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Journal of Virology, May 2008, p. 4955-4964, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.00211-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi{triangledown}

B. Costes,1 G. Fournier,1 B. Michel,1 C. Delforge,1 V. Stalin Raj,1 B. Dewals,1 L. Gillet,1 P. Drion,2 A. Body,3 F. Schynts,3 F. Lieffrig,3 and A. Vanderplasschen1*

Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium,1 Animal Facility (B23), University of Liège, B-4000 Liège, Belgium,2 CER Groupe, rue du Carmel 1, B-6900 Marloie, Belgium3

Received 30 January 2008/ Accepted 29 February 2008

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

* Corresponding author. Mailing address: Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium. Phone: 32-4-366 42 64. Fax: 32-4-366 39 08. E-mail: A.vdplasschen{at}ulg.ac.be

{triangledown} Published ahead of print on 12 March 2008.

Journal of Virology, May 2008, p. 4955-4964, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.00211-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Costes, B., Raj, V. S., Michel, B., Fournier, G., Thirion, M., Gillet, L., Mast, J., Lieffrig, F., Bremont, M., Vanderplasschen, A. (2009). The Major Portal of Entry of Koi Herpesvirus in Cyprinus carpio Is the Skin. J. Virol. 83: 2819-2830 [Abstract] [Full Text]  


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