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Journal of Virology, January 2001, p. 420-428, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.420-428.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos

Teshome Mebatsion,1,* Stefan Verstegen,1 Leonarda T. C. De Vaan,1 Angela Römer-Oberdörfer,2 and Carla C. Schrier1

Department of Virology, Intervet International B.V., 5830 AA Boxmeer, The Netherlands,1 and Institute of Molecular Biology, Friedrich-Loeffler Institute, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany2

Received 10 May 2000/Accepted 29 September 2000

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

* Corresponding author. Mailing address: Department of Virology, Intervet International B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands. Phone: 31 485 587 351. Fax: 31 485 587 339. E-mail: teshome.mebatsion{at}intervet.com.

Journal of Virology, January 2001, p. 420-428, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.420-428.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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