Signal peptide and N-glycosylation of N-terminal-CD2v determine the hemadsorption of African swine fever virus.

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 Virus Induces STAT1 and STAT2 Degradation to Counteract IFN-I Signaling.

African swine fever virus (ASFV) causes a serious disease in domestic pigs and wild boars and is currently expanding worldwide. No safe and efficacious vaccines against ASFV are available, which threats the swine industry worldwide. African swine fever virus (ASFV) is a complex dsDNA virus that displays multiple mechanisms to counteract the host innate immune response, whose efficacy might determine the different degrees of virulence displayed by attenuated and virulent ASFV strains. Here we report that infection with both virulent Arm/07/CBM/c2 and attenuated NH/P68 strains prevents interferon-stimulated gene (ISG) expression in interferon (IFN)-treated cells by counteracting the JAK/STAT pathway. This inhibition results in an impaired nuclear translocation of the interferon-stimulated gene factor 3 (ISGF3) complex, as well as in the proteasome-dependent STAT2 degradation and caspase 3-dependent STAT1 cleavage. The existence of two independent mechanisms of control of the JAK/STAT pathway, suggests the importance of preventing this pathway for successful viral replication. As ASFV virulence is likely associated with the efficacy of the IFN signaling inhibitory mechanisms, a better understanding of these IFN antagonistic properties may lead to new strategies to control this devastating pig disease.

African Swine Fever Virus Armenia/07 Virulent Strain Controls Interferon Beta Production through the cGAS-STING Pathway.

African swine fever virus (ASFV) is a complex, cytoplasmic double-stranded DNA (dsDNA) virus that is currently expanding throughout the world. Currently, circulating virulent genotype II Armenia/07-like viruses cause fatal disease in pigs and wild boar, whereas attenuated strains induce infections with various levels of chronic illness. Sensing cytosolic dsDNA, mainly by the key DNA sensor cyclic GMP-AMP synthase (cGAS), leads to the synthesis of type I interferon and involves signaling through STING, TBK1, and IRF3. After phosphorylation, STING translocates from the endoplasmic reticulum to the Golgi compartment and to the perinuclear region, acting as an indispensable adaptor connecting the cytosolic detection of DNA to the TBK1-IRF3 signaling pathway. We demonstrate here that attenuated NH/P68, but not virulent Armenia/07, activates the cGAS-STING-IRF3 cascade very early during infection, inducing STING phosphorylation and trafficking through a mechanism involving cGAMP. Both TBK1 and IRF3 are subsequently activated and, in response to this, a high level of beta interferon (IFN-ß) was produced during NH/P68 infection; in contrast, Armenia/07 infection generated IFN-ß levels below those of uninfected cells. Our results show that virulent Armenia/07 ASFV controls the cGAS-STING pathway, but these mechanisms are not at play when porcine macrophages are infected with attenuated NH/P68 ASFV. These findings show for the first time the involvement of the cGAS-STING-IRF3 route in ASFV infection, where IFN-ß production or inhibition was found after infection by attenuated or virulent ASFV strains, respectively, thus reinforcing the idea that ASFV virulence versus attenuation may be a phenomenon grounded in ASFV-mediated innate immune modulation where the cGAS-STING pathway might play an important role.IMPORTANCE African swine fever, a devastating disease for domestic pigs and wild boar, is currently spreading in Europe, Russia, and China, becoming a global threat with huge economic and ecological consequences. One interesting aspect of ASFV biology is the molecular mechanism leading to high virulence of some strains compared to more attenuated strains, which produce subclinical infections. In this work, we show that the presently circulating virulent Armenia/07 virus blocks the synthesis of IFN-ß, a key mediator between the innate and adaptive immune response. Armenia/07 inhibits the cGAS-STING pathway by impairing STING activation during infection. In contrast, the cGAS-STING pathway is efficiently activated during NH/P68 attenuated strain infection, leading to the production of large amounts of IFN-ß. Our results show for the first time the relationship between the cGAS-STING pathway and ASFV virulence, contributing to uncover the molecular mechanisms of ASFV virulence and to the rational development of ASFV vaccines.

African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses.

The risk of spread of African swine fever virus (ASFV) from Russia and Caucasian areas to several EU countries has recently emerged, making it imperative to improve our knowledge and defensive tools against this important pathogen. The ASFV genome encodes many genes which are not essential for virus replication but are known to control host immune evasion, such as NFκB and the NFAT regulator A238L, the apoptosis inhibitor A224L, the MHC-I antigen presenting modulator EP153R, and the A276R gene, involved in modulating type I IFN. These genes are hypothesized to be involved in virulence of the genotype I parental ASFV NH/P68. We here describe the generation of putative live attenuated vaccines (LAV) prototypes by constructing recombinant NH/P68 viruses lacking these specific genes and containing specific markers.