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.

Advanced virtual screening enables the discovery of a host-targeting and broad-spectrum antiviral agent.

We employed an advanced virtual screening (AVS) approach to identify potential inhibitors of human dihydroorotate dehydrogenase (DHODH), a validated target for development of broad-spectrum antivirals. We screened a library of 495118 compounds and identified 495 compounds that exhibited better binding scores than the reference ligands involved in the screening. From the top 100 compounds, we selected 28 based on their consensus docking scores and structural novelty. Then, we conducted in vitro experiments to investigate the antiviral activity of selected compounds on HSV-1 infection, which is susceptible to DHODH inhibitors. Among the tested compounds, seven displayed statistically significant antiviral effects, with Comp 19 being the most potent inhibitor. We found that Comp 19 exerted its antiviral effect in a dose-dependent manner (IC50 = 1.1 µM) and exhibited the most significant antiviral effect when added before viral infection. In the biochemical assay, Comp 19 inhibited human DHODH in a dose-dependent manner with the IC50 value of 7.3 µM. Long-timescale molecular dynamics simulations (1000 ns) revealed that Comp 19 formed a very stable complex with human DHODH. Comp 19 also displayed broad-spectrum antiviral activity and suppressed cytokine production in THP-1 cells. Overall, our study provides evidence that AVS could be successfully implemented to discover novel DHODH inhibitors with broad-spectrum antiviral activity.

Antiviral activity of brequinar against African swine fever virus infection in vitro.

African swine fever virus (ASFV) is a double-stranded DNA virus that causes an acute and hemorrhagic disease in domestic swine, resulting in significant economic losses to the global porcine industry. The lack of vaccines and antiviral drugs highlights the urgent need for antiviral studies against ASFV. Here, we report that brequinar (BQR), which is a specific inhibitor of dihydroorotate dehydrogenase, robustly inhibits ASFV replication in Vero cells, as well as in porcine macrophages. We demonstrate that BQR exerts its antiviral activity in a dose-dependent manner through the depletion of pyrimidine pool. Although BQR does not affect the synthesis of an early viral protein, pI215L, the synthesis of late viral proteins, p17 and p72, is suppressed in the presence of BQR. We also show that BQR is able to induce cellular antiviral response in ASFV-infected macrophages by enhancing the expression of interferon-stimulated genes. Taken together, our study reveals that targeting nucleotide biosynthesis represents a promising strategy for developing antiviral agents against ASFV.