Exploring antiviral strategies to combat African swine fever

Abstract

African swine fever (ASF), caused by the highly contagious African swine fever virus (ASFV), poses a significant threat to domestic and wild pigs worldwide. Despite its limited host range and lack of zoonotic potential, ASF has severe socio-economic and environmental consequences. Current control strategies primarily rely on early detection and culling of infected animals, but these measures are insufficient given the rapid spread of the disease. Developing effective therapeutics against ASFV is crucial to prevent further spread and mitigate economic losses. Although vaccination remains critical, recent vaccine approvals in Vietnam have raised safety and efficacy concerns. Moreover, as challenges persist in vaccine development and deployment, particularly in complex field conditions, antiviral agents have emerged as a critical complementary approach. These agents have the potential to mitigate side effects and control viral spread when vaccines alone are insufficient or when animals face simultaneous exposure to vaccine strains and wild-type viruses. However, advancing them from proof-of-concept to widespread practical application entails a significant interdisciplinary effort, given the logistical and economic constraints of in vivo testing. In this review, we examine emerging antiviral approaches and highlight key ASFV replication mechanisms and therapeutic targets to guide rational drug design amidst an evolving viral landscape.

This review provides an updated synthesis of recent advances in ASF antiviral strategies, identifying key viral replication mechanisms and therapeutic targets, and emphasizes the importance of coordinated efforts to overcome experimental constraints and translate these findings into effective means of disease control.

African swine fever virus: global impact and genetic complexity

Since its initial detection in Kenya in 1921 (Montgomery and Eustace Montgomery 1921), African swine fever (ASF) has emerged as a significant concern for both domestic and wild pig populations worldwide. This devastating disease, caused by the African swine fever virus (ASFV), perpetuates through an ancient sylvatic cycle primarily involving African warthogs (Phacochoerus africanus) and soft ticks of the Ornithodoros moubata complex as vectors (Plowright et al. 1969, Wilkinson et al. 1988). Though domestic pigs and Eurasian wild boars (Sus scrofa) are the exclusive hosts susceptible to ASFV infection outside of Africa, with no identified zoonotic potential, the virus’s high lethality in these species (Blome et al. 2013, Penrith et al. 2019) inflicts severe socio-economic and environmental consequences in affected regions.

The global spread of ASF has extended its impact beyond Africa to Europe and Asia, thus reaching territories that had previously been unaffected by the disease. Notably, an ASF outbreak in 2018 in China, a key player in the global pork market, had significant spill-over effects on global meat and animal feed markets, overshadowing in some respects the COVID-19 pandemic that soon followed (Frezal et al. 2021, Schmidhuber 2021). The perturbation of Chinese pork output resulted in record imports, leading to higher world prices for pork and other meats, along with a reduction in grain and oil meal imports, the main components of pig rations (Frezal et al. 2021, Schmidhuber 2021). Although some countries have reported successful ASF eradication (Danzetta et al. 2020), the disease continues to spread worldwide, impacting both domestic and wild pig populations and causing trade restrictions and disruptions in the global pork market, as described by the World Organization for Animal Health (WOAH 2024).

Beyond the widespread impacts on commercial pig production and international trade, smallholders in low- and middle-income countries have experienced even more severe consequences of ASF (Penrith et al. 2022, 2023). These farmers often rely on pig farming as a primary source of income and food security, with limited resource availability further exacerbating their vulnerability and resulting in increased food insecurity and severe disruptions to their livelihoods (Penrith et al. 2022, 2023). In these settings, free-ranging (and feral) pigs are common, yet the role of such animals in disease transmission varies significantly by region. In eastern and southern Africa, transmission via the classic sylvatic cycle to domestic pigs occurs almost exclusively indirectly, mainly through infected ticks that domestic pigs pick up where they are sympatric with warthogs (Mulumba-Mfumu et al. 2019, Penrith et al. 2019). Under natural conditions, warthogs and bushpigs (Potamochoerus larvatus) harbor little if any ASFV and they are unlikely to shed the virus, making the risk of transmission from their carcasses very low (Thomson et al. 1980, Anderson et al. 1998). In Europe, the transmission and spread of ASFV through Eurasian wild boars has resulted in a different sylvatic cycle (Blome et al. 2013). Being conspecific with domestic pigs, wild boar share the same susceptibility to ASFV and they are efficient transmitters of the virus, with both presenting similar clinical symptoms and high fatality rates (Blome et al. 2013, Penrith et al. 2023). Before ASF was introduced into the European Union in 2014, wild boars were not considered likely to sustain ASFV infection in the absence of domestic pigs as reservoirs (Penrith et al. 2023). However, recent studies have demonstrated independent circulation in wild boars, and geographic overlap between wild boar cases and domestic pig outbreaks has been reported in several Eastern European countries (Gervelmeyer 2017). In Asia, spill-over of ASFV from domestic pigs to wild boars has also been reported (Denstedt et al. 2021), and there is some evidence of sustained circulation in these latter (Jo and Gortázar 2020, Lee et al. 2021).

The ASF virus is a 200-nm diameter icosahedral DNA virus consisting of an envelope, capsid, inner capsule membrane, core shell, and inner core. Its linear double-stranded DNA (dsDNA) genome, typically 170–190 kb in length, encodes ~150–200 viral proteins, including 68 structural proteins and over 100 nonstructural proteins (Alejo et al. 2018, Karger et al. 2019). Historically, ASFV has been viewed as genetically stable, evidenced by its similarity to ancient viral elements in the soft tick genome (Forth et al. 2020). Strain classification has primarily focused on variations in the B646 L gene, responsible for encoding the major capsid protein p72, which has been used to categorize ASFV isolates into genotypes and, more recently, biotypes (Dinhobl et al. 2024).

Since its introduction into Eurasia in 2007, only genotype 2 has been found outside of Africa (Gallardo et al. 2014, Gonzales et al. 2021, Sauter-Louis et al. 2021, Iscaro et al. 2022, Ramirez-Medina et al. 2022a, Kim et al. 2023). Numerous reports have documented the existence of variations stemming from the highly virulent genotype 2 strain, yet despite more than a decade of epidemic circulation, it has accumulated few mutations (Gallardo et al. 2014, Zani et al. 2018, Forth et al. 2019, Mazur-Panasiuk et al. 2020). Notable exceptions include the detection of five distinct lineages in wild boars in Germany, which are clearly different from the Eurasian genotype 2 strain and harbor previously undocumented high-impact mutations (Forth et al. 2023). In addition, a hybrid virus combining a newly detected low-virulent genotype 1 virus, closely resembling a historical vaccine strain (Sun et al. 2021a), and the genotype 2 strain has been identified in China (Zhao et al. 2023). These unexpected genetic divergences challenge previous assumptions about ASFV evolution and epidemiology, potentially having a significant impact on the course of the current ASFV pandemic.

The German scenario is particularly worrisome. The emergence of distinct genotype 2 variants there evidences higher ASFV sequence variability than previously observed (Forth et al. 2023). This development has been plausibly linked to a mutation in the O174 L gene that encodes DNA repair polymerase (PolX), an enzyme believed to be a strategic mutagenase (Lamarche et al. 2006), interfering with its activity (Forth et al. 2023). Assuming a direct correlation between heightened mutational activity and the emergence of new variants, the mutation rate in this geographic region may be elevated (Forth et al. 2023). Given the recent identification of positive disease cases near the borders of countries free from the disease (Belgium, France, and Luxembourg) (WOAH 2024), there is a very real risk of further spread of these variants across the EU.

In China, the emergence of the genotype I–II hybrid variant, along with low-virulence genotype II viruses that resemble potentially illegally copied vaccines (Patton and Patton 2021, Sun et al. 2021b), raises intriguing questions about ASFV evolution and the interaction between naturally occurring and vaccine-derived strains. Since they are capable of causing chronic infection and are highly transmissible, these low-virulence strains are characterized by ill-defined clinical symptoms and a long incubation period (Sun et al. 2021b), which can delay detection of infected animals. Under inadequate monitoring conditions, this scenario could lead to further outbreaks silently spreading within the country (Ito et al. 2022). The identification of ASF high-risk areas around eight major airports and three significant seaports in China (Ito et al. 2022), coupled with the detection of ASFV in pork products imported from China, some hosting infective virus (Jurado et al. 2019), underscores the transmission risk posed by international travel. The hybrid variant has already spread across the border to Vietnam, exacerbating the pandemic threat (Arter-Hazzard et al. 2024). Moreover, the growing number of Chinese-owned pig farms in various continents (Cook et al. 2016, Gálvez 2021, Mishler 2023) further amplifies the potential for dissemination of these atypical viruses into new territories, heightening the risk of international transmission.

Effective control measures against ASF remain elusive, despite current strategies focused on early detection and culling of infected animals. While vaccination serves as a cornerstone of ASF control, concerns regarding safety and efficacy, as observed with recent vaccine approvals in Vietnam (Guarascio et al. 2023, WOAH 2023a, van den Born et al. 2025), and the potentially complex interplay between natural strains and vaccine-derived strains, as described above for mainland China, emphasize the necessity of exploring alternative therapeutic approaches. Doing so is particularly crucial given the potential for an accelerated rate of mutation within ASFV, which could give rise to variants with novel characteristics, potentially altering the trajectory of the ASFV pandemic dramatically.

Overview of the current treatment landscape

At present, there is no specific treatment available for ASF. Instead, disease management largely depends on preventive measures, early detection, and stringent control protocols (World Organisation for Animal Health 2024). Preventive strategies include the implementation of strict biosecurity measures to avoid introducing ASFV into ASF-free areas or farms. That includes regulating the movement of pigs and pork products, as well as decontamination of potential fomites that could carry the virus. In regions where ASF is endemic or where there is a high risk of incursion, the emphasis is on early detection and disease containment through surveillance of pig populations for clinical signs, as well as the implementation of control measures, such as culling of infected and at-risk animals to prevent further spread of the virus (WOAH 2024).

However, the implementation of effective biosecurity measures to control ASFV presents significant challenges. High costs related to biosecurity, coupled with the low immediate return on investment, mean that there is little economic incentive for farmers to invest in comprehensive biosecurity (Penrith et al. 2023). This is particularly true in resource-limited settings and for smallholder farms, where the financial cost of biosecurity can represent a heavy burden (Ebata et al. 2020). A previous study reported that many farmers do not fully appreciate the importance of stringent biosecurity, often due to a lack of knowledge or awareness of the dynamics of disease transmission (Aliro et al. 2022). Additionally, structural issues such as poor-quality perimeter fencing, inadequate sanitation protocols, and the challenges inherent in managing multiple farm sites further complicate biosecurity efforts (Dione et al. 2020).

Given these limitations, development and implementation of effective vaccines are considered cornerstones of long-term disease control and potential eradication (Rock 2021). However, progress is challenged by several factors, including the complexity of the ASFV genome, lack of a full understanding of its pathogenesis, limited knowledge about innate immune evasion strategies, and the complex interactions between the virus and its host (Rock 2021).

Currently, several vaccine candidates are being developed using different strategies: modified live vaccines using attenuated viral forms (Vu and McVey 2024); subunit vaccines targeting specific viral proteins (Gaudreault and Richt 2019); and viral vector vaccines using modified viruses to deliver ASFV genes and trigger an immune response (Gaudreault and Richt 2019). However, only two ASFV vaccines, NAVET-ASFVAC (ASFV-G-ΔI177 L strain) and AVAC ASF LIVE (ASFV-G-ΔMGF strain), have obtained commercial approval (Reuters 2023). Developed in Vietnam, both of these live-virus vaccines target genotype II Vietnamese strains.

Although the approval of these two vaccines is a major step in ASF control efforts, it has also prompted warnings from WOAH regarding the safety and efficacy of ASF vaccines (WOAH 2023b). Although these warnings are not directed at any specific vaccine, they point out the necessity of extensive testing before widespread use and highlight the need for careful evaluation to minimize the risks of inferior vaccines. Indeed, recent evidence shows that the ASFV-G-ΔI177 L strain can revert to virulence after passaging and significantly affects sow reproductive performance (van den Born et al. 2025), factors that may be driving the recent spike in infections across Vietnam (Arter-Hazzard et al. 2024) and implying limited effectiveness of the vaccination campaign. This situation underscores the inherent risks of using live-virus vaccines and stresses the importance of rigorous safety and efficacy trials before relying on them for ASF control.

Exploring alternative approaches

Deployment of ASF vaccines, especially those based on modified live virus formulations such as those approved in Vietnam, is associated with a number of challenges and concerns. There is a risk of reversion to a virulent form, especially in pigs with concurrent infections or other health conditions (Rock 2021). Additionally, administering vaccines during active ASF outbreaks complicates efficacy/safety assessments, as pigs could be exposed simultaneously to both the attenuated vaccine virus and the wild-type virus. Such dual exposure renders it difficult to distinguish between immunity conferred by the vaccine and natural infection. Conditions in the field can also vary widely, making standardized evaluations problematic (Madewell et al. 2021). More importantly, the benefits of vaccines, especially in the smallholder sector, can be constrained by genotype variability with respect to homologous vaccine protection, thermostability, costs, and practical implementation. These factors can significantly influence both the effectiveness and feasibility of vaccine deployment in different agricultural contexts (Penrith et al. 2023).

