Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning

Abstract

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.

Bovine Herpesvirus 4 Induces Apoptosis of Human Carcinoma Cell Lines In vitro and In vivo

“…Future studies on the identification of the proapoptotic BoHV-4 gene product(s) involved in this phenomenon, or eventually the development of BoHV-4 as a viro-oncoapopoptotic vector, will be greatly facilitated by the recent bacterial artificial chromosome cloning of BoHV-4 (47). By using random transposon mutagenesis, a library of mutant viruses could be generated with minimal effort.…”

Ex Vivo Bioluminescence Detection of Alcelaphine Herpesvirus 1 Infection during Malignant Catarrhal Fever

“…Bovine turbinate fibroblasts (BT; ATCC CRL-1390) and embryonic bovine lung (EBL)-nuclear localization signal (NLS)-Cre cells (18) were cultured in Dulbecco's modified essential medium (DMEM; Invitrogen Corporation). Madin-Darby bovine kidney cells (MDBK; ATCC) were cultured in modified essential medium (MEM).…”

“…Singlecell suspensions were frozen on dry ice for 30 min and then thawed at 37°C. After removal of cell debris by centrifugation (350 ϫ g, 10 min) the virions contained in the supernatant were titrated by plaque assay as described previously (18). In each assay, syncytia were revealed 4 days later by indirect immunofluorescent staining using a rabbit anti-AlHV-1 polyserum as the primary antibody.…”

“…Ten-fold dilution series of cell suspensions were seeded on MDBK cell monolayers. After overnight incubation, the cocultures were overlaid with complete medium containing 0.6% (wt/vol) carboxymethylcellulose (CMC) as described previously (18). In some experiments, cells were counted before coculture to determine the number of infectious centers per 10 6 cells.…”

“…Infected cultures (cells and supernatant) in triplicate were harvested at successive intervals after infection and stored at Ϫ80°C. The amount of infectious virus was then determined by plaque assay on MDBK cells as described previously (18). Syncytia were revealed by indirect immunofluorescent staining using a rabbit anti-AlHV-1 polyserum as the primary antibody.…”