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Journal of Virology, November 2000, p. 10737-10744, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Establishment of a Rescue System for Canine Distemper Virus

Uta Gassen, Fergal M. Collins, W. Paul Duprex, and Bert K. Rima*

School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland

Received 25 April 2000/Accepted 10 August 2000

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

* Corresponding author. Mailing address: School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Rd., Belfast BT9 7BL, Northern Ireland, United Kingdom. Phone: 44 2890 335858. Fax: 44 2890 236505. E-mail: b.rima{at}qub.ac.uk.

Journal of Virology, November 2000, p. 10737-10744, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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