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. 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.