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。 我们已经追求了以前发表的观察到E2F转录因子的作用，该因子的作用着重于11 kD的非结构蛋白，该蛋白诱导CD36红细胞祖细胞靶细胞中的凋亡。 我们用siRNA构建了重组慢病毒，以靶向EF2K。这些siRNA击倒EF2K的水平。 我们目前正在测试EF2K缺乏是否可以预防CD36细胞中的凋亡。 此外，序列分析表明，11 kD蛋白富含脯氨酸，包含四个SH3结合基序（PPXP）。 我们创建了具有变化的PPXP域的串行11 kD突变体。 在两个远端SH3结合基序中取代突变消除了11 kD凋亡活性。 我们假设B19 11-KD蛋白与宿主SH3结构域之间的相互作用导致EF2K激活和B19靶细胞的凋亡。 该假设正在功能上检验。 我们先前发现，B19衣壳基因的密码子优化增加了非抗药性细胞中病毒capsid蛋白的产生。 为了产生大量的B19空病毒包囊进行疫苗开发，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表达在Qmate-诱导的启动子的控制之下。 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，我们通过构建突变体VP1基因取代D175a来消除酶活性。 我们已经建立了两种哺乳动物细胞系，它们稳定地表达了天然或PLA2阴性突变体VP1或VP2衣壳蛋白。 在这些细胞线中，免疫印迹显示1：5的VP1/VP2 rratio。 B19空的衣壳已通过OptiPrep密度分级核心纯化，直接的电显微镜显示出典型的细小病毒样颗粒。 为了再次产生其他主要病毒病原体，我们通过插入血凝集素区域或流感病毒的M2蛋白来修饰构建体中。 这些流感蛋白会引起广泛中和抗体。 嵌合空的衣壳可能可用于开发普遍的流感疫苗，以及可引起对流感和细小病毒的抗体的疫苗。 早期结果表明，来自转染细胞的纯化的空衣壳含有带有两个病毒抗原的颗粒。 我们目前正在努力建立稳定的CHO细胞系，能够产生大量的空帽质以进行动物免疫。 病毒感染和性贫血：已经反复出现了识别血清神经肝炎病毒病因（非A，非B和非C）。 尽管在美国很少有血清肝炎，但在亚洲诊所中，多达20％的肝炎病例具有血清症。 血清症急性肝炎与已知的病毒肝炎不同，其人口特征和临床后果。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：患者通常是男性，通常是年轻的，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。由于无法通过使用多种分子，免疫学和生化方法分离出推定的感染剂，从骨髓或血液的血液或血液的血液中或从接受肝脏移植的患者中获得的肝炎患者获得的肝脏样本中，我们已经与其他患者进行了绩效，从而获得了劳累的疗程。 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。 使用Solexa深层测序，我们从中国西部的一家大型传染病医院获得的样品中有分离的病毒序列。 重叠群已组装成大约3500个碱基对的完整病毒序列。 序列分析表明一种非结构蛋白和衣壳蛋白结构域，类似于细小病毒，但在核苷酸或氨基酸序列中的同源性相对较低。 非capsid蛋白具有圆环病毒的特征，而衣壳蛋白更类似于细小病毒。 大多数血清肝炎患者中，病毒序列平均存在约1,000至10,000个基因组副本。 在当代健康的血库供体中，无法通过基因扩增来检测序列。 免疫印迹显示了大多数肝炎患者中对衣壳蛋白的IgM抗体的存在，并且在患者和对照组中都存在IgG抗体。 在蔗糖密度垫上纯化后，通过电显微镜检查可视化颗粒。 我们的结果表明，在Circoviridae和Parvoviridae之间的遗传学上是一种新型病毒，并可能与疾病相关。 当前的努力针对各种细胞中病毒蛋白的免疫染色和表达，包括建造传染性克隆。 需要使用来自中国和其他地方的样本来确定该病毒的作用，称为NIH-CQ，在人类肝炎和其他疾病中，需要使用血清杂种学研究。