Aptamer-mediated inhibition of cellular apoptosis and ROS induced by the p54 protein of African swine fever virus.

African swine fever (ASF) is a viral disease of domestic pigs and wild boars with a high fatality rate. The causative agent of this disease is the African swine fever virus (ASFV), which continues to represent a major threat to the global swine industry, for which therapeutic options remain scarce. Among ASFV's structural proteins, p54 plays a crucial role in host-virus interactions, although its involvement in host cell stress responses and apoptosis remains incompletely elucidated. In the present study, we examined the functional characteristics of the ASFV p54 protein and assessed a nucleic acid aptamer-based approach to mitigate its pathogenic effects. The ASFV p54 gene was cloned into a mammalian expression vector (pcDNA 3.1-p54) and transfected into the porcine macrophage cell line 3D4/31 to assess its effects on host cells. p54 expression strongly induced apoptosis, accompanied by increased intracellular and mitochondrial ROS levels, which are indicative of cell death and mitochondrial stress. To corroborate these results in a viral context, a recombinant Newcastle disease virus (NDV) expressing ASFV p54 (rNDV-p54) was constructed and employed to infect porcine macrophages. The rNDV-p54 infection mirrored the aforementioned cellular responses, affirming pro-apoptotic and oxidative stress-inducing properties of ASFV p54 protein. Subsequently, for therapeutic purposes, the ASFV p54 protein-targeted aptamer was screened and evaluated. The selected aptamer inhibited p54-induced apoptosis in 3D4/31 cells, accompanied by reduced ROS levels. Collectively, our findings identify ASFV p54 protein as a major inducer of oxidative stress-dependent apoptosis in host cells and show that targeted nucleic acid aptamers can potently neutralize these impacts. The findings highlight an aptamer-based approach with high binding affinity for the ASFV p54 protein, underscoring its potential for diagnostic applications and as a platform for subsequent antiviral research. Nevertheless, additional research is essential to assess its antiviral effectiveness in ASF infections via appropriate in vitro and in vivo models.