Genetic mutant organisms pervade all certain specific areas of Biology. early stage. Id of viral strains with uncommon properties, e.g. not capable of initiating lytic replication, such as for example Raji, or of changing B cells, such as for example P3HR1, later combined to sequencing allowed the id of genes or of several genes apt to be involved with these features [1-3]. Although these early EBV mutants spontaneously made an appearance, they provided a significant device for EBV analysis. Recently, strategies have already been developed to permit researchers to immediate mutagenesis from the EBV genome to be able to style particular mutants appealing. The capability to associate particular genes with original mutant phenotypes was a significant step, nevertheless, definitive proof that such phenotypes are connected with particular genes needed the structure of revertants. For instance, proof which the P3HR1 phenotype was due to the increased loss of EBNA2 needed the reintroduction of the gene back to the mutant Xdh genome through transfection of the EBV DNA fragment that spans the EBNA2 area as well as the observation a effectively recombined trojan acquired regained its transforming capability [4,5]. Not merely did this observation determine EBNA2 as a key transforming gene, it also provided an elegant method to select for recombinants from the background of defective P3HR1 viruses. Indeed, lymphoblastoid cell lines (LCL) generated with supernatants from EBNA-2 transfected P3HR1 cells contained predominantly, if not exclusively, recombinant viruses [4,5]. Consequently, the intro of EBNA2 offered a potent selection method that may be used to construct mutant viruses. Recombination with a combination of cosmid that contained EBNA2 and of overlapping cosmids that carried a mutated version SL 0101-1 of another EBV gene, e.g. EBNA3, allowed the generation of EBV mutants that experienced both re-acquired EBNA2 SL 0101-1 and integrated the mutated gene [6]. This technology, based on homologous recombination in eukaryotic cells, offers proven priceless for our understanding of EBV-driven B cell transformation. A related but unique strategy for generating EBV mutants consisted of exchanging a viral gene of interest located on the EBV Akata genome with a selection marker such as neomycin [7]. Neomycin resistant Akata cell clones must then become screened to identify those comprising successfully recombined mutants. In a further step, mutants often had to be purified from crazy type EBV genomes present in the same cell clones. This was usually obtained by inducing the lytic cycle in the clones of interest and subsequently exposing an EBV-negative cell line to the supernatants from these cells. This was performed at a low multiplicity of infection to ensure that every newly infected cell would carry either the mutant or the wild type viruses [7]. The B cell clones would then be screened for the presence of the mutant and selected for phenotypic characterization. This purification step can only be performed if the mutant has retained its ability to lytically replicate and to infect target cells from which they can be expanded. Therefore, mutant viruses that lack the genetic elements essential for either replication or infection cannot, in SL 0101-1 principle, be obtained by this method. These limitations, combined with the tedious sequential screening steps required by this method, led to the development of a quicker and more versatile strategy for the construction of recombinant viruses [8]. This new method, known as HV BAC technology, was developed in the late 1990 s in several laboratories in Munich for murine cytomegalovirus, EBV, human cytomegalovirus, and murine gammaherpesvirus 68 [9-12]. Since then, several human and animal HV genomes, including herpes simplex virus type 1 [13,26], varicella-zoster virus [14], Kaposi’s sarcoma-associated herpesvirus (KSHV) [15,16], rhesus cytomegalovirus SL 0101-1 [17], rhesus rhadinovirus [18], pseudorabies virus [19], herpesvirus saimiri [20], and Marek’s disease virus [21], have been cloned as BACs. The rationale of the HV BAC approach, which represented an abrupt change of tack from.