METHODS: Mice were intraperitoneally-infected with a mouse-adapted EV-A71 strain and treated with a dose of monoclonal antibody (MAb) daily for 3 days on day 1, 2 and 3 post-infection or for 3 days on 3, 4 and 5 post-infection. Treatment effectiveness was evaluated by signs of infection and survival rate. Histopathology and qPCR analyses were performed on mice sacrificed a day after completing treatment.

RESULTS: In mock-treated mice, CNS infection was established from day 3 post-infection. All mice treated before established CNS infection, survived and recovered completely without CNS infection. All mice treated after established CNS infection survived with mild paralysis, and viral load and antigens/RNA at day 6 post-infection were significantly reduced.

METHODS: The cytotoxic effects of three flavonoids towards rhabdomyosarcoma (RD) cells were first examined using cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Compounds found to be non-cytotoxic in RD cells were evaluated for their in vitro antiviral properties against the EV-A71 subgenotype B4 strain 41 (5865/SIN/000009) using antiviral assays. Viral infectivity was determined by reduction of the formation of plaques in RD cells. For the measurement of RNA copy number, the real time quantitative reverse transcription PCR (qRT-PCR) was used. The most potent compound was further evaluated to determine the mode of action of inhibition by time course, virus attachment and entry assays in Vero cells.

RESULTS: Silymarin was shown to exert direct extracellular virucidal effects against EV-A71 at 50% inhibitory concentration (IC50) of 15.2 ± 3.53 μg/mL with SI of 10.53. Similarly, baicalein exhibited direct extracellular virucidal effects against EV-A71 at a higher IC50 value of 30.88 ± 5.50 μg/mL with SI of 13.64. Besides virucidal activity, silymarin was shown to block both viral attachment and entry of EV-A71 to inhibit infection in Vero cells.