Shifts in receptors during submergence of an encephalitic arbovirus.

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 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) 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 receptors4. 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.

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

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.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

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.

The transmission dynamic of Madariaga Virus by bayesian phylogenetic analysis: Molecular surveillance of an emergent pathogen.

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.

Emerging arboviruses in Quebec, Canada: assessing public health risk by serology in humans, horses and pet dogs.

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.

[EPIDEMIOLOGIC ANALYSIS OF OUTBREAKS OF DISEASES CAUSED BY AMERICAN EQUINE ENCEPHALITIS CAUSATIVE AGENTS IN ENDEMIC REGIONS].

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.

FIRST RECORDS OF AEDES TORMENTOR AND CULEX PANOCOSSA AS A RESULT OF VECTOR SURVEILLANCE ACTIVITIES CONDUCTED DURING THE CHARACTERIZATION OF FOCI OF EASTERN EQUINE ENCEPHALITIS IN TAMAULIPAS, MEXICO.

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.

Uncertainties Surrounding Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex.

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.

Interactions of Equine Viruses with the Host Kinase Machinery and Implications for One Health and Human Disease.

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.

Co-occurrence probabilities between mosquito vectors of West Nile and Eastern equine encephalitis viruses using Markov Random Fields (MRFcov).

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.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

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.

Eastern Equine Encephalitis Virus Diversity in Massachusetts Patients, 1938-2020.

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.

Use of Cervid Serosurveys to Monitor Eastern Equine Encephalitis Virus Activity in Northern New England, United States, 2009-2017.

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.

Ecology of Eastern Equine Encephalitis Virus in the Southeastern United States: Incriminating Vector and Host Species Responsible for Virus Amplification, Persistence, and Dispersal.

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.

Ecology and Epidemiology of Eastern Equine Encephalitis Virus in the Northeastern United States: An Historical Perspective.

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.

Winter biology of wetland mosquitoes at a focus of eastern equine encephalomyelitis virus transmission in Alabama, USA.

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.

Eastern Equine Encephalitis Virus in Visibly Affected Ruffed Grouse (Bonasa umbellus) in Michigan, Minnesota, and Wisconsin, USA.

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.

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.

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil.

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.