title: Generation and characterization of West Nile pseudo-infectious reporter virus for antiviral screening.
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 293TNS1cell line expressing NS1 was selected for trans-supplying NS1 protein to support the replication of WNV-ΔNS1 virus and WNV-ΔNS1-Gluc reporter virus with large-fragment deletion of NS1. WNV-ΔNS1 virus and WNV-Gluc-ΔNS1 reporter virus were confined to complete their replication cycle in this 293TNS1cell 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 293TNS1cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo-infectious WNV-Gluc-ΔNS1 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.
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Hong-Lei
authors.initials: HL
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Ye
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China.
authors.forename: Cheng-Lin
authors.initials: CL
authors.lastname: Deng
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China.
authors.forename: Si-Qing
authors.initials: SQ
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA; Department of Pharmacology and Toxicology, Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
authors.forename: Cheng-Feng
authors.initials: CF
authors.lastname: Qin
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China. Electronic address: zhangbo@wh.iov.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D017930:Genes, Reporter; D016679:Genome, Viral; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D014902:West Nile virus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*
chemical_list: D000998:Antiviral Agents; D017361:Viral Nonstructural Proteins; C531936:nonstructural protein 1, West Nile virus
keywords: Antiviral screening; BSL-3; Pseudo-infectious virus; Reporter virus; West Nile virus