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.
