Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

Use of temperature to improve West Nile virus forecasts.

Ecological and laboratory studies have demonstrated that temperature modulates West Nile virus (WNV) transmission dynamics and spillover infection to humans. Here we explore whether inclusion of temperature forcing in a model depicting WNV transmission improves WNV forecast accuracy relative to a baseline model depicting WNV transmission without temperature forcing. Both models are optimized using a data assimilation method and two observed data streams: mosquito infection rates and reported human WNV cases. Each coupled model-inference framework is then used to generate retrospective ensemble forecasts of WNV for 110 outbreak years from among 12 geographically diverse United States counties. The temperature-forced model improves forecast accuracy for much of the outbreak season. From the end of July until the beginning of October, a timespan during which 70% of human cases are reported, the temperature-forced model generated forecasts of the total number of human cases over the next 3 weeks, total number of human cases over the season, the week with the highest percentage of infectious mosquitoes, and the peak percentage of infectious mosquitoes that on average increased absolute forecast accuracy 5%, 10%, 12%, and 6%, respectively, over the non-temperature forced baseline model. These results indicate that use of temperature forcing improves WNV forecast accuracy and provide further evidence that temperature influences rates of WNV transmission. The findings provide a foundation for implementation of a statistically rigorous system for real-time forecast of seasonal WNV outbreaks and their use as a quantitative decision support tool for public health officials and mosquito control programs.

Population genetics of an invasive mosquito vector, Aedes albopictus in the Northeastern USA.

The Asian tiger mosquito (Aedes albopictus) arrived in the USA in the 1980's and rapidly spread throughout eastern USA within a decade. The predicted northern edge of its overwintering distribution on the East Coast of the USA roughly falls across New York, Connecticut, and Massachusetts, where the species has been recorded as early as 2000. It is unclear whether Ae. albopictus populations have become established and survive the cold winters in these areas or are recolonized every year. We genotyped and analyzed populations of Ae. albopictus from the northeast USA using 15 microsatellite markers and compared them with other populations across the country and to representatives of the major global genetic clades to investigate their connectivity and stability. Founder effects or bottlenecks were rare at the northern range of the Ae. albopictus distribution in the northeastern USA, with populations displaying high levels of genetic diversity and connectivity along the East Coast. There is no evidence of population turnover in Connecticut during the course of three consecutive years, with consistent genetic structure throughout this period. Overall, these results support the presence of established populations of Ae. albopictus in New York, Connecticut, and Massachusetts, successfully overwintering and migrating in large numbers. Given the stability and interconnectedness of these populations, Ae. albopictus has the potential to continue to proliferate and expand its range northward under mean warming conditions of climate change. Efforts to control Ae. albopictus in these areas should thus focus on vector suppression rather than eradication strategies, as local populations have become firmly established and are expected to reemerge every summer.

Meteorological and hydrological influences on the spatial and temporal prevalence of West Nile virus in Culex mosquitoes, Suffolk County, New York.

The factors determining the spatial and temporal distribution of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) activity are not well understood. Here, we explore the effects of hydrological and meteorological conditions on WNV infection among Culex genus mosquitoes collected during 2001-2009 in Suffolk County, Long Island, NY. We show that WNV infection rates in assayed pools of Culex mosquitoes are associated in both space and time with hydrological and meteorological variability. Specifically, wet winter, warm and wet spring conditions, and dry summer conditions are associated with the increased local prevalence of WNV among Culex mosquitoes during summer and fall. These findings indicate that within Suffolk County, and for a given year, areas at risk for heightened WNV activity may be identified in advance by using hydrology model estimates of land surface wetness and observed meteorological conditions.

Susceptibility of Ixodes scapularis (Acari: Ixodidae) to Permethrin Under a Long-Term 4-Poster Deer Treatment Area on Shelter Island, NY.

