Establishment Of The Screening Model With Anti-zika Virus Drugs And Research Of Anti-zika Virus Tranditional Chinese

Purpose:In order to establish the cells and mice models of Zika virus(ZIKV)infection for our laboratory,determination of the anti-Zik V activity of various major active compounds of Scutellaria baicalensis Georgi have been done.We expect to establish an evaluation of traditional Chinese medicine extracts and small molecule compounds against Zik V infection and their pharmacological efficacy in the cells and mice aspects,screening the anti-Zik V drug with high efficiency and low toxicity,which laid the foundation for further research on the pharmacodynamic mechanism of anti-Zik V.Method:1.Incubate C6/36 cells to expand Zik V and determine the titer of ZIKV by plaque assay.2.Vero and Hela cells were infected with the ZIKV,detect the RNA level at 48h after virus infection by real-time quantitative PCR.Western blotting was used to detect the expression of viral protein levels,and appropriate infection titers were confirmed.3.ZIKV infected intraperitoneally in BALB/c and C57BL/6immunocompetent mice and C57BL/6 immunodeficient mice.Daily weight and status were recorded.Fundus venous plexus blood was taken to detect viremia from 1-4d after challenged.4.CCK-8 was used to determine the cytotoxicity of drugs in Vero.and real-time quantitative PCR was used to determine the activity of the drug against Zika virus.Vero cells were incubated with drugs for 1h.After incubation,Zika virus with MOI=0.05 was added.After infection for 1h,the drugs were added again and the virus RNA was determined after 48h.Result:1.The titer of Zik V amplified by C6/36 was 1×10~7 pfu/m L determine by plaque assay.2.Vero and Hela can be infected with the ZIKV,the expression of RNA and viral protein levels were remarkable determining by q PCR and Western blotting after 48h infection in Vero with MOI=0.05 and Hela with MOI=0.5.3.BALB/c and C57BL/6 immunocompetent mice infected with ZIKV infection status and weight were not significantly changed,nor viremia.C57BL/6immunodeficiency mice infected with ZIKV showed the viremia present in 1d and arrived peak in 2d,the weight gradually decreased with days of infection,until the death or the end of the experiment.4.Baicalein CC50=43.3μM except,both Baicalin,Wogonoside and Scutellarin CC50>400μM.Baicalin,Baicalein and Scutellarin showed anti-ZIKV activity except Wogonoside which could not inhibit the replication of ZIKV.The EC50 of Baicalein,Baicalein and Scutellarin were 0.1μM,5.6μM and 58.0μM respectively.Conclusion:1.ZIKV can infect Vero and Hela cells,and can be detected the replication of Zika virus by q PCR and Western blotting after 48h infection.The suitable titers of anti-ZIKV screening in Vero and Hela cells are(multiplicity of infection)MOI=0.05,MOI=0.5.2.ZIKV can not infect BALB/c and C57BL/6 immunocompetent mice,it can infect C57BL/6 immunodeficiency mice and present with a series of infection symptoms such as weight loss,viremia and even death.3.Baicalein,Baicalin showed remarkable anti-Zik V infection activity.Further animal experiments should be done for screening the frist choice to reseach anti-ZIKV pharmacological effects of drugs.