A dual-gene-deleted ASFV Lv17/WB/Rie1-ΔCD candidate administered orally to wild boar confers DIVA-compatible protection against virulent challenge.

African swine fever (ASF) continues to expand worldwide. Recent detection in wild boar in Spain highlights the urgent need for effective control tools, with oral vaccination as a key priority. Following previous evaluation of the attenuated Lv17/WB/Rie1 strain, we assess an improved derivative, Lv17/WB/Rie1-ΔCD, lacking EP402R (CD2v) and EP153R, replaced by GFP to abrogate haemadsorption and enable Differentiating-Infected-from-Vaccinated-Animals (DIVA) diagnostics. Vaccinated animals received either a single high dose (104 TCID₅₀) or a prime and re-exposure regimen (102 TCID₅₀ plus a 104 TCID₅₀). Animals were challenged intramuscularly with the virulent Armenia07 genotype II strain. The ΔCD vaccine was well tolerated, inducing only transient low-grade fever. Prime-re-exposure vaccination induced earlier seroconversion (mean 12 ± 4 dpv) and sterilizing immunity in 5/6 animals in the high dose group. Overall protection reached 90%, while all unvaccinated controls died within 7 days. Quantitative PCR revealed >10³-fold reductions in viral genome copies in blood and tissues versus controls. DIVA ELISA reliably distinguished vaccine-induced antibodies from infection-derived responses. These findings identify Lv17/WB/Rie1-ΔCD as a safer oral ASFV vaccine candidate, addressing concerns raised with the parental Lv17/WB/Rie1 by increasing attenuation and supporting multi-gene deletion strategies. Further studies on safety, transmission, genetic stability, and environmental behaviour are required before large-scale field trials.