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Georgia Veterinary Scholar	Faculty Mentor	Faculty Mentor
		Dr. Bruce Seal   Southeast Poultry Research Laboratory, USDA-ARS
Jessica Robertson University of Georgia Class of 2005	Dr. Corrie Brown

Use of Anti-Peptide Antibodies and the Expressed Gene to Detect Newcastle Disease Phosphoproteins in Infected and Transfected Mammalian Cells

Highly virulent Newcastle disease virus (NDV) is a list A pathogen and requires reporting to the Office International des Epizooties. NDV, a negative sense single stranded RNA virus belonging to the Rubulavirus genus of the Paramyxovirinae, causes Newcastle disease in poultry. The NDV genome contains a 1.7 kb nucleoprotein (N) gene and a 1.4 kb phosphoprotein (P) gene. Located within the conserved central region of the P gene is a second potential in frame start site. This second ATG site could encode a predicted X protein and this protein was the focus of our study. The N protein (Seal et. al., 2002) and P protein genes (Locke et. al., 2000) from more than twenty NDV isolates were cloned and sequenced. From N and P consensus amino acid sequences an internal N peptide and an X peptide were synthesized. Subsequently, affinity purified antibodies from rabbits were obtained for detection purposes. Two proteins corresponding to the predicted molecular weights of P and X were detected by Western blot of NDV infected Vero cells using X antibodies. Western blot analysis of mammalian cells transfected with P and X gene segments also produced bands corresponding to the P and X proteins. Anti-peptide antibodies were utilized immunohistochemically to detect N and P peptides in NDV infected chicken tissues. The N and X peptides were reliably utilized as antigens in enzyme linked immunosorbent assays (ELISA) for detection of serum antibodies to NDV among chickens. Evaluation of ELISA results demonstrated that whole antigen ELISA is less sensitive than peptide antigen ELISA. Whole antigen ELISA resulted in false negative tests. Our results demonstrate that the second ATG site in the NDV phosphoprotein gene does serve as a functional start site for production of the X protein, and that it is expressed in NDV infected cells.