Pesticide resistance in medically significant disease vectors can negatively impact the efficacy of control efforts. Resistance research on ticks has focused primarily on species of veterinary significance that experience relatively high degrees of control pressure. Resistance in tick vectors of medical significance has received little attention, in part because area-wide pesticide applications are not used to control these generalist tick species. One of the few effective methods currently used for area-wide control of medically important ticks, including Ixodes scapularis Say (Acari: Ixodidae), is deployment of 4-poster devices. Deer self-apply a topical acaricide (permethrin) while feeding on corn from the devices. A 4-poster program using permethrin has been deployed on Shelter Island, NY to control I. scapularis populations since 2008. We collected engorged female ticks from deer in this management area and a location in the Mid-Hudson River Valley, NY without area-wide tick control. Larvae were reared from egg masses and their susceptibility to permethrin was tested. Larvae originating from a long-term laboratory colony were used as a susceptible baseline for comparison. Compared against the laboratory colony, resistance ratios at LC-50 for Shelter Island and Hudson Valley I. scapularis were 1.87 and 1.51, respectively. The susceptibilities of the field populations to permethrin were significantly lower than that of the colony ticks. We provide the first data using the larval packet test to establish baseline susceptibility for I. scapularis to permethrin along with information relevant to understanding resistance emergence in tick populations under sustained control pressure from 4-poster devices.

Using supervised machine learning to identify efficient blocking schemes for record linkage.

Record linkage enables survey data to be integrated with other data sources, expanding the analytic potential of both sources. However, depending on the number of records being linked, the processing time can be prohibitive. This paper describes a case study using a supervised machine learning algorithm, known as the Sequential Coverage Algorithm (SCA). The SCA was used to develop the join strategy for two data sources, the National Center for Health Statistics' (NCHS) 2016 National Hospital Care Survey (NHCS) and the Center for Medicare & Medicaid Services (CMS) Enrollment Database (EDB), during record linkage. Due to the size of the CMS data, common record joining methods (i.e. blocking) were used to reduce the number of pairs that need to be evaluated to identify the vast majority of matches. NCHS conducted a case study examining how the SCA improved the efficiency of blocking. This paper describes how the SCA was used to design the blocking used in this linkage.

NEVBD Pesticide Resistance Monitoring Network: Establishing a Centralized Network to Increase Regional Capacity for Pesticide Resistance Detection and Monitoring.

Pesticide resistance in arthropod vectors of disease agents is a growing issue globally. Despite the importance of resistance monitoring to inform mosquito control programs, no regional monitoring programs exist in the United States. The Northeastern Regional Center for Excellence in Vector-Borne Diseases (NEVBD) is a consortium of researchers and public health practitioners with a primary goal of supporting regional vector control activities. NEVBD initiated a pesticide resistance monitoring program to detect resistant mosquito populations throughout the northeastern United States. A regionwide survey was distributed to vector control agencies to determine needs and refine program development and in response, a specimen submission system was established, allowing agencies to submit Culex pipiens (L.) (Diptera:Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) for pesticide resistance testing. NEVBD also established larvicide resistance diagnostics for Bacillus thuringiensis israelensis (Bti) and methoprene. Additional diagnostics were developed for Cx. pipiens resistance to Lysinibacillus sphaericus. We received 58 survey responses, representing at least one agency from each of the 13 northeastern U.S. states. Results indicated that larvicides were deployed more frequently than adulticides, but rarely paired with resistance monitoring. Over 18,000 mosquitoes were tested from six states. Widespread low-level (1 × LC-99) methoprene resistance was detected in Cx. pipiens, but not in Ae. albopictus. No resistance to Bti or L. sphaericus was detected. Resistance to pyrethroids was detected in many locations for both species. Our results highlight the need for increased pesticide resistance testing in the United States and we provide guidance for building a centralized pesticide resistance testing program.

Aedes japonicus and West Nile Virus in New York.

Identifying the array of vectors that play a role in perpetuating West Nile virus (WNV) infection in endemic foci will help in controlling the disease. Aedes japonicus has the potential to be a vector in the wild of at least 3 kinds of encephalitis, including WNV. Aedes japonicus is a nonnative species in the USA that is temperature tolerant and a potential human biter. Detection of WNV in mosquito pools of this field-collected invasive species, combined with their ability to feed on humans, make this mosquito species a possible public health concern. In this study, we collected mosquito abundance data and tested them for WNV-positive mosquito samples from 3 counties in New York State. We found a significant association between the season and land demography and the likelihood of the virus in Ae. japonicus.

