West Nile Virus Amplification Research Articles

The effect of temperature on the boundary conditions of West Nile virus circulation in Europe.

West Nile virus (WNV) is a vector-borne flavivirus that causes an increasing number of human and equine West Nile fever cases in Europe. While the virus has been present in the Mediterranean basin and the Balkans since the 1960s, recent years have witnessed its northward expansion, with the first human cases reported in Germany in 2018 and the Netherlands in 2020. WNV transmission and amplification within mosquitoes are temperature-dependent. This study applies a mathematical modelling approach to assess the conditions under which WNV circulation occurs based on the proportion of mosquito bites on WNV-competent birds (dilution), vector-host ratios, mosquito season length and the observed daily temperature data. We modelled five distinct European regions where previous WNV circulation has been observed within the Netherlands, Germany, Spain, Italy, and Greece. We observed that the number of days in which the basic reproduction number (R0) is above one, increased over the last 40 years in all five regions. In the Netherlands, the number of days in which the R0 is above one, is 70% lower than in Spain. The temperature in Greece, Spain and Italy allowed for circulation under low vector-host ratios, and at a high dilution. On the other hand in the Netherlands and Germany, given the observed daily temperature, the thresholds for circulation requires a lower dilution and higher vector-host ratios. For the Netherlands, a short window of introductions between late May and mid-June would result in detectable outbreaks. Our findings revealed that the temperate maritime climate of the Netherlands allows WNV circulation primarily during warmer summers, and only under high vector-host ratios. This research contributes valuable insights into the dynamic relationship between temperature, vector properties, and WNV transmission, offering guidance for proactive strategies in addressing this emerging health threat in Europe.

A One Health view of the West Nile virus outbreak in Andalusia (Spain) in 2020

ABSTRACT Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds (Turdus merula) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

American alligators are capable of West Nile virus amplification, mosquito infection and transmission

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

The development of a spatially resolved retrospective ensemble forecast model of West Nile virus in Long Island, New York

The development of a spatially resolved retrospective ensemble forecast model of West Nile virus in Long Island, New York Nicholas DeFelice, Meytar Sorek-Hamer, Scott R Campbell and Krishna Vemuri West Nile virus (WNV) emerged in the western hemisphere in 1999, and has established itself as the leading cause of domestically acquired arboviral disease in the United States. Though the transmission of WNV exhibits a pronounced sensitivity to a complex seasonal ecology along with hydrological and meteorological conditions, our ability to predict the timing, duration, and magnitude of local WNV outbreaks remains limited. In Long Island, New York, annual peak estimates of WNV infected mosquitos have ranged from 1 in 300 to 1 in 50 resulting in anywhere from 0 to 24 reported human WNV cases, respectively. Here we report the development of a spatially refined model that uses ECOsystem Spaceborne Thermal Radiometer Experiment on Space Station (ECOSTRESS) data to capture the variability in physical environmental factors (e.g., temperature and hydrology) along with a compartmental model describing WNV transmission dynamics, to retrospectively forecast WNV outbreaks in Long Island, New York. The inclusion of ECOSTRESS’ high spatial (70m) and temporal (revisits every 4 days) resolution data will allow us to capture changes in the micro-ecosystem. Ensemble simulations with the WNV model are iteratively optimized using data assimilation methods and observations of human incidence, mosquito abundance, and mosquito infection rates. The ECOSTRESS meteorological and hydrological indicators are included in the core model structure to better constrain WNV amplification and transmission dynamics. The model-inference system can be used to better understand the spatial variability of the outbreak and estimate the relationship between zoonotic amplification and human outbreaks. This work represents an initial step in the development of a statistically rigorous system for a spatially resolved real-time forecast of seasonal outbreaks of West Nile virus.

The role of different Culex mosquito species in the transmission of West Nile virus and avian malaria parasites in Mediterranean areas

Vector-borne diseases, especially those transmitted by mosquitoes, have severe impacts on public health and economy. West Nile virus (WNV) and avian malaria parasites of the genus Plasmodium are mosquito-borne pathogens that may produce severe disease and illness in humans and birds, respectively, and circulate in an endemic form in southern Europe. Here, we used field-collected data to identify the impact of Culex pipiens, Cx. perexiguus and Cx. modestus, on the circulation of both WNV and Plasmodium in Andalusia (SW Spain) using mathematical modelling of the basic reproduction number (R0 ). Models were calibrated with field-collected data on WNV seroprevalence and Plasmodium infection in wild house sparrows, presence of WNV and Plasmodium in mosquito pools, and mosquito blood-feeding patterns. This approach allowed us to determine the contribution of each vector species to pathogen amplification. Overall, 0.7% and 29.6% of house sparrows were positive to WNV antibodies and Plasmodium infection, respectively. In addition, the prevalence of Plasmodium was higher in Cx. pipiens (2.0%), followed by Cx. perexiguus (1.8%) and Cx. modestus (0.7%). Three pools of Cx. perexiguus were positive to WVN. Models identified Cx. perexiguus as the most important species contributing to the amplification of WNV in southern Spain. For Plasmodium models, R0 values were higher when Cx. pipiens was present in the population, either alone or in combination with the other mosquito species. These results suggest that the transmission of these vector-borne pathogens depends on different Culex species, and consequently, their transmission niches will present different spatial and temporal patterns. For WNV, targeted surveillance and control of Cx. perexiguus populations appear as the most effective measure to reduce WNV amplification. Also, preventing Culex populations near human settlements, or reducing the abundance of these species, are potential strategies to reduce WNV spillover into human populations in southern Spain.

The influence of weather and weather variability on mosquito abundance and infection with West Nile virus in Harris County, Texas, USA

Early warning systems for vector-borne diseases (VBDs) prediction are an ecological application where data from the interface of several environmental components can be used to predict future VBD transmission. In general, models for early warning systems only consider average environmental conditions ignoring variation in weather variables, despite the prediction from Schmalhausen's law about the importance of environmental variability for biological systems. We present results from a long-term mosquito surveillance program from Harris County, Texas, USA, where we use time series analysis techniques to study the abundance and West Nile virus (WNV) infection patterns in the local primary vector, Culex quinquefasciatus Say. We found that, as predicted by Schmalhausen's law, mosquito abundance was associated with the standard deviation and kurtosis of environmental variables. By contrast, WNV infection rates were associated with 8-month lagged temperature, suggesting environmental conditions during overwintering might be key for WNV amplification during summer outbreaks. Finally, model validation showed that seasonal autoregressive models successfully predicted mosquito WNV infection rates up to 2 months ahead, but did rather poorly at predicting mosquito abundance, a result that might reflect impacts of vector control for mosquito population reduction, geographic scale, and other artifacts generated by operational constraints of mosquito surveillance systems.

Comparative fitness of West Nile virus isolated during California epidemics.

West Nile virus (WNV) has been circulating in California since its first detection in 2003, causing repeated outbreaks affecting public, wildlife and veterinary health. Epidemics of WNV are difficult to predict due to the multitude of factors influencing transmission dynamics among avian and mosquito hosts. Typically, high levels of WNV amplification are required for outbreaks to occur, and therefore associated viral strains may exhibit enhanced virulence and mortality in competent bird species resulting in increased mosquito infection prevalence. In our previous study, most WNV isolates made from California during 2007–08 showed increased fitness when competed in House Finches (HOFI, Haemorhous mexicanus) and Culex tarsalis Coquillett mosquitoes against COAV997-5nt, a genetically marked recombinant virus derived from a 2003 California strain. Herein, we evaluated the competitive fitness of WNV strains isolated during California epidemics in 2004, 2005, 2007, 2011 and 2012 against COAV997-5nt. These outbreak isolates did not produce elevated mortality in HOFIs, but replicated more efficiently than did COAV997-5nt based on quantification of WNV RNA copies in sera, thereby demonstrating increased competitive fitness. Oral co-infections in Cx. tarsalis resulted in similar virus-specific infection and transmission rates, indicating that outbreak isolates did not have a fitness advantage over COAV997-5nt. Collectively, WNV isolates from outbreaks demonstrated relatively greater avian, but not vector, replicative fitness compared to COAV997-5nt, similar to previously characterized non-outbreak isolates of WNV. Our results indicated that ecological rather than viral factors may facilitate WNV amplification to outbreak levels, but monitoring viral phenotypes through competitive fitness studies may provide insight into altered replication and transmission potential among emerging WNV strains.

