Studies of the interaction of hematopoietic cells and viruses have mainly concentrated on members of the Parvoviridae and with our interest in hepatitis-associated aplastic anemia, novel putative hepatitis agents. Parvovirus B19 infects erythroid progenitor cells and infection in humans causes both the hematologic syndromes transient aplastic crisis and pure red cell aplasia as well as the common childhood exanthem fifth disease. In order to understand more about disease pathogenesis, we have developed new methods to extract and detect infectious virus by realtime PCR and real time RT-PCR. In addition, we have developed an improved method for obtaining primary cells that are fully permissive for B19, and are working on developing cell lines based on these cells. Using our recently described infectious clone of B19, we have continued our studies of the molecular biology of narrow permissiveness of cells that can be infected with B19, and show that there appears to be multiple blocks, including at viral entry and capsid protein trafficking. In addition, the plasmid has been manipulated to knock out all the known and putative proteins, and the effects on protein trafficking, DNA replication and production of an infectious virus examined, including a previously unrecognized key role fo the small proteins encoded by the virus. With this new information, and our new method for culturing virus w e are investigating ways to produce a packaging system to produce recombinant B19 for further studies and possible gene therapy. Studies on the role of the capsid protein on viral entry, and the the role of the potent phospholipase A2 activity, continue.? ? Two new and distinctly different parvoviruses have been reported in the literature over the past year. The first, Parv4, has closest similarity to the erythrovirus group. We have cloned and expressed the capsid protein, and developed both DNA and antibody tests for detection of this new virus. Studies are ongoing to determine the viruses transcription map, site of replication in the body, and association with disease. The second virus, related to the animal bocaviruses is similarly being studied? ? Our studies to identify the etiological agent of hepatitis-associated aplastic anemia continue with the collection of epidemiological data and animal studies. This syndrome is more common in young males, and may be associated with soil or rodent contact. Distinctly different correlations of HAA with HLA types are observed than with aplastic anemia (AA) or PNH, with a decreased representation of HLA DR2. In contrast, there is an increased representation of the alleles of the ancestral haplotype 8.1, HLA B8, Cw7 and DQ2. There is clear evidence of an inflammatory response in the liver, with aberrant cytokine production confirmed by our RNAse protection and realtime PCR assays, and distinctly different from those observed in the livers of patients with viral hepatitis (hepatitis B and/or C). In addition, in collaboration with the BioInformatics group we continue to work on the design of a viral chip, to potently identify known and novel viruses in hepatitis-associated aplasia and other disease syndromes.
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