Deletion of CD2-Like (CD2v) and C-Type Lectin-Like (EP153R) Genes from African Swine Fever Virus Georgia- increment 9GL Abrogates Its Effectiveness as an Experimental Vaccine

African swine fever virus (ASFV) is currently the most dreaded infectious disease affecting the global swine production industry. There is no commercial vaccine available, making the culling of infected animals the current solution to control outbreaks. Effective experimental vaccines have been developed by deleting virus genes associated with virulence. Deletion of the ASFV 9GL gene ( increment 9GL) has resulted in the attenuation of different ASFV strains, although the degree of attenuation varies across isolates. Here, we investigated the possibility of the increased safety of the experimental vaccine strain ASFV-G-Delta 9GL by deleting two additional virus genes involved in pathogenesis, CD2v, a CD2 like viral encoded gene from the EP402R open reading frame (ORF), and C-type lectin-like viral gene, encoded from the EP153R ORF. Two new recombinant viruses were developed, ASFV-G-Delta 9GL/Delta CD2v and ASFV-G-Delta 9GL/Delta CD2v/Delta EP153R, harboring two and three gene deletions, respectively. ASFV-G-Delta 9GL/Delta CD2v/Delta EP153R, but not ASFV-G-Delta 9GL/Delta CD2v, had a decreased ability to replicate in vitro in swine macrophage cultures when compared with parental ASFV-G-Delta 9GL. Importantly, ASFV-G-Delta 9GL/Delta CD2v and ASFV-G-Delta 9GL/Delta CD2v/Delta EP153R induced almost undetectable viremia levels when inoculated into domestic pigs and failed to protect them against challenge with parental virulent ASFV-Georgia, while ASFV-G-Delta 9GL offered robust protection during challenge. Therefore, the deletion of CD2-like and C-type lectin-like genes significantly decreased the protective potential of ASFV-G-Delta 9GL as a vaccine candidate. This study constitutes an example of the unpredictability of genetic manipulation involving the simultaneous deletion of multiple genes from the ASFV genome.