METHODS: The dengue infection in mouse model was established by inoculation of non-mouse adapted New Guinea C strain dengue virus (DEN-2) in AG129 mice. The freeze-dried CPLJ compounds were identified by Ultra-High Performance Liquid Chromatography High Resolution Accurate Mass Spectrometry analysis. The infected AG129 mice were orally treated with 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ, starting on day 1 post infection for 3 consecutive days. The blood samples were collected from submandibular vein for plasma NS1 assay and quantitation of viral RNA level by quantitative reverse transcription PCR.
RESULTS: The AG129 mice infected with dengue virus showed marked increase in the production of plasma NS1, which was detectable on day 1 post infection, peaked on day 3 post-infection and started to decline from day 5 post infection. The infection also caused splenomegaly. Twenty-four compounds were identified in the freeze-dried CPLJ. Oral treatment with 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ did not affect the plasma NS1 and dengue viral RNA levels. However, the morbidity level of infected AG129 mice were slightly decreased when treated with freeze-dried CPLJ.
CONCLUSION: Oral treatment of 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ at the onset of viremia did not affect the plasma NS1 and viral RNA levels in AG129 mice infected with non-mouse adapted New Guinea C strain dengue virus.
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.