In vitro Assessment of Antiviral Effect of Natural Compounds on Porcine Epidemic Diarrhea Coronavirus

Abstract

Organic acid and essential oils (EOs), well-known antimicrobials, could also possess antiviral activity, a characteristic which has not been completely addressed up to now. In this study, the effect of two organic acids (formic acid and sodium salt of coconut fatty acid distillates) and two single EO compounds (thymol and cinnamaldehye) was evaluated against porcine epidemic diarrhea virus (PEDV). The concentration used for each compound was established by cytotoxicity assays in Vero cells. The antiviral activity was then evaluated at three multiplicities of infection (MOIs) through visual cytopathic effect (CPE) evaluation and an alamarBlue assay as well as real-time reverse-transcription PCR (RT-qPCR) and viral titration of cell supernatants. Formic acid at at a dose of 1,200 ppm was the only compound which showed antiviral activity, with a weak reduction of CPE caused by PEDV. Through the alamarBlue fluorescence assay, we showed a significant anti-CPE effect of formic acid which could not be observed by using an inverted optical microscope. RT-qPCR and infectivity analysis also showed that formic acid significantly reduced viral RNA and viral titers in a PEDV MOI-dependent manner. Our results suggest that the antiviral activity of formic acid could be associated to its inhibitory effect on viral replication. Further studies are required to explore the anti-PEDV activity of formic acid under field conditions alone or together with other antiviral agents.

Keywords: antiviral activity; cytopathic effect; organic acids; pig; single essential oil compounds.

Figure 1

Relative cell viability rate [(mean fluorescence of treated well/mean fluorescence of control well) × 100] measured using an alamarBlue assay after exposure of Vero cell monolayers for 48 h to different concentrations of each compound tested. Data are expressed as mean of the three independent replicates ± standard deviations.

Figure 2

Anti-PEDV activity of two organic acids, formic acid and sodium salt of coconut fatty acid distillates, and two single EO compounds, thymol and cinnamaldehyde, determined in Vero cells infected with three different PEDV MOIs. (A) Proportion (%) of wells (n = 16) with CPE obtained after visual inspection of cells under an inverted light microscope. (B) Mean values ± standard deviations of log₁₀ viral RNA molecules/μl estimated using RT-qPCR. (C) Mean values ± standard deviations of viral titer expressed as log₁₀TCID₅₀/ml and calculated following the Reed and Muench method. *Statistically significant differences with respect to controls (p < 0.05).

Figure 3

Anti-PEDV activity of two organic acids, formic acid and sodium salt of coconut fatty acid distillates, and two single EO compounds, thymol and cinnamaldehyde, determined in Vero cells by means of an alamarBlue assay for estimating cell viability. (A) Standard curve generated from fluorescence intensity associated with exposition to serial dilutions of PEDV (TCID₅₀/cell). Linear trend line and R² value obtained are shown. (B) Relative cell viability rate [(mean fluorescence of treated-PEDV infected wells/mean fluorescence of treated non-infected wells) × 100]. Data are expressed as mean of the independent replicates ± standard deviations. *Statistically significant differences with regard to controls (p < 0.05).

Figure 4

Effect of a treatment with 1,200 ppm of formic acid on the different steps of the infection cycle of PEDV: replication (A), viral inactivation (B), attachment (C) and entry (D). Data are expressed as mean ± standard deviations of log₁₀ viral RNA molecules/μl) estimated using RT-qPCR. *Statistically significant differences with regard to controls (p < 0.05).

Figure 5

Effect of a treatment with 1,200 ppm of formic acid on the different steps of the infection cycle of PEDV: replication (A), viral inactivation (B), attachment (C) and entry (D). Data are expressed as mean ± standard deviations of viral titer expressed as log₁₀TCID₅₀/ml estimated using an infectivity assay following the Reed and Muench method. *Statistically significant differences with regard to controls (p < 0.05).