Virology, ecology, epidemiology, pathology, and treatment of eastern equine encephalitis.

Eastern equine encephalitis (EEE) was one of the first-recognized neuroinvasive arboviral diseases in North America, and it remains the most lethal. Although EEE is known to have periodic spikes in infection rates, there is increasing evidence that it may be undergoing a change in its prevalence and its public health burden. Numerous factors shape the scope of EEE in humans, and there are important similarities with other emergent viral diseases that have surfaced or strengthened in recent years. Because environmental and ecological conditions that broadly influence the epidemiology of arboviral diseases also are changing, and the frequency, severity, and scope of outbreaks are expected to worsen, an expanded understanding of EEE will have untold importance in coming years. Here we review the factors shaping EEE transmission cycles and the conditions leading to outbreaks in humans from an updated, multidomain perspective. We also provide special consideration of factors shaping the virology, host-vector-environment relationships, and mechanisms of pathology and treatment as a reference for broadening audiences.

Multi-season transmission model of Eastern Equine Encephalitis.

Eastern Equine Encephalitis (EEE) is an arbovirus that, while it has been known to exist since the 1930's, recently had a spike in cases. This increased prevalence is particularly concerning due to the severity of the disease with 1 in 3 symptomatic patients dying. The cause of this peak is currently unknown but could be due to changes in climate, the virus itself, or host behavior. In this paper we propose a novel multi-season deterministic model of EEE spread and its stochastic counterpart. Models were parameterized using a dataset from the Florida Department of Health with sixteen years of sentinel chicken seroconversion rates. The different roles of the enzootic and bridge mosquito vectors were explored. As expected, enzootic mosquitoes like Culiseta melanura were more important for EEE persistence, while bridge vectors were implicated in the disease burden in humans. These models were used to explore hypothetical viral mutations and host behavior changes, including increased infectivity, vertical transmission, and host feeding preferences. Results showed that changes in the enzootic vector transmission increased cases among birds more drastically than equivalent changes in the bridge vector. Additionally, a 5% difference in the bridge vector's bird feeding preference can increase cumulative dead-end host infections more than 20-fold. Taken together, this suggests changes in many parts of the transmission cycle can augment cases in birds, but the bridge vectors feeding preference acts as a valve limiting the enzootic circulation from its impact on dead-end hosts, such as humans. Our what-if scenario analysis reveals and measures possible threats regarding EEE and relevant environmental changes and hypothetically suggests how to prevent potential damage to public health and the equine economy.

Eastern Equine Encephalomyelitis in Michigan: Historical Review of Equine, Human, and Wildlife Involvement, Epidemiology, Vector Associations, and Factors Contributing to Endemicity.

Eastern equine encephalomyelitis (EEE) is a mosquito-borne viral disease that is an emerging public health concern in the state of Michigan. Although Michigan has one of the highest incidence rates of EEE in the United States, much of the information known about cases in humans, equines, and other animals residing in Michigan is unpublished. This article summarizes such information and explores spatial trends in the historic distribution of EEE in Michigan. Outbreaks in Michigan have occurred over an 80-yr interval, involving only horses in 1942-1943 and 1973-1976, and then episodically from 1980 to 2020, and involving horses, humans, and wild and domestic animals. An estimated 1,036 equine cases (confirmed and suspected) and 36 confirmed human cases have occurred, including 10 in 2019 (6 deaths) and 4 in 2020 (2 deaths). Human cases ranged in age from 1 to 81 yr; 70% were male, and fatality rate of 34.3%. Equine and human cases occurred from July to October, peaked in August, and cluster in space in southwestern and southeastern lower Michigan. Cases occurred in glacial outwash and ice-contact landscapes in glacial interlobate zones. EEE virus (EEEV) was recovered from Culiseta melanura, Coquillettidia perturbans, five species of Aedes, and other mosquito species near horse and human case sites. Virus isolations or presence of neutralizing antibodies in several passerine species of birds suggest broad EEEV-bird associations. White-tailed deer and other wildlife were also affected. Geographic spread to northern areas of the state suggests expansion of this disease system into new and unsuspected foci.

Magnetic resonance imaging, clinicopathologic findings, and clinical progression of a puppy with confirmed Eastern equine encephalitis virus.

