title: Infectious cDNA clone of the epidemic west nile virus from New York City.
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 x 10(9) to 5 x 10(9) 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.
authors.affiliation: Wadsworth Center, New York State Department of Health. Department of Biomedical Sciences, University at Albany, State University of New York, Albany, New York 12201, USA. ship@wadsworth.org
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.forename: Mark
authors.initials: M
authors.lastname: Tilgner
authors.forename: Michael K
authors.initials: MK
authors.lastname: Lo
authors.forename: Kim A
authors.initials: KA
authors.lastname: Kent
authors.forename: Kristen A
authors.initials: KA
authors.lastname: Bernard
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics; D004196:Disease Outbreaks; D006801:Humans; D051379:Mice; D009519:New York City / Q000453:epidemiology; D012367:RNA, Viral / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells; D014901:West Nile Fever / Q000453:epidemiology / Q000821:virology*; D014902:West Nile virus / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral