Infectious cDNA clone of the epidemic West Nile virus from New York City

Pei-Yong Shi, Mark Tilgner, Michael K. Lo, Kim A. Kent, Kristen A. Bernard

Research output: Contribution to journalArticle

161 Citations (Scopus)

Abstract

We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 × 109 to 5 × 109 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3′-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

Original languageEnglish (US)
Pages (from-to)5847-5856
Number of pages10
JournalJournal of Virology
Volume76
Issue number12
DOIs
StatePublished - 2002
Externally publishedYes

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West Nile virus
Complementary DNA
Clone Cells
nucleotides
clones
Nucleotides
RNA
viruses
Viruses
transfection
Viral RNA
plasmids
Viral Load
virulence
Genetic Markers
pathogenicity
pathogenesis
reverse transcriptase polymerase chain reaction
promoter regions
Reverse Transcription

ASJC Scopus subject areas

  • Immunology

Cite this

Infectious cDNA clone of the epidemic West Nile virus from New York City. / Shi, Pei-Yong; Tilgner, Mark; Lo, Michael K.; Kent, Kim A.; Bernard, Kristen A.

In: Journal of Virology, Vol. 76, No. 12, 2002, p. 5847-5856.

Research output: Contribution to journalArticle

Shi, Pei-Yong ; Tilgner, Mark ; Lo, Michael K. ; Kent, Kim A. ; Bernard, Kristen A. / Infectious cDNA clone of the epidemic West Nile virus from New York City. In: Journal of Virology. 2002 ; Vol. 76, No. 12. pp. 5847-5856.
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