A 5-month-old puppy was evaluated for rapidly progressive neurologic signs and pyrexia. Magnetic resonance imaging showed multifocal meningoencephalitis with transtentorial and foramen magnum herniation. A cerebrospinal fluid tap revealed highly cellular fluid, and the puppy was euthanized. Histopathology showed lymphoplasmacytic and neutrophilic meningoencephalitis. Viral polymerase chain reaction testing for Eastern equine encephalitis was positive. Rapid progression of neurologic signs and respiratory arrest necessitated mechanical ventilation. Severe hypernatremia, most consistent with central diabetes insipidus, developed. Key clinical message: Transtentorial and foramen magnum herniation and high cerebrospinal fluid cell counts may be indicators of poor prognosis. Brain death, respiratory arrest, and central diabetes insipidus may also ensue with Eastern equine encephalitis infection.

Imagerie par résonance magnétique, résultats clinicopathologiques et progression clinique d'un cas confirmé d'infection par le virus de l'encéphalite équine de l'Est chez un chiot. Un chiot de 5 mois a été évalué pour des signes neurologiques progressant rapidement et une pyrexie. L'imagerie par résonance magnétique a montré une méningo-encéphalite multifocale avec hernies transtentorielle et au foramen magnum. Un prélèvement de liquide céphalo-rachidien a révélé un liquide hautement cellulaire et le chiot a été euthanasié. L'histopathologie a montré une méningo-encéphalite lymphoplasmocytaire et neutrophilique. Un test de réaction en chaîne par la polymérase pour l'encéphalite équine de l'Est était positif. La progression rapide des signes neurologiques et l'arrêt respiratoire ont nécessité une ventilation mécanique. Une hypernatrémie sévère, plus compatible avec un diabète insipide central, s'est développée.Message clinique clé:Une hernie transtentorielle et au foramen magnum de même qu'un nombre élevé de cellules dans le liquide céphalorachidien peuvent être des indicateurs de mauvais pronostic. La mort cérébrale, l'arrêt respiratoire et le diabète insipide central peuvent également s'ensuivre avec une infection par l'encéphalite équine de l'Est.(Traduit par Dr Serge Messier).

Eastern Equine Encephalitis Virus: A Scoping Review of the Global Evidence.

Background: Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that is primarily found in North America and the Caribbean. Over the past decade there has been an increase in virus activity, including large outbreaks in human and horse populations. Predicted climate change is expected to affect the range of mosquitoes including vectors of EEEV, which may alter disease risk posing a public health concern. Methods: A scoping review (ScR) was conducted to identify and characterize the global evidence on EEEV. A thorough search was conducted in relevant bibliographic databases and government websites. Two reviewers screened titles and abstracts for relevance and the characteristics of relevant articles were extracted using a uniformly implemented data collection form. The study protocol was developed a priori and described the methods and tools used and this article follows the PRISMA-ScR guidelines for reporting ScRs. Results: The ScR included 718 relevant research articles. The majority of the articles originated from North America (97%) between 1933 and 2019. EEEV has been identified in 35 species of mosquitoes, over 200 species of birds, various domestic animals, wild mammals, reptiles, and amphibians. Articles identified in this ScR primarily covered three topic areas: epidemiology of hosts and vectors (344 articles) including surveillance results (138), pathogenesis of EEEV in hosts (193), and in vitro studies characterizing EEEV (111). Fewer articles evaluated the accuracy of diagnostic tests (63), the efficacy of mitigation strategies (62), transmission dynamics (56), treatment of EEEV in hosts (10), societal knowledge, attitudes, and perceptions (4), and economic burden (2). Conclusion: With the projected impact of climate change on mosquito populations, it is expected that the risk of EEEV could change resulting in higher disease burden or spread into previously unaffected areas. Future research efforts should focus on closing some of the important knowledge gaps identified in this ScR.

Eastern Equine Encephalitis Virus Seroprevalence in Maine Cervids, 2012-2017.

