| The genus Ebolavirus, in the family Filoviridae, is divided into four species: Zaire, Sudan, Ivory Coast and Reston. With the exception of Reston ebolavirus (REBOV), all filoviruses cause severe haemorrhagic fever in both human and non-human primates. A molecular basis for this difference in pathogenicity has not yet been established.; Preliminary work for this study involved sequence determination for REBOV. As expected, based on the dramatic phenotypic differences, the full-length sequence of REBOV differed substantially at both the nucleotide and amino acid levels from more pathogenic filoviruses; however, it is difficult to speculate on the role of such differences, particularly in light of the limited functional data available for filovirus proteins. Based on these data we were able to develop a minigenome system for REBOV, which models many aspects of the viral lifecycle (i.e. encapsidation, transcription, replication and packaging). Using this system it was observed that the ability of the RNP proteins to mediate transcription from a given template correlated with the in vivo pathogenicity of the virus from which they were derived. Data also suggest that the transcriptional requirement for VP30 by Ebola viruses may be dependent on characteristics of the RNP complex, and not solely on the presence of genomic hairpins, which have been previously reported to play a role in this process.; We have also applied our minigenome system as a screening tool for siRNAs that inhibit transcription/replication of either REBOV, or the highly pathogenic Zaire ebolavirus (ZEBOV). We found that the ZEBOV siRNAs were also capable of inhibiting virus growth in cell culture as well as in a mouse model of infection. Here, treatment with siRNA decreased weight loss, a sensitive indicator of illness, suggesting that further testing in a lethal model is warranted and that siRNAs have potential for development as a therapeutic measure.; Finally, based on information obtained during this study, an infectious clone system for REBOV is now being constructed. Once this system becomes available, it will be possible to generate REBOV entirely from cloned cDNA. This will make it possible to study recombinant viruses and thereby allowing us to determine the role of different virus proteins and/or genomic elements in pathogenesis, particularly when complementary recombinants are evaluated in the pathogenic ZEBOV infectious clone system. |
