The Fusion Protein Gene Of Newcastle Disease Virus Strain F₄₈E₈:Sequence Analysis And Expression By A Recombinant Fowlpox Virus

The genomic RNA of Newcastle disease virus(NDV) strain F48E8,a typical Chinese virulent NDV, was extracted with phenolDS,then used as template for reverse transcriptionolymerase chain reaction (RT-PCR) to amplify the fusion (F) protein gene. The specific amplicons about 1. 7kb in length were cloned into pUC19, and a positive recombinant plasmid named pF37 was obtained by RE digestion analysis and southern blot hybridization. The nucleotide sequence of the F gene of NDV F48E8 strain was determined. A single open reading frame in the sequence encoded a polypeptide of 553 amino acids which had essential features of the F protein of virulent NDV strains previously established. These included the presence of three major hydrophobic regions and the structure of Arg-Arg-Gin-Arg-Arg in the cleavage ? activation site. Six potential glycosylation sites ,twelve cystein residues and two heptad repeats were also found. A comparison was made among amino acid sequence of the F protein of F48E8 strain and previously published sequences of Miyadera strain and Texas GB strain, the homology of 93. 64% and 92. 41% were observed respectiyely. F48E8 was phylogenetically related to the two Middle ast strains (AUS Victoria and Miyadera) and showed high genetic similarity to four NDV strins isolated from chickens in China currently suffered from Newcastle disease. In the present study, the fowlpox virus(FPV) transfer vector pFGF1175? was constructed by cloning the F gene of NDV F48E8 strain in the FPV insertion vector pFG1175 ?1, downstream of P7. 5 promoter. DOSPER liposome ? mediated transfection with pFGF1175 ?1 was performed on chicken embryo fibroblast (CEF) cell cultures which had been infected with FPV Chinese vaccine strain 282E4 for 3 4 hours to produce recombinant viruses. A recombinant FPV named rFPVDF was selected and purified on CEF cell clutures overlaid agar containing X-gal, and was confirmed expressing NDV fusion protein by indirected immunofluorescence assay. Vaccination of SPF chickens with rFPVDF induced significant levels of neutralizing antibody and good protection against challenge with the virulent NDV strain. In commercial broiler chickens that were inoculated in the presence of maternally derived NDV immunity, the protective rate of rFPV-NDF was 80%. Two inoculations of rFPV ?NDF with 10 days interval in commercial chickens could provide complete protection against challenge.