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In 2006, Tahyna virus was isolated from Culex spp. mosquitoes collected in Xinjiang, People's Republic of China. In 2007, to determine whether this virus was infecting humans, we tested serum from febrile patients. We found immunoglobulin (Ig) M and IgG against the virus, which suggests human infection in this region. [ABSTRACT FROM AUTHOR]

In July and August of 2018, a field trial was conducted to examine the effectiveness of the North Shore Mosquito Abatement District's operational ultra-low volume (ULV) adulticide program. Two study sites were selected in Skokie, IL, and treated by truck-based ULV with d-phenothrin and prallethrin synergized with piperonyl butoxide over the course of a month. Natural mosquito populations were sampled via Biogents (BG)-counter baited with CO 2 or Alfalfa infusion. The results from this study demonstrate that host-seeking mosquitoes were reduced by 65.3% after ULV treatment while gravid mosquitoes were reduced by only 29.2%. In addition, host-seeking mosquitoes rebounded dramatically (303.1%) 3 days posttreatment while gravid mosquitoes did not (5.7%). Based on the differential effect between gravid and host-seeking mosquitoes, we concluded that the gonotrophic cycle and timing of ULV adulticide operations are important factors affecting the resistance of West Nile virus vectors to pyrethroid exposures.

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 Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Effectiveness in controlling mosquitoes in storm water catch basins in the North Shore Mosquito Abatement District (northeastern Cook County, Illinois) was determined for 3 formulations of methoprene-based larvicides (Altosid XR 150-day Briquets, Altosid 30-day Pellets, Altosid 30-day Granules) in 2017 using a pass/fail evaluation criterion, in which emergence of a single adult from pupae collected from the basin constituted a control failure. Over the course of the 16-week study, basins receiving the 150-day briquets were treated once and basins receiving the pellet and granular formulations were treated every 4 weeks, with the first treatment occurring during the last week of May. Untreated basins were also observed for comparison with the treated basins. Over the course of the study, adult mosquitoes emerged from pupae collected in 94.2% of the untreated basins that contained pupae. All of the formulations evaluated in the study demonstrated some degree of control compared with the untreated basins, with pupae successfully emerging as adults in 64.6%, 55.5%, and 21.8% of samples from 150-day briquet, 30-day tablet, and 30-day pellet-treated basins that contained pupae, respectively. Pellets reapplied every 28 days provided significantly more effective control than the other formulations. The simple pass/fail criterion for evaluating control effectiveness proved to be a useful procedure for comparing effectiveness to untreated basins and among treatments., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Effectiveness in controlling mosquito larvae in stormwater catch basins in the North Shore Mosquito Abatement District (northeastern Cook County, IL) was determined for 2 extended-duration larvicides indicating up to 180 days of control on their labels (Natular™ XRT, FourStar® Briquet) and 3 larvicides indicating up to 30 days of control (Natular™ T30, Natular™ G30, and VectoLex® FG). Over the course of the 26-wk study, catch basins receiving the extended-release products were treated twice (an initial treatment in early April followed by a re-treatment after 16 wk), and catch basins receiving the shorter-duration products were treated every 28 days, with the 1st treatment occurring during the 1st week in April. Control in an individual catch basin was considered to have failed if late-stage larvae or pupae were found in 2-dip samples taken from the catch basin. Control for a treatment was considered to have failed if >25% of catch basins treated with the product failed at a given time period posttreatment. All of the products evaluated in the study demonstrated some degree of control; however, the Natular XRT-, FourStar Briquet-, and Natular T30-treated basins rarely achieved the effectiveness threshold of ≤25% of catch basins failing. By comparison, basins treated with Natular G30 were below that threshold for 3 of 4 wk every treatment round and VectoLex® FG was consistently below that threshold for all 4 wk posttreatment for every treatment round. Compared with untreated catch basins, the total season pupal production was reduced by approximately 48% in the Natular XRT-, FourStar Briquet-, and Natular T30-treated basins, and by 87% and 99% in the Natular G30- and VectoLex FG-treated basins, respectively. Operational quality control observations indicated that effective control (i.e., ≤25% of catch basins failing) ranged from 3 to 12 wk posttreatment for catch basins treated with Natular XRT and from 5 to 9 wk with VectoLex FG, and that there was considerable geographic variation in the duration of effectiveness. The results indicate that 30-day re-treatments with granular formulations in difficult-to-control areas may provide a more cost-effective outcome than using 1 or 2 applications of extended-duration larvicides.

Since the mid-1990s, the North Shore Mosquito Abatement District (NSMAD) has applied extended-release formulations of mosquito larvicides to approximately 50,000 catch basins in the suburbs north of Chicago, IL, USA. This is performed as part of NSMAD's efforts to reduce local populations of the West Nile virus vector, Culex pipiens. Analyses from NSMAD's monitoring of larvicide-treated basins throughout the District over the 2014 and 2015 seasons suggest that larvicides intended to provide extended durations of control (30-180 days) failed to provide control for the maximum duration specified on the product label in approximately 25% of the District's basins. For larvicides designed to last up to 180 days (or about 26 weeks), failures were found at 1-15 weeks after treatment with most found at five weeks posttreatment. For larvicides formulated to last up to 30 days, failures were found at one to four weeks after applications with most found at three weeks posttreatment. The highest percentages of failing basins (ie, containing late-stage mosquito larvae or pupae during the specified product effectiveness period) were found in communities on the eastern side of the District, bordering Lake Michigan. As the larvicides appeared to function properly in the majority of monitored basins, it appears that the failures likely resulted from basin-specific physical factors (ie, basin volume, sediment content, and hydrology) that cause either product removal or a reduction in the concentration of the larvicide's active ingredient below the effective levels in these basins.

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 disease 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., (© The American Society of Tropical Medicine and Hygiene.)

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.

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: 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 multiorgan 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., Results: HRTV antigens were detected in postmortem spleen and lymph nodes by immunohistochemistry, and HRTV was detected in premortem blood by reverse transcription polymerase chain reaction 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., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

We evaluated the commercially available Rapid Analyte Measurement Platform (RAMP) 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 log10 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 > or =4 log10 PFU/ml scored > or =50 RAMP units. Real-time RT-PCR results varied among the confirmation laboratories. With few exceptions, pools returning a RAMP score of > or =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.

Objective: The western borderland between Yunnan Province, China, and Myanmar is characterized by a climate that facilitates year-round production of mosquitoes. Numerous mosquito-transmitted viruses, including Japanese encephalitis virus circulate in this area. This project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations., Methods: Mosquitoes were collected in Mangshi and Ruili cities of Dehong Prefecture near the border of China and Burma in Yunnan Province, the Peoples Republic of China in 2010. We monitored mosquito species abundance for a 12-month period using ultraviolet light, carbon dioxide baited CDC light and gravid traps; and tested the captured mosquitoes for the presence of virus to evaluate mosquito-virus associations in rural/agricultural settings in the area., Results: A total of 43 species of mosquitoes from seven genera were collected, including 15 Culex species, 15 Anopheles spp., four Aedes spp., three Armigeres spp., one Mimomyia spp., two Uranotaenia spp. and three Mansonia spp.. Species richness and diversity varied between Mangshi and Ruili. Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Anopheles peditaeniatus were the most abundant species in both sampling sites. Ultraviolet light traps collected more specimens than CDC light traps baited with dry ice, though both collected the same variety of mosquito species. The CDC gravid trap was the most effective trap for capture of Culex quinquefasciatus, a species underrepresented in light trap collections. A total of 26 virus strains were isolated, which included 13 strains of Japanese encephalitis virus, four strains of Getah virus, one strain of Oya virus, one strain from the orbivirus genus, and seven strains of Culex pipien pallens densovirus., Conclusions: The present study illustrates the value of monitoring mosquito populations and mosquito-transmitted viruses year-round in areas where the climate supports year-round adult mosquito activity.

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 CO2-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.

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.

