Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro

Abstract

Background: An antiviral drug is needed for the treatment of patients suffering from yellow fever. Several compounds present in plants can inactive in vitro a wide spectrum of animal viruses.

Aim: In the present study the inhibitory effect of essential oils of Lippia alba, Lippia origanoides, Oreganum vulgare and Artemisia vulgaris on yellow fever virus (YFV) replication was investigated.

Methods: The cytotoxicity (CC(50)) on Vero cells was evaluated by the MTT reduction method. The minimum concentration of the essential oil that inhibited virus titer by more than 50% (MIC) was determined by virus yield reduction assay. YFV was incubated 24 h at 4 degrees C with essential oil before adsorption on Vero cell, and viral replication was carried out in the absence or presence of essential oil. Vero cells were exposed to essential oil 24 h at 37 degrees C before the adsorption of untreated-virus.

Results: The CC(50) values were less than 100 microg/mL and the MIC values were 3.7 and 11.1 microg/mL. The CC(50)/MIC ratio was of 22.9, 26.4, 26.5 and 8.8 for L. alba, L origanoides, O. vulgare and A. vulgaris, respectively. The presence of essential oil in the culture medium enhances the antiviral effect: L. origanoides oil at 11.1 microg/mL produced a 100% reduction of virus yield, and the same result was observed with L. alba, O. vulgare and A. vulgaris oils at 100 microg/mL. No reduction of virus yield was observed when Vero cells were treated with essential oil before the adsorption of untreated-virus.

Conclusion: The essential oils evaluated in the study showed antiviral activities against YFV. The mode of action seems to be direct virus inactivation.

Figure 1

Direct inactivation of essential oils from Colombian plants on yellow fever virus (YFV). About 9.5 × 10⁴ PFU of YFV were incubated for 24 h at 4°C with concentrations of essential oil and then were adsorbed on Vero cells. Virus titers in supernatants of cell cultures were determined by plaque assays method at 48 h after adsorption. The data represent the means for three replicate samples of two independent experiment. Error bars indicate standard deviations.

Figure 2

Increase of direct yellow fever virus (YFV) inactivation by the presence of essential oil in the supernatant of virus-infected cells culture. YFV previously incubated with serial dilutions of essential oil (Figure 1) was replicated in Vero cells at 37°C in M-199 medium containing varied concentration of essential oil. Virus titers in supernatants of cell cultures were determined by plaque assays method at 48 h after adsorption. The data represent the means for three replicate samples of two independent experiment. Error bars indicate standard deviations.

Figure 3

Absence of antiviral effect of essential oils from Colombian plants on YFV replication by treatment of host cell. Vero cells were exposed to varied concentration of essential oil for 24 h at 37°C before YFV infection (MOI = 1). Virus titers in supernatants of cell cultures were determined by plaque assays at 48 h after adsorption. The data represent the means for three replicate samples of two independent experiment. Error bars indicate standard deviations.