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.

Non-Invasive Sampling in the Aspect of African Swine Fever Detection-A Risk to Accurate Diagnosis.

African swine fever remains one of the most economically important and dangerous diseases of the Suidae family. Until now, neither a safe vaccine nor a treatment against ASF has been available, which is why prevention of the disease involves biosecurity measures and early recognition based on accurate diagnosis. Nowadays, different strategies for ASF detection are discussed to reduce both animal suffering and the costs of ASF surveillance. This article aims to indicate the risk, with regard to non-invasive sampling, for the detection of ASFV. In this study, we analyzed data from three independent animal trials, in the framework of the detection of positive samples in different matrices (blood, sera, oral and rectal swabs) collected from nineteen domestic pigs infected with similar doses but under different scenarios, including different ASFV strains or routes of infection. Genetic material of ASFV was found in all matrices, but detection occurred earlier in the blood samples than in the oral and the rectal swabs. Furthermore, analyses revealed that at relevant sampling timepoints, PCR-positive blood samples were detected more frequently and reached higher percentages (up to 100% during fever) than oral and rectal swabs. Moreover, mean Ct values in blood samples collected from animals infected with virulent strains were significantly lower than in oral and rectal swabs, ensuring a higher probability of ASFV detection. High Ct values and occasional shedding in all tested matrices, in the cases of animals infected by an attenuated ASFV-strain, showed that blood sampling may be necessary to confirm the presence of anti-ASFV antibodies in sera. This study showed that during veterinary surveillance, blood sampling (for both PCR and serological analyses) is essential for the accurate diagnosis of ASF and provides the highest probability of detection of the disease.

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.

Long amplicon sequencing for improved genetic characterization of African swine fever virus.

African Swine Fever Virus (ASFV) causes a transmissible and fatal disease in pigs that is currently devastating global swine production. Efficient and economical collection of genetic data from ASFV field isolates is essential for bio-surveillance, to limit and control its spread, and to better understand ASF disease ecology. Standard genotyping and subtyping of ASFV field isolates is currently limited to a few variable regions within the ASFV genome. However, more extensive sequencing is necessary to better understand ASFV molecular evolution and identify regions relevant to genetic diversity. In this study, we developed a method for rapid and efficient next generation sequencing of approximately 40% of the ASFV genome using long PCR amplification of six different genomic regions. The amplified regions contain all segments currently used for genotyping and additional genes predicted to contribute to ASFV diversity. The primers used for amplification are broadly compatible with published ASFV genomes, permitting their use on relevant ASFV isolates. This methodology provides the enhanced depth of coverage of amplicon-based sequencing while mitigating complications associated with ASFV whole-genome sequencing. Implementation of this methodology could substantially increase the scale of ASFV genetic data collection, which is necessary to effectively monitor and combat this critical agricultural disease.

An Update on African Swine Fever Virology.

Animal diseases constitute a continuing threat to animal health, food safety, national economy, and the environment. Among those, African swine fever (ASF) is one of the most devastating viruses affecting pigs and wild suids due to the lack of vaccine or effective treatment. ASF is endemic in countries in sub-Saharan Africa, but since its introduction to the Caucasus region in 2007, a highly virulent strain of ASF virus (ASFV) has continued to circulate and spread into Eastern Europe and Russia, and most recently into Western Europe, China, and various countries of Southeast Asia. Given the importance of this disease, this review will highlight recent discoveries in basic virology with special focus on proteomic analysis, replication cycle, and some recent data on genes involved in cycle progression and viral-host interactions, such as I215L (E2 ubiquitin-conjugating enzyme), EP402R (CD2v), A104R (histone-like protein), QP509L, and Q706L (RNA helicases) or P1192R (Topoisomerase II). Taking into consideration the large DNA genome of ASFV and its complex interactions with the host, more studies and new approaches are to be taken to understand the basic virus-host interaction for ASFV. Proteomic studies are just paving the way for future research.

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.

Development of vaccines against African swine fever virus.

An outbreak in the Caucasus in 2007 initiated the spread of ASFV through Russia and Eastern Europe, subsequently affecting Ukraine, Belarus, Poland, the Baltic States, the Czech Republic, Moldova, Romania and Bulgaria. The declaration of outbreaks in China and Central Europe in August 2018, definitely confirms the serious threat that the extension of ASF represents for the global swine industry and the environment. Despite the efforts of several groups to generate a vaccine against ASFV, currently only control and eradication measures are available based mainly on the early detection and implementation of strict sanitary procedures, including the mass slaughter of animals, both domestic and wild boar. However, the rapid spread of the disease shows that these actions are clearly insufficient to control the current pandemic situation, and the development of a vaccine is urgently required.

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.

DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain.

