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Journal of Clinical Microbiology, September 2001, p. 3171-3178, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3171-3178.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Use of a Heteroduplex Mobility Assay To Detect Differences in the Fusion Protein Cleavage Site Coding Sequence among Newcastle Disease Virus Isolates

Analia Berinstein,1,2 Holly S. Sellers,3,dagger Daniel J. King,3 and Bruce S. Seal3,*

Instituto de Biotecnologia, Centro de Investigacion en Ciencias Veterinarias, Instituto Nacional de Tecnologia Agropecuria, CC7725 Castelar (1712), Buenos Aires,1 and CONICET, Rivadavia 1917 (1033), Capital Federal,2 Argentina, and Southeast Poultry Research Laboratory, USDA Agricultural Research Service, Athens, Georgia 306053

Received 19 March 2001/Returned for modification 17 June 2001/Accepted 1 July 2001

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

* Corresponding author. Mailing address: Southeast Poultry Research Laboratory, A.R.S., U.S.D.A., 934 College Station Rd., Athens, GA 30605. Phone: (706) 546-3463 or (706) 546-3434. Fax: (706) 546-3161. E-mail: bseal{at}seprl.usda.gov.

dagger Present address: Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

Journal of Clinical Microbiology, September 2001, p. 3171-3178, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3171-3178.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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