Development of high-throughput and high sensitivity capillary gel electrophoresis platform method for Western, Eastern, and Venezuelan equine encephalitis (WEVEE) virus like particles (VLPs) purity determination and characterization.

In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 µg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 µg/mL for VEE and 20-250 µg/mL for EEE and WEE VLPs.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Mosquitoes in Moose Country: A Mosquito Survey of Northern Minnesota.

An adult mosquito survey was conducted at 12 sites using carbon dioxide traps in northern Minnesota throughout the summer of 2012. Specimens were counted, identified to species, sorted into pools, and tested for eastern equine encephalitis (EEEV) and West Nile virus (WNV). Our findings extend the known range of Culiseta melanura, Anopheles barberi, and An. quadrimaculatus and document the presence and abundance of 27 other mosquito taxa in the region. None of the pools tested positive for EEEV or WNV.

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.

Enhancing Eastern Equine Encephalitis Virus Surveillance in New Jersey: Optimized Collection of Culiseta melanura.

Eastern equine encephalitis virus (EEEV) causes the most clinically severe neuroinvasive arboviral disease in the United States. The virus is endemic in eastern and Gulf Coast states and the Great Lakes region, causing cases annually. To detect EEEV circulation in its enzootic cycle before the virus infects humans and other mammals, mosquito control agencies in New Jersey have conducted mosquito surveillance using a series of permanent wooden resting box sites since 1975. We conducted 2 field studies, 1 evaluating resting traps and 1 evaluating efficacy of CO2 lures, to optimize collection of Culiseta melanura, the primary enzootic vector of EEEV. Resulting mosquito samples were subjected to molecular analysis to determine EEEV infection rates. Corrugated plastic boxes trapped more bloodfed Cs. melanura than other resting trap types (resting boxes, Centers for Disease Control and Prevention [CDC] resting traps, or fiber pots) and were similar to resting boxes in total number of female Cs. melanura caught. Further, non-baited CDC light traps were more successful in trapping host-seeking Cs. melanura than those baited with dry ice, a CO2 lure. The EEEV RNA was identified in Cs. melanura, Aedes vexans, Anopheles quadrimaculatus, and Uranotaenia sapphirina. Our findings indicate that corrugated plastic boxes and non-CO2 baited traps could improve detection of Cs. melanura. Mosquito control agencies are encouraged to periodically assess their surveillance strategy for EEEV.

Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

Leukocyte response to eastern equine encephalomyelitis virus in a wild passerine bird.

Leukocyte counts are frequently used to assess the immunologic status of animals; however, few studies have directly looked at the predictive value of leukocyte counts and an animal's ability to respond to an infection with a pathogen. Understanding how an animal's leukocyte profile is altered by an active infection can assist with interpretation of leukocyte profiles in animals for which infection status is not known. In this study we examine the leukocyte counts of gray catbirds (Dumetella carolinensis) infected with eastern equine encephalomyelitis virus (EEEV). Blood smears were collected from infected catbirds on -4, 2, 5, and 14 days postinoculation (dpi) with EEEV, and from a corresponding uninfected control group, to monitor leukocyte counts. Although we found that preinfection leukocyte counts were not a reliable predictive of a catbird's viremia, we did find that infected catbirds exhibited significant hematologic changes in response to EEEV infection. We observed a significant drop in all subpopulations of leukocytes (i.e., lymphocytes, monocytes, and granulocytes) following infection. Lymphocytes and granulocytes still had not recovered to preinfection levels at 14 dpi. Uninfected catbirds also exhibited statistically significant changes in leukocyte counts, but this was due to a slight increase at 14 dpi and was not considered biologically relevant. Studies such as this can provide important information for field ecoimmunologists that use leukocyte counts to assess immunocompetence in free-living animals.

Development of an ELISA-array for simultaneous detection of five encephalitis viruses.

Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.

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.

Alphavirus Identification in Neotropical Bats.

Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.

Test of recrudescence hypothesis for overwintering of eastern equine encephalomyelitis virus in gray catbirds.

