4.1.1. Temperature Stability

We begin by covering recent thermal stability studies that describe how natural temperature variations affect viral persistence in different settings such as animal carcasses and tissues, biological matrices, fomite surfaces, and feed materials. It has been reported that skin samples from ASFV-infected pigs and wild boars can remain infectious at room temperature for up to 3 months [75]. Kinetic analysis of ASFV contamination in pig tissues supports that the half-lives of viral infectivity vary from ~32 days at −20°C to ~9 h at +23°C [77]. Higher moisture content and the presence of organic matter also tended to accelerate ASFV inactivation in the tissue samples. These effects were attributed to greater tissue preservation in matrices with low moisture content (e.g., straw, hay), while organic matter such as water, soil, and leaf litter caused quicker virus inactivation, which may be related to environmental pH effects or the types of reactive molecules present in the organic matter. Similar temperature-dependent results have been observed for ASFV contamination in soil samples, with infectious virus detectable after 112 and 42 days incubation at +4 and +22°C, respectively [78]. In addition, ASFV-infected blood samples from an infected wild boar were incubated in different types of soil and high stability was observed in sandy soils but no infectious virus remained in acidic forest soils [79].

Another study reported that the half-life of ASFV at 4°C in serum-containing media was around 36–119 days and the half-life decreased to around 1 day at 37°C and to around 1 min at 60°C [80]. Interestingly, PCR testing has demonstrated that ASFV nucleic acid levels remain largely stable for at least 5 days at temperatures up to 70°C, whereas ELISA results showed temperature-dependent loss of ASFV antigen conformation after 56°C treatment for 1 h [81]. This finding supports that the loss of viral infectivity mainly stems from structural damage to the virus particle (e.g., protein conformational changes). A combination of moderately elevated temperature (48°C), alkaline conditions (pH 10.2), and high hydrogen peroxide concentration (103 mM) has also been reported to inhibit ASFV infectivity in porcine plasma [82]. Separately, groundwater contamination studies have shown that ASFV at 4°C can remain infectious in river water for greater than 42 days but is rendered noninfective after 28 and 14 days at 15 and 21°C, respectively [83].

Additionally, the persistence of dried infectious ASFV on porous and non-porous surfaces such as glass, metal, rubber, and cellulose paper was investigated at different environmental temperatures between 25 and 42°C [84]. In this case, ASFV inactivation kinetics were analyzed by measuring the decimal reduction time (D_T), which is defined as the time required to reduce ASFV infectivity per 1 log at a certain temperature (T). A smaller D_T value means quicker inactivation. A similar trend was observed on all surfaces whereby higher temperature led to quicker inactivation and the corresponding D₂₅, D₃₃, and D₄₂ values were around 1.42–2.42, 0.72–1.94, and 0.07–0.23 days, respectively.

The persistence of infectious ASFV in these different environments has prompted detailed examination of ASFV infectivity in feed and feed ingredients, which is an important transmission risk for livestock viruses in general. By mimicking storage conditions associated with transoceanic shipping over 30 days, it was determined that infectious ASFV remained viable in 9 out of 11 feed ingredients and had a half-life of ~1–2 days [85]. A more detailed analysis of ASFV stability in the different feed ingredients over a 30-day simulated shipping period provided a revised estimate that infectious ASFV has a half-life of around 9–14 days [86]. In another study, ASFV was incubated in complete feed, soybean meal, and ground corncobs at 4.4, 20, or 35°C for up to 1 year, and it was identified that viral infectivity was maintained in soybean meal at 4.4°C for at least 112 days, at 20°C for 21 days, and at 35°C for 7 days [87]. These results demonstrate that ASFV is more sensitive to higher temperatures and have prompted the development of thermal inactivation strategies based on elevated temperature treatment.

4.1.2. Thermal Treatment

While extended storage of feed and feed ingredients can help to reduce contamination risk, thermal treatment of these materials at elevated temperatures is a complementary strategy that provides quicker and more robust inactivation. For this reason, several recent studies have investigated how elevated temperatures affect ASFV infectivity in complete feed, feed ingredients, crops, and swill, which has also provided scientific evidence to guide regulatory policy developments. These studies are summarized in Table 1 and discussed below.

The combined effect of drying and heat treatment has been reported, whereby ASFV-contaminated field crops—a potential source of material for feed and bedding—were dried for 2 h at room temperature and then incubated for 1 h at elevated temperatures ranging between 40 and 75°C [88]. The drying step reduced viral infectivity of ASFV-infected field crops in cell-based assays to undetectable levels, even at room temperature. However, compared to drying alone, ASFV viral nucleic acids were more degraded but still present after subsequent thermal treatment according to PCR testing. Additional testing with blood samples demonstrated loss of viral infectivity at ≥55°C, supporting that elevated temperatures in that range are most effective at viral inactivation in biologically relevant matrices. The use of thermal treatment at elevated temperatures (≥55°C) is important to ensure robust ASFV inactivation because lack of viral infectivity in cell-based assays may be due to insufficient assay sensitivity and infectious ASFV residues can still persist in some cases. For example, it has been reported that dry-cured meat products from ASFV-infected pigs were noninfective according to cell-based assays but still caused infection in healthy pigs that consumed the meat products [94].

For thermal inactivation of ASFV in feed ingredients such as meat and bone meal, soybean meal, and maize grain, the corresponding D₆₀, D₇₀, D₈₀, and D₈₅ values were around 5–7, 2–3, 1–2, and 0.2–1 min, respectively [89]. Another study reported that a significant reduction in ASFV infectivity in three different swill formula occurred starting at 60°C and the corresponding D₆₀, D₇₀, D₇₅, and D₈₀ values were around 23–33, 6–11, ~2, and ~1 min, respectively [90]. Importantly, based on analyzing the viral inactivation kinetics, it was concluded that complete inactivation of ASFV contamination in swill could require up to 119 and 4 min at 70 and 90°C, respectively, which is important for guiding policy recommendations that are further discussed below. Another recent study reported that infectious ASFV can remain viable in porcine serum for >60 days at temperatures up to 37°C, 2 days at 50°C, 1 day at 60°C, and ≤5 min at 70°C, which supports a similar trend of temperature-dependent ASFV inactivation [91]. As discussed above, the presence of organic matter such as feed, straw, or wood shavings also accelerated virus degradation, which was attributed to acidic conditions and reactive chemicals within the materials.

Such findings fit well with ASFV-related regulatory guidelines regarding the import of plant-based feed ingredients for use in livestock feed [95]. The Canadian Food Inspection Agency (CFIA) has mandated that, for imports from regions not recognized as free of ASF, thermal treatment must be performed whereby the product temperature reaches at least 70°C for 30 min or 85°C for 5 min in order to ensure sufficient virus inactivation. Alternatively, the imported feed ingredients can be stored for at least 20 days at 20°C or 100 days at 10°C. More broadly, the United States Department of Agriculture (USDA) published an ASF Response Plan that describes how heat application is an effective strategy for cleaning and disinfection as part of biosecurity efforts and is also useful in disease response contexts [96]. The plan cited WOAH recommendations that effective ASFV inactivation requires thermal treatment at 56°C for 70 min or at 60°C for 20 min.

The WOAH has also issued detailed guidelines advising that ASFV inactivation in swill requires thermal treatment at a minimum of 90°C for at least 60 min or 121°C for at least 10 min, whereas ASFV inactivation in meat products requires a minimum temperature of 70°C throughout the meat for at least 30 min, and ASFV inactivation in pig litter or manure requires a minimum temperature of 55°C for at least 60 min or a minimum temperature of 70°C for at least 30 min [97]. The FAO has more generally recommended that effective ASFV inactivation in porcine-related materials (e.g., meat products, carcasses, blood, abattoir swill) can be achieved by incubating at 70°C for 30 min [98].

The Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF) has also advised that food waste-derived swill must be treated at 90°C for at least 60 min [99] in line with the WOAH guidelines. Conversely, the South Korean Ministry of Agriculture, Food and Rural Affairs (MAFRA) only requires thermal treatment at 80°C for 30 min, which is below the WOAH recommendations, and it has been suggested that these mandated treatment conditions are potentially insufficient for effective ASFV inactivation [100]. This deviation raises concerns about incomplete ASFV inactivation, potentially increasing transmission risks and highlighting the importance of formulating evidence-backed biosecurity practices. While this practical example demonstrates that there can be some quantitative differences in recommended thermal treatment conditions depending on the regulatory agency, it is generally recognized that thermal treatment at >55°C is effective for ASFV inactivation. Combined with the latest scientific evidence described above, the overall body of scientific evidence supports that thermal treatment in the >70°C range is effective in most cases, while swill processing demands more stringent conditions for precaution.

There has also been extensive interest in preventing ASFV contamination of spray-dried animal plasma (SDAP) products, including spray-dried porcine plasma (SDPP), which are popular feed ingredients for pig production. SDAP manufacturing involves a short (< 1 s) spray-drying step at 80°C outlet temperature, and it was found that this step causes an ~2 log₁₀ drop in ASFV infectivity [92]. Furthermore, to mimic the longer residence times found in commercial spray dryers, an extended incubation (1 min) at 80°C after drying was found to decrease ASFV infectivity by over a 4 log₁₀ drop, which is considered suitable for effective ASFV inactivation. Another study investigated the effects of spray drying on ASFV infectivity in SDPP samples with the outlet temperature set at 80 or 71°C and a 3.2–4.2 log₁₀ or 2.5–2.8 log₁₀ drop in ASFV infectivity was achieved at the two respective temperatures [93]. After a subsequent 14-day storage period at 4 or 20°C, complete inactivation of all ASFV-contaminated SDPP samples was achieved and no infectious ASFV could be detected. In addition, a quantitative risk assessment model was applied to SDPP processing of ASFV-contaminated plasma and supported the feasibility of ensuring ASFV decontamination in large batches up to 20 tons [101].

