We have established a reverse genetics system that allows the generation of recombinant influenza viruses from plasmid cDNA. This system, together with the identification of gene sequences of the 1918 influenza isolates makes it now possible to study the molecular basis for the high pathogenesis of the 1918 pandemic virus. We will generate recombinant influenza viruses containing different genes of the 1918 virus in a high containment facility at the USDA. We will then focus our research efforts on characterizing the 1918 nueleoprotein, polymerase proteins and the regulatory non-coding regions with regard to viral replication and on understanding the potential role of these factors in determining pathogenicity. We will also investigate the molecular mechanism of the newly described pro-apoptotic influenza virus-encoded protein, PB1-F2 and the potential contribution of this protein (and specifically that of the 1918 virus) to determining virulence. Finally, we will investigate the molecular properties of genes from viruses circulating before and after 1918, in order to gain insight into their contribution towards the unusually high pathogenesis of the 1918 virus. Therefore, using innovative techniques such as allele-specific quantitative PCR, proteomics and microarray analyses, our goal is to further the understanding of the biological properties of the 1918 virus on a molecular level, which together with data from the other projects will provide us with a more complete picture of what determinants are critical for virulence.