Spatial and temporal expansions of Eastern equine encephalitis virus and phylogenetic groups isolated from mosquitoes and mammalian cases in New York State from 2013 to 2019.

Surveillance for the emerging infectious disease Eastern equine encephalitis, and its causative virus in mosquitoes, continued within New York State from 2013 to 2019. There were increases in geographic area and number of consecutive years, with cases in four mammalian species, and virus in 11 mosquito species. The first cases in a goat and in an emu were reported. The first detection of virus in Aedes cinereus was reported. Virus in phylogenetic group NY4 was isolated from a horse and from mosquitoes 6 kilometers and 13 days apart in 2013. Phylogenetic groups NY4 and NY5 were found 15 days apart in two towns 280 kilometers distant in 2013. Within four adjacent counties there was a pattern of overlap, where four had NY5, two adjacent counties had NY6, two adjacent counties had NY7, and one county had NY5, NY6, and NY7, reducible to a Euler diagram. Virus in phylogenetic group NY5, found within an 11-kilometer wide area in New York State, was related to FL4 found in Florida 1,398 kilometers distant. This was consistent with a phylogenetic group originating in Florida, then being moved to a specific location in New York State, by migratory birds in consecutive years 2013 and 2014.

First glimpse into the origin and spread of the Asian longhorned tick, Haemaphysalis longicornis, in the United States.

Established populations of Asian longhorned ticks (ALT), Haemaphysalis longicornis, were first identified in the United States (US) in 2017 by sequencing the mitochondrial cytochrome c oxidase subunit I (cox1) 'barcoding' locus followed by morphological confirmation. Subsequent investigations detected ALT infestations in 12, mostly eastern, US states. To gain information on the origin and spread of US ALT, we (1) sequenced cox1 from ALT populations across 9 US states and (2) obtained cox1 sequences from potential source populations [China, Japan and Republic of Korea (ROK) as well as Australia, New Zealand and the Kingdom of Tonga (KOT)] both by sequencing and by downloading publicly available sequences in NCBI GenBank. Additionally, we conducted epidemiological investigations of properties near its initial detection locale in Hunterdon County, NJ, as well as a broader risk analysis for importation of ectoparasites into the area. In eastern Asian populations (China/Japan/ROK), we detected 35 cox1 haplotypes that neatly clustered into two clades with known bisexual versus parthenogenetic phenotypes. In Australia/New Zealand/KOT, we detected 10 cox1 haplotypes all falling within the parthenogenetic cluster. In the United States, we detected three differentially distributed cox1 haplotypes from the parthenogenetic cluster, supporting phenotypic evidence that US ALT are parthenogenetic. While none of the source populations examined had all three US cox1 haplotypes, a phylogeographic network analysis supports a northeast Asian source for the US populations. Within the United States, epidemiological investigations indicate ALT can be moved long distances by human transport of animals, such as horses and dogs, with smaller scale movements on wildlife. These results have relevant implications for efforts aimed at minimizing the spread of ALT in the United States and preventing additional exotic tick introductions.

Modeling and Surveillance of Reporting Delays of Mosquitoes and Humans Infected With West Nile Virus and Associations With Accuracy of West Nile Virus Forecasts.