Eastern equine encephalitis virus (EEEV) first emerged in Maine in the early 2000s and resulted in an epizootic outbreak in 2009. Since 2009, serum samples from cervids throughout Maine have been collected and assessed for the presence of neutralizing antibodies to EEEV to assess EEEV activity throughout the state. We tested 1,119 Odocoileus virginianus (white-tailed deer) and 982 Alces americanus (moose) serum samples collected at tagging stations during the hunting seasons from 2012 to 2017 throughout the state of Maine. Odocoileus virginianus from all 16 counties were EEEV seropositive, whereas A. americanus were seropositive in the northwestern counties of Aroostook, Somerset, Piscataquis, and Franklin counties. Seroprevalence in O. virginianus ranged from 6.6% to 21.2% and in A. americanus from 6.6% to 10.1%. Data from this report in conjunction with findings previously reported from 2009 to 2011 indicate that EEEV is endemic throughout Maine.

National Seroprevalence and Risk Factors for Eastern Equine Encephalitis and Venezuelan Equine Encephalitis in Costa Rica.

Eastern equine encephalitis and Venezuelan equine encephalitis are endemic neglected tropical diseases in the Americas, causing encephalitis in both horses and humans. In 2013, a cross-sectional study was performed in 243 horses located in the highlands and lowlands throughout Costa Rica. Serum samples were analyzed with an IgG ELISA and confirmed by the plaque-reduction neutralization test (PRNT80). Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) overall seroprevalences by the PRNT80 were 36% (95% confidence interval [CI]: 29.9-42.5; 78/217 horses) and 3% (95% CI: 1.3-5.9; 6/217 horses), respectively. Both the viruses occurred in the lowlands and highlands. Rainfall and altitude were associated with VEEV seropositivity in the univariate analysis, but only altitude <100 meters above sea level was considered a risk factor in the multivariate analysis. No risk factors could be identified for the EEEV in the multivariate analysis. This is the first study that estimates the seroprevalence of the EEEV and VEEV in Costa Rican horses. The VEEV is widely distributed, whereas the EEEV occurs at a much lower frequency and only in specific areas. Clinical cases and occasional outbreaks of both viruses are to be expected.

Impact of the Southern Oscillation Index, Temperature, and Precipitation on Eastern Equine Encephalitis Virus Activity in Florida.

Eastern equine encephalitis virus (EEEV), an Alphavirus from family Togaviridae, is a highly pathogenic arbovirus affecting the eastern United States, especially Florida. Effects of the Southern Oscillation Index (SOI), precipitation, and cooling degree days on EEEV horse case data in Florida from 2004 to 2018 were modeled using distributed lag nonlinear models (DLNMs). The analysis was conducted at statewide and regional scales. DLNMs were used to model potential delayed effects of the covariates on monthly counts of horse cases. Both models confirmed a seasonal trend in EEEV transmission and found that precipitation, cooling degree days, and the SOI were all predictors of monthly numbers of horse cases. EEEV activity in horses was associated with higher amounts of rainfall during the month of transmission at the statewide scale, as well as the prior 3 mo at the regional scale, fewer cooling degree days during the month of transmission and the preceding 3 mo and high SOI values during the month and the previous 2 mo, and SOI values in the prior 2 to 8 mo. Horse cases were lower during El Niño winters but higher during the following summer, while La Niña winters were associated with higher numbers of cases and fewer during the following summer. At the regional scale, extremely low levels of precipitation were associated with a suppression of EEEV cases for 3 mo. Given the periodicity and potential predictability of El Niño Southern Oscillation (ENSO) cycles, precipitation, and temperature, these results may provide a method for predicting EEEV risk potential in Florida.

Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants.

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.

Wetland characteristics linked to broad-scale patterns in Culiseta melanura abundance and eastern equine encephalitis virus infection.

