些细胞系中 VP1/VP2 的比例为 1:5。 B19空衣壳已通过Optiprep密度分级离心纯化，直接电镜检查显示典型的细小病毒样颗粒。 为了生产针对其他主要病毒病原体的嵌合疫苗，我们通过将甲型流感病毒的血凝素区域或 M2 蛋白插入 VP1 独特区域来修改构建体。 这些流感蛋白会引发广泛的中和抗体。 嵌合空衣壳可能有助于开发通用流感疫苗，以及引发针对流感和细小病毒的抗体的疫苗。 早期结果表明，从转染细胞中纯化的空衣壳含有带有两种病毒抗原的颗粒。 我们目前正在努力建立稳定的 CHO 细胞系，能够产生大量用于动物免疫的空衣壳。 病毒感染和再生障碍性贫血：血清阴性肝炎（非甲型、非乙型和非丙型）的病毒病因多次未能确定。 虽然血清阴性肝炎在美国很少见，但亚洲诊所多达 20% 的肝炎病例血清阴性。 血清阴性急性肝炎在人口特征和临床后果方面与已知的病毒性肝炎不同；特别是，血清阴性急性肝炎后暴发性肝炎和肝炎后再生障碍性贫血的严重晚期并发症发生率较高。对于骨髓衰竭，模式是刻板的：患者通常是男性，通常是年轻人，并且没有已知的肝炎病毒暴露危险因素；肝炎是短暂但严重的，胆红素和血清转氨酶显着升高；全血细胞减少症是严重的，历史上几乎总是致命的。由于无法使用多种分子、免疫学和生化方法从肝炎后再生障碍性贫血患者的骨髓或血液中或从因暴发性肝炎接受肝移植的患者获得的肝脏样本中分离出假定的传染源，我们有与其他机构合作，从进入血清阴性肝炎急性期的患者身上采集血液。 这些样本也可能比肝炎发作并可能被免疫系统清除几个月后获得的样本更可能含有传染性物质。 使用 Solexa 深度测序，我们从中国西部重庆一家大型传染病医院获得的样本中分离出了病毒序列。 重叠群已组装成大约 3500 个碱基对的完整病毒序列。 序列分析表明非结构蛋白和衣壳蛋白结构域与细小病毒相似，但在核苷酸或氨基酸序列上具有相对较低的同源性。 非衣壳蛋白具有圆环病毒的特征，而衣壳蛋白更类似于细小病毒。 在大多数血清阴性肝炎患者中，病毒序列平均存在约 1,000 至 10,000 个基因组拷贝。 在当代健康血库捐献者中，无法通过基因扩增检测到序列。 免疫印迹显示大多数肝炎患者存在针对衣壳蛋白的 IgM 抗体，而患者和对照者均存在 IgG 抗体。 在蔗糖密度垫上纯化后，通过电子显微镜观察颗粒。 我们的结果表明，这是一种新型病毒，从谱系学角度来看，它介于圆环病毒科和细小病毒科之间，并且可能与疾病相关。 目前的工作致力于免疫金电镜染色和病毒蛋白在多种细胞中的表达，包括构建感染性克隆。 需要对来自中国和其他地方的样本进行血清流行病学研究，以更确定地确定这种称为 NIH-CQ 的病毒在人类肝炎和其他疾病中的作用。