TY - JOUR T1 - Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning35 JF - J.Gen.Virol. Y1 - 2005 A1 - Gillet,L. A1 - Daix,V. A1 - Donofrio,G. A1 - Wagner,M. A1 - Koszinowski,U.H. A1 - Bernard China A1 - Ackermann,M. A1 - Markine-Goriaynoff,N. A1 - Vanderplasschen,A. KW - 0 KW - a KW - an KW - Animals KW - article KW - AS KW - bacteria KW - Belgium KW - Biology KW - Cattle KW - Cell KW - cells KW - Chromosomes,Artificial,Bacterial KW - Cloning,Molecular KW - Complement Inactivator Proteins KW - Development KW - disease KW - Diseases KW - Escherichia coli KW - EVALUATION KW - expression KW - Faculty KW - Features KW - Functional KW - Genetic Vectors KW - genetics KW - Genome KW - Herpesvirus 4,Bovine KW - i KW - im KW - immunology KW - Infectious KW - IS KW - IT KW - Ixodes KW - journal KW - LEVEL KW - levels KW - Medicine KW - metabolism KW - MODEL KW - mutagenesis KW - ON KW - physiology KW - present KW - Print KW - production KW - protein KW - Proteins KW - recombinant KW - recombination KW - Recombination,Genetic KW - Research KW - Research Support KW - SB - IM KW - Sites KW - study KW - technology KW - Universities KW - university KW - use KW - Vector KW - veterinary KW - Veterinary Medicine KW - VIRUS KW - Virus Replication AB - 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 VL - 86 CP - Pt 4 U1 - 38735 M3 - 86/4/907 [pii];10.1099/vir.0.80718-0 [doi] ER -