Viruses and Hematopoiesis

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。

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.