Detection of West Nile virus-infected mosquitoes and seropositive juvenile birds in the vicinity of virus-positive dead birds.

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.

Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East.

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.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

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.