Background
African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is a highly infectious hemorrhagic disease of domestic pigs and wild boars with mortality rates close to 100% [
1,
2]. ASF causes heavy losses to the pork industry of affected countries due to the lack of effective vaccines [
3,
4]. In 2018, ASF outbreaks were reported in China and neighboring countries that are collectively responsible for almost half of the world’s pork production, thereby presenting a global threat to the pork supply [
5]. This issue has led to strict biosecurity practices to prevent ASF spread as well as the search for natural compounds with antiviral properties that can mitigate ASFV in potential transmission vectors such as water and feed [
6‐
8]. In addition to these preventative measures, there is high interest in discovering natural compounds that might also be capable of curbing ASFV infection in pigs based on inhibiting viral genome replication along with reducing disease symptoms such as inflammation [
9].
Pigs infected with highly virulent ASFV strains display high fever, with temperatures up to 42 °C, lethargy, inactivity, and anorexia [
10]. The affected animals usually show cyanosis, nasal discharges, vomiting, and diarrhea. Animals die within 7 days from the onset of disease. Hemorrhagic splenomegaly and lymphadenitis are recorded during
post-mortem observations. Hemorrhagic lesions may also be observed with less frequency in other organs. While various pathogenic mechanisms have been proposed as primary contributors to hemorrhages, the current consensus identifies pro-inflammatory cytokine production as the initial cause of lesions in ASF [
11,
12]. Serum analysis of infected pigs revealed a significant elevation of pro-inflammatory cytokines, including TNF-α, IFN-α, IL-1β, IL-6, IL-8, and IL-12, which is commonly referred to as “cytokine storm” [
13]. The “cytokine storm” effect is common for several highly pathogenic viruses such as SARS-CoV-2, influenza virus, and Ebola virus, and can result in multiple organ failure and mortality [
14]. Thus, controlling pro-inflammatory cytokine production may prevent disease progression and reduce the mortality rate associated with ASF and other viral diseases.
Previously, the antiviral efficacy of some natural compounds possessing generally recognized anti-inflammatory properties was studied against ASFV. For instance, we have reported that the flavonoid apigenin significantly inhibited ASFV replication in immortalized Vero cells [
15]. Genkwanin, a natural derivative of apigenin, also exerted potent anti-ASFV activity in Vero cells and porcine macrophages (PAMs) [
16]. These results prompted us to consider the potential of other anti-inflammatory natural compounds as inhibitors of ASFV infection. Such compounds may have the potential to act as dual-purpose antiviral drugs, which are capable of not only inhibiting ASFV replication but also mitigating the “cytokine storm” associated with ASFV infection.
Herein, we applied a small library screening approach to evaluate the antiviral activity of 297 natural compounds with generally recognized anti-inflammatory properties. The screen was conducted using the laboratory-adapted, non-virulent ASFV BA71V strain [
17] and led us to identify five natural compounds that inhibited virus-induced cytopathic effect (CPE) by greater than 50%. Virus yield reduction experiments identified that two of these anti-inflammatory compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. The antiviral effects of tetrandrine and berbamine on mitigating virulent ASFV infection in PAMs were also confirmed. Notably, during ASFV infection in PAM cells, both compounds, especially tetrandrine, markedly reduced the production of pro-inflammatory cytokines such as TNF-α that plays a key role in ASF disease pathogenesis [
18].
Methods
Cells, viruses, and compounds
Eagle’s minimum essential medium (EMEM; Lonza, Switzerland) with 10% fetal bovine serum (Lonza, Switzerland), 2 mM L-glutamine (Lonza, Switzerland), 100 IU mL− 1 penicillin (Reyoung, China), and 100 µg mL− 1 streptomycin (Arterium, Ukraine) were used to grow Vero (African green monkey kidney) cells at 37 °C. The antiviral screening process and some antiviral assays were conducted by using the Vero-adapted ASFV BA71V strain as specified. We measured the titer of the Vero-adapted ASFV BA71V strain by cytopathic effect (CPE) assay on Vero cells. We used the Spearman-Kärber endpoint method to calculate the titer, which was expressed in log TCID50 mL− 1 units.
Experiments with the highly virulent ASFV Arm/07 isolate involved porcine alveolar macrophages (PAMs) that were prepared as previously reported [
19]. The porcine macrophages were maintained at 37 °C in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Germany) that was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU mL
− 1 penicillin, and 100 µg mL
− 1 streptomycin. Titration of the ASFV Arm/07 isolate was conducted using the hemadsorption (HAD) assay and expressed in log HADU
50 mL
− 1 units.
