In vitro viral inactivation photosensitized by a natural anthraquinones

MUGAS L; KONIGHEIM BS; AGUILAR JJ; CABRERA JL; CONTIGIANI MS; NÚÑEZ MONTOYA SC

Villa Carlos Paz

Encuentro; XIII ENCUENTRO LATINOAMERICANO DE FOTOQUÍMICA Y FOTOBIOLOGÍA (ELAFOT); 2017

Grupo Argentino de Fotobiología

From the phototoxic plant, Heterophyllaea pustulata Hook. f. (Rubiaceae), a bush that grows in the Andean northwest of Argentina, we have obtained 9,10-anthraquinone derivatives (AQs) with photosensitizing properties [1-3]. Previously, we have demonstrated that these AQs show in vitro virucidal effect against Herpes Simplex virus Type 1 (HSV-1), which means an inactivation of viral particles before their entry into the host cells, and this inhibition was increased by light action (photostimulation) [4]. The aim of this work was study the in vitro inactivation of HSV-1 once the virus entered into the host-cells, by the photosensitized action of two natural AQs: soranjidiol (Sor) and 5,5´-bisoranjidiol (Bisor), a soranjidiol dimer.Isolated AQs were identified by their RMN spectra. To test the viral inactivation effect of each AQ on infected cells, it is previously necessary to find the concentrations of each AQ that are not toxic for the host cells (cytotoxic assay). In the two assays, morphological cellular alterations were evaluated by optical microscopic (cytopathic effect, CPE) and cellular viability (CV) was determined by uptake NR assay, under two simultaneous conditions: darkness and irradiation (actinic lamp 380-480 nm, Philips TL/03). Cytotoxic test was performed in a monolayer of Vero cells (host cells) treated with different concentrations of each AQ, dissolved in MEM with DMSO <1%. Non cytotoxic concentrations were estimated from a dose-response curve (% CV vs. concentrations) [5]. Thus, a Subtoxic Concentration (SubTC, CC20 concentration that ensures 80 % of viable cells) and the Maximum Non-Cytotoxic Concentration (MNCC, CC10) were established. Subsequently, infected cells with HSV-1 (KOS strain) were incubated 4 h with each AQs at SubTC and MNCC, and then were irradiated 15 min. Active viral particles after both treatments (darkness and irradiation) were detected by extraction of cellular content and its subsequent inoculation in a new Vero cell monolayer to evaluate by Plaque reduction assay after 72 h of incubation [6]. Cellular, viral, toxicity and positive (Acyclovir) controls were included. The inactivation results were expressed as percentage of plaque inhibition (%I) by comparison with viral controls. Bisor was more toxic than Sor, and the toxicity was increased by the irradiation. For Sor the SubTC was 393M and the MNCC was 137M, whereas for Bisor they were 79M and 59M, respectively. In darkness, the virus was not inactivated by any AQ, but a significant inactivation was observed under irradiation. Sor inactivated 95% and 57% of viral particles at SubTC and MNCC respectively. On the other hand, Bisor inactivate 38% only at SubTC. The cellular controls show that the irradiation not produces significant changes in the cell control, viral control and the toxicity controls had the same effects which were observed in the cytotoxicity assay.In conclusion, Sor was better than its dimer Bisor, since it photo-inactivated 95% of HSV-1 particles and also it is less cytotoxic. This result motivates us to continue to deepen studies with purified AQs, with the aim to provide a basis for the development of new treatments for viral etiology lesions.