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.

FIG. 1.

Construction of the full-length cDNA clone of WNV. Genome organization and unique restriction sites as well as their nucleotide numbers are shown at the top. Four cDNA fragments represented by thick lines were synthesized from genomic RNA through RT-PCR to cover the complete WNV genome. Individual fragments were assembled to form the full-length cDNA clone of WNV (pFLWNV) as described in Materials and Methods. The complete WNV cDNA is positioned under the control of T7 promoter elements for in vitro transcription. Three silent mutations (shown in lowercase) were engineered to create a StyI site (∗) and to knock out an EcoRI site (Δ) in the NS5 gene. The numbers are the nucleotide positions based on the sequence from GenBank accession no. AF404756.

FIG. 2.

Transcription of WNV RNA. Formaldehyde-denaturing 1.0% agarose electrophoresis of RNA transcript together with genomic RNA purified from WNV.

FIG. 3.

IFA of viral protein expression in cells transfected with full-length WNV RNA transcript. BHK-21 cells transfected with full-length WNV RNA transcript were analyzed by IFA at the indicated times posttransfection. Photomicrographs were taken at magnifications of ×400. The left and right panels represent the same field of view for each time point. The left panels were visualized with differential interference contrast (DIC), and the right panels were visualized with a fluorescein isothiocyanate (FITC) filter set. For the IFA, WNV immune mouse ascites fluid and goat anti-mouse immunoglobulin G antibody conjugated with FITC were used as primary and secondary antibodies, respectively.

FIG. 4.

Recombinant WNV retains the genetic markers engineered during cDNA construction. A StyI site was created and an EcoRI site was knocked out in the NS5 gene of the recombinant virus to serve as genetic markers to distinguish recombinant virus from parental virus. A 388-bp fragment (from nt 8706 to 9093) spanning the StyI or EcoRI site was amplified through RT-PCR from RNA extracted from either recombinant virus or parental virus. The RT-PCR fragments were subjected to StyI and EcoRI digestion. The 388-bp fragment derived from recombinant virus should be cleaved by StyI but not by EcoRI; the RT-PCR fragment amplified from parental viral RNA should be digested by EcoRI but not StyI. (A) Schematic drawing of genetic marker analysis. The expected sizes of the digestion products are indicated. (B) Agarose gel analysis of genetic markers. Expected digestion pattern as depicted in panel A was observed. As a negative control, no RT-PCR products were detected from the extracted supernatant collected from cells 5 days after transfection with a mutant RNA containing a deletion of the 3′-terminal 199 nt of the genome (lane 9, 3′ dltn). A 100-bp ladder was loaded on lanes 1, 5, and 10 as a standard.

FIG. 5.

Plaque morphology of parental and clone-derived WNV on Vero cells. Vero cells in six-well plates were infected with 100 PFU of parental WNV 3356 (A) or 100 PFU of WNV derived from pFLWNV (B). Plaques were visualized 3 days postinoculation by staining for 24 h with neutral red.

FIG. 6.

Comparison of the growth kinetics of recombinant and parental WNV. The growth of recombinant and parental viruses was compared at high and low MOIs on BHK-21 or Aedes albopictus C6/36 cells. (A) Growth in BHK-21 cells. (B) Growth in C6/36 cells. Viruses were inoculated at an MOI of 5.0 (filled symbols) or an MOI of 0.05 (open symbols) in triplicate in 12-well plates. Recombinant virus is designated by squares along a solid line. Parental virus is designated by triangles along a dashed line. Error bars represent ± standard deviation of triplicate wells. Dotted line indicates the limit of detection (500 PFU/ml).