Antiviral screening of natural, anti-inflammatory compound library against African swine fever virus.

BACKGROUND: African swine fever virus (ASFV) is a major threat to pig production and the lack of effective vaccines underscores the need to develop robust antiviral countermeasures. Pathologically, a significant elevation in pro-inflammatory cytokine production is associated with ASFV infection in pigs and there is high interest in identifying dual-acting natural compounds that exhibit antiviral and anti-inflammatory activities. METHODS: Using the laboratory-adapted ASFV BA71V strain, we screened a library of 297 natural, anti-inflammatory compounds to identify promising candidates that protected Vero cells against virus-induced cytopathic effect (CPE). Virus yield reduction, virucidal, and cell cytotoxicity experiments were performed on positive hits and two lead compounds were further characterized in dose-dependent assays along with time-of-addition, time-of-removal, virus entry, and viral protein synthesis assays. The antiviral effects of the two lead compounds on mitigating virulent ASFV infection in porcine macrophages (PAMs) were also tested using similar methods, and the ability to inhibit pro-inflammatory cytokine production during virulent ASFV infection was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The screen identified five compounds that inhibited ASFV-induced CPE by greater than 50% and virus yield reduction experiments showed that two of these compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. Mechanistic analysis confirmed that both compounds potently inhibited early stages of ASFV infection and that the compounds also inhibited infection of PAMs by the virulent ASFV Arm/07 isolate. Importantly, during ASFV infection in PAM cells, both compounds markedly reduced the production of pro-inflammatory cytokines involved in disease pathogenesis while tetrandrine had a greater and more sustained anti-inflammatory effect than berbamine. CONCLUSIONS: Together, these findings support that dual-acting natural compounds with antiviral and anti-inflammatory properties hold promise as preventative and therapeutic agents to combat ASFV infection by simultaneously inhibiting viral replication and reducing virus-induced cytokine production.

Glycerol Monolaurate Inhibits Wild-Type African Swine Fever Virus Infection in Porcine Macrophages.

Naturally abundant antimicrobial lipids, such as fatty acids and monoglycerides, that disrupt membrane-enveloped viruses are promising mitigants to inhibit African swine fever virus (ASFV). Among mitigant candidates in this class, glycerol monolaurate (GML) has demonstrated particularly high antiviral activity against laboratory-adapted ASFV strains. However, there is an outstanding need to further determine the effects of GML on wild-type ASFV strains, which can have different virulence levels and sensitivities to membrane-disrupting compounds as compared to laboratory-adapted strains. Herein, we investigated the antiviral effects of GML on a highly virulent strain of a wild-type ASFV isolate (Armenia/07) in an in vitro porcine macrophage model. GML treatment caused a concentration-dependent reduction in viral infectivity, and there was a sharp transition between inactive and active GML concentrations. Low GML concentrations had negligible effect on viral infectivity, whereas sufficiently high GML concentrations caused a >99% decrease in viral infectivity. The concentration onset of antiviral activity matched the critical micelle concentration (CMC) value of GML, reinforcing that GML micelles play a critical role in enabling anti-ASFV activity. These findings validate that GML can potently inhibit wild-type ASFV infection of porcine macrophages and support a biophysical explanation to guide antimicrobial lipid performance optimization for pathogen mitigation applications.

Flavonoid Library Screening Reveals Kaempferol as a Potential Antiviral Agent Against African Swine Fever Virus.

