Over 50,000 human West Nile virus (WNV) (Flaviviridae: Flavivirus) clinical disease cases have been reported to the CDC during the 20 yr that the virus has been present in the United States. Despite the establishment and expansion of WNV-focused mosquito surveillance and control efforts and a renewed emphasis on applying integrated pest management (IPM) principles to WNV control, periodic local and regional WNV epidemics with case reports exceeding 2,000 cases per year have occurred during 13 of those 20 yr in the United States. In this article, we examine the scientific literature for evidence that mosquito control activities directed at either preventing WNV outbreaks or stopping those outbreaks once in progress reduce WNV human disease or have a measurable impact on entomological indicators of human WNV risk. We found that, despite a proliferation of research investigating larval and adult mosquito control effectiveness, few of these studies actually measure epidemiological outcomes or the entomological surrogates of WNV risk. Although many IPM principles (e.g., control decisions based on surveillance, use of multiple control methodologies appropriate for the ecosystem) have been implemented effectively, the use of action thresholds or meaningful public health outcome assessments have not been used routinely. Establishing thresholds for entomological indicators of human risk analogous to the economic injury level and economic thresholds utilized in crop IPM programs may result in more effective WNV prevention.

The National Ecological Observatory Network (NEON) intends to monitor mosquito populations across its broad geographical range of sites because of their prevalence in food webs, sensitivity to abiotic factors, and relevance for human health. We describe the design of mosquito population sampling in the context of NEON's long-term continental scale monitoring program, emphasizing the sampling design schedule, priorities, and collection methods. Freely available NEON data and associated field and laboratory samples, will increase our understanding of how mosquito abundance, demography, diversity, and phenology are responding to land use and climate change.

Parasites and pathogens are increasingly recognized as significant drivers of ecological and evolutionary change in natural ecosystems. Concurrently, transmission of infectious agents among human, livestock, and wildlife populations represents a growing threat to veterinary and human health. In light of these trends and the scarcity of long-term time series data on infection rates among vectors and reservoirs, the National Ecological Observatory Network (NEON) will collect measurements and samples of a suite of tick-, mosquito-, and rodent-borne parasites through a continental-scale surveillance program. Here, we describe the sampling designs for these efforts, highlighting sampling priorities, field and analytical methods, and the data as well as archived samples to be made available to the research community. Insights generated by this sampling will advance current understanding of and ability to predict changes in infection and disease dynamics in novel, interdisciplinary, and collaborative ways.

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.

Before 1999, the United States had no appropriated funding for arboviral surveillance, and many states conducted no such surveillance. After emergence of West Nile virus (WNV), federal funding was distributed to state and selected local health departments to build WNV surveillance systems. The Council of State and Territorial Epidemiologists conducted assessments of surveillance capacity of resulting systems in 2004 and in 2012; the assessment in 2012 was conducted after a 61% decrease in federal funding. In 2004, nearly all states and assessed local health departments had well-developed animal, mosquito, and human surveillance systems to monitor WNV activity and anticipate outbreaks. In 2012, many health departments had decreased mosquito surveillance and laboratory testing capacity and had no systematic disease-based surveillance for other arboviruses. Arboviral surveillance in many states might no longer be sufficient to rapidly detect and provide information needed to fully respond to WNV outbreaks and other arboviral threats (e.g., dengue, chikungunya).

West Nile virus (WNV) is a leading cause of mosquito-borne disease in the United States. Annual seasonal outbreaks vary in size and location. Predicting where and when higher than normal WNV transmission will occur can help direct limited public health resources. We developed models for the contiguous United States to identify meteorological anomalies associated with above average incidence of WNV neuroinvasive disease from 2004 to 2012. We used county-level WNV data reported to ArboNET and meteorological data from the North American Land Data Assimilation System. As a result of geographic differences in WNV transmission, we divided the United States into East and West, and 10 climate regions. Above average annual temperature was associated with increased likelihood of higher than normal WNV disease incidence, nationally and in most regions. Lower than average annual total precipitation was associated with higher disease incidence in the eastern United States, but the opposite was true in most western regions. Although multiple factors influence WNV transmission, these findings show that anomalies in temperature and precipitation are associated with above average WNV incidence. Readily accessible meteorological data may be used to develop predictive models to forecast geographic areas with elevated WNV disease risk before the coming season.