To investigate the infection status and the spatial distribution of Tahyna virus infection among unknown fever cases in Xinjiang, China. Sera samples of unknown fever cases from Kashi in southern Xin-jiang and Yili in northern Xinjiang were tested against Tahyna virus by IFA. Partial positive cases were tested against Tahyna virus/Snowshoe hare virus/Inkoo virus parrelled. Finally, 742 sera samples of unknown fever cases were collected from Kashi, Southern Xinjiang in 2007-2008, the positive rate of IgM antibody against Tahyna virus was 5.3%, the positive rate of IgG antibody against Tahyna virus was 18.3%. 222 sera samples of unknown fever cases were collected from Yili, Northern Xinjiang in 2008, no positive case of IgM antibody against Tahyna was found. 10 cases showed antibody neutralization against Tahyna virus by plaque reduction neutralization test. Our results demonstrate that there is current infection and past infection of Tahyna virus among Southern Xinjiang residents.

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.

An investigation was conducted to identify the distribution of mosquitoes and mosquito-borne arboviruses in the Qinghai-Tibet Plateau, China from July to August in 2007. A total of 8,147 mosquitoes representing six species from three genera (Aedes, Culex, and Anopheles) were collected in three locations (Geermu city, altitude of 2,780 m; Xining city, 2,200 m; Minhe county, 1,700 m). Six virus isolates were obtained including Tahyna virus (TAHV), Liaoning virus, and Culex pipiens pallens Densovirus. A serosurvey showed immunoglobulin G antibodies by immunofluorescence assay (IFA) against TAHV in residents of all three locations. The IFA-positive human samples were confirmed by 90% plaque-reduction neutralization tests (PRNT(90)) against TAHV with titers ranging from 1:20 to 1:10,240. In addition, TAHV seropositive cows, sheep, and swine were found in these locations. This investigation represents the first isolation of TAHV from Ae. (Och.) detritus and the first evidence of TAHV infection in residents and livestock in the Qinghai-Tibet Plateau.

A rapid assessment was conducted in July-August 2007 to determine the impact of heavy rains and early summer floods on the mosquitoes and arbovirus activity in 4 southeastern Kansas counties. During 10 days and nights of collections using different types and styles of mosquito traps, a total of 10,512 adult female mosquitoes representing 29 species were collected, including a new species record for Kansas (Psorophora mathesoni). High numbers of Aedes albopictus were collected. Over 4,000 specimens of 4 Culex species in 235 species-specific pools were tested for the presence of West Nile, St. Louis, and western equine encephalitis viruses. Thirty pools representing 3 Culex species were positive for West Nile virus (WNV). No other arboviruses were detected in the samples. Infection rates of WNV in Culex pipiens complex in 2 counties (10.7/1,000 to 22.6/1,000) and in Culex salinarius in 1 county (6.0/1,000) were sufficiently high to increase the risk of transmission to humans. The infection rate of WNV in Culex erraticus was 1.9/1,000 in one county. Two focal hot spots of intense WNV transmission were identified in Montgomery and Wilson counties, where infection rates in Cx. pipiens complex were 26/ 1,000 and 19.9/1,000, respectively. Despite confirmed evidence of WNV activity in the area, there was no increase in human cases of arboviral disease documented in the 4 counties for the remainder of 2007.

We conducted a laboratory evaluation of the ability of commercial antigen-capture assays, the Rapid Analyte Measurement Platform (RAMP) and the VecTest wicking assay, as well as Real Time reverse transcriptase polymerase chain reaction (RT-PCR, Taqman) and Vero cell plaque assay to detect West Nile virus (WNV) in large mosquito pools. Real-Time PCR (Taqman) was the most sensitive, detecting WNV ribonucleic acid (RNA) in 100% of samples containing a single infected mosquito in pool sizes of up to 500 mosquitoes. Mosquito body tissues minimally impacted the ability of Real Time RT-PCR to detect WNV in a pool size of 500, reducing sensitivity by 0.6 log10 plaque-forming units (PFU)/ml. Vero cell plaque assay detected live virus from a single infected mosquito in 100% of pools containing up to 200 mosquitoes, but was unreliable at larger pool sizes. VecTest detected 100% of positive pools containing 50 mosquitoes with 5.8 log10 PFU/ml virus, 100 mosquitoes with 5.9 log10 PFU/ml, and 200 mosquitoes with 5.2 log10 PFU/ ml. The RAMP assay detected 100% of positive pools containing 50 mosquitoes with 3.3 log10 PFU/ml virus, 100 mosquitoes with 3.7 log10 PFU/ml, and 200 mosquitoes with 4.0 log10 PFU/ml. Results indicate that WNV can be reliably detected by all 4 assays in pools of mosquitoes exceeding 50 specimens, though there is some loss of sensitivity with very large pool sizes.

