Viruses and Hematopoiesis

病毒和造血作用

• 批准号: 8746643
• 负责人: NEAL S YOUNG
• 金额: $ 111.28万
• 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/8746643
• 关键词: Achievement; Acute; Acute Hepatitis; Adult; Anemia; Animals; Annual Reports; Antibodies; Antibody Formation; Antigens; Aplastic Anemia; Asians; Baculoviruses; Base Pairing; Bilirubin; Biochemical; Biological Assay; Blast Cell; Blood; Bone Marrow; CD34 gene; Capsid; Capsid Proteins; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Separation; Cells; Centers for Disease Control and Prevention (U.S.); Cerebrospinal Fluid; Child; Childhood; Chimera organism; China; Chinese Hamster Ovary Cell; Chiroptera; Chronic; Circoviridae; Circovirus; Cities; Clinic; Clinical; Collaborations; Communicable Diseases; Congestive Heart Failure; Data; Disease; Encephalitis; Epidemic; Epidemiologic Studies; Epitopes; Erythema Infectiosum; Etiology; Exanthema; Failure; Family; Fetus; Functional disorder; Gene Amplification; Genes; Genetic; Genetic Transcription; Genome; Globosides; Goals; Hematology; Hematopoiesis; Hematopoietic; Hepatitis; Hepatitis C; Hepatitis Viruses; Hospitals; Human; Human Parvovirus B19; Human Volunteers; Hydrops Fetalis; Immune; Immune system; Immunoblotting; Immunoglobulin G; Immunoglobulin M; In Vitro; Infection; Infectious Agent; Influenza; Institution; Kenya; Laboratories; Liver; Maps; Mediating; Methodology; Methods; Modification; Molecular; Mothers; Mutate; Nucleotides; Open Reading Frames; Pancytopenia; Parvoviridae; Parvovirus; Parvovirus Infections; Pathogenesis; Patients; Pattern; Phase; Phase I Clinical Trials; Phase II/III Trial; Phospholipase; Phylogenetic Analysis; Physiological; Plasmids; Plasmodium; Porcine Parvovirus; Prevention; Production; Proteins; Publishing; Pure Red-Cell Aplasia; Recombinant Vaccines; Recombinants; Research; Research Personnel; Risk Factors; Saint Jude Children' s Research Hospital; Sampling; Second Pregnancy Trimester; Serological; Serum; Sickle Cell Anemia; Single Stranded DNA Virus; Specimen; Structural Protein; Structure; Structure-Activity Relationship; Surface; Suspension substance; Suspensions; Syndrome; System; Toxic effect; Transaminases; United States; United States National Institutes of Health; Vaccines; Viral; Viral Proteins; Viral hepatitis; Virus; Virus Diseases; Yeasts; Zinc; arthropathies; base; deep sequencing; erythroid differentiation; in utero; in vivo; influenza virus vaccine; interest; liver transplantation; male; member; neutralizing antibody; particle; phase 1 study; receptor; research clinical testing; self assembly; stillbirth; tissue culture; tool; vaccine candidate

