Virus reconstituted from infectious bacterial artificial chromosome (BAC)-Cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gamma-herpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus R gamma HV68A98.01 , containing BAC vector sequences, is attenuated in vivo as determined by ( i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen ce lls reactivating virus in an ex vivo reactivation assay. Since the BAC vect or sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus R gamma HV68A98.02, with biological properties comparable to those of wild -type MHV-68, On the basis of these data we conclude (i) that excision of B AC vector sequences from cloned MHV-68 genomes is critical for reconstituti on of the wild-type phenotypic properties of this virus and (ii) that the B AG-cloned MHV-68 genome is suitable for the construction of mutants and mut ant libraries whose phenotypes can be reliably assessed in vivo.