West Nile Virus Screening Method

Our cell-based panels of assays were developed to screen for inhibitors of flaviviruses. Specifically, our assays could be used to examine if inhibitors of West Nile virus (WNV) also inhibit other flaviviruses such as Dengue (DENV), Yellow fever (YFV), Hepatitis C (HCV), Saint Louis encephalitis (SLEV), Japanese encephalitis (JEV), Murray Valley encephalitis (MVEV) and Tick-borne encephalitis viruses (TBEV). Currently, there is only one type of cell-based assay available for screening flaviviral inhibitors and it is limited in overall efficiency since only a single enzyme or protein can be tested and monitored for any potential assays. Additionally, the assay is highly labor-intensive and impossible to use when screening compound libraries in large quantities.

Our screening assays are high throughput and sensitive which makes for rapid screening and identification of potential inhibitors in libraries with a large amount of compounds possible. We use “reverse genetic systems”, which are cDNA clones of RNA viruses, and developed them for use in our genetic cell-based screening assays. Comparisons were performed on three cell based high throughput screening assays for WNV drug discovery:  1) an assay with a replicon-containing cell line that allows screening for inhibitors of viral replication; 2) a replicon bearing VLP infection assay that allows screening for inhibitors of viral entry as well as replication; and 3) a full-length reporting virus infection assay that allows screening for inhibitors of any step(s) of the viral life cycle, including entry, replication, and virion assembly.  All three assays were converted into a 384-well format, validated with known WNV inhibitors, and now offer speed and sensitivity which make them ideal for screening libraries with large numbers of compounds.

Screening Assays Available

A. WNV replicon with both Renilla luciferase (Rluc) and Neomycin Phosphotransferase (Neo)

B. WNV VLPs containing a Renilla luciferase expressing replicon (Rluc-VLP)

C. SFV expression system expressing WNV structural proteins CprME

D. Plasmid for DENV-1 replicon containing Renilla luciferase reporter

E. Vero cell line containing DENV-1 Renilla luciferase-Neomycin gene-replicon

F. SFV expression system expressing DENV-1 structural proteins CprME


  • Rapid primary screening of large compound libraries for potential inhibitors of WNV and other flaviviruses.
  • Secondary screening to characterize functional activity as well as activity against the target species being tested.
  • Engineering targeted mutations into WNV, resulting in attenuated viral strains that could potentially be used for vaccine development.


  • As a primary screening assay, to analyze multiple viral protein targets simultaneously through large compound libraries.
  • As a secondary screening assay, to study the mode of action of inhibitors or the assay can be adapted to screen for inhibitors targeting individual viral proteins i.e. target-based secondary profiling
  • cDNA clones from human pathogenic RNA of West Nile viruses, therefore compounds identified from such an assay may have direct relevance to human disease.

State of Development

  • The assay system is established.
  • Our panel of assays have been developed for analysis of potential mode of action of potential inhibitors.


USPTO no. 7,355,033


Pei-Yong_Shi_photoPei-Yong Shi, PhD
Pei-Yong Shi is currently Head of the Dengue Unit at Novartis Institute for Tropical Diseases. He received his Ph.D. in flavivirus replication in 1995 from Georgia State University, USA. After postdoctoral training at Yale University, he joined Bristol-Myers Squibb as a Principal Scientist from 1998 to 2000. He then moved to the Wadsworth Center, New York State Department of Health. His group at the Wadsworth Center developed the first infectious clone of the epidemic strain of West Nile virus, discovered two cap methylation activities of flavivirus NS5 protein, identified essential RNA elements for flavivirus replication, and established various novel platforms that have been used to screen and study inhibitors of flaviviruses. He an Editor for Journal of General Virology and a member of the Editorial Board for Journal of Virology. He joined Novartis in August 2008.


Min-Qing, Wei Liu, Zhiming Yuan, Feng Gu and Pei-Yong Shi.  A High-Throughput Assay using Dengue-1 Virus-Like Particles for Drug Discovery.  Antiviral Research (2010) doi:  10.1016/j.antiviral.2010.02.313

Francesc Puig-Basagoiti, Mark Tilgner, Brett M. Forshey, Sean M. Philpott, Noel G. Espina, David E. Wentworth, Scott J. Goebel, Paul S. Masters, Barry Falgout, Ping Ren, David M. Ferguson and Pei-Yong Shi. Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication.  Antimicrobial Agents and Chemotherapy, Apr. 2006 p. 1320-1329  doi:10.1128/AAC50.4.1320-1329  2006

Francesc Puig-Basagoiti, Tia S. Deas, Ping Ren, Mark Tilgner, David M. Ferguson, and Pei-Yong Shi.  High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery.  Antimicrobial Agents and Chemotherapy, Dec 2005 p. 4980-4988  doi:  10.1128/AAC.49.12.4980-4988.2005


Diane L. Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
Health Research, Inc. – 150 Broadway – Suite 560, Menands, New York 12204-2719 U.S.A.
Phone 518-431-1213  Fax 518-431-1234
E-mail:  diane.borghoff@healthresearch.org  Website:  www.healthresearch.org