Surveillance systems for West Nile virus (WNV) combine several methods to determine the location and timing of viral amplification. The value of each surveillance method must be measured against its efficiency and costs to optimize integrated vector management and suppress WNV transmission to the human population. Here we extend previous comparisons of WNV surveillance methods by equitably comparing the most common methods after standardization on the basis of spatial sampling density and costs, and by estimating optimal levels of sampling effort for mosquito traps and sentinel chicken flocks. In general, testing for evidence of viral RNA in mosquitoes and public-reported dead birds resulted in detection of WNV approximately 2-5 weeks earlier than serological monitoring of sentinel chickens at equal spatial sampling density. For a fixed cost, testing of dead birds reported by the public was found to be the most cost effective of the methods, yielding the highest number of positive results per $1000. Increased spatial density of mosquito trapping was associated with more precise estimates of WNV infection prevalence in mosquitoes. Our findings also suggested that the most common chicken flock size of 10 birds could be reduced to six to seven without substantial reductions in timeliness or sensitivity. We conclude that a surveillance system that uses the testing of dead birds reported by the public complemented by strategically timed mosquito and chicken sampling as agency resources allow would detect viral activity efficiently in terms of effort and costs, so long as susceptible bird species that experience a high mortality rate from infection with WNV, such as corvids, are present in the area.

Chikungunya virus (CHIKV) is an alphavirus transmitted in an urban epidemic cycle by the mosquitoes Aedes aegypti and Ae. albopictus. CHIKV outbreaks are characterized by rapid spread and infection rates as high as 75%; 72%–93% of infected persons become symptomatic. The disease manifests as acute fever and potentially debilitating polyarthralgia. In a variable proportion of cases, polyarthritis and fatigue can persist for 2 years or longer (1). During outbreaks, the large percentage of symptomatic infections places a considerable strain on resources of local health care providers and hospitals. Fortunately, death from chikungunya is rare. | CHIKV was first identified in Tanganyika (now Tanzania) in 1952 (2). The virus was later found to be widely distributed and to cause sporadic, mostly small outbreaks in Africa and Asia through the 1960s and 1970s. Little activity was reported from the mid-1980s until June 2004, when an epidemic occurred on Lamu Island, Kenya, then spread during 2005 to Comoros, La Reunion, and to other Indian Ocean islands, causing ≈500,000 cases (3). This was followed in 2006–2009 by an epidemic in India that produced >1.5 million cases in 17 of the country’s 28 states and subsequently spread through Southeast Asia to the islands of the Pacific Ocean (4). The public health community has come to recognize CHIKV as a major emerging, epidemic-prone pathogen.

BACKGROUND: Heartland virus (HRTV) is a tick-borne phlebovirus recently described in Missouri that is associated with fever, leukopenia and thrombocytopenia. The virus has also been detected in Ambylomma americanum ticks. METHODS AND RESULTS: Here we report the first fatal case of HRTV disease in an 80 year-old Tennessee resident. He was hospitalized with fever, confusion, leukopenia, and thrombocytopenia and developed multi-organ failure and hemorrhage. A tick-borne illness was suspected and testing for ehrlichiosis was negative. He died on hospital day 15 and autopsy specimens were tested for various pathogens as part of an unexplained death evaluation. HRTV antigens were detected in post-mortemspleen and lymph nodes by immunohistochemistry, and HRTV was detected in pre-mortem blood by RT-PCR and by isolation in cell culture. CONCLUSIONS: This case demonstrates that HRTV infection can cause severe disease and death and expands the geographic range of HRTV within the United States.

We evaluated the commercially available Rapid Analyte Measurement Platform (RAMP (R)) West Nile virus (WNV) antigen detection test for sensitivity and consistency with real-time reverse transcriptase polymerase chain reaction (RT-PCR) confirmation testing. Panels of samples consisting of WNV-spiked mosquito pools and negative control pools were sent to 20 mosquito abatement districts (MADs) that processed the pools using the RAMP assay. The samples were then sent to the reference laboratories used by the MADs for confirmation by real-time RT-PCR. Positive pools with virus titers of roughly 1-3 log(10) PFU/ml had RAMP scores above the RAMP test positive cutoff score of 30 RAMP units, but these virus-positive samples could not be reliably confirmed by real-time RT-PCR testing. Pools with virus titers >= 4 log(10) PFU/ml scored >= 50 RAMP units. Real-time RT-PCR results varied among the confirmation laboratories. With few exceptions, pools returning a RAMP score of >= 100 were confirmed with real-time RT-PCR, while pools returning a RAMP score of 50-99 appeared to be at the limit of real-time RT-PCR detection. Therefore, we recommend using a positive cutoff of 50 RAMP units with no real-time RT-PCR confirmation to maximize speed, efficiency, and economy of the RAMP assay. A more conservative approach would be to implement a "gray zone'' range of 50-100 RAMP units. Pools scoring within the gray zone could be submitted for real-time RT-PCR confirmation with the understanding that positive pools may not confirm due to the inhibitory effect of the RAMP buffer on the real-time RT-PCR assay. We also conducted a series of experiments using laboratory-prepared mosquito pools spiked with WNV to compare mosquito homogenization buffers, pool sizes, and grinding methods in order to determine how these variables affect the RAMP and real-time RT-PCR assay results.