Antiviral agents can play a significant role as an alternative or adjunct to vaccination programs, especially in controlling viral infections where vaccines are either unavailable, only partially effective, or face practical deployment challenges. For instance, several compounds such as recombinant interferons (IFNs) and small-molecule antivirals have been investigated for their potential use against economically significant veterinary pathogens, e.g. bovine herpesvirus type 1 and respiratory syncytial virus (Newcomer et al. 2014). As an adjunct to vaccination, antivirals can represent a more robust and comprehensive approach to managing outbreaks of viral diseases. Recently, antiviral drugs were crucial in reducing disease severity and transmission during the COVID-19 pandemic, and they complemented vaccination programs by providing early protection and reducing disease severity, particularly in populations less responsive to vaccination, such as older adults (Cheung et al. 2024). In pigs, combination of an inactivated foot-and-mouth disease (FMD) vaccine with an antiviral vector expressing porcine IFNs and small interfering RNAs (siRNAs) endowed rapid protection within 1–2 days postinoculation, i.e. well before vaccine-induced immunity alone would have taken effect (You et al. 2017). Similarly, in poultry, coadministration of antiviral drugs, such as baloxavir marboxil with vaccination against highly pathogenic avian influenza was shown to improve survival and reduce viral shedding (Twabela et al. 2020).

In the context of ASF, antiviral agents could be deployed strategically alongside vaccination efforts to improve outcomes in various production systems. For instance, antivirals might be administered during the early stages of an outbreak to lower viral load and transmission risk while vaccine-induced immunity develops. This concept has already been explored in the context of FMD, with a previous study indicating that antiviral agents may bridge the immunity gap between the onset of an outbreak and the time required for effective vaccine-induced protection (You et al. 2017). In FMD-free regions facing sudden outbreaks, delays in vaccine strain selection or deployment can exacerbate disease spread, and coadministration of antivirals with emergency vaccines has been proposed as an efficient strategy to contain fast-spreading infections (You et al. 2017). Applying similar principles to ASF, combined antiviral-vaccine strategies may be particularly useful in high-density commercial farms or smallholder systems, where rapid containment is essential and vaccine logistics may be limited. Moreover, this combined approach could also help mitigate adverse reactions to vaccines, particularly during vaccination campaigns when animals are likely to have concurrent viral infections, which can either exacerbate reactions or mask immune responses. This issue is particularly relevant under conditions when animals are exposed to both vaccine strains and wild-type viruses, which may complicate immune responses and result in more severe reactions. In such situations, antiviral agents might help manage viral load, reduce the severity of infections, and improve the efficacy of the vaccine. In wild or free-ranging populations, antiviral-laced baits could serve both therapeutic and prophylactic purposes when deployed in tandem with oral vaccines. Although regulatory frameworks for such integrated approaches are still evolving, their flexibility and potential to reduce overreliance on any single intervention make them a valuable area for future research.

For these reasons, there is a growing interest in antiviral compounds with demonstrated in vitro and/or in vivo activity against ASFV. Such antivirals can be divided into two categories based on their mechanisms of action: direct-acting antivirals (DAAs) that target viral proteins to inhibit key processes of ASFV replication, and host-targeting antivirals (HTAs) that interfere with cellular pathways essential to the virus (Fig. 1). Designing such antiviral strategies requires a detailed understanding of the ASFV replication cycle, as both viral and host components represent essential targets. In the following section, we briefly review the process of virus replication, thereby providing a basis for pinpointing potential targets of antiviral interventions.

Figure 1.

ASFV infectious cycle and antiviral intervention points. ASFV enters host cells through receptor-mediated endocytosis (1), with several candidate receptors proposed (CD163, CD45, and MHC II) though none have been definitively confirmed. While Fc receptors do not appear to mediate infection directly, antibody-dependent enhancement (ADE) has been observed, whereby virus–antibody complexes are internalized via Fc receptors, increasing viremia and disease severity. Additional entry routes include clathrin-mediated endocytosis (2), macropinocytosis (3), and efferocytosis (4). After internalization, virions are transported along microtubules within endosomes to the perinuclear region. Acidification in late endosomes triggers capsid disassembly (5) and enables fusion and uncoating (6), releasing the viral core into the cytoplasm. Replication and assembly (7) take place in perinuclear viral factories located at the microtubule-organizing center, where early and late gene expression supports genome replication and morphogenesis. Mature virions are assembled using modified endoplasmic reticulum membranes, then undergo transport (8) to the plasma membrane, where they are released by budding (9), acquiring an outer envelope. Alternatively, ASFV can exit the cell via apoptotic body-mediated release (10), a process that may aid immune evasion and viral spread. Text boxes indicate known or proposed antiviral compounds and inhibitors that disrupt specific stages of the infectious cycle, including entry, trafficking, genome replication, virion egress, and suppression of host immune responses. Adapted from Dixon et al. (2025). Advances in African swine fever virus molecular biology and host interactions contributing to new tools for control. J Virol. https://doi.org/10.1128/jvi.00932-24 Created in https://BioRender.com.

Alt text: A schematic illustrating the lifecycle of ASFV within a host cell. The diagram follows the virus from entry to release, showing receptor-mediated endocytosis, internalization via clathrin-mediated endocytosis, macropinocytosis, and efferocytosis. It depicts endosomal trafficking and uncoating (pH < 5), followed by replication and assembly in perinuclear viral factories located at the microtubule-organizing center, and concludes with release via apoptotic bodies. Text boxes identify inhibitors targeting specific stages of the infectious cycle, including entry, trafficking, genome replication, virion egress, and suppression of host immune responses.

ASFV replication cycle and therapeutic targets

ASFV replicates in the mononuclear phagocyte system, with macrophages and monocytes being the primary sites of infection (Gómez-Villamandos et al. 2013). The replication process starts with attachment and entry into the host cell using a range of mechanisms, including receptor-mediated pathways influenced by low pH and temperature-sensitive events (Valdeira and Geraldes 1985, Alcamí et al. 1989, 1990). Although specific ASFV entry receptors have not yet been identified, a number of candidates have been proposed, such as CD163, CD45, and MHC II (Sánchez-Torres et al. 2003, Lithgow et al. 2014). Additionally, recent research has implicated a role for laminin receptor 1 (RPSA), either as an entry receptor or a restriction factor (Chen et al. 2022). RPSA is known to be involved in other viral infections (e.g. FMD), where it interacts with viral proteins to alter host cell pathways (such as MAPK) (Zhu et al. 2020), thus promoting viral replication and having potential implications for ASFV entry. Furthermore, structural proteins like p12, p30, p72, and p54 are important mediators in viral attachment to permissive cells. The highly expressed p30 phosphoprotein facilitates viral internalization, and previous studies have shown that p72 binds to host proteins, such as CD63, B2M, and YTHDF2 that are involved in different processes of virus attachment, invasion, and immune regulation (Carrascosa et al. 1991, Angulo et al. 1993, Gómez-Puertas et al. 1996, 1998, Chen et al. 2022, Weng et al. 2024). Though Fc receptors have been suggested as a possible viral entry route, one study reported that they do not mediate ASFV infection in macrophages (Alcamí and Viñuela 1991). However, other evidence supports antibody-dependent enhancement (ADE), whereby IgG-bound virus-antibody complexes are internalized via Fc receptors and result in heightened viremia and disease exacerbation among vaccinated swine (Gaudreault and Richt 2019). Similar ADE mechanisms have been documented in infections caused by porcine reproductive and respiratory syndrome virus (PRRSV) and dengue virus (Taylor et al. 2015).

ASFV also utilizes nonreceptor-mediated mechanisms for infectivity, such as macropinocytosis and phagocytosis (Basta et al. 2010, Sánchez et al. 2012, Hernáez et al. 2016). Macropinocytosis, an actin-dependent process involving the uptake of extracellular fluid, has been described previously as a pathway for ASFV entry into swine macrophages, as has clathrin-mediated endocytosis (CME) (Andrés 2017, Sánchez et al. 2017). Both mechanisms are modulated by phosphoinositide-3-kinase (PI3K) and Rho-GTPase signaling, which are critical for actin reorganization and viral internalization (Sánchez et al. 2017). DAB2, which facilitates receptor recruitment to clathrin-coated pits, may also be involved in ASFV infection by promoting CME through the protein’s interaction with the RPSA receptor, with this latter potentially activated by the viral p30 protein (Figliuolo da Paz et al. 2020, Chen et al. 2022). This binding process may simultaneously suppress innate immune responses and enhance viral replication (Chen et al. 2022). In addition, ASFV has been shown to exploit apoptotic mimicry to enhance entry into macrophages via efferocytosis, a specialized form of phagocytosis. During late stages of infection, ASFV induces apoptosis in infected cells, leading to the exposure of phosphatidylserine on the plasma membrane. Virions can associate with apoptotic bodies or directly incorporate phosphatidylserine, facilitating recognition and uptake by macrophages through phosphatidylserine receptors such as TIM4. This route of entry is supported by studies showing that blocking phosphatidylserine or TIM4 significantly reduces infection (Chen et al. 2023a, Gao et al. 2023, Dixon 2025). Key structural components involved in core detachment and release include the pp220 polyprotein, which is cleaved by the viral protease pS273R into four major core proteins necessary for viral uncoating (Andrés et al. 2002a, b, Alejo et al. 2003), and the internal envelope protein pE248R that is required for fusion of the viral envelope to endosomal membranes, thereby allowing core release (Rodríguez et al. 2009, Hernáez et al. 2016, Gaudreault et al. 2020).

After internalization, ASFV particles are trafficked to late endosomes, a step in which Rab7 plays a pivotal role (Cuesta-Geijo et al. 2012, Hernáez et al. 2016). The acidic conditions inside endosomes trigger viral uncoating (Hernáez et al. 2016), leading to fusion of the inner viral envelope with endosomal membranes and release of the viral core into the cytoplasm, where subsequent replication occurs. In addition to its role in core release, the pE248R protein, together with pE199L, facilitates viral core penetration by interacting with endosomal proteins, such as Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and Lamp-2), thus allowing penetration of the viral core into the host cytoplasm (Matamoros et al. 2020, Cuesta-Geijo et al. 2022). Consistent with this process, genome-wide CRISPR-Cas9 screens on porcine cells have identified genes coding for proteins in late endosome membranes as being essential for ASFV replication, including the MHC II-specific transcription factors RFXAP and CIITA, components of nonclassical MHC II SLADM molecules (SLA_DMA and SLA_DMB), and the transmembrane protein 239 (TMEM239) (Pannhorst et al. 2023, Shen et al. 2024, Dixon 2025). Similarly, the ASFV p54 protein interacts with the cellular dynein light chain (LC8), which is essential for the viral transport machinery (Alonso et al. 2001). Moreover, host cell phosphoinositides, including phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns (4,6) biphosphate, play a critical role in promoting early infection events toward the initiation of ASFV replication (Cuesta-Geijo et al. 2012, Muñoz-Moreno et al. 2015).

ASFV gene expression is highly regulated, with early gene transcription occurring 4–6 hours postinfection (hpi) and supporting viral replication in perinuclear viral factories (Dixon et al. 2013, Muñoz-Moreno et al. 2015, Gaudreault et al. 2020). The virus encodes a unique DNA polymerase B (G1211R) responsible for initiating replication, as well as enzymes like PolX (O174L), which may assist in replication within the oxidative macrophage cytoplasm potentially through a base excision repair (BER) pathway (Dixon et al. 2013, Gaudreault et al. 2020). DNA replication begins 6–8 hpi, followed by the expression of intermediate and late genes (Gaudreault et al. 2020). These latter encode structural proteins essential for virion assembly, including an E2 ubiquitin-conjugating enzyme (pI215L) (Freitas et al. 2018), a histone-like protein (pA104R) (Frouco et al. 2017a), RNA helicases (QP509 L and Q706L) (Freitas et al. 2019), and a type II topoisomerase (pP1192R) (Coelho et al. 2015, Freitas et al. 2016). Although ASFV replicates primarily in the cytoplasm, there is evidence to indicate that an early phase of DNA replication may occur in the nucleus (Simões et al. 2019), though its biological relevance remains unclear. After assembly, mature virus particles are transported through the cytoplasm to the plasma membrane via microtubules (MTs). The interaction between p54 and the dynamin light chain (LC8) regulates the intracellular transport of this MT-based dynamic complex (Alonso et al. 2001, Rodríguez et al. 2004). Binding of structural proteins, including pE120R and the major shell protein p72, is required to transport newly synthesized viruses from the assembly site to the host cell membrane, where they are coupled to kinesin to facilitate their egress (Andrés et al. 2001b, Cuesta-Geijo et al. 2017). The entire ASFV infection cycle, from attachment and entry to the budding of mature virus particles, is typically completed within 24 hpi (Muñoz-Moreno et al. 2015).

ASFV employs a multifaceted strategy to evade host immunity by targeting both innate and adaptive immune responses. A core part of this strategy involves disrupting the cGAS-STING pathway, which effectively inhibits type I IFN production, particularly through the suppression of IFN-β (García-Belmonte et al. 2019, Chen et al. 2024). Viral proteins, such as DP96R and pI215 L interfere with key steps in this pathway, with pI215 L notably blocking the K63-linked polyubiquitination of TANK-binding kinase 1 (TBK1) that is essential for cGAS-STING-mediated IFN production (Wang et al. 2018, Huang et al. 2021a). Proteins encoded by ASFV, including I329 L and members of the MGF360 and MGF505/530 multigene families, further inhibit IFN responses, most significantly by blocking toll-like receptor 3 (TLR3) signaling and suppressing IFN (Alfonso et al. 2004, de Oliveira et al. 2011, Correia et al. 2013, Golding et al. 2016, Li et al. 2021). Furthermore, viral protein A137R downregulates IFN-β production, whereas K421R, EP364R, C147L, A137R, and notably pA238 L all inhibit NF-κB, another downstream component of cGAS-STING, to enhance viral replication (Silk et al. 2007, Dixon et al. 2019, Wu et al. 2023). CD2v contributes to this process by blocking transcription of immunoregulatory genes and disrupting the trafficking of cellular proteins (Pérez-Núñez et al. 2015), while other viral proteins such as DP71L, A179L, A224L, EP153R, and S273R promote host cell survival by inhibiting host cell death pathways (Wang et al. 2018, 2021b, Zhao et al. 2022). ASFV also modulates proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), another key component of the antiviral response, by blocking its transcription and expression (Zhu et al. 2019, Wang et al. 2021b), thereby further compromising the ability of the host to mount an effective response to the infection. Additionally, ASFV reduces the expression of major histocompatibility complex (MHC) molecules, limiting cytotoxic T-cell activation and, hence, adaptive immunity (Wang et al. 2018, Zhu et al. 2019).

