Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral 20 disease of domestic pigs that has significant economic consequences for the swine industry. The control 21 of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Successful 22 experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically 23 modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific 24 virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of 25 the 9GL (B119L) gene in highly virulent ASFVs Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL 26 viruses) resulted in complete protection when challenged with parental isolates. When similar deletions 27 were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the 28 protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another 29 gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. 30 Here we report the construction of a double gene deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. 31 When administered intramuscularly (IM) to swine there is no induction of disease even at high doses (106 HAD50). Importantly, animals infected with 104 32 HAD50 of ASFV-G-Δ9GL/ΔUK were protected as 33 early as 14 days post-inoculation when challenged with ASFV-G. Presence of protection correlates with 34 appearance of serum anti-ASFV antibodies but not with virus specific circulating ASFV-specific INF-γ 35 producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that 36 protects against the highly virulent ASFV Georgia 2007 isolate as soon as 2 weeks post-vaccination.