<i>In vitro</i> comparison of three common essential oils mosquito repellents as inhibitors of the Ross River virus

<div><p>Background</p><p>The essential oils of <i>Cymbopogon citratus</i> (CC), <i>Pelargonium graveolens</i> (PG) and <i>Vetiveria zizanioides</i> (VZ) are commonly used topically to prevent mosquito bites and thus the risk of infection by their vectored pathogens such as arboviruses. However, since mosquito bites are not fully prevented, the effect of these products on the level of viral infection remains unknown.</p><p>Objectives</p><p>To evaluate <i>in vitro</i> the essentials oils from Reunion Island against one archetypal arbovirus, the Ross River virus (RRV), and investigate the viral cycle step that was impaired by these oils.</p><p>Methods</p><p>The essential oils were extracted by hydrodistillation and analyzed by a combination of GC-FID and GC×GC-TOF MS techniques. <i>In vitro</i> studies were performed on HEK293T cells to determine their cytotoxicity, their cytoprotective and virucidal capacities on RRV-T48 strain, and the level of their inhibitory effect on the viral replication and residual infectivity prior, during or following viral adsorption using the reporter virus RRV-<i>ren</i>Luc.</p><p>Results</p><p>Each essential oil was characterized by an accurate quantification of their terpenoid content. PG yielded the least-toxic extract (CC₅₀ > 1000 μg.mL^-1). For the RRV-T48 strain, the monoterpene-rich CC and PG essential oils reduced the cytopathic effect but did not display virucidal activity. The time-of-addition assay using the gene reporter RRV-<i>ren</i>Luc showed that the CC and PG essential oils significantly reduced viral replication and infectivity when applied prior, during and early after viral adsorption. Overall, no significant effect was observed for the low monoterpene-containing VZ essential oil.</p><p>Conclusion</p><p>The inhibitory profiles of the three essential oils suggest the high value of the monoterpene-rich essential oils from CC and PG against RRV infection. Combined with their repellent activity, the antiviral activity of the essential oils of CC and PG may provide a new option to control arboviral infection.</p></div

<i>In vitro</i> comparison of three common essential oils mosquito repellents as inhibitors of the Ross River virus - Fig 2

<p><b>Infectious capacity of RRV-T48</b> determined by plaque assay on infected Vero cells after pre-treatment of the viruses (1×10⁵ PFU) with the essential oils: <b>A:</b> residual infectivity; pre-treatment of the cells with the essential oils <b>B</b>: entry inhibition. Controls are infected cells and virus without treatment by the essential oils and values are expressed as mean ± SEM (n = 5).</p

Time course of infection from RRV determined by plaque assay on infected Vero cells and luciferase activity on infected HEK293T cells.

<p>Results are expressed as a mean ± SEM (n = 5).</p

Inhibition of RRV-<i>ren</i>Luc replication by the 3 essential oils under the experimental conditions shown in Fig 5.

<p>HEK293T was exposed to RRV-<i>ren</i>Luc at MOI 2 and treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196757#pone.0196757.g005" target="_blank">Fig 5</a> with essential oils (<b>A</b>) luciferase activity on HEK293T cells (<b>B</b>) Viral growth by plaque assay on Vero cells. Controls are RRV-<i>ren</i>Luc-infected cells infected without treatment by the essential oils. Values are expressed as relative percentage ± SEM (n = 5). (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005).</p

Comparative effect of the essential oils on HEK293T against RRV.

<p>Viability of HEK293T cells infected with RRV-T48 (MOI 2) upon treatment with chloroquine at the concentration 20 μg.mL^-1and essential oil at the concentration 1× CC₁₀ at 24 h post-infection (<b>A</b>); Inhibition of RRV-<i>ren</i>Luc replication at MOI 2 using co-treatment of chloroquine at the concentration 20 μg.mL^-1and essential oil at the concentration 1× CC₁₀ (<b>B</b>); Viral growth by plaque assay on Vero cells (<b>C</b>). Controls are RRV-infected cells without treatment by chloroquine and the essential oils. Values are expressed as mean ± SEM (n = 5). (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005).</p