Development And Application Of Antivirals High Throughput Screening System For Zika Virus And Chikungunya Virus

Arboviruses(Arthropod-borne viruses)are infectious virus transmitted by mosquitoes,ticks and other arthropod vectors.Arboviruses are among the most serious international infectious threats to the human health and can lead to epidemics in both human and animal populations.Since the early 2000 s,two new/re-emerging mosquitoborne viruses,Zika virus(ZIKV)and Chikungunya Virus(CHIKV)have led to global outbreaks and millions of infections that posed a major public health challenge to the world.With no vaccines or antiviral drugs licensed for these two viruses,there is an urgent need to discover and develop specific and efficient antiviral drugs.It is a very effective method to identify antiviral drugs by rapid screening from drug libraries.In this study,an antiviral drug screening platform based on ZIKV replicon cell lines.By using the CHIKV-Δns P3-e GFP high throughput screening platform for antiviral drugs established in our laboratory,we screened different small molecule compound libraries and successfully screened out 6 effective anti-CHIKV inhibitors from 2244 compounds.Furthermore,the antiviral mechanism of two prospective compounds was studied.This study provides technical and theoretical support for the research and development of new drugs for ZIKV and CHIKV,and is of great significance for the research and development of antiviral drugs for these two important arbovirusesThe first part of this thesis deals with antiviral drug screening system based on ZIKV replicon cell lines as an antiviral drug system.In this study,the Zika virus replicon(ZIKV-Pac-Rluc-rep)containing a dual reporter gene was constructed by inserting a PAC-2A-Rluc-2A gene fragment with a replacement structural protein gene between the 5’UTR and NS1 of the ZIKV virus genome.The Zika virus replicon can replicate efficiently in the cell,and Rluc signal can effectively characterize replication.The puromycin screen enabled the establishment of cell lines stably expressing high levels of Zika virus replicon.The optimized high-throughput drug screening system was further validated with several known flavivirus replication inhibitors and finally,the FDA drug library was screened for a reported drug candidate using the highthroughput drug screening system.The acquisition of ZIKV replicant cell lines provides a platform for further study of ZIKV replication mechanism.Meanwhile,the establishment of a high-throughput antiviral drug screening system provides a platform basis and technical support for the new use of old drugs and the research and development of new drugs.The second part of this thesis is the screening of the drug molecular library based on an attenuated CHIKV-e GFP reporter virus-based anti-CHIKV drug screening platform,and the validation of the resulting drug candidates.Firstly,1443 drugs in FDA drug library and 801 small molecule compounds in MCE compound library were screened,and drugs with inhibition rate of more than 90% were selected as preliminary drug candidates.After the first round of preliminary screening and the second round of wild-type virus level verification and cytotoxicity detection,a total of 6 drugs with no obvious toxicity and inhibitory effect on CHIKV-WT were screened from the two drug library.Further through concentration gradient inhibition test,we selected sorafenib,CHL,Atovaquone and Rupatadine,four drugs with good inhibitory effect,to test the virulence of CHIKV pathogenic model C57BL/6 mice.It was found that sorafenib and CHL could effectively inhibit CHIKV infection in mice,and the viremia and foot joint swelling were significantly reduced,indicating that these two drugs have good potential as patent drugs.Therefore,we conducted further studies on the inhibitory effects of sorafenib and CHL,and found that the inhibition of sorafenib and CHL on CHIKV was not cell dependent.Meanwhile,these two drugs had obvious inhibitory effects on different CHIKV strains and a variety of alphavirus viruses.It is suggested that it has the potential to be developed as a broad-spectrum anti-alphavirus inhibitor and has a good application prospect.On the basis of the above studies,we further study the inhibition mechanism of sorafenib and CHL,which is the third part of this paper.We first detected the possible action periods of sorafenib and CHL,and found that sorafenib mainly acted on the replication stage of CHIKV,while CHL acted on multiple stages of CHIKV infection,such as the early translation stage and the replication stage.Further,we screened the resistant strains of sorafenib and CHL in order to explain the drug targets through the resistance sites.After continuous action of drugs,drug-resistant virus strains specific to different drugs were obtained.Sequencing and functional verification of drug-resistant strains showed that mutations of ns P2-N437H+ns P3-90 aa and ns P1-P249 L could effectively improve the replication efficiency of the virus.E2-H351P-K337M-E1-A392 V can improve the entry ability of virus.It has been reported that sorafenib can block the properties of RAF MEK/ERK-mediated cell signaling pathway.We take this opportunity to explore the relationship between CHIKV infection and MAPK signaling pathway.It was found that CHIKV infection can promote the activation of MAPK signaling pathway,and the degree of activation is related to the replication efficiency of CHIKV.Inhibitors of MAPK signaling pathway can effectively inhibit the replication of CHIKV infection.In conclusion,sorafenib and CHL,two drugs with inhibitory effects in both cell and animal levels,were screened from the two drug libraries,and mechanism studies showed that sorafenib acts on the replication stage of the virus,targets ns P2 and ns P3,and is related to MAPK signaling pathway.CHL acts on multiple stages of the virus,targeting the ns P1 and E proteins.The above study results indicate that sorafenib and CHL are expected to be used as new examples of old drugs to develop into broad spectrum antiviral inhibitors.