Forage Ratio Analysis of the Southern House Mosquito in College Station, Texas.

Culex quinquefasciatus is the principal vector of West Nile virus (WNV) in the South Central United States, yet limited data on host utilization are available. We evaluated host utilization over a 3-month period in 2013 in a residential landscape in College Station, Texas. PCR sequencing of the mitochondrial cytochrome oxidase 1 gene permitted molecular identification of vertebrate bloodmeals to the species level. Forage ratio analysis identified bird species that were overutilized and underutilized by comparing community feeding index values to expected relative abundance values of bird species, derived from eBird data. Community feeding index values were also used in conjunction with reservoir competence data from the literature to generate reservoir capacity index values, a means of identifying relative importance of vertebrate reservoir hosts. Of 498 blood-engorged Cx. quinquefasciatus, 313 (62.9%) were identified to vertebrate species. The majority (95.5%) of bloodmeals originated from avian species with the remainder from mammals, but not humans. Northern mockingbird (Mimus polyglottos) was the principal host for mosquito feeding in June and July, but northern cardinal (Cardinalis cardinalis) became primary host in August. Forage ratio analysis revealed the overutilization of house finch (Haemorhous mexicanus), American robin (Turdus migratorius), northern mockingbird, northern cardinal, white-winged dove (Zenaida asiatica), and mourning dove (Zenaida macroura). Great-tailed grackle (Quiscalus mexicanus), blue jay (Cyanocitta cristata), and Carolina wren (Thryothorus ludovicianus) were under-utilized relative to availability. Reservoir capacity calculations suggested that northern mockingbird and northern cardinal were the principal amplifiers in the study area. These data identify the primary avian species contributing to the enzootic amplification of WNV in East-Central Texas and reveal that the heavy feeding on moderately competent hosts and no feeding on humans likely limit epidemics in this region.

An Integrative Eco-Epidemiological Analysis of West Nile Virus Transmission

West Nile disease, caused by the West Nile virus (WNV), is a mosquito-borne zoonotic disease affecting humans and horses that involves wild birds as amplifying hosts. The mechanisms of WNV transmission remain unclear in Europe where the occurrence of outbreaks has dramatically increased in recent years. We used a dataset on the competence, distribution, abundance, diversity and dispersal of wild bird hosts and mosquito vectors to test alternative hypotheses concerning the transmission of WNV in Southern France. We modelled the successive processes of introduction, amplification, dispersal and spillover of WNV to incidental hosts based on host–vector contact rates on various land cover types and over four seasons. We evaluated the relative importance of the mechanisms tested using two independent serological datasets of WNV antibodies collected in wild birds and horses. We found that the same transmission processes (seasonal virus introduction by migratory birds, Culex modestus mosquitoes as amplifying vectors, heterogeneity in avian host competence, absence of ‘dilution effect’) best explain the spatial variations in WNV seroprevalence in the two serological datasets. Our results provide new insights on the pathways of WNV introduction, amplification and spillover and the contribution of bird and mosquito species to WNV transmission in Southern France.

West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks.

