Antiviral Activity Of Remdesivir Against Enterovirus 71 In Vitro And In Vivo

ObjectiveFirstly,a screening model of against enterovirus 71（EV71）drugs in vitro was constructed based on live virus level,which was used to evaluate the anti-enterovirus activity of FDA drug library compounds.On this basis,we carried out evaluation of compounds activity.The actived remdesivir was selected to further explore its preclinical pharmacodynamic studies at the cellular level and animal level.Methods1.The suitable cell line which can support virus replication and produce obvious cytopathic effect was selected.The cells that have a good condition were inoculated on the white background 96-well plate according to a certain cell density.Different cells were infected with different titers of EV71.The degree of cytopathic effect was observed every day,and the cell survival rate was detected by Cell Titer-Glo^?Luminescent cell viability detection kit.The cell inoculation dose,infection dose and infection time were cross-tested to determine the suitable parameters for high throughput screening of small molecular drugs.2.In order to find potential candidate active small molecular compounds,the above parameters for high throughput screening of anti-EV71 small molecular drug were used to evaluate the antiviral activity of FDA library in vitro.The active remdesivir was selected to further verify the anti-EV71 activity.3.RD cells infected with EV71 were treated with different concentrations of remdesivir（2.5,0.8,0.3,0.1,0.03,0μM）and used as 2.5,0.8,0.3,0.1,0.03μM remdesivir groups and virus control group.Samples were collected at 30h,24h,and q RT-PCR（Quantitative real-time PCR）,Western Blotting and immunofluorescence assays were used to systematically evaluate and verify anti-EV71 activity of remdesivir in vitro at RNA and protein levels.4.The RD cells infected with EV71 were treated respectively with 0.8μM remdesivir and 3μM NITD008 at different stages of virus infection,and 3μM NITD008 was used as positive control.Intracellular RNA was collected at 30h and EV71 RNA copy numbers were detected by q RT-PCR assay.5.The lethal infection model of 1-day-old ICR sucking mouse infected with EV71 was established and divided into the placebo group and remdesivir groups（3mg/kg and 1.5mg/kg groups）and administered continuously for two weeks.The morbidity and survival of mice were observed and recorded.In addition,the tissue viral load was detected by q RT-PCR assay to further evaluate its antiviral ability in vivo.6.ADMET Predictor 8.5 software was used to predict the blood-brain barrier permeability of Remdesivir.The ability of the drug to pass through the blood-brain barrier was predicted qualitatively and quantitatively.Results1.The RD cell line,which is a sensitive to enterovirus,was infected with100 TCID₅₀virus.By controlling the two key parameters of S/B value≥3 and0.5≤Z’factor value≤1,the detection end point was determined on 72h after virus infection.The parameters suitable for high throughput screening of anti-enterovirus small molecular drugs were as follows:the number of cells inoculated:1×10⁵cells/well;infection dose:100 TCID₅₀;detection time:72 hours after infection.2.Based on the successful screening model of anti-EV71 drugs in vitro,30 compounds with good anti-EV71 activity were found.Remdesivir selected and it′s anti-EV71 activities in vivo and in vitro were explored in depth.3.Compared with virus control group,RD cells infected with EV71 were treated with different concentrations of Remdesivir（2.5,0.8,0.3,0.1,0.03μM）.The expression of intracellular EV71 RNA was reduced in 2.5,0.8,0.3,0.1 drug treatment group.The results of Western Blotting detection showed that2.5,0.8,0.3,0.1μM remdesivir inhibited the expression of EV71 VP1 in a concentration-dependent manner at 24h,and immunofluorescence assay showed that 2.5,0.8μM remdesivir could significantly inhibit the expression of EV71 VP1 at 30 h.4.Compared with virus control group,0.8μM remdesivir could significantly inhibit the expression of EV71 RNA at the virus replication stage.5.Compared with the placebo group,the survival rate of mice in 3mg/kg and 1.5mg/kg drug treatment group has a certain improvement,but there was no significant difference between any two groups.Compared with the placebo group,the viral load in the lung and muscle tissue was decreased significantly in the 3mg/kg group,while the viral load in the brain tissue was not decreased.6.The prediction results of ADMET Predictor 8.5 software show that:1)the qualitative prediction result is Low（43%）,that is,it is not easy for remdesivir to pass through the blood-brain barrier;2)the quantitative prediction result is Log BB=-1.637,Log BB<-0.1,indicating that the blood-brain barrier permeability of the compound is low.ConclusionsThe in vitro screening model of against EV71 drugs was successfully constructed based on RD cell,which laid the foundation for high throughput screening of against EV71 compounds and searching for potential against EV71 drugs.Based on ICR suckling mice,a lethal model of EV71 infection was successfully established to evaluate the efficacy of drugs against virus infection in vivo.Remdesivir has a good antiviral activity against enteroviruses represented by EV71 in vitro,and it mainly plays an antiviral role in the stage of virus replication.After treatment with remdesivir,the survival rate of Suckling mice was improved to a certain extent,and the viral load in the lung and muscle tissue of infected Suckling mice was significantly reduced,suggesting that remdesivir has a certain therapeutic potential in animal level.