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In this study, a viral-cDNA-based replicon of Japanese encephalitis virus (JEV), which is the causative agent of Japanese encephalitis, was constructed by replacing the viral structural proteins with a green fluorescent protein (JEV-GFP replicon). The resulting JEV-GFP replicon was used as a tool to screen antiviral drugs targeting JEV nonstructural proteins, and the five compounds JNJ-A07, HZ-1157, NITD-2, quinine, and NITD008 were obtained, which significantly inhibited the replication of the JEV-GFP replicon and JEV in vitro, and the properties of these five compounds were also analyzed. The CC50, EC50, and SI indices of these five compounds were analyzed. In addition, the JEV-GFP replicon was used as a tool to identify the residues of viral nonstructural proteins involved in RNA replication, and the cysteine residue at position 4 of nonstructural protein 1 was found to be essential for JEV RNA replication. These data suggested that the noninfectious JEV-GFP replicon could be used as tool for different purposes, such as antiviral drug screening and gene function studies.<\/jats:p>","DOI":"10.3390\/v17060759","type":"journal-article","created":{"date-parts":[[2025,5,29]],"date-time":"2025-05-29T03:30:19Z","timestamp":1748489419000},"page":"759","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development of a Noninfectious Japanese Encephalitis Virus Replicon for Antiviral Drug Screening and Gene Function Studies"],"prefix":"10.3390","volume":"17","author":[{"given":"Yang","family":"Yang","sequence":"first","affiliation":[{"name":"Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, 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