In Vitro Antiviral Activities of Salinomycin on Porcine Epidemic Diarrhea Virus

Abstract

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. Owing to the lack of effective vaccines and specific therapeutic options for PEDV, it is pertinent to develop new and available antivirals. This study identified, for the first time, a salinomycin that actively inhibited PEDV replication in Vero cells in a dose-dependent manner. Furthermore, salinomycin significantly inhibited PEDV infection by suppressing the entry and post-entry of PEDV in Vero cells. It did not directly interact with or inactivate PEDV particles, but it significantly ameliorated the activation of Erk1/2, JNK and p38MAPK signaling pathways that are associated with PEDV infection. This implied that salinomycin inhibits PEDV replication by altering MAPK pathway activation. Notably, the PEDV induced increase in reactive oxidative species (ROS) was not decreased, indicating that salinomycin suppresses PEDV replication through a pathway that is an independent pathway of viral-induced ROS. Therefore, salinomycin is a potential drug that can be used for treating PEDV infection.

Keywords: MAPK pathway; PEDV; ROS; salinomycin.

Figure 1

Cytotoxicity of salinomycin in Vero cells. Vero cells treated with salinomycin at indicated concentrations for 24 h at 37 °C or incubated with the culture medium containing DMSO as a control and subjected to CCK8 assay to detect cell viability. Cell viability ranged from 0% to 100%. Data are expressed as three independent experiments. * p < 0.05. ** p < 0.01.

Figure 2

Antiviral activity of salinomycin on porcine epidemic diarrhea virus (PEDV) infection in Vero cells. Vero cells mock pretreated with DMSO or pretreated with salinomycin at concentration of 0.01, 0.1 and 1 μM, respectively. These cells were subjected to PEDV infection for 1 h with or without salinomycin treatment. After extensive washing using PBS, cells were further cultured for 24 h with the medium containing salinomycin or blank medium. Viral yields were then titrated by (a) western blot, (b) plaque assays and (d) indirect immunofluorescence assay (IFA). Green: PEDV N protein; Blue: DAPI; Scale bar, 20 μm. (c) Statistical of plaque assays results. The assays were performed in duplicate and data present as means ± SD. * p < 0.05, ** p < 0.01.

Figure 3

Viricidal effects of salinomycin on PEDV. Viral stocks exposed to salinomycin at 1 μM at 37 °C for 1 h. Subsequently, the viral yields were titrated by the TCID₅₀ assays in Vero cells (a). The virus propagation in Vero cells after infection for 12 h was checked by western blot (b). The assay was performed in duplicate and data presented as the means ± SD.

Figure 4

Antiviral effects of salinomycin on different stages of PEDV infectious cycle. Vero cells infected with PEDV were mock-treated with DMSO or treated with salinomycin at different stages of infection. (a) Viral titers (log10 TCID50/mL) from cells treated with salinomycin at the viral binding, (b) entry and replication stages as calculated by the method of Reed and Muench. The assays were performed in duplicate and data presented as means ± SD. * p < 0.05. ** p < 0.01.

Figure 5

Effect of salinomycin on MAPK signaling in response to PEDV infection. (a) Serum starved Vero cells pretreated with salinomycin at the indicated concentrations for 1 h, respectively, then infected with PEDV in the presence of salinomycin for 24 hpi. The cell lysate was prepared and subjected to western blotting analysis. (b) Serum starved Vero cells were exposed to salinomycin at the indicated concentrations for 24 h. Cell lysate was prepared and subjected to western blotting analysis. The band intensity was analyzed with software image J. These results represented three independent experiments and data presented as means ± SD. * p < 0.05. ** p < 0.01.

Figure 6

Inhibition of PEDV replication by salinomycin is independent of reactive oxidative species (ROS) generation. Vero cells subjected to pretreatment with NAC (5 mM) or salinomycin(1 μM) for 1 h, were infected with PEDV along with NAC or salinomycin. At 24 hpi cellular ROS were detected using H₂DCFDA (5 μM, 30 min). Values represent three independent experiments. Significant differences compared to untreated infected cells are indicated by *, (* p < 0.05), ns, no significance.