While thermal inactivation strategies play an important role in combating potential ASFV contamination of feed and feed ingredients, these strategies are typically one-time treatments and re-exposure to ASFV after treatment is possible. For example, the postmanufacturing exposure of SDPP to infectious ASFV has been investigated and room temperature storage of the ASFV-contaminated SDPP for 1 or 2 weeks led to >2.8 log₁₀ and >5.7 log₁₀ drops in viral infectivity, respectively, indicating that complete inactivation can be achieved in 2 weeks [102]. On the other hand, storage at 4°C was insufficient to inactivate ASFV even after 5 weeks. This issue has also been recently raised in the context of persistent ASFV contamination in a pilot-scale feed mill facility [103] and points to the utility of employing additional inactivation strategies, especially fast-acting ones that support repeated use.

4.2.1. Comparison Studies

Many studies have comparatively investigated disinfectant candidate panels in aqueous suspensions, and we first cover these results before commenting on more specific examples. In one study, the antiviral effects of four commercial disinfectants—comprising sodium hypochlorite, potassium peroxymonosulfate, glutaraldehyde, or quaternary ammonium compounds as the active ingredient—on the infectivity of the cell-culture-adapted, nonvirulent ASFV BA71V strain was investigated using Vero cells [108]. Products based on glutaraldehyde or quaternary ammonium compounds showed high cytotoxicity to the Vero cells, which precluded further examination. Conversely, products based on sodium hypochlorite and potassium peroxymonosulfate demonstrated more favorable virucidal properties. The sodium hypochlorite-based product was active at 0.5% and 1% concentrations and caused ASFV infectivity drops of >4 log₁₀ but was less effective in the presence of soil-mimicking organic compounds. On the other hand, the potassium peroxymonosulfate-based product at 1% concentration was more effective in highly soiled conditions but was cytotoxic in standard conditions. A follow-up study with a modified protocol to enable cytotoxic compound evaluation was performed to test the antiviral activity of eight active compounds—formaldehyde, sodium hypochlorite, caustic soda, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate, and acetic acid—and it was reported that sodium hypochlorite, caustic soda, and potassium peroxymonosulfate had the best inactivation performance [109]. By contrast, benzalkonium chloride, which is a quaternary ammonium compound, glutaraldehyde, and formaldehyde also exhibited virucidal properties but had high cytotoxicity.

Another recent study reported testing a panel of 24 commercial disinfectants against the ASFV BA71V strain and included compounds categorized as oxidizing agents, acids, aldehydes, formic acids, and phenols [110]. It was found that disinfectants based on formic acid, phenolic compounds, and oxidizing agents decreased ASFV infectivity by >4 log₁₀, whereas other disinfectants based on hydrogen peroxide, aldehyde, and quaternary ammonium compounds also inhibited ASFV to varying extents but had high cytotoxicity. These results are consistent with the reported efficacy of oxidizing agents such as sodium hypochlorite and potassium peroxymonosulfate described in the aforementioned studies and other oxidizing agents such as chlorine dioxide [111] and hydrogen peroxide [81] have also been reported to inhibit ASFV. In addition, another oxidizing agent, iodine, has been reported to have high inactivation efficiency in a complexed form [112] and works synergistically with organic acids [113]. The results of the panel study further support that aldehydes like glutaraldehyde are effective at ASFV inactivation but are cytotoxic. Even so, it should also be noted that certain ones, especially formaldehyde, are still being explored additionally as mitigants for ASFV inactivation in more complex biomatrices such as feed and display rapid inactivation kinetics in suspension tests [114].

Several recent studies have also evaluated disinfectants against virulent ASFV strains using PAM cells. For example, it has been reported that various disinfectants—an iodine and acid mixed solution, potassium peroxymonosulfate, citric acid, sodium dichloroisocyanurate, glutaraldehyde/deciquam, and deciquam—wereeffective at inactivating ASFV in immersion and spraying formats [115]. However, in the presence of organic matter, the disinfectants were appreciably less active, indicating they are best suited for decontaminating clean surfaces rather than mitigation in biomatrices. Interestingly, it has also been reported that a mixture of quaternary ammonium compounds inactivates ASFV better than potassium hydrogen peroxymonosulfate, suggesting that different ASFV strains may have varying susceptibilities and also that compositions of different molecule types can be tailored to improve virucidal performance [116]. In terms of applications testing, four commercial disinfectants—composed of a cationic surfactant, glutaraldehyde + cationic/anionic surfactants, iodine, and glutaraldehyde + cationic surfactant – were tested in the absence of interfering organic species and were all found to rapidly reduce virulent ASFV infectivity by >4 log₁₀ within 15 min [117]. Another study tested five commercial disinfectants comprising glutaraldehyde or quaternary ammonium compounds as the active ingredient and noted that all had similarly high antiviral activity against a virulent ASFV strain, while it was further reported that these disinfectants had high cytotoxicity that likely restricts their use to surface decontamination applications [118].

