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.
CONCLUSIONS: Silymarin has a stronger inhibition activity against EV-A71 in comparison to baicalein. It could serve as a promising antiviral drug to treat EV-A71 infections.
MAIN METHODS: A pull-down assay was performed to identify the binding partner of the L-SP40 peptide. Co-immunoprecipitation and co-localization assays with the L-SP40 peptide were employed to confirm the receptor partner in RD cells. The outcomes were validated using receptor knockdown and antibody blocking assays. The L-SP40 peptide was further evaluated for the protection of neonatal mice against lethal challenge by mouse-adapted EV-A71.
KEY FINDINGS: The L-SP40 peptide was found to interact and co-localize with nucleolin, the key attachment receptor of Enteroviruses A species, as demonstrated in the pull-down, co-immunoprecipitation and co-localization assays. Knockdown of nucleolin from RD cells led to a significant reduction of 3.5 logs of viral titer of EV-A71. The L-SP40 peptide demonstrated 80% protection of neonatal mice against lethal challenge by the mouse-adapted virus with a drastic reduction in the viral loads in the blood (~4.5 logs), skeletal muscles (1.5 logs) and brain stem (1.5 logs).
SIGNIFICANCE: L-SP40 peptide prevented severe hind limb paralysis and death in suckling mice and could serve as a potential broad-spectrum antiviral candidate to be further evaluated for safety and potency in future clinical trials against EV-A71.
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.
CONCLUSIONS: Passive immunization with our MAb could prevent CNS infection in mice if given early before the establishment of CNS infection. It could also ameliorate established CNS infection if optimal and repeated doses were given.