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.

Eastern Equine Encephalitis Virus Taxonomy, Genomics, and Evolution.

Eastern equine encephalitis virus (EEEV; Togaviridae, Alphavirus) is an arthropod-borne virus (arbovirus) primarily maintained in an enzootic cycle between Culiseta melanura (Coquillett) and passerine birds. EEEV, which has the highest reported case- fatality rate among arbovirus in the Americas, is responsible for sporadic outbreaks in the Eastern and Midwest United States. Infection is associated with severe neurologic disease and mortality in horses, humans, and other vertebrate hosts. Here, we review what is known about EEEV taxonomy, functional genomics, and evolution, and identify gaps in knowledge regarding the role of EEEV genetic diversity in transmission and disease.

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.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

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.

Transmission of Eastern Equine Encephalitis Virus From an Organ Donor to 3 Transplant Recipients.

BACKGROUND: In fall 2017, 3 solid organ transplant (SOT) recipients from a common donor developed encephalitis within 1 week of transplantation, prompting suspicion of transplant-transmitted infection. Eastern equine encephalitis virus (EEEV) infection was identified during testing of endomyocardial tissue from the heart recipient. METHODS: We reviewed medical records of the organ donor and transplant recipients and tested serum, whole blood, cerebrospinal fluid, and tissue from the donor and recipients for evidence of EEEV infection by multiple assays. We investigated blood transfusion as a possible source of organ donor infection by testing remaining components and serum specimens from blood donors. We reviewed data from the pretransplant organ donor evaluation and local EEEV surveillance. RESULTS: We found laboratory evidence of recent EEEV infection in all organ recipients and the common donor. Serum collected from the organ donor upon hospital admission tested negative, but subsequent samples obtained prior to organ recovery were positive for EEEV RNA. There was no evidence of EEEV infection among donors of the 8 blood products transfused into the organ donor or in products derived from these donations. Veterinary and mosquito surveillance showed recent EEEV activity in counties nearby the organ donor's county of residence. Neuroinvasive EEEV infection directly contributed to the death of 1 organ recipient and likely contributed to death in another. CONCLUSIONS: Our investigation demonstrated EEEV transmission through SOT. Mosquito-borne transmission of EEEV to the organ donor was the likely source of infection. Clinicians should be aware of EEEV as a cause of transplant-associated encephalitis.

Using Bloodmeal Analysis to Assess Disease Risk to Wildlife at the New Northern Limit of a Mosquito Species.

The historically southeastern mosquito species Culex erraticus has over the last 30 years undergone a marked expansion north. We evaluated this species' potential to participate in local disease cycles in the northeastern USA by identifying the vertebrate sources of blood in Cx. erraticus specimens from New Jersey. We found that the majority of bloodmeals (92.6%) were derived from birds, followed by 6.8% from mammals (of which half were human), and a single amphibian bloodmeal from a spring peeper (0.56%). Medium- and large-sized water birds from the order Pelecaniformes made up 60.4% of the bird species and 55.9% of all identified hosts. This group of birds is known enzootic hosts of arboviruses such as eastern equine encephalitis virus, for which Cx. erraticus is a competent vector. Additionally, we screened blooded mosquitoes for avian malaria parasites and identified three different lineages of Plasmodium, including what may represent a new Plasmodium species (likely a wetland bird specialist) in bloodmeals from Green Herons, a Great Egret, and a Double-Crested Cormorant. Our results support the utility of mosquito bloodmeals as sources of information about circulating wildlife pathogens and reveal the potential of range-expanding species to intensify local zoonoses and bridge enzootic pathogens to humans.

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

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

Validation of a Risk Index Model for Predicting Eastern Equine Encephalitis Virus Transmission to Horses in Florida.

Eastern Equine Encephalitis Virus (EEEV) is the most pathogenic arbovirus endemic to the United States. EEEV primarily infects birds but can be fatal to humans, horses, and some other mammals. Although EEEV transmission occurs in the Northeastern, Southeastern, and Midwestern United States, the largest number of horse and human cases have been reported in Florida, the only state where transmission occurs year round. Currently, a GIS-based risk index (RI) model is used to map EEE transmission risk to horses in Florida. This study validates that RI model using a 5-yr dataset of horse cases in Florida. RI values were similar between summer (N = 152, x¯ = 0.59) and winter (N = 25, x¯ = 0.66) cases, suggesting the model is effective for mapping risk during both transmission seasons. These risk values were larger and remained similar when a 100-m buffer was applied to the case locations to account for modest spatial errors in case reporting (summer x¯ = 0.73, winter x¯ = 0.77). In both comparisons, RI values for summer and winter cases were higher than expected at random in the Panhandle, North, and Central regions of the state, although the analysis was inconclusive in the South, where only two cases were observed. This suggests the RI map could be used to target EEEV surveillance, prevention, and control efforts in both transmission seasons in Florida.