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 continued efforts toward production and clinical testing of a parvovirus vaccine for humans. We are collaborating with Phillip Dormitzer of Novartis and investigators at St. Jude Childrens Hospital in utilizing the Novartis yeast methodology for empty capsid production. The eventual aim is a Phase I study of yeast derived empty capsids, enriched for VP1 structural protein, in normal adults to be conducted at the Clinical Center by the Hematology Branch, utilizing the method recently published by Novartis and the GMP facility in Memphis. If no serious toxicity were observed and neutralizing antibodies were elicited, the phase I trial would be followed a phase II/III trial at St. Jude in children with sickle cell disease. Financial constraints have retarded moving forward on this project to date. In our laboratory, we have successfully established four suspension CHO cell lines for stable expression of B19 parvovirus-like particles. These include particles containing VP1, VP1 with the phospholipase gene mutated, and two B19-influenza chimeras: VP1-M2 and VP1-HA2. The latter are developed with the goal of a universal influenza vaccine on a platform of parvovirus capsids. Optimization of expression of the empty capsids was achieved through multiple rounds cell sorting. B19 capsids have been purified using Opti-Prep gradient centrification. Antigenicity and structure were confirmed with immunoblot and by electromicroscopy. To enhance and adjust for expression levels of VP1, which are critical for neutralizing antibody production, we are currently modifying the cell lines using zinc-inducible recombinant plasmids for the appropriate genes. 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 Chongqing in the west of China. Ninety-two serum specimens were collected from patients with seronegative hepatitis between 1999 and 2007. Ten serapools were screened by Selexa deep sequencing. We discovered a 3780 base pair contig present in all ten pools that yielded high blast xe scores against paroviruses. The complete sequence of the assembled contigs was confirmed by gene amplification of overlapping regions over almost the entire genome. This virus has been designated NIH-CQV provisionally. The contig is composed of two major open reading frames. ORF1 was most homologous to the replication associated protein of bat circovirus, and ORF2 to the capsid protein of porcine parvovirus. Phylogenetic analysis shows that NIH-CQV is at the interface of the Parvoviridae and Circoviridae. The prevelance of NIH-CQV in patients was determined by quantitative PCR: 63 of 90 patient samples (70%) were positive while all 45 healthy controls were negative for virus by this sensitive method. The average virus titer in patient specimens was approximately 1x104copy/ml. Specific antibodies directed to NIH-CQV were detected by immunoblotting. In patients, 84% were positive for IgG and 31% were positive for IgM; in contrast, 78% of healthy controls were positive IgG but none for IgM. These data are highly suggestive that NIH-CQV is a pathogenic virus in humans, and epidemiologic studies in China will be conducted in collaboration with the Chongqing group and others in various cities in order to determine the presence and prevelance of virus and antibodies. Using the same methods, we are currently analyzing specimens from Kenya in collaboration with Joel Montgomery of the CDC in Nairobi. Results to date are suggestive that plasmodium species may be implicated in epidemics of undiagnosed acute encephalitis, based on deep sequencing of cerebral spinal fluid 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。 我们一直在为人类的细节疫苗生产和临床测试而努力。 我们正在与诺华的Phillip Dormitzer合作，并在圣裘德儿童医院的调查员使用Novartis酵母方法来进行空的Capsid生产。 最终的目的是对酵母衍生的空衣壳的第一阶段研究，富含VP1结构蛋白，在正常的成年人中，由血液学分支在临床中心进行，利用诺华最近出版的方法和孟菲斯的GMP设施。 如果未观察到严重的毒性并引起中和抗体，则将在圣裘德（St. Jude）进行镰状细胞病儿童的II/III期试验之后。 迄今为止，财务限制已延续了该项目。 在我们的实验室中，我们成功地建立了四种悬浮液细胞系，用于稳定地表达B19细小病毒样颗粒。 这些包括含有VP1，VP1突变的VP1，VP1的颗粒，以及两个B19-Influenza Chimeras：VP1-M2和VP1-HA2。 后者的开发是在细小病毒capsids平台上进行通用流感疫苗的目标。 通过多个圆形细胞分类实现了空衣壳的表达的优化。 B19的衣壳已使用Opti-Prep梯度中心化纯化。 通过免疫印迹和电显微镜证实抗原性和结构。 为了增强和调整VP1的表达水平，这对于中和抗体的产生至关重要，我们目前正在使用适当基因的锌诱导重组质粒修饰细胞系。 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深度测序，我们从中国西部的重庆大型传染病医院获得的样品中有分离的病毒序列。 在1999年至2007年之间，从血清肝炎的患者中收集了92个血清标本。通过SELEXA深测序筛选了十个Serapools。 我们在所有十个池中发现了一个3780个基对重叠群，该重叠群在针对parovirus的所有十个池中产生了高XE XE得分。 通过在几乎整个基因组上重叠区域的基因扩增，组装重叠群的完整序列被证实。 该病毒已被临时指定为NIH-CQV。 重叠群由两个主要的开放式阅读帧组成。 ORF1与蝙蝠病毒的复制蛋白和猪capsid蛋白的复制蛋白是最同源的。 系统发育分析表明，NIH-CQV位于parvoviridae和Circoviridae的界面。 通过定量PCR确定NIH-CQV在患者中的良好症状：90例患者样本中的63个（70％）为阳性，而通过这种敏感方法，所有45个健康对照对病毒均为阴性。 患者标本中的平均病毒滴度约为1x104copy/ml。 通过免疫印迹检测到针对NIH-CQV的特异性抗体。 在患者中，IgG的84％为阳性，IgM为阳性31％。相比之下，78％的健康对照是阳性IgG，但不适合IgM。 这些数据高度暗示，NIH-CQV是人类中的一种致病病毒，并且将与重庆群体以及各个城市的其他人合作进行中国的流行病学研究，以确定病毒和抗体的存在和prect。 使用相同的方法，我们目前正在与内罗毕的CDC的Joel Montgomery合作分析肯尼亚的标本。 迄今为止，基于脑脊髓液样本的深层测序，迄今为止的结果暗示了疟原虫可能与未诊断的急性脑炎的流行相关。