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Western equine encephalitis virus (WEEV) is a mosquito-borne arbovirus (genus Alphavirus , family Togaviridae ) that has re-emerged in South America in late 2023, causing severe disease in both horses and humans after a nearly 40-year intermission period. We here describe the virological, serological, pathological, and molecular features of WEEV infection in horses during the 2023-2024 outbreak in Argentina. WEEV-infected horses developed neurological signs with mild to severe encephalitis associated with minimal to abundant WEEV-infected cells, as demonstrated by WEEV-specific in situ hybridization. The distribution of viral RNA was multifocal, with predominance within neuronal bodies, neuronal processes, and glial cells in the medulla oblongata and thalamic regions. Phylogenetic analysis of partial nsP4 sequences from three viral isolates obtained from three different provinces of Argentina support grouping with other temporally current WEEV strains from Uruguay and Brazil under a recently proposed novel lineage.

Background: Madariaga virus (MADV), a member of the eastern equine encephalitis virus (EEEV) complex, circulates in Latin America and exhibits distinct evolutionary and ecological features compared to the North American EEEV. While published data have shed light on MADV ecology, several key aspects remain unknown. Methods: In this study, we compiled data on virus isolation, vector competence, and animal serology collected over six decades in Latin America to identify critical knowledge gaps on MADV transmission and ecology. Results: Specific vertebrate animals serving as amplifying hosts and the mosquito species acting as enzootic and epizootic vectors have not yet been identified. Other aspects that remain unclear are the virus current geographic distribution, the role of equines as hosts in epizootic cycles, and the full impact of MADV on human health in endemic regions. Conclusions: The numerous knowledge gaps surrounding MADV, its widespread distribution in Latin America, and its potential to cause severe disease in animals and humans emphasize the urgent need for increased research efforts, heightened awareness, and intensified surveillance towards this potential emerging threat.

We report the 1st records of Aedes tormentor and Culex panocossa throughout vector surveillance events carried out in putative foci of eastern equine encephalitis in Tamaulipas, Mexico. Formerly, Ae. tormentor had been reported in, at least, 2 Central American countries and Mexico. In Mexico, reports were from the states of Campeche, Chiapas, Quintana Roo, and Veracruz. Records of Ae. tormentor in these 4 Neotropical states were recently reviewed and eliminated; thus, the southernmost geographic distribution for this species is considered to be the state of Tamaulipas Mexico in its neotropical zone. Further, Cx. panocossa had been collected in Guerrero, Tabasco, and Veracruz. In Tamaulipas, there are 82 species of mosquitoes, being the 4th state accounting for the highest mosquito species diversity of 11 states in which comprehensive studies have been conducted on the subject of mosquito distribution., (Copyright © 2024 by The American Mosquito Control Association, Inc.)

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

We report the genomic analysis from early equine cases of the Western equine encephalitis virus outbreak during 2023-2024 in Uruguay. Sequences are related to a viral isolate from an outbreak in 1958 in Argentina. A viral origin from South America or continuous enzootic circulation with infrequent spillover is possible.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s 1-3 . The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence 3 ) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors 4 . However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains., (© 2024. The Author(s).)

Eastern equine encephalitis virus (EEEV) is a relatively little-studied alphavirus that can cause devastating viral encephalitis, potentially leading to severe neurological sequelae or death. Although case numbers have historically been low, outbreaks have been increasing in frequency and scale since the 2000 s. It is critical to investigate EEEV evolutionary patterns, especially within human hosts, to understand patterns of emergence, host adaptation, and within-host evolution. To this end, we obtained formalin-fixed paraffin-embedded tissue blocks from discrete brain regions from five contemporary (2004-2020) patients from Massachusetts, confirmed the presence of EEEV RNA by in situ hybridization (ISH) staining, and sequenced viral genomes. We additionally sequenced RNA from scrapings of historical slides made from brain sections of a patient in the first documented EEE outbreak in humans in 1938. ISH staining revealed the presence of RNA in all contemporary samples, and quantification loosely correlated with the proportion of EEEV reads in samples. Consensus EEEV sequences were generated for all six patients, including the sample from 1938; phylogenetic analysis using additional publicly available sequences revealed clustering of each study sample with like sequences from a similar region, whereas an intrahost comparison of consensus sequences between discrete brain regions revealed minimal changes. Intrahost single nucleotide variant (iSNV) analysis of four samples from two patients revealed the presence of tightly compartmentalized, mostly nonsynonymous iSNVs. This study contributes critical primary human EEEV sequences, including a historic sequence as well as novel intrahost evolution findings, contributing substantially to our understanding of the natural history of EEEV infection in humans.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control., Competing Interests: Declaration of interests The authors declare no conflicts of interest related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Zoonotic pathogens that are vector-transmitted have and continue to contribute to several emerging infections globally. In recent years, spillover events of such zoonotic pathogens have increased in frequency as a result of direct contact with livestock, wildlife, and urbanization, forcing animals from their natural habitats. Equines serve as reservoir hosts for vector-transmitted zoonotic viruses that are also capable of infecting humans and causing disease. From a One Health perspective, equine viruses, therefore, pose major concerns for periodic outbreaks globally. Several equine viruses have spread out of their indigenous regions, such as West Nile virus (WNV) and equine encephalitis viruses (EEVs), making them of paramount concern to public health. Viruses have evolved many mechanisms to support the establishment of productive infection and to avoid host defense mechanisms, including promoting or decreasing inflammatory responses and regulating host machinery for protein synthesis. Viral interactions with the host enzymatic machinery, specifically kinases, can support the viral infectious process and downplay innate immune mechanisms, cumulatively leading to a more severe course of the disease. In this review, we will focus on how select equine viruses interact with host kinases to support viral multiplication.

