A prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization.

Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction.

Temporal and Spatial Variability of Entomological Risk Indices for West Nile Virus Infection in Northern Colorado: 2006-2013.

West Nile virus (WNV) is enzootic in northern Colorado. Annual surveillance activities in Fort Collins, CO, include collecting female Culex mosquitoes and testing them for the presence of WNV RNA in order to calculate 1) Culex female abundance, 2) WNV infection rate, and 3) the vector index (VI). These entomological risk indices inform public policy regarding the need for emergency adulticiding. Currently, these are calculated on a city-wide basis. In this study, we present descriptive data from historical surveillance records spanning 2006-2013 to discern seasonal and yearly patterns of entomological risk for WNV infection. Also, we retrospectively test the hypothesis that entomological risk is correlated with human transmission risk and is heterogeneous within the City of Fort Collins. Four logistically relevant zones within the city were established and used to test this hypothesis. Zones in the eastern portion of the city consistently had significantly higher Culex abundance and VI compared with zones in the west, leading to higher entomological risk indicators for human WNV infection in the east. Moreover, the relative risk of a reported human case of WNV infection was significantly higher in the eastern zones of the city. Our results suggest that a more spatially targeted WNV management program may better mitigate human risk for WNV infection in Fort Collins, and possibly other cities where transmission is enzootic, while at the same time reducing pesticide use.

Modeling Culex tarsalis abundance on the northern Colorado front range using a landscape-level approach.

Remote sensing and Geographic Information System (GIS) data can be used to identify larval mosquito habitats and predict species distribution and abundance across a landscape. An understanding of the landscape features that impact abundance and dispersal can then be applied operationally in mosquito control efforts to reduce the transmission of mosquito-borne pathogens. In an effort to better understand the effects of landscape heterogeneity on the abundance of the West Nile virus (WNV) vector Culex tarsalis, we determined associations between GIS-based environmental data at multiple spatial extents and monthly abundance of adult Cx. tarsalis in Larimer County and Weld County, CO. Mosquito data were collected from Centers for Disease Control and Prevention miniature light traps operated as part of local WNV surveillance efforts. Multiple regression models were developed for prediction of monthly Cx. tarsalis abundance for June, July, and August using 4 years of data collected over 2007-10. The models explained monthly adult mosquito abundance with accuracies ranging from 51-61% in Fort Collins and 57-88% in Loveland-Johnstown. Models derived using landscape-level predictors indicated that adult Cx. tarsalis abundance is negatively correlated with elevation. In this case, low-elevation areas likely more abundantly include habitats for Cx. tarsalis. Model output indicated that the perimeter of larval sites is a significant predictor of Cx. tarsalis abundance at a spatial extent of 500 m in Loveland-Johnstown in all months examined. The contribution of irrigated crops at a spatial extent of 500 m improved model fit in August in both Fort Collins and Loveland-Johnstown. These results emphasize the significance of irrigation and the manual control of water across the landscape to provide viable larval habitats for Cx. tarsalis in the study area. Results from multiple regression models can be applied operationally to identify areas of larval Cx. tarsalis production (irrigated crops lands and standing water) and assign priority in larval treatments to areas with a high density of larval sites at relevant spatial extents around urban locations.

Aedes (Stegomyia) aegypti in the continental United States: a vector at the cool margin of its geographic range.

