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Journal of Virology, November 2005, p. 14330-14338, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14330-14338.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

David D. Brown,1 Bertus K. Rima,1 Ingrid V. Allen,1 Michael D. Baron,2 Ashley C. Banyard,2 Thomas Barrett,2 and W. Paul Duprex1*

School of Biomedical Sciences, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom,1 Institute for Animal Health, Pirbright Laboratories, Ash Road, Pirbright, Woking GU24 0NF, United Kingdom2

Received 29 June 2005/ Accepted 8 August 2005

Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPVKO) can be rescued. A recombinant virus, rRPVKOL-RREGFPR, which grew at an indistinguishable rate and to an identical titer as rRPVKO in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 104 TCID50 of the rRPVKO parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPVKOL-RREGFPR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

* Corresponding author. Mailing address: School of Biomedical Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom. Phone: 44 28 9097 2060. Fax: 44 28 9097 5877. E-mail: p.duprex{at}qub.ac.uk.

Journal of Virology, November 2005, p. 14330-14338, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14330-14338.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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