Mosquito vectors of eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) in the USA reside within broad multi-species assemblages that vary in spatial and temporal composition, relative abundances and vector competence. These variations impact the risk of pathogen transmission and the operational management of these species by local public health vector control districts. However, most models of mosquito vector dynamics focus on single species and do not account for co-occurrence probabilities between mosquito species pairs across environmental gradients. In this investigation, we use for the first time conditional Markov Random Fields (CRF) to evaluate spatial co-occurrence patterns between host-seeking mosquito vectors of EEEV and WNV around sampling sites in Manatee County, Florida. Specifically, we aimed to: (i) quantify correlations between mosquito vector species and other mosquito species; (ii) quantify correlations between mosquito vectors and landscape and climate variables; and (iii) investigate whether the strength of correlations between species pairs are conditional on landscape or climate variables. We hypothesized that either mosquito species pairs co-occur in patterns driven by the landscape and/or climate variables, or these vector species pairs are unconditionally dependent on each other regardless of the environmental variables. Our results indicated that landscape and bioclimatic covariates did not substantially improve the overall model performance and that the log abundances of the majority of WNV and EEEV vector species were positively dependent on other vector and non-vector mosquito species, unconditionally. Only five individual mosquito vectors were weakly dependent on environmental variables with one exception, Culiseta melanura, the primary vector for EEEV, which showed a strong correlation with woody wetland, precipitation seasonality and average temperature of driest quarter. Our analyses showed that majority of the studied mosquito species' abundance and distribution are insignificantly better predicted by the biotic correlations than by environmental variables. Additionally, these mosquito vector species may be habitat generalists, as indicated by the unconditional correlation matrices between species pairs, which could have confounded our analysis, but also indicated that the approach could be operationalized to leverage species co-occurrences as indicators of vector abundances in unsampled areas, or under scenarios where environmental variables are not informative., (© 2023. The Author(s).)

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is a mosquito-borne pathogen found in eastern North America that causes severe disease in humans and horses. The mosquito Culiseta melanura (Coquillett) (Diptera: Culicidae) is the primary enzootic vector of EEEV throughout eastern North America while several mosquito species belonging to diverse genera serve as bridge vectors. The ecology of EEEV differs between northern and southern foci, with respect to phenology of outbreaks, important vertebrate hosts, and bridge vector species. Active transmission is limited to roughly half of the year in northern foci (New York, New Hampshire, Massachusetts, Connecticut), while year-round transmission occurs in the southeastern region (particularly Florida). Multiple phylogenetic analyses indicate that EEEV strains circulating in northern foci are likely transported from southern foci by migrating birds. Bird species that overwinter or migrate through Florida, are bitten by Cs. melanura in late spring, and arrive at northern breeding grounds in May are the most likely candidates to disperse EEEV northward. Available data indicate that common yellowthroat and green heron satisfy these criteria and could serve as virus dispersers. Understanding the factors that drive the phenology of Cs. melanura reproduction in the south and the timing of avian migration from southern foci could provide insight into how confluence of these biological phenomena shapes outbreaks of EEE throughout its range. This information could be used to develop models predicting the likelihood of outbreaks in a given year, allowing vector control districts to more efficiently marshal resources necessary to protect their stakeholders., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vertebrate surveillance for eastern equine encephalitis virus (EEEV) activity usually focuses on three types of vertebrates: horses, passerine birds, and sentinel chicken flocks. However, there is a variety of wild vertebrates that are exposed to EEEV infections and can be used to track EEEV activity. In 2009, we initiated a pilot study in northern New England, United States, to evaluate the effectiveness of using wild cervids (free-ranging white-tailed deer and moose) as spatial sentinels for EEEV activity. In Maine, New Hampshire, and Vermont during 2009-2017, we collected blood samples from hunter-harvested cervids at tagging stations and obtained harvest location information from hunters. U.S. Centers for Disease Control and Prevention processed the samples for EEEV antibodies using plaque reduction neutralization tests (PRNTs). We detected EEEV antibodies in 6 to 17% of cervid samples in the different states and mapped cervid EEEV seropositivity in northern New England. EEEV antibody-positive cervids were the first detections of EEEV activity in the state of Vermont, in northern Maine, and northern New Hampshire. Our key result was the detection of the antibodies in areas far outside the extent of documented wild bird, mosquito, human case, or veterinary case reports of EEEV activity in Maine, New Hampshire, and Vermont. These findings showed that cervid (deer and moose) serosurveys can be used to characterize the geographic extent of EEEV activity, especially in areas with low EEEV activity or with little or no EEEV surveillance. Cervid EEEV serosurveys can be a useful tool for mapping EEEV activity in areas of North America in addition to northern New England., (Published by Oxford University Press on behalf of Entomological Society of America 2021.)

