LAUSR

ABSTRACT West Nile virus (WNV), a mosquito‐borne flavivirus, is an important neurotropic human pathogen. As a biosafety level‐3 (BSL‐3) agent, WNV is strictly to BSL‐3 laboratories for experimentations, thus greatly hindering the development of vaccine and antiviral drug. Here, we developed a novel pseudo‐infectious WNV reporter virus expressing the Gaussia luciferase (Gluc). A stable 293TNS1 cell line expressing NS1 was selected for trans‐supplying NS1 protein to support the replication of WNV‐&Dgr;NS1 virus and WNV‐&Dgr;NS1‐Gluc reporter virus with large‐fragment deletion of NS1. WNV‐&Dgr;NS1 virus and WNV‐Gluc‐&Dgr;NS1 reporter virus were confined to complete their replication cycle in this 293TNS1 cell line, displaying nearly identical growth kinetics to WT WNV although the viral titers were lower than those of WT WNV. The reporter gene was stably maintained in virus genome at least within three rounds of passage in 293TNS1 cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo‐infectious WNV‐Gluc‐&Dgr;NS1 could be used for antiviral screening. Furthermore, a high‐throughput screening (HTS) assay in a 96‐well format was optimized and validated using several known WNV inhibitors, indicating that the optimized HTS assay was suitable for high‐throughput screening WNV inhibitors. Our work provides a stable and safe tool to handle WNV outside of a BSL‐3 facility and facilitates high throughput screening for anti‐WNV drugs. HighlightsA stable 293TNS1 cell line expressing NS1 was established for trans‐supplying NS1 protein.Pseudo‐infectious WNV‐Gluc‐&Dgr;NS1 reporter viruses were confined to complete replication cycle in the 293TNS1 cell line.The Gluc reporter gene was stably maintained in genome of the WNV‐Gluc‐&Dgr;NS1 reporter virus during passage.The WNV‐Gluc‐&Dgr;NS1 reporter virus could be used for antiviral screening.