2009 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 The project is related to objective 2 of the in-house project: Development of improved Newcastle disease control strategies addressing issues important to virus transmission, vaccines and vaccination, diagnostics, or international trade. Develop models to show vaccine is a viable method of controlling avian paramyxovirus outbreaks. During the last year a number of advances have been made in the development of our technology for imaging ribonucleic acid (RNA) at the single molecule level[1]. Just as a review, multiply-labeled tetravalent RNA imaging probes were delivered into live cells via reversible cell membrane permeabilization that bind rapidly to RNA (<10 minutes) and allow for single RNA sensitivity using conventional fluorescence microscopy techniques. We have used a streptavidin linkage to increase the brightness of the probe four-fold. The achieved brightness of these probes is imperative for RNA imaging. These probes can also be used in fixed cells or tissue to detect RNA using standard fluorescence in-situ hybridization approaches, but are single RNA sensitive, a major advantage over other techniques. A goal for the coming year will be to apply these probes to the detection of Newcastle disease virus (NDV) viral genomic RNA in both live cells and fixed tissue samples. One possible difficulty in staining RNA in formaldehyde fixed tissue is the lack of accessibility of the RNA, especially if it is in inclusion bodies. Our strategy to overcome this challenge will be to employ antigen retrieval strategies, including the use of proteases, and to increase the affinity of our probes. MONITORING: Continued communication between Southeast Poultry Research Laboratory and Georgia Technical Institute has been accomplished through e-mail, phone calls and visits.