BACKGROUND: Eastern equine encephalitis virus (EEEV) is an expanding mosquito-borne threat to humans and domestic animal populations in the northeastern United States. Outbreaks of EEEV are challenging to predict due to spatial and temporal uncertainty in the abundance and viral infection of Cs. melanura, the principal enzootic vector. EEEV activity may be closely linked to wetlands because they provide essential habitat for mosquito vectors and avian reservoir hosts. However, wetlands are not homogeneous and can vary by vegetation, connectivity, size, and inundation patterns. Wetlands may also have different effects on EEEV transmission depending on the assessed spatial scale. We investigated associations between wetland characteristics and Cs. melanura abundance and infection with EEEV at multiple spatial scales in Connecticut, USA. RESULTS: Our findings indicate that wetland vegetative characteristics have strong associations with Cs. melanura abundance. Deciduous and evergreen forested wetlands were associated with higher Cs. melanura abundance, likely because these wetlands provide suitable subterranean habitat for Cs. melanura development. In contrast, Cs. melanura abundance was negatively associated with emergent and scrub/shrub wetlands, and wetland connectivity to streams. These relationships were generally strongest at broad spatial scales. Additionally, the relationships between wetland characteristics and EEEV infection in Cs. melanura were generally weak. However, Cs. melanura abundance was strongly associated with EEEV infection, suggesting that wetland-associated changes in abundance may be indirectly linked to EEEV infection in Cs. melanura. Finally, we found that wet hydrological conditions during the transmission season and during the fall/winter preceding the transmission season were associated with higher Cs. melanura abundance and EEEV infection, indicating that wet conditions are favorable for EEEV transmission. CONCLUSIONS: These results expand the broad-scale understanding of the effects of wetlands on EEEV transmission and help to reduce the spatial and temporal uncertainty associated with EEEV outbreaks.

Characterization and pathogenesis of aerosolized eastern equine encephalitis in the common marmoset (Callithrix jacchus).

BACKGROUND: Licensed antiviral therapeutics and vaccines to protect against eastern equine encephalitis virus (EEEV) in humans currently do not exist. Animal models that faithfully recapitulate the clinical characteristics of human EEEV encephalitic disease, including fever, drowsiness, anorexia, and neurological signs such as seizures, are needed to satisfy requirements of the Food and Drug Administration (FDA) for clinical product licensing under the Animal Rule. METHODS: In an effort to meet this requirement, we estimated the median lethal dose and described the pathogenesis of aerosolized EEEV in the common marmoset (Callithrix jacchus). Five marmosets were exposed to aerosolized EEEV FL93-939 in doses ranging from 2.4 × 101 PFU to 7.95 × 105 PFU. RESULTS: The median lethal dose was estimated to be 2.05 × 102 PFU. Lethality was observed as early as day 4 post-exposure in the highest-dosed marmoset but animals at lower inhaled doses had a protracted disease course where humane study endpoint was not met until as late as day 19 post-exposure. Clinical signs were observed as early as 3 to 4 days post-exposure, including fever, ruffled fur, decreased grooming, and leukocytosis. Clinical signs increased in severity as disease progressed to include decreased body weight, subdued behavior, tremors, and lack of balance. Fever was observed as early as day 2-3 post-exposure in the highest dose groups and hypothermia was observed in several cases as animals became moribund. Infectious virus was found in several key tissues, including brain, liver, kidney, and several lymph nodes. Clinical hematology results included early neutrophilia, lymphopenia, and thrombocytopenia. Key pathological changes included meningoencephalitis and retinitis. Immunohistochemical staining for viral antigen was positive in the brain, retina, and lymph nodes. More intense and widespread IHC labeling occurred with increased aerosol dose. CONCLUSION: We have estimated the medial lethal dose of aerosolized EEEV and described the pathology of clinical disease in the marmoset model. The results demonstrate that the marmoset is an animal model suitable for emulation of human EEEV disease in the development of medical countermeasures.

Characterizing areas of potential human exposure to eastern equine encephalitis virus using serological and clinical data from horses.

Eastern equine encephalitis (EEE) is a rare but severe emerging vector-borne disease affecting human and animal populations in the northeastern United States where it is endemic. Key knowledge gaps remain about the epidemiology of EEE virus (EEEV) in areas where its emergence has more recently been reported. In Eastern Canada, viral activity has been recorded in mosquitoes and horses throughout the 2000s but cases of EEEV in humans have not been reported so far. This study was designed to provide an assessment of possible EEEV human exposure by modelling environmental risk factors for EEEV in horses, identifying high-risk environments and mapping risk in the province of Quebec, Canada. According to logistic models, being located near wooded swamps was a risk factor for seropositivity or disease in horses [odds ratio (OR) 4·15, 95% confidence interval (CI) 1·16-14·8) whereas being located on agricultural lands was identified as protective (OR 0·75, 95% CI 0·62-0·92). A better understanding of the environmental risk of exposure to EEEV in Canada provides veterinary and public health officials with enhanced means to more effectively monitor the emergence of this public health risk and design targeted surveillance and preventive measures.