One recent study further demonstrated that sodium hypochlorite, glutaraldehyde, potassium peroxysulfate, sodium hydroxide, phenol, and formaldehyde maintain ASFV inactivation efficacy across a wide range of temperatures from +21 to −20°C [119]. Conversely, benzalkonium chloride and acetic acid were less effective at sub-zero temperatures, and these findings provide guidance regarding which disinfectants would be useful for surface decontamination in cold environments.

A practical example of a disinfectant application in an ASF outbreak setting was described for a medium-sized farm in Uganda [125]. After the outbreak started, workers rinsed their boots in a water bath when moving from the pig slaughter facilities to the pig pens. The active ingredient in the water bath was described as a commercial disinfectant containing ammonium chloride, which likely refers to a quaternary ammonium compound although the exact formulation was not specified. While boot rinsing was performed, it was noted that the boots were not physically cleaned prior to immersion. As such, organic material was likely still present on the boots, which would limit ASFV inactivation efficacy as discussed above. This example illustrates both the feasibility of using quaternary ammonium compounds for surface decontamination and the need for improved biosecurity practices, such as removing organic material before disinfection rinsing, to enhance efficacy.

These findings have also provided a scientific foundation to support regulatory decisions regarding the use of particular disinfectants for ASF biosecurity. The USDA Animal and Plant Health Inspection Service (APHIS) has approved several disinfectants for emergency use against ASFV, including those containing citric acid, acetic acid, sodium hypochlorite, and thymol, for surface decontamination [126]. Other active ingredients found in disinfectants approved by the USDA APHIS for use against ASFV in farm settings include potassium peroxymonosulfate, several quaternary ammonium compounds, and oxidizing agents. Interestingly, the oxidizing agent sodium dichloro-striazinetrione that releases hypochlorous acid is approved for use in disinfection baths for boots, while the listed quaternary ammonium compounds are not described for this application [127].

The WOAH has also provided recommendations that include sodium hydroxide, hypochlorites, formaldehyde, and oxidizing iodine compounds while noting that virucidal efficacy can vary depending on pH, incubation time, and the presence of organic matter [128]. In some cases, disinfectants can be used for soil decontamination as well, and there are affordable options such as lime products based on calcium hydroxide, which can be ~20 times cheaper than surfactant options [129]. At the same time, there has been discussion about whether applying basic disinfectants to acidic soils is appropriate, whereas acidic disinfectants like citric acid might be more useful [79]. In addition to these cost and compatibility factors, other practical considerations for disinfectant selection include handling safety, corrosiveness, chemical stability, and efficacy in the presence of organic matter.

Overall, these comparison studies have also highlighted the importance of developing disinfectants that balance high virucidal activity with low cytotoxicity for broad usage and have prompted efforts to find more natural and sustainable disinfectant options. Recently, a powdered disinfectant containing thymol as the active ingredient was also reported to effectively inhibit ASFV [130] and highlights the push towards more natural and sustainable disinfectant options. Within this scope, we introduce two examples of natural disinfectants that are being considered for ASFV disinfectant applications.

4.2.2. Water

Oxidizing agents are one of the most effective types of disinfectants to inactivate ASFV, although many compounds in this class are synthetic and not ideal for uses involving human or animal contact. As such, there is high interest in developing safer, natural versions for broader application usage. Ozonized water is a promising candidate that can be produced at >40 mg/L concentrations by simple electrolysis of water and contains ozone molecules in the water. Ozone is a nontoxic oxidizing agent that can rapidly inactivate viruses and bacteria and safely decomposes into oxygen within a few days. It was recently demonstrated that 1-min treatment with 5–20 mg/L ozonized water could reduce virulent ASFV infectivity by up to 3 log₁₀ (99.9%) in a dose-dependent manner in PAM cell experiments [120].

In addition, there has been exploration of chlorinated water for inactivating ASFV, which involves electrolysis of acidic water to generate the HOCl oxidizing agent [121]. Electrolyzed water containing 40–60 ppm free chlorine was effective at reducing ASFV infectivity by >4 log₁₀ whereas lower concentrations had appreciably less activity. In water containing 5% fetal bovine serum (FBS) to mimic soil conditions, effective ASFV inactivation of 4 log₁₀ or greater required at least 80–100 ppm free chlorine in the water. These findings support that chlorinated water could be useful for decontaminating contact surfaces and soil and may also be used in drinking water.

4.2.3. Natural Plant Extracts

Inspired by plants used in African traditional medicine, 28 plant extracts from various natural sources were tested for ASFV disinfectant properties [122]. Four extracts inhibited ASFV replication in infected cells by >80% (i.e., antiviral), while six different extracts directly inhibited ASFV virus particles by >80% (i.e., virucidal). These findings support that extracts can have different mechanisms of antiviral activity and an extract from the Sarcocephalus latifolius root, which has been found to contain medicinally active quinovic acid glycosides and monoterpene indole alkaloids [131], had the highest inactivating effect on ASFV infectivity (~100% drop).

