Molecular Changes of the Fusion Protein Gene of Chicken Mesenchymal Stem Cells–Adapted Velogenic Newcastle Disease Virus

Abstract

The susceptibility of the chicken mesenchymal stem cells to velogenic Newcastle disease virus (NDV) was characterized after twenty consecutive passages in chicken mesenchymal stem cells. Virus replication was monitored by cytopathic effect observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction (RT-PCR). At 120 h post-infection (p.i.) in forth passage, the cytopathic effect was characterized by rounding up of cell, monolayer detachment and syncytium construction, intracytoplasmic brownish coloration was readily observed by from 72 h p.i in third passage. Tissue culture effective dose50 (TID50) was used to measure virus titration performed on chicken mesenchymal stem cells and the titers in twenty passage was 109.6 TID50/ml. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription–polymerase chain reaction. Sequence analysis revealed a total of 135 nucleotide substitutions which resulted in the change of 42 amino acids between the velogenic virus and the 20th passage virus. The predicted amino acid motif present at the cleavage site of the virulent virus was 15SRRRRQRRFVG25and the corresponding region of the adapted virus was 15SGGRRQKRFIG25.The results obtained in this study suggested that the chicken mesenchymal stem cells can be used for adaptation and attenuation NDV and may be considered a step forward for the use of these cells in the future for NDV vaccine production.