Eastern equine encephalomyelitis virus infection in six captive southern cassowaries (Casuarius casuarius).

CASE DESCRIPTION Within a 2-week period, 4 southern cassowaries (Casuarius casuarius) at an exhibit at a Virginia zoo died acutely subsequent to eastern equine encephalitis virus (EEEV) infection. This prompted a search for other EEEV outbreaks in cassowaries, which resulted in the identification of 2 additional cassowaries that died of EEEV infection at a conservation center in Florida. CLINICAL FINDINGS Both juvenile and adult birds were affected. Three of the 6 birds died acutely with no premonitory signs. Clinical disease in the other 3 birds was characterized by lethargy and ataxia. Clinicopathologic findings typically included leukocytosis, hyperuricemia, abnormally high liver enzyme activities, and hyper-ß globulinemia, which was indicative of acute inflammation. TREATMENT AND OUTCOME The 3 birds with clinical disease died despite supportive treatment. Gross abnormalities commonly observed during necropsy included coelomitis and evidence of diarrhea. Frequently observed histologic abnormalities were encephalitis, vasculitis, hepatitis, nephritis, and splenitis. The diagnosis of EEEV infection was confirmed by detection of serum anti-EEEV antibodies or detection of viral RNA in brain tissue by use of a reverse-transcriptase PCR assay. CLINICAL RELEVANCE Findings suggested that EEEV can cause high morbidity and mortality rates in southern cassowaries. Clinical disease might be reduced or prevented by vaccination, isolation of ill birds, and mosquito control strategies.

Widespread Detection of Antibodies to Eastern Equine Encephalitis, West Nile, St. Louis Encephalitis, and Turlock Viruses in Various Species of Wild Birds from Across the United States.

Wild birds serve as amplifying hosts for many arboviruses, and are thought to be responsible for introducing these viruses into new areas during migration as well as reintroducing them to places where winter temperatures disrupt mosquito-borne transmission. To learn more about four mosquito-borne arboviruses of concern to human or animal health, we tested sera from 997 wild birds of 54 species and 17 families across 44 states of the United States collected from January 1, 2013, through September 30, 2013. Samples were tested for antibody against eastern equine encephalitis, St. Louis encephalitis, West Nile, and Turlock viruses using plaque reduction neutralization tests with an endpoint of 80% or greater. Of the 333 (33.4%) birds that tested positive for antibody to at least one arbovirus, 29.7% were exposed to two or more arboviruses. Exposure to all four arboviruses was detected in Canada geese, double-crested cormorants, mallards, mute swans, laughing gulls, and American coots. Our results suggest that exposure to arboviruses is widespread in the United States across a diversity of wild bird species.

Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.

An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure.

Geography and Timing of Cases of Eastern Equine Encephalitis in New York State from 1992 to 2012.

INTRODUCTION: In New York State (NYS), Eastern equine encephalitis (EEE) was first reported in a human in 1971, in horses in 1970, and in pheasants in 1952. MATERIAL AND METHOD: Following work for the interval from 1970 to 1991, we identified cases in vertebrates from 1992 to 2012, through a passive surveillance system involving veterinarians in clinical practice, county health departments, and the Departments of Agriculture and Markets, Environmental Conservation, and Health, of the State of New York. RESULT: During an 11-year hiatus, from 1992 to 2002, no case in any vertebrate was observed. In a re-emergence, from 2003 to 2012, disease occurred in 12 counties, including 7 counties where disease had never been documented. Vertebrate cases included 4 cases in humans and 77 nonhuman occurrences; in 58 horses, Equus ferus caballus L.; 2 deer, Odocoileus virginianus Zimmermann; 6 dogs, Canis familiaris; 10 birds; and 1 flock of pheasants, Phasianus colchicus L. These were the first reported cases in NYS in white-tailed deer, the domestic dog, and in five species of birds: American crow, Corvus brachyrhynchos Brehm; American goldfinch, Carduelis tristis L.; bald eagle, Haliaeetus leucocephalus L.; blue jay, Cyanocitta cristata (L.); and red-tailed hawk, Buteo jamaicensis Gmelin. One crow was dually infected with EEE virus and West Nile virus. The northern, southern, and southeastern borders of the state were newly affected. CONCLUSION: The geographic area, time periods, and vertebrate species with risk of EEE disease expanded from 1992 to 2012.