In the current review, we examine the regional history, ecology, and epidemiology of eastern equine encephalitis virus (EEEV) to investigate the major drivers of disease outbreaks in the northeastern United States. EEEV was first recognized as a public health threat during an outbreak in eastern Massachusetts in 1938, but historical evidence for equine epizootics date back to the 1800s. Since then, sporadic disease outbreaks have reoccurred in the Northeast with increasing frequency and northward expansion of human cases during the last 20 yr. Culiseta melanura (Coquillett) (Diptera: Culicidae) serves as the main enzootic vector that drives EEEV transmission among wild birds, but this mosquito species will occasionally feed on mammals. Several species have been implicated as bridge vectors to horses and humans, with Coquilletstidia perturbans (Walker) as a leading suspect based on its opportunistic feeding behavior, vector competence, and high infection rates during recent disease outbreaks. A diversity of bird species are reservoir competent, exposed to EEEV, and serve as hosts for Cs. melanura, with a few species, including the wood thrush (Hlocichia mustelina) and the American robin (Turdus migratorius), contributing disproportionately to virus transmission based on available evidence. The major factors responsible for the sustained resurgence of EEEV are considered and may be linked to regional landscape and climate changes that support higher mosquito densities and more intense virus transmission., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

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 (EEEV) infects many avian species but has rarely been described in Ruffed Grouse (Bonasa umbellus). Between September and December 2019, 40 Ruffed Grouse, most in poor physical condition, were submitted to the Michigan, Wisconsin, and Minnesota (US) Departments of Natural Resources; eight were positive for EEEV., (© Wildlife Disease Association 2021.)

A total of 102 free-range wild boars, 170 hunting dogs, and 49 hunters from 3 Brazilian regions were sampled and tested for antibodies to eastern equine encephalitis virus (EEEV), western equine encephalitis virus, and Venezuelan equine encephalitis virus. Three of the 102 (2.9%) wild boars were positive for antibodies against EEEV by microplate serum neutralization test. Based on our data, free-range wild boars from central-western Brazil may be exposed to EEEV, and further studies are needed to evaluate the potential of incorporating serosurveys in routine arbovirus activity surveillance specifically to identify arbovirus activity foci and to help establish thresholds for epidemic transmission.

Neurological disorders represent an important sanitary and economic threat for the equine industry worldwide. Among nervous diseases, viral encephalitis is of growing concern, due to the emergence of arboviruses and to the high contagiosity of herpesvirus-infected horses. The nature, severity and duration of the clinical signs could be different depending on the etiological agent and its virulence. However, definite diagnosis generally requires the implementation of combinations of direct and/or indirect screening assays in specialized laboratories. The equine practitioner, involved in a mission of prevention and surveillance, plays an important role in the clinical diagnosis of viral encephalitis. The general management of the horse is essentially supportive, focused on controlling pain and inflammation within the central nervous system, preventing injuries and providing supportive care. Despite its high medical relevance and economic impact in the equine industry, vaccines are not always available and there is no specific antiviral therapy. In this review, the major virological, clinical and epidemiological features of the main neuropathogenic viruses inducing encephalitis in equids in Europe, including rabies virus ( Rhabdoviridae ), Equid herpesviruses ( Herpesviridae ), Borna disease virus ( Bornaviridae ) and West Nile virus ( Flaviviridae ), as well as exotic viruses, will be presented.

