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Journal of Virology, June 1999, p. 5001-5009, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence
that Cleavability of the Fusion Protein Is a Major Determinant
for Virulence
Ben P. H.
Peeters,*
Olav S.
de Leeuw,
Guus
Koch, and
Arno L. J.
Gielkens
Department of Avian Virology, Institute for
Animal Science and Health, 8200 AB Lelystad, The Netherlands
Received 18 November 1998/Accepted 24 February 1999
A full-length cDNA clone of Newcastle disease virus (NDV) vaccine
strain LaSota was assembled from subgenomic overlapping cDNA fragments
and cloned in a transcription plasmid between the T7 RNA polymerase
promoter and the autocatalytic hepatitis delta virus ribozyme.
Transfection of this plasmid into cells that were infected with a
recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in
the synthesis of antigenomic NDV RNA. This RNA was replicated and
transcribed by the viral NP, P, and L proteins, which were expressed
from cotransfected plasmids. After inoculation of the transfection
supernatant into embryonated specific-pathogen-free eggs, infectious
virus derived from the cloned cDNA was recovered. By introducing three
nucleotide changes in the cDNA, we generated a genetically tagged
derivative of the LaSota strain in which the amino acid sequence of the
protease cleavage site (GGRQGR
L) of the fusion protein F0 was
changed to the consensus cleavage site of virulent NDV strains
(GRRQRR
F). Pathogenicity tests in day-old chickens showed that the
strain derived from the unmodified cDNA was completely nonvirulent
(intracerebral pathogenicity index [ICPI] = 0.00). However, the
strain derived from the cDNA in which the protease cleavage site was
modified showed a dramatic increase in virulence (ICPI = 1.28 out
of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the
F protein showed that the efficiency of cleavage of the F0 protein was
greatly enhanced by the amino acid replacements. These results
demonstrate that genetically modified NDV can be recovered from
cloned cDNA and confirm the supposition that cleavage of the F0
protein is a key determinant in virulence of NDV.
*
Corresponding author. Mailing address: Institute for
Animal Science and Health (ID-DLO), Department of Avian Virology, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Phone: 31 320-238238. Fax:
31-320-238668. E-mail: b.p.h.peeters{at}id.dlo.nl.
Journal of Virology, June 1999, p. 5001-5009, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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