Antiviral agents

DDAs

Nucleoside/nucleotide analogs

Nucleoside and nucleotide analogs have become critical elements in antiviral therapies due to their ability to interfere with viral replication. Through their incorporation into viral DNA or RNA, nucleoside and nucleotide analogues impede replication processes, either by halting chain elongation or inducing mutations, while also acting as inhibitors of essential viral and cellular enzymes, such as ribonucleotide reductases and polymerases. Iododeoxyuridine was the first of these analogs to demonstrate activity against ASFV, but its utility is limited by cytotoxicity at higher concentrations (Gil-Fernández et al. 1979).

Subsequently, acyclic nucleoside phosphonate (ANP) analogs—including (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], cidofovir (HPMPC), and adefovir (PMEA)—showed promising inhibition of ASFV replication in vitro (Gil-Fernández and De Clercq 1987, Gil-Fernández et al. 1987). Specifically, cyclic cidofovir (cHPMPC), an improved derivative of cidofovir, demonstrated the capacity to block ASFV replication at early stages of infection, indicating that the viral DNA polymerase PolX or late RNA polymerase might be likely targets. Oral administration of cHPMPC to pigs delayed or prevented the onset of clinical signs and reduced replication of the highly virulent genotype II Georgia 2007/1 strain (Goulding et al. 2022). This cidofovir derivative exhibited a selectivity index (SI, representing the effectiveness and safety of a drug substance) >120 (Table 1; Table S1). Molecular docking and computational studies have revealed that a second nucleotide analog, Cangrelor, inhibits ASFV by specifically targeting PolX, though it has yet to be tested in cell-based or in vivo assays (Choi et al. 2021).

Table 1.

Antiviral compounds with high selectivity index. This table summarizes the nine compounds identified with a Selectivity Index (SI) > 100, selected based on reported CC₅₀ and IC₅₀ values. Compounds are categorized by mechanism of action—direct-acting (DAA), host-targeting (HTA), or unknown—in alignment with the main text sections. Full compound data can be found in Tables S1–S3. CC₅₀, 50% cytotoxic concentration; IC₅₀, half maximal inhibitory concentration; and CPE, cytopathic effect.

Compound	Mechanism	Pharmaceutical class	Specific target	Target phase of infection	Experimental model	Inhibitory phase	CC₅₀a	IC₅₀a	SI	References
(S)-HPMPA	DAA	Nucleoside/nucleotide (ANP analogue)	Viral polymerase (PolX or late RNA polymerase)b	Viral replicationb	In vitro	Mid to late stagesb	150 μg/ml (MTC50)	0.01 μg/ml (MIC₅₀)	15 000	Gil-Fernández and De Clercq (1987), Gil-Fernández et al. (1987)
Cyclic cidofovir (cHPMPC)	DAA	Acyclic nucleoside phosphonate (ANP) analogues	Viral polymerase (PolX or late RNA polymerase)	Viral replication	In vitro and in vivo	Early stageb	39.43–44.29 μM (in PBMs depending on assay type)	<1 μM	> 120c	Goulding et al. (2022)
Brincidofovir	DAA	Nucleoside/nucleotide (ANP analogue)	DNA repair polymerase (PolX)	Viral replication	In vitro and in vivo	Early and late stages	58 μM	2.76 nM (0.001 μM)	21 014.5	Guo et al. (2023a)
Triapine	DAA	Ribonucleotide reductase inhibitor	Viral pF334 L ribonucleotide reductase	Viral replication	In vitro	Early and late stages	158.3 μM	0.44 μM	359.7	Li et al. (2024a)
Cytarabine hydrochloride	DAA	Antimetabolite antineoplastic agent	DNA repair polymerase (PolX)	Viral replication	In vitro	Early and late stages	229.6 μM	0.23 μM	998.2	Li et al. (2024b)
3-deazaneplanocin A	HTA	Adenosine analogue/AdoHcy hydrolase inhibitors	Viral mRNA capping (RNAP complex modulator)	Viral replicationb	In vitro	Early	30 µg/ml	0.01 µg/ml (EC₅₀)	3000	Villalón et al. (1993)
Genkwanin	HTA	Natural flavonoid found in several plants	Microtubule network	Viral replication	In vitro	Early and late stages	595.1 μM	2.9 μM	205.2	Hakobyan et al. (2019)
Toosendanin	HTA	Triterpenoid saponin from Melia toosendan Sieb. Et Zucc	Host gene IRF1	Attachment and Internalizationb	In vitro	Early stageb	31 μM	0.085 μM (EC₅₀)	365	Liu et al. (2022)
Lambda carrageenan	Unknown	Sulphated polysaccharide found in several plants	Unknown	Unknown	In vitro	Data unavailable	3000 µg/ml	25 µg/ml (EC₅₀)	120	García-Villalón and Gil-Fernández (1991)

^aThis table incorporates the most relevant data available from each study; in cases where CC₅₀ and IC₅₀ values were not provided, alternative metrics reported in the original research have been used. Different units were used in the table to reflect the original measurements reported in the various scientific articles; these units are preserved to maintain the accuracy and context of the reported data.

^bThe study does not explicitly verify the proposed targets, phases of infection, and/or inhibitory phases. These assertions are based on comparative studies of similar viruses or inferred from the broader scientific context.

^cCC₅₀ not reached at highest concentration tested; actual SI may be higher.

Of the newer candidates, brincidofovir, a lipid-conjugated derivative of cidofovir developed to improve bioavailability and reduce toxicity, has shown substantial efficacy against ASFV. In both in vitro and in vivo studies, brincidofovir has demonstrated potent inhibition of ASFV replication in porcine alveolar macrophages (PAMs) by significantly reducing viral titers in a dose-dependent manner with minimal cytotoxicity (Guo et al. 2023a). Notably, this compound has shown particular efficacy during postentry stages of the viral replication cycle, lowering viral loads in blood and tissues, reducing viral shedding, and improving survival rates in in vivo trials. Molecular docking studies revealed that brincidofovir competitively binds to PolX at the 42ARG domain, indicating an interaction with the viral polymerase. However, this interaction alone cannot fully explain brincidofovir’s potent inhibitory activity, implying that it may also interfere with ASFV replication through additional mechanisms or pathways (Guo et al. 2023a). Indeed, brincidofovir has displayed the highest SI yet reported for anti-ASFV agents (SI = 21 014.5) (Guo et al. 2023a), highlighting its strong potential for further development. A high SI of 15 000 has also been determined for its parent compound, (S)-HPMPA (Gil-Fernández et al. 1987) (Table 1; Table S1).

In addition to ANP analogs, the nucleoside analog GS-441524 has also been investigated for anti-ASFV activity in PAMs (Huang et al. 2021b). GS-441524 effectively inhibited ASFV replication in a dose-dependent manner when administered at the early stages of infection, with reductions observed in viral mRNA levels and protein expression, although viral attachment and entry were unaffected. These inhibitory effects are most likely attributable to competition with adenosine triphosphate (ATP) at viral transcription sites since low ATP concentrations counteracted the antiviral activity of GS-441524. Notably, levels of the antiviral cytokines IFN-α, IFN-β, TNF-α, and IL-6 were not affected, indicating that inhibition may occur directly at viral transcription sites rather than through immune modulation.

Rigid amphipathic fusion inhibitors (RAFIs) that were developed initially for enveloped viruses have also been evaluated for their ability to prevent ASFV infection. RAFIs such as aUY11 and cm1UY11 have exhibited dose-dependent inhibition of ASFV in vitro, particularly during the virus internalization stage (Hakobyan et al. 2018). Given that these nucleosides do not affect nonenveloped viruses, their mechanism of action is thought to involve interaction with viral lipids, compromising the structural integrity of the viral envelope necessary for successful infection (St Vincent et al. 2010, Vigant et al. 2014, Arabyan et al. 2019).

Finally, although they do not directly target viral components, adenosine analogues represent another promising approach to tackle ASFV by disrupting the viral mRNA capping process (Villalón et al. 1993). This host-targeting mechanism is explored further below in the section on HTAs.

In addition to the broad-spectrum activity of nucleoside and nucleotide analogues, several classes of antivirals adopt a more targeted approach by specifically inhibiting viral proteins critical to ASFV replication. These inhibitors are designed to interfere directly with essential viral enzymes and structural proteins, thereby hindering key stages of the viral lifecycle. In targeting viral components such as the viral topoisomerase II pP1192R (Topo II), helicases, RNA polymerase complex (RNAP), PolX, and the protease pS273R, these agents aim to block replication, assembly, or release processes, which may increase specificity while reducing adverse effects on host cells. In the following sections, we examine currently available and emerging compounds that disrupt ASFV at these targeted stages, providing a focused strategy for antiviral intervention.

Topoisomerase inhibitors

Topoisomerases are essential enzymes in the lifecycle of viruses and they are responsible for regulating DNA supercoiling during replication and transcription. By relieving torsional strain and resolving DNA tangles, these enzymes ensure that the viral replication machinery can proceed efficiently. ASFV encodes its own Topo II that performs these critical functions (Coelho and Leitão 2020).

Topo II inhibitors have shown promise in disrupting viral replication. In an early study, Mottola et al. (2013) screened 30 fluoroquinolones (bacterial DNA topoisomerase inhibitors) against ASFV-infected Vero cells and identified six that significantly reduced cytopathic effects when administered early in infection. After a 7-day treatment, the ASFV genome was undetectable by polymerase chain reaction, and treated supernatants failed to infect new cultures. Pulsed-field gel electrophoresis revealed a reduction in viral DNA replication and altered viral protein synthesis patterns, indicating that fluoroquinolones might interfere with ASFV replication by targeting Topo II.

Based on these observations, Coelho et al. (2016) cloned and expressed Topo II in yeast and characterized its biochemical activity, showing that it was able to decatenate and relax DNA. Then, they screened various Topo II inhibitors and found that coumermycin A1, doxorubicin, m-AMSA, and genistein significantly inhibited Topo II activity, highlighting the therapeutic potential of targeting this enzyme. Around the same time, Freitas et al. (2016) conducted experiments to further understand the role of Topo II in viral DNA replication and gene expression. By knocking down Topo II in ASFV using siRNAs, they confirmed its critical role in genome replication. Additionally, the authors confirmed that fluoroquinolones could inhibit Topo II, reducing viral DNA replication and inducing fragmentation of the viral genome when administered at specific stages of infection.

In a study of flavonoids as ASFV inhibitors, Hakobyan et al. (2016) evaluated several compounds for their efficacy against ASFV infection in Vero cells. From this screening, apigenin and genistein emerged as the most effective flavonoids, with apigenin significantly reducing viral factories, viral protein expression, ASFV-induced cytopathic effects, and viral DNA in supernatants when administered early in infection. Although the specific mechanism of action was not determined in that study, apigenin has been postulated as modulating eukaryotic type II topoisomerases by enhancing Topo II-mediated DNA cleavage, indicating that it may exert its antiviral effects against ASFV by targeting the virus’s topoisomerase (Constantinou et al. 1995, Coelho and Leitão 2020). Arabyan et al. (2018) later demonstrated that genistein similarly disrupted ASFV infection in both Vero cells and porcine macrophages at noncytotoxic concentrations. The most significant antiviral effect was observed at the mid-stage of infection, ∼8 hpi, when it disrupted viral DNA replication. Single-cell electrophoresis revealed fragmented ASFV genomes in genistein-treated cells, supporting interference of Topo II activity. Molecular docking analysis lent further support to this mode of action, indicating that genistein may function as an ATP-competitive inhibitor by binding with higher affinity than ATP to four key residues in the Topo II ATP-binding site.

Most recently, Lv et al. (2023) investigated natural compounds isolated from Bacillus subtilis bacterial strains for their potential to inhibit Topo II in ASFV. The screening of 138 B. subtilis strains identified four that could effectively inhibit ASFV replication in vitro. The antiviral effects were mediated by the action of small-molecule metabolites, rather than secretory proteins, with arctiin and, again, genistein emerging as the most effective inhibitors. These flavonoids inhibited ASFV replication at the mid-stage of infection by competitively binding to the ATP-binding domain of Topo II, disrupting its catalytic activity. Molecular docking, biolayer interferometry, and competitive decatenation assays confirmed this mechanism. Pigs treated orally with arctiin or genistein showed reduced viral loads, decreased tissue damage, and improved survival rates, positioning these compounds as promising candidates for use as antivirals and demonstrating the potential of B. subtilis metabolites as preventative biologics against ASFV. Although the reported selectivity indices in the Lv et al. (2023) study were relatively low (SI > 2.41 for arctiin and SI > 2.27 for genistein) (Table S1), these values reflect a lower bound based on the maximum concentration tested (10 μM), which did not induce cytotoxicity in PAMs. Previous studies using other cell types have reported higher SI values for genistein, ranging from 5.59 to 22 (Table S1), indicating that the actual selectivity of these compounds may be substantially greater than yet reported.

In a novel approach to antiviral development, Muturi et al. (2021) targeted G-quadruplexes (G4s) within the ASFV genome to explore their potential to disrupt viral replication. Through bioinformatics-based prediction and experimental screening, they identified two functionally significant putative G4-forming sequences, one of which was located in the gene encoding Topo II. Targeting these G4s with ligands, such as N-Methyl Mesoporphyrin (NMM) and pyridostatin significantly repressed viral gene expression in 293T cells, with NMM also demonstrating a dose-dependent inhibitory effect on ASFV replication in PAMs. G4s are an attractive target for antiviral intervention, as stabilizing these secondary DNA structures has the potential to disrupt transcriptional and replication processes while simultaneously impacting a large number of essential genes, regardless of whether or not their products can be directly inhibited. Although the efficacy in vivo of G4 ligands against ASFV has yet to be determined, the Muturi et al. (2021) study has demonstrated that G4 structures in the ASFV genome can be stabilized by ligands such as NMM, resulting in the disruption of viral gene expression.

