Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system

A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus VΔMGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus VΔMGFΔGUS. The EP402R and EP153R genes were subsequently deleted from the genome of VΔMGFΔGUS, using the same GUS marker gene, to construct virus VΔMGFΔCD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.