Madariaga virus (MADV), previously known as South American eastern equine encephalitis virus (SA EEEV; family Togaviridae, genus Alphavirus ), is a mosquito-borne virus associated mainly with equine disease. In 2010, the first human outbreak by MADV was reported in Central America, but the mosquito vectors and vertebrate hosts involved in the outbreak were not identified. In Argentina, the first epizootic of MADV was in 1930, and since then, several epizootics by MADV have been reported. However, the potential vectors and hosts involved in the transmission cycle remain unknown. In the present study, MADV was detected in Culex ( Culex ) spp. mosquitoes and the phylogenetic analysis showed that the MADV fragment amplified grouped with the lineage/subtype III of the SA EEEV complex. Our results provide information about the natural infection with MADV in mosquitoes collected in a wild environment of Argentina and its genetic relatedness.

Madariaga Virus (MADV) is an emergent Alphavirus of the eastern equine encephalitis virus (EEEV) strain complex causing epizootic epidemics. In this study the genetic diversity and the transmission dynamics of Madariaga virus has been investigated by Bayesian phylogenetics and phylodynamic analysis. A database of 32 sequences of MADV group structural polyprotein were downloaded from GenBank, aligned manually edited by Bioedit Software. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Neighbor-joining tree was generated using MEGA7. The phylogenetic signal of the dataset was tested by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. Selective pressure analysis revealed one positive selection site. The phylogenetic trees showed two main clusters. In particular, Lineage II showed an epizootic infection in monkeys and Lineage III, including 2 main clusters (IIIa and IIIB), revealing an epizootic infection in humans in Haiti and an epizootic infection in humans in Venezuela during the 2016, respectively. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor estimates, showed that the root of the tree dated back to the year 346 with the probable origin in Brazil. Gene flow analysis revealed viral exchanges between different neighbor countries of South America. In conclusion, Bayesian phylogenetic and phylodynamic represent useful tools to follow the transmission dynamic of emergent pathogens to prevent new epidemics spreading worldwide., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Between February and June 2011, more than 300 horses with unexplained neurological disease were observed in New South Wales, Australia. A virulent strain of West Nile virus (WNV NSW2011 ), of Australian origin, was shown to be the cause of many of these cases., Methods: We reviewed the clinical descriptions provided by veterinary practitioners and the associated laboratory results. Although there was a range of clinical signs described, ataxia was the only sign that was consistently described in laboratory-confirmed cases., Results: WNV was detected in brain samples by real-time reverse transcription PCR assay and virus isolation. For serological confirmation of clinical cases, an equine IgM ELISA specific for WNV was shown to be the most effective tool., Conclusion: A state-wide serological survey undertaken after the outbreak indicated that, contrary to expectation, although infection had been widespread, the seroprevalence of antibodies to WNV was very low, suggesting that there could be a significant risk of future disease outbreaks., (© 2019 State of New South Wales.)

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.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region. IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas., (Copyright © 2018 American Society for Microbiology.)

Periodic outbreaks of West Nile virus (WNV), Eastern equine encephalitis virus (EEEV) and to a lesser extent, California serogroup viruses (CSGV), have been reported in parts of Canada in the last decade. This study was designed to provide a broad assessment of arboviral activity in Quebec, Canada, by conducting serological surveys for these arboviruses in 196 horses, 1442 dogs and 485 humans. Sera were screened by a competitive enzyme linked immunosorbent assay and positive samples confirmed by plaque reduction neutralisation tests. The percentage of seropositive samples was 83·7%, 16·5%, 7·1% in horses, 18·8%, 0·6%, 0% in humans, 11·7%, 3·1%, 0% in adult dogs and 2·9%, 0·3%, 0% in juvenile dogs for CSGV, WNV and EEEV, respectively. Serological results in horses and dogs appeared to provide a meaningful assessment of risk to public health posed by multiple arboviruses.

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.

Epidemiologic analysis of epidemic outbreaks caused by American equine encephalitis causative agents is carried out in the review. Eastern equine encephalomyelitis (EEE), Western equine encephalomyelitis (WEE) and Venezuela equine encephalomyelitis (VEE) viruses are etiologic agents of dangerous transmissive diseases that are usually accompanied by fever and neurologic symptoms. Among the New World alphaviruses, VEE virus has the most potential danger for humans and domestic animals. Currently, enzootic strains of VEE play an increasing role as etiologic agents of human diseases. Most of the VEE cases in humans in endemic regions during inter-epidemic period are caused by infection with VEE subtype ID virus. A possibility of emergence of novel epidemic outbreaks of VEE is determined by mutations of ID subtype strains into IC subtype, and those currently pose a potential threat as an etiologic agent of the disease. Despite low morbidity, EEE and WEE are a problem for healthcare due to a relatively high frequency of lethal outcomes of the disease.