Anecdotally, there have also been stories about how rural farmers use plants for ASFV disease management, including unsubstantiated reports that feeding Ancistrocladus uncinatus plant preparations to pigs helps ward off ASF disease [123]. This background context motivated researchers to extract compounds from the leaf, root, and stem portions of the plant, which included alkaloids, glycosides, steroids, saponins, flavonoids, and tannins, and explore how the extracts might be used as disinfectants. At ~5 mg/mL concentration, the crude acetone extract was cytotoxic to a primary bone marrow culture (PBMC), while lower extract concentrations in the range of 0.0078–1 mg/mL concentration were subsequently tested in antiviral assays. Extracts prepared using different organic solvents were tested for inhibitory activity against a virulent ASFV strain from Nigeria by using a PBMC cell culture system, and it was discovered that the acetone, dichloromethane, and methanol extracts had potent inhibitory activities, which were confirmed by PCR experiments. On the other hand, the hexane and chloroform extracts had weaker antiviral effects. These results support the validity that plant extracts can inhibit ASFV according to antiviral and/or virucidal mechanisms, while high cytotoxicity was noted and solubility improvements are needed.

A more recent study investigated 14 plant extracts and found that only peppermint extract at 1.05% concentration was effective at reducing ASFV infectivity by >4 log₁₀ while maintaining acceptable cell viability [124]. Other test materials like strawberry and raspberry extracts are known to be active against other viruses [132] but were not effective against ASFV, emphasizing the challenges of inactivating its complex envelope structure. In general, it was also noted that organic contaminants representative of soil conditions tended to decrease the antiviral activity of plant extracts, and it was discussed how effective use of plant extracts as disinfectants should be preceded by pre-cleaning contact surfaces to physically remove organic debris.

Collectively, these findings indicate that a wide range of synthetic and natural disinfectants exhibit high ASFV inactivation properties with fast-acting mechanisms and are important components of biosecurity efforts. It should be emphasized that disinfectants work optimally on pre-cleaned surfaces devoid of organic contaminants. On the other hand, most disinfectants, even natural ones, have relatively high cell cytotoxicity levels or other disadvantageous properties like causing irritation or corrosion and are generally not ideal for mitigation in more complex biomatrices like feed due to safety and efficacy issues. While disinfectants will continue to play a key role in ASFV biosecurity, these limitations have also increased attention to develop mitigants, which are generally classified as physically disruptive antiviral molecules that are designed to inhibit ASFV in more complex biological matrices such as feed as well as exhibit a broader set of health-promoting functions.

4.3.1. Monoglycerides

Widely found in natural sources such as tropical oils and milk, monoglycerides (MGs) are nonionic molecules that are approved by regulatory agencies for food and agricultural applications [145, 146]. They have long been studied as broad-spectrum antimicrobial agents that can inhibit membrane-enveloped viral and bacterial pathogens [147]. It has been reported that medium-chain MGs with saturated hydrocarbon tails, especially 12-carbon-long glycerol monolaurate (GML), have potent inhibitory activity against enveloped viruses such as human immunodeficiency virus (HIV) and herpes simplex virus (HSV) [148]. MGs exhibit antiviral properties by physically disrupting the lipid bilayer surrounding enveloped virus particles [149] and can also exhibit additional mechanisms of antiviral activity, such as stabilizing cellular membranes to prevent infectious pathogen entry [150] and influencing cellular signaling networks [151]. Furthermore, certain MGs exhibit anti-inflammatory properties as well [152–154]. All of these mechanisms stem from the physical interaction of MGs with phospholipid membranes, which is distinct from the chemical reactivity of typical disinfectants.

In the ASFV context, the antiviral activity of GML has been explored in liquid and feed environments [134]. The non-virulent ASFV BA71V strain was used with Vero cells, and initial virucidal experiments in aqueous buffer conditions identified that GML was more effective at reducing ASFV infectivity than equivalent concentrations of different medium-chain fatty acids (MCFAs), including 8-carbon-long caprylic acid, 10-carbon-long capric acid, and 12-carbon-long lauric acid, and GML caused an ~98% drop in viral infectivity. Additional experiments revealed that GML exhibited multiple mechanisms of antiviral activity, leading to an overall 99.8% reduction in viral infectivity. Mitigation experiments in feed showed that GML reduced ASFV infectivity in a dose-dependent manner, and a 2 wt% inclusion rate caused an 88% drop in viral infectivity after 30-min incubation. The inclusion of 2 wt% GML markedly accelerated ASFV mitigation overall, leading to a 98% drop in viral infectivity compared to the initial condition. By contrast, a free-flowing, dry MCFA blend containing silica carrier had no effect on ASFV infectivity. More detailed analysis of the feed samples showed that GML did not affect ASFV DNA levels measured by PCR but did affect ASFV p72 capsid protein conformational properties according to ELISA measurements (consistent with reported effects of GML on altering the conformation of membrane-associated structural proteins of other viruses [155]), supporting that GML mitigation damages ASFV particle integrity but does not affect viral nucleic acids.

Follow-up studies have verified that GML can also inhibit a virulent, wild-type ASFV strain (Armenia/07) and mitigate infection of PAM cells in vitro [156]. Highly active GML concentrations had only minor effects on PAM cell viability, supporting that GML is potentially useful to inhibit ASFV infection in pigs. A solid-phase blend of GML and sodium diformate, which is a known antimicrobial compound [157], has also been reported to inhibit ASFV infectivity in feed [135]. In that study, a longer time scale of feed mitigation was evaluated whereby the feed additive at a 0.3 wt% inclusion rate inhibited ASFV infectivity by 82%, 92%, 99%, and 100% within <30 min, 1 day, 3 days, and 7 days postincubation, respectively.

Recently, there has also been exploration of a liquid-phase mitigant mixture composed of GML and medium-chain lactylates to inhibit ASFV and other enveloped viruses such as influenza A virus (IAV) [136]. The mixture could be added directly to aqueous solution, and 1:50 dilutions upward demonstrated virucidal activity against ASFV and IAV based on viral infectivity measurements. Notably, for the ASFV case, the virucidal doses also caused reduced levels of intact viral nucleic acids according to PCR experiments, which is consistent with virus particle disruption and was further supported by measured changes in membrane ionic permeability. The mixture also maintained cell viability at concentrations up to 1:10 dilution, supporting that there is an effective dose range that inhibits ASFV and is non-cytotoxic. In terms of potential utility, the mixture could be added to drinking water lines for rapid response to ASFV outbreaks, while it was also shown to be effective at mitigating ASFV in feed.

Together, these findings support that GML is capable of mitigating ASFV infectivity in liquid and feed conditions while calling for further evaluation of potential antiviral effects in animal models. These possibilities are supported by recent findings that orally administered GML can reduce mortality, clinical symptoms, viral loads, and inflammation in PEDV-infected piglets [158] and can also reduce clinical symptoms, viral loads, and inflammation in Seneca Valley virus (SVV)-infected piglets [159]. While GML has been the focus of ASFV mitigation studies, other MGs, especially 8-carbon-long glycerol monocaprylate and 10-carbon-long glycerol monocaprate, have been reported to effectively inhibit PEDV and PRRSV in feed mitigation and in vivo therapeutic contexts [160, 161] and merit further attention for ASFV applications.

4.3.2. MCFAs

MCFAs are naturally occurring lipid amphiphiles that have a single hydrocarbon chain and are also widely approved for food and agricultural applications [145]. Defined as fatty acids with 6- to 12-carbon-long chains, MCFAs are a key building block of MGs and exhibit membrane-disruptive properties against various types of membrane-enveloped viruses and bacteria [162, 163]. Past studies have shown that 10- and 12-carbon-long fatty acids called capric acid and lauric acid, respectively, are highly active and corresponding 6- and 8-carbon-long fatty acid chains called caproic and caprylic acids also have advantages such as liquid phase properties around room temperature [164].

Inspired by past efforts at PEDV mitigation [165, 166], early work on testing MCFAs against ASFV was done in comparison to formaldehyde. The antiviral activity of an MCFA blend composed of caproic acid, caprylic acid, and capric acid in a 1:1:1 ratio was tested along with a 37% aqueous formaldehyde and propionic acid mixture and involved measuring mitigation effects on ASFV BA71V strain infectivity in a Vero cell model [137]. The MCFA blend reduced ASFV infectivity by around 82% at concentrations as low as 0.13%, and 0.6% MCFA caused >99.9% reduction. At higher MCFA concentrations, ASFV infectivity was reduced below limits of detection. Similar trends were observed with formaldehyde, which was effective at reducing ASFV infectivity by around 82% at concentrations as low as 0.03% and caused >99.9% inhibition at 0.3% concentration. Adjusting for the different molecular weights of the two mitigants, the data support that MCFA had similar, if not better, performance to formaldehyde in terms of molar concentrations.

In the same study, various feed ingredients were then spiked with the virulent ASFV Georgia 2007 strain and stored for 30 days according to transoceanic shipping conditions. The feed samples were pre- or posttreated with 1% MCFA or 0.33% formaldehyde and infected ASFV was not detected in any treated sample according to PAM cell infection experiments. Interestingly, however, ASFV nucleic acid levels remained unchanged in the MCFA-treated feed samples, whereas formaldehyde treatment caused marked reductions in some feed ingredients. The lack of nucleic acid damage caused by MCFA agrees well with GML mitigation data, supporting that membrane-disruptive mitigants do not necessarily damage viral nucleic acids in the ASFV case. The feed samples were then fed to pigs in a bioassay format [167] and no pigs exhibited clinical symptoms of ASFV infection. Out of the 24 pigs in the treatment groups, only one pig in the MCFA treatment group acquired ASFV infection based on virological testing after euthanasia.

