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United States Department of Agriculture

Agricultural Research Service

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

Authors
item Berinstein, Analia - INST DE BIOTECNOLOGIA
item Sellers, Holly - UNIV OF GEORGIA - ATHENS
item King, Daniel
item Seal, Bruce

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2001
Publication Date: N/A

Interpretive Summary: 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. Infections with virulent NDV strains are required to be reported to the Office of International Epizootes (OIE) and may result in severe trade restrictions. The amino acid dsequence of the fusion (F) protein is an important reason why some NDV isolates are more virulent than others. Consequently, techniques such as reverse-transcription, polymerase chain reaction (RT-PCR) have been used to amplify genomic nucleotide sequences to determine the F protein amino acid sequence. Since many diagnostic laboratories do not have the capability to complete genomic sequence analysis following RT-PCR, an alternative approach was tested. Using a common NDV vaccine virus as a standard the RT PCR product from this virus was compared with other more virulent viruses by a method known as the heteroduplex mobility assay (HMA). When the RT-PC products are mixed together and heated the strands separate. The strands are allowed to come back together and the annealed bands are visualized following electrophoresis in gels. If the two viruses differ in genomic sequence they can be clearly distinguished by having a pattern distinct from a control. The HMA could be used for rapid identification of potentially virulent NDV that continue to threaten commercial poultry.

Technical Abstract: 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. 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, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criteria. 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. We were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. 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.

Last Modified: 12/22/2014
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