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Journal of Virology, July 2002, p. 6660-6668, Vol. 76, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.13.6660-6668.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction and Manipulation of an Infectious Clone of the Bovine Herpesvirus 1 Genome Maintained as a Bacterial Artificial Chromosome

Timothy J. Mahony,1* Fiona M. McCarthy,2 Jennifer L. Gravel,2 Lani West,1 and Peter L. Young1

Queensland Agricultural Biotechnology Centre, Agency for Food and Fibre Sciences, Gehrmann Laboratories, Brisbane 4072,1 Queensland Beef Industry Institute, Agency for Food and Fibre Sciences, Brisbane 4105, Queensland, Australia2

Received 26 December 2001/ Accepted 8 April 2002

The complete genome of bovine herpesvirus 1 (BoHV-1) strain V155 has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli strain DH10B, the BoHV-1 BAC was stably propagated over multiple generations of its host. BAC DNA recovered from DH10B cells and transfected into bovine cells produced a cytopathic effect which was indistinguishable from that of the parent virus. Analysis of the replication kinetics of the viral progeny indicated that insertion of the BAC vector into the thymidine kinase gene did not affect viral replication. Specific manipulation of the BAC was demonstrated by deleting the gene encoding glycoprotein E by homologous recombination in DH10B cells facilitated by GET recombination. These studies illustrate that the propagation and manipulation of herpesviruses in bacterial systems will allow for rapid and accurate characterization of BoHV-1 genes. In turn, this will allow for the full utilization of BoHV-1 as a vaccine vector.

* Corresponding author. Mailing address: Queensland Agricultural Biotechnology Centre, Agency for Food and Fibre Sciences, Gehrmann Laboratories, Research Rd., St. Lucia, Queensland 4072, Australia. Phone: 61 7 3365 4960. Fax: 61 7 3365 4980. E-mail: Timothy.Mahony{at}dpi.qld.gov.au.

Journal of Virology, July 2002, p. 6660-6668, Vol. 76, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.13.6660-6668.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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