O-2-Alkylated Cytosine Acyclic Nucleoside Phosphonamidate Prodrugs Display Pan-Genotype Antiviral Activity against African Swine Fever Virus

African swine fever virus (ASFV) causes a highly contagious hemorrhagic disease with case fatality rates approaching 100% in domestic pigs. ASFV is responsible for substantial economic losses, but despite ongoing efforts, no vaccine or antiviral agent is currently available. Attempts to control the spread of ASFV are dependent on early detection, adherence to biosecurity measures, and culling of infected herds. However, an effective antiviral agent may be used in lieu of or in conjunction with a vaccine to effectively curb ASFV outbreaks. The dose-dependent antiviral activities of two amidate prodrugs (compounds 1a and 1b) of O-2-alkylated 3-fluoro-2-(phosphonomethoxy)propyl cytosine [(R)-O-2-alkylated FPMPC] against ASFV isolates of four different genotypes were determined. Both compounds were found to inhibit ASFV progeny virus output by >90% at noncytotoxic concentrations (<25 μM) in primary porcine macrophages. Analysis of viral transcription and viral protein synthesis indicated that these acyclic nucleotide analogues inhibited late gene expression. Interestingly, time-of-addition studies suggest different viral targets of the compounds, which may be attributed to their differing amino acid prodrug moieties. In view of their promising antiviral activity, these nucleotide analogues merit further evaluation as potential prophylactic and/or therapeutic agents against ASFV infection and their antiviral efficacy in vivo should be considered. IMPORTANCE African swine fever virus is a highly contagious hemorrhagic viral disease. Since its transcontinental spread to Georgia in 2007, ASFV has continued to spread across the globe into countries previously without infection. It is responsible for substantial losses in the domestic pig population and presents a significant threat to the global swine industry. Despite ongoing efforts, there are no vaccines currently available; in their absence, antiviral agents may be a viable alternative. The significance of our research is in identifying the pan-genotype antiviral activity of prodrugs of O-2-alkylated 3-fluoro-2-(phosphonomethoxy)propyl cytosine, which will drive further research on the development of these compounds as antivirals against ASFV.