PROJECT SUMMARY: Humans are the only natural reservoir for the extremely contagious measles virus (MeV). Thus, a critical challenge for MeV study is identification of representative human model systems. For decades, MeV was thought to enter the human host through the apical surface of airway cells, a misconception based on studies in immortalized cell lines. Well-differentiated primary cultures of airways epithelial cells from human donors (HAE) provide a more physiological relevant model of human airways. Using HAE, we found that MeV exclusively enters the basolateral membrane. This observation lead to a completely new paradigm for how MeV enters the human host. In addition to basolateral entry, we observed that MeV infection of HAE results in the formation of infectious centers that retain intact plasma membranes and are substantially different than the syncytia observed in immortalized cells. Infectious centers differ from syncytia in two important ways: 1) infectious centers stop growing 3-4 days post-infection and 2) infectious centers disappear after ~10 days leaving the cell layer intact. Why infectious centers stop growing and how they disappear in HAE remain a mystery and is the focus of this application. We hypothesize that infectious center formation in the respiratory epithelium is a vital step in the final amplification process before release to the next host. In Aim 1, we define the innate immune response pathways in the airways. We quantify 14 antiviral sentinel genes at 12 timepoints ranging from 6 hours to 2 weeks. In addition, laser capture of infectious centers is used to isolate infected cells from uninfected cells within an epithelial sheet and deep sequencing is used to map the cellular response to MeV. In Aim 2, we address how MeV is released from HAE. Preliminary data suggest that infectious centers are shed, intact, from the HAE (rather than rupturing). The timecourse and frequency of shedding will be defined. The roles of cell proliferation and cell death pathways will be probed by cell labeling to discern how large infectious centers can be released yet the epithelial integrity is maintained. MeV mediated cytoskeletal modifications are likely to be mechanistically involved in infectious center release, which we test using inhibitors of F-actin treadmilling. In Aim 3, we hypothesize that shed infectious centers are physiologically relevant vectors for MeV delivery. We will deliver cell-associated and cell-free MeV to the airways of rhesus macaques and quantify the time-course of infection. This aim has the potential to reshape a fundamental dogma of how MeV is spread host-to-host. In summary, these studies use an appropriate model system to study MeV entry, spread, and luminal release. Our research will elucidate mechanisms by which the most contagious human respiratory virus undergoes its final amplification step before release to its next host.