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 viral 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 continued efforts toward production and clinical testing of a parvovirus vaccine for humans. In collaboration with Phillip Dormitzer of Novartis (now GlaxoSmithKline) and Jane Hankins and Julia Horowitz of St. Jude Childrens Research Hospital, we are pursuing commercial development of a B19 parvovirus vaccine. Dormitzer and colleagues, based on previously published work from the Hematology Branch which is U.S. Government patent protected, have utilized a yeast expression system to generate empty capsids enriched in VP1 minor protein. These modified capsids elicit neutralizing antibodies in mice, as predicted from our earlier work with baculovirus-generated empty capsids. A three party agreement allowing production of empty capsids at St. Judes in their GMP facility will be funded by a novel consortium of the United States Government (industry, and an academic medical institution; shared by in-kind and direct contributions from these parties. The immediate Hematology Branch role will be a phase I/II trial in normal volunteers of yeast-generated empty capsids at multiple doses, with comparison to control and with and without MF59 adjuvant. A prediction is that, at adequate capsid concentrations, immunogenicity will be elicited in the absence of an adjuvant. The phase I/II trial will be conducted in the Hematology Branch clinic. If successful, especially if local cutaneous eruptions are not serious or frequent, and pending FDA tentative approval, empty capsids would be used to inoculate sickle cell disease patients, beginning with young adults and eventually enrolling older children, with a surrogate endpoint of neutralizing antibody production rather than clinical events. This study would be conducted in Memphis with collaboration on the part of the Hematology Branch in performing neutralizing antibody testing with our validated assays. 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. We reported isolation of putative viral sequences in samples of seronegative hepatitis obtained from a large infectious disease hospital in Chongqing, western China: utilizing Solexa deep sequencing, a novel virus was assembled in silico. Approximately 3800 base pairs in length, the virus was unusual in that the first open reading frame was related to circoviruses, while the second open reading frame shared homologies to parvoviruses. Unfortunately, NIH-CQV, a novel hybrid virus, proved to be a contaminant of the DNA isolation procedure. We have pursued the extent of the contamination and also its source. NIH-CQV sequences at low concentrations can be eluted from Qiagen purification columns, but contamination is highly variable, even within a single batch of columns. The virus is not present in human plasma or serum when ultra-pure Qiagen columns or other methods of DNA isolation are used. Although it has been speculated that source of the virus might be the silica itself, which is of diatomic biological origin, this is impossible given the extreme heating intrinsic to production process. However, the silica is extensively washed with water. In exhaustive searches of databases, we found NIH-CQV sequences within species of oomycetes, an organism that shares features with plant viruses, protozoa, and fungi. Of particular note, sequences were present in species of Phytophtora, which causes diseases such as potato blight and other similar syndromes in oak, chestnut, and other plants. Preliminary data suggests that the integrated sequences are secondary to active infection, as viral DNA sequences can be obtained from supernatants of oomycetes cultures. A second contaminating virus was also assembled in silico. Smaller than NIH-CQV, also not present in human samples, this virus has unusual features suggesting that it is a DNA/RNA hybrid. Extensive Phylogenetic analysis is underway in collaboration with Eugene Koonins laboratory in NIAID. However, overall results with deep sequencing of seronegative hepatitis do not suggest that this syndrome can be readily associated with a pathogen, even by the most sensitive methods, and these efforts will be abandoned.