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.

Enzootic mosquito vector species at equine encephalitis transmission foci in the República de Panamá.

The identification of mosquito vector species present at arboviral enzootic transmission foci is important to understand transmission eco-epidemiology and to propose and implement prevention and control strategies that reduce vector-borne equine encephalitis transmission. The goal of this study was to identify mosquito species potentially involved in the transmission of enzootic equine encephalitis, in relation to their abundance and diversity at three endemic regions in the República de Panamá. We sampled adult mosquitoes during the dry and rainy season of Panamá. We employed CDC light traps with octanol, EV traps with CO2 and Trinidad 17 traps baited with live hamsters. Traps were deployed in the peridomicile and extradomicile of houses from 18:00 to 6:00 h. We estimated the abundance and diversity of sampled species. We collected a total of 4868 mosquitoes, belonging to 45 species and 11 genera, over 216 sampling nights. Culex (Melanoconion) pedroi, a major Venezuelan equine encephalitis vector was relatively rare (< 2.0% of all sampled mosquitoes). We also found Cx. (Mel) adamesi, Cx. (Mel) crybda, Cx. (Mel) ocossa, Cx. (Mel) spissipes, Cx. (Mel) taeniopus, Cx. (Mel) vomerifer, Aedes scapularis, Ae. angustivittatus, Coquillettidia venezuelensis, Cx. nigripalpus, Cx. declarator, Mansonia titillans, M. pseudotitillans and Psorophora ferox all species known to be vectorially competent for the transmission of arboviruses. Abundance and diversity of mosquitoes in the sampled locations was high, when compared with similar surveys in temperate areas. Information from previous reports about vectorial competence / capacity of the sampled mosquito species suggest that sampled locations have all the elements to support enzootic outbreaks of Venezuelan and Eastern equine encephalitides.

Insights into the recent emergence and expansion of eastern equine encephalitis virus in a new focus in the Northern New England USA.

BACKGROUND: Eastern equine encephalomyelitis virus (EEEV) causes a highly pathogenic zoonosis that circulates in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine birds in freshwater hardwood swamps in the northeastern U.S. Epidemic/epizootic transmission to humans/equines typically occurs towards the end of the transmission season and is generally assumed to be mediated by locally abundant and contiguous mammalophagic "bridge vector" mosquitoes. METHODS: Engorged mosquitoes were collected using CDC light, resting box, and gravid traps during epidemic transmission of EEEV in 2012 in Addison and Rutland counties, Vermont. Mosquitoes were identified to species and blood meal analysis performed by sequencing mitochondrial cytochrome b gene polymerase chain reaction products. Infection status with EEEV in mosquitoes was determined using cell culture and RT-PCR assays, and all viral isolates were sequenced and compared to other EEEV strains by phylogenetic analysis. RESULTS: The host choices of 574 engorged mosquitoes were as follows: Cs. melanura (n = 331, 94.3 % avian-derived, 5.7 % mammalian-derived); Anopheles quadrimaculatus (n = 164, 3.0 % avian, 97.0 % mammalian); An. punctipennis (n = 56, 7.2 % avian, 92.8 % mammalian), Aedes vexans (n = 9, 22.2 % avian, 77.8 % mammalian); Culex pipiens s.l. n = 6, 100 % avian); Coquillettidia perturbans (n = 4, 25.0 % avian, 75.0 % mammalian); and Cs. morsitans (n = 4, 100 % avian). A seasonal shift in blood feeding by Cs. melanura from Green Heron towards other avian species was observed. EEEV was successfully isolated from blood-fed Cs. melanura and analyzed by phylogenetic analysis. Vermont strains from 2012 clustered with viral strains previously isolated in Virginia yet were genetically distinct from an earlier EEEV isolate from Vermont during 2011. CONCLUSIONS: Culiseta melanura acquired blood meals primarily from birds and focused feeding activity on several competent species capable of supporting EEEV transmission. Culiseta melanura also occasionally obtained blood meals from mammalian hosts including humans. This mosquito species serves as the primary vector of EEEV among wild bird species, but also is capable of occasionally contributing to epidemic/epizootic transmission of EEEV to humans/equines. Other mosquito species including Cq. perturbans that feed more opportunistically on both avian and mammalian hosts may be important in epidemic/epizootic transmission under certain conditions. Phylogenetic analyses suggest that EEEV was independently introduced into Vermont on at least two separate occasions.

