B19 Parvovirus: B19 parvovirus is a small, nonenveloped, single-stranded DNA virus, the only member of the Parvoveridae 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 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, laboratory successes concerning B19 parvovirus ranged from detailed analysis of its molecular biology to vaccine studies in humans. For the former, we pursued our observations, made using extensive microarray analysis for gene transcription, of the hijacking of the erythroid program in native human CD36 erythroid progenitor cells shortly after B19 infection. We found that B19 parvovirus exploits the E2F master family of transcription factors, down-regulating activating E2Fs (E2F1-E2F3a) and up-regulating repressive E2Fs (E2F4-E2F8). These effects are mediated by the major nonstructural protein (NS1), which is expressed early after B19 parvovirus entry into the cell. NS1 altered E2F1-E2F5 expression;interaction between NS1 and E2F4 or E2F5 enhanced nuclear transport of these repressive E2Fs and induced stable G2 arrest of the cell cycle. G2 arrest was independent of P53 activation and increased viral replication. Downstream E2F4/E2F5 targets, involved in progression from G2 to M phase and subsequent erythroid differentiation, were identified by microarray analysis. These results provide new insights into molecular events following of B19 parvovirus infection of its native target cells. Second, as an extension of our previous findings that CD34 cell-derived human CD36 erythroid progenitor cells were highly permissive for B19 parvovirus infection, we derived a transformed cell line by manipulation of CD34 cells. CD34 cells were propagated in serum-free media and exposed to appropriate growth factors, including erythropoietin, to force erythroid differentiation. Lentiviral vectors carrying SV40T antigen, the telomerase gene, or human papilloma virus type 16 E6 and E7 viral oncogenes were introduced into CD36 cells, singularly or in combination. HPV16E6-E7 genes successfully transformed CD36 erythroid progenitor cells to a continuous cell line with immunophenotypic, colony forming, and hemoglobinization properties of normal erythroid cells, although with marked differences by transcriptional array profiling. Such a cell line should be useful in many studies of erythropoiesis, including for B19 parvovirus infection. Finally, studies have continued of B19 empty capsids as vaccine reagents. We obtained samples from an NIAID-sponsored phase I protocol, in which human volunteers were inoculated with B19 parvovirus empty capsids in order to elicit a neutralizing antibody response. We showed that such antibodies were indeed formed in volunteers who received 5 or 25 micrograms of B19 empty capsids in SF59 adjuvent. In other work, towards rapid introduction of a vaccine into the clinic, codon usage was altered for transfected B19 parvovirus sequence to allow its full expression in mammalian cell lines, including Chinese hamster ovary cells. Alteration of codons to more favorably exploit tRNAs abundant in mammalian cells was accomplished, with production of empty capsids;these capsids appear to be physically and biochemically identical to those made in insect cells. A patent has been filed for this novel modification of a strategy to more conveniently generate recombinant parvovirus vaccine. 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 serumtransaminases;pancytopenia is profound and historically almost always fatal. Due to inability to isolate, using a wide variety of molecular, immunological and biochemical methods, a putative infectious agent from either bone marrow or blood in 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 with acute 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. The number of sequences present in both control normal sera and in patient material was large and not easily distinguished. Further efforts based on Solexa sequencing with more refined computational ability are underway.
