The Construction Of Reporter Recombinant Zika Virus And Its Application In Screening Antiviral Drugs

Zika virus?ZIKV?is an important zoonotic pathogen,which is transmitted by Aedes mosquitoes.Rabbits,pigs and human beings are susceptible to it.After infection,there are no obvious symptoms.However,if ZIKV infection occurs in the period of pregnancy,it may lead to birth defects in the newborn and contribute serious clinical symptoms such as miscarriage and premature birth.At present,ZIKV is a worldwide disease.Studies have shown that newborn piglets are susceptible to ZIKV,which may pose a imperative economic loss to the pig breeding industry.There are no specific therapeutic drugs and vaccines for ZIKV on a global scale.Therefore,it is extremely urgent to take actions to prevent the prevalence of ZIKV.Here,the ZIKV C,prM and E genes,which come from the ZIKV?FSS13025 strain?gene published by GenBank,were cloned into pMAL-c5x vector by codon optimization technique.The above three plasmids were transformed into E.coli ER2523 cells,respectively.After propagating the transformed cells to reach an OD₆₀₀ number between 0.6 and 0.8,adding the IPTG of 1.0 mM into it,and then keeping it for 4 hours.The protein of ZIKV C,prM and E were verified by SDS-PAGE and western blot trials and purified by MBP affinity chromatography column.After being fully emulsified with MONTANIDETM ISA 61 VG adjuvant at a weight ratio of 6:4,it immunized New Zealand white rabbits with an immunization dose of 300 ug each occasion.The results of IFA and Western blot showed that the polyclonal antibodies were successfully prepared.In order to rescue recombinant ZIKV and reporter ZIKV-GFP virus,here,we successfully constructed the replicon of the recombinant plasmid that based on the ZIKV gene sequence of pZIKVrepdCME-GFP and the plasmid of pCIneo-ZIKV-CME.The PCR amplification products of pCIneo-ZIKV-CME and the pZIKVrepdCME-GFP replicon plasmid linearized by Xhol I were co-transfected into 293T cells.Electron microscopy,plaque assay,western blot and IFA experiments showed that the recombinant ZIKV was rescued successful,and the recombinant ZIKV virus growth curve assay showed that the titer of the recombinant ZIKV virus showed an upward trend between 24 h and 72 h and reached the peak at 72 h,decreasing from 96 h to144 h.Reviews showed that the integrity of flavivirus C protein plays an significant role in replication,we explored the effect of the length of the C-terminal amino acid sequence of ZIKV C protein on viral replication.The retention of the 38-amino acid sequence at the C-terminus of ZIKV C protein can promote the rescuing of the reporter ZIKV-GFP virus.Therefore,we constructed the plasmid of pCIneo-ZIKV-C38-GFP-CME,and the PCR amplification of the plasmid of pCIneo-ZIKV-C38-GFP-CME was transfected into 293T cells with the replicon plasmid of pZIKVrepdCME-GFP.The results of electron microscopy and plaque assay showed that the reporter ZIKV-GFP virus was successfully rescued,and the growth curve assay of the ZIKV-GFP virus showed that the titer of the ZIKV-GFP virus showed an upward trend between 24 h and 72 h and reached the peak at 72 h,decreasing from 96 h to 144 h,too.In this study,the intensity of green fluorescence of the ZIKV-GFP virus showed that the reporter ZIKV-GFP virus showed an uniform fluorescence intensity in BHK-21 cells and stronger than that in Vero cells.However,the reporter ZIKV-GFP virus inoculated A549 and Huh-7 cells that not only required large doses but also generated uneven fluorescence intensity.Therefore,we construct a cell line?BHK-DR?that contains an adhesion factor on the cell surface using the plasmid of pCAG-DC-SIGN in BHK-21 cells,which can enhance the infectivity of envelope virus.The reporter ZIKV-GFP virus has a strong fluorescence intensity in BHK-DR cells,which provides favorable conditions for screening anti-ZIKV drugs by using a high content screening system.The chloroquine,6-Azauridine and ribavirin drugs were validated by using a high-content screening system,and the results were in agreement with the previously description.After carring out antiviral drug screening experiments,we identified that five drugs play an importane role in the aspect of anti-ZIKV from 974 compounds of library.To sum up,The polyclonal antibodies prepared in this study can be used not only for the study of the structure and function of M and E proteins,but also for the establishment of rapid diagnostic methods for ZIKV infection.The successful establishment of platform of reverse genetics for ZIKV laid the foundation for vaccine research,screening and development of antiviral drugs,and exploring the mechanisms between ZIKV and host-related factors.An antiviral drug screening platform was established,and five drugs of natural plant were identified.This study provides an important basis for further research on ZIKV prevention and control.