After more than a half century without recognized local dengue outbreaks in the continental United States, there were recent outbreaks of autochthonous dengue in the southern parts of Texas (2004-2005) and Florida (2009-2011). This dengue reemergence has provoked interest in the extent of the future threat posed by the yellow fever mosquito, Aedes (Stegomyia) aegypti (L.), the primary vector of dengue and yellow fever viruses in urban settings, to human health in the continental United States. Ae. aegypti is an intriguing example of a vector species that not only occurs in the southernmost portions of the eastern United States today but also is incriminated as the likely primary vector in historical outbreaks of yellow fever as far north as New York, Philadelphia, and Boston, from the 1690s to the 1820s. For vector species with geographic ranges limited, in part, by low temperature and cool range margins occurring in the southern part of the continental United States, as is currently the case for Ae. aegypti, it is tempting to speculate that climate warming may result in a northward range expansion (similar to that seen for Ixodes tick vectors of Lyme borreliosis spirochetes in Scandinavia and southern Canada in recent decades). Although there is no doubt that climate conditions directly impact many aspects of the life history of Ae. aegypti, this mosquito also is closely linked to the human environment and directly influenced by the availability of water-holding containers for oviposition and larval development. Competition with other container-inhabiting mosquito species, particularly Aedes (Stegomyia) albopictus (Skuse), also may impact the presence and local abundance of Ae. aegypti. Field-based studies that focus solely on the impact of weather or climate factors on the presence and abundance of Ae. aegypti, including assessments of the potential impact of climate warming on the mosquito's future range and abundance, do not consider the potential confounding effects of socioeconomic factors or biological competitors for establishment and proliferation of Ae. aegypti. The results of such studies therefore should not be assumed to apply in areas with different socioeconomic conditions or composition of container-inhabiting mosquito species. For example, results from field-based studies at the high altitude cool margins for Ae. aegypti in Mexico's central highlands or the Andes in South America cannot be assumed to be directly applicable to geographic areas in the United States with comparable climate conditions. Unfortunately, we have a very poor understanding of how climatic drivers interact with the human landscape and biological competitors to impact establishment and proliferation of Ae. aegypti at the cool margin of its range in the continental United States. A first step toward assessing the future threat this mosquito poses to human health in the continental United States is to design and conduct studies across strategic climatic and socioeconomic gradients in the United States (including the U.S.-Mexico border area) to determine the permissiveness of the coupled natural and human environment for Ae. aegypti at the present time. This approach will require experimental studies and field surveys that focus specifically on climate conditions relevant to the continental United States. These studies also must include assessments of how the human landscape, particularly the impact of availability of larval developmental sites and the permissiveness of homes for mosquito intrusion, and the presence of other container-inhabiting mosquitoes that may compete with Ae. aegypti for larval habitat affects the ability of Ae. aegypti to establish and proliferate. Until we are armed with such knowledge, it is not possible to meaningfully assess the potential for climate warming to impact the proliferation potential for Ae. aegypti in the United States outside of the geographic areas where the mosquito already is firmly established, and even less so for dengue virus transmission and dengue disease in humans.

Irrigated agriculture is an important risk factor for West Nile virus disease in the hyperendemic Larimer-Boulder-Weld area of north central Colorado.

This study focused on two West Nile virus (WNV) disease outbreak years, 2003 and 2007, and included a three-county area (Larimer, Boulder, and Weld) in North Central Colorado that is hyperendemic for WNV disease. We used epidemiological data for reported WNV disease cases at the census tract scale to: (1) elucidate whether WNV disease incidence differs between census tracts classified as having high versus lower human population density (based on a threshold value of 580 persons/km2) and (2) determine associations between WNV disease incidence and habitat types suitable as development sites for the larval stage of Culex mosquito vectors. WNV disease incidence was significantly elevated in census tracts with lower human population density, compared with those with high density of human population, in both 2003 (median per census tract of 223 and 143 cases per 100,000 population, respectively) and 2007 (median per census tract of 46 and 19 cases per 100,000 population). This is most likely related, in large part, to greater percentages of coverage in less densely populated census tracts by habitats suitable as development sites for Culex larvae (open water, developed open space, pasture/hay, cultivated crops, woody wetlands, and emergent herbaceous wetlands) and, especially, for the subset of these habitats made up by irrigated agricultural land (pasture/hay and cultivated crops) that presumably serve as major producers of the locally most important vector of WNV to humans: Culex tarsalis. A series of analyses produced significant positive associations between greater coverage of or shorter distance to irrigated agricultural land and elevated WNV disease incidence. As an exercise to produce data with potential to inform spatial implementation schemes for prevention and control measures within the study area, we mapped the spatial patterns, by census tract, of WNV disease incidence in 2003 and 2007 as well as the locations of census tracts that had either low (<25th percentile) or high (>75th percentile) WNV disease incidence in both outbreak years (relative to the incidence for each year). This revealed substantial changes from 2003 to 2007 in the spatial pattern for census tracts within the study area with high WNV disease incidence and suggests a dynamic and evolving scenario of WNV transmission to humans that needs to be taken into account for prevention and control measures to stay current and represent the most effective use of available resources.

