RESEARCH the patient’s hematologic parameters improved, G-CSF was discontinued on day 9 of hospitalization, and the patient con- tinued to be afebrile. Because of the clinical picture of enceph- alitis and because the patient lived in an area where WNV was endemic, lumbar puncture was performed on day 8, and a CSF specimen was sent to the New York State Department of Health laboratories for comprehensive PCR testing. Renal tubular necrosis developed in the patient, leading to acute renal failure by day 10, with further deterioration of her mental status. Dialysis was initiated on day 16, and antibiotic therapy was discontinued on day 17 as the patient remained afebrile and the neutropenia resolved. The patient remained unrespon- sive, staphylococcal septicemia developed, and she died on day 35 of hospitalization. Autopsy showed a small focus of perivascular lymphocyte cuffing in the mamillary bodies of the brain, consistent with viral encephalitis. No evidence of residual lymphoma was found. Materials and Methods The PCR Laboratory associated with the Virology Diagnos- tic 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) infec- tions. 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, Califor- nia 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, Carls- bad, 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 µ L) 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. Primer CU9093 CL9279 D87F D156R Forward Reverse Probe 1368 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 fur- ther 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 mini- mal 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 mono- clonal antibody H5-46 (provided by CDC, Fort Collins, CO). This monoclonal immunoglobulin (Ig) M antibody is specific for a glycoprotein epitope on WNV. Results Diagnosis of WNV Infection CSF was simultaneously examined for a panel of 11 viruses by RT-PCR/PCR as described in Material and Methods. No amplification product was observed for any other virus in the PCR battery except WNV. Two primer pairs (CU9093/CL9279 and D87F/D156R) in the NS5 region (5,6) of the WNV genome were used in the initial screening by standard RT-PCR (Table 1). PCR products of appropriate size for both primer pairs were obtained (Figure 1). RT-PCR was independently repeated on another aliquot of CSF by using four primer pairs located in var- Sequence (5′–3′) AGYMGRGCHATHTGGTWYATGTGG TTCCAVCCDGCKGTRTCATC GCTCCGCTGTCCCTGTGA CACTCTCCTCCTGCATGGATG TCAGCGATCTCTCCACCAAAG GGGTCAGCACGTTTGTCATTG TGCCCGACCATGGGAGAAGCTC RT-PCR product size (bp) 206 70 70 NS5 NS5 NS5 NS5 ENV ENV ENV 9097–9120 9302–9283 10034–10051 10103–10083 1160–1180 1229–1209 1186–1207 aRT-PCR, reverse transcription–polymerase chain reaction. bGenome position according to GenBank accession no. AF196835. Table 1. Oligonucleotide primers and probe used in the standard RT-PCR and TaqMan assaysa Genome target Genome positionb Emerging Infectious Diseases • Vol. 8, No. 12, December 2002