Two commercially available West Nile virus (WNV) detection assays (RAMP WNV test, Response Biomedical Corp., Burnaby, British Columbia, Canada; and VecTest WNV antigen assay, Medical Analysis Systems, Inc., Camarillo, CA) were compared for sensitivity, specificity, and ability to detect WNV in field-collected mosquito pools. Serially diluted stock seed WNV and St. Louis encephalitis virus (SLEV) were used to determine sensitivity and specificity. The RAMP WNV test detected WNV at concentrations as low as 3.17 log10 plaque-forming units per milliliter (PFU/ml), whereas the VecTest assay detected WNV at concentrations as low as 5.17 log10 PFU/ml. Neither test cross-reacted with SLEV. A WNV-specific reverse transcriptase polymerase chain reaction was used to identify positives among field-collected mosquito pools. The RAMP WNV test detected 94% of positive pools and the VecTest assay detected 65% of the positive field-collected pools. Despite these differences, both assays have characteristics that make them useful in WNV surveillance programs.

Culex quinquefasciatus, Cx. restuans, Cx. pipiens complex, and Cx. nigripalpus were collected as larvae or egg rafts from the southern USA. Adult female mosquitoes were intrathoracically inoculated with approximately 1,000 plaque-forming units of West Nile virus (WNV) and saliva was collected from them 5 days later. The amount of infectious WNV in the saliva samples was quantified by plaque assay and WNV RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). More than 90% of the mosquitoes had either infectious virus or viral RNA in their saliva. The RT-PCR assay detected a greater percent of samples with WNV RNA than the plaque assay detected infectious virus. Pairwise comparisons revealed 6 significant differences between the 7 groups surveyed. The Cx. nigripalpus secreted lower mean amounts of WNV than 3 other groups, and a difference was found between early- and late-season Cx. quinquefasciatus collected in Louisiana.

We compared the VecTest WNV antigen assay with standard methods of West Nile virus (WNV) detection in swabs from American Crows (Corvus brachyrhynchos) and House Sparrows (Passer domesticus). The VecTest detected WNV more frequently than the plaque assay and was comparable to a TaqMan reverse transcription-polymerase chain reaction.

From 1937 until 1999, West Nile virus (WNV) garnered scant medical attention as the cause of febrile illness and sporadic encephalitis in parts of Africa, Asia, and Europe. After the surprising detection of WNV in New York City in 1999, the virus has spread dramatically westward across the United States, southward into Central America and the Caribbean, and northward into Canada, resulting in the largest epidemics of neuroinvasive WNV disease ever reported. From 1999 to 2004, >7,000 neuroinvasive WNV disease cases were reported in the United States. In 2002, WNV transmission through blood transfusion and organ transplantation was described for the first time, intrauterine transmission was first documented, and possible transmission through breastfeeding was reported. This review highlights new information regarding the epidemiology and dynamics of WNV transmission, providing a new platform for further research into preventing and controlling WNV disease.

In total, 1,324 Culex pipiens pipiens L. female mosquitoes were collected at Ft. Hancock, Monmouth County, New Jersey, from January to March 2001-2003. Mosquitoes were held in an insectary at 27 degrees C and a photoperiod of 16:8 (L:D) h for 6 to 21 d after which they were tested in 34 pools. West Nile viral RNA was detected in one pool by a TaqMan reverse transcription-polymerase chain reaction assay; however, infectious virus could not be isolated using either Vero cell plaque assay or C6/36 mosquito cells. Twenty females dissected in January and March 2003 confirmed ovarian diapause status. We suggest that the mode of infection in this pool of overwintering females may have been due to vertical (transgenerational) transmission.