Importance: West Nile virus (WNV) is the leading cause of domestically acquired arboviral disease. Objective: To develop real-time WNV forecasts of infected mosquitoes and human cases. Design, Setting, and Participants: Real-time forecasts of WNV in 4 geographically dispersed locations in the United States were generated using a WNV model-inference forecasting system previously validated with retrospective data. Analysis was performed to evaluate how observational reporting delays of mosquito WNV assay results and human medical records were associated with real-time forecast accuracy. Exposures: Mosquitoes positive for WNV and human cases. Main Outcomes and Measures: Delays in reporting mosquito WNV assay results and human medical records and the association of these delays with real-time WNV forecast accuracy. Results: Substantial delays in data reporting exist for both infected mosquitoes and human WNV cases. For human cases, confirmed data (n = 37) lagged behind the onset of illness by a mean (SD) of 5.5 (2.3) weeks (range, 2-14 weeks). These human case reporting lags reduced mean forecast accuracy for the total number of human cases over the season in 110 simulated outbreaks for 2 forecasting systems by 26% and 14%, from 2 weeks before to 3 weeks after the predicted peak of infected mosquitoes. This period is the time span during which 47% of human cases are reported. Of 7064 mosquito pools, 500 (7%) tested positive; the reporting lag for these data associated with viral testing at a state laboratory was a mean (SD) of 6.6 (2.6) days (range, 4-11 days). This reporting lag was associated with decreased mean forecast accuracy for the 3 mosquito infection indicators, timing, magnitude, and season, by approximately 5% for both forecasting systems. Conclusions and Relevance: Delays in reporting human WNV disease and infected mosquito information are associated with difficulties in outbreak surveillance and decreased real-time forecast accuracy. Infected mosquito lags were short enough that skillful forecasts could still be generated for mosquito infection indicators, but the human WNV case lags were too great to support accurate forecasting in real time. Forecasting WNV is potentially an important evidence-based decision support tool for public health officials and mosquito abatement districts; however, to operationalize real-time forecasting, more resources are needed to reduce human case reporting lags between illness onset and case confirmation.

Salt marsh as Culex salinarius larval habitat in coastal New York.

Culex salinarius is considered one of the most likely bridge vectors involved in the human transmission cycle of West Nile virus (WNV) and eastern equine encephalomyelitis virus (EEEV) in the northeastern USA. The larval habitats of this species in the coastal region of New York State are currently poorly known. Between 2005 and 2007, a larval survey was carried out to identify and characterize possible larval habitats in Suffolk County, encompassing natural and man-made freshwater wetlands, artificial containers, and salt marshes. Only relatively undisturbed salt marsh yielded Cx. salinarius larvae in considerable numbers from several sites over a period of 2 years. The immature stages of this species were found associated with Spartina patens and S. alterniflora of the upper marsh at salinities ranging from 4.3 to 18.8 parts per thousand. Both heavily impacted and relatively undisturbed salt marshes produced several hundreds of adult Cx. salinarius per Centers for Disease Control and Prevention (CDC) light trap per night, an order of magnitude higher than CDC light traps deployed at upland sites. The ability of Cx. salinarius to use both heavily impacted and relatively undisturbed salt marshes for reproduction has significant repercussions for marsh restoration and vector control practices.

Twenty years of surveillance for Eastern equine encephalitis virus in mosquitoes in New York State from 1993 to 2012.

BACKGROUND: The year 1971 was the first time in New York State (NYS) that Eastern equine encephalitis virus (EEEV) was identified in mosquitoes, in Culiseta melanura and Culiseta morsitans. At that time, state and county health departments began surveillance for EEEV in mosquitoes. METHODS: From 1993 to 2012, county health departments continued voluntary participation with the state health department in mosquito and arbovirus surveillance. Adult female mosquitoes were trapped, identified, and pooled. Mosquito pools were tested for EEEV by Vero cell culture each of the twenty years. Beginning in 2000, mosquito extracts and cell culture supernatant were tested by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: During the years 1993 to 2012, EEEV was identified in: Culiseta melanura, Culiseta morsitans, Coquillettidia perturbans, Aedes canadensis (Ochlerotatus canadensis), Aedes vexans, Anopheles punctipennis, Anopheles quadrimaculatus, Psorophora ferox, Culex salinarius, and Culex pipiens-restuans group. EEEV was detected in 427 adult mosquito pools of 107,156 pools tested totaling 3.96 million mosquitoes. Detections of EEEV occurred in three geographical regions of NYS: Sullivan County, Suffolk County, and the contiguous counties of Madison, Oneida, Onondaga and Oswego. Detections of EEEV in mosquitoes occurred every year from 2003 to 2012, inclusive. EEEV was not detected in 1995, and 1998 to 2002, inclusive. CONCLUSIONS: This was the first time in NYS that EEEV was detected in Cx. salinarius, Ps. ferox and An. punctipennis. The detection of EEEV in mosquitoes every year for 10 years was the longest time span since surveillance began in 1971. The calendar date of the earliest annual appearance of EEEV in mosquitoes did not change during surveillance spanning 42 years.

Improved high-throughput method for molecular identification of Culex mosquitoes.