A library comprising 297 natural compounds (Additional file 1. Table
S1) was obtained from ChemFaces (China). The compounds were selected based on their known anti-inflammatory properties and undocumented efficacy against ASFV. Each compound was dissolved in dimethyl sulfoxide (DMSO) as a 5 mg mL
− 1 stock solution and diluted in EMEM. Dilutions in the cell culture medium were made to maintain a final DMSO concentration below 1% (v/v).
Library screening
Confluent Vero cells were seeded in 96-well cell culture plates at a density of 2 × 10
4 cells per well. The cells were subsequently infected with the ASFV BA71V strain (at 0.2 TCID
50 per cell) and immediately treated with test compounds at a concentration of 50 µM, except for some compounds that were tested at 25 µM due to cytotoxic effects (see Table
S1) for 72 h at 37 °C. Following the incubation period, once complete CPE (as indicated by cell rounding, detachment, and extensive destruction of cell monolayer) was observed in the untreated wells (Additional file 1. Fig.
S1), cell viability was assessed using the MTT assay. In brief, Vero cells were washed with cold PBS and MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide from Sigma-Aldrich, Germany] was added. The cells were then allowed to incubate at 37 °C for 2 h, followed by the extraction of purple formazan using the MTT test solvent (DMSO). The optical density (OD) was measured colorimetrically at 570 nm by using a microplate reader (BioTek Epoch 2, Agilent Technologies, USA). The percentage of CPE inhibition was calculated by the following formula: (OD
tv-OD
v/OD
c-OD
v) × 100% where OD refers to the optical density of each well,
tv refers to ASFV-infected cells treated with compound,
c refers to mock-infected cells without treatment, and
v refers to ASFV-infected cells without treatment.
ASFV-infected cells treated with a minimal concentration of DMSO (< 0.5%) were utilized as the negative control, while ASFV-infected cells treated with 50 µM of apigenin were used as the positive control. Each plate contained six samples of both negative and positive controls. The OD values obtained from these controls were used to calculate the Z’-factor, which is a statistical parameter to assess the variability of the screening assay [
20]. The Z’-factor was calculated by using the following formula: 1-[(3σ
p + 3σ
n)/(µ
p-µ
n)], where σ refers to the standard deviation, µ refers to the mean,
p refers to the positive control, and
n refers to the negative control. If the Z’-factor value is between 0.5 and 1.0, then the screening assay performance is considered consistent and robust.
Compound validation assays
For the yield reduction assay, Vero cells were cultivated in 24-well cell culture plates with a seeding density of 2 × 105 cells per well. The cells were subsequently exposed to the ASFV BA71V strain (at 0.2 TCID50 per cell) and treated with selected compounds at concentrations of 50 µM or 25 µM (for tetrandrine). Following a 72-h incubation period, the supernatant was harvested and titrated.
For the virucidal assay, a virus suspension containing 2 × 105 TCID50 ASFV particles was incubated with selected compounds at indicated concentrations for 1 h at room temperature. Then, Vero cells seeded in 96-well cell culture plates (seeding density: 2 × 104 cells per well) were exposed to 20-fold dilutions of the treated viral suspension to eliminate potential virostatic effects of the test compounds on ASFV infection. After a 24-h post-infection, the supernatant was harvested and subjected to titration.
Dose-dependent assay
Vero cells (2 × 105 cells per well) or PAMs (4 × 104 cells per well) cultured in 24-well plates were subjected to infection with the ASFV BA71V strain [multiplicity of infection (MOI) of 0.2 TCID50] or ASFV Arm/07 isolate (MOI of 0.5 HADU50), respectively. Following infection, cells were treated with selected compounds at different concentrations in a two-fold dilution format. The infection was allowed to progress for 24 h, after which the supernatant was harvested and titrated.
MOI-dependent assay
Vero cells cultured in 24-well plates (2 × 105 cells per well) were infected with the ASFV BA71V strain at different MOI values ranging from 0.1 to 1 TCID50 per cell and treated with selected compounds at indicated concentrations. The virus was collected and titrated at 24 h post-infection.
Virus replication stage and treatment assays
For the ASFV replication cycle experiments, Vero cells (2 × 105 cells per well) cultured in 24-well plates were infected with the ASFV BA71V strain (MOI of 0.2 TCID50) and then treated with selected compounds at indicated concentrations. The infection was allowed to progress for 24, 48, or 72 h, after which the supernatants were harvested and titrated.
For time-of-addition experiments, Vero cells (2 × 105 cells per well) or PAM cells (4 × 104 cells per well) were grown in 24-well cell culture plates. The wells were marked as − 2, 0, 2, 8, and 16 h, corresponding to the time period relative to the onset of ASFV infection (inoculation). These time points cover the early, middle, and late stages of ASFV infection during one replication cycle. In the pre-infection format, Vero cells or macrophages were exposed to the selected compounds before (− 2 h) infection with the ASFV BA71V strain (0.2 TCID50 per cell) or ASFV Arm/07 isolate (0.5 HADU50 per cell). In the co-infection format, Vero cells were simultaneously exposed to compounds and ASFV. In the post-infection format, cells were infected with ASFV and the compounds were then added at 2, 8, or 16 h post-infection. The supernatants were collected at 24 h post-infection and titrated.