Ecology of Culiseta Melanura and Other Mosquitoes (Diptera: Culicidae) from Walton County, FL, During Winter Period 2013-2014.

Winter ecology of putative vectors of eastern equine encephalomyelitis virus (EEEV) in northern Florida was investigated at field locations with evidence of historic EEEV winter transmission. Light traps and resting shelters were used to sample the mosquito community in the vicinity of eight sentinel flocks throughout the winter period (November-April) of 2013 and 2014 in Walton County, FL. Overall mosquito activity was relatively low, although mosquitoes were captured during each week of the study period. Mosquito activity was linked to morning temperature, and females were captured when ambient morning temperatures were quite low (1-5°C). Anopheles crucians Wiedemann, Culex erraticus (Dyar and Knab), Culex territans Walker, and Culiseta melanura (Coquillett) were the most commonly collected mosquito species (of 20 total species). Analysis of blood-engorged mosquitoes revealed a number of mosquito species feeding upon chickens, other birds, amphibians, and domestic and wild mammals. Cs. melanura fed primarily upon chickens and songbirds (Passeriformes), suggesting that this mosquito species is the likely winter vector of EEEV to sentinel chickens in northern Florida. Both resident and nonresident songbird species were fed upon, constituting 63.9 and 36.1% of total songbird meals, respectively. Our results suggest important roles for Cs. melanura and songbird hosts for the winter transmission of EEEV in northern Florida.

Arboviruses in North Dakota, 2003-2006.

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.

[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.

Field investigations of winter transmission of eastern equine encephalitis virus in Florida.

Studies investigating winter transmission of Eastern equine encephalitis virus (EEEV) were conducted in Hillsborough County, Florida. The virus was detected in Culiseta melanura and Anopheles quadrimaculatus in February 2012 and 2013, respectively. During the winter months, herons were the most important avian hosts for all mosquito species encountered. In collections carried out in the summer of 2011, blood meals taken from herons were still common, but less frequently encountered than in winter, with an increased frequency of mammalian- and reptile-derived meals observed in the summer. Four wading bird species (Black-crowned Night Heron [Nycticorax nycticorax], Yellow-crowned Night Heron [Nyctanassa violacea], Anhinga [Anhinga anhinga], and Great Blue Heron [Ardea herodias]) were most frequently fed upon by Cs. melanura and Culex erraticus, suggesting that these species may participate in maintaining EEEV during the winter in Florida.

Las encefalitis esquinas: Una Revisión / The equine encephalitis: a review

El grupo de las encefalitis Equina Venezolana, del Este, del Oeste y del Nilo Occidental (EEV, EEE, EEO y ENO respectivamente) son zoonosis transmitidas por mosquitos a humanos y equinos. Este grupo de enfermedades se mantienen en la naturaleza en ciclos enzoóticos de transmisión entre mosquitos y roedores silvestres o aves que son sus hospedadores naturales. La circulación de estos virus es enzoótica y ocurre en áreas geográficas definidas y tienen capacidad de causar epidemias/epizootias con morbilidad y mortalidad que puede ser de importancia significativa. Este artículo describe detalladamente todas las encefalitis virales que afectan a los équidos y sus aspectos clínicos, patológicos y diagnósticos.

The encephalitis of Venezuelan Equine, Eastern, Western and West Nile (VEE, EEE, WEE and ENO respectively) are zoonotic encephalitis transmitted by mosquitoes to humans and horses. This group of diseases is maintained in nature in cycle’s enzootic transmission between mosquitoes and wild rodents or birds are their natural hosts. The circulation of this virus is enzootic and occurs in defined geographical areas but is capable of causing epidemics / epizootics with morbidity and mortality can be significant importance. This article describes all viral encephalitis affecting equines and their clinical, pathological and diagnostic aspects.