We report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.

Saliva was collected from 4 species of mosquitoes intrathoracically inoculated with West Nile virus (WNV). The amount of infectious virus in the saliva was quantified by plaque assay and the number of WNV genomic equivalents (GE) was measured by reverse transcriptase-polymerase chain reaction. Ochlerotatus triseriatus had the greatest mean amount of infectious virus per saliva collection, followed by Aedes albopictus, Culex pipiens, and Cx. quinquefasciatus. The mean GE/saliva collection was also greatest in Oc. triseriatus, followed by Cx. quinquefasciatus, Cx. pipiens, and Ae. albopictus. The variance of log GE/saliva collection for Ae. albopictus was significantly lower than the variance for the other 3 species. This study provides a basis for comparing this component of vector competence and for determining the amounts of virus inoculated into vertebrates in experimental host competence studies.

VecTest assays for detecting eastern equine encephalitis virus (EEE) and western equine encephalitis virus (WEE) antigen in mosquito pools were evaluated to determine their sensitivity and specificity by using a range of EEE, WEE, St. Louis encephalitis virus (SLE), and West Nile virus (WN) dilutions as well as individual and pooled mosquitoes containing EEE or WEE. The EEE test produced reliable positive results with samples containing > or = 5.3 log10 plaque-forming units (PFU) of EEE/ml, and the WEE test produced reliable positive results with samples containing > or = 4.7 log10 PFU WEE/ml. Both assays detected the respective viral antigens in single virus-positive mosquitoes and in pools containing a single positive mosquito and 49 negative specimens. The SLE and WN assays also contained on the dipsticks accurately detected their respective viruses. No evidence was found of cross reaction or false positives in any of the tests. The VecTest assays were less sensitive than the EEE- and WEE-specific TaqMan reverse transcriptase polymerase chain reaction and Vero cell plaque assay, but appear to be useful for detecting arboviruses in mosquito-based arbovirus surveillance programs.

Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed.

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.

The host-feeding patterns of mosquitoes (n = 247) collected in the Borough of Queens in New York City in July and August 2000 were investigated using an indirect ELISA and a polymerase chain reaction (PCR)-heteroduplex assay. Culex pipiens L. and Cx. restuans Theobald fed primarily on birds, and their feeding habits support their implication as enzootic vectors of West Nile virus. Culex salinarius Coquillett and Coquillettidia perturbans (Walker) fed mainly on mammals, with fewer blood meals taken from birds, and these two species are potential bridge vectors of West Nile virus. Culex mosquitoes took blood meals (n = 54) from 11 different avian species. Only the northern cardinal (Cardinalis cardinalis), American robin (Turdus migratorius), and Brown-headed cow bird (MolIothrus ater) were fed upon by all three Culex species. Multiple blood feedings on avian hosts were detected in Cx. pipiens and Cx. restuans. Species identifications of Culex mosquitoes made using morphological characteristics were confirmed with a PCR assay that employed species-specific primers. All Cx. pipiens (n = 20) and Cx. salinarius (n = 10) specimens were correctly identified, but three (20%) of 15 Cx. restuans were misidentified as Cx. pipiens.

Surveillance of container-inhabiting mosquitoes was conducted from June 17 through November 9, 1998, at 2 1997 La Crosse virus (LAC) human case sites (Knox and Cocke counties, Tennessee). Mosquitoes were collected weekly with 2 dry ice-baited Centers for Disease Control miniature light traps, 2 omnidirectional Fay traps, and 40 oviposition traps at each site. A total of 8,408 mosquitoes, composed of Ochlerotatus triseriatus (n = 2,095) and Aedes albopictus (n = 6,313), were reared or collected and assayed for virus. The majority of host-seeking Ae. albopictus (n = 567) collected from July through October from both sites were dissected to determine parity status. Monthly parity rates ranged from 0.78 to 0.85 and 0.79 to 0.92 in Knox and Cocke counties, respectively. The high parity rates indicate that this population of Ae. albopictus has a high daily survival rate and may have a high vector potential. The temporal patterns in Ae. albopictus and Oc. triseriatus egg collections from both of the human case sites were significantly correlated, suggesting that the populations fluctuate in a similar manner across the eastern Tennessee region. Although LAC was not isolated from either species, one isolation of a California serogroup virus, most likely a subtype of Jamestown Canyon virus (JC), was recovered from a pool of 50 male Ae. albopictus reared from eggs collected at the Knox County site (minimum field infection rate of 1.89 per 1,000). This is the 1st report of a very closely related JC-like virus in Ae. albopictus and from Tennessee, as well as the 1st time this potential human pathogen has been isolated from transovarially infected field populations of Ae. albopictus.

