Generation of a transposon insertion mutant library for bovine herpesvirus 4 cloned as a bacterial artificial chromosome by in vitro MuA based DNA transposition system.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although the BoHV-4 genome has been sequenced, the function of the majority of putative genes is elusive. Several features make BoHV-4 attractive as a backbone for use as a viral expression vector and/or as a model to study gamma herpesvirus biology and determining which genes are essential for its replication is a very important task. Starting from BoHV-4 genome cloned as infectious bacterial artificial chromosome (BAC-BoHV-4) in Escherichia coli. A random insertion mutant library for BoHV4 was generated by the use of MuA transposase-catalyzed in vitro transposition reaction. Viral mutant transfection and direct sequencing allow the rapid determination of which BoHV-4 genes are essential for viral growth in a permissive eukaryotic cell line. BoHV-4 functional analysis information is fundamental when the BoHV-4 genome is modified for vector purposes.