For time-of-removal experiments, Vero cells grown in 24-well cell culture plates were infected with the ASFV BA71V strain and treated with compounds at indicated concentrations. Afterwards, the compound was removed after incubation at 2-, 4-, 8-, 12-, or 16-h post-treatment. This removal step was done by washing the cells with 1× PBS and replacing the media. The supernatant was then collected and titrated 24 h post-infection.
Virus entry assays
In the virus attachment experiments, Vero cells (2 × 105 cells per well) or macrophages (4 × 104 cells per well) were grown in 24-well cell culture plates. The cells were incubated with the ASFV BA71V strain (0.2 TCID50) or ASFV Arm/07 isolate (0.5 HADU50), respectively, along with the test compounds at 4°C for 1 h. This step was done to facilitate virus binding while preventing internalization. Following this procedure, the unbound virus and compound were removed by thoroughly washing the cells with 1× PBS. Fresh medium containing 3% FBS was then added. Virus in the supernatant was collected and titrated 24 h post-infection.
In the internalization experiments, Vero cells or macrophages were infected with ASFV BA71V or ASFV Arm/07, respectively, at 4 °C for 1 h. Following this step, unbound virus was removed by thoroughly washing the cells with 1× PBS. The compounds were then added to the cells and incubated at 37 °C, allowing virus entry to proceed for 1 h. Subsequently, the compounds were removed by washing with 1× PBS to prevent their effect on later stages of infection. The infection was then allowed to continue for 24 h, after which the supernatants were collected and titrated.
Cytotoxicity assay
The cytotoxicity of natural compounds was examined in Vero cells and PAMs by using the crystal violet staining method [
21]. Cells (2 × 10
4 cells per well) reached confluence and were then exposed to increasing compound concentrations, which ranged from 3.1 µM to 50 µM. The cells were incubated for 72 h at 37 °C in 5% CO
2. Following incubation, the cell culture medium was discarded and a 4% crystal violet solution (in ethanol) was added to the wells. The solution was allowed to incubate for 40 min at room temperature. Then, the wells were washed with distilled water, and 200 µL of methanol was added to each well for 20 min. The OD of each well was measured at 570 nm using a plate reader (BioTek Epoch 2, Agilent Technologies, USA). The relative viability of the treated cells compared to mock-treated cells (no compound) was expressed as a percentage and calculated at each concentration using the following formula: OD
t/OD
c × 100%, where OD
t and OD
c correspond to the absorbance of treated and negative control (mock-treated) cells, respectively.
Western blotting
Vero cells (5 × 105 cells per well) were grown in 6-well cell culture plates and infected with ASFV at a MOI of 2 TCID50 in the presence or absence of test compounds at two different compound concentrations. After 24 h, the cells were dissociated using Laemmli buffer and heated for 5 min at 95 °C, electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and transferred onto nitrocellulose membranes (GE Healthcare). The membranes were incubated with mouse monoclonal antibodies p30, p72, or tubulin (Sigma Aldrich) at dilutions of 1:1000, 1:1000, and 1:2000, respectively. Antibodies were detected using horseradish peroxidase (HRP) conjugated secondary antibodies. Bands were visualized by using a Chemidoc XRS imaging system from BioRad.
Cytokine quantification
The release of TNF-α, IL-1β, and IL-6 cytokines into the culture medium was quantified using commercially available ELISA kits (R&D Systems, USA). Experiments were performed according to the instructions provided by the manufacturer. For each cytokine, a standard curve was plotted, which represented the cytokine concentration as a function of the OD value. The standard curve was used to derive a linear regression equation, which was then utilized to determine the concentration of each cytokine released.
Statistical analysis
All experiments were conducted in triplicate. Data are expressed as mean ± standard deviation (s.d.) of three independent experiments. The unpaired Student’s t-test (versus virus-only positive control) and one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (versus other test groups) were used for antiviral and anti-inflammatory experiments, respectively. Statistical significance was computed in terms of standard or multiplicity-adjusted P values as appropriate, and P < 0.05, P < 0.01, and P < 0.001 indicate the levels of statistical significance (*, **, ***) while ns indicates a non-significant effect (P > 0.05). The GraphPad Prism software program (version 10.0.2, GraphPad Software, USA) was used to calculate 50% inhibitory concentration (IC50) values by variable slope analysis (four-parameter logistic curve) of absolute virus quantities with appropriate units as a function of compound concentration. The IC50 values correspond to the compound concentrations needed to reduce viral infectivity by 50%.
