Entry of coronaviruses into host cells is an under-explored area of virology research. In this application, we propose to use infectious bronchitis virus (IBV) of chickens as a model that would be directly applicable to highly pathogenic human coronaviruses, such as SARS-CoV. The major goal of this application is to define basic information regarding the fusion mechanism and route of internalization of IBV. Certain important gaps remain in our understanding of coronavirus entry: specifically 1) the fact that, to date, no virus--cell fusion assays have been employed to study coronavirus entry, and 2) although coronaviruses are generally considered to undergo cell--cell fusion at neutral pH, previous data also suggest a pH-dependent route of entry through endosomes. To address these issues, we have two specific aims: 1 - To develop a virus-cell fusion assay for coronaviruses. Using protocols in our laboratory to study influenza virus--cell fusion, a principal goal is to develop a fluorescence-based virus-cell fusion assay for the coronavirus IBV. 2 - To determine the internalization route of IBV into host cells. We have previously characterized the entry of other viruses using a combination of molecular, pharmacological and morphological approaches. Using similar techniques, a second principal goal is to define the role of endocytosis during IBV entry. Our work is designed not only to elucidate the basic entry mechanism of IBV, but also to serve as a model for coronaviruses in general--especially highly pathogenic human coronaviruses, such as SARS-CoV. IBV would serve as an excellent model system to understand coronavirus entry and fusion, and facilitate the development of future anti-viral drugs. ? ?