Collectively, these studies pinpoint ASFV’s Topo II as a key target for antiviral intervention. More traditional approaches, such as administration of fluoroquinolones and flavonoids, disrupt replication by directly inhibiting enzymatic activity or competitively binding to critical domains. In contrast, the approach of Muturi et al. (2021) exploits G4 stabilization to interfere with viral processes by targeting functional DNA structures within key genes such as P1192R. This latter strategy bypasses the need for druggable gene products and allows for the exploration of a broader range of targets by stabilizing G4s in essential genomic regions. These findings open up the prospect of multiple therapeutic interventions to disrupt ASFV replication, which could hasten the development of antivirals targeting topoisomerases and new strategies based on genome architecture.

Helicase inhibitors

The ASFV genome encodes six putative SF2 RNA helicases (A859L, F1055L, B962L, D1133L, Q706L, and QP509L), alongside an additional NTPase (C962R) that resembles the D5 protein of vaccinia virus (VACV) and likely plays a role in DNA replication. As a member of the AAA subfamily of superfamily III DNA helicases, C962R may function at the replication fork and it contains motifs associated with DNA primase activity, indicating that it may play a role in initiating DNA replication (Dixon et al. 2013, Shao et al. 2023). The F1055 L helicase is homologous to the UL-9 helicase of herpesviruses, indicating a possible role in binding replication origins and facilitating DNA synthesis (Dixon et al. 2013). Although the specific functions of these ASFV helicases in processes like DNA replication, repair, transcription, and RNA metabolism have not yet been established, studies on gene-deleted viruses have revealed that not all of them are essential for virulence. For instance, deletion of A859L, C962R, or QP509 L did not impair viral replication in swine macrophages nor did it significantly affect virulence in domestic pigs (Ramirez-Medina et al. 2020, 2021, 2022b). However, targeting those ASFV helicases that are essential for the viral lifecycle could offer a promising strategy for antiviral therapy by selectively disrupting critical stages of viral replication.

In a recent study, Cui et al. (2023) explored cyproheptadine hydrochloride (periactin) as an inhibitor of ASFV that specifically targets the D1133 L helicase. Identified via in silico screening methods, this compound demonstrated a high affinity for D1133L. Experimental results showed that it inhibited ASFV replication in a dose-dependent manner, significantly reducing transcription and translation of the viral D1133 L gene in vitro. Moreover, cyproheptadine displayed minimal cytotoxicity at effective concentrations, further confirming its potential as a viable candidate for antiviral development.

Further screening of compound libraries will be essential to identify additional ASFV helicase inhibitors. Although advances in molecular modeling and docking simulations are expected to facilitate the identification of ASFV helicase inhibitors, obtaining crystal structures for key helicases such as F1055 L would significantly increase precision and accuracy in designing inhibitors. In the case of herpes simplex virus, helicase–primase inhibitors such as pritelivir (BAY 57–1293) have shown effectiveness by targeting essential replication functions (Shadrick et al. 2013), with ASFV’s F1055 L helicase representing a promising target for analogous approaches. Future research should also examine the effects of these inhibitors within a cellular environment, as conditions in vivo may differ substantially from those of in vitro assays.

Polymerase inhibitors

RNAP

ASFV encodes its own unique RNAP, which enables it to transcribe its mRNAs independently of a host RNA polymerase II. This transcriptional machinery includes at least seven subunits with similarities to elements of eukaryotic RNA polymerase II (i.e. EP1242L, C147L, NP1450L, H359L, D205R, CP80R, and D339L) and it is supported by various viral transcription factors that regulate stage-specific gene expression (C315R, G1340L, I243L, B175 L, and B385R) (Dixon et al. 2013). By producing viral mRNAs directly inside infected cells, ASFV ensures precise temporal control over its gene expression that is independent of host regulatory mechanisms. Experiments in which siRNAs were deployed to inhibit viral H359 L and C315R have demonstrated reduced viral replication efficiency in PAMs, highlighting the potential of this complex as an antiviral target (Yang et al. 2024).

In a novel approach to inhibitor screening, Zhang et al. (2023) recently developed a luciferase-based system to identify compounds targeting the ASFV RNAP. This system featured a p72 promoter-driven luciferase reporter construct, with transcriptional activity dependent on coexpression of the seven ASFV RNAP subunits and the transcription factor C315R. Successful transcription by the viral RNAP resulted in luciferase expression, which was quantified using a luminescence reader, enabling high-throughput screening of a natural product library. From a library of 3200 compounds, they identified three potent candidate inhibitors, i.e. ailanthone (AIL), gartanin (GAR), and walsuralactam A (WAL). These compounds significantly reduced luciferase activity, highlighting their ability to suppress RNAP-driven transcription. Of the three, AIL was the most effective, inhibiting infection in PAMs at nanomolar concentrations, while also demonstrating low cytotoxicity. Furthermore, it was able to inhibit replication of a wildtype ASFV strain HLJ-2018 in a dose-dependent manner. The transcriptional suppression by these inhibitors was validated through diminished p72 mRNA levels, underscoring the system’s accuracy in mimicking ASFV transcription. Subsequent assays showed that overexpression of p23 partially rescued the inhibitory effect of AIL and promoted ASFV replication. HSP90 and its cochaperone p23 form a complex that is responsible for stabilizing and ensuring the functionality of numerous client proteins. By inhibiting the interaction between HSP90 and p23, AIL caused misfolding or degradation of viral RNAP subunits and this mode of action was evidenced by a reduction in p72-driven luciferase activity. Even though AIL was found not to target the ASFV RNAP directly, its effects underline the importance of this polymerase for viral replication and highlight the chaperone machinery as another promising target for intervention. It should be noted that since the mechanisms of GAR and WAL were not elucidated in the study, it remains possible that these compounds could directly target RNAP subunits, which warrants further investigation.

Complementing this work is a study by Yaru et al. ( 2022), who explored the role of BET family proteins, such as BRD4, in facilitating ASFV transcription by interacting with the viral RNAP. In that study, 12 BET inhibitors were evaluated for their effects on ASFV replication in PAMs. Four inhibitors showed significant, dose-dependent inhibition of ASFV replication, RNA transcription, and protein synthesis. ZL0580 and ARV-825, both BRD4-specific inhibitors, were particularly effective, with ZL0580 pretreatment of PAMs nearly completely suppressing ASFV transcription. The BET inhibitors are host-targeting compounds that interfere with the interaction between BRD4 and the ASFV RNAP. This interference significantly limits the expression of ASFV RNAP subunits, which in turn disrupts viral transcription and replication. Thus, inhibitors that directly target subunits of the ASFV RNAP could potentially achieve comparable disruption of transcriptional processes.

Whereas RNAP inhibitors directly block RNA synthesis, mRNA capping inhibitors disrupt the essential modifications required for RNA stability and translation, offering a complementary approach to targeting the ASFV transcription cycle. The capping of viral mRNA, which encompasses addition of a guanosine cap and subsequent methylation, is crucial for ensuring mRNA stability, efficient translation, and protection from host immune responses. The ASFV pNP868R enzyme, a guanylyltransferase, catalyses the addition of the guanosine cap, with methylation being mediated by methyltransferases that rely on S-adenosylmethionine (SAM) as a methyl donor. In a recent study, Pandarangga et al. (2024) effectively demonstrated in silico that hyperoside, a flavonoid derivative of quercetin, binds strongly and stably to pNP868R, indicating its potential as an mRNA capping inhibitor. Although they are not directly acting antivirals per se, adenosine analogs, such as 3-deazaneplanocin A have also demonstrated an ability to interfere with this mechanism. By targeting the host enzyme S-adenosylhomocysteine hydrolase (AdoHcy), which is critical for regenerating SAM, these compounds have exhibited potent antiviral activity against ASFV in vitro (Villalón et al. 1993). The compound 3-deazaneplanocin A has exhibited a particularly high SI of 3000 (Villalón et al. 1993), placing it among the most promising antiviral candidates identified in this review (Table 1; Table S2).

Research into inhibitors that directly target ASFV RNA polymerase subunits is still in its early stages. Studies like that of Zhao et al. (2022) have shed light on the interactions between viral proteins and host factors, such as BRD4, furthering our understanding of transcriptional regulation in ASFV. Similarly, the work by Pandarangga et al. (2024) represents a first step toward targeting mRNA capping. Future studies should aim to validate these findings in vitro and in vivo to confirm their potential as antiviral strategies.

DNA Repair polymerase

Compared to the relatively early-stage research on other aspects of ASFV transcriptional regulation, PolX has drawn the most attention as a target for antiviral development. PolX is central to the virus’s BER pathway, which repairs oxidative DNA damage caused by the reactive oxygen species (ROS) produced by macrophages. As the smallest known DNA polymerase, PolX evolved for short-patch BER, filling single-nucleotide gaps, while cooperating with other viral enzymes like the apurinic/apyrimidinic (AP) endonuclease pE296R and the DNA ligase pNP419L. Its low fidelity and error-prone nature may contribute to viral antigenic variation, highlighting its critical role in maintaining genome integrity (Dixon et al. 2013). Among the class of nucleotide analogues discussed above, both cangrelor and brincidofovir have shown potential as PolX inhibitors. Brincidofovir, a lipid-conjugated cidofovir derivative, demonstrates strong inhibition of ASFV replication both in vitro and in animal models by competitively binding to the PolX enzyme, although additional mechanisms may contribute to its antiviral potency (Guo et al. 2023a). Cangrelor, identified through molecular docking studies, binds to the active site of PolX and consequently it may function as a competitive inhibitor of PolX activity, though this has yet to be tested in cellulo or in vivo (Choi et al. 2021).

In addition to cangrelor, Choi et al. (2021) also identified pentagastrin, fostamatinib, and, most recently, polygalic acid as potential inhibitors of ASFV replication through their targeting of PolX. Fostamatinib, primarily known as a spleen tyrosine kinase inhibitor, was shown by molecular docking to bind to the active site of PolX in a mode of action similar to that observed for cangrelor. This interaction suggests that fostamatinib may inhibit the enzyme’s activity, thus affecting ASFV’s DNA repair processes and overall replication. Although fostamatinib is mainly used in the treatment of immune-related conditions, its potential antiviral activity against ASFV makes it an attractive candidate for further investigation. Docking simulations also revealed that polygalic acid, a bioactive natural compound possessing antiinflammatory and antioxidant properties, is efficiently bound to the GTP-binding site of PolX, forming hydrogen bonds and interactions within the enzyme’s DNA-binding pocket. This high-affinity binding suggests that polygalic acid may function as a competitive inhibitor of PolX, thereby potentially compromising ASFV genome stability and replication. As with cangrelor, further in vitro and in vivo studies are needed to validate the efficacy of fostamatinib and polygalic acid as ASFV therapeutics and to fully elucidate their mechanisms of action within the viral replication cycle.

A comprehensive study by Li et al. (2024b) screened 2580 FDA-approved drugs in PAMs in order to systematically assess their antiviral activity against ASFV. This high-throughput screening identified cytarabine hydrochloride and triapine as effective inhibitors of ASFV replication in vitro. The active metabolite of cytarabine hydrochloride, cytarabine triphosphate, seems to inhibit PolX by being incorporated into the viral DNA, leading to disruption of DNA synthesis and termination of elongation. Meanwhile, triapine targets the viral ribonucleotide reductase pF334L, a key enzyme for DNA synthesis, by coordinating with its Fe²⁺ ion. Both compounds were found to significantly reduce ASFV replication in a dose-dependent manner, decreasing both viral titers and DNA levels, and significantly affecting ASFV gene transcription and protein synthesis. Notably, cytarabine hydrochloride and triapine exhibited high selectivity indices of 998.2 and 359.7, respectively (Table 1; Table S1), supporting their potential as promising antiviral candidates for further development.

In another study, Macalalad and Orosco (2024) employed advanced computer-aided drug design (CADD) to screen medicinal fungal metabolites as potential multitarget inhibitors against the BER proteins of ASFV, specifically targeting PolX, pE296R, and pNP419L. After assessing the safety and bioavailability of 1830 metabolites, they selected 319 compounds, several of which presented strong binding affinity and stability, implying potential multitarget inhibition of BER proteins. Notably, methyl ganoderate E and antcamphin M showed promising inhibitory activities against PolX and pNP419L, and cochlactone A demonstrated broad antagonistic effects against all three BER proteins. These findings indicate that fungal compounds may serve as effective antiviral agents against ASFV, although further in vitro and in vivo testing is needed to validate their efficacies.

Most recently, Wu et al. (2024) identified D-132 as a potent PolX inhibitor exhibiting a novel slow-on-fast-off binding mechanism targeting key residues in the enzyme’s palm and finger domains. This interaction interferes with the enzyme’s DNA-binding affinity, drastically inhibiting ASFV replication in vitro. In line with most of the aforementioned recent research, this study also used CADD to accelerate identification of potential ASFV inhibitors targeting specific viral components, thereby efficiently prioritizing candidates for further experimental validation. Further in vivo and in vitro research will be essential to confirm the antiviral efficacy and safety profile of these promising compounds.

