Gonzalo Vigara-Astillero

African swine fever virus (ASFV) encodes numerous multigene family (MGF) proteins whose individual contributions to virulence and immune modulation remain poorly understood. Here, we characterize the role of MGF110-2L in shaping innate immunity and disease outcome in vivo . Deletion of MGF110-2L from the virulent Arm/07/CBM/c2 isolate markedly attenuated clinical disease and conferred complete protection against homologous challenge. In macrophages, the MGF110-2L-deficient recombinant induced enhanced type I interferon (IFN) responses, consistent with a role for MGF110-2L in antagonizing antiviral signaling. However, transcriptomic profiling of peripheral blood mononuclear cells revealed that the absence of MGF110-2L prevents the excessive IFN and inflammatory hyperactivation triggered by the parental virus in vivo , establishing a controlled antiviral program associated with reduced pathology. These findings identify MGF110-2L as a functional virulence factor that drives dysregulated innate immune responses during ASFV infection. Our work advances the molecular understanding of ASFV-host interactions and highlights the contribution of MGF110-2L protein to immune evasion and pathogenesis. IMPORTANCE: Finding safe and effective vaccines is essential to impair the spread of African swine fever virus (ASFV), responsible for the largest animal pandemic. Identifying ASFV virulence factors and understanding the mechanisms of pathogenesis and protection are crucial. By using RNAseq from in vivo peripheral blood mononuclear cells, together with immunological studies, we showed that MGF110-2L regulates type I IFN production and cytokine storm in pigs. Deletion of the MGF110-2L gene results in attenuation and induces protection against the parental virulent strain. These results support the induction of an alert immune state together with dampened systemic inflammation but preserved cytotoxic readiness in vaccinated animals. Altogether, our data shed light on the mechanisms underlying protection against ASFV and contribute to the development of new protective tools.

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo.

African swine fever virus (ASFV) is the etiological agent causing African swine fever (ASF), affecting domestic pigs and wild boar, which is currently the biggest animal epidemic in the world and a major threat to the swine sector. At present, some safety concerns about using LAVs against ASFV still exist despite a commercial vaccine licensed in Vietnam. Therefore, the efforts to identify virulence factors and their mechanisms, as well as to generate new vaccine prototypes, are of major interest. In this work, we have identified the MGF505-2R gene product as an inhibitor of the cGAS/STING pathway, specifically through its interaction with STING protein, controlling IFN-β production. In addition, immunization of a recombinant virus lacking this gene, Arm/07-ΔMGF505-2R, resulted in complete attenuation, demonstrating its involvement in ASFV virulence. Finally, immunization with Arm/07-ΔMGF505-2R induced the generation of antibodies and proved to be partially protective against virulent ASFV strains. These results identify MGF505-2R, as well as its mechanism of action, as a gene contributing to understanding the molecular mechanisms of ASFV virulence, which will be of great value in the design of future vaccine prototypes.

African swine fever virus (ASFV) is the cause of African swine fever (ASF), a devastating disease that affects domestic pigs and wild boar and is currently responsible for the largest animal epidemic. One of the characteristics of ASFV infection, but absent in naturally attenuated strains, is hemadsorption (HAD), a phenomenon that has been linked to virulence. In this study, we have shown that ASFV HAD depends exclusively on the Nt domain of the ASFV CD2v protein during infection. CD2v is a highly glycosylated protein, and we found that glycosylation is essential for HAD. However, despite the higher degree of CD2v glycosylation, only simultaneous N-glycosylation of two Asp residues on the Nt region is the determinant for HAD. On the contrary, we have demonstrated that the presence of a specific signal peptide sequence on CD2v not only influences the degree of CD2v glycosylation but is also critical for HAD, although not for CD2v localization. Finally, we have shown that the CD2v expression during infection of the non-HAD NH/P68 strain is not sufficient for HAD, despite its glycosylation and cell surface localization. Complementation studies of CD2v from NH/P68 with CD2v signal peptides from HAD+ strains indicate (i) that different signal peptides from different genotypes are able to restore HAD and (ii) the existence of a HAD-inhibitory sequence in NH/P68-CD2v. This study lays the molecular basis for ASFV HAD, which could be key for the study of virulent and pathogenic aspects of the virus, as well as the rational development of new vaccines against ASFV. IMPORTANCE: African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines.

African swine fever (ASF) is an obligated declaration swine disease, provoking farm isolation measures and the closing of affected country boarders. ASF virus (ASFV) is currently the cause of a pandemic across China and Eurasia. By the end of 2019, ASF was detected in nine EU Member States: Bulgaria, Romania, Slovakia, Estonia, Hungary, Latvia, Lithuania, Poland and Belgium. The affected area of the EU extended progressively, moving mostly in a southwestern direction (EFSA). Inactivated and/or subunit vaccines have proven to fail since certain virus replication is needed for protection. LAVs are thus the most realistic option, which must be safe, effective and industrially scalable. We here generated a vaccine prototype from the Arm/07/CBM/c2 genotype II strain, in which we have deleted the EP402R (CD2v) and A238L genes by CRISPR/Cas9 in COS-1 cells, without detectable further genetic changes. The successful immunization of pigs has proven this vaccine to be safe and fully protective against the circulating Korean Paju genotype II strain, opening the possibility of a new vaccine on the market in the near future.

No efficient vaccines exist against African swine fever virus (ASFV), which causes a serious disease in wild boars and domestic pigs that produces great industrial and ecological concerns worldwide. An extensive genetic characterization of the original ASFV stocks used to produce live attenuated vaccine (LAV) prototypes is needed for vaccine biosecurity and control. Here, we sequenced for the first time the Arm/07 stock which was obtained from an infected pig during the Armenia outbreak in 2007, using an improved viral dsDNA purification method together with high coverage analysis. There was unexpected viral heterogeneity within the stock, with two genetically distinct ASFV subpopulations. The first, represented by the Arm/07/CBM/c2 clone, displayed high sequence identity to the updated genotype II Georgia 2007/1, whereas the second (exemplified by clone Arm/07/CBM/c4) displayed a hemadsorbing phenotype and grouped within genotype I based on a central region conserved among all members of this group. Intriguingly, Arm/07/CBM/c4 contained a unique EP402R sequence, produced by a single mutation in the N-terminal region. Importantly, Arm/07/CBM/c4 showed in vitro features of attenuated strains regarding innate immune response pathway. Both Arm/07/CBM/c2 and c4 represent well-characterized viral clones, useful for different molecular and virus-host interaction studies, including virulence studies and vaccine development.