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.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Deoxycholic acid inhibits ASFV replication by inhibiting MAPK signaling pathway.

African swine fever (ASF) is an acute, febrile, highly contagious infection of pigs caused by the African swine fever virus (ASFV). The purpose of this study is to understand the molecular mechanism of ASFV infection and evaluate the effect of DCA on MAPK pathway, so as to provide scientific basis for the development of new antiviral drugs. The transcriptome analysis found that ASFV infection up-regulated the IL-17 and MAPK signaling pathways to facilitate viral replication. Metabolome analysis showed that DCA levels were up-regulated after ASFV infection, and that exogenous DCA could inhibit activation of the MAPK pathway by ASFV infection and thus inhibit viral replication. Dual-luciferase reporter assays were used to screen the genes of ASFV and revealed that I73R could significantly up-regulate the transcription level of AP-1 transcription factor in the MAPK pathway. Confocal microscopy demonstrated that I73R could promote AP-1 entry into the nucleus, and that DCA could inhibit the I73R-mediated nuclear entry of AP-1, inhibiting MAPK pathway, and I73R interacts with AP-1. These results indicated that DCA can inhibit ASFV-mediated activation of the MAPK pathway, thus inhibiting ASFV replication. This study provides a theoretical basis for research on ASF pathogenesis and for antiviral drug development.

Tetrandrine (TET) inhibits African swine fever virus entry into cells by blocking the PI3K/Akt pathway.

African Swine Fever Virus (ASFV) infection causes an acute and highly contagious disease in swine, resulting in significant economic losses and societal harm worldwide. Currently, there are no effective vaccines or antiviral drugs available for ASFV. Tetrandrine (TET) is extracted from the traditional Chinese herb Stephania tetrandrae, possesses diverse biological functions such as anti-inflammatory, anti-tumor, and antiviral activities. The study comprehensively evaluated the anti-ASFV effect of TET and validated it through biological assays. The dose-dependent inhibition of TET against ASFV was confirmed and a novel mechanism of TET's anti-ASFV activity was elucidated. TET effectively inhibits ASFV during internalization by blocking macropinocytosis through the inhibition of the PI3K/Akt pathway. The specific inhibitor LY294002, targeting the PI3K/Akt pathway, exhibits similar antiviral activity against ASFV as TET. Furthermore, the inhibitory effect of TET against other viruses such as Lumpy Skin Disease Virus (LSDV) and Porcine Epidemic Diarrhea Virus (PEDV) was also identified. Our findings suggest that TET effectively inhibits ASFV and reveal the potential for broad-spectrum antiviral drugs targeting the PI3K/Akt pathway.

Structural Analysis, Multi-Conformation Virtual Screening and Molecular Simulation to Identify Potential Inhibitors Targeting pS273R Proteases of African Swine Fever Virus.

The African Swine Fever virus (ASFV) causes an infectious viral disease in pigs of all ages. The development of antiviral drugs primarily aimed at inhibition of proteases required for the proteolysis of viral polyproteins. In this study, the conformation of the pS273R protease in physiological states were investigated, virtually screened the multi-protein conformation of pS273R target proteins, combined various molecular docking scoring functions, and identified five potential drugs from the Food and Drug Administration drug library that may inhibit pS273R. Subsequent validation of the dynamic interactions of pS273R with the five putative inhibitors was achieved using molecular dynamics simulations and binding free energy calculations using the molecular mechanics/Poison-Boltzmann (Generalized Born) (MM/PB(GB)SA) surface area. These findings demonstrate that the arm domain and Thr159-Lys167 loop region of pS273R are significantly more flexible compared to the core structural domain, and the Thr159-Lys167 loop region can serve as a "gatekeeper" in the substrate channel. Leucovorin, Carboprost, Protirelin, Flavin Mononucleotide, and Lovastatin Acid all have Gibbs binding free energies with pS273R that were less than -20 Kcal/mol according to the MM/PBSA analyses. In contrast to pS273R in the free energy landscape, the inhibitor and drug complexes of pS273R showed distinct structural group distributions. These five drugs may be used as potential inhibitors of pS273R and may serve as future drug candidates for treating ASFV.

