Construction Of A Replicon And Pseudovirus Packaging System For Nienokoue Virus

Nienokoue virus(NIEV)is a mosquito flavivirus belonging to the genus Flavivirus in the family Flaviviridae.Flaviviruses are dicided based on their mode of transmission as insect-specific flaviviruses,mosquito-borne flaviviruses,tick-borne flaviviruses,and noknown vectors flaviviruses.NIEV are classical insect-specific flaviviruses whose infection hosts are restricted to insects;mosquito-borne flaviviruses and tick-borne flaviviruses use insects as vectors for transmission of infection.At present,the pathogens of some important epidemics affecting animal breeding industry belong to mosquito-borne flaviviruses,among which Japanese encephalitis virus and duck Tembusu virus have caused incalculable losses to our breeding industry.Among many means of prevention and control,blocking the transmission route of the virus can be a new idea for prevention and control.Since NIEV only proliferates in mosquitoes,resolving the replication mechanism of NIEV can provide key information for the prevention and treatment of mosquito-borne flavivirus.Replicons,pseudovirus packaging systems and infectious clones,as molecular tools for flavivirus reverse genetic manipulation,can be used for vaccine strain development and antiviral drug screening for mosquito-borne transmission interruption.To serve the needs of molecular design platform for mosquito-borne transmission-blocking vaccine strains of flaviviruses,this thesis establishes a series of laboratory tools for NIEV,including: NIEV NS1 polyclonal mouse antiserum,NIEV reporter replicon carrying Nano Luc luciferase or m Cherry fluorescent protein,and NIEV pseudovirus packaging system,which are briefly described as follows:1.Preparation of NIEV NS1 polyclonal mouse antiserumTo prepare the NIEV NS1 polyclonal mouse antiserum,the NS1 prokaryotic expression plasmid p ET28-NS1 was constructed in this thesis.p ET28-NS1 was transformed into E.coli BL21 for induction expression,and then the induction expression time,temperature and IPTG concentration of NS1 expressing bacteria were optimized.The results showed that the highest expression of NS1 recombinant protein was induced at 37℃and 0.4 m M IPTG for 2 h;the NS1 recombinant protein was expressed in the inclusion bodies.The NS1 recombinant protein was solubilized with 8 M urea and affinity purified using a nickel column.The purified protein was immunized to SPF Kunming mice,and the mouse serum was collected at the end of 4 immunizations to obtain NS1 polyclonal antiserum.Finally,the antibodies were specifically detected by Western blot and indirect immunofluorescence assay,and the results showed that the NIEV NS1 polyclonal mouse antiserum could specifically recognize the eukaryotic expression of NIEV NS1 protein.2.Construction and application of NIEV repliconIn order to monitor the genomic RNA replication of NIEV,NIEV replicons were constructed in this thesis.For the flavivirus replicon to initiate transcription and form true5’ ends in mosquitoes and mammalian cells,respectively,it is necessary to screen for a usable promoter.In this thesis,reporter plasmids were constructed to initiate the expression of reporter genes e CFP and Nano Luc with CMV,EF1α,SV40,Ubi and Op IE2 promoters,respectively,and the efficiency of the different promoters was clarified by detecting the expression of the reporter genes.The results showed that the most suitable promoters on C6/36 and BHK21 cells were CMV and Op IE2,respectively.Next,replicons carrying different promoters and different reporter genes were constructed(Op IE2-NIEV-NLuc-rep,Op IE2-NIEV-m Cherry-rep,CMV-NIEV-NLuc-rep,CMV-NIEV-m Cherry-rep)and the corresponding replication-deficient replicons.The results showed that the NIEV replicon could be translated and replicated on C6/36 cells,and only translated but not replicated in BHK21 cells.The NIEV replicon was further applied to antiviral drug screening,and the results showed that saquinavir and ribavirin could inhibit the NIEV replicon work,and the inhibition effect was positively correlated with the dose.The above indicates that the NIEV replicon system constructed in this thesis can respond to the replication of NIEV genomic RNA and can be applied to the screening of antiviral drugs.3.Construction of NIEV pseudovirus packaging systemIn order to monitor the adsorption and entry of NIEV,a NIEV pseudovirus packaging system was constructed in this thesis.NIEV replicon and viral structural protein NIEVCpr ME eukaryotic expression plasmid were co-transfected into cells and assembled to form single round infectious particles(SRIPs).To enhance the packaging efficiency of SRIPs,the ratio of the two plasmids and the collection time after transfection were optimized in this thesis.The results showed that the highest packaging efficiency was achieved by transfecting the packaging plasmid and replicon into cells with a 1:5 plasmid ratio and collecting SRIPs after 120 h of transfection.Based on this,this thesis further constructed the stable cell lines expressing NIEV-Cpr ME,and packaging using the stable cell line is more conducive to the mass production of SRIPs.The SRIPs were infected with different cells and found to infect C6/36 cells but not BHK21 cells,indicating that the SRIPs packaged in this thesis can work properly.The establishment of the NIEV pseudovirus packaging system provides the necessary research tools for the development of flavivirus mosquito-borne transmission-blocking vaccine strains.In summary,the NIEV replicon and pseudovirus packaging system were successfully established by optimizing the conditions of promoter,transfection ratio and transfection time.It is clear that it can be applied to the screening of NIEV antiviral drugs and the study to reveal the proliferation characteristics of NIEV,which can provide the necessary research tools for the development of flavivirus mosquito-borne transmission blocking vaccine strains.