2008 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research project is to develop new fluorescent-based viral detection reagents for the study of Newcastle disease virus. 1b.Approach (from AD-416) Fluorescent molecular beacon-based probes targeted to the viral RNA will be used to analyse Newcastle Disease Virus replication. This approach allows for the analysis of the basic replication and assembly processes in four dimensions; involving space (x,y,z) and time. Initial studies will focus on the use of vaccine strain of NDV investigated in chicken embryo fibroblasts, while future studies will progress to mesogenic and velogenic strains for comparison. 3.Progress Report This project is related to Objective 1 of the in-house project: Identify determinants of virulence, tissue tropism and host range of avian paramyxoviruses. In this cooperative relationship we are working to create imaging probes that will allow the study and detection of Newcastle disease virus (NDV) ribonucleic acid (RNA) in living cells. Our initial choice of probe was the molecular beacon. Due to insufficient sensitivity, we sought to design a new class of RNA imaging probes which have single molecule sensitivity, and are capable of imaging multiple species of RNA using standard microscopy technology. To achieve these goals, we sought to significantly improve on the general approach of using exogenous oligonucleotides as probes for imaging endogenous or non-plasmid derived RNA in live cells. The new probe we designed consists of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage. This approach allowed for the development of a multivalent, highly sensitive and versatile imaging probe we’ve designated MTRIP (multiply-labeled tetravalent RNA imaging probe) when delivered via cell membrane permeabilization with streptolysin O (SLO), bound rapidly to RNA (<10 minutes) and allowed for single RNA sensitivity using conventional fluorescence microscopy techniques, both epifluorescence and spinning disk confocal. Target RNA was identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA or through the natural accumulation of RNA in granules. In this way, the identification of target was achieved in an analogous way to that of the green-fluorescent protein (GFP)-RNA binding protein systems, but utilized native target sequences and significantly fewer binding sites. In addition, due to the low molecular weight, small (<10 nm, estimated from molecular structures) diameter, and single probe sensitivity, interference with normal RNA processes and dynamics, compared with other methods, should be mitigated. We have also shown the ability to image single probes in the cytoplasm of living cells. Next, we will make an MTRIP probe against the NDV RNA genome and demonstrate the ability to image viral RNA with single molecule sensitivity. MONITORING: The Authorized Departmental Officer's Deisgnated Representative (ADODR) had regular email and telephone conversations with the cooperator at the University of Georgia on research planning and twice a year visits to discuss planning in detail.