bioRxiv2025
African Swine Fever Virus (ASFV) represents a looming threat to animal health, food safety and to the livestock industry. Virulent strains of ASFV cause a severe and often fatal illness, while attenuated strains are usually associated with mild symptoms. Naturally-occurring attenuated strains are typically deleted of more than 20 genes located at the viral genomes extremities. Whether other key differences between virulent and attenuated ASFV strains may contribute to the virulence phenotype remains however largely unexplored. In this work, we sought to determine how the dynamics of viral gene expression may shape the hosts innate immune response to ASFV infection and contribute to ASFV virulence. We conducted a medium-throughput transcriptomic study to characterize the viral transcriptome of a panel of virulent and attenuated strains (171 viral genes), as well as the host response of ASFV-infected macrophages (92 host genes). Confocal imaging allowed further characterization of cellular response to infection, by assessing the dynamics of IFN and NF-{kappa}B pathway activation in ASFV-infected cells. Our results indicate that the two types of viral pathotypes exhibit global differences in the dynamics of genome replication and viral transcription. Virulent ASFV strains displayed a burst of viral transcription early on, while attenuated strains tended to replicate to higher levels at late time points. The host response was much more pronounced in cells infected with attenuated strains compared to virulent ones, with higher expression levels of interferon-stimulated genes, some innate immunity sensors, and the inducible chaperone HSP70.2. Unexpectedly, genotype I and genotype II virulent strains exhibited some notable differences in their kinetics of viral genome replication and in the host response they provoked, with higher levels of pro-inflammatory cytokines being induced by genotype II strains. Confocal imaging analysis of ASFV-infected primary macrophages revealed that attenuated strains, but not virulent ones, caused the translocation of both p65 and STAT2 to the nucleus. Strikingly, we identified a group of 26 viral genes that were either expressed at higher levels or at an earlier stage of infection by virulent strains. Several of these genes, such as R298L, H233R, DP71L and MGF505-7R encode for proteins that inhibited the type I Interferon response in a reporter cell line system. This work sheds new light on the mechanistical drivers of ASFV virulence and will in the long run help to better understand the protection offered by ASFV Live-Attenuated Vaccine candidates. Author summaryAfrican Swine Fever (ASF), a severe infectious disease affecting domestic pigs and wild boars, presents a global threat to the livestock industry. It is caused by African Swine Fever Virus (ASFV), a large DNA virus encoding between 150 and 200 genes. While virulent ASFV strains cause a fatal illness in infected animals, attenuated strains induce only minor symptoms and some can confer subsequent protection against a pathogenic infection. While Live-Attenuated Vaccines for ASFV are under development and represent a promising tool in the fight against ASF, the mechanisms of ASFV virulence (and conversely, attenuation) are not fully understood. In particular, it is unclear whether key differences may exist between attenuated and virulent ASFV strains, beyond the extensive genomic deletions harbored by the former. In this work, we explored for the first time how the dynamics of viral gene expression may influence the innate immune response to different ASFV strains. We found that attenuated ASFV strains trigger a stronger host response compared to virulent ASFV strains, with higher expression levels of innate immune genes and a stronger activation of key signaling hubs. Finally, we identified a group of 26 ASFV genes that may drive this phenomenon and represent novel virulence factors.