Deletion of MGF505-2R Gene Activates the cGAS-STING Pathway Leading to Attenuation and Protection against Virulent African Swine Fever Virus.

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-ß 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.

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.

Recombinant African Swine Fever Virus Arm/07/CBM/c2 Lacking CD2v and A238L Is Attenuated and Protects Pigs against Virulent Korean Paju Strain.

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.

African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation.

African swine fever virus (ASFV) is the causative agent of one of the most lethal diseases affecting domestic pig and wild boar, which is endangering the swine industry due to its rapid expansion. ASFV has developed different mechanisms to evade the host immune response, including inhibition of type I IFN (IFN-I) production and signaling, since IFN-I is a key element in the cellular antiviral response. Here, we report a novel mechanism of evasion of the IFN-I signaling pathway carried out by the ASFV ubiquitin-conjugating enzyme pI215L. Our data showed that pI215L inhibited IFN-stimulated response element (ISRE) activity and the consecutive mRNA induction of the IFN-stimulated genes ISG15 and IFIT1 through the ubiquitination and proteasomal degradation of STAT2. Additionally, by immunofluorescence, co-immunoprecipitation and nucleus-cytoplasm fractionation approaches, we have confirmed the interaction and colocalization of STAT2 and pI215L, in ectopic experiments and during ASFV infection. Moreover, expression of the catalytic mutant (I215L-C85A) did not inhibit the induction of ISG15 and IFIT1, nor the activity of ISRE. Furthermore, we confirmed that STAT2 degradation by pI215L is dependent on its catalytic activity, since expression of the pI215L-C85A mutant did not affect STAT2 levels, compared to the wild-type protein. Yet, our data reveal that the interaction of pI215L with STAT2 does not require the integrity of its catalytic domain since the pI215L-C85A mutant co-immunoprecipitates with STAT2. All these findings reveal, for the first time, the involvement of E2-ubiquitin-conjugating enzyme activity of pI215L in the immune response modulation.

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.

Identification and Isolation of Two Different Subpopulations Within African Swine Fever Virus Arm/07 Stock.

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.

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.

Evaluation of a viral DNA-protein immunization strategy against African swine fever in domestic pigs.

African swine fever virus (ASFV) causes serious disease in domestic pigs for which there is no vaccine currently available. ASFV is a large DNA virus that encodes for more than 150 proteins, thus making the identification of viral antigens that induce a protective immune response difficult. Based on the functional roles of several ASFV proteins found in previous studies, we selected combinations of ASFV recombinant proteins and pcDNAs-expressing ASFV genes, to analyze their ability to induce humoral and cellular immune responses in pigs. Pigs were immunized using a modified prime-boost approach with combinations of previously selected viral DNA and proteins, resulting in induction of antibodies and specific cell-mediated immune response, measured by IFN-γ ELISpots. The ability of antibodies from pigs immunized with various combinations of ASFV-specific antigens to neutralize infection in vitro, and antigen-specific activation of the cellular immune response were analyzed.