Protease inhibitors

The protease pS273R plays a vital role in the ASFV lifecycle by processing two large polyprotein precursors, pp220 and pp62, which are required for virion assembly and infectivity. Synthesized in the late stages of infection, pS273R cleaves these precursors into structural proteins, including p5, p14, p34, and others that form the virus’s core-shell (Andrés et al. 2001a, 2002a). As a cysteine protease with unique structural features, pS273R not only drives viral maturation but also modulates host immune responses by cleaving gasdermin D (GSDMD), a protein associated with apoptosis induction, and it possibly interferes with IFN signaling pathways (Zhao et al. 2022). All these functions make pS273R an attractive target for ASFV therapeutic intervention. Recent studies have centered on designing inhibitors that can effectively block pS273R activity in order to impair virus maturation and reduce the production of infectious viruses, a strategy similar to the drug targeting of proteases in HIV and SARS-CoV-2 (Mahdi et al. 2020, Hattori et al. 2021).

Various approaches have been used to identify effective inhibitors of pS273R. In an early study by Jo et al. (2020), a FRET-based protease assay was used to screen a flavonoid library, with myricetin along with its derivative myricitrin found to be potent pS273R inhibitors. In 2020, Guobang et al. (2020) solved the crystal structure of pS273R, providing crucial insights into this viral target. Using this information, the authors further synthesized three peptidomimetic aldehyde inhibitors designed to target the protease’s active site. Notably, ZW60 demonstrated the highest inhibitory potency, effectively suppressing pS273R enzymatic activity with a submicromolar IC₅₀ (half-maximal inhibitory concentration) value. Although the work by Jo et al. (2020) predated the publication of the pS273R crystal structure, this recent breakthrough now provides an opportunity to determine structures of the enzyme in complex with myricetin derivatives that could enhance our understanding of their inhibitory mechanisms.

More recently, computational techniques, including virtual screening and molecular dynamics simulations, have been applied to this critical ASFV protease. Wang et al. (2021a) found that OPTX-1, a defensin-like peptide derived from the tick Ornithodoros papillipes, competitively inhibits pS273R, resulting in a significant reduction of ASFV replication in vitro. Subsequently, Liu et al. (2021) reported that E-64, a cysteine protease inhibitor, covalently binds to the catalytic triad of pS273R, effectively blocking its activity and preventing virus maturation. Building on these observations, a more recent study identified five FDA-approved drugs—leucovorin, carboprost, protirelin, flavin mononucleotide, and lovastatin acid—that bind stably to the active site of pS273R, revealing their potential as ASFV antiviral candidates by impairing virus maturation and reducing infectivity (Lu et al. 2023a). Although these studies have identified promising inhibitors of pS273R, further validation in vitro and in vivo is needed to fully confirm their efficacies and potential as ASFV therapeutic agents.

Gene-specific inhibition using RNA-based strategies

RNA-targeting approaches, including siRNAs, antisense oligonucleotides (ASOs), and CRISPR/Cas9 systems, have been emphasized recently for their potential to inhibit viruses (Musa et al. 2024). siRNAs work by binding to complementary viral mRNA sequences, thus promoting their degradation and effectively silencing genes critical for viral replication. ASOs are single-stranded DNA or RNA molecules that bind to viral mRNA, blocking translation or promoting mRNA degradation, thereby reducing the synthesis of viral proteins. CRISPR/Cas9 is an adaptive bacterial immune system that has been repurposed for precise genome editing. In antiviral applications, it can be programmed to target and cleave viral DNA, which disrupts the genome and consequently prevents replication.

In the context of ASFV control, siRNAs have been used to inhibit viral replication in vitro by targeting two viral genes, A151R and B646 L (encoding p72) (Keita et al. 2010). That study demonstrated that the siRNAs directed against these genes significantly reduced ASFV replication, with up to a 4-log reduction in viral titers and a 3-log decrease in transcript levels. Although a combination of multiple siRNAs did not substantially improve the antiviral effect compared to the siRNAs administered individually, the results indicate that siRNA-based approaches may represent a targeted strategy for controlling ASFV infection.

Building on these results, Hakobyan et al. (2021) investigated the antiviral activity of ASOs against ASFV by specifically targeting p72 mRNA. In order to enhance cellular delivery and antiviral efficacy, the ASOs were conjugated with amino-modified mesoporous silicon dioxide (mSiO₂) nanoparticles, forming nanocomplexes that exhibited a greater antiviral effect when compared to free oligonucleotides. Among the ASOs evaluated, two sequences achieved a significant reduction in viral titers, with an observed one-log decrease in infected Vero cells. This outcome indicates that nanoparticle-based ASO delivery may be a promising strategy for ASFV inhibition, although the study noted high cytotoxicity at higher ASO concentrations.

Expanding on the scope for RNA-based inhibition with siRNAs and ASOs, Hübner et al. (2018) applied CRISPR/Cas9 to target the ASFV gene encoding p30 (CP240L), aiming to achieve a gene-specific reduction in viral replication. In that study, a wild boar lung-derived cell line (WSL) was engineered to constitutively express Cas9 together with guide RNAs directed at a conserved region within CP240L, which resulted in a significant reduction in ASFV plaque formation and a 4-log decrease in viral yields. This CRISPR/Cas9 system achieved almost complete inhibition of viral spread and replication in the modified WSL cells, for both avirulent (BA71V) and virulent (Armenia) ASFV strains. Specificity was further confirmed by analyzing escape mutants harboring nucleotide variations within the target site, which were not inhibited, underscoring the precision of this approach. Cas9 expression in the cell line remained stable for over fifty passages, with no adverse effects on cell growth.

In an attempt to adapt this approach to an in vivo setting, Zheng et al.( 2024) employed a multiplexed CRISPR/Cas9 system targeting nine distinct loci within the ASFV genome to improve antiviral efficacy. By cleaving the viral genome at multiple sites, this strategy was designed to limit viral escape through mutations. In vitro tests demonstrated a significant reduction in ASFV replication using this multitargeted approach, prompting further testing in transgenic pigs with germline-encoded Cas9 and guide RNAs. Although the modified pigs exhibited a delay in onset of ASFV symptoms compared to wild-type pigs, they did not present increased survival upon direct infection. However, cohabitation experiments indicated a herd-level effect, as some wild-type pigs exposed to infected transgenic pigs exhibited delays in infection. These findings underscore the potential of the CRISPR/Cas9 system as an element of ASFV control strategies, but further optimization is required to achieve stronger protection in vivo.

Though the strategies outlined herein have shown promise in targeting ASFV, several limitations persist. Challenges such as efficient delivery, potential cytotoxicity, and the risk of viral escape mutations highlight the need for their further refinement. Additionally, studies in vivo are crucial to fully determine efficacy and safety. Continued research into optimizing delivery mechanisms, reducing off-target effects, and improving the specificity of viral targeting will all be essential to take full advantage of RNA-based antivirals for ASFV control.

HTAs

Endosomal pathway inhibitors

Strategies that target the inhibition of viral proteins or interfere directly with specific viral processes may, unintentionally, promote the selection of drug-resistant virus populations through evolutionary selective pressures (Irwin et al. 2016). A promising alternative is to target host cell pathways crucial for viral replication, such as entry and replication. Doing so not only reduces the likelihood of resistance, but also has the potential to endow broad-spectrum antiviral efficacy by exploiting host dependencies shared among different viruses.

Entry of ASFV into host cells begins with viral adsorption and entry into the host cell, followed by internalization through clathrin/dynamin-mediated endocytosis and macropinocytosis. In a critical step of the ASFV lifecycle, the virion undergoes uncoating elicited by molecular cues from endosomes, then decapsidation, and finally fusion of the inner viral membrane to the endosomal membrane. The process of endosomal maturation required for decapsidation involves steady acidification of the endosomal compartment, driven by the vacuolar-type H+-ATPase (v-ATPase) proton pump, with cholesterol efflux being necessary for fusion. Endosomal cholesterol efflux is mediated by specific ASFV proteins, including E248R and E199L, which interact with host cell endosomal proteins such as Niemann-Pick C type 1 (NPC1) and lysosomal-associated membrane proteins (Lamp-1 and -2) (Cuesta-Geijo et al. 2022). Given the crucial role of endosomal trafficking in ASFV infection, antivirals targeting this pathway can be particularly effective. Inhibitors designed to interfere with acidification of the endosomal compartment or that block the interactions between ASFV proteins and the endosomal proteins that mediate cholesterol transport can cause retention of virions inside endosomes, thereby inhibiting the progress of infection. In support of this approach, various studies have shown that antiviral drugs targeting endosomal processes can inhibit ASFV effectively.

For instance, in parallel studies by Galindo et al. (2021), the calcium channel blockers tetrandrine (TET) and verapamil were shown to disrupt ASFV entry by modulating endosomal functions required for the viral lifecycle. Notably, TET was shown to inhibit the PI3K/AKT signaling pathway required for macropinocytosis during viral internalization. Activation of PI3K occurs early after virus uptake and it plays an important role in infection, regulating the translational machinery through the AKT-mTOR pathway, among others (Sánchez et al. 2012). Jackman et al. (2024) confirmed TET’s significant antiviral activity against ASFV. In that study, TET was found to be the most potent of 297 natural compounds screened for antiinflammatory properties, achieving a 3.6-log reduction in viral titers in Vero cells without inducing cytotoxicity. Berbamine, another compound identified in this screening, also demonstrated antiviral efficacy, although its effects were less pronounced compared to those of TET.

Berbamine hydrochloride is a bisbenzylisoquinoline alkaloid (BBA) that has been independently confirmed as displaying strong antiviral activity against ASFV (Zhu et al. 2022). In PAMs, berbamine hydrochloride inhibited ASFV in a dose-dependent manner, reducing viral titers by 4.14-log at noncytotoxic concentrations. A time-of-addition analysis revealed this inhibition to operate across the entire viral lifecycle, with particular efficacy in blocking the early stages of ASFV infection. Findings from other experimental systems have indicated that berbamine hydrochloride may act by inhibiting lysosomal acidification (Zhan et al. 2023), providing a rational basis for its early-stage antiviral effects.

In a study on the anti-ASFV activities of four other BBAs, cepharanthine (CEP) showed the highest activity and selectivity index (Zhu et al. 2024). CEP was actually confirmed to inhibit acidification of late endosomes, to hinder ASFV endosomal transport, and to disrupt virus uncoating signals, thereby preventing viral internalization. Although the precise mechanism underlying this effect remains unclear, a separate study by Su et al. (2024) demonstrated that CEP disrupts the interaction between Hsp90 and its cochaperone Cdc37, a complex critical for stabilizing and activating host proteins essential for ASFV replication. During infection, Cdc37 initially binds to AKT to form a binary complex, which subsequently associates with Hsp90 to create a ternary Hsp90–Cdc37–AKT complex. This interaction facilitates AKT phosphorylation via PIP3 generated by ASFV-activated PI3K. Phosphorylated AKT promotes glycolysis through HK2 and LDHA, leading to increased lactate production and IL-1β release via NF-κB signaling, both of which synergistically enhance ASFV replication. By suppressing Hsp90–Cdc37–AKT complex formation, CEP reduces AKT phosphorylation, disrupting these glycolytic and inflammatory pathways. Interestingly, AKT also plays a key role in regulating vesicular trafficking and endosomal acidification. By disrupting AKT phosphorylation, CEP may impair the activity of v-ATPase, the proton pump responsible for endosomal acidification, leading to alkalization of late endosomes and lysosomes (Soliman et al. 2018).

Recent studies have highlighted Rab7 as another critical host target in the ASFV endocytic pathway (Cuesta-Geijo et al. 2012, Guo et al. 2023b). Rab7, a small GTPase predominantly associated with late endosomes and lysosomes, regulates endosomal maturation, vesicular trafficking, and transport of viral particles to the cytoplasm. Guo et al. (2023b) recently demonstrated that emodin (EM) and rhapontigenin (RHAG), two natural compounds extracted from the flowering plant genus Polygonum, can effectively inhibit ASFV replication by downregulating Rab7 expression. This interference disrupts the late endosomal functions necessary for viral uncoating and genome release. Silencing Rab7 through siRNA-mediated or pharmacological inhibition similarly prevented ASFV replication, confirming its essential role in the virus lifecycle. In addition to Rab7 targeting, EM and RHAG induced accumulations of free cholesterol in endosomes, disrupting the cholesterol efflux critical for ASFV uncoating and membrane fusion. This dual mechanism effectively traps the virus within late endosomes, preventing it from reaching the perinuclear region. The same study reported that both compounds inhibited endosomal acidification. This effect could arise directly by interfering with v-ATPases or indirectly via Rab7 downregulation. Rab7 recruitment, linked to the collapse in endosomal pH during endosome maturation, may regulate v-ATPase activity through its effectors or involve other mechanisms such as phosphorylation or proton channel modulation (Podinovskaia et al. 2021). If Rab7 interference is indeed a key driver of acidification inhibition, it further highlights the importance of targeting this GTPase in antiviral strategies. However, if EM and RHAG act through other as yet undefined mechanisms, it opens up new avenues for discovering complementary or synergistic targets within the endocytic pathway. Consequently, further studies on this topic are warranted.

The lipid signaling pathway—including cellular cholesterol, the cholesterol biosynthesis pathway, and phosphoinositides—is a closely related molecular network central to successful viral infection (Ángel et al. 2016, Galindo et al. 2019). Whereas endosomal inhibitors act on late-stage trafficking events, lipid biosynthesis inhibitors target upstream metabolic pathways, offering an alternative way of disrupting ASFV replication. Inhibitors of genes related to lipid synthesis, such as that encoding fatty acid synthase (FASN), can be particularly effective in preventing the virus from establishing and sustaining infection. Cerulenin (CRL), a FASN inhibitor, was first shown by Bernardes et al. (1998) to inhibit ASFV replication by disrupting cholesterol metabolism, which is critical for fusing ASFV to endosomal membranes. Though CRL does not appear to affect viral binding or internalization, it effectively blocked the fusion process, preventing the establishment of infection and subsequent viral replication. More recently, de León et al. (2019) confirmed CRL’s potent antiviral activity against ASFV, demonstrating a ∼3-log reduction in viral production by cell monolayers infected with the nonpathogenic ASFV isolate BA71V. CRL inhibited the synthesis of late ASFV proteins, including p17 and p12, and it markedly reduced the expression of the early protein p32, consistent with the overall inhibition of infective virus yield. Interestingly, though the study by Bernardes et al. (1998) indicated that CRL inhibits ASFV fusion activity without affecting viral binding or internalization, the time-of-addition assays reported in de León et al. (2019) revealed that CRL was most effective when applied prior to viral adsorption, highlighting a possible effect on cell membranes before virus adsorption.