Relationship between distance from major larval habitats and abundance of adult mosquitoes in semiarid plains landscapes in Colorado.

We examined the relationship between distance from major larval habitats and abundance of adult mosquitoes in the semiarid plains landscape characteristic of eastern Colorado. Mosquito collection was conducted from late June to early August 2007 and included trap locations at distances ranging from <10 m up to 20-150 m and 160-373 m from three major larval habitats: one area along a river corridor and two small reservoirs. The study yielded 65,140 mosquitoes of 14 species, and five species were sufficiently abundant to be included in statistical analyses: Aedes vexans (Meigen), Culex tarsalis Coquillett, Ochlerotatus dorsalis (Meigen) (=Ae. dorsalis), Ochlerotatus melanimon (Dyar) (=Ae. melanimon), and Culex pipiens L. Distance to nearest major larval habitat was not strongly related to Culex abundance within the approximately = 400-m range from larval habitats examined in this study. Abundance of Ae. vexans declined significantly with distance from the larval habitat, whereas abundance was significantly higher in the 20-150- and 160-373-m classes compared with areas within 10 m of the larval habitat for both Ochlerotatus species. Except for Ae. vexans, however, we did not find monotonic increasing or decreasing abundance trends associated with distance from larval habitats for the 400-m range examined. This, combined with a finding that fine-scale habitat heterogeneity influenced abundance for most of the mosquitoes examined, underscores the importance of considering not only distance from larval habitat but also fine-scale habitat heterogeneity to understand how important nuisance-biters and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) vectors use the landscape. We also discuss how these results relate to previous studies from western North America and explore their relevance to operational implementation of adulticides to suppress mosquito vectors during WNV disease outbreaks in the Great Plains.

Seasonal patterns for entomological measures of risk for exposure to Culex vectors and West Nile virus in relation to human disease cases in northeastern Colorado.

We examined seasonal patterns for entomological measures of risk for exposure to Culex vectors and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in relation to human WNV disease cases in a five-county area of northeastern Colorado during 2006-2007. Studies along habitat/elevation gradients in 2006 showed that the seasonal activity period is shortened and peak numbers occur later in the summer for Culex tarsalis Coquillett females in foothills-montane areas >1600 m compared with plains areas <1600 m in Colorado's Front Range. Studies in the plains of northeastern Colorado in 2007 showed that seasonal patterns of abundance for Cx. tarsalis and Culex pipiens L. females differed in that Cx. tarsalis reached peak abundance in early July (mean of 328.9 females per trap night for 18 plains sites), whereas the peak for Cx. pipiens did not occur until late August (mean of 16.4 females per trap night). During June-September in 2007, which was a year of intense WNV activity in Colorado with 578 reported WNV disease cases, we recorded WNV-infected Cx. tarsalis females from 16 of 18 sites in the plains. WNV infection rates in Cx. tarsalis females increased gradually from late June to peak in mid-August (overall maximum likelihood estimate for WNV infection rate of 8.29 per 1000 females for the plains sites in mid-August). No WNV-infected Culex mosquitoes were recorded from sites >1600 m. The vector index for abundance of WNV-infected Cx. tarsalis females for the plains sites combined exceeded 0.50 from mid-July to mid-August, with at least one site exceeding 1.00 from early July to late August. Finally, we found that abundance of Cx. tarsalis females and the vector index for infected females were strongly associated with weekly numbers of WNV disease cases with onset 4-7 wk later (female abundance) or 1-2 wk later (vector index).

Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus.

The global population at risk from mosquito-borne diseases-including dengue, yellow fever, chikungunya and Zika-is expanding in concert with changes in the distribution of two key vectors: Aedes aegypti and Aedes albopictus. The distribution of these species is largely driven by both human movement and the presence of suitable climate. Using statistical mapping techniques, we show that human movement patterns explain the spread of both species in Europe and the United States following their introduction. We find that the spread of Ae. aegypti is characterized by long distance importations, while Ae. albopictus has expanded more along the fringes of its distribution. We describe these processes and predict the future distributions of both species in response to accelerating urbanization, connectivity and climate change. Global surveillance and control efforts that aim to mitigate the spread of chikungunya, dengue, yellow fever and Zika viruses must consider the so far unabated spread of these mosquitos. Our maps and predictions offer an opportunity to strategically target surveillance and control programmes and thereby augment efforts to reduce arbovirus burden in human populations globally.

Publisher Correction: Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus.

In the version of this Article originally published, the affiliation for author Catherine Linard was incorrectly stated as '6Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK'. The correct affiliation is '9Spatial Epidemiology Lab (SpELL), Universite Libre de Bruxelles, Brussels, Belgium'. The affiliation for author Hongjie Yu was also incorrectly stated as '11Department of Statistics, Harvard University, Cambridge, MA, USA'. The correct affiliation is '15School of Health, Fudan University, Key Laboratory of Public Health Safety, Ministry of Education, Shanghai, China'. This has now been amended in all versions of the Article.

Publisher Correction: Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus.

This Article was mistakenly not made Open Access when originally published; this has now been amended, and information about the Creative Commons Attribution 4.0 International License has been added into the 'Additional information' section.

Use of Google Earth to strengthen public health capacity and facilitate management of vector-borne diseases in resource-poor environments.

OBJECTIVE: Novel, inexpensive solutions are needed for improved management of vector-borne and other diseases in resource-poor environments. Emerging free software providing access to satellite imagery and simple editing tools (e.g. Google Earth) complement existing geographic information system (GIS) software and provide new opportunities for: (i) strengthening overall public health capacity through development of information for city infrastructures; and (ii) display of public health data directly on an image of the physical environment. METHODS: We used freely accessible satellite imagery and a set of feature-making tools included in the software (allowing for production of polygons, lines and points) to generate information for city infrastructure and to display disease data in a dengue decision support system (DDSS) framework. FINDINGS: Two cities in Mexico (Chetumal and Merida) were used to demonstrate that a basic representation of city infrastructure useful as a spatial backbone in a DDSS can be rapidly developed at minimal cost. Data layers generated included labelled polygons representing city blocks, lines representing streets, and points showing the locations of schools and health clinics. City blocks were colour-coded to show presence of dengue cases. The data layers were successfully imported in a format known as shapefile into a GIS software. CONCLUSION: The combination of Google Earth and free GIS software (e.g. HealthMapper, developed by WHO, and SIGEpi, developed by PAHO) has tremendous potential to strengthen overall public health capacity and facilitate decision support system approaches to prevention and control of vector-borne diseases in resource-poor environments.

Mosquito species richness, composition, and abundance along habitat-climate-elevation gradients in the northern Colorado Front Range.

