Establishment and characterization of a replication-restricted modified African swine fever virus

African swine fever is a fatal, febrile, infectious disease that affects pigs and wild boars and is caused by infection with African swine fever virus (ASFV). The current pandemic strain, belonging to genotype II, first emerged in Georgia in 2007 and subsequently spread across Russia, Europe, Asia, and the Caribbean. Developing live attenuated viruses represents the most promising vaccine strategy; however, biosafety and biosecurity concerns due to long-term persistence of attenuated viruses in immunized animals may lead to secondary transmission or reversion to virulence. To address this concern, we developed a replication-restricted ASFV by deleting the S273R gene that encodes an essential factor for core shell formation and viral maturation within host cells. The resulting strain, AQSΔS273R, was unable to replicate in natural host cells because these cells lack a gene that complements the function of S273R. To enable propagation of infectious progeny <i>in vitro</i>, we established S273R gene-complemented immortalized porcine kidney macrophages, which are competent to support virus production. In animal trials, pigs immunized with AQSΔS273R exhibited no clinical signs or viremia. In the challenge study, vaccination with replication-restricted ASFV (AQSΔS273R) conferred approximately 30% protection, with partial reduction of clinical signs compared with controls. These findings demonstrate the feasibility of a novel approach for developing replication-restricted ASFV vaccine candidates with enhanced biological safety.IMPORTANCETo date, no reliable African swine fever (ASF) vaccines are available. Although some attenuated African swine fever viruses (ASFVs) have been approved for field applications and have attracted attention as potential vaccine candidates, their long-term persistence in inoculated animals raises concerns about virulence reversion or genetic recombination in field settings. In this study, we developed a novel approach to generate safer vaccines by creating a replication-restricted, S273R gene-deleted virus in combination with genetically modified host cells stably expressing the S273R gene. Not only did this mutant virus fail to induce any clinical signs in immunized pigs, but it also partially protected them against challenge with virulent ASFV. These results demonstrate that this newly developed replication-restricted ASFV strain is expected to be a promising and biologically safe vaccine candidate against ASF.