West Nile virus (WNV) infection is a non-contagious disease mainly transmitted by the bites of infected mosquitoes from the genus Culex. The virus is maintained in a mosquito-bird-mosquito cycle, and can accidentally be transmitted to mammalian hosts. Among mammalian hosts, equines and humans are the most sensitive to WNV infection and can develop severe meningoencephalitis. As WNV infections are zoonotic and can be severe in humans and equines, West Nile fever is considered to be a public and animal health concern. After a silent period of almost ten years, WNV re-emerged in France at the periphery of the Camargue area during the summer of 2015, underlining the fact that the Camargue area creates favourable conditions for WNV emergence and amplification in France. The French Network for Epidemiological Surveillance of Equine Diseases (Réseau d'Épidémio-Surveillance en Pathologie Équine [RESPE]) facilitated the early detection of WNV cases in horses. In total, 49 horses were found to be infected; among them, 44 presented clinical signs, 41 with meningoencephalitis and three with hyperthermia only. Six horses among the 41 with nervous symptoms died from the disease or were euthanised (a case fatality rate of 14.6%). The authors describe the characteristics of the 2015 WNV epizootics, the early detection of the first WNV equine cases via the RESPE network and the coordination of WNV surveillance in France.

Transmission Dynamics of the West Nile Virus in Mosquito Vector Populations under the Influence of Weather Factors in the Danube Delta, Romania.

Mosquitoes were collected in the Danube Delta during the active seasons of 2011-2013. For Culex spp. mosquitoes, the abundance was calculated. Culex pipiens (sensu lato), (s.l.) and Culex modestus pools were tested for the presence of West Nile virus (WNV) genome, and the maximum likelihood of the infection rate was established. Mean daily temperatures and precipitation were obtained for the closest meteorological station. A negative binominal model was used to evaluate linkages between the temperature/precipitation and mosquito population size. A zero-inflated negative binomial model was used to test the relationship between the temperature and the infection rate. A single complex model for infection rate prediction was also used. The linkages were calculated for lag 0 and for 10days earlier (lag 1), 20days earlier (lag 2), and 30days earlier (lag 3). Significant positive linkages (P<0.001) were detected between temperature and mosquito population size for lag 1, lag 2, and lag 3. The linkages between temperature and infection rates were positive and significant for lag 2 and lag 3. Negative significant (P<0.001) results were detected between precipitation and infection rates for lags 1, 2, and 3. The complex model showed that the best predictors for infection rate are the temperature, 20days earlier (positive linkage) and the precipitation, 30days earlier (negative linkage). Positive temperature anomalies in spring and summer and rainfall decrease contributed to the increase in the Culex spp. abundance and accelerated the WNV amplification in mosquito vector populations in the following weeks.

Supersuppression: Reservoir Competency and Timing of Mosquito Host Shifts Combine to Reduce Spillover of West Nile Virus.

In the eastern United States, human cases of West Nile virus (WNV) result from spillover from urban epizootic transmission between passerine birds and Culex mosquitoes. In Atlanta, GA, substantial WNV presence in hosts and vectors has not resulted in the human disease burden observed in cities with similar infection pressure. Our study goal was to investigate extrinsic ecological conditions that potentially contribute to these reduced transmission rates. We conducted WNV surveillance among hosts and vectors in urban Atlanta and recorded an overall avian seroprevalence of nearly 30%, which was significantly higher among northern cardinals, blue jays, and members of the mimid family, and notably low among American robins. Examination of temporal Culex feeding patterns showed a marked feeding shift from American robins in the early season to northern cardinals in the late season. We therefore rule out American robins as superspreaders in the Atlanta area and suggest instead that northern cardinals and mimids act as WNV "supersuppressor" species, which slow WNV transmission by drawing many infectious bites during the critical virus amplification period, yet failing to amplify transmission due to low host competencies. Of particular interest, urban forest patches provide spillover protection by increasing the WNV amplification fraction on supersuppressor species.

Bionomics and Vector Potential of Culex thriambus (Diptera: Culicidae) Mosquitoes in Lake County, California.

