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1 Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
2 Dipartimento di Salute Animale, Facoltà di Medicina Veterinaria, Sezione di Malattie Infettive degli Animali, Università degli Studi di Parma, I-43100 Parma, Italy
3 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Virology, Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany
5 Food Sciences Department (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
6 Institute for Virology, University of Zurich, CH-8057 Zurich, Switzerland
Correspondence
A. Vanderplasschen
A.vdplasschen{at}ulg.ac.be
Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.
These authors contributed equally to this work.
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