Culex pipiens, Cx. restuans, and Cx. salinarius play important and most likely different roles in transmission of West Nile virus (WNV) in the northeastern United States. While Cx. pipiens and Cx. restuans are considered the main enzootic vectors of WNV, Cx. salinarius may be involved in epizootic cycles due to its broader host preferences. Accurate morphological identification of field-collected Culex specimens may be difficult, and therefore the New York State Department of Health arbovirus surveillance program allows combined Cx. pipiens and Cx. restuans pools to be tested for WNV. We have developed a modified and improved DNA isolation protocol using proteinase K digestion without traditional mosquito trituration and nucleic acid extraction to permit high-throughput screening of a large number of Culex specimens for species identification using polymerase chain reaction (PCR). This method utilizes a 96-well-plate format and a novel 1-step crude extraction procedure using proteinase K to obtain genomic DNA template from 1 mosquito leg in sufficient quantity for at least 2 standard 50-microl PCR reactions. Proteinase K digestion of legs from individual Culex mosquitoes was performed and used for PCR amplification with previously described species-specific ribosomal DNA primers. Using these rDNA primers, our modified proteinase K method successfully identified 91% to 100% of the Culex samples.

Spatiotemporal modeling of ecological and sociological predictors of West Nile virus in Suffolk County, NY, mosquitoes.

Suffolk County, New York, is a locus for West Nile virus (WNV) infection in the American northeast that includes the majority of Long Island to the east of New York City. The county has a system of light and gravid traps used for mosquito collection and disease monitoring. In order to identify predictors of WNV incidence in mosquitoes and predict future occurrence of WNV, we have developed a spatiotemporal Bayesian model, beginning with over 40 ecological, meteorological, and built-environment covariates. A mixed-effects model including spatially and temporally correlated errors was fit to WNV surveillance data from 2008 to 2014 using the R package "R-INLA," which allows for Bayesian modeling using the stochastic partial differential equation (SPDE) approach. The integrated nested Laplace approximation (INLA) SPDE allows for simultaneous fitting of a temporal parameter and a spatial covariance, while incorporating a variety of likelihood functions and running in R statistical software on a home computer. We found that land cover classified as open water and woody wetlands had a negative association with WNV incidence in mosquitoes, and the count of septic systems was associated with an increase in WNV. Mean temperature at two-week lag was associated with a strong positive impact, while mean precipitation at no lag and one-week lag was associated with positive and negative impacts on WNV, respectively. Incorporation of spatiotemporal factors resulted in a marked increase in model goodness-of-fit. The predictive power of the model was evaluated on 2015 surveillance results, where the best model achieved a sensitivity of 80.9% and a specificity of 77.0%. The spatial covariate was mapped across the county, identifying a gradient of WNV prevalence increasing from east to west. The Bayesian spatiotemporal model improves upon previous approaches, and we recommend the INLA SPDE methodology as an efficient way to develop robust models from surveillance data to develop and enhance monitoring and control programs. Our study confirms previously found associations between weather conditions and WNV and suggests that wetland cover has a mitigating effect on WNV infection in mosquitoes, while high septic system density is associated with an increase in WNV infection.

Ensemble forecast of human West Nile virus cases and mosquito infection rates.

West Nile virus (WNV) is now endemic in the continental United States; however, our ability to predict spillover transmission risk and human WNV cases remains limited. Here we develop a model depicting WNV transmission dynamics, which we optimize using a data assimilation method and two observed data streams, mosquito infection rates and reported human WNV cases. The coupled model-inference framework is then used to generate retrospective ensemble forecasts of historical WNV outbreaks in Long Island, New York for 2001-2014. Accurate forecasts of mosquito infection rates are generated before peak infection, and >65% of forecasts accurately predict seasonal total human WNV cases up to 9 weeks before the past reported case. This work provides the foundation for implementation of a statistically rigorous system for real-time forecast of seasonal outbreaks of WNV.

High levels of local inter- and intra-host genetic variation of West Nile virus and evidence of fine-scale evolutionary pressures.

