Confirmation of gamma irradiation mediated inactivation of a Vero adapted African swine fever virus Lisbon 60 strain for molecular assays

African swine fever virus (ASFV), the etiologic agent of African Swine Fever (ASF), is a high-consequence pathogen requiring experiments to be conducted in containment in non-endemic countries, thereby restricting diagnostic development, the creation of reference standards, and proficiency testing (PT). Safe and reliable inactivation methods are essential to expand diagnostic capacity while preserving nucleic acid integrity for molecular assays in unaffected countries. This study employed gamma irradiation to achieve complete inactivation of ASFV without compromising downstream molecular detection, as gamma irradiation offers deep penetration and uniform dose delivery. ASFV-cell culture supernatants were subjected to gamma irradiation doses ranging from 2 to 50 kGy. Viral replication was evaluated using TCID{square}{square} and serial passages, revealing a consistent dose{square}dependent reduction in infectivity across increasing irradiation dose levels and a complete loss of ASFV infectivity at 30 and 50 kGy. Molecular detection remained unaffected at all of the tested doses as confirmed by qPCR Ct values and sequence identity of the p72 gene. Whole genome sequencing demonstrated >99% genome coverage and consistent read depth profiles across irradiated and non-irradiated samples, indicating preservation of genomic integrity at all tested doses. These findings demonstrate that gamma irradiation at 50 kGy fully inactivates ASFV-cell supernatants while maintaining nucleic acid quality suitable for molecular diagnostics. The resulting inactivated material meets quality assurance requirements for molecular reference standards and PT panels and can be safely distributed to laboratories outside high containment facilities, supporting broader diagnostic readiness and harmonization of ASFV testing.