Structural Insight into Molecular Inhibitory Mechanism of InsP6 on African Swine Fever Virus mRNA-Decapping Enzyme g5Rp.

Removal of 5' cap on cellular mRNAs by the African swine fever virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate-linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and the hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer is composed of a unique N-terminal helical domain and a C-terminal classic Nudix domain. As g5Rp is an mRNA-decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp which are important to RNA binding and decapping enzyme activity. Furthermore, the g5Rp-mediated mRNA decapping was inhibited by InsP6. The g5Rp-InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp. IMPORTANCE ASF is a highly contagious hemorrhagic viral disease in domestic pigs which causes high mortality. Currently, there are still no effective vaccines or specific drugs available against this particular virus. The protein g5Rp is the only viral mRNA-decapping enzyme, playing an essential role in the machinery assembly of mRNA regulation and translation initiation. In this study, we solved the crystal structures of g5Rp dimer and complex with InsP6. Structure-based mutagenesis studies revealed critical residues involved in a candidate RNA binding region, which also play pivotal roles in complex with InsP6. Notably, InsP6 can inhibit g5Rp activity by competitively blocking the binding of substrate mRNA to the enzyme. Our structure-function studies provide the basis for potential anti-ASFV inhibitor designs targeting the critical enzyme.

Effects of the NF-κB Signaling Pathway Inhibitor BAY11-7082 in the Replication of ASFV.

African swine fever virus (ASFV) mainly infects the monocyte/macrophage lineage of pigs and regulates the production of cytokines that influence host immune responses. Several studies have reported changes in cytokine production after infection with ASFV, but the regulatory mechanisms have not yet been elucidated. Therefore, the aim of this study was to examine the immune response mechanism of ASFV using transcriptomic and proteomic analyses. Through multi-omics joint analysis, it was found that ASFV infection regulates the expression of the host NF-B signal pathway and related cytokines. Additionally, changes in the NF-κB signaling pathway and IL-1ß and IL-8 expression in porcine alveolar macrophages (PAMs) infected with ASFV were examined. Results show that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1ß and IL-8. The NF-κB inhibitor BAY11-7082 inhibited the expression profiles of phospho-NF-κB p65, p-IκB, and MyD88 proteins, and inhibited ASFV-induced NF-κB signaling pathway activation. Additionally, the results show that the NF-κB inhibitor BAY11-7082 can inhibit the replication of ASFV and can inhibit IL-1ß and, IL-8 expression. Overall, the findings of this study indicate that ASFV infection activates the NF-κB signaling pathway and up-regulates the expression of IL-1ß and IL-8, and inhibits the replication of ASFV by inhibiting the NF-κB signaling pathway and interleukin-1 beta and interleukin-8 production. These findings not only provide new insights into the molecular mechanism of the association between the NF-κB signaling pathway and ASFV infection, but also indicate that the NF-κB signaling pathway is a potential immunomodulatory pathway that controls ASF.

Identification of African Swine Fever Virus Inhibitors through High Performance Virtual Screening Using Machine Learning.

African swine fever virus (ASFV) is a highly contagious virus that causes severe hemorrhagic viral disease resulting in high mortality in domestic and wild pigs, until few antiviral agents can inhibit ASFV infections. Thus, new anti-ASFV drugs need to be urgently identified. Recently, we identified pentagastrin as a potential antiviral drug against ASFVs using molecular docking and machine learning models. However, the scoring functions are easily influenced by properties of protein pockets, resulting in a scoring bias. Here, we employed the 5'-P binding pocket of AsfvPolX as a potential binding site to identify antiviral drugs and classified 13 AsfvPolX structures into three classes based on pocket parameters calculated by the SiteMap module. We then applied principal component analysis to eliminate this scoring bias, which was effective in making the SP Glide score more balanced between 13 AsfvPolX structures in the dataset. As a result, we identified cangrelor and fostamatinib as potential antiviral drugs against ASFVs. Furthermore, the classification of the pocket properties of AsfvPolX protein can provide an alternative approach to identify novel antiviral drugs by optimizing the scoring function of the docking programs. Here, we report a machine learning-based novel approach to generate high binding affinity compounds that are individually matched to the available classification of the pocket properties of AsfvPolX protein.