Dynamics of Vector-Host Interactions in Avian Communities in Four Eastern Equine Encephalitis Virus Foci in the Northeastern U.S.

BACKGROUND: Eastern equine encephalitis (EEE) virus (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching) birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors. METHODOLOGY AND PRINCIPLE FINDINGS: Contacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an empirically informed mathematical model for EEE virus transmission using Cs. melanura abundance and preferred and non-preferred avian hosts. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. We measured strong feeding preferences for a number of avian species based on the proportion of mosquito blood meals identified from these bird species in relation to their observed frequencies. These included: American Robin, Tufted Titmouse, Common Grackle, Wood Thrush, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo. We found that these bird species, most notably Wood Thrush, play a dominant role in supporting EEE virus amplification. It is also noteworthy that the competence of some of the aforementioned avian species for EEE virus has not been established. Our findings indicate that heterogeneity induced by mosquito host preference, is a key mediator of the epizootic transmission of vector-borne pathogens. CONCLUSION AND SIGNIFICANCE: Detailed knowledge of the vector-host interactions of mosquito populations in nature is essential for evaluating their vectorial capacity and for assessing the role of individual vertebrates as reservoir hosts involved in the maintenance and amplification of zoonotic agents of human diseases. Our study clarifies the host associations of Cs. melanura in four EEE virus foci in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study identifies Wood Thrush, American Robin and a few avian species that may serve as superspreaders of EEE virus. Our study elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host community. This research provides a basis to better understand the involvement of Cs. melanura and avian hosts in the transmission and ecology of EEE virus and the risk of human infection in virus foci.

Vector-Host Interactions of Culiseta melanura in a Focus of Eastern Equine Encephalitis Virus Activity in Southeastern Virginia.

Eastern equine encephalitis virus (EEEV) causes a highly pathogenic mosquito-borne zoonosis that is responsible for sporadic outbreaks of severe illness in humans and equines in the eastern USA. Culiseta (Cs.) melanura is the primary vector of EEEV in most geographic regions but its feeding patterns on specific avian and mammalian hosts are largely unknown in the mid-Atlantic region. The objectives of our study were to: 1) identify avian hosts of Cs. melanura and evaluate their potential role in enzootic amplification of EEEV, 2) assess spatial and temporal patterns of virus activity during a season of intense virus transmission, and 3) investigate the potential role of Cs. melanura in epidemic/epizootic transmission of EEEV to humans and equines. Accordingly, we collected mosquitoes at 55 sites in Suffolk, Virginia in 2013, and identified the source of blood meals in engorged mosquitoes by nucleotide sequencing PCR products of the mitochondrial cytochrome b gene. We also examined field-collected mosquitoes for evidence of infection with EEEV using Vector Test, cell culture, and PCR. Analysis of 188 engorged Cs. melanura sampled from April through October 2013 indicated that 95.2%, 4.3%, and 0.5% obtained blood meals from avian, mammalian, and reptilian hosts, respectively. American Robin was the most frequently identified host for Cs. melanura (42.6% of blood meals) followed by Northern Cardinal (16.0%), European Starling (11.2%), Carolina Wren (4.3%), and Common Grackle (4.3%). EEEV was detected in 106 mosquito pools of Cs. melanura, and the number of virus positive pools peaked in late July with 22 positive pools and a Maximum Likelihood Estimation (MLE) infection rate of 4.46 per 1,000 mosquitoes. Our findings highlight the importance of Cs. melanura as a regional EEEV vector based on frequent feeding on virus-competent bird species. A small proportion of blood meals acquired from mammalian hosts suggests the possibility that this species may occasionally contribute to epidemic/epizootic transmission of EEEV.