Naturally occurring plant flavonoids are a promising class of antiviral agents to inhibit African swine fever virus (ASFV), which causes highly fatal disease in pigs and is a major threat to the swine industry. Currently known flavonoids with anti-ASFV activity demonstrate a wide range of antiviral mechanisms, which motivates exploration of new antiviral candidates within this class. The objective of this study was to determine whether other flavonoids may significantly inhibit ASFV infection in vitro. We performed a cell-based library screen of 90 flavonoids. Our screening method allowed us to track the development of virus-induced cytopathic effect by MTT in the presence of tested flavonoids. This screening method was shown to be robust for hit identification, with an average Z-factor of 0.683. We identified nine compounds that inhibit ASFV Ba71V strain in Vero cells. Among them, kaempferol was the most potent and exhibited dose-dependent inhibition, which occurred through a virostatic effect. Time-of-addition studies revealed that kaempferol acts on the entry and post-entry stages of the ASFV replication cycle and impairs viral protein and DNA synthesis. It was further identified that kaempferol induces autophagy in ASFV-infected Vero cells, which is related to its antiviral activity and could be partially abrogated by the addition of an autophagy inhibitor. Kaempferol also exhibited dose-dependent inhibition of a highly virulent ASFV Arm/07 isolate in porcine macrophages. Together, these findings support that kaempferol is a promising anti-ASFV agent and has a distinct antiviral mechanism compared to other anti-ASFV flavonoids.

Inhibition of African swine fever virus in liquid and feed by medium-chain fatty acids and glycerol monolaurate.

BACKGROUND: The ongoing African swine fever virus (ASFv) epidemic has had a major impact on pig production globally and biosecurity efforts to curb ASFv infectivity and transmission are a high priority. It has been recently identified that feed and feed ingredients, along with drinking water, can serve as transmission vehicles and might facilitate transboundary spread of ASFv. Thus, it is important to test the antiviral activity of regulatory compatible, antiviral feed additives that might inhibit ASFv infectivity in feed. One promising group of feed additive candidates includes medium-chain fatty acids (MCFA) and monoglyceride derivatives, which are known to disrupt the lipid membrane surrounding certain enveloped viruses and bacteria. RESULTS: The antiviral activities of selected MCFA, namely caprylic, capric, and lauric acids, and a related monoglyceride, glycerol monolaurate (GML), to inhibit ASFv in liquid and feed conditions were investigated and suitable compounds and inclusion rates were identified that might be useful for mitigating ASFv in feed environments. Antiviral assays showed that all tested MCFA and GML inhibit ASFv. GML was more potent than MCFA because it worked at a lower concentration and inhibited ASFv due to direct virucidal activity along with one or more other antiviral mechanisms. Dose-dependent feed experiments further showed that sufficiently high GML doses can significantly reduce ASFv infectivity in feed in a linear manner in periods as short as 30 min, as determined by infectious viral titer measurements. Enzyme-linked immunosorbent assay (ELISA) experiments revealed that GML treatment also hinders antibody recognition of the membrane-associated ASFv p72 structural protein, which likely relates to protein conformational changes arising from viral membrane disruption. CONCLUSION: Together, the findings in this study indicate that MCFA and GML inhibit ASFv in liquid conditions and that GML is also able to reduce ASFv infectivity in feed, which may help to curb disease transmission.

Medium-chain fatty acids and monoglycerides as feed additives for pig production: towards gut health improvement and feed pathogen mitigation.

Ongoing challenges in the swine industry, such as reduced access to antibiotics and virus outbreaks (e.g., porcine epidemic diarrhea virus, African swine fever virus), have prompted calls for innovative feed additives to support pig production. Medium-chain fatty acids (MCFAs) and monoglycerides have emerged as a potential option due to key molecular features and versatile functions, including inhibitory activity against viral and bacterial pathogens. In this review, we summarize recent studies examining the potential of MCFAs and monoglycerides as feed additives to improve pig gut health and to mitigate feed pathogens. The molecular properties and biological functions of MCFAs and monoglycerides are first introduced along with an overview of intervention needs at different stages of pig production. The latest progress in testing MCFAs and monoglycerides as feed additives in pig diets is then presented, and their effects on a wide range of production issues, such as growth performance, pathogenic infections, and gut health, are covered. The utilization of MCFAs and monoglycerides together with other feed additives such as organic acids and probiotics is also described, along with advances in molecular encapsulation and delivery strategies. Finally, we discuss how MCFAs and monoglycerides demonstrate potential for feed pathogen mitigation to curb disease transmission. Looking forward, we envision that MCFAs and monoglycerides may become an important class of feed additives in pig production for gut health improvement and feed pathogen mitigation.