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. B19 DNA can be detected in other tissues, including liver and synovium and we extended our studies to define diseases caused by parvovirus B19, especially the role of B19 as a cause of hepatitis and rheumatoid arthritis. We previously identified a new variant of B19, A6, with ~10 % sequence variability from other B19 sequences, including the recently described V9 variant, and showed that these variants can be detected in liver tissue from patients with or without seronegative hepatitis. We continued our studies of the molecular biology of B19 replication, especially the marked narrow permissiveness of cells that can be infected with B19. We show that this does not appear to be at the level of transcription, but at the level of viral entry and/or DNA replication, and suggest the presence of a second B19 receptor in addition to globoside. We have expressed the VP1-unique region of the capsid protein and confirmed that it has potent phospholipase A2 activity. We are performing structural and binding studies on this protein. Previous investigations of B19 have been hampered by inability to culture the virus efficiently or lack of an infectious clone. Using low temperature culture and bacteria without recombinases we successfully produced a plasmid containing the full-length B19 genome, including the long terminal repeat sequences that have not been cloned previously. We confirmed that the plasmid produced infectious B19 in a series of transfection and infection studies of permissive cell lines. In addition we showed that we could manipulate the plasmid by the insertion of an artificial mutation in the B19 genome, and that this sequence change is present in infectious virus progeny. This plasmid will be a valuable tool for reverse genetics to enable more studies of the molecular biology of B19, and for the production of infectious recombinant B19 particles for further investigation of viral entry. 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, and marked Va skewing by spectratype analysis. Treatment with immunosuppression returns the skewed pattern to normal. Animal and tissue culture studies to identify the etiological factor continue, including the inoculation of tissue and serum into rodents and primates. Currently we are looking at the immune profile of livers from patients with hepatitis-aplasia and controls using a variety of methods including gene chip analysis, RNAse protection assays, immuno-spectratyping and realtime PCR.
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