Construction Of Zika Virus Replicon Trans-Packing System And Drug Screening Applications

Zika virus(ZIKV)is an insect-borne virus,Dengue virus,Yellow Fever virus,and West Nile virus are all single positive-strand RNA viruses,which belong to the genus Flavivirus,Flaviviridae.Since it has caused outbreaks and epidemics in many countries and regions and has been associated with severe clinical manifestations and congenital malformations.However,no effective antiviral drugs and vaccines are available for the clinical treatment of ZIKV at present.Therefore,the research on safe and effective ZIKV antiviral drugs and vaccines are urgent.Replicon system is a useful tool for antiviral drug screening.In this paper,we mainly carry out the following three experiments: Construction of ZIKV replicon stably expressing Renilla luciferase(Rluc),Replicon for anti-viral drug screening experiments,Development of ZIKV replicon trans-packaging system.I.Construction of ZIKV replicon stably expressing Renilla luciferase(Rluc)In this study,we constructed a ZIKV replicon(Rluc-ZIKV-Rep)based on the cDNA of the ZIKV infectious clone.Renilla luciferase gene was introduced into the replicon.The expression and replication of the replicon in the cells can be easily detected by measuring the Rluc activity and RT-PCR.II.Replicon for anti-viral drug screening experimentsSeveral small-molecule compounds,particularly Manidipine and Cilnidipine,were used in this antiviral screening.The replication of Rluc-ZIKV-Rep was inhibited dose dependently with increasing concentration of these compounds.In conclusion,we demonstrated that the ZIKV replicon is a useful tool for antiviral screening.III.Development of ZIKV replicon trans-packaging systemIn this article,we used the reverse genetics method to construct an eukaryotic expression vector containing the ZIKV structural proteins —— CprME and verified its expression in two different cells(293T and Vero)by Western Blot.To prepare replicon packaging particles,we transfected the replicon and the plasmid expressing the structural proteins into the cells by co-transfection and sequential transfection,so that the genes that encode structural and non-structural proteins are co-expressed in the cells.Vero cells are then continuously infected by the supernatant of the collected cells,and then the production of defective virus packaged by the replicon was examined by measuring the luciferase activity in the cells.On this basis,we can prepare defective virus-like particles through 293 T cells,which will lay a foundation for the research of the new ZIKV vaccine.