There has also been interest in exploring how the antiviral activity of different MCFA mixtures compares, which has led researchers to explore three compositions consisting of (1) caprylic acid alone, (2) a 1:1:1 mixture of caproic acid, caprylic acid, and capric acid, and (3) a 1:1:1 mixture of caprylic acid, capric acid, and lauric acid [138]. All candidate formulations maintained high PAM cell viability at 100 µg/mL mitigant concentrations whereas mixture (2) was more cytotoxic at 200 µg/mL concentration. The different MCFA mixtures were added to complete feed at inclusion rates of 0.125, 0.25, 0.375, or 0.5 wt% and inhibitory effects on the virulent ASFV Pig/Hanoi/2019/01 strain were investigated. According to PCR data, all mixtures exhibited dose-dependent inhibitory effects in feed. Among them, mixture (2) was the best-performing one and caused a decrease in ASFV nucleic acid levels at mitigant inclusion rates as low as 0.125%, whereas mixtures (1) and (3) were active starting from inclusion rates of 0.375 and 0.25 wt%, respectively. However, none of the mixtures affected ASFV infectivity in a PAM cell culture system based on viral titer measurements.

4.3.3. Formaldehyde

Formaldehyde is industrially produced at large scales and is widely used as an antimicrobial disinfectant and additive in agricultural applications. Formaldehyde has been reported to inhibit a wide range of enveloped viruses and mainly works by crosslinking viral proteins and nucleic acids [168, 169]. This mechanism has contributed to the role of formaldehyde and other mechanistically similar aldehydes as chemically reactive disinfectants and their established use in surface decontamination applications has led to exploring broader use in feed decontamination. While formaldehyde is still widely used in major markets worldwide, there is growing recognition of its carcinogenic risks [170] and some regulatory jurisdictions like the European Union have restricted its use in applications such as pathogen feed mitigation [171]. Nevertheless, due to its perceived high antiviral efficacy and low cost, formaldehyde is still widely explored for inhibiting swine viruses such as PEDV in feed [172], and recent studies have focused on evaluating its ASFV mitigation properties. The direct comparison with MCFA is reported above, and this section focuses on other formaldehyde-based products.

In one study, 3 kg/ton inclusion rates of different formaldehyde-based products (2 liquid and 1 dry) were evaluated for ASFV feed mitigation. Using the virulent ASFV VN/Pig/HN/19 strain, PCR measurements were performed to determine ASFV nucleic acid levels at 1, 3, or 7 days postcontamination and one liquid product reduced nucleic acid levels after only 1 day whereas the other two products acted more slowly [139]. The top-performing product was also effective at reducing ASFV infectivity in feed by day 3 postcontamination according to viral titration measurements in PAM cells and the maximum effect resulted in a 79% drop in viral infectivity. The dry product also reduced viral infectivity to a smaller extent by day 7 postcontamination while the other liquid product was ineffective at reducing viral infectivity.

Another formaldehyde-based product was also evaluated as a feed mitigant to inhibit the virulent ASFV VN/Pig/Hue/1270 strain [140]. Different inclusion rates of 1, 2, or 3 kg/ton were tested, and PCR measurements at 1, 3, or 7 days postcontamination demonstrated that reductions in ASFV nucleic acid level were greater with higher inclusion rates and longer incubation times. All inclusion rates were also effective at reducing viral infectivity and the greatest effect was observed with the highest inclusion rate, which equated to an 88% decrease in viral infectivity. Judging from these results, another interesting observation is that GML (as described above) and formaldehyde had comparable levels of feed mitigation based on infectivity reduction levels.

4.3.4. Other Mitigant Candidates

Another promising mitigant option involves a 1:1:1 blend of natural oils extracted from Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia, which was found to contain terpenoid-based antiviral molecules such as cineole, linalool, and isobornyl acetate [173]. Initially, different dilutions of the natural oil blend ranging from 250,000 to 8 ppm (starting at 25 v/v% downwards) were tested for PAM cell cytotoxicity, and high viability without cell death was maintained for 72 h at mitigant concentrations up to 1000 ppm [174]. Different oil dilutions were incubated with the virulent ASFV VNUA-ASFV-L01/HN/04/19 strain in aqueous solution before infecting PAM cells with the virus–mitigant mixture and dilutions down to 63 ppm natural oil blend were effective at fully preventing ASFV infection in vitro. The latter result was confirmed by PCR results.which demonstrated markedly lower ASFV nucleic acid levels compared to the virus-only positive control.

While other mitigants have been mainly explored for feed applications, this natural oil blend has been uniquely tested as a drinking water additive at 80 ppm concentration (equivalent to 80 mL/ton) to combat ASFV infection in pigs [141]. The experiments involved the virulent ASFV VNUA-ASFV-L01/HN/04/19 strain and it was found that the drinking water additive prevented lateral transmission from ASFV-infected pigs to cohoused, healthy pigs according to observed clinical symptoms and PCR diagnostics. On the other hand, in the untreated group that did not receive the drinking water additive, ASFV-infected pigs caused cohoused, healthy pigs to also become infected. Among ASFV-infected pigs in the treatment group, the drinking water additive did not prevent ASFV infection but did delay the progression of clinical symptoms.

