MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development

African swine fever (ASF) is a highly contagious and lethal disease, but few therapeutic options are available for its treatment. Therefore, there is an urgent need for the development of safe and effective vaccines. In this regard, we described the differential effect of deletion of whole MGF100 and MGF300 families from genotype II highly virulent strains on African swine fever virus (ASFV) replication, virulence, and induction of protection. The resulting ASFV-Δ100 and ASFV-Δ300 mutants demonstrated reduced growth kinetics in vitro, with the former displaying aberrant virus morphogenesis. ASFV-Δ100 was efficiently attenuated, whereas ASFV-Δ300 retained its virulence. In the homologous lethal challenge, the two mutants achieved the same protection rate, with the former providing more protection against pathology in organs. Mechanistically, we found that ASFV-Δ100 was capable of inducing a robust innate immune response in vitro and a consistent P30 antibody response in vivo. In conclusion, the MGF100 family is a potential multigene-deleted target for ASFV live-attenuated vaccine development.