The growth of arthralgic Ross River virus is restricted in human monocytic cells

International audienceAlphaviruses such as Chikungunya and Ross River (RRV) viruses are associated with persistent arthritisand arthralgia in humans. Monocytes and macrophages are believed to play an important role in alphaviralarthritides. In this study, we evaluated RRV permissiveness of the human acute leukemia MM6 cellline. Viral growth analysis showed that RRV infection of MM6 cells resulted in a very low virus progenyproduction with daily output. Using recombinant RRV expressing the reporter gene Renilla luciferase, aweak viral replication level was detected in infected cells at the early stages of infection. The infectionrestriction was not associated with type-I interferon and pro-inflammatory cytokines release. Apoptosishallmarks (i.e. mitochondrial BAX localisation and PARP cleavage) were observed in infected MM6cells indicating that RRV can trigger apoptosis at late infection times. The long-term persistence of RRVgenomic RNA in surviving MM6 cells identifies human monocytic cells as potential cellular reservoirs ofviral material within the infected host

Caractérisation chimique et biologique de trois huiles essentielles répulsives issues de la biodiversité régionale contre l'alphavirus du Ross River

Essential oils of citronella (Cymbopogon citratus), geranium (Pelargonium graveolens) and vetiver (Vetiveria zizanioides) are used worldwide as topical repellent against the main vectors (mosquitoes, ticks) of human infectious diseases (Malaria, chikungunya, …). Skin treatment with these natural products, initially to avoid contact with the vector had not yet been considered as a way to disrupt the early stages of infection when the repelling action fails. To check this hypothesis, a structured framework has been performed for the chemical and biological re-evaluation of the three essential oils. The latter was tested against Ross River virus (alphavirus) that belongs to the same family of Chikungunya virus. Analysis of essential oils using a high-resolution technique (GC × GC / TOF-MS) resulted in a more accurate chemotypical profile of the local production. The use of specific markers (molecular clones of the virus, Saclay CEA) allowed to establish the inhibition of viral replication depending of the conditions of geranium and citronella essential oils application. These results suggest the great interest of an essential oil topical repellent in the early stages of a vector infection. The comparative study established the high value of geranium essential oil and gave future direction to the discovery of new anti-infectious solutions from monoterpenes-rich natural complexes.Les huiles essentielles de citronnelle (Cymbopogon citratus), de géranium (Pelargonium graveolens) et de vétiver (Vetiveria zizanioides) sont utilisées partout dans le monde pour leur activité répulsive contre les principaux vecteurs (moustiques, tiques) de maladies infectieuses chez l'Homme (paludisme, chikungunya, …). L'application cutanée de ces produits naturels pour éviter le contact avec un vecteur n'avait pas été encore envisagée comme moyen de limiter les premiers stades de l’infection par l'agent pathogène transmis par le vecteur. Pour vérifier cette hypothèse, les travaux ont été consacrés à la mise en place d'un cadre structuré pour la réévaluation chimique et biologique des trois huiles essentielles sur le modèle du virus du Ross River (alphavirus) de la même famille que le virus du Chikungunya. La caractérisation chimique des huiles essentielles avec une technique de haute résolution (GC×GC/TOF-MS) a permis d'établir leur profil chémotypique précis. L'utilisation de marqueurs spécifiques (clones moléculaires du virus) a permis d'établir l'inhibition de la réplication virale en fonction des conditions d'application des huiles essentielles de géranium et citronnelle. Ces résultats suggèrent l'intérêt d’une huile essentielle répulsive dans les premiers stades d'une infection par un vecteur. À ce titre, l'étude comparative établit la haute valeur ajoutée de l'huile essentielle de géranium et oriente la recherche de nouveaux anti-infectieux naturels vers des complexes riches en monoterpènes

Chemical and biological characterization of three repellent essential oils from regional biodiversity against Ross River alphavirus

Les huiles essentielles de citronnelle (Cymbopogon citratus), de géranium (Pelargonium graveolens) et de vétiver (Vetiveria zizanioides) sont utilisées partout dans le monde pour leur activité répulsive contre les principaux vecteurs (moustiques, tiques) de maladies infectieuses chez l'Homme (paludisme, chikungunya, …). L'application cutanée de ces produits naturels pour éviter le contact avec un vecteur n'avait pas été encore envisagée comme moyen de limiter les premiers stades de l’infection par l'agent pathogène transmis par le vecteur. Pour vérifier cette hypothèse, les travaux ont été consacrés à la mise en place d'un cadre structuré pour la réévaluation chimique et biologique des trois huiles essentielles sur le modèle du virus du Ross River (alphavirus) de la même famille que le virus du Chikungunya. La caractérisation chimique des huiles essentielles avec une technique de haute résolution (GC×GC/TOF-MS) a permis d'établir leur profil chémotypique précis. L'utilisation de marqueurs spécifiques (clones moléculaires du virus) a permis d'établir l'inhibition de la réplication virale en fonction des conditions d'application des huiles essentielles de géranium et citronnelle. Ces résultats suggèrent l'intérêt d’une huile essentielle répulsive dans les premiers stades d'une infection par un vecteur. À ce titre, l'étude comparative établit la haute valeur ajoutée de l'huile essentielle de géranium et oriente la recherche de nouveaux anti-infectieux naturels vers des complexes riches en monoterpènes.Essential oils of citronella (Cymbopogon citratus), geranium (Pelargonium graveolens) and vetiver (Vetiveria zizanioides) are used worldwide as topical repellent against the main vectors (mosquitoes, ticks) of human infectious diseases (Malaria, chikungunya, …). Skin treatment with these natural products, initially to avoid contact with the vector had not yet been considered as a way to disrupt the early stages of infection when the repelling action fails. To check this hypothesis, a structured framework has been performed for the chemical and biological re-evaluation of the three essential oils. The latter was tested against Ross River virus (alphavirus) that belongs to the same family of Chikungunya virus. Analysis of essential oils using a high-resolution technique (GC × GC / TOF-MS) resulted in a more accurate chemotypical profile of the local production. The use of specific markers (molecular clones of the virus, Saclay CEA) allowed to establish the inhibition of viral replication depending of the conditions of geranium and citronella essential oils application. These results suggest the great interest of an essential oil topical repellent in the early stages of a vector infection. The comparative study established the high value of geranium essential oil and gave future direction to the discovery of new anti-infectious solutions from monoterpenes-rich natural complexes

<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

<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

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