IMPORTANCE: Since its introduction in North America in 1999, West Nile virus has produced the 3 largest arboviral neuroinvasive disease outbreaks ever recorded in the United States. OBJECTIVE: To review the ecology, virology, epidemiology, clinical characteristics, diagnosis, prevention, and control of West Nile virus, with an emphasis on North America. EVIDENCE REVIEW: PubMed electronic database was searched through February 5, 2013. United States national surveillance data were gathered from the Centers for Disease Control and Prevention. FINDINGS: West Nile virus is now endemic throughout the contiguous United States, with 16,196 human neuroinvasive disease cases and 1549 deaths reported since 1999. More than 780,000 illnesses have likely occurred. To date, incidence is highest in the Midwest from mid-July to early September. West Nile fever develops in approximately 25% of those infected, varies greatly in clinical severity, and symptoms may be prolonged. Neuroinvasive disease (meningitis, encephalitis, acute flaccid paralysis) develops in less than 1% but carries a fatality rate of approximately 10%. Encephalitis has a highly variable clinical course but often is associated with considerable long-term morbidity. Approximately two-thirds of those with paralysis remain with significant weakness in affected limbs. Diagnosis usually rests on detection of IgM antibody in serum or cerebrospinal fluid. Treatment is supportive; no licensed human vaccine exists. Prevention uses an integrated pest management approach, which focuses on surveillance, elimination of mosquito breeding sites, and larval and adult mosquito management using pesticides to keep mosquito populations low. During outbreaks or impending outbreaks, emphasis shifts to aggressive adult mosquito control to reduce the abundance of infected, biting mosquitoes. Pesticide exposure and adverse human health events following adult mosquito control operations for West Nile virus appear negligible. CONCLUSIONS AND RELEVANCE: In North America, West Nile virus has and will remain a formidable clinical and public health problem for years to come.

In 2010, Arizona experienced an unusually early and severe outbreak of West Nile virus (WNV) centered in the southeast section of Maricopa County. Entomological data were collected before and during the outbreak, from May 25 through July 31, 2010, using the CO-baited light trap monitoring system maintained by Maricopa County Vector Control. In the outbreak area, the most abundant species in the Town of Gilbert and in the area covered by the Roosevelt Water Conservation District was Culex quinquefasciatus, constituting 75.1% and 71.8% of the total number of mosquitoes collected, respectively. Vector index (VI) profiles showed that the abundance of infected Cx. quinquefasciatus peaked prior to human cases, suggesting that this species was involved in the initiation of the outbreak. In contrast, the VI profiles for Cx. tarsalis were consistently low, suggesting limited involvement in initiating and sustaining transmission. Taken together, the higher abundance and the VI profiles strongly suggest that Cx. quinquefasciatus was the primary vector for this outbreak. The VI profiles consistently showed that the abundance of infected mosquitoes peaked 1 to 2 wk before the peaks of human cases, suggesting that VI could have successfully been utilized to predict the WNV outbreak in Maricopa County, AZ, in 2010.

In a remarkable demonstration of the potential for invasive organisms to spread | and establish in new, permissive habitats. West Nile virus (WNV) expanded from | a small area in New York City in 1999 and | is now found across much of the western | hemisphere from central Canada to southern Argentina. Enzootic WNV transmission | and human WNV disease have been reported | from all 48 of the continental United States. | The disease burden imposed by this new addition to the U.S. public health scene has | been equally remarkable. During 1999-2012, | more than 36,000 cases of human disease | were reported to the Centers for Disease | Control and Prevention (CDC), including | approximately 16,000 cases of neuroinvasive disease and 1,500 deaths (Petersen & | Fischer, 2012). Some models estimate that | between two million and four milUon people | have been infected with WNV since 1999, | and 400,000 to 1 million people may have | experienced some degree of illness due to the | infection (Petersen et al., 2012).

