ELUCIDATING THE EFFECTS OF ORIENTIN AND MADECASSOSIDE ON SAFFOLD VIRUS INFECTION IN BV2 MICROGLIAL AND NE-4C NEURAL STEM CELLS

Abstract

Introduction: Saffold virus (SAFV), of the Picornaviridae family is currently associated with unknown pathogenicity although the virus had been detected in infants with neurological disorders, including meningitis and cerebellitis. Exposure to SAFV infection mouse models showed that the virus is able to induce apoptosis of neuronal and glial cells. Orientin and madecassoside are potential compounds having an effect against SAFV infection due to their anti-viral, anti-inflammatory, antioxidative and anti-apoptotic abilities. Therefore, the aim of this study is to investigate if orientin and madecassoside provide preventive, direct inhibitory or a rescue effect on BV2 microglia cells and NE-4C neural stem cells infected with SAFV. Methods: The effects of orientin and madecassoside on BV2 and NE-4C cells infected with SAFV were examined at three different time-points: prevention (cells were treated with the two compounds respectively first, followed by SAFV infection), direct inhibition (the two compounds respectively were administered onto SAFV first and then added to the two cell lines) and rescue (cells were first infected with SAFV followed by administration of the two compounds respectively). The IC50 dose of orientin and madecassoside, and the CCID50 of SAFV on BV2 and NE-4C cells were determined via MTT assays. Subsequently, the two compounds were administered on the BV2 and NE-4C cells infected with SAFV at the three aforementioned time-points. The cells’ viability was measured via the MTT assay while the virus titer was determined by real-time PCR. Effectiveness of the compounds was determined based on improvements in cell viability and reductions in virus titer compared to the negative control. Results: Results from the efficacy experiments indicated that orientin was more effective than madecassoside in inhibiting SAFV as shown in the direct inhibition and rescue modes. However, both compounds were ineffective when administered in a preventive manner. Direct inhibition was selected as the optimum method as it provided more consistent results. Orientin significantly improved cell viability in BV2 cells and NE-4C cells by 94% and 90% respectively and had significantly lower virus titer when administered via direct inhibitory mode at a dose of 1μg. Whereas, madecassoside was only effective in NE-4C cells during direct inhibition with a significant improvement in cell viability of 87% and significantly lower virus titer compared to the negative control at a dose of 1μg. Conclusion: This study enabled the development of a cell-based assay with suitable methodology and criteria for analysis of results for drug testing against SAFV. Positive results were obtained for both rescue and direct inhibition modes but were more consistent for latter mode of action. Hence, direct inhibition of SAFV was the preferred mode of action and results indicated that orientin was superior to madecassoside in improving cell viability and reducing SAFV virus titer.