A blinded cohort study was conducted in 2000 to better understand the emergence of La Crosse virus infection in eastern Tennessee, with special emphasis on the potential mosquito vector(s). Children with suspected central nervous system infection were enrolled at the time of clinical presentation at a large pediatric referral hospital. Clinical, environmental, and entomological data were collected prior to case confirmation. Sixteen of the 40 children included in the final analysis were confirmed to have La Crosse infection by a fourfold increase in antibody titers between collection of acute- and convalescent-phase sera. Factors significantly associated with La Crosse infection included average number of hours per day spent outdoors (5.9 for La Crosse virus cases vs. 4.0 for noncases, p = 0.049); living in a residence with one or more tree holes within 100 m (relative risk = 3.96 vs. no tree holes within 100 m, p = 0.028); and total burden of Aedes albopictus (number of female and male larvae and adults collected at a site), which was more than three times greater around the residences of La Crosse virus cases versus noncases (p = 0.013). Evidence is accumulating that the newly introduced mosquito species Ae. albopictus may be involved in the emergence of La Crosse virus infection in eastern Tennessee.

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.

La Crosse (LAC) virus, a California serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the United States and an emerging disease in Tennessee, West Virginia, and North Carolina. Human cases of LAC encephalitis in Tennessee and North Carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. This report describes the isolation of LAC virus from the exotic mosquito Aedes albopictus. The discovery of LAC virus in wild populations of Ae. albopictus coupled with its expanding distribution in the southeastern United States, suggests that this mosquito may become an important accessory vector, potentially increasing the number of human cases in endemic foci or expanding the range of the disease.

Surveillance of mosquito populations for virus activity is not often performed by small, vector-control districts because they do not have the financial resources to use virus isolation, or newer methods such as the polymerase chain reaction. Consequently, development and refinements of rapid, sensitive, and simple enzyme-linked immunosorbent assays (ELISAs) applicable to a wide variety of public health settings are justified. We have developed an antigen-capture ELISA for the detection of eastern equine encephalitis (EEE) virus in mosquitoes that uses both monoclonal capture and detector antibodies. The sensitivity of this assay is 4.0-5.0 log10 plaque-forming units/ml, which is comparable to previously published EEE antigen-capture assays developed with polyclonal antibody reagents. This test identifies only North American strains of EEE virus and does not react with either western equine encephalitis or Highlands J viruses. Test sensitivity was enhanced by sonicating mosquito pools, treating them with Triton X-100, and increasing the time and temperature of antigen incubation. The conversion of this ELISA to a monoclonal antibody-based format should result in a readily standardizable and transferable assay that will permit laboratories lacking virus isolation facilities to conduct EEE virus surveillance.

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.

West Nile (WN) virus transmission in the United States during 2000 was most intense on Staten Island, New York, where 10 neurologic illnesses among humans and 2 among horses occurred. WN virus was isolated from Aedes vexans, Culex pipiens, Cx. salinarius, Ochlerotatus triseriatus, and Psorophora ferox, and WN viral RNA was detected in Anopheles punctipennis. An elevated weekly minimum infection rate (MIR) for Cx. pipiens and increased dead bird density were present for 2 weeks before the first human illness occurred. Increasing mosquito MIRs and dead bird densities in an area may be indicators of an increasing risk for human infections. A transmission model is proposed involving Cx. pipiens and Cx. restuans as the primary enzootic and epizootic vectors among birds, Cx. salinarius as the primary bridge vector for humans, and Aedes/Ochlerotatus spp. as bridge vectors for equine infection.