We exploited elevation gradients (1,500-2,400 m) ranging from plains to montane areas along the Poudre River and Big Thompson River in the northern Colorado Front Range to determine how mosquito species richness, composition, and abundance change along natural habitat-climate-elevation gradients. Mosquito collections in 26 sites in 2006 by using CO2-baited CDC light traps yielded a total of 7,136 identifiable mosquitoes of 27 species. Commonly collected species included Aedes vexans (Meigen) (n = 4,722), Culex tarsalis Coquillett (n = 825), Ochlerotatus increpitus (Dyar) (n = 546), Ochlerotatus trivittatus (Coquillett) (n = 303), Aedes cinereus Meigen (n = 280), Ochlerotatus melanimon (Dyar) (n = 146), Ochlerotatus dorsalis (Meigen) (n = 67), Culiseta inornata (Williston) (n = 52), Ochlerotatus pullatus (Coquillett) (n = 38), Ochlerotatus spencerii idahoensis (Theobald) (n = 37), and Culex pipiens L. (n = 29). Species richness was highest in plains habitats at elevations below 1,600 m. Numerous species were found exclusively or predominantly at low elevations below 1,700 m [Anopheles earlei Vargas, Anophelesfreeborni Aitken, Coquilletidia perturbans (Walker), Culex erythrothorax (Dyar), Cx. pipiens, Culex territans Walker, Oc. dorsalis, Ochlerotatus hendersoni (Cockerell), Oc. melanimon, and Oc. trivittatus], whereas others occurred predominantly at high elevations above 2,300 m [Ae. cinereus, Culiseta incidens (Thomson), Culiseta morsitans (Theoblad), Ochlerotatus cataphylla (Dyar), Ochlerotatus intrudens (Dyar), Oc. pullatus, and Ochlerotatus punctor (Kirby)]. Ae. vexans and Cx. tarsalis were abundant in the plains (< 1,600 m; mean June-August temperature > 19.5 degrees C), occurred at low abundances in foothills and low montane areas (1,610-1,730 m; 18.0-19.5 degrees C), and they were collected only sporadically in montane areas above 1,750 m (mean June-August temperature < 17.5 degrees C). These findings suggest that future climate warming may lead to shifts in distribution patterns of West Nile virus vectors (e.g., Cx. tarsalis) toward higher elevations in Colorado.

Interdisciplinary research in the ecology of vector-borne diseases: opportunities and needs.

In addition to their importance to human and animal health, vector-borne diseases are fascinating systems to study. The involvement of multiple species whose biologies and life cycles cover differing space and time scales makes it extremely difficult to predict epidemics. A single environmental factor may have opposite impacts on the system at different points in time. Patchiness at different geographical scales may have very different causes, so it is important to identify the proper scale for a particular study. New developments in remote sensing, GIS, and spatial analysis make it easier to tease out causes of observed patchiness. Interdisciplinary collaboration is essential for many of the projects we carry out, but this requires awareness of the differences between disciplines and the ability to effectively communicate with each other. It is only by forming multi-disciplinary groups to focus on specific vector-host-pathogen systems that we will be able to answer the most interesting (and pressing) problems in our field.

Dengue fever seroprevalence and risk factors, Texas-Mexico border, 2004.

Reported autochthonous dengue fever transmission in the United States has been limited to 5 south Texas border counties since 1980. We conducted a cross-sectional serosurvey in Brownsville, Texas, and Matamoros, Tamaulipas, Mexico (n = 600), in 2004 to assess dengue seroprevalence. Recent dengue infection was detected in 2% (95% confidence interval [CI] 0.5%-3.5%) and 7.3% (95% CI 4.3%-10.3%) of residents in Brownsville and Matamoros, respectively. Past infection was detected in 40% (95% CI 34%-45%) of Brownsville residents and 78% (95% CI 74%-83%) of Matamoros residents. For recent infection, only weekly family income

Molecular evidence for trypanosomatids in Culex mosquitoes collected during a West Nile virus survey.

