| The family Filoviridae is comprised of two genera: Marburgvirus and Ebolavirus. To date minigenome systems have been developed for two Ebolavirus species (Reston ebolavirus and Zaire ebolavirus [ZEBOV]) as well as for Lake Victoria marburgvirus, the sole member of the Marburgvirus genus. The use of these minigenome systems has helped characterize functions for many viral proteins in both genera as well as having provided valuable insight towards the development of an infectious clone system in the case of ZEBOV. The recent development of two such infectious clone systems, one for ZEBOV and MARV now allow effective strategies for experimental mutagenesis to study the biology and pathogenesis of one of the most lethal human pathogens.;We also utilized this genetic system to characterize the gene mutation seen within the ZEBOV guinea pig adapted virus. In characterizing the gene mutations the nuclear protein in combination with VP24 mutations produced a viral variant, which was 100% lethal in guinea pigs. We also demonstrated that signal mutations could not produce lethal virus genotypes. We also generated a full length ZEBOV guinea pig adapted cDNA construct. This reverse genetic system is 100% lethal in guinea pig and is now a valuable tool in the study of filovirus pathogenicity.;In the development of diagnostic tools to help in the study of filoviruses, we generated a ZEBOV-GFP cDNA construct which expresses GFP in infected cells. Using this newly generated virus we determined the ability of ZEBOV-GFP to be detected in vivo and in vitro.;In order to better understand and optimize the reverse genetic system, we studied the relatedness of VP35, VP30 NP and viral polymerase (L) for their role in transcription and replication. We expressed the above mentioned proteins derived from Reston ebolavirus and Marburg virus, strain Musoke, using the chicken beta actin promoter. After optimizing the reverse genetic system (nearly 100% rescue), we studied the capacity of heterologous support proteins in virus rescue of Zaire ebolavirus, strain Mayinga. This was done by determining the expression of the heterologous protein(s) in the cell compared to virus rescue as determined by the cytopathogenic effect and virus characterization. |
