Replicación viral y producción de óxido nítrico y malondialdehído en cultivos celulares y ratones tratados preventivamente con melatonina, minociclina y ácido ascórbico infectados por el virus de encefalitis equina venezolana

Résumé

The Venezuelan Equine Encephalitis virus (VEE) has caused extensive epidemics throughout South America, especially Venezuela. Affecting, with high morbidity, the central nervous system (CNS) in humans and horses. On the other hand, there are antioxidants that interact with free radicals slowing the oxidation; among them are the Melatonin (MLT), Minocycline (MIN) and Ascorbic acid (AA). It is proposed to determine the effect in vitro and in vivo of the MLT, MIN and AA on viral replication, production of nitric oxide (NO) and malondialdehyde (MDA) in cell cultures and mice infected with VEE virus. For in vitro assays murine neuroblastoma cells (Na2) were used, which received preventive treatment of individual and combined antioxidants doses as follows: MLT at 0.5 mM, 1.0 mM and 5.0 mM 30 minutes before infection, doses of MIN at 0.1 µM, 0.2 µM and 2.0 µM 30 minutes before infection and dose of 25.0 µM of AA, 50.0 µM and 75.0 µM 1 hour before viral infection. Infection with VEE virus was performed at a concentration of 1x10-6 PFU / ml in an atmosphere of 5% CO2 and 37 °C. Supernatants were taken to determine NO, MDA and viral titers at 24 hours post-infection. In vivo assays were performed in NMRI albino mice which received individual and combined preventive treatment subcutaneously with 500 micrograms of MLT / kg of body weight, MIN 50 mg / kg body weight and 50 mg AA /kg body weight, from 3 days before viral infection. Mice were injected intraperitoneally with the Guajira strain (E-100) of the VEE virus as the experimental protocol. Animals on 1st, 3rd and 5th post-infection days, five mice per experimental group for each trial were sacrificed. Whole blood was recolected by orbital sinus puncture to obtain serum in which the IgM anti-VEE antibody titer NO concentration by Griess reaction and MDA by the thiobarbituric acid colorimetric method were determined. Subsequently, animals were sacrificed by cervical dislocation to obtain their brains after perfusion and make the determination of viral titers by plaque forming unit technique, NO and MDA. In cell cultures infected in vitro, a decrease (p<0.01) in the production of NO and, MDA we observed, likewise, it was shown a synergistic effect when using antioxidants in combination. These effects were not dose dependent. On the other hand, the viral replication in cells when treated with drugs was decreased. In brain homogenates and serum of infected animals there was an increase in the generation of NO (p <0.001) in addition to the increased production of MDA, which decreased their levels when were treated with MLT, MIN and MLT combinations with AA and AA and MIN and MIN. In conclusion, prophylactic treatment with MLT, MIN and AA decreased viral titers in cell cultures, brain homogenates and serum being the combination of MLT and MIN the most effective, in turn, preventive treatment with MLT and AA but not with MIN, caused a decrease in the production of NO and MDA in experimental models of VEE virus infection in vitro and in vivo. These results support the development of preventive therapeutic strategies for VEE virus infection being MLT the preferred candidate because of its high tolerance.