Discussion
ASFV remains a significant threat to the global swine industry and is characterized by its devastating impact on pig populations and the absence of approved vaccines or specific antiviral therapies [
33]. The absence of preventive measures against ASFV highlights the critical need to discover effective interventions to control and manage this highly contagious disease in pigs. Within this context, the exploration of natural compounds with potential antiviral properties has gained immense importance and highlights the opportunity to identify novel inhibitory strategies to combat ASFV infection [
9].
In this work, the screening of a library of natural compounds with anti-inflammatory properties led to the identification of five promising candidates—paeonol, pinoresinol, curcumin, berbamine, and tetrandrine—that showed substantial inhibition of ASFV-induced CPE without affecting cell monolayers. These compounds belong to distinct classes, with berbamine and tetrandrine exhibiting more complex chemical structures compared to the other lead candidates. Subsequent validation experiments in Vero cells confirmed the antiviral efficacy of these compounds against ASFV replication. Tetrandrine displayed the most potent effect, achieving a 3.6 log reduction in viral titer, while paeonol and pinoresinol exhibited weaker inhibitory effects. Notably, none of these five compounds demonstrated virucidal activity or cytotoxic effects at tested concentrations, confirming the specificity of their antiviral activities. Recently, Zhu et al. and Qian et al. independently reported the antiviral activity of berbamine and tetrandrine against ASFV in vitro [
34,
35], thereby additionally validating the robustness of our screening results.
In the present context, further mechanistic investigations focused on berbamine and tetrandrine revealed their dose-dependent antiviral activities, inhibiting the synthesis of ASFV proteins and consistently reducing viral yields across different MOIs. Time-of-addition experiments elucidated the compounds’ action points within the viral replication cycle, highlighting their significant impact during pre- and co-treatment phases. Berbamine and tetrandrine significantly inhibited ASFV replication by interfering with early infection stages, particularly virus attachment and internalization into host cells, that in turn disrupted viral entry. Interestingly, both compounds exhibited sustained antiviral effects over multiple replication cycles (cf. Figure
4A). This finding fits with the relatively long half-lives that have been reported for both compounds [
36], which supports their capability to impact ASFV replication over several cycles. Given that both compounds also exhibited antiviral activity during early stages of viral infection post-internalization in the present experiments (cf. Figure
4C), it is reasonable that the most significant antiviral effect was observed during the prolonged presence of compounds in the cell culture medium (cf. Figure
4B) and the results are consistent with their effects on virus replication at various stages, especially viral entry.
Furthermore, extending the study to primary target cells, porcine macrophages, revealed consistent antiviral effects of berbamine and tetrandrine. Notably, these compounds inhibited ASFV infection by disrupting viral entry mechanisms, suggesting that their antiviral mechanism is irrespective of host cell type or virus strain. It has been reported that tetrandrine inhibits Ebola virus entry into human monocyte-derived macrophages by disrupting endosomal Ca
2+ channels [
37]. In addition, berbamine suppresses the replication of Japanese encephalitis virus by blocking Ca
2+ permeable non-selective cation channels in endosomes [
38]. Therefore, it is reasonable to hypothesize that comparable inhibitory mechanisms might play a role in exerting antiviral effects against ASFV. Conducting further experiments could potentially unveil the involvement of endosomal Ca
2+ channels in ASFV infection in future work.
Importantly, we also demonstrated that berbamine and tetrandrine effectively reduced the production of pro-inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in ASFV-infected macrophages, which had not been explored before. Among these cytokines, TNF-α is considered to play an essential role in driving pathological changes observed in ASFV-infected pigs [
13,
39]. Therefore, compounds such as berbamine and tetrandrine can not only suppress viral replication but also demonstrate the capability to mitigate pathological conditions associated with the heightened production of pro-inflammatory cytokines due to viral infection. Of note, tetrandrine exhibited a particularly high and sustained level of anti-inflammatory activity in the ASFV infection context and, together with its high antiviral activity, supports that it has promising merits.
These findings are particularly significant because berbamine and tetrandrine, along with other bis-benzylisoquinoline alkaloids, have also been recently reported to inhibit porcine epidemic diarrhea virus (PEDV) with similar potency [
40,
41], suggesting their broad utility as porcine virus countermeasures. At the same time, previous studies have focused on validating the antiviral activity of these compounds and did not evaluate potential infection-related anti-inflammatory effects. Our findings expand on this viewpoint and demonstrate proof-of-principle to show that the anti-inflammatory properties of berbamine and tetrandrine are translatable to inhibiting production of pro-inflammatory cytokines during viral infection along with reducing viral load.
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