Project Summary/Abstract The emergence of pathogenic viruses represents continuous infectious disease threats to public health. Among these, the paramyxoviruses, which include many important human and animal pathogens, also include two excellent examples of emerged, zoonotic pathogens of importance: the henipaviruses; Hendra virus (HeV) and Nipah virus (NiV). HeV and NiV have a uniquely broad host tropism capable of infecting at least 18 animal species across 6 orders of mammals. HeV and NiV can also cause a systemic and often fatal respiratory and/or neurological disease in 11 mammalian species including humans. These henipaviruses remain significant biothreats to humans and economically important livestock in Australia and throughout South East Asia, and there are no vaccines or antivirals approved for human use. The henipaviruses are single-stranded, negative sense, enveloped RNA viruses and possess two membrane anchored glycoproteins involved in virus entry, one for host cell receptor attachment (G glycoprotein) and the other a fusion (F) glycoprotein which facilitates virion and host cell membrane fusion. The G and F glycoproteins are the major antigenic targets of neutralizing antibodies and also the focus of several vaccine strategies. In contrast however, antiviral drug discovery for HeV and NiV has been significantly hampered because of the requirements of biosafety level-4 (BSL-4) containment, and presently there is a complete lack of any effective antiviral therapeutics against henipaviruses for human use. We have been extensively characterizing the recently identified non-pathogenic species of henipavirus, Cedar virus (CedPV). Using recombinant viral glycoprotein mediated cell-cell fusion assays, we have discovered that CedPV-mediated membrane fusion is similar to HeV and NiV, however, the ephrin receptor tropism of CedPV was found to be remarkably broad and fusion could be triggered by ephrin- B1 and -B2 as well as the glycophosphatidylinositol-anchored A subtype ephrins-A1, -A2, and -A5. The cell- cell fusion activity supported by each of these ephrin receptors also correlated with CedPV G binding ability as measured by surface plasmon resonance. Further, we have recently rescued recombinant CedPV encoding eGFP (rCedPV-GFP) using a reverse genetics approach. Our rCedPV platform represents a new virological system that can be used in a variety of applications to study various aspects of henipavirus cell biology and host cell interactions safely under BSL-2 containment. But of further importance, it is also an authentic henipavirus infection and replication reporter system now suitable for high-throughput screening (HTS) for the discovery of potentially pan-anti-henipavirus molecules. Using this new live-henipavirus platform, our objectives here will be to develop and optimize a rCedPV reporter system and utilize it in an HTS assay to discover antiviral molecules capable of inhibiting the infection of henipaviruses. Specifically, we will: 1) Adapt and develop CedPV reporters for the HTS assay; 2) Optimize the HTS parameters of recombinant virus infection and reporter activities; and 3) Pilot a HTS assay using the Prestwick Chemical Library.