Japanese Encephalitis Virus Reporter Virus For High-content Antiviral Screening

Japanese encephalitis virus(JEV)is an arbovirus that belongs to the genus Flavivirus of the Flaviviridae and is the main pathogen of Japanese encephalitis.JEV is mainly popular in Asia Pacific,more than 3 billion people in the region are at risk of JEV transmission.Although the use of vaccines has greatly reduced the incidence of epidemic encephalitis,there are still 67,000 cases of encephalitis in the world every year.The death rate of the disease is as high as 30%,and most survivors are accompanied by severe neurological diseases,which are a serious threat to human health.At present,there are still no drugs and special treatments that can treat JEV infection.Therefore,it is especially important for the screening and development of antiviral drugs.The reported virus is a virus with a reporter gene in the viral genome,which has a similar life cycle as the wild type virus.The growth of the virus can be observed by detecting the expression of the reporter gene.Therefore,it is possible to screen antiviral drugs against the entire life cycle of a virus by detecting the expression of the reporter gene,which is more convenient and faster than the conventional method.In addition,the reported virus can also be used for in vivo imaging to observe the growth and spread of the virus,which plays an important role in studying the pathogenic mechanism of the virus and the evaluation of the drug.In this study,we inserted the eGFP gene into the 5’ UTR and Capsid of the viral genome by fusion PCR to construct a full-length infectious clone of the JEV-eGFP reporter virus.The viral RNA is then transfected into sensitive cells,and the rescued JEV-eGFP reporter virus is able to replicate and spread in the cells.Studies on the growth characteristics and stability of JEV-eGFP revealed that the growth curve of the reporter virus was similar to that of the wild type and was stable in cells for 4 generations.Inhibitory effects of NITD008 on JEV-eGFP reporter virus showed that the JEV-eGFP reporter virus was similar to the NITD008 inhibition curve of JEV-WT.It was demonstrated that the JEV-eGFP reporter virus can be used for antiviral drug screening and can characterize the inhibitory effect of the drug on the virus by observing the change in the fluorescence intensity of eGFP and the number of positive cells expressing eGFP.To establish a high-throughput drug screening method that is resistant to JEV virus,we constructed a high-content antiviral drug screening platform using JEV-eGFP.Using this platform,we conducted a high-content screening of anti-JEV viruses based on FDA-approved drug libraries.The FDA drug library contains 1,443 antiviral compounds,all of which are clinically available,so the drugs that are screened against the JEV virus are highly likely to be used clinically.Through initial screening and re-validation,we screened some drugs that could potentially inhibit JEV from the FDA drug library.Six of these drugs have been shown to have potential antiviral effects against wild-type viruses.Prove the reliability of the screening system we built.In addition,the newly discovered seven drugs showed better antiviral effects for JEV infection.The above results demonstrate that the reporter virus and high-content antiviral drug screening platform we constructed can be well applied to the screening of antiviral drugs.