Mechanism Of Lycorine Inhibiting Zika Virus Replication

Objectives:Zika virus is neurotrophic and teratogenic.Infection with the virus during pregnancy may lead to neonatal microcephaly,and may also cause guillain-barre syndrome,which seriously harms human health^[1-3].However,there are currently no special drugs for the prevention and treatment of ZIKV infection,and there is an urgent need to develop safe and effective antiviral drugs.In recent years,according to the research on antiviral drugs,it has been found that drugs derived from natural plants have great advantages in antiviral.Therefore,this topic mainly studies the antiviral effect through in vivo and in vitro experiments and mechanism of lycorine on zika virus.Methods:1.Study on anti-ZIKV activity of lycoris extractAfter 48h of ZIKV infection in Vero cells,the content of viral RNA?vRNA?in the cells was detected by quantitative real time polymerase chain reaction?qRT-PCR?,and the antiviral activity of lycoris extract was verified.2.Study on anti-ZIKV activity of lycorine in vitroqRT-PCR was used to detect the content of vRNA in Vero,Huh7 and A549 cells infected with ZIKV for 48h.The effects of lycorine on viral proteins were detected by western blot and immunofluorescence assay,and the effects of lycorine on the number of viral progeny were detected by plaque assay,so as to verify the antiviral activity of lycorine.3.Study on the mechanism of lycorine inhibiting ZIKV replicationAfter the cells were infected with the virus,the drugs were added at different times?-2,0,1,2,4,6,10 hpi?.qRT-PCR was used to detect the level of vRNA in the cells,the number of viral progeny was detected by plaque assay,and the expression of viral proteins was detected by western blot,respectively,to verify the effect of lycorine on the replication stage of ZIKV.Lycorine was added at 6h and 24h after infection and withdrawn at 6,9,12,15,18,21,24 hpi and 12,18,36 hpi,respectively,use the qRT-PCR detection of vRNA levels in cell.ZIKV E and NS5 protein as an index,by detecting the expression of viral proteins,indicating the lycorine effects on virus replication.qRT-PCR was used to detect the positive and negative strand content of vRNA and to prove the effect of lycorine on the positive and negative strand of vRNA.Cellular Thermal Shift Assay?CETSA?was performed to elucidate the interaction between lycorine and ZIKV NS5protein.In vitro RNA dependent RNA polymerase?Rd Rp?assay was used to detect Rd Rp activity to elucidate the effect of lycorine on Rd Rp.Autodock molecular docking technology was used to predict the molecular docking between lycorine and Rd Rp,indicating the possible binding sites of lycorine and Rd Rp.4.Study on the antiviral activity of lycorine in mice infected with ZIKVA model of virus-infected AG6 mice was constructed by injecting into the foot pad and treated with high,medium and low dose of lycorine every day for 14 days,and a blank control group and a virus group were set.During the experiment,the changes in body weight and survival rate were observed.The fundus venous plexus blood was given for 1 and 3 days to detect the virus content in the blood.The contents of vRNA were detected in the brain and liver tissues of mice.Results:1.Use qRT-PCR to prove that the extract of lycoris shows dose-dependent inhibition of viral RNA content in cells and has anti-Zika virus activity.2.Study on anti-ZIKV activity of lycorine in vitro?1?The CC₅₀ of lycorine in Vero,A549 and Huh7 cells were21,5.39and 27.4?M,respectively.?2?The EC₅₀of lycorine in Vero,A549 and Huh7 cells were 0.39,0.35and 0.24?M,respectively.?3?Immunofluorescence assay and western blot results showed that lycorine inhibited the synthesis of ZIKV proteins,and complete protein synthesis was achieved when the concentration was greater than 5?M.?4?Plaque experiment results showed that lycorine inhibited the formation of viral progeny,and no plaque was formed when the concentration was greater than 1.75?M.3.Study on the anti-ZIKV mechanism of lycorine?1?Time-of-addition assay showed that lycorine acted on the late stage of viral infection.?2?Lycorine inhibited vRNA replication and protein synthesis at 6h after viral infection,and did not inhibit vRNA replication and protein synthesis at 24h after infection,indicating that lycorine acted on the replication stage of ZIKV genome.?3?Lycorine inhibited the formation of positive and negative chains of vRNA,and the inhibition rate of positive and negative chains was 93%and 95%respectively at 48h after viral infection.?4?CETSA showed that lycorine increased the value of NS5 Tagg from 44.6?to 47.8?,increasing protein stability.?5?In vitro enzyme activity experiments proved that lycorine inhibited Rd Rp activity.4.Study on the antiviral activity of lycorine in mice infected with ZIKV?1?The survival rate of mice in the high,medium and low dose groups increased by 83%,66%and 33%,respectively.?2?Lycorine reduced vRNA of blood at day 1 and 3 of viral infection.?3?Lycorine was reduce vRNA in brain and liver tissues.Conclusion:This study found that lycorine has anti-zika virus activity both in vivo and in vitro.Lycorine inhibits zika virus replication by inhibiting Rd Rp activity and exerts anti-viral effects.Our research proved the anti-zika virus activity and the anti-zika virus mechanism of lycorine,which provides a basis for lycorine as a candidate drug against zika virus and provides a basis for elucidating the mechanism of lycorine against zika virus.