The BHV-1 strain will be propagated in Madin-Darby bovine kidney cells, which will be grown in Dulbecco’s modified essential medium supplemented with 10% fetal calf serum. To obtain BHV-1 BACs, viral DNA will be prepared from infected cells and co-transfected with recombinant transfer plasmids by the calcium phosphate precipitation method. Recombinant transfer plasmid pgC_HA2 will be generated by cloning of PCR fragments flanking the targeted locus within the gC (UL44) gene and insertion of the PacI fragment of plasmid pHA2 into plasmid pTZ18R. The resulting construct will contain a mini-F origin of replication, the enhanced green fluorescent protein (GFP) ORF, and the E. coli guanosine phosphoribosyl transferase (gpt) gene. Progeny fluorescing virus resulting from the co-transfections of viral and pgc_HA2 DNA will be purified to homogeneity and checked for the presence of pHA2 sequences by Southern blot analysis. Virus DNA will be prepared at various times after infection and used to electroporate E. coli DH10B cells, which will be spread on chloramphenicol (cam)-containing agar plates. Resistant colonies will be grown in liquid medium and DNA will be analyzed by restriction enzyme digestion and Southern blotting. Finally, bovine kidney or lung cells will be transfected with various amounts of BHV-1 BAC DNA isolated from E. coli GFP-expressing cells developing into BHV-1-specific plaques should be visible 24 hours after transfection. To generate the deletion mutants in the generated BHV-1 BAC, a strategy named en passant mutagenesis will be applied. The method is based on Red-catalyzed seamless insertions of sequences into BAC clones without retention of unwanted foreign sequences. BAC DNA will be electroporated into the required E. coli EL250 cells, which harbour a defective prophage supplying the exo and betenzyme genes needed for recombination, and whose expression is controlled by a heat-inducible promoter. The point mutations will be carried out in a two-step