Differentiation between Virulent and Avirulent Newcastle Disease Virus Isolates Using the Cleavage Site Motif of the Fusion Protein

Abstract

Six Newcastle disease virus (NDV) strains, Dikheinat-1990 (DIK-90), Dongola, Gedarif.Elsoufi.1 (GD.S.1), I2, Lasota and Clone 30 were propagated in chicken embryonated eggs and identified by hemagglutination inhibition test (HI). RNA was extracted from allantoic fluid harvests of the various strains. Two step reverse transcription-polymerase chain reactions (RT-PCR) were carried out, which amplified 805 base pair (bp) of the first part, including the cleavage site, of the fusion protein gene (F gene) of NDV strains using a set of gene specific primers. Following sequencing, alignment of the resulted gene sequences were carried out using ClustalX program. The annotation and subsequent steps were implemented using MEGA 4 program. 714 bp were translated to protein sequence and used to analyze the cleavage site motives of the F protein, which is a known virulent determinant of NDV strains, in order to differentiate between virulent and a virulent NDV strains. Genetic analyses of NDVs have demonstrated a relationship between the amino acid sequence at the F protein cleavage site and virulence among various strains. Results indicated that the three test strains Dongola, GD.S.1, DIK-90 and the reference vaccinal strain Clone 30 have a motive of avirulent NDV 112G-R-Q-G-R-L117, while the I2 and Lasota possessed the motif of a typical low virulent NDV 112R-K-Q-G-R-L117 represented by lentogenic strains. The two velogenic strains DIK-90 and GD.S.1 have the cleavage site motive of lentogenic strain, unexpectedly. This suggests that the cleavage site motif of the F protein is not the only factor that determines the virulence of NDV strains. Phylogenetically from this study, it could be shown that all the strains cluster into genotype II