The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number (≈500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

The complete nucleotide sequences of eight West Nile (WN) virus strains (Egypt 1951, Romania 1996-MQ, Italy 1998-equine, New York 1999-equine, MD 2000-crow265, NJ 2000MQ5488, NY 2000-grouse3282, and NY 2000-crow3356) were determined. Phylogenetic trees were constructed from the aligned nucleotide sequences of these eight viruses along with all other previously published complete WN virus genome sequences. The phylogenetic trees revealed the presence of two genetic lineages of WN viruses. Lineage 1 WN viruses have been isolated from the northeastern United States, Europe, Israel, Africa, India, Russia, and Australia. Lineage 2 WN viruses have been isolated only in sub-Saharan Africa and Madagascar. Lineage 1 viruses can be further subdivided into three monophyletic clades.

The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States.

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After the 1999 West Nile (WN) encephalitis outbreak in New York, 2,300 overwintering adult mosquitoes were tested for WN virus by cell culture and reverse transcriptase-polymerase chain reaction. WN viral RNA and live virus were found in pools of Culex mosquitoes. Persistence in overwintering Cx. pipiens may be important in the maintenance of WN virus in the northeastern United States.

Widespread deaths of American Crows (Corvus brachyrhynchos) were associated with the 1999 outbreak of West Nile (WN) virus in the New York City region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive target organ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance.

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10 2.1 to 10 3.7 PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n ‫؍‬ 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10 3 PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Abstract. Mosquitoes and wild birds were collected from three sites near locations in the New York City metropolitan area where single, West Nile (WN) virus−positive dead birds were found early in the 2000 transmission season. The mosquitoes were tested for the presence of infectious virus with a Vero cell culture assay and for WN viral RNA by using reverse transcriptase−polymerase chain reaction (RT-PCR) protocols. Serum samples from wild birds were tested for the presence of neutralizing antibodies against WN virus. Infectious WN virus and WN viral RNA were found in Culex species adult mosquitoes from each of the three sites, and a seropositive hatch-year house sparrow (Passer domesticus) was found in one of the three sites. Molecular techniques used to identify the species in the positive mosquito pools found that most of the pools contained a combination of Culex pipiens and Cx. restuans. The minimum infection rate in Culex species mosquitoes from the sites ranged from 0.2 to 6.0 per 1,000 specimens tested. The results demonstrated that, at least early in the transmission season, detection of a WN virus−positive dead bird indicates a local WN virus transmission cycle. This information is valuable in focusing subsequent surveillance and vector management programs. In addition, the RT-PCR procedure for detecting WN viral RNA in mosquito pools detected more positive pools than did the Vero cell plaque assay.