During the fall of 2010, 332 deer serum samples were collected from 15 of the 16 (93.8%) Maine counties and screened for eastern equine encephalitis virus (EEEV) antibodies using plaque reduction neutralizing tests (PRNTs). The aim was to detect and map EEEV activity in the state of Maine. Forty-seven of the 332 (14.2%) sera were positive for EEEV antibodies, showing a much wider distribution of EEEV activity in Maine than previously known. The percentage of EEEV antibody-positive deer sera was ≥10% in six counties-Piscataquis (100%), Somerset (28.6%), Waldo (22.2%), Penobscot (21.7%), Kennebec (13.7%), and Sagadahoc (10%). Positive sera were detected in all the six counties (Somerset, Waldo, Penobscot, Kennebec, Cumberland, and York) that were positive in 2009, suggesting endemic EEEV activity in these counties. EEEV antibodies were not detected in sera collected in five counties-Franklin, Knox, Lincoln, Oxford, and Washington-which was either due to low sample size or lack of EEEV activity in these counties. Our data suggest higher EEEV activity in central Maine compared to southern Maine, whereas EEEV activity in Maine has historically been associated with the southern counties of York and Cumberland.

To investigate arbovirus transmission in North Dakota, we collected and screened mosquitoes for viral infection by Vero cell culture assay. Seven viruses were isolated from 13 mosquito species. Spatial and temporal distributions of the important vectors of West Nile virus (WNV), Cache Valley virus, Jamestown Canyon virus (JCV), and trivittatus virus are reported. Snowshoe hare virus, Potosi virus, and western equine encephalomyelitis virus were also isolated. The risks of Culex tarsalis and Aedes vexans transmitting WNV to humans were 61.4% and 34.0% in 2003-2006, respectively, but in 2003 when the largest epidemic was reported, risks for Ae. vexans and Cx. tarsalis in Cass County were 73.6% and 23.9%, respectively. Risk of humans acquiring an infectious bite was greatest from about the second week of July through most of August. West Nile virus sequences were of the WN02 genotype. Most JCV strains belonged to a single clade of genetically related strains. Cache Valley virus and JCV were prevalent during August and early September and during July and August, respectively., (© The American Society of Tropical Medicine and Hygiene.)

Moose sera were collected from harvested animals during the 2010 hunting season in Maine. Of the 145 serum samples screened by plaque reduction neutralization test (PRNT), 16 (11%) had antibodies to eastern equine encephalitis virus (EEEV). Positive samples were collected from Aroostook County (n=13), Somerset County (n=2), and Piscataquis County (n=1) in northern and central Maine. Preliminary mosquito surveillance revealed the presence of enzootic and bridge vectors mosquitoes, including Culiseta (Climacura) melanura (Coquillett), Aedes (Aedimorphus) vexans (Meigen), and Coquillettidia (Coquillettidia) perturbans (Walker). Select mosquito species were tested by RT-PCR for the presence of EEEV. None were positive. This is the first report of EEEV in moose from Maine.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic arbovirus that infects humans, horses, and other animals. There has been a significant increase in EEEV activity in southeastern Canada since 2008. Few data are available regarding nonlethal EEEV infections in mammals, and consequently the distribution and pathogenicity spectrum of EEEV infections in these hosts is poorly understood. This cross-sectional study focuses on the evaluation of viral activity in southern Quebec's horses by seroprevalence estimation. A total of 196 horses, 18 months and older, which had never been vaccinated against EEEV and have never traveled outside Canada, were sampled from 92 barns distributed throughout three administrative regions of southern Quebec. Blood samples were taken from each horse and titrated for EEEV antibodies by plaque reduction neutralization test (PRNT). Equine population vaccination coverage was estimated by surveying horse owners and equine practitioners. PRNT results revealed an EEEV seroprevalence up to 8.7%, with 95% confidence limits ranging from 4.4% to 13.0%. Vaccination coverage was estimated to be at least 79%. Our study reveals for the first time in Canada a measure of EEEV seroprevalence in horses. High seroprevalence in unvaccinated animals challenges the perception that EEEV is a highly lethal pathogen in horses. Monitoring high-risk vector-borne infections such as EEEV in animal populations can be an important element of a public health surveillance strategy, population risk assessment and early detection of epidemics.

West Nile Virus (WNV) and Eastern Equine Encephalitis Virus (EEEV) are both primarily bird viruses, which can be transmitted by several mosquito species. Differences in larval habitats, flight, and biting patterns of the primary vector species result in substantial differences in epidemiology, with WNV more common, primarily occurring in urban areas, and EEEV relatively rare, typically occurring near swamp habitats. The complex transmission ecology of these viruses complicates prediction of disease outbreaks. The Rhode Island Department of Environmental Management (DEM) and Department of Health (DoH) provide prevention assistance to towns and maintain a mosquito surveillance program to identify potential disease risk. Responses to potential outbreaks follow a protocol based on surveillance results, assessment of human risk, and technical consultation.

