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Abstract Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.