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

Abstract

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.

The essential and non-essential genes of Bovine herpesvirus 1

“…The BoHV-1 strain V155, maintained as a bacterial artificial chromosome (BAC) (pBACBHV37) in Escherichia coli DH10B cells (Mahony et al, 2002), was used for this study. pBACBHV37 has a thymidine kinase deficient (TK 2 ) phenotype.…”

“…TK 2 BoHV-1 have been extensively investigated and utilized as vaccine candidates due to their reduced virulence in vivo (Bello et al, 1992;Chowdhury, 1996;Kaashoek et al, 1996). pBACBHV37 shows no apparent attenuation and displays indistinguishable replication kinetics to that of a wild-type BoHV-1 in vitro (Mahony et al, 2002).…”

“…CRIB-1 cells were cultured as described previously (Mahony et al, 2002). Standard liquid and solid preparations of Luria-Bertani (LB) media containing 25 mg chloramphenicol (CAP) ml 21 and/or 30 mg kanamycin (KAN) ml 21 and/ or 100 mg ampicillin (AMP) ml 21 were used where appropriate to propagate BAC clones.…”

“…Targeted deletion of selected BoHV-1 genes was catalysed by homologous recombination using the GETrec system (Narayanan et al, 1999) and analysed as described previously (Mahony et al, 2002). Modified pBACBHV37GETD clones were identified by colony formation on dual selective media and confirmed by sequencing.…”

“…In this study, a transposon insertion mutagenesis system (Mahony et al, 2004) and homologous recombination (using the GETrec system; Narayanan et al, 1999) have been utilized to examine the requirement of individual genes for the in vitro viability of an infectious clone of BoHV-1 (Mahony et al, 2002). A map has been established that identifies BoHV-1 genes that are essential or nonessential for in vitro viability.…”