Searching for a methodology for assess in vitro antiviral activity photosentitized

MUGAS M., KONIGHEIM B.S, AGUILAR J., MARIONI J., COMINI L., CONTIGIANI M., CABRERA J., NÚÑEZ MONTOYA S.

Córdoba

Congreso; 3º REUNIÓN INTERNACIONAL DE CIENCIAS FARMACÉUTICAS (RICiFa); 2014

Our research group studies the activity of photosensitizing anthraquinones (AQs), purified from a phototoxic plant, against several viruses. Previously, we have demonstrated that these AQs exhibit virucidal effect against HSV-1 and JUNV (by inactivating viral particles before they enter into the host cell), and this inhibition is increased by light (photostimulation). To study the antiviral activity of these AQs (on the infected cells, at some stage of viral replication), which can in turn be photostimulated, an appropriate methodology is needed. Therefore, the aim of this work was to assess two techniques, Neutral Red uptake assay (NR) and reduction of plaque forming units test (PFU), and establishing the conditions necessary to quantify the in vitro antiviral effect photosensitized. An extract enriched in AQs (benzene extract) was assayed in order to ensure a photostimulated effect against HSV-1. Seven different concentrations were tested (≤CC50, which was estimated from the curve of cellular viability vs. concentrations of extract on Vero cells, by NR assay). The antiviral effect was performed by both techniques in Vero cells, under two simultaneous conditions: darkness and irradiation (actinic lamp 380-480 nm, Philips TL/03). The following variables were evaluated: culture medium during irradiation, irradiation time on virus-cells-extract, culture medium post-irradiation and incubation time. The optimal conditions for both methodologies were: PBS 1% DMSO (culture medium during irradiation), 15 minutes (irradiation time on virus-cells-extract), MEM supplemented with 2% FBS (culture medium post-irradiation), 36 h incubation. The NR assay was chosen because it allowed determining the inhibition percentage, which could not be estimated by the PFU test, since it only showed the qualitative toxicity of the photostimulated extract. Besides, during the NR assay, the cytopathic effect (microscopic observation of morphological alterations in cells) could be simultaneously assessed. This is very important