The Study Of West Nile Virus Infectious Replicon Particles With Green Fluorescent Protein Reporter Gene Generated Using A Packaging-restricted Cell Line

West Nile virus(WNV)is a mosquito transmitted arbovirus which belonging to genus Flavivirus in the family Flaviviridae.It is an arthropod-transmitted virus that mainly affects birds,humans and horses,which is an acute zoonotic infectious disease,Birds especially the crows are the natural hosts of West Nile virus,people generally are susceptible of virus.Its clinical symptoms include fever,encephalitis,even to death.There is currently no vaccine or totally effective treatment for human,we need to do some study for WNV.However,the research on WNV must be under BSL-3 conditions,and it demands the professional quality of personnel and increases serious health risk and cost,restricting the research progress of vaccine and drugs to treat WNV.This study intend to develop West Nile virus infectious replicon particles with green fluorescent protein reporter gene instead of wild virus for studying the vaccine development and antiviral research.To generate a replication-defective West Nile virus expressing green fluorescent protein(GFP)report gene,the full length cDNA of WNV was chemically synthesized with the gfp gene replaced the viral structural proteins(C/PrM/E)encoding sequence according to the sequence of NY99 strain and inserted into the eukaryotic expression plasmid pCI-neo to construct the WNV replicon plasmid of pWNVrepdCME-GFP.Then the virus was rescued by co-transfecting the pWNVrepdCME-GFP with a recombinant plasmid pCAG-WNV-C expressing capsid protein of WNV into envelope protein prM-E expressing BWNV-ME cell line or directly infected the C-prM-E expressing cell line BWNV-CME,replicon defective virus named RRPs capable of initiating a single round of infection were released,yielding titers of up to(>10~6 PFU/mL)in the supernatant of these cells.which displayed GFP expression in the cytoplasm.In addition,the rescued virus in the medium collected from transfected cells was able to infect BHK-21 cells.However,the cell medium from rescued WNV infected and replicated BHK-21cells failed to infect or pass in fresh BHK-21 cells,indicating the RRPs was the replication-defective virus,which only possessed single-round infection.Moreover,directly infected the C-prM-E expressing cell line BWNV-CME,the RRPs was able to replicate,form green fluorescence foci,and exhibit cytopathic plaques similar to that induced by the wild type virus.The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells.The infectious properties of RRPs were tested on flavivirus susceptible cells,including Vero,HEK-293,BHK-21 and SK-N-SH cells.For mouse sera,the results obtained by RRPs PRNT and WNV PRNT demonstrated good concordance,the virus neutralizing antibody level were about:1:80 to 1:160.All goose and horse sera positive in the ELISA were also positive by PRNT,antibody level were about:1:160 to 1:320.For the antiviral assay,ribavirin and6-Azauridine both inhibited the viral titre in a dose-responsive manner.Therefore,the establishment of stable BWNV-CME cell lines facilitates the usage of this WNV replicon system,rendering it a more suitable tool for studying the mechanisms of WNV invasion,vaccine development and antiviral research.