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Update: Surveillance for West Nile Virus in Overwintering Mosquitoes --- New York, 2000

Following the 1999 West Nile encephalitis outbreak in New York, guidelines were developed to direct surveillance, prevention, and control efforts in the eastern United States (1). As recommended in the guidelines, the New York City and New York state departments of health developed comprehensive West Nile virus (WNV) surveillance and control programs, which included collecting overwintering Culex mosquitoes to determine whether WNV might persist throughout the winter and initiate a zoonotic transmission cycle in the spring of 2000. As part of this surveillance effort, adult Culex mosquitoes were collected from structures in New York City during January-February 2000 to determine whether overwintering mosquitoes were infected with WNV. This report summarizes the results of this analysis, which documented WNV RNA in some mosquito pools.

Mosquitoes were sought from sites within the city's storm and sanitary sewer system, historic sites at Fort Totten in northeastern Queens, hangars and other locations at the abandoned Flushing Airport, and utility rooms under the Whitestone Bridge and under municipal swimming pools. Collection sites were selected based on location of WNV-infected humans and mosquitoes during the 1999 outbreak (2). Mosquitoes were pooled and then tested for the presence of WNV using vero cell plaque assay (3) and a fluorogenic real-time polymerase chain reaction (PCR) assay (TaqMan™, Perkin-Elmer Biosystems, Foster City, California*) that focused on three different primer pairs: the envelope protein and the NS-1 and NS-5 regions (4).

No pools produced live virus isolates in the plaque assay. However, three of the 67 pools containing Culex spp. mosquitoes, all of which were collected from Fort Totten, reproducibly demonstrated low but detectable levels of WNV RNA.

Reported by: J Cooper, MPA, New York City Dept of Parks and Recreation; J Miller, MD, New York City Dept of Health; P Bennett, MPH, New York City Dept of Environmental Protection; D White, PhD, P Smith, State Epidemiologist, New York State Dept of Health. Arbovirus Diseases Br, Div of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, CDC.

Editorial Note:

The standard technique for detecting virus in mosquitoes is the cell culture plaque assay, which detects only live virus. The real-time PCR technique was first used to detect WNV RNA in mosquitoes in the outbreak investigation during September-November 1999, and produced results consistent with those obtained by plaque assay (CDC, unpublished data, 1999). This experimental assay is highly sensitive for detecting the nucleic acids of pathogens and represents a novel approach for detecting and quantifying viruses.

In the positive pools described in this report, the intensity of the TaqMan signal was in the range consistent with approximately one plaque forming unit (vero cell plaque assay equivalent) according to a standard curve generated in the assay. The ability to detect WNV RNA in the absence of infectious viral particles might be because 1) the virus titer in the overwintering mosquito may be near or below the detectable limits of the plaque assay method; 2) the virus may be noninfectious because of biologic changes in overwintering mosquitoes; 3) the virus may have been killed during the collection and processing of specimens; 4) noninfectious viral RNA may persist in the mosquitoes; or 5) the results were false positives. Attempts to isolate virus from these pools are continuing using other isolation systems.

It is unknown whether WNV will persist in the New York area. Overwintering mosquitoes were difficult to locate, and intact WNV has not been identified. Three fourths of all specimens were obtained from the Fort Totten site. Surveillance of overwintering mosquitoes will continue.

WNV can be transmitted from parent to offspring mosquitoes (5), and this vertical transmission has been documented in field populations of Culex univittatus in Kenya (6). The role of vertical transmission in the maintenance cycles of this virus is uncertain. A related flavivirus (St. Louis encephalitis virus) may persist through the winter in vertically infected, diapausing Culex mosquitoes, but it is probably a rare occurrence if it occurs in nature (7).

The findings in this report demonstrate the value of continued vigilance in detecting the re-emergence of WNV. Counties where WNV transmission occurred in 1999 should monitor closely for WNV and conduct mosquito-control activities in the spring to reduce the potential for recurrence and amplification of WNV. Mosquito-control activities include reducing the number of mosquito breeding sites, particularly around homes and suburban and urban areas, and applying larvicide to Culex larval habitats early.

In December 1999, CDC announced availability of funds to support WNV surveillance, prevention, and control programs. The 19 state and local health departments eligible to apply for these funds represent areas where WNV transmission already has occurred or where transmission would be more likely to occur based on bird migration patterns. The focus of these cooperative agreements enables state and local health departments to increase surveillance activities and enhance laboratory capacity for detecting WNV and other arboviruses. In 2000, surveillance activities will be focused on determining whether WNV survived the winter and, if so, to ascertain its geographic distribution along the Atlantic and Gulf coasts.


  1. CDC. Guidelines for surveillance, prevention, and control of West Nile virus infection---United States. MMWR 2000;49:25-8.
  2. CDC. Update: West Nile virus encephalitis---New York, 1999. MMWR 1999;48:944-6,955.
  3. Beaty BJ, Calisher CH, Shope RS. Arboviruses. In: Schmidt NJ, Emmons RW, eds. Diagnostic procedures for viral, rickettsial and chlamydial infections. Washington, DC: American Public Health Association, 1989:797-856.
  4. Perkin-Elmer Biosystems. TaqMan one-step RT-PCR master mix reagents kit protocol [Product information sheet]. Foster City, California: Perkin-Elmer Biosystems, 1999.
  5. Baqar S, Hayes CG, Murphy JR, Watts DM. Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes. Am J Trop Med Hyg 1993;48:757-62.
  6. Miller BR, Nasci RS, Godsey MS, et al. First field evidence for natural vertical transmission of West Nile virus in Culex univitattus mosquitoes from Rift Valley Province, Kenya. Am J Trop Med Hyg 2000 (in press).
  7. Bailey CL, Eldridge BF, Hayes DE, Watts DM, Tammariello RF, Dalrymple JM. Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes. Science 1978; 199:1346-9.

* The use of trade names is for identification only and does not imply endorsement by CDC or the U.S. Department of Health and Human Services. 

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