Construction And Application Of Reverse Genetics System Of Genotype Ⅰ And Ⅲ Japanese Encephalitis Viruses Through Transformation Associated Recombination In Yeast

The Japanese encephalitis virus(JEV)is a mosquito-borne natural focal zoonotic pathogen that can cause reproductive disorders in pigs and nervous system diseases in humans.In pigs,it mainly manifests as orchitis in boars and abortion,stillbirth,and mummified fetus in pregnant sows.In humans,it can cause fever,vomiting,memory loss and cognitive impairment.JEV infection poses a great threat to the global pig industry and human public health safety.JEV has five genotypes,including genotypes GⅠ,GⅡ,GⅢ,GⅣ,and GⅤ.Before the 1990s,the main epidemic JEV strains in Asia belonged to GⅢ,especially in China.However,in the past 20 years,there has been a phenomenon of JEV epidemic strains shifting from GⅢ to GⅠ in many Asian regions.All marketed vaccines are developed based on GⅢstrains and can only provide partial cross-protection against GI strains.Currently,there is no specific treatment or medication for JEV.Therefore,it is urgent to develop new vaccines and antiviral drugs for GⅠ and GⅢ JEV strains.Reverse genetics is an efficient tool for the investigation of viral replication and pathogenesis,vaccine development,and novel reporter viruses for antiviral drug screening.To establish efficient and stable JEV reverse genetics systems for the GⅠ and GⅢ JEV strains,four pairs of primers anchoring the genomic regions conserved between the GⅠ JEV strain(YZ-1)and the GⅢ JEV strain(HSY-1)were designed to amplify four overlapping DNA fragments covering the complete viral genomes.For each virus,four cDNA fragments were assembled with a linearized TAR vector through transformation-associated recombination(TAR)technology in yeast.The assembled full-length cDNA infectious clones of GⅠ and GⅢJEV were named TAR-rGⅠ and TAR-rGⅢ.Subsequently,using linearized TAR-rGⅠ and TARrGⅢ as templates,the full-length genomic RNAs were synthesized by in vitro transcription driven by the T7 promoter.Based on the cytopathic effect and viral protein expression detected by IFA,both rGⅠ and rGⅢ were successfully rescued by RNA transfection of BHK-21 cells.The results of multi-step growth curves showed that the rescued viruses(rGⅠ and rGⅢ)had similar growth characteristics to their parental viruses.The size and morphology of plaques formed by the rescued viruses were also consistent with those of their parental viruses.Furthermore,the yeast and E.coli.containing the full-length infectious clone plasmids were serially passaged ten times,and the genetic stability of TAR-rGⅠ and TAR-rGⅢ was confirmed by RFLP analysis and Sanger sequencing.These results indicate that the GⅠ and GⅢ JEV reverse genetic based on TAR technology are efficient,stable,and easy to operate.Since viral replication could be monitored by virus-encoded fluorescent protein,reporter viruses expressing a fluorescent protein are powerful tools for high-throughput screening of antiviral drugs.To generate JEV reporter viruses,we inserted an EGFP reporter gene into TAR-rGⅠ and TAR-rGⅢ by homologous recombination respectively.The in vitro transcribed viral RNA was transfected into BHK-21 cells to rescue reporter viruses rGⅠ-EGFP and rGⅢEGFP.The recovery of the reporter viruses was indicated by green fluorescence and the cytopathic effect of the transfected BHK-21 cells.Compared with their parental viruses,reporter viruses grew to lower virus titers and formed smaller plaques.Reporter viruses rGⅠEGFP and rGⅢ-EGFP produced strong green fluorescence in infected BHK-21 cells which correlated with virus titers.In addition,rGⅠ-EGFP and rGⅢ-EGFP remain stable after ten passages in vitro.In conclusion,the green fluorescence produced by the reporter viruses could be used to monitor viral replication of GⅠ and GⅢ JEV.By optimizing infection dose and timing to end infection,an antiviral screening platform based on rGⅠ-EGFP and rGⅢ-EGFP was successfully established.The platform was validated by using the known JEV antiviral drug Nitroxoline.Moreover,we further used this platform to analyze the antiviral effect of four small molecule drugs(dihydroartemisinin,ARDP006,melatonin and digitonin)on JEV,and dihydroartemisinin and ARDP0006 exhibited antiviral effects on GⅠ and GⅢ JEV.In conclusion,we successfully established stable reverse genetics for GⅠ and GⅢ JEV using the TAR technology.With these reverse genetics,the reporter viruses rGⅠ-EGFP and rGⅢ-EGFP stably expressing green fluorescent protein were obtained for the antiviral screening platform for GⅠ and GⅢ JEV.Using the fluorescence-readout screening system,we identified dihydroartemisinin and ARDP0006 as antiviral compounds against GⅠ and GⅢ JEV.This study provides an important basis for the prevention and control of multiple JEV genotypes.