Grant: $325,042 - National Institutes of Health - Jul. 15, 2009
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Award Description: The broad long-term objectives of the application are to determine the mechanisms by which the Severe Acute Respiratory Syndrome (SARS)-associated coronavirus (SCoV) causes acute lung injury and to identify new therapeutic strategies. The central hypothesis of this application is that the pathogenicity of SCoV is caused, at least in part, by the expression of factor(s) that interfere with the host response to viral lung injury. This hypothesis is based on our combined observations that a) expression of surfactant protein-B and -C, which is essential for lung function, is decreased in the lungs of SARS patients, b) SCoV inhibits both virus- and interferon-dependent signaling, and c) a non-structural protein of SCoV recapitulates in isolation the inhibitions observed in SCoV-infected cells, and also inhibits cell proliferation, which is required for alveolar repair. Aim#1: analyze the response of lung alveolar cells to SCoV infection; Aim #2: identify the mechanism by which SCoV impedes the function of lung alveolar cells; and Aim #3: produce and validate attenuated SCoVs. The research design involves the analysis of viral replication, virus- and interferon- dependent signaling, surfactant protein expression and cellular proliferation in SCoV-infected cells. This analysis will be conducted in vivo, using lung tissues from SARS cases and from experimentally infected cynomolgus macaques; and in vitro, using primary human alveolar cells and lung cell lines. The role of the non-structural protein in virulence will be tested by introducing inactivating mutations in the context of recombinant SCoVs by reverse genetics. Viable mutants will be propagated in interferon-deficient cells. Their attenuated character will be determined by comparison to wild-type virus in vitro and in vivo, using cynomolgus macaques as the model organism. Together, the proposed investigations are expected to uncover how SCoV causes acute lung injury and to identify promising therapeutic targets. We expect that our studies on SCoV virulence will serve as a model for other human coronaviruses and respiratory viruses that evade the innate immune response. Understanding this mechanism of SCoV virulence is significant for public health because it will provide knowledge essential for the development of new therapeutic strategies to fight SARS
Project Description: Constructed tetracyclin-regulated retroviral vectors for expression of SARS-CoV nsp1 and other interferon antagonists (EBOV VP35, EBOV VP24, BUNV NSs), and for expression of T7 RNA polymerase. Produced retroviral vector preparations in GP2-293 cells for Tet-Off regulation, T7 RNA polymerase, SARS-CoV nsp1, EBOV VP35, EBOV VP24 and BUNV NSs expression. Transduced cell lines of interest for SARS-CoV production first with Tet-Off vector, then with T7 RNA polymerase vector (cell lines transduced so far: VeroE6, Calu3, 17Cl1, BHK21). Selected cell lines with antibiotics (G418 and Hygromycin B), all successfully obtained. Testing of the cell lines for inducible T7 RNA polymerase expression and for suitability to produce recombinant SARS-CoV from transfected DNA is in progress.
Jobs Summary: Dr. Savalia will help produce SARS-CoV mutants using reverse genetics and analyze their phenotype in terms of replication and ability to evade the host antiviral response; in addition, Dr. Savalia will help purify and culture human alveolar epithelial cells and characterize their ability to be infected by and support the replication of SARS-CoV. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 15, 2009. Help expand these official descriptions using the wiki below.