Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates

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, 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.

FIG. 1

HMA for reference NDV isolates. (A) Utilization of the B1 vaccine type virus as the reference. (B) Utilization of the Ulster vaccine type virus as the reference. Virus isolates are presented in Table 1.

FIG. 2

HMA for NDV isolates not previously characterized. (A) Utilization of the B1 vaccine type virus as the reference. (B) Utilization of the Ulster vaccine type virus as the reference. Virus isolates are presented in Table 1.

FIG. 3

Phylogenetic relationships and predicted amino acid sequences of the F protein cleavage site among NDV isolates examined by HMA. (A) Nucleotide sequences of the amplification products used for HMAs were aligned. Subsequently, an unrooted tree was generated by parsimony analysis. The number of nucleotide differences is provided for each branch point. Bootstrap confidence limits following 1,000 replications are presented in parentheses for the major informative branches, while the predominant NDV clades are designated I and II. (B) Alignment of the predicted amino acid sequences obtained from the amplification products for each isolate.