2007 Annual Report 1a.Objectives (from AD-416) The long-term goal of this research is the development of an assay system for viral infectious agents that is highly portable, relatively inexpensive, and easy to use by relatively untrained personnel. 1b.Approach (from AD-416) Develop of a number of assays for viral targets using Raman immunoassays and solution RNA hybridization assays. The basic principle of the Raman assays is that Raman resonance is enhanced when the dye is close proximity to a metal colloid. Standard ELISA and in solution hybridization assays will be modified to take utilize this novel detection system. West Nile virus and the related Saint Louis Enchephalitits viruses will be the specific targets for this project. 3.Progress Report This report documents research conducted under a Reimbursable Cooperative Agreement between ARS and Colorado State University. Additional details of research can be found in the report for the in-house associated projects 5410-32000-011-00D & 5410-32000-016-00D (new), Molecular Biology and Pathogenesis of Arboviruses. The goals of this project are to develop assay methods for the clinically similar West Nile Virus (WNV) and St. Louis Encephalitis (SLE) viruses, as well as the instrumentation that allows the assays to be carried out in the field and in laboratory settings. The basis of the assay methods to be investigated is surface enhanced Raman spectroscopy (SERS). It was proposed that the extraordinarily large signal enhancement attendant upon localization of some dyes at silver or gold surfaces would provide the sensitivity necessary for detection of WNV and SLE. The two assay formats chosen for examination were a nucleic acid hybridization (NAH) assay and an immunoassay. The overall project involves a three way collaboration: organic synthesis (UW), assay development (Animal Borne Diseases Research Lab (ABDRL) and the University of Wyoming (UW) and Raman instrumentation development to complement the assays (DeltaNu). We have demonstrated successful capture of a nucleic acid ternary (DNA/DNA/RNA) complex which contained hybridizing sequences identical to those proposed for the SERS-based NAH assay. However, as capture levels of the complex were considerably reduced relative to maximum calculated levels under the conditions employed and we encountered several technical problems endemic to working with Dynal™ paramagnetic beads, we saw no reason to pursue the optimization of a model system which differs in several critical features from the actual proposed assay. Therefore we developed a modified RNase Protection assay to evaluate specific oligonucleotides for use in the SERS WNV genome detection. This system has allowed us to improve hybridization conditions and identify optimal hybridization target sites in the genome. High background signals were noted in our initial SERS assays and modifications of the assay are in progress. Two WNV recombinant constructs have also been made expressing truncated regions of WNV envelope glycoprotein. These truncated proteins have been expressed and protein purification protocols developed. The assembled reagents for the WNV SERS immunoassay have been validated using ELISA techniques and "proof-of-concept" experiments are underway.