[Impact of modification of cleavage site of fusion protein and foreign gene insertion on the virulence of Newcastle Disease Virus LaSota vaccine strain].
Newcastle disease virus (NDV) cause a highly contagious and economically loss in poultry. The amino acid sequence at the protease cleavage site of the fusion (F) protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F protein cleavage site sequence in NDV virulence by use reverse genetics technology. The sequence G-R-Q-G-R-L present at the cleavage site of the F protein of avirulent strain LaSota was mutated to R-R-Q-R-R-F or R-R-Q-R-R-L. The resultant mutated rL-FmF and rL-FmL virus were evaluated by mean death test (MDT) in eggs, intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. The results showed that the modification of the F protein cleavage site resulted in a dramatic in crease in virulence from an ICPI value of 0.36 for LaSota to a value of 1.18 for rL-FmF and 1.06 for rL-FmL, respectively. In addition, the mutational viruses showed increase MDT and identical IVPI values to parent virus, the virulence of rescued viruses was greatly enhanced by the amino acid replacements, whether the amino acid on the N terminus of F2 was F or L. On this base, we constructed another two NDVs that expressed a H5 subtype avian influenza virus hemagglutinin (rL-FmF-HA) and green fluorescent protein (rL-FmF-EGFP). the ICPI value of recombinant virus rL-FmF-HA and rL-FmF-EGFP were 0.67 and 1.10, respectively lower than that of rL-FmF. the IVPI of both recombinant viruses still keep 0.00. The values of MDT for rL-FmF-HA and rL-FmF-EGFP were 117h and 101h, greater than 90h. thus, Introduction of a foreign gene into NDV genome resulted in growth retardation and attenuation, the decrease degree depended on the nature of foreign protein expressed. These results also indicate that cleavability of the F0 protein is an important determinant for virulence of NDV. NDV can be manipulated in the future for use as a vaccine vector.
Wang
Yong
Y
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China. nmnqwy@163.com
Ge
Jinying
J
Xie
Xidi
X
Ding
Yulin
Y
Bu
Zhigao
Z
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D005818:Genetic Engineering; D016679:Genome, Viral; D009154:Mutation; D009522:Newcastle disease virus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014765:Viral Vaccines / Q000009:adverse effects* / Q000235:genetics* / Q000276:immunology; D014774:Virulence
D004274:DNA, Recombinant; D014760:Viral Fusion Proteins; D014765:Viral Vaccines