Eastern equine encephalitis (EEE) virus is a highly pathogenic mosquito-borne zoonosis that is responsible for outbreaks of severe disease in humans and equines, resulting in high mortality or severe neurological impairment in most survivors. In the northeastern United States, EEE virus is maintained in an enzootic cycle involving the ornithophilic mosquito, Culiseta melanura (Coquillett) and passerine birds in freshwater swamp habitats. To evaluate the role of Cs. melanura and Culiseta morsitans (Theobald) in recent episodes of EEE virus activity in Massachusetts, we collected blood-fed mosquitoes between June, 2007, and October, 2008, from virus foci in 6 counties, and identified the source of blood meals by PCR amplification of mitochondrial cytochrome b gene and sequencing. Analysis of 529 Cs. melanura and 25 Cs. morsitans revealed that nearly 99% and 96% of mosquitoes, respectively, acquired blood meals solely from avian hosts. American Robin, Turdus migratorius Linnaeus was identified as the most common vertebrate host for Cs. melanura (21.7%, n=115), followed by Tufted Titmouse, Baeolophus bicolor (L.) (8.7%, n=46), Black-capped Chickadee, Poecile atricapillus (L.) (8.5%, n=45), Scarlet Tanager, Piranga olivacea (Gmelin) (6.8%, n=36), Field Sparrow, Spizella pusilla (Wilson) (6.2%, n=33), Northern Cardinal, Cardinalis cardinalis (L.) (5.7%, n=30), and other mostly Passeriformes birds. Mammalian-derived blood meals were identified as white-tailed deer, Odocoileus virginianus Zimmermann, domestic cow, Bos taurus L., and human, Homo sapiens L. There were 4 isolations of EEE virus, West Nile virus, and Highland J virus from Cs. melanura. Our results in conjunction with other lines of evidence, including reservoir competency, prevalence of antibody, and infection in nature, suggest that the American Robin, Tufted Titmouse, Black-capped Chickadee, and a few other passerine birds may play key roles in supporting EEE virus transmission in Massachusetts. Infrequent blood feeding of Cs. melanura on mammalian hosts, including humans, also indicates that this mosquito may occasionally contribute to epidemic/epizootic transmission of EEE virus in this region.

From July to September, 2009, an outbreak of eastern equine encephalitis virus (EEEv) occurred in five counties in Maine. The virus was isolated from 15 horses, 1 llama, and pheasants in three separate captive flocks. One wild turkey, screened before translocation, also showed exposure to the virus in January 2010. Two pools of Culiseta melanura (Coquillett) tested positive for EEEv during routine seasonal surveillance in York County in September, but none of the mosquitoes collected during rapid response surveys tested positive. There were more Cs. melanura in July, August, and September 2009 than in preceding (2006-08) and subsequent (2010-11) years. August and September Cs. melanura abundances were correlated with July rainfall, and abundance of all species combined was correlated with total rainfall for the meteorological summer. This outbreak represents a substantial expansion of the range of EEEv activity in northern New England.

Two strains of the Western equine encephalitis virus (WEEV) were first detected and isolated in China in 2001. The maintenance and transmission cycles of WEEV in China are currently not well understood, and the mosquito vectors involved in these cycles are unknown. To understand the ability of the local mosquitoes in China to transmit WEEV, the vector competence of five mosquito species, namely, Culex pipiens pallens Coquillett, Cx. p. quinquefasciatus Say, Aedes (Stegomyia) albopictus Skuse, Ae. (Stegomyia) aegypti Linnaeus, and C. tritaeniorhynchus Giles, for WEEV were evaluated. Infection rates for Cx. p. pallens, Cx. p. quinquefasciatus, Cx. tritaeniorhynchus, Ae. Albopictus, and Ae. aegypti were 46%, 60%, 80%, 37%, and 25%, respectively. Dissemination rates for the same species were 60%, 61%, 75%, 55%, and 50%, respectively. Transmission rates were 41%, 53%, 57%, and 45% for Cx. p. pallens, Cx. p. quinquefasciatus, Ae. Albopictus, and Ae. Aegypti, respectively. Infection rates were significantly different between species, but the difference between dissemination and transmission rates were nonsignificant. These results suggest that several local mosquito species in China are competent laboratory vectors for WEEV.