In addition to natural oils, benzoic acid is a widely used antimicrobial in food applications and has been recently explored as an ASFV mitigant [142]. Various dilutions of pure benzoic acid or combined with a cocktail of natural flavoring agents (thymol, eugenol, piperine, and curcumin) were tested in terms of PAM cell viability and the antiviral properties of a non-toxic dilution were then further evaluated. Benzoic acid alone and in the mixture was effective at inhibiting the virulent ASFV China/GZ20180 strain in aqueous solution based on viral infectivity experiments. The inhibitory effect was further confirmed by reported reductions in ASFV nucleic acid levels. In feed mitigation experiments, both additives were effective at a 0.5% inclusion rate while benzoic acid combined with the natural flavoring agents was the most effective one in terms of speed and magnitude of mitigation. While further research is needed to understand why the inclusion of natural flavoring agents improves ASFV inhibition, similar effects have been reported for pig growth performance gains [175], and it has been discussed how benzoic acid mainly acts as an acidifier whereas the natural flavoring agents contain hydrophobic structural features that may aid membrane disruption [176].

Since ASFV is susceptible to highly acidic environments, a buffered formic acid solution with a pH around 2.6–3.2 has also been explored as a less hazardous alternative to formaldehyde in order to inhibit ASFV contamination of feed ingredients [143]. Different feed ingredients were mixed with a fixed volume of 1 or 2 v/v% formic acid solution and then spiked with the virulent ASFV Georgia 2007/01 strain. There was a dose-dependent effect of the formic acid feed additive on mitigating ASFV nucleic acid levels in maize according to PCR measurements and similar results were also obtained with compound feed and rice bran. However, the mitigant was not effective in all feed ingredients. There was no change in soybean meal and the ASFV nucleic acid levels were unexpectedly higher in meat/bone meal compared to the virus-only positive control. These findings were corroborated by viral titer quantification results obtained using PBMC cultures, and it was discussed how the additive was less effective when treating feed ingredients with higher protein and fat contents, which may be attributed to a greater intrinsic buffering capacity of these ingredients.

There have also been recent efforts to compare the virucidal efficacy of a powdered organic acid blend (OAB)—consisting of formic acid, lactic acid, calcium formate, calcium lactate, and citric acid with pH 2—with bioactive plant extracts from H. lupulus flowers (Phyto.A04, liquid form with pH 11) and from G. glabra licorice (Phyto.B, powder form with pH 5) to inhibit a virulent ASFV strain (VNUA/HY-ASF1/Vietnam/2019) in spiked feed [144]. PCR test results showed that 0.3% OAB, 1% Phyto.A04, and 0.1–0.5% Phyto. B were all effective at reducing viral nucleic acid levels and dual and triple combination blends of these feed additives had quicker effects. Viral infectivity analysis of the treated feed sample supernatants was performed on PAM cells in vitro and showed that Phyto.A04 and Phyto. B treatments completely inhibited infectious ASFV by day 3 while OAB treatment alone reduced viral infectivity more slowly. Among single treatments, Phyto.A04 was the most effective, while the triple combination treatments caused the largest reduction in viral infectivity after 1-day storage. Overall, the body of evidence from recent mitigation tests supports that physically disruptive mitigants demonstrate high performance based on the combination of observed antiviral efficacy and regulatory acceptable safety profiles worldwide.

To summarize, the three main classes of mitigation strategies covered herein, including thermal treatments, chemically reactive disinfectants, and physically disruptive mitigants, all demonstrate effective ASFV inactivation in different application contexts and can support integrated control and prevention strategies. For example, thermal treatments are typically one-time procedures to inactivate potential ASFV contaminants in feed, feed ingredients, and swill. These treatments can help to prevent transboundary spread of ASFV via feed and feed ingredients into ASF-free regions, while also ensuring safe use of swill and other food wastes in ASF-endemic regions and other areas with strict biosecurity controls to reduce transmission risk. As discussed above, regulatory guidance supports these uses and growing scientific evidence is providing well-defined protocols to facilitate effective decontamination with high confidence. On the other hand, chemically reactive disinfectants offer a more robust approach for routine decontamination of material surfaces around farm settings and can also help to prevent ASFV persistence in soil and animal carcasses. These disinfectants are widely recommended by regulatory agencies, while different disinfectants have particular advantages such as high efficacy, low cost, chemical stability, and safe handling depending on the context. They can be broadly applied in ASF-free and endemic regions to support routine biosecurity practices. Even so, it should be considered that disinfectants often exhibit reduced efficacy in the presence of organic matter and can pose cytotoxicity-related safety concerns that limit their use in direct animal exposure scenarios. Complementing these options, physically disruptive mitigants offer favorable regulatory profiles and can inhibit ASFV in complex biological matrices while also supporting pig health through immune enhancement and prevention of lateral transmission. Unlike thermal treatment and chemical disinfectant strategies, these mitigants can be integrated more readily into feed and drinking water, making them valuable for long-term biosecurity efforts. Hence, there is strong motivation to develop effective mitigation protocols based on these strategies. Towards this goal, recent advances in developing ASFV surrogate models for more widespread testing possibilities are helping to accelerate future innovation and support practical implementation.