California statewide West Nile virus (WNV) minimum infection rates in Culex thriambus Dyar mosquitoes are high; however, few specimens are submitted and tested each year, as their distribution seems limited to larval habitats along riparian systems. To evaluate the role of Cx. thriambus in the amplification, maintenance, and overwintering of WNV in Lake County, CA, the bionomics and vector potential of the species was investigated during 2014 and 2015. Culex thriambus was the most abundant mosquito species, with 1,153 adults and 7,624 immatures collected by vacuum aspiration and dip sampling, respectively, at the primary study site. Detection of WNV in four mosquito pools during September through November coincided with peak seasonality. Females entered and maintained a reproductive diapause during winter under field and seminatural conditions. Diapause was initiated in the majority of Cx. thriambus females by October and was terminated by 30 March. Some parous females (7.1%) and those in host-seeking arrest (7.1%) were collected throughout the winter period. An accrual of 679.51 degree-days (°D) was necessary for diapause termination under seminatural conditions. Culex thriambus females fed on 16 different avian species during spring and summer, and no mammalian feeds were detected. West Nile viral RNA was detected in four of 42 Cx. thriambus pools tested during June through November and infection rates ranged from 3.53–28.15/1,000 tested. In summary, WNV transmission may be increased along riparian corridors throughout California where Cx. thriambus mosquitoes remain relatively abundant.

The Impact of Cycling Temperature on the Transmission of West Nile Virus.

West Nile virus (WNV) is an important cause of disease in humans and animals. Risk of WNV infection varies seasonally, with the greatest risk during the warmest parts of the year due in part to the accelerated extrinsic incubation rate of the virus in mosquitoes. Rates of extrinsic incubation have been shown in constant-temperature studies to increase as an approximately linear function of temperature, but for other vector-borne pathogens, such as malaria or dengue virus, nonlinear relationships have been demonstrated under cycling temperatures near the thermal limits of pathogen replication. Using typical daily air temperature profiles from three key periods of WNV amplification in a hyperendemic area of WNV activity in California's Central Valley, as well as a fourth temperature profile based on exposures that would result from daily mosquito host-seeking and resting behavior, we explored the impacts of cycling temperatures on WNV transmission by Culex tarsalis Coquillett, one of the principal vectors in the western United States. The daily cycling temperature ranges studied were representative of those that occur across much of California, but they did not significantly alter the extrinsic incubation period of WNV compared with estimates from mean temperatures alone. This suggests that within the relatively broad range we studied, WNV incubation rates are a simple function of mean temperature. Realistic daily temperature patterns that reflected mosquitoes' avoidance of daytime high temperatures during summer reduced transmission over time compared with air temperatures, indicating that adjustment for mosquito exposure temperatures would be prudent for calculating risk.

SUSCEPTIBILITY AND ANTIBODY RESPONSE OF VESPER SPARROWS (POOECETES GRAMINEUS) TO WEST NILE VIRUS: A POTENTIAL AMPLIFICATION HOST IN SAGEBRUSH-GRASSLAND HABITAT.

West Nile virus (WNV) spread to the US western plains states in 2003, when a significant mortality event attributed to WNV occurred in Greater Sage-grouse ( Centrocercus urophasianus ). The role of avian species inhabiting sagebrush in the amplification of WNV in arid and semiarid regions of the North America is unknown. We conducted an experimental WNV challenge study in Vesper Sparrows ( Pooecetes gramineus ), a species common to sagebrush and grassland habitats found throughout much of North America. We found Vesper Sparrows to be moderately susceptible to WNV, developing viremia considered sufficient to transmit WNV to feeding mosquitoes, but the majority of birds were capable of surviving infection and developing a humoral immune response to the WNV nonstructural 1 and envelope proteins. Despite clearance of viremia, after 6 mo, WNV was detected molecularly in three birds and cultured from one bird. Surviving Vesper Sparrows were resistant to reinfection 6 mo after the initial challenge. Vesper sparrows could play a role in the amplification of WNV in sagebrush habitat and other areas of their range, but rapid clearance of WNV may limit their importance as competent amplification hosts of WNV.