An outbreak of fever and meningitis/encephalitis occurred in Xinjiang, China, from August 5 to September 3, 2004. In preliminary diagnostic testing, several cerebrospinal fluid (CSF) and serum samples showed positive immunoglobulin M (IgM) antibody to Japanese encephalitis virus. Here, the CSF and serum samples of 6 cases collected at that time were tested by immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization assay (PRNT) for the existence of IgM antibody or neutralization antibody against West Nile virus (WNV) or other arboviruses. The results demonstrate the evidence of West Nile infection in Xinjiang, China.

Tahyna virus (TAHV) is widely distributed in Europe and Asia. A previous study reported a high level of conservation of the TAHV genome in isolates from Europe. During 2006 and 2007, three Tahyna virus isolates from mosquitoes were obtained from various locations in Xinjiang, People's Republic of China. We analyzed the complete coding sequence of full-length small, medium, and large segments of these isolates. Molecular and phylogenetic analyses of the three complete TAHV genomes showed that sequence identity between isolates from China and Europe was more divergent, and an unexpected level of medium segment diversity was found among isolates from China compared with high levels of sequence conservation for the small and large segments. This study indicated that effects of genotypic diversity on the ecology, transmission, and pathogenicity of TAHV in China should be studied.

Early identification of increasing mosquito activity is critical to effective mosquito control, particularly when increasing host-seeking behavior may be associated with increased risk of mosquito-borne disease. In this paper, we analyzed the temporal abundance pattern of the West Nile Virus vector, Culex tarsalis, in Fort Collins, CO, using an autoregressive integrated moving average model. We determined that an autoregressive model order 5 with lagged minimum temperatures was best at describing the seasonal abundance of Cx. tarsalis. We then tested the effect of using both temporal and spatial subsets of the data to determine the effect of reduced sampling effort on abundance predictions. We found that, if reduced trapping is necessary due to limited resources, removal of the least productive 1/3 or 1/4 of the traps produced the least erroneous predictions of seasonality represented in the observed data. We show that this productivity-based subset scheme performs better than other sampling effort reductions in generating the best estimate of Cx. tarsalis abundance per trap-night.

In 2006-2007, stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), were suspected of being enzootic vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) during a die-off of American white pelicans (Pelecanus erythrorhynchos Gmelin) (Pelecanidae) in Montana, USA. WNV-positive stable flies were observed feeding en masse on incapacitated, WNV-positive pelicans, arousing suspicions that the flies could have been involved in WNV transmission among pelicans, and perhaps to livestock and humans. We assessed biological transmission by infecting stable flies intrathoracically with WNV and testing them at 2-d intervals over 20 d. Infectious WNV was detected in fly bodies in decreasing amounts over time for only the first 6 d postinfection, an indication that WNV did not replicate within fly tissues and that stable flies cannot biologically transmit WNV. We assessed mechanical transmission using a novel technique. Specifically, we fed WNV-infected blood to individual flies by using a cotton swab (i.e., artificial donor), and at intervals of 1 min-24 h, we allowed flies to refeed on a different swab saturated with WNV-negative blood (i.e., artificial recipient). Flies mechanically transmitted viable WNV from donor to recipient swabs for up to 6 h postinfection, with the majority of the transmission events occurring within the first hour. Flies mechanically transmitted WNV RNA to recipient swabs for up to 24 h, mostly within the first 6 h. Given its predilection to feed multiple times when disturbed, these findings support the possibility that the stable fly could mechanically transmit WNV.

We used epidemiologic data for human West Nile virus (WNV) disease in Colorado from 2003 and 2007 to determine 1) the degree to which estimates of vector-borne disease occurrence is influenced by spatial scale of data aggregation (county versus census tract), and 2) the extent of concordance between spatial risk patterns based on case counts versus incidence. Statistical analyses showed that county, compared with census tract, accounted for approximately 50% of the overall variance in WNV disease incidence, and approximately 33% for the subset of cases classified as West Nile neuroinvasive disease. These findings indicate that sub-county scale presentation provides valuable risk information for stakeholders. There was high concordance between spatial patterns of WNV disease incidence and case counts for census tract (83%) but not for county (50%) or zip code (31%). We discuss how these findings impact on practices to develop spatial epidemiologic data for vector-borne diseases and present data to stakeholders.