Serum from 226 free-ranging white-tailed deer (Odocoileus virginianus) was screened for Eastern Equine Encephalitis Virus (EEEV) antibodies using plaque reduction neutralization tests. EEEV antibodies were detected in 7.1% of samples. This is the first time EEEV antibodies have been detected in O. virginianus populations in the state of Maine (ME). The highest percentage of EEEV positive sera was in Somerset County (19%) in central ME, and this is the first time that EEEV activity has been detected in that County. EEEV RNA was not detected in any of the 150 harvested deer brain samples submitted to the ME Department of Inland Fisheries and Wildlife as a part of screening for Chronic Wasting Disease. This suggests that screening deer brains is not an efficient method to detect EEEV activity. For each serum sample tested, the geographic location in which the deer was harvested was recorded. Significant spatial clustering of antibody-positive sera samples was not detected. Relative to seronegative deer, seropositive deer were slightly more likely to be harvested in nonforested areas compared with forested areas. Results indicate that screening of free-ranging deer sera can be a useful tool for detecting EEEV activity in ME and other parts of North America.

At temperate latitudes, vectors and pathogens must possess biological mechanisms for coping with cold temperatures and surviving from one transmission season to the next. Mosquitoes that overwinter in the adult stage have been proposed as winter maintenance hosts for certain arboviruses. In the cases of West Nile virus (family Flaviviridae, genus Flavivirus) and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus), discovery of infected overwintering females lends support to this hypothesis, but for other arboviruses, in particular Eastern equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, EEEV), overwintering of the virus in mosquito hosts as not been demonstrated. In the current study, we collected overwintering mosquitoes from a focus of EEEV transmission in the southeastern United States to determine whether mosquitoes serve as winter maintenance hosts for EEEV and to document overwintering biologies of suspected vectors. No virus was detected via reverse transcription-polymerase chain reaction of > 500 female mosquitoes collected during three winters. Investigation into the winter biologies indicated that Anopheles punctipennis (Say), Culex erraticus (Dyar & Knab), Culex peccator Dyar & Knab, and Uranotaenia sapphirina (Osten Sacken) overwinter as females. Females of these species were collected from hollow trees and emergence traps placed over ground holes. Southern magnolia, Magnolia grandiflora L., trees were preferred overwintering sites of culicine mosquitoes. Emergence from underground overwintering sites peaked in mid-March, when air temperatures reached 18-22 degrees C, and the first blood-engorged females of Cx. erraticus and Cx. peccator were collected during this same period. Blood-fed Culex territans Walker females were collected as early as mid-February. This work provides insight into the overwintering biologies of suspected virus vectors at a site of active EEEV transmission and provides limited evidence against the hypothesis that EEEV persists through intertransmission periods in overwintering mosquitoes.

The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20-30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for SLEV in equines regardless of vaccine status, and 36.4% for WEEV and 47.7% for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil.

In 2006, 2,817 blood-fed mosquitoes were collected from the site of a 2005 eastern equine encephalitis outbreak in Chester County, TN. Using a polymerase chain reaction-based assay, 264 vertebrate hosts were identified from seven mosquito species. Culex erraticus and Cx. nigripalpus fed on a diversity of mammalian, avian, and reptilian hosts, whereas Anopheles quadrimaculatus and An. punctipennis were predominantly mammalophagic. Overall, 27% of Cx. nigripalpus, 16% of Cx. erraticus, and 7% of An. quadrimaculatus blood meals were acquired from avian hosts. No avian-derived blood meals were identified from An. punctipennis. The house finch, Carolina wren, and mourning dove were the most commonly identified avian host species. By incorporating this study with flight range, vector competence, and virus field isolation data, we assessed certain aspects of the enzootic and epizootic vectorial capacity of the mosquito species present at this outbreak site.

Historically, western equine encephalomyelitis virus (WEEV) caused large equine and human epidemics in the Americas from Canada into Argentina. Despite recent enhanced surveillance for West Nile virus, there have been few reports of equine or human cases and little documented enzootic activity of WEEV. During the past three years, WEEV has been active again in California, but without human or equine cases. In the current study, we compared host and vector competence of representative WEEV isolates made during each decade over the past 60 years using white-crowned sparrows, house sparrows, and Culex tarsalis Coquillett as representative hosts. Results indicated limited time-related change in virulence among WEEV strains in birds and little difference in vector competence in Cx. tarsalis. Although temporal and spatial genetic changes have been documented, these seem to present limited phenotypic change in host competence and cannot explain the absence of equine and human cases.