The Efficacy of Disinfection on Modified Vaccinia Ankara and African Swine Fever Virus in Various Forest Soil Types.

African swine fever (ASF) has become a global threat to the pig industry and wild suids. Within Europe, including Germany, affected wild boar populations play a major role. Fencing and carcass removal in combination with the reduction in environmental contamination are key to control further spread. The handling of the ASF virus (ASFV) is restricted to high-containment conditions in Germany. According to the regulation of the German Veterinarian Society (DVG), modified vaccinia Ankara virus (MVAV) is the virus of choice to determine the efficacy of disinfection for enveloped viruses. The aim of this study was to use the MVAV as a guide to select the best possible disinfectant solution and concentration for the inactivation of ASFV in soil. Both viruses were tested simultaneously. In this study, two layers (top and mineral soil) of soil types from six different locations in Saxony, Germany, were collected. The tenacity of ASFV and MVAV were tested at various time points (0.5 to 72 h). The capabilities of different concentrations of peracetic acid and citric acid (approx. 0.1 to 2%) to inactivate the viruses in the selected soil types with spiked high protein load were examined under appropriate containment conditions. Around 2-3 Log10 (TCID50) levels of reduction in the infectivity of both ASFV and MVAV were observed in all soil types starting after two hours. For MVAV, a 4 Log10 loss was recorded after 72 h. A total of 0.1% of peracetic acid (5 L/m2) was sufficient to inactivate the viruses. A 4 log10 reduction in the infectivity of MVAV was noticed by applying 1% citric acid, while a 2 log10 decline was recorded with ASFV. In conclusion, comparing MVAV to ASFV for efficacy screening of disinfectant solutions has revealed many similarities. Peracetic acid reduced the infectivity of both viruses independently of the soil type and the existence of a high organic soiling.

Efficient inactivation of African swine fever virus by a highly complexed iodine.

African swine fever (ASF) is a highly lethal contagious disease of swine caused by African swine fever virus (ASFV). Cleaning and disinfection remain one of the most effective tools to prevent the ASFV spread in pig holdings. This study evaluated the inactivation effect of a highly complexed iodine (HPCI) disinfectant against ASFV. A commercially available povidone-iodine (PVP-I) was used as reference for comparison. The results showed that 5% HPCI and 5% PVP-I did not exhibit cytotoxicity in primary porcine alveolar macrophages, and 107.0 and 105.0 TCID50/mL ASFV were completely inactivated by 5% and 0.25% HPCI, respectively, in 5 min via either immersion or spray disinfection. However, 5% PVP-I required at least 15 min to completely inactivate 107.0 TCID50/mL ASFV, whereas 0.25% PVP-I failed to completely inactivate 105.0 TCID50/mL ASFV. This study demonstrated that HPCI could rapidly and efficiently inactivate ASFV, representing an effective disinfectant for ASF control.

Disinfection to control African swine fever virus: a UK perspective.

A review of African swine fever (ASF) was conducted, including manifestations of disease, its transmission and environmental persistence of ASF virus. Findings on infectious doses of contemporary highly-pathogenic strains isolated from outbreaks in Eastern Europe were included. Published data on disinfectant susceptibility of ASF virus were then compared with similar findings for selected other infectious agents, principally those used in the UK disinfectant approvals tests relating to relevant Disease Orders for the control of notifiable and zoonotic diseases of livestock. These are: swine vesicular disease virus, foot and mouth disease virus, Newcastle disease virus and Salmonella enterica serovar Enteritidis. The comparative data thus obtained, presented in a series of charts, facilitated estimates of efficacy against ASF virus for some UK approved disinfectants when applied at their respective General Orders concentrations. Substantial data gaps were encountered for several disinfectant agents or classes, including peracetic acid, quaternary ammonium compounds and products based on phenols and cresols.