Adult mosquitoes were previously collected and tested for West Nile virus during an intense WNV outbreak in 2003-2004 along the Cache la Poudre River in Colorado, USA. A subset of these mosquitoes was also tested for infection with trypanosomatids using nested PCR to amplify 18S rRNA. Of the 69 pools of Culex pipiens that were screened for both pathogens, 4.3% were positive for WNV and 11.6% tested positive for trypanosomes; no pools were found to be co-infected with both pathogens. One hundred and forty-three pools of Culex tarsalis, considered to be the principal WNV vector in this area, were tested in the same manner. 7.7% were positive for WNV and 20.3% of these pools tested positive for trypanosomes. Five pools of C. tarsalis were found to be co-infected with both pathogens, which was approximately 2.2 times more frequent than would be expected if these pathogens are independent of each other. Sequencing and maximum parsimony analysis of 18S rRNA revealed that four of the isolates arise in or near clades of described avian trypanosomes, likely indicating that these are vectored pathogens between birds and mosquitoes. Unexpectedly, the majority (24/28, 86%) of our positive samples form their own separate clade within the order Trypanosomatida with 100% bootstrap support. We have identified a potential new clade of trypanosomatids that exist within important mosquito vectors and discuss the potential ecological connections between these trypanosomes, arboviruses and mosquitoes.

Reported Distribution of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus in the United States, 1995-2016 (Diptera: Culicidae).

Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) transmit arboviruses that are increasing threats to human health in the Americas, particularly dengue, chikungunya, and Zika viruses. Epidemics of the associated arboviral diseases have been limited to South and Central America, Mexico, and the Caribbean in the Western Hemisphere, with only minor localized outbreaks in the United States. Nevertheless, accurate and up-to-date information for the geographical ranges of Ae. aegypti and Ae. albopictus in the United States is urgently needed to guide surveillance and enhance control capacity for these mosquitoes. We compiled county records for presence of Ae. aegypti and Ae. albopictus in the United States from 1995-2016, presented here in map format. Records were derived from the Centers for Disease Control and Prevention ArboNET database, VectorMap, the published literature, and a survey of mosquito control agencies, university researchers, and state and local health departments. Between January 1995 and March 2016, 183 counties from 26 states and the District of Columbia reported occurrence of Ae. aegypti, and 1,241 counties from 40 states and the District of Columbia reported occurrence of Ae. albopictus. During the same time period, Ae. aegypti was collected in 3 or more years from 94 counties from 14 states and the District of Columbia, and Ae. albopictus was collected during 3 or more years from 514 counties in 34 states and the District of Columbia. Our findings underscore the need for systematic surveillance of Ae. aegypti and Ae. albopictus in the United States and delineate areas with risk for the transmission of these introduced arboviruses.

The global compendium of Aedes aegypti and Ae. albopictus occurrence.

Aedes aegypti and Ae. albopictus are the main vectors transmitting dengue and chikungunya viruses. Despite being pathogens of global public health importance, knowledge of their vectors' global distribution remains patchy and sparse. A global geographic database of known occurrences of Ae. aegypti and Ae. albopictus between 1960 and 2014 was compiled. Herein we present the database, which comprises occurrence data linked to point or polygon locations, derived from peer-reviewed literature and unpublished studies including national entomological surveys and expert networks. We describe all data collection processes, as well as geo-positioning methods, database management and quality-control procedures. This is the first comprehensive global database of Ae. aegypti and Ae. albopictus occurrence, consisting of 19,930 and 22,137 geo-positioned occurrence records respectively. Both datasets can be used for a variety of mapping and spatial analyses of the vectors and, by inference, the diseases they transmit.

The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus.

Dengue and chikungunya are increasing global public health concerns due to their rapid geographical spread and increasing disease burden. Knowledge of the contemporary distribution of their shared vectors, Aedes aegypti and Aedes albopictus remains incomplete and is complicated by an ongoing range expansion fuelled by increased global trade and travel. Mapping the global distribution of these vectors and the geographical determinants of their ranges is essential for public health planning. Here we compile the largest contemporary database for both species and pair it with relevant environmental variables predicting their global distribution. We show Aedes distributions to be the widest ever recorded; now extensive in all continents, including North America and Europe. These maps will help define the spatial limits of current autochthonous transmission of dengue and chikungunya viruses. It is only with this kind of rigorous entomological baseline that we can hope to project future health impacts of these viruses.

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.