Eastern equine encephalitis (EEE) virus has been recognized as affecting horses and humans in the eastern United States for 70 yr. Evidence of exposure with EEE virus has been reported in a variety of free-ranging wild birds and mammals but cases of clinical disease are much less commonly reported. In Michigan, reports of outbreaks of EEE virus in equine species extend back more than a half century. We report diagnosis of EEE virus infection of multiple free-ranging white-tailed deer (Odocoileus virginianus) from three Michigan counties during late summer of 2005. Infection was confirmed in seven of 30 deer collected based on reported neurologic signs and results from immunohistochemistry, polymerase chain reaction, and/or virus isolation. One of the deer also was infected with West Nile virus and an eighth deer had microscopic lesions in the cerebrum consistent with those reported for EEE. To our knowledge, this is the first report of multiple cases of EEE in free-ranging white-tailed deer, and highlights several issues of significance to wildlife managers and public health officials.

Recent outbreaks of West Nile virus-associated (WNV) diseases, both in the old World and Americas, underline the importance for early warning systems that rapidly identify emerging and re-emerging diseases and thus help in their control. Traditional approaches of disease monitoring become less reliable and increasingly costly when used for rare health-related events, such as WNV outbreaks in southern France. The objective of this work was to discuss methodological issues related to syndromic monitoring of WNV-associated disease in Camargue horses by veterinary practitioners. Tracking cases of equine encephalitis by veterinarians is an example of such syndromic monitoring of an emerging disease. Signs of illness, observed prior diagnostic confirmation, can be of interest because they may provide an early warning for WNV circulation in a given area and allow authorities to take appropriate preventive measures for public health.

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.

Human eastern equine encephalitis (EEE) is a life-threatening mosquito-borne disease. To determine whether mosquito abundance and EEE virus infection rates are associated with human EEE disease, we evaluated retrospectively a total of 592,637 mosquitoes and onset dates for 20 confirmed human cases over 26 years in Massachusetts. Annual Culiseta melanura populations at 10 defined sites decreased over the study period (P = 0.002). Weekly infection rates and number of infected Culiseta melanura captured per trap night were positively associated EEE cases (P < 0.023 and P < 0.001, respectively), whereas abundance was not (P = 0.077). The infection rate for Culiseta melanura of 0.39 per 1,000 tested mosquitoes identified human cases with a sensitivity of 0.87, a specificity of 0.82, a positive predictive value of 0.14, and a negative predictive value of 0.995. Timely mosquito testing and infection rate calculation are critical for disease risk estimation and outbreak control efforts.

Gambel's and California quail were infected repeatedly whenever western equine encephalomyelitis virus (WEEV), St. Louis encephalitis virus (SLEV), and (WNV) West Nile virus were active during summer in California. The timing of virus appearance and quail infection coincided well with the appearance of chicks in nature, leading us to hypothesize that large coveys containing these non-immune birds could be important in focal virus amplification in rural settings. However, experimental infection studies with chicks, juveniles, and adults of both quail species using sympatric strains of WEEV, SLEV, and WNV indicated that only immature birds were competent hosts for WEEV, producing viremias sufficiently elevated to efficiently infect Culex tarsalis mosquitoes. Quail were less competent hosts for WNV and were incompetent for SLEV. Large populations of quail that frequently are infected with SLEV or WNV, but produce low to moderate viremias, may serve as dead end hosts for these viruses. Due to their abundance and repeated infection, these birds may attenuate virus amplification in rural areas of California and possibly could be one reason why WNV epidemics seem to occur more frequently in urban and periurban than in rural landscapes.

Eastern equine encephalitis (EEE) was diagnosed in a flock of African penguins. Diagnosis was based on history and clinical signs and confirmed via serologic testing, virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and histologic examination. Clinical signs in penguins included anorexia, behavior changes, depression, regurgitation, ataxia, recumbency, and seizures, and some penguins did not have any clinical signs. Mean +/- SD number of days that affected penguins had clinical signs was 12 +/- 5 days. Abnormalities initially detected on CBC included heterophilic leukocytosis and anemia; lymphocytosis and monocytosis were detected later. Plasma biochemical abnormalities included high activities of aspartate amino-transferase and creatine kinase, hyponatremia, hypochloremia, hyperglycemia, and high concentrations of globulin, triglycerides, and cholesterol. Mean +/- SD number of days required for resolution of CBC and plasma biochemical abnormalities was 67 +/- 24 days after the onset of clinical signs. Treatment consisted of supportive therapy. All penguins survived with the exception of one that was euthanatized; histopathologic findings were consistent with encephalitis. Results of RT-PCR assays performed on tissue from the right cerebrum of the penguin that was euthanatized were positive for EEE viral RNA. An inability to isolate virus several weeks after illness suggested successful viral clearance in recovered penguins. To the authors' knowledge, EEE infection in any penguin species has not been reported.