Prediction of African Swine Fever Virus Inhibitors by Molecular Docking-Driven Machine Learning Models.

African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and k-means clustering in molecular docking studies of FDA-approved drugs. These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.

GS-441524 inhibits African swine fever virus infection in vitro.

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes serious socio-economic consequences in endemic countries for which no safe and effective vaccine is currently available. GS-441524, a 1-cyano-substituted adenine C-nucleoside ribose analogue, inhibits viral RNA transcription by competing with natural nucleosides (ATP, TTP, CTP, and GTP) and effectively inhibits viral RNA-dependent RNA polymerase activity. However, whether GS-441524 can inhibit the replication of DNA viruses is unknown. In this study, we confirmed that GS-441524 inhibits ASFV infection in porcine alveolar macrophages (PAMs) in a dose-dependent manner; GS-441524 significantly inhibited ASFV replication at different time points after ASFV infection, particularly at the early stages of viral replication. Notably, GS-441524 did not increase the levels of antiviral cytokines or ATP in PAMs. However, an increase in the concentration of natural ATP in PAMs promoted the replication of ASFV and attenuated the inhibitory effect of GS-441524 in a dose-dependent manner. Our results suggest that GS-441524 is an effective antiviral against ASFV.

A new microtubule-stabilizing agent shows potent antiviral effects against African swine fever virus with no cytotoxicity.

African swine fever virus (ASFV) is the causal agent of a fatal disease of domestic swine for which no effective antiviral drugs are available. Recently, it has been shown that microtubule-targeting agents hamper the infection cycle of different viruses. In this study, we conducted in silico screening against the colchicine binding site (CBS) of tubulin and found three new compounds with anti-ASFV activity. The most promising antiviral compound (6b) reduced ASFV replication in a dose-dependent manner (IC50 = 19.5 µM) with no cellular (CC50 > 500 µM) and animal toxicity (up to 100 mg/kg). Results also revealed that compound 6b interfered with ASFV attachment, internalization and egress, with time-of-addition assays, showing that compound 6b has higher antiviral effects when added within 2-8 h post-infection. This compound significantly inhibited viral DNA replication and disrupted viral protein synthesis. Experiments with ASFV-infected porcine macrophages disclosed that antiviral effects of the compound 6b were similar to its effects in Vero cells. Tubulin polymerization assay and confocal microscopy demonstrated that compound 6b promoted tubulin polymerization, acting as a microtubule-stabilizing, rather than a destabilizing agent in cells. In conclusion, this work emphasizes the idea that microtubules can be targets for drug development against ASFV.

Mitigating the risk of African swine fever virus in feed with anti-viral chemical additives.

African swine fever (ASF) is currently considered the most significant global threat to pork production worldwide. Disease caused by the ASF virus (ASFV) results in high case fatality of pigs. Importantly, ASF is a trade-limiting disease with substantial implications on both global pork and agricultural feed commodities. ASFV is transmissible through natural consumption of contaminated swine feed and is broadly stable across a wide range of commonly imported feed ingredients and conditions. The objective of the current study was to investigate the efficacy of medium-chain fatty acid and formaldehyde-based feed additives in inactivating ASFV. Feed additives were tested in cell culture and in feed ingredients under a transoceanic shipment model. Both chemical additives reduced ASFV infectivity in a dose-dependent manner. This study provides evidence that chemical feed additives may potentially serve as mitigants for reducing the risk of ASFV introduction and transmission through feed.

Antiviral drugs targeting endosomal membrane proteins inhibit distant animal and human pathogenic viruses.

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds.

Virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus and avian influenza virus.