African swine fever virus (ASFV) causes high morbidity and mortality in swine (Sus scrofa), for which there is no commercially available vaccine. Recent outbreaks of the virus in Trans-Caucasus countries, Eastern Europe, Belgium and China highlight the urgent need to develop effective vaccines against ASFV. Previously, we evaluated the immunogenicity of a vaccination strategy designed to test various combinations of ASFV antigens encoded by DNA plasmids and recombinant proteins with the aim to activate both humoral and cellular immunity. Based on our previous results, the objective of this study was to test the combined DNA-protein vaccine strategy using a cocktail of the most immunogenic antigens against virulent ASFV challenge. Pigs were vaccinated three times with a cocktail that included ASFV plasmid DNA (CD2v, p72, p32, +/-p17) and recombinant proteins (p15, p35, p54, +/-p17). Three weeks after the third immunization, all pigs were challenged with the virulent ASFV Armenia 2007 strain. The results showed that vaccinated pigs were not protected from ASFV infection or disease. Compared to the non-vaccinated controls, earlier onset of clinical signs, viremia, and death were observed for the vaccinated animals following virulent ASFV challenge. ASFV induced pathology was also enhanced in the vaccinated pigs. Furthermore, while the vaccinated pigs developed antigen-specific antibodies, immunized pig sera at the time of challenge lacked the capacity to neutralize virus, and instead was observed to enhance ASFV infection in vitro. The results of this work points to a putative immune enhancement mechanism involved in ASFV pathogenesis that warrants further investigation. This pilot study provides insight for the selection of appropriate combinations of ASFV antigens for the development of a rationally-designed, safe, and efficacious vaccine for ASF.

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis.

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from ß-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.

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.

African Swine Fever Virus Biology and Vaccine Approaches.

African swine fever (ASF) is an acute and often fatal disease affecting domestic pigs and wild boar, with severe economic consequences for affected countries. ASF is endemic in sub-Saharan Africa and the island of Sardinia, Italy. Since 2007, the virus emerged in the republic of Georgia, and since then spread throughout the Caucasus region and Russia. Outbreaks have also been reported in Belarus, Ukraine, Lithuania, Latvia, Estonia, Romania, Moldova, Czech Republic, and Poland, threatening neighboring West European countries. The causative agent, the African swine fever virus (ASFV), is a large, enveloped, double-stranded DNA virus that enters the cell by macropinocytosis and a clathrin-dependent mechanism. African Swine Fever Virus is able to interfere with various cellular signaling pathways resulting in immunomodulation, thus making the development of an efficacious vaccine very challenging. Inactivated preparations of African Swine Fever Virus do not confer protection, and the role of antibodies in protection remains unclear. The use of live-attenuated vaccines, although rendering suitable levels of protection, presents difficulties due to safety and side effects in the vaccinated animals. Several African Swine Fever Virus proteins have been reported to induce neutralizing antibodies in immunized pigs, and vaccination strategies based on DNA vaccines and recombinant proteins have also been explored, however, without being very successful. The complexity of the virus particle and the ability of the virus to modulate host immune responses are most likely the reason for this failure. Furthermore, no permanent cell lines able to sustain productive virus infection by both virulent and naturally attenuated African Swine Fever Virus strains exist so far, thus impairing basic research and the commercial production of attenuated vaccine candidates.

Mechanisms of Entry and Endosomal Pathway of African Swine Fever Virus.

African Swine Fever Virus (ASFV) causes a serious swine disease that is endemic in Africa and Sardinia and presently spreading in Russia and neighboring countries, including Poland and recently, the Czech Republic. This uncontrolled dissemination is a world-wide threat, as no specific protection or vaccine is available. ASFV is a very complex icosahedral, enveloped virus about 200 nm in diameter, which infects several members of pigs. The virus enters host cells by receptor-mediated endocytosis that depends on energy, vacuolar pH and temperature. The specific receptor(s) and attachment factor(s) involved in viral entry are still unknown, although macropinocytosis and clathrin-dependent mechanisms have been proposed. After internalization, ASFV traffics through the endolysosomal system. The capsid and inner envelope are found in early endosomes or macropinosomes early after infection, colocalizing with EEA1 and Rab5, while at later times they co-localize with markers of late endosomes and lysosomes, such as Rab7 or Lamp 1. A direct relationship has been established between the maturity of the endosomal pathway and the progression of infection in the cell. Finally, ASFV uncoating first involves the loss of the outer capsid layers, and later fusion of the inner membrane with endosomes, releasing the nude core into the cytosol.

Characterization of the African Swine Fever Virus Decapping Enzyme during Infection.

African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts.IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.

CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection.

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.

African swine fever virus uses macropinocytosis to enter host cells.

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.