Construction and characterization of subgenomic replicons of New York strain of West Nile virus

Pei-Yong Shi, Mark Tilgner, Michael K. Lo

Research output: Contribution to journalArticle

110 Citations (Scopus)

Abstract

The lineage I strain of West Nile virus (WNV) frequently causes human epidemics, including the recent outbreak in North America (Lanciotti et al., 1999, Science 286:2333-2337). As an initial step in studying the replication and pathogenesis of WNV, we constructed several cDNA clones of a WNV replicon derived from an epidemic strain (lineage I) isolated from the epicenter of New York City in the year 2000. Replicon RNAs were in vitro transcribed from cDNA plasmids and transfected into BHK-21 cells. RNA replication in transfected cells was monitored by immunofluorescence analysis (IFA) and 5′ nuclease real-time RT-PCR (TaqMan). The replicon RNAs contained large in-frame deletions (greater than 92%) of the C-prM-E structural region yet still replicated efficiently in BHK-21 cells. 5′ nuclease real-time RT-PCR showed that a great excess of plus-sense replicon RNA over the minus-sense RNA was synthesized in transfected cells. Replication efficiency decreased upon insertion of a green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the upstream end of the 3′ untranslated region of the replicon. Strong GFP expression was detected in cells transfected with a replicon containing IRES-GFP positioned in the plus-sense orientation. IFA showed that GFP and viral proteins were exclusively coexpressed in transfected cells. In contrast, no GFP fluorescence was observed in cells transfected with a replicon containing IRES-GFP positioned in the minus-sense orientation, despite high levels of synthesis of viral proteins and RNA in the cells. Substitution of the GFP gene in the plus-sense GFP replicon with the neomycin phosphotransferase gene allowed selection of geneticin-resistant cells in which WNV replicons persistently replicated without apparent cytopathic effect. These results suggest that WNV replicons may serve as a noncytopathic RNA virus expression system and should provide a valuable tool to study WNV replication.

Original languageEnglish (US)
Pages (from-to)219-233
Number of pages15
JournalVirology
Volume296
Issue number2
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

West Nile virus
Replicon
Green Fluorescent Proteins
RNA
Viral Proteins
Fluorescent Antibody Technique
Real-Time Polymerase Chain Reaction
Complementary DNA
Kanamycin Kinase
RNA Viruses
Viral RNA
3' Untranslated Regions
Virus Replication
North America
Reporter Genes
Genes
Disease Outbreaks
Plasmids
Clone Cells
Fluorescence

Keywords

  • Flavivirus replication
  • Replicon
  • West Nile virus

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Construction and characterization of subgenomic replicons of New York strain of West Nile virus. / Shi, Pei-Yong; Tilgner, Mark; Lo, Michael K.

In: Virology, Vol. 296, No. 2, 2002, p. 219-233.

Research output: Contribution to journalArticle

Shi, Pei-Yong ; Tilgner, Mark ; Lo, Michael K. / Construction and characterization of subgenomic replicons of New York strain of West Nile virus. In: Virology. 2002 ; Vol. 296, No. 2. pp. 219-233.
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