Field Methods and Sample Collection Techniques for the Surveillance of West Nile Virus in Avian Hosts.

Avian hosts play an important role in the spread, maintenance, and amplification of West Nile virus (WNV). Avian susceptibility to WNV varies from species to species thus surveillance efforts can focus both on birds that survive infection and those that succumb. Here we describe methods for the collection and sampling of live birds for WNV antibodies or viremia, and methods for the sampling of dead birds. Target species and study design considerations are discussed.

Regional variation of climatic influences on West Nile virus outbreaks in the United States.

The national resurgence of human West Nile virus (WNV) disease in 2012 raised questions about the factors responsible for WNV outbreaks. Interannual climatic variations may influence WNV amplification and transmission to humans through multiple pathways, including mosquito breeding habitats, gonotrophic cycles, extrinsic incubation, avian communities, and human behavior. We examined the influences of temperature and precipitation anomalies on interannual variation in human WNV cases in three regions of the United States. There were consistent positive influences of winter temperatures, weaker and more variable positive effects of spring and summer temperatures, and highly variable precipitation effects that ranged from positive to negative. The overwintering period may be a particularly important climatic constraint on the dynamics of WNV in cold-temperate regions of North America. Geographic differences in the seasonal timing and relative importance of climatic drivers of WNV risk likely reflect underlying variability in key ecological and social characteristics.

Diverse host feeding on nesting birds may limit early-season West Nile virus amplification.

Arboviral activity tracks vector availability, which in temperate regions means that transmission ceases during the winter and must be restarted each spring. In the northeastern United States, Culex restuans Theobald resumes its activity earlier than Culex pipiens L. and is thought to be important in restarting West Nile virus (WNV) transmission. Its role in WNV amplification, however, is unclear, because viral levels commonly remain low until the rise of Cx. pipiens later in the season. Because a vector's feeding habits can reveal key information about disease transmission, we identified early-season (April-June) blood meals from Cx. restuans collected throughout New Jersey, and compared them to published datasets from later in the season and also from other parts of the country. We found significantly higher avian diversity, including poor WNV hosts, and fewer blood meals derived from American Robins (17% versus over 40% found in later season). Critically, we identified blood meals from significantly more female than male birds in species where females are the incubating sex, suggesting that Cx. restuans is able to feed on such a wide variety of hosts in early spring because incubating birds are easy targets. Because WNV amplification depends on virus consistently reaching competent hosts, our results indicate that Cx. restuans is unlikely to be an amplifying vector of WNV in the early season. As the season progresses, however, changes in the availability of nesting birds may make it just as capable as Cx. pipiens, although at somewhat lower abundance as the summer progresses.

Vector Contact Rates on Eastern Bluebird Nestlings Do Not Indicate West Nile Virus Transmission in Henrico County, Virginia, USA

Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV). Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae) across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.

Isolations of Cache Valley virus from Aedes albopictus (Diptera: Culicidae) in New Jersey and evaluation of its role as a regional arbovirus vector.

The Asian tiger mosquito, Aedes albopictus (Skuse), is an invasive species and a major pest problem in urban and suburban locales in New Jersey. To assess its potential role as an arbovirus vector, we sampled Ae. albopictus from two New Jersey counties over a 3-yr period and estimated the prevalence of virus infection by Vero cell culture and reverse transcription-polymerase chain reaction assays. Three virus isolates were obtained from 34,567 field-collected Ae. albopictus, and all were identified as Cache Valley virus by molecular methods. Ae. albopictus (N = 3,138), collected in Mercer County from late July through early September 2011, also were retested for West Nile virus (WNV) by reverse transcription-polymerase chain reaction, and all were negative. These results corroborate previous findings showing that Ae. albopictus may occasionally acquire Cache Valley virus, a deer-associated arbovirus, in nature. In contrast, we did not detect WNV infection in Ae. albopictus despite concurrent WNV amplification in this region.