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细小病毒：B19细小病毒是一种小的、无包膜的单链DNA病毒，是已知对人类致病性的唯一细小病毒科成员。B19细小病毒感染在儿童中很常见，大多数成年人都暴露于病毒，通过抗病毒IgG血清学检测确定。B19细小病毒是第五种疾病的病原，是一种儿童疾病；第五种疾病在成年期表现为慢性关节病。在血液学上，B19细小病毒引起几种疾病：溶血综合征的短暂性再生危象，导致严重的，有时是致命的急性贫血，如镰状细胞病患者；妊娠中期母亲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胎；慢性纯红细胞发育不全是由持续感染引起的，宿主无法产生足够的中和抗体反应。血液学分支在B19细小病毒研究方面的显著成就包括首次在细胞培养中繁殖；详细的转录图谱的阐明，导致病毒重新分类到一个新的属；细胞受体、糖苷或P抗原的鉴定，并确定受体的遗传缺失导致体外和体内的不敏感；描述了VP1独特区域上存在的中和性表位，这些表位位于衣壳表面外；以及基于杆状病毒系统中B19衣壳蛋白的表达和随后将蛋白自组装成空衣壳的重组候选疫苗的生产，通过调整VP1的含量来最大限度地中和动物和人类的抗体反应。近年来，该科的研究人员还开发了强大的工具，用于研究组织培养中的B19细小病毒：这是一种传染性克隆，可以在核苷酸水平上对病毒蛋白进行修饰，从而对结构-功能关系进行详细的分子定位，并利用从正常人类志愿者身上获得的CD34细胞进行红系分化，作为生产细胞培养系统的基础，允许病毒在生理条件下繁殖。