Establishment Of Drug Screening System For Zika Virus And Dengue Virus And Efficacy Evaluation Of DAA Drugs

Zika virus(ZIKV)and dengue virus(DENV)are the most common arboviruses,which infect mammals and humans majorly through the bite of arthropods,especially Aedes aegypti or Aedes albopictus,and spread by blood and sex among people.In recent years,ZIKV and DENV have shown a severe global epidemic.It was reported that more than 100 countries and regions around the world have reported cases of zika virus transmission,and nearly 4 billion people in 128 countries are at risk of dengue virus infection.Yunnan Province on the southwest border of China is constantly threatened by imported and locally infected ZIKV and DENV from neighbored Southeastern Asian countries.So far,there are no vaccines against ZIKV and DENV,and no specific therapeutic drugs have been approved.Mosquito eradication anting biting and clinically indicated treatment are the main countermeasures to deal with these virus infections.Thus,the development of specific drugs for ZIKV and DENV is urgent.The use of approved direct-acting antiviral(DAA)drug is one of the important strategies in the research of new virus antiviral drug.It is possible that DAA drugs expand its applicability to the flaviviruses for treating the Zika and Dengue fever.In this study,we aim to establish a method system for drug screening in vitro against ZIKV and DENV,and compare target proteins through bioinformatics and conduct molecular docking with candidate DAA compounds.Based on the analysis results,anti-virus effect of the tested DAA compound is carried out in this established screening system to provide experimental data for clinical medication.First of all,a method system for drug screening in vitro against ZIKV and DENV was established.BHK-21 cells were plated with 1×10~4cells/well and cultured overnight,the positive drug Ribavirin was added.It was showed the 50%cell cytotoxic(CC50)for 48 h and 72 h were 240.5μM and 289μM.After the cells were plated overnight,the optimal infection amount of Zika virus and Dengue virus with 1×10~5copies/well incubated for 2 h,and then Ribavirin was added.It was showed the 50%effective concentration(EC50)of anti-ZIKV for 48 h and 72 h were 1.616μM and3.439μM.The EC50 of anti-DENV2 for 48 h and 72 h were 5.919μM and 0.2739μM.The selection index(SI)of Ribavirin to ZIKV was 148.5 at 48 h and 86.25 at 72 h,and the SI of Ribavirin to DENV2 was 40.63 at 48 h and 1087.99 at 72 h.Furthermore,the basic characteristics and structural comparison of the target proteins in common flaviviridae virus was theoretically analyzed,and the candidate DAA compounds with the target proteins of ZIKV and DENV2 were subjected to molecular docking and interaction analysis.The results showed that the secondary structure and distribution of the polymerase proteins in the flavivirus were more similar than the protease proteins.The tertiary structure of the protease of ZIKV,YFV,and WNV were similar,and the tertiary structure of the polymerase of ZIKV,DENV2,and JEV were similar.The docking bag of HCV protease and polymerase target protein binding with small molecule ligand was suitable for the flavivirus.The proteases of ZIKV,WNV,and JEV had a close evolutionary relationship,and the proteases of DENV2 and YFV had a close evolutionary relationship,and the polymerases of ZIKV,DENV2,HCV,and YFV had a close evolutionary relationship.Lopinavir,Paritaprevir,Sofosbuvir and Dasabuvir were identified as the tested DAA compounds by the molecular docking results.With interaction analysis,the binding of ZIKV protease with its inhibitors were more stable than that of DENV2,and the binding of Sofosbuvir to the polymerase target proteins of ZIKV and DENV2 were more stable than that of Dasabuvir.At last,the anti-ZIKV and anti-DENV2 effects of the tested DAA compounds were evaluated in vitro.The drug selection index was initially obtained,and the virus inhibition of different drug action time,virus infection time,virus infection dose and other aspects were conducted by the optimal drug concentration,and the effect of multi-target combination medication were evaluated by the drug half effective concentration.The results showed that the SI of Lopinavir,Paritaprevir,Dasabuvir,and Sofosbuvir to ZIKV at 48 h were 1466.92,1214.67,949.15,5.87,and the SI of Lopinavir,Paritaprevir,and Dasabuvir to DENV2 at 48 h were 540.68,721.13,1173.40,and the SI of Sofosbuvir to DENV2 at 72 h was 14.98.These four DAA compounds showed low toxicity and high efficiency for ZIKV and DENV2.The optimal drug concentration of the compound was further analyzed.As the drug action time prolonged,the four DAA compounds still showed good antiviral effect on ZIKV,while the viral inhibition rate of DENV2 showed a downward trend,in which the anti-DENV2 efficiency of the polymerase inhibitors were better.As the virus infection time prolonged,the inhibition rate of the four DAA compounds to ZIKV and DENV2were decreased.Sofosbuvir was the best drug for long-term infection.With the increase of the virus concentration,the inhibition rate of four DAA compounds to ZIKV decreased,and the polymerase inhibitors still had obvious antiviral effects at high dose of DENV2.The combination of Lopinavir and Ribavirin or Dasabuvir had the best therapeutic effect on ZIKV,the combination of Paritaprevir and Sofosbuvir had better anti-ZIKV effect,the combination of Lopinavir is not effective to anti-DENV2,and the combination of Paritaprevir and Ribavirin or Sofosbuvir had a better inhibitory effect on DENV2.In summary,this study has successfully established a method system for drug screening in vitro against Zika virus and Dengue virus.From a theoretical point of view,it is revealed that the flaviviridae virus uses protease and polymerase as drug targets,and their structures are similar,which indicates that DAA drugs can be used to treat the flavivirus.The experimental data verified the drug effects of Lopinavir,Paritaprevir,Dasabuvir,and Sofosbuvir to ZIKV and DENV.Provide reference for the clinical medication of ZIKV and DENV.