Another agent recently found to interfere with host lipid metabolism is theaflavin (TF), a compound derived from tea (Camellia sinensis) (Chen et al. 2023d). Pretreatment of either ASFV-infected or uninfected PAMs with TF upregulated levels of phosphorylated AMPK (p-AMPK), and its antiviral effects were partially reversed by the AMPK inhibitor dorsomorphin, confirming the involvement of this pathway in ASFV inhibition. TF was also found to suppress the expression of lipid synthesis-related genes, including FASN, while reducing intracellular cholesterol and triglyceride accumulation, both critical for viral replication. Thus, TF appears to inhibit ASFV replication by disrupting lipid metabolism by activating the AMPK signaling pathway. Despite its potent anti-ASFV activity, TF’s poor bioavailability is a major limiting factor to clinical application, though it may still serve as a lead compound for the development of other antiviral drugs against ASFV.

Other compounds such as apilimod (a PIKfyve inhibitor) and selective estrogen receptor modulators (SERMs) including raloxifene and tamoxifen exhibit significant antiviral activity by similarly interfering with lipid metabolism (Galindo et al. 2021). PIKfyve inhibition blocks the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P2), a lipid essential for late endosome function and ASFV replication. ASFV has been shown to upregulate PIKfyve in infected cells, underscoring the virus’s dependency on this kinase for its lifecycle (Cuesta-Geijo et al. 2012, 2017). Galindo et al. (2021) reported that apilimod potently inhibits ASFV infection by inducing endosomal vacuolization and preventing maturation. Similarly, SERMs disrupt cholesterol distribution in endosomes, leading to the retention of ASFV virions within vesicles and preventing their cytoplasmic release.

Overall, compounds such as tetrandrine, cepharanthine, and emodin demonstrate the strategic potential of targeting ASFV’s reliance on endosomal and lipid signaling pathways. By disrupting key processes such as Rab7-mediated trafficking, phosphoinositide metabolism, cholesterol synthesis and efflux, and Hsp90–Cdc37 complex activity, these inhibitors effectively block viral uncoating and genome release. Such findings emphasize the value of endosome-targeting antivirals, not only as a direct means to combat ASFV, but also as a framework for exploiting viral dependencies on the host cell machinery. Nevertheless, it is important to highlight that some of the compounds discussed in this section, including tamoxifen, verapamil, cerulenin, and apilimod, exhibit relatively low selectivity indices (ranging from ~3.7 to 9.9) (Table S2). In some cases, such as verapamil and cerulenin, the CC₅₀ threshold (50% cytotoxic concentration) was not reached at the highest tested concentrations, indicating that the true SI could be higher. However, such low SI values imply a limited therapeutic window and warrant cautious interpretation of their potential antiviral utility until further toxicity and efficacy data become available.

MT inhibitors

A key aspect of endosomal maturation that ASFV exploits is movement of endosomes along MTs to the perinuclear area in a process mediated by motor proteins, such as dynein and kinesin. This transport is critical for early infection and it is disrupted by MT depolymerizing agents (Alonso et al. 2001). Furthermore, Rac1 activation during early ASFV infection coincides with MT acetylation, stabilizing the MT network for efficient intracellular transport (Quetglas et al. 2012). After ASFV entry, endosomes containing virus particles are transported to perinuclear regions adjacent to the MT organizing center (MTOC), from which core particles are subsequently released (Alonso et al. 2018). Viral replication mainly occurs in viral factories that, unsurprisingly given the importance of MTs in this process, are found in the MTOC (Alonso et al. 2001). ASFV exploits the MT acetylation that facilitates formation of viral factories (Jouvenet et al. 2004), and it has been shown to redistribute γ-tubulin from the MTOC, thereby inhibiting MT nucleation and influencing the host cell’s cytoskeletal architecture to suit its replication needs (Jouvenet and Wileman 2005). The transmembrane structural protein p54, located in the internal viral membrane, is critical for the recruitment and remodeling of endoplasmic reticulum membranes into precursors of the viral envelope, a function likely involving interaction with the dynein motor (Alonso et al. 2001, Rodríguez et al 2004). Golgi disruption by ASFV, a process critical for viral assembly, has also been reported as MT-dependent (Netherton et al. 2006). After virus particles have been assembled, kinesin adopts a multiterminal MT motor to facilitate their transport from the viral factory to the cell membrane (Wang et al. 2021b). Transport to the plasma membrane is highly dependent on MT integrity and it can also be disrupted by depolymerizing agents (Jouvenet et al. 2004).

Building on their earlier work on apigenin, Hakobyan et al. (2019) screened commercially available apigenin derivatives for their ability to inhibit ASFV. Among them, they identified genkwanin, an O-methylated flavone abundant in the seeds of Alnus glutinosa, as significantly reducing viral titers by nearly 2-log in a dose-dependent manner, with a selectivity index of 205.2 that underscores a favorable balance between potency and safety (Table 1; Table S2). Further experiments indicated that genkwanin inhibits both ASFV entry and egress, processes that rely heavily on MTs. By means of in silico docking, the authors demonstrated that genkwanin binds to the colchicine-binding site of β-tubulin (CBS), indicating that it may destabilize MT assembly and thus interfere with viral transport. Though genkwanin’s MT-linked mechanism of action was supported by these findings, its parent compound, apigenin, has not been linked to MT disruption in the context of ASFV infection. Instead, apigenin’s antiviral activity has been hypothesized as involving Topo II modulation based on its ability to stabilize the eukaryotic Topo II–DNA complex (Coelho and Leitão 2020). Thus, genkwanin displays a novel mechanism of action among apigenin derivatives by specifically targeting the MT network that ASFV exploits.

Sirakanyan et al. (2021) presented a contrasting perspective to MT targeting by demonstrating the antiviral potential of 6b, an MT-stabilizing compound that like genkwanin also targets the CBS. However, unlike genkwanin, 6b enhances tubulin polymerization, potentially interfering with ASFV processes that rely on dynamic MT remodeling. Initial experiments with classical MT-targeting agents—including colchicine, paclitaxel, nocodazole, and vinblastine—revealed that these compounds reduced ASFV titers significantly, supporting the idea that MTs are critical for ASFV infection. However, the cytotoxicity observed with prolonged exposure makes them undesirable as therapeutic agents. Virtual screening subsequently identified 6b as a novel compound that reduced ASFV replication in a dose-dependent manner with no cellular or animal toxicity. The results further showed that 6b disrupts multiple stages of the ASFV lifecycle, including viral attachment, internalization, and egress, by promoting tubulin polymerization and stabilizing MTs. This stabilization effect also impaired the formation of viral factories within the MTOC and reduced viral DNA replication and protein synthesis. Thus, it may serve as a promising compound for anti-ASFV therapy that can be further modified to develop analogues with improved therapeutic effectiveness.

Taken together, these results underscore the critical role played by MTs in ASFV infection and demonstrate the potential for MT-targeting agents as a valuable antiviral strategy, provided their cytotoxicity can be minimized. However, the seemingly contradictory data regarding the mechanisms of action of apigenin and its derivatives underlines the risks of inferring mechanisms from studies on other viruses or homologous systems. Rigorous experimental validation remains essential to elucidate specific mechanisms in the context of ASFV.

Innate immunity modulators

Several host-directed immunomodulatory approaches, such as administration of IFNs, have been evaluated for their antiviral effects against ASFV. Although the results have been mixed, these strategies may serve as adjuncts to vaccination or direct antiviral therapies. Early investigations by Esparza et al. (1988) demonstrated that recombinant bovine IFN-α1 and porcine IFN-γ successfully inhibited ASFV replication in porcine monocytes and alveolar macrophages. That study showed that IFN-γ exhibited stronger inhibition than IFN-α1, particularly in PAMs, and that the inhibitory effect was associated with a marked reduction in late viral protein synthesis, likely due to inhibition of viral DNA polymerase activity. Similarly, Paez et al. (1990) reported that human IFN-α and IFN-γ, separately and in combination, suppressed ASFV replication in vitro in Vero cells. Their study also noted enhanced antiviral effects when these IFNs were combined with TNF, further implicating IFNs as potential modulators of viral protein and DNA synthesis.

More recent studies have built upon these findings, focusing on the use of porcine-derived IFNs. Fan et al. (2020) demonstrated that recombinant porcine IFN-α and IFN-γ effectively inhibited ASFV replication in PAMs. These IFNs were found to trigger the expression of interferon-stimulated genes (ISGs) including IFIT1, IFITM3, MX1, and OASL, which play critical roles in establishing an antiviral environment. Interestingly, that study uncovered dose-dependent effects, with lower doses eliciting more significant reductions in viral load in infected pigs​. Corroborating these findings, Jiao et al. (2023) tested a recombinant porcine IFN-α and IFN-γ cocktail, which delayed ASF symptom onset and reduced disease severity in infected pigs. Although the cocktail did not prevent mortality, it enhanced expression of ISGs and modulated inflammatory responses, reducing tissue damage associated with cytokine storms​.

Contrasting findings on TNF-α and the dose-dependent effects of IFNs against ASFV both underline the complex dynamics of host–pathogen interactions shaped by ASFV’s immune evasion strategies. Esparza et al. (1988) demonstrated that administration of TNF-α alone increased viral production in porcine monocytes, whereas Paez et al. (1990) reported its antiviral effects in synergy with IFN-α and IFN-γ in Vero cells. These differences may reflect ASFV’s exploitation of the host-specific immune response, particularly in primary porcine immune cells, where TNF-α might increase inflammation in a way that ASFV can manipulate to its benefit. In contrast, in nonhost Vero cells, TNF-α might amplify IFN-driven antiviral mechanisms, such as activation of ISGs, without the same degree of viral exploitation. ASFV’s modulation of TNF-α, in addition to its broad suppression of type I IFN signaling through cGAS-STING inhibition by viral proteins pI215 L and DP96R, underlines how the virus calibrates host cytokine environments. High doses of exogenous IFNs could overstimulate immune responses, triggering ASFV’s immune evasion pathways, such as NF-κB inhibition via pA238 L or IFN-β suppression by A137R. This scenario may explain why low doses of IFN elicit a more sustained antiviral response, as they trigger the immune system without overwhelming it, thereby potentially bypassing some of the counterstrategies developed by ASFV.

Regarding the selective inhibition of late viral proteins in IFN-γ-treated cells reported by Esparza et al. (1988), the link to ASFV DNA polymerase activity needs to be investigated further. ASFV’s DNA polymerase is essential for replication of the viral genome, which serves as a template for late gene expression, and includes structural proteins essential for virion assembly (Wang et al. 2021b). IFN-γ-induced ISGs, such as MX1, may directly inhibit DNA polymerase activity or indirectly disrupt associated replication complexes, cascading into a series of failures in late-stage protein synthesis. In fact, MxA, one of the two main myxovirus resistance (Mx) proteins in humans, encoded by the MX1 gene, has demonstrated potent antiviral effects against ASFV. Netherton et al. (2009) showed that stably transfected Vero cells expressing human MxA protein significantly reduced ASFV replication by up to 100-fold compared to control cells and prevented the formation of virus plaques. This inhibition was associated with a dramatic reduction in late ASFV protein synthesis, resembling the effects observed following IFN-γ treatment. Furthermore, MxA localizes to the perinuclear viral assembly sites surrounding viral factories, likely disrupting their formation or functioning. Porcine Mx1 could theoretically inhibit ASFV in a manner similar to human MxA, but this supposition requires experimental verification. Alternatively, IFN-γ might enhance cellular stress responses or proteasome activity, which would result in the degradation of viral components and impair their translation. It might also modify host gene expression, creating an environment unfavorable to ASFV replication. However, the inhibition of ASFV late protein synthesis observed in both the Esparza et al. (1988) and Netherton et al. (2009) studies points to a converging mechanism. ASFV late protein synthesis depends on intact genome replication and the formation of viral factories. If Mx proteins interfere with perinuclear viral assembly, as is the case with MxA, this disruption could similarly interfere with the production or translation of late-stage proteins, a scenario in line with the findings of Esparza et al. (1988).

Expanding on the effect of IFN-γ-induced ISGs, studies by Muñoz-Moreno et al. (2016) and, more recently, Sun et al. (2023) have elucidated important antiviral roles for the IFITM family and OAS1, respectively, in the context of ASFV infection. OAS1 was found to promote activation of the JAK-STAT signaling pathway and it increased phosphorylation of STAT1/2, thus activating innate immune responses. Like cGAS, OAS1 is activated upon detection of AT-rich dsDNA from the ASFV genome to produce OAS, which can promote the antiviral function of RNase L and further degrade virus-derived mRNA (Sun et al. 2023). Furthermore, OAS1 interacts directly with the ASFV capsid protein p72, recruiting TRIM21 to mediate its K63-linked polyubiquitination and degradation, thereby impairing assembly of ASFV mature particles and reducing the production of mature virions (Sun et al. 2023). OAS1 may interact with additional viral proteins, including the major capsid protein p30 and I329L, with this latter acting as a structural protein capable of inhibiting OAS1 expression and its activation of NF-κB (de Oliveira et al. 2011, Chen et al. 2022).