This study evaluated the virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus (ASFV) and avian influenza virus (AIV), according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. AEW (pH 5.0-6.5) was prepared using a commercially available "Electrolyzed Water Generator" with a free chlorine concentration (FCC) of 5-140 ppm, and its efficiency in reducing the titer of ASFV and AIV was tested in a suspension under low- and high-level organic soiling. Under low-level organic soiling conditions, AEW with FCC ≥40 ppm was effective against ASFV; under high-level organic soiling conditions, AEW with FCC ≥80 ppm was effective against ASFV. Under low-level organic soiling conditions, AEW with FCC ≥60 ppm was effective against AIV; under high-level organic soiling conditions, AEW with FCC ≥100 ppm was effective against AIV. The virucidal effect of AEW seemed dependent on the FCC and the presence of organic soiling. Based on these data, we recommend the following minimum FCCs in AEW treatment for routine disinfection in veterinary field under low- and high-level organic soiling conditions: for ASFV, 50 ppm and 100 ppm; and for AIV, 75 ppm and 125 ppm, respectively. In conclusion, the virucidal effects of AEW against ASFV and AIV emphasize its potential utility as a disinfectant, and we suggest considering organic soiling conditions while using AEW for implementing effective control measures for field applications.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal ß-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 µM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

Efficient inactivation of African swine fever virus by ozonized water.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating disease of domestic pigs and wild boar, causing significant economic losses to the pig industry worldwide. To evaluate the ability of ozonized water as a disinfectant to inactivate ASFV, ozonized water of different concentrations was tested, and the viral reduction was determined by infectivity assay on porcine primary alveolar macrophages. The results showed that 2 log10 (99 %) reduction in viral titer was observed when 104.0 TCID50/mL wild-type or reporter ASFV was inactivated with ozonized water as lower as 5 mg/L within 1 min at room temperature; while a viral reduction of approximately 2 log10 (99 %) was observed when 105.0 TCID50/mL wild-type or reporter ASFV was inactivated with 5 mg/L ozonized water within 1 min, and 3 log10 (99.9 %) virus was inactivated by 10 or 20 mg/L ozonized water within 3 or 1 min, respectively; furthermore, 5 mg/L ozonized water inactivated 2 log10 (99 %) reporter ASFV as higher as 106.75 TCID50/mL in 1 min, and a viral reduction of approximately 3 log10 (99.9 %) in reporter ASFV or 2 log10 (99 %) in wild-type virus was observed when inactivated with 10 mg/L ozonized water in 1 min; meanwhile, a viral reduction of 3 log10 (99.9 %) was observed when 20 mg/L ozonized water was applied to the wild-type ASFV of 106.75 TCID50/mL in 3 min. Overall, ozonized water can rapidly and efficiently inactivate ASFV, representing an effective disinfectant for ASF control.

Recovery and chemical disinfection of foot-and-mouth disease and African swine fever viruses from porous concrete surfaces.

AIMS: Develop an effective laboratory method to consistently recover viral loads from porous concrete coupons sufficient for disinfectant efficacy testing. Investigate the role of concrete matrix pH on the recovery of foot-and-mouth disease virus (FMDV) and African Swine Fever virus (ASFV) from porous concrete. Compare parameters off FMDV and ASFV inactivation on porous and nonporous surfaces in quantitative carrier tests of a liquid chemical disinfectant. METHODS AND RESULTS: Concrete test coupons were fabricated from commercial and industrial sources and carbonated by exposure to 5% CO2 in a humidified incubator, lowering the matrix pH. Neither dried FMDV nor ASFV were recovered from high-pH concrete control coupons. Recovery of infectious virus from lower pH carbonated concrete was similar to stainless steel coupon controls. Exposure to the liquid disinfectant Virkon™ S inactivated FMDV and ASFV on porous concrete. CONCLUSIONS: Concrete matrix pH had a greater impact than surface porosity on the ability to recover viable virus from unsealed concrete. SIGNIFICANCE AND IMPACT OF THE STUDY: Concrete is commonly found in environments where virus decontamination is required. This study demonstrates a reproducible method to recover sufficient viral loads from porous concrete coupons to facilitate quantitative carrier testing. This method provides a basis for evidence-based validation testing of chemical disinfectants to inactivate pH-sensitive viruses on unsealed concrete.