First Isolation of West Nile Virus from a Patient With Encephalitis in the United States

Cinnia Huang, Brett Slater, Robert Rudd, Nandakishore Parchuri, Rene Hull, Michelle Dupuis, Alexander Hindenburg


Emerging Infectious Diseases. 2002;8(12) 

In This Article

Materials and Methods

The PCR Laboratory associated with the Virology Diagnostic Services, Wadsworth Center, New York State Department of Health, has developed a panel of PCR and RT-PCR assays that allow tests on CSF and brain tissue for a wide range of viruses associated with human central nervous system (CNS) infections. Specifically, the test battery includes herpes simplex viruses (types 1 and 2), varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (Human herpesvirus 4), enteroviruses, and the following arboviruses: Eastern equine encephalitis, California serogroup (LaCrosse virus [LACV], Jamestown Canyon, and others), Powassan virus (POWV), St. Louis encephalitis virus (SLEV), WNV, and Cache Valley virus.

RNA and DNA were simultaneously extracted from 0.25 mL of CSF sample with Trizol LS reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, RNA was first transcribed into cDNA with random primers (Roche Diagnostics Corp., Indianapolis, IN), and an aliquot of this cDNA (5 μL) was used in PCR reactions with primers for detecting viruses in the test panel. Aliquots of DNA were also examined for the presence of the herpesviruses in the panel. Amplification products were analyzed on a 2% agarose (EM Science, Gibbstown, NJ) gel containing ethidium bromide. Routinely, for any virus for which a band corresponding to a positive result is observed, the band is run into low melting agarose, and sequenced directly on the gel slice without further purification. DyeTerminator sequencing was performed on an Applied Biosystems Model 373A automated sequencer (Foster City, CA). For the TaqMan assay, one-step RT-PCR Ready-Mix Kit (Applied Biosystems) was used. The primers and probe for the quantification of the RNA copy number of WNV used in this study are listed in Table 1 .

Virus was isolated by using monolayers of Vero cells grown in tubes. Aliquots of 0.01 and 0.05 mL of whole blood were pretreated with antibiotics (penicillin and streptomycin) for 30 min at room temperature (22°C); 1 mL of Eagle's minimal essential medium containing 2% fetal bovine serum was added to the treated blood samples, and the mixtures were used to inoculate the Vero cell monolayers.

After incubation for 24 hr at 37°C, the monolayers were rinsed with phosphate-buffered saline, 2 mL of fresh medium was added, and the culture was subsequently monitored daily for cytopathic effects (CPE). Cell monolayers that showed CPE were harvested. To confirm that the infectious agent was WNV, an aliquot of the supernatant serially diluted (10-4-10-6) was used to infect fresh Vero cell monolayers. Virus-infected monolayers from the second passage were examined for WNV antigen by immunofluorescence assay (IFA) by using monoclonal antibody H5-46 (provided by CDC, Fort Collins, CO). This monoclonal immunoglobulin (Ig) Mantibody is specific for a glycoprotein epitope on WNV.