In a mechanism similar to that elaborated on above for EM and RHAG (Guo et al. 2023b), expression of IFITM1, 2, and 3 was found to reduce virus infectivity in Vero cells by impacting viral entry and uncoating (Muñoz-Moreno et al. 2016). Upon IFN treatment, IFITM proteins were observed to redistribute into a perinuclear vesicular pattern resembling endosomes, with IFITM2 and IFITM3 localizing predominantly to late endosomes and lysosomes, whereas IFITM1 was found primarily at the plasma membrane and early endosomes. While IFITM2 was found to reduce the number of decapsidated virions, indicating that it inhibits ASFV exit from late endosomes, IFITM3 increased the retention of encapsidated virions, preventing them from progressing to productive infection. Both proteins were also associated with cholesterol accumulation in late endosomes, further disrupting endosomal functions critical for ASFV uncoating and genome release. This ability of IFITMs to alter endosomal physiology highlights their potential for deployment in concert with pharmacological inhibitors that disrupt endosomal trafficking.

Overall, innate immunity modulators, particularly IFNs, have demonstrated notable potential in inhibiting ASFV replication. IFN-γ-induced ISGs, such as those encoding porcine Mx1, may interfere with viral DNA polymerase and disrupt late protein synthesis and viral factory formation. Similarly, IFITM proteins impede ASFV entry and uncoating by altering endosomal dynamics. Taken together, these findings provide a rationale for deploying IFNs and associated modulators as part of a broader strategy against ASFV infection. Future studies should focus on optimizing their application and exploring their potential in combination therapies.

Cell death pathway modulators

To sustain its replication and evade host immune defenses, ASFV encodes several proteins that target and modulate host cell death pathways (Suresh et al. 2017, Zhao et al. 2022, Niu et al. 2023, Suraweera et al. 2024). For example, the virus homolog of apoptosis-associated Bcl-2 family proteins, i.e. pA179L, inhibits autophagy by binding to Beclin-1, thereby preventing it from initiating autophagosome formation. Other viral proteins such as MGF505-7R and MGF360-11 L mediate autophagosome-associated degradation of immune regulators, including STING and TBK1, in order to abrogate IFN responses. pA179 L also counteracts apoptosis by sequestering proapoptotic proteins, thus inhibiting caspase activation and ultimately preserving cell viability. In a similar fashion, the viral protease pS273R downregulates JAK-STAT signaling by degrading STAT2, further impairing host defenses. The viral protease pS273R counteracts pyroptosis by cleaving GSDMD into a fragment incapable of triggering inflammatory cell death, and H240R inhibits the NLRP3 inflammasome and NF-κB signaling. Late in infection, ASFV may switch to promoting necroptosis to help with viral release. However, rather than acting directly on these viral proteins, the following antiviral agents target the host pathways they manipulate, turning the virus’s own strategies against it.

Flavonoids are a class of plant secondary metabolites frequently found in fruits and vegetables and they exert diverse biological effects. We have already reported above on the inhibitory actions of apigenin and genistein against Topo II in ASFV, as well as myricetin and its derivative myricitrin, both strong inhibitors of pS273R. In one of their most recent studies, Arabyan et al. (2021) screened a cell-based library of 90 flavonoids, identifying kaempferol as a potent dose-dependent ASFV inhibitor having a virostatic effect. Further analysis revealed that kaempferol interferes with ASFV replication at both the entry and postentry stages, significantly reducing viral DNA and protein synthesis. Notably, kaempferol’s mode of action was linked to autophagy induction in infected cells. This mechanism may involve its interaction with the AMPK/mTOR signaling pathway, as has been reported elsewhere (Li et al. 2024a). By activating AMPK and/or inhibiting mTOR, kaempferol could potentially disrupt the ASFV-induced inhibition of autophagy triggered by the Beclin-1/pA179 L interaction, freeing Beclin-1 to drive autophagosome formation (Suresh et al. 2017).

Similarly, another flavonoid, dihydromyricetin (DHM), derived from the plant Ampelopsis grossedentata, was found to have antiviral effects against ASFV by targeting the TLR4/MyD88/MAPK/NF-κB signaling pathway (Chen et al. 2023c). DHM blocks TLR4 signaling, which prevents NLRP3 inflammasome priming and thus limits pyroptosis. This mechanism is particularly notable as ASFV manipulates pyroptosis via pS273R-mediated cleavage of GSDMD, a key mediator of this process. In the study by Chen et al. (2023c), DHM activity was further confirmed by the partial reversion of its effects upon treatment with TLR4 and pyroptosis agonists. Furthermore, targeting TLR4 with a specific inhibitor and siRNAs impaired ASFV replication in a comparable manner to that observed following DHM treatment.

Lauryl gallate (LG), a derivative of gallic acid, also exhibits notable antiviral activity against ASFV by modulating pathways involved in host cell death. In complementary studies by de Léon et al. (2019) and Hurtado et al. (2008), LG was shown to display potent antiviral activity at noncytotoxic concentrations, significantly reducing viral replication in a dose-dependent manner. One of the key mechanisms involved inhibiting caspase-3 activation, a critical step in the apoptotic cascade. This inhibition prevented the virus-induced apoptosis typically exploited by ASFV to facilitate its replication and spread. Although LG blocked apoptosis-associated caspase activation, it did not interfere with early viral protein synthesis or virus-induced activation of p53, implying a relatively selective effect on the downstream apoptotic machinery.

More recently, Luo et al. (2023) and Song et al. (2023) identified Aloe-emodin (Ae) and Rhein, naturally occurring anthraquinone compounds, as potent inhibitors of ASFV replication. ASFV infection induces early activation of the NF-κB signaling pathway, followed by late-stage induction of apoptosis in PAMs (Luo et al. 2023). Ae significantly reduced ASFV replication by downregulating key elements of NF-κB signaling, mirroring the antiinflammatory effects observed for DHM. Additionally, both Ae and Rhein were found to promote apoptosis in infected cells by reducing the expression of antiapoptotic Bcl-2 and increasing the levels of proapoptotic Bax and cleaved caspase-3 proteins. Thus, they effectively counteract the antiapoptotic action of ASFV’s Bcl-2 homologue, pA179L, which sequesters Bax to prevent cell death. This mode of action contrasts with LG’s inhibition of caspase-3 activation to block virus-induced apoptosis, highlighting how different compounds can target the same pathway with opposing effects and still hinder viral replication.

In yet another approach targeting cell death pathways, brequinar, a synthetic DHODH inhibitor, was found to inhibit ASFV replication by inducing ferroptosis in ASFV-infected PAMs (Chen et al. 2023b). Ferroptosis is a form of regulated cell death driven by the accumulation of intracellular and mitochondrial iron, along with lipid peroxides, which causes oxidative damage to cellular membranes. Brequinar was shown to enhance these processes, creating an environment that is hostile to ASFV replication. This effect was dose-dependent and remained consistent across all treatment timings, i.e. pre-, co-, and postinfection, sustaining ASFV inhibition for up to 72 hpi. Brequinar’s antiviral activity was confirmed by the partial reversal of its effects with ferroptosis inhibitors like ferrostatin-1, whereas exogenous uridine supplementation had no impact, demonstrating that this mechanism is independent of its inhibition of pyrimidine biosynthesis.

The cross-talk between viral proteins and mediators of cell death pathways represents a delicate balance that is exploited by ASFV. Agents like those discussed herein tip this balance in favor of the host, disrupting these processes to restore cellular defenses and hindering the virus’s ability to propagate effectively.

Other host pathway inhibitors

Although IFNs and ISGs effectively combat ASFV by amplifying host immune defenses, the virus can counter these mechanisms by manipulating host cellular pathways to suppress antiviral gene expression and promote its replication. A major strategy involves the recruitment of histone deacetylases (HDACs), specifically HDAC1, -2, and -3, to viral factories. This recruitment leads to the hypoacetylation of histone H3 that promotes heterochromatinization, a tightly packed chromatin state that silences host genes, including those crucial for antiviral defenses (Simões et al. 2015). This critical role of HDACs has been further supported by a study demonstrating the antiviral activity of HDAC inhibitors against ASFV. In that screening study of four HDAC inhibitors—trichostatin A, vorinostat, valproic acid (VPA), and sodium phenylbutyrate (NaPB)—NaPB was the most effective, completely abrogating viral replication in a dose-dependent manner (Frouco et al. 2017b). NaPB disrupted ASFV-induced hypoacetylation of H3K9/K14, thereby restoring an open chromatin state that likely facilitated the expression of genes nonconducive to viral progression. Additionally, NaPB significantly inhibited late ASFV protein synthesis and reduced the size and number of viral factories, highlighting the potential of HDAC inhibitors, particularly NaPB, as antiviral agents against ASFV. Similar findings were reported by de Léon et al. (2019) for VPA treatments, which again significantly reduced ASFV yields in infected cells. VPA lowered virus yields by ~3-log and inhibited early protein expression and the synthesis of late ASFV proteins. Time-of-addition assays further indicated that VPA was most effective when added during the first 8 hpi, and while it had mostly additive effects when used in combination with other inhibitors, it sometimes showed enhanced inhibition when coadministered with LG.

Toosendanin (TSN), a triterpenoid saponin, also exhibited potent anti-ASFV activity at submicromolar concentrations through a mechanism that closely resembles that of HDAC inhibitors (Liu et al. 2022). That study demonstrated that TSN upregulates interferon regulatory factor 1 (IRF1), a transcription factor critical for activating antiviral gene expression in PAMs. By enhancing IRF1 levels, TSN restored the expression of ISGs that are crucial for the host’s antiviral response. This led to significant reductions in ASFV replication, synthesis of viral proteins such as p30, and the production of infectious particles. Thus, much like HDAC inhibitors that counteract ASFV-induced gene silencing by reopening chromatin, TSN combats ASFV by reversing the virus’s suppression of host immune defenses. Interestingly, TSN was also found to inhibit ASFV internalization. Since both IRF1 activation and inhibition of ASFV internalization occur at early stages of infection, it is possible that IRF1 plays a role in preventing the virus from entering cells. However, this connection requires further investigation. Toosendanin demonstrated a selectivity index of 365, indicating strong antiviral activity with relatively limited cytotoxicity in vitro, placing it among the more compelling host-targeting candidates identified in the current review (Table 1; Table S2).

ASFV has been shown to speed up viral replication by enhancing the host’s energy and amino acid metabolisms in the early stages of infection, with lactic acid accumulation during the later stages inhibiting IFN-β and consequently further facilitating replication (Qiao et al. 2022, Zhu et al. 2024). Similar findings from PRRSV have shown that blocking glucose and glutamine metabolism can effectively inhibit viral replication. Recently, Dai et al. (2024) reported that ASFV infection induces metabolic reprogramming in the host and that levels of the metabolite phenyllactic acid (PLA), an important broad-spectrum antimicrobial compound, were significantly increased in infected cells. Pretreatment with PLA significantly inhibited the increased concentration of glutamine induced by ASFV infection and the metabolic flux of related pathways involved in glutamine metabolism, indicating that PLA can be used as a natural small-molecule compound for antiviral purposes. The authors speculated that PLA accumulation inhibits glutamine metabolism through the feedback of host metabolic reprogramming. However, the authors also cautioned that given the increased energy requirements of immune cells, PLA could have an inhibitory effect on their activation and proliferation, which could lead to immunosuppression and potentially aggravate the disease.

Deoxycholic acid (DCA), a secondary bile acid, has also shown interesting antiviral effects against ASFV by targeting the MAPK signaling pathway, which is important for viral replication (Gao et al. 2024). I73R is a recently characterized nucleic-acid-binding viral protein with a Zα domain that localizes to the nucleus and inhibits host protein synthesis by blocking the nuclear export of cellular mRNAs (Liu et al. 2023). Gao et al. (2024) further demonstrated that I73R plays a key role in ASFV’s ability to hijack host transcription factors, such as AP-1, by promoting its nuclear translocation. This process leads to the activation of MAPK signaling and facilitates viral genome replication. However, DCA can inhibit this pathway, blocking AP-1 nuclear entry and reducing the phosphorylation of ERK1/2 and JNK. As a result, ASFV-induced viral factories were disrupted and viral replication was significantly reduced. Similar to the mechanism of HDAC inhibitors and TSN, both of which counteract ASFV’s suppression of host genes, DCA appears to interfere with another viral strategy to manipulate the host cellular machinery for its benefit, further emphasizing the complexity of ASFV–host interactions. Interestingly, DCA was one of the eight small-molecule metabolites identified in the study by Lv et al. (2023) that highlighted arctiin and genistein as Topo II inhibitors. Although DCA’s mechanism of action differs from these latter, it exhibited comparable levels of viral inhibition, with an IC₅₀ of 6.369 μM. Though the authors reported a relatively modest selectivity index of >1.57 (Table S2) based on a maximum tested concentration of 10 μM, data from Gao et al. (2024) indicate a CC₅₀ closer to 175 μM in PAMs. This value corresponds to an estimated SI of ∼27, indicating that DCA may have a broader therapeutic window than initially assumed and lends further support to its potential as a viable antiviral candidate.

Inhibitors with unknown targets

In addition to the well-characterized inhibitors outlined above, some other antiviral agents have proven active against ASFV, despite the mechanism of action remaining obscure. Although their exact targets are unknown, these agents can inhibit viral replication effectively, making them promising candidates for ASFV control. Among them, plant-derived agents form the largest category, offering a diverse range of bioactive compounds with possible antiviral activity. In this section, we also discuss other agents not traditionally deployed in viral inhibition, yet they have shown efficacy against ASFV.

Plant-derived agents

In an early demonstration of the potential of natural compounds as ASFV inhibitors, García-Villalón and Gil-Fernández (1991) reported the anti-ASFV activity of sulfated polysaccharides lambda and kappa carrageenan, pentosan polysulfate, and fucoidan in Vero cells. Among them, lambda carrageenan presented the best activity in terms of reducing virus yield and inhibiting virus adsorption, with a favorable selectivity index of 120 that reflects low cytotoxicity (Table 1; Table S3). Nearly a decade later, Fabregas et al. (1999) screened a panel of polysaccharide-rich fractions from 10 marine microalgae for their ability to inhibit viral infection. Extracts from Porphyridium cruentum, Chlorella autotrophica, and Ellipsoidon sp. exerted significant dose-dependent inhibition of ASFV replication in vitro, an effect the authors suggested was linked to the presence of sulfated polysaccharides within the extracts. Although the inhibitory activities of these compounds were mostly attributed to blocking viral adsorption, direct action on the virus could not be ruled out.

Building on these early findings, Galindo et al. (2011) further explored the inhibitory potential of plant-derived agents against ASFV by analyzing the stilbenes resveratrol and oxyresveratrol present in grape skin and more than 70 other plants. Their study showed that both compounds strongly inhibited ASFV replication in a dose-dependent manner, achieving up to 98%–100% inhibition of viral titers at noncytotoxic concentrations. Unlike the previous studies on polysaccharides in which inhibition primarily occurred during viral adsorption, resveratrol and oxyresveratrol interfered specifically with viral DNA replication, late-stage viral protein synthesis, and viral factory formation, indicating that these compounds act at critical stages of the viral lifecycle beyond initial entry. This antiviral activity was observed for both synthetic resveratrol and oxyresveratrol, as well as natural oxyresveratrol extracted from mulberry twigs. Recently, Liu et al. (2020) demonstrated that the stilbene derivatives SD1 and SD4, which were previously shown to inhibit HU protein of Mycobacterium tuberculosis (Suarez et al. 2017), an analogue of ASFV’s pA104R protein, similarly disrupted pA104R interactions and inhibited ASFV replication in PAMs. Thus, it is plausible that the antiviral effects of resveratrol and oxyresveratrol against ASFV might partly involve targeting pA104R.

Although the potential of natural antivirals such as oxyresveratrol is undeniable since they offer benefits such as low cost and a broad spectrum of biochemical and pharmacological effects that can be advantageous in combating viral resistance, these agents sometimes come with significant limitations. One such example is the study by Fasina et al. (2013), who evaluated the antiviral effects of Ancistrocladus uncinatus extracts against ASFV in vitro. Though various solvent extracts significantly reduced ASFV titers and prevented virus replication, cytotoxicity was found to be a major problem. Furthermore, the active components in the plant varied based on which part was sampled and the extraction method used, making it potentially difficult to ensure consistent dosages and reproducibility. Ancistrocladus uncinatus was chosen for that study because of anecdotal reports from West African farmers, who had observed reduced morbidity and mortality, and in some cases, complete freedom from illness, in animals treated with preparations of the plant. Even though the results of the study did not completely align with these claims, it remains essential to systematically analyze and peer-review ethno-veterinary reports such as these, especially as ASF disproportionately affects smallholder farms with limited resources, where the cost of conventional treatments can be prohibitive.

Other agents

Chlorine dioxide (ClO₂), a strong oxidant widely used as a biocide, has exhibited strong antiviral activity against ASFV. Wei et al. (2022) reported that ClO₂ inhibited ASFV replication in PAMS by targeting various steps of the viral lifecycle. The inhibitory effect occurred during the attachment phase rather than entry, indicating that ClO₂ acts at an early stage of the viral replication process. Furthermore, ClO₂ was shown to degrade viral nucleic acids and proteins at concentrations as low as 1.2 µg/ml. However, this degradative ability was temperature-sensitive, potentially limiting its utility in practical settings. Interestingly, ClO₂ also suppressed ASFV-induced inflammatory cytokines, such as IL-6 and IFN-β, which are otherwise elevated during infection. Though its precise molecular targets remain unclear, the multifaceted antiviral nature of ClO₂ still renders it an attractive candidate for ASFV control strategies.

Glycerol monolaurate (GML) is a natural monoglyceride that has shown potent antiviral activity against ASFV by destabilizing the viral lipid envelope. Jackman et al. (2023) demonstrated that GML inhibits ASFV replication in PAMs in a concentration-dependent manner related to its critical micelle concentration. At concentrations above 63 µM, GML micelles disrupt the viral phospholipid bilayer, resulting in a striking reduction of viral infectivity by more than 99%. Unlike agents that specifically target viral proteins or nucleic acids, the mode of action of GML seems to be purely biophysical, disrupting the structural integrity of the viral envelope. This unique mode of action underscores its potential for application in feed or water sanitization. However, more studies are needed to ascertain its interaction with ASFV particles and to determine its efficacy in the field.

Several additional compounds have been tested in various screenings for their antiviral activity against ASFV, though they were not a primary focus of the respective studies. These include plant-derived compounds such as luteolin, which may act on MAPK, PI3K-AKT, TLR4/8, and NF-κB signaling pathways (Hakobyan et al. 2016, Chen et al. 2023c, Lu et al. 2023b); ivermectin, an antiparasitic drug that may block nuclear transport of viral proteins through the importin α/β1 heterodimer (Yang et al. 2020, Guo et al. 2023a); and the antimicrobials niclosamide and salinomycin, which may affect acidification of endosomal compartments, blocking ASFV trafficking and uncoating (Jurgeit et al. 2012, Jang et al. 2018, Guo et al. 2023a). Although these mechanisms of action have been proposed based on studies of other viruses, further research is necessary to confirm their specific relevance and efficacy against ASFV. Additionally, it is important to note that, aside from luteolin, these compounds present relatively low SI values, implying limited therapeutic windows and warranting cautious interpretation of their potential as antiviral agents (Table S3).

Overall then, inhibitors with unknown targets present a diverse array of antiviral strategies against ASFV, ranging from plant-derived compounds to unconventional agents such as ClO₂ and GML. Even though their exact mechanisms are not fully understood, these agents show significant potential to stop infection by disrupting ASFV’s structural integrity or interfering in key stages of viral replication. Their multifaceted antiviral effects make them interesting candidates for further research, particularly in developing affordable and more accessible solutions for ASFV control.

Discussion

The complexity of ASFV transmission dynamics requires strategies tailored to the specific circumstance of affected regions. For example, ASF is currently the most common viral disease in animal species in Europe, accounting for 71.7% of outbreaks reported in the last 6 years (Sasse et al. 2024). Moreover, the involvement of wild boars has led to a sylvatic cycle being established, with 81.4% of outbreaks attributable to this species, which act as efficient transmitters of the virus and contribute to further outbreaks in domestic pig populations. In contrast, in Africa and many low- and middle-income countries, ASFV spreads primarily through domestic pigs kept in traditional free-ranging systems, where low biosecurity and socio-economic constraints amplify the risks. The situation is even more complex in countries such as China, where modern high-biosecurity farms coexist alongside traditional farming practices that remain vulnerable to ASF outbreaks. In all of these contexts, the imposition of quarantine measures and trade restrictions often discourages farmers from reporting disease outbreaks for fear of economic losses, making it even harder to manage ASF. Exacerbating these challenges, the increased genetic variability of ASFV reported in Germany and China heightens the need for novel and varied approaches to combat the virus. The emergence of vaccine-derived strains and hybrid viruses further complicates control efforts, reinforcing that additional antiviral strategies are needed to complement existing biosecurity and vaccination efforts.

As elaborated on in this review, a wide range of antiviral agents, from nucleoside analogues to host-targeting therapies, offer hope for controlling ASFV (Fig. 1). By targeting distinct stages of the viral lifecycle or host–virus interactions, these agents can improve the efficacy of existing control measures, mitigate vaccine side effects, and/or reduce the risk of silent outbreaks spreading in areas with inadequate surveillance. Furthermore, they could play a vital role in addressing the challenges posed by wild boar and feral pig populations. Worldwide, bait-delivered vaccination programs have been deployed successfully to manage and prevent zoonotic diseases in feral pigs, as well as to deliver contraceptives and toxicants for population control (Campbell and Long 2007, Massei et al. 2010, Rossi et al. 2015). These programs provide a model for integrating antiviral agents into baits or other delivery systems to reduce ASFV transmission. Such strategies are potentially feasible in a number of ecological and agricultural contexts, including resource-limited settings. For example, including an antiviral agent like GML into feed and water sources could complement or even replace vaccination in some circumstances. Unlike vaccines, which require specific formulations and depend on an immune response, GML employs a biophysical mechanism that is less dependent on the immune status of individual animals, thus making it possibly more effective across different populations. This approach could provide a faster response, which would be particularly useful in outbreak scenarios where vaccine-induced immune responses require time to develop. Furthermore, targeting shared resources such as waterholes could ensure broad coverage of wild boars and feral pig populations with minimal requirements for additional infrastructure. Although the ecological and logistical challenges associated with treating wildlife populations are substantial, the demonstrated success of oral vaccination campaigns provides a solid foundation for considering such strategies. Nonetheless, formulation-specific barriers remain. For instance, antiviral compounds must remain stable and effective under fluctuating environmental conditions, and bait systems must be selective, palatable, and durable—all characteristics recently explored in a study on oral vaccine baits for wild boar (Relimpio et al. 2024).

A critical consideration in deploying antiviral strategies is how to balance the strengths and limitations of DAAs and HTAs. DAAs target specific viral proteins such as the PolX DNA polymerase with high selectivity, which contributes to lower toxicity and reduces the risk of side effects. Notable examples include cytarabine hydrochloride and brincidofovir, with this latter exhibiting the highest selectivity index (SI ∼21 000) among all of the compounds reviewed herein. However, ASFV can utilize redundant mechanisms to suppress immune pathways, meaning that targeting a single viral protein can be ineffective in clinical settings. For instance, the virus inhibits production of IFN-β by targeting several molecules in the cGAS-STING signaling pathway through pI215 L and DP96R viral proteins. In contrast, HTAs target host cellular pathways that are crucial for ASFV replication, such as lipid metabolism and endosomal trafficking. Compounds like cerulenin that disrupts fatty acid synthase and endosomal inhibitors like tetrandrine demonstrate the potential of this approach to broadly inhibit viral replication across strains. Although HTAs risk increased cytotoxicity by interfering with essential host functions, several candidates—such as toosendanin (SI = 365), genkwanin (SI = 205), and most notably 3-deazaneplanocin A (SI = 3000)—demonstrate a favorable therapeutic window. Nonetheless, careful optimization remains essential to ensure efficacy while avoiding harmful side effects, as many HTAs still operate within a narrow margin of safety.

New computational methods driven by high-throughput molecular modeling have aided in this optimization, speeding up the identification of promising antiviral candidates. For example, targeting ASFV’s G-quadruplex structures (G4s) has the potential to impact multiple essential genes irrespective of whether their protein products can be directly inhibited. Similarly, drug repurposing, which builds on the already established safety and pharmacokinetic profiles of approved pharmaceutical drugs, has benefited from computational methods. This approach provides a cost-effective alternative to traditional de novo drug development and was recently employed in the rapid identification of viable candidates for treating emerging infectious diseases, such as COVID-19 (Galindez et al. 2021), in a shift towards more targeted and efficient methodologies. In the search for new and effective antivirals against ASFV, researchers have likewise employed advanced CADD techniques to screen vast libraries of compounds for their potential multitarget inhibition against the BER proteins of ASFV, enabling efficient prioritization of candidates, for example methyl ganoderate E and D-132, for experimental validation. Such strategies not only accelerate the drug discovery process, but also emphasize the importance of advanced analytics in the development of precision medicine for the management of viral diseases. As ASFV continues to evolve, these approaches could prove essential for adapting to and potentially controlling new viral variants with unpredictable characteristics.

Despite the progress made in ASFV antiviral research, significant gaps remain in translating laboratory findings into practical applications. For instance, very few compounds have been evaluated for efficacy in vivo and those subjected to such experiments still face substantial regulatory hurdles before they can be approved for therapeutic use in livestock. Moreover, many in vitro studies use recombinant viral proteins expressed in heterologous systems that often lack the proper modifications and cellular contexts necessary to accurately assess their efficacy. Issues such as poor solubility and interactions with cellular lipid membranes further complicate evaluations. Agents targeting unique viral functions, such as the BER pathway, hold promise due to their low risk of cytotoxicity, as demonstrated by the dose-dependent inhibition of PolX by polygalic acid, cytarabine hydrochloride, and triapine. Natural products such as apigenin and myricetin offer additional advantages by targeting multiple stages of the viral lifecycle, potentially reducing the likelihood of resistance development. However, challenges including cytotoxicity, variability in active compound concentrations, and limited scalability hinder their use in the field. Beyond these scientific hurdles, practical barriers also remain significant. Strict regulatory environments, particularly in regions, such as the European Union, prohibit the use of antiviral drugs in food-producing animals over safety concerns and the risk of promoting drug-resistant viral strains that could impact human health (Sasse et al. 2024). While regulatory frameworks differ worldwide, many countries maintain similarly cautious or restrictive policies, which limit the availability and approved use of antiviral agents in livestock. Cost is another significant challenge, particularly for smallholder systems in low- and middle-income countries, where veterinary infrastructure is limited and pharmaceutical interventions may be unaffordable or unavailable. In summary, antiviral therapeutic approaches, including direct-acting agents, host-targeting therapies, immunomodulators, and gene-editing technologies, offer hope in managing ASF. However, bridging the gap from proof-of-concept to real-world implementation demands robust and interdisciplinary collaboration among molecular virologists, veterinarians, epidemiologists, pharmacologists, and public health stakeholders. Future research should prioritize the development of combination strategies that integrate antiviral tools with existing biosecurity and vaccination measures to create multilayered protection, especially in high-risk or resource-limited settings. These efforts must also include rigorous safety and pharmacokinetic assessments to address concerns relating to drug residues and regulatory compliance, alongside feasibility and cost-benefit analyses tailored to different production systems. Such integrated strategies are increasingly urgent given the accelerating genetic variability of ASFV and its potential to alter the course of the current pandemic.

Conflict of interest

None declared.

Funding

This work was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through projects UIDB/00276/2020 (CIISA-Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon) and LA/P/0059/2020-AL4ANIMALS (AL4AnimalS).