Studies On The Action And Mechanism Of Ginsenoside Rb1 And Its Derivatives Against Zika Virus

Objective:The Zika virus（ZIKV）,which causes microcephaly and Guillain-Barre syndrome in newborns,has no effective control.In order to find and find safe and effective anti-Zik V drugs,the anti-Zikv activities of Rb1 and its derivatives were studied,from which compounds with good activity were screened for mechanism study and in vivo activity verification,so as to lay the foundation for the development of effective anti-Zi KV drugs.Methods:In this study,the broad-spectrum antiviral drug ribavirin was used as a positive control drug,and the compounds with good anti-ZIKV activity were screened from Rb1 and its derivatives（XCQ）by plaque assay.Then the cell viability was determined by MTT assay,and the compounds with low cytotoxicity and high activity were selected for further study.The in vitro activity of the compounds was verified by RT-q PCR,Western blot and cell-based ELISA experiments.On this basis,different treatments（Pre-treatment,Post-treatment,Co-treatment,Cell treatment）,attachment and entry experiment and protease activity assay were used to investigate the anti-ZIKV mechanism.Finally,a mouse model of ZIKV infection was established to study the in vivo activity of the compounds with better activity against ZIKV.Results:In this study,the anti-ZIKV activity and toxicity of ribavirin and Rb1 were detected by plaque assay and MTT cytotoxicity assay,and then the compounds with anti-ZIKV activity and low cytotoxicity were screened from 19 XCQ compounds.The anti-ZIKV active compounds were confirmed and the mechanism was studied.The EC₅₀of Rb1 and ribavirin to ZIKV SZ-WIV01 was 125.03±0.11μmol/L and24.83±0.06μmol/L,respectively.The EC₅₀of Rb1 and ribavirin to ZIKV MR766 was501.23±1.88μmol/L and 24.84±0.04μmol/L,respectively.Among the 19 XCQ compounds,6 showed significant anti-ZIKV activity,including XCQ-5,XCQ-6,XCQ-8,XCQ-9,XCQ-11 and XCQ-13.In the plaque assay,the EC₅₀of the six compounds against ZIKV SZ-WIV01 were 7.25±5.19μmol/L,4.93±1.77μmol/L,5.29±0.44μmol/L,4.08±1.80μmol/L,7.55±3.71μmol/L and 4.98±1.77μmol/L.The EC₅₀to MR766 were 9.93±0.02μmol/L,9.89±0.03μmol/L,19.79±0.006μmol/L,4.91±0.12μmol/L,9.86±0.2μmol/L and 2.47±0.04μmol/L.These results indicate that the six compounds are more effective than ribavirin and Rb1 in inhibiting the Asian and African strains of ZIKV.RT-q PCR was used to verify that the six compounds inhibited ZIKV replication at the molecular level,and their EC₅₀were3.46±2.35μmol/L,5.86±1.37μmol/L,1.72±0.08μmol/L,1.27±0.32μmol/L,4.04±0.13μmol/L and 2.67±0.39μmol/L.Western blotting and cell-based ELISA were used to confirm the inhibition of ZIKV E protein expression.The EC₅₀values in cell-based ELISA were 19.54±1.31μmol/L,19.22±0.69μmol/L,20.59±5.09μmol/L,9.69±0.10μmol/L,18.39±0.52μmol/L and 13.31±5.09μmol/L,respectively.XCQ-9with a TI value higher than that of the other five compounds was used for mechanism study.The inhibition rate of 10μmol/L XCQ-9 on ZIKV was 79%and 93%in attachment and entry experiments,respectively.Furthermore,the protease activity of NS2B-NSB and Rd RP was inhibited by 14%and 39%,respectively.In vivo anti-ZIKV activity study showed that XCQ-9 significantly inhibited ZIKV replication in A129 mice and improved the survival rate of the mice.Conclusion:This study shows for the first time that Rb1 and its six derivatives XCQ-5,XCQ-6,XCQ-8,XCQ-9,XCQ-11 and XCQ-13 have anti-ZIKV activity.XCQ-9 can inhibit ZIKV attachment and entry and may exert anti-ZIKV effect in vitro at the post-entry stage of ZIKV.XCQ-9 can inhibit virus replication and improve the survival rate of ZIKV-infected A129 mice,which is expected to be a potential drug for anti-ZIKV drug research.