In the winter of 2002-2003 a new viral pathogen, severe acute respiratory syndrome coronavirus (SARS-CoV), rapidly spread across 5 continents, infected over 8,000 people, and caused 774 deaths. The re-emergence of this virus is likely, and much remains to be learned about the molecular basis for its pathogenesis. The most significant organ system affected in fatal SARS cases is the respiratory tract, and of existing animal models, the cynomolgus macaque (Macaca fascicularis) best replicates the pulmonary pathogenesis associated with human SARS. The macaque model was used to prove that SARS-CoV is the primary etiologic agent of SARS, and that type-1 and -2 pneumocytes may be important target cells for SARS-CoV. In this application, our goal is to use the macaque model, primary cell culture systems, infectious molecular SARS-CoV clones, and advanced genomic technologies, to increase our knowledge of SARS-CoV pulmonary pathogenesis. In Specific Aim 1 we will use the macaque model to evaluate the pathogenesis of SARS-CoV and the development of lung disease. We intend to refine the identification of target cells permissive for SARS-CoV and track possible changes in their distribution at different times after infection. The role of age,an important host factor in the severity SARS, will be examined by comparing the development of SARS in macaques of different ages. Macaques will be infected with molecularly cloned wild-type SARS-CoV and SARS-CoV containing mutations in specific accessory genes that may contribute to virulence. Specific Aim 2 will focus on the establishment of an appropriate cell culture system in which to evaluate the cellular response to wild-type and engineered SARS-CoV. This will include studies with < [unreadable][unreadable] i primary human bronchial and small airway epithelial cells. We also will attempt to infect -macaque primary . type-2 pneumocytes. Finally, Specific Aim 3 will determine molecular meehanisms<and.pathogehic;<:,, So ti signatures of SARS using global gene expression profiling. We will use humaitand;macaque using ck-oai .-; oligonucleotide microarrays and sophisticated information technologies to examin&igene;expression; -UKJ :o; changes in response to SARS-CoV infection. Experiments will be performed using RNA-isolated from in vitro infections and from cells and tissues from experimentally infected macaques. Collectively, data obtained from these studies will provide a better understanding of how SARS-CoV causes lung disease. Such knowledge may lead to new diagnostic, therapeutic, or vaccine strategies, which may serve as important countermeasures to future SARS outbreaks.