West Nile virus (WNV; Flaviviridae, Flavivirus) has been endemic in New York State (NYS) since its 1999 introduction, yet prevalence in Culex mosquitoes varies substantially over small spatial and temporal scales. It is unclear if viral genetics plays a role in this variability, as genetic and phenotypic characterization on local scales has generally been lacking. In addition, intrahost diversity of circulating strains have not been fully characterized despite the documented role of minority variants in viral fitness and virulence. In an effort to characterize WNV variability within epidemiologically relevant scales, we performed phylogenetic analyses on NYS isolates from 1999 to 2012. In addition, we performed full-genome, deep-sequencing and genetic analyses on 15 WNV strains isolated in 2012 from Cx. pipiens in an endemic focus of Suffolk County, NY. Our results indicate continued evolution and seasonal maintenance in NYS, yet also widespread mixing and high levels of genetic diversity within geographic foci and individual seasons. Well supported local clusters with shared amino acid differences were identified and suggest local evolutionary pressures and the potential for phenotypic variability. Intrahost diversity of focal isolates was also high, with polymorphism at levels >1.0% identified in approximately 10% of the WNV genome. Although most minority mutations were unique, mutational hotspots shared among local isolates were identified, particularly in C, NS1 and NS2A genes. The most polymorphic region, positions 3198-3388 of the NS1 gene, was comprised predominately of non-synonymous mutations, suggesting a selective advantage for amino acid diversity in this region.

Prevalence of Ehrlichia, Borrelia, and Rickettsial agents in Amblyomma americanum (Acari: Ixodidae) collected from nine states.

Ambyomma americanum (L.) (Acari: Ixodidae) is an aggressive tick that feeds on humans during all postembryonic life stages. In many regions of the United States, it is the tick most commonly found attached to humans. Public health interest has grown recently, due to the recognition of new human pathogens transmitted by A. americanum and the expanding distribution of the tick. A. americanum is a vector of several bacteria pathogenic to humans. Ehrlichia chaffeensis and Ehrlichia ewingii cause moderate-to-severe febrile illness. "Rickettsia amblyommii," a member of the spotted fever group Rickettsia, also has recently been implicated as a possible human pathogen based on serologic evidence from persons recovering from illness after a tick bite. We have determined the prevalence of infection of Ehrlichia chaffeensis, E. ewingii, "Borrelia lonestari", and R. amblyommii within A. americanum ticks from 29 sites in nine states. Overall infection prevalences were 4.7% for E. chaffeensis (range, 0-27%), 3.5% for E. ewingii (range, 0-18.6%), 2.5% for B. lonestari (range, 0-12.2%), and 41.2% for R. amblyommii (range, 0-84.0%). In addition, 87 ticks (4.3%) were infected with two or more bacteria. This report documents new distribution records for E. ewingii, B. lonestari, and R. amblyommii and underscores the nonhomogeneous distribution of pathogen foci of infection. Additional surveillance throughout the range ofA. americanum is warranted to increase physician and public awareness of the risk of disease to humans from exposure to the agents transmitted by this tick.

Development and validation of a climate-based ensemble prediction model for West Nile Virus infection rates in Culex mosquitoes, Suffolk County, New York.

BACKGROUND: West Nile Virus (WNV) is an endemic public health concern in the United States that produces periodic seasonal epidemics. Underlying these outbreaks is the enzootic cycle of WNV between mosquito vectors and bird hosts. Identifying the key environmental conditions that facilitate and accelerate this cycle can be used to inform effective vector control. RESULTS: Here, we model and forecast WNV infection rates among mosquito vectors in Suffolk County, New York using readily available meteorological and hydrological conditions. We first validate a statistical model built with surveillance data between 2001 and 2009 (m09) and specify a set of new statistical models using surveillance data from 2001 to 2012 (m12). This ensemble of new models is then used to make predictions for 2013-2015, and multimodel inference is employed to provide a formal probabilistic interpretation across the disparate individual model predictions. The findings of the m09 and m12 models align; with the ensemble of m12 models indicating an association between warm, dry early spring (April) conditions and increased annual WNV infection rates in Culex mosquitoes. CONCLUSIONS: This study shows that real-time climate information can be used to predict WNV infection rates in Culex mosquitoes prior to its seasonal peak and before WNV spillover transmission risk to humans is greatest.