Study On The Inhibition Of Ebola Virus Entry And Its Mechanism By Extracts Of Geranium Strictipes R. Knuth And Sophora Davidii (Franch.) Skeels

Objective:Ebola virus can cause the highly fatal Ebola hemorrhagic fever in humans,for which there is no effective and approved vaccine or treatment.The ongoing outbreak in West Africa demonstrates the urgent need to develop drugs and therapeutic regimens that target the infection and pathogenesis of Ebola.Plant natural ingredients have increasingly become one of the sources for new drug development due to their chemical diversity and easy access.This project will screen a portion of natural ingredient drugs with anti-Ebola effect by constructing Ebola pseudovirus screening model.To study and analyze the main mechanism of action of the drug and to investigate whether the drug can be studied as an effective inhibitor of Ebola entry.Methods:1.A Zaire/Sudan type Ebola pseudovirus screening model was constructed based on BSL-2 grade conditions using the pseudovirus system plasmids p NL4-3.Luc.R-E-,pc DNA3.1-Zaire GP,and pc DNA3.1-Sudan GP.2.The Ebola pseudovirus model was used to screen seven drug candidates and analyze the anti-Ebola virus inhibition effect of each drug.Calcu Syn,Graph Pad Prism（version 9）and other software were used to study and calculate the IC₅₀and IC₉₀of each drug to evaluate and screen the inhibition effect of each drug.3.The cytotoxicity of candidate drugs was evaluated by using CCK-8 reagent.By adding drugs at different times or treating target cells under different conditions,the antiviral mode of action of each drug was studied and analyzed,and the mechanism of action of each drug was preliminarily predicted and evaluated.4.The drugs are pre-incubated with host cells to study whether their drug targets act on the host target cells.The drug candidates were subjected to"inactivation"experiments and their results were verified using western blot assay to study and analyze the drug interactions with Ebola GP proteins.Results:1.The Zaire/Sudan Ebola pseudovirus drug screening model was successfully constructed.2.The ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels had better inhibition effect on Ebola virus.The IC₅₀for Zaire and Sudan pseudoviruses were 2.501±0.392μg/m L and 2.162±0.075μg/m L,2.992±0.385μg/m L and 2.443±0.498μg/m L,3.379±0.330μg/m L and 4.050±0.339μg/m L.The IC50 values of these three drugs were low,and they had good antiviral effect,and they were preliminarily identified as candidate Ebola entry inhibitors.3.The experiment of adding drugs at different times showed that ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels mainly acted on the early stage of Ebola virus infection.The toxic effects of the three drugs on host cells were low at the effective inhibitory concentration.4.The results of the synchronized infection experiments showed that the ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth acted mainly on the attachment and binding process of Ebola virus.Ethanol extracts of Sophora davidii（Franch.）Skeels acted mainly on the membrane fusion process with host target cells in the entry of Ebola virus infection.5.The drug target of ethyl acetate and n-butanol extract of Geranium strictipes R.Knuth and ethanol extract of Sophora davidii（Franch.）Skeels was not Huh-7 cells.6.Ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels had no"inactivation"effect on Ebola pseudovirus particles at the effective inhibition concentration.Direct interaction of the three extracts with Ebola virus did not result in lysis of virus particles.Conclusion:1.Ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels had good anti-Ebola virus inhibition effect.2.Ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels prevented Ebola virus infection from entering host cells in different ways.3.Ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels showed low cytotoxicity at effective inhibitory concentrations,and host target cells Huh-7 cells were not the target of the drug.4.Preliminary studies on the anti-Ebola effects of three plant extracts and their mechanisms of action were conducted,and ethyl acetate and n-butanol extracts of Geranium strictipes R.Knuth and ethanol extracts of Sophora davidii（Franch.）Skeels were able to be further investigated as anti-Ebola drug candidates.