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.