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) epizootics are infrequent, but they can lead to high mortality in infected horses and humans. Despite the importance of EEEV to human and animal health, little is known about how the virus overwinters and reinitiates transmission each spring, particularly in temperate regions where infected adult mosquitoes are unlikely to survive through the winter. One hypothesis to explain the mechanism by which this virus persists from year to year is the spring recrudescence of latent virus in avian reservoir hosts. In this study, we tested the recrudescence hypothesis with gray catbirds (Dumatella carolinensis) captured in northern Ohio (July-August 2007). Birds were experimentally infected with EEEV on 1 October 2007. In January 2008, they were then exposed to exogenous testosterone and/or extended photoperiod to initiate reactivation of latent EEEV infection. All birds became viremic with EEEV, with mean viremia of 6.0 log10 plaque-forming units/ml serum occurring at 1 d postinoculation. One male in the testosterone, long-day treatment group had EEEV viral RNA in a cloacal swab collected on 18 January 2008. Otherwise, no other catbirds exhibited reactivated infections in cloacal swabs or blood. Antibody titers fluctuated over the course of the study, with lowest titers observed in January 2008, which corresponded with the lowest mean weight of the birds. No EEEV viral RNA was detected in the blood, kidney, spleen, brain, liver, and lower intestine upon necropsy at 19 wk postinfection.

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 in mosquitoes and their role as bridge vectors.

Eastern equine encephalitis virus (EEEV) is maintained in an enzootic cycle involving Culiseta melanura mosquitoes and avian hosts. Other mosquito species that feed opportunistically on mammals have been incriminated as bridge vectors to humans and horses. To evaluate the capacity of these mosquitoes to acquire, replicate, and potentially transmit EEEV, we estimated the infection prevalence and virus titers in mosquitoes collected in Connecticut, USA, by cell culture, plaque titration, and quantitative reverse transcription-PCR. Cs. melanura mosquitoes were the predominant source of EEEV (83 [68%] of 122 virus isolations) and the only species to support consistently high virus titers required for efficient transmission. Our findings suggest that Cs. melanura mosquitoes are primary enzootic and epidemic vectors of EEEV in this region, which may explain the relative paucity of human cases. This study emphasizes the need for evaluating virus titers from field-collected mosquitoes to help assess their role as vectors.

Cotton rats and house sparrows as hosts for North and South American strains of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is an arbovirus that causes severe disease in humans in North America and in equids throughout the Americas. The enzootic transmission cycle of EEEV in North America involves passerine birds and the ornithophilic mosquito vector, Culiseta melanura, in freshwater swamp habitats. However, the ecology of EEEV in South America is not well understood. Culex (Melanoconion) spp. mosquitoes are considered the principal vectors in Central and South America; however, a primary vertebrate host for EEEV in South America has not yet been identified. Therefore, to further assess the reservoir host potential of wild rodents and wild birds, we compared the infection dynamics of North American and South American EEEV in cotton rats (Sigmodon hispidus) and house sparrows (Passer domesticus). Our findings suggested that each species has the potential to serve as amplification hosts for North and South America EEEVs.

A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses.

In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.

Spatial and temporal expansions of Eastern equine encephalitis virus and phylogenetic groups isolated from mosquitoes and mammalian cases in New York State from 2013 to 2019.

Surveillance for the emerging infectious disease Eastern equine encephalitis, and its causative virus in mosquitoes, continued within New York State from 2013 to 2019. There were increases in geographic area and number of consecutive years, with cases in four mammalian species, and virus in 11 mosquito species. The first cases in a goat and in an emu were reported. The first detection of virus in Aedes cinereus was reported. Virus in phylogenetic group NY4 was isolated from a horse and from mosquitoes 6 kilometers and 13 days apart in 2013. Phylogenetic groups NY4 and NY5 were found 15 days apart in two towns 280 kilometers distant in 2013. Within four adjacent counties there was a pattern of overlap, where four had NY5, two adjacent counties had NY6, two adjacent counties had NY7, and one county had NY5, NY6, and NY7, reducible to a Euler diagram. Virus in phylogenetic group NY5, found within an 11-kilometer wide area in New York State, was related to FL4 found in Florida 1,398 kilometers distant. This was consistent with a phylogenetic group originating in Florida, then being moved to a specific location in New York State, by migratory birds in consecutive years 2013 and 2014.

Case report: Eastern equine encephalitis virus imported to the UK.

Eastern equine encephalitis (EEE) is rare, but the most severe of the mosquito-borne encephalitides in the United States with a high case fatality rate of 30%. Here, we present a patient with EEE. EEE virus causes sporadic human disease in the Eastern parts of the United States, but the case we describe was a Scottish tourist who acquired the disease from mosquito bites while in holiday in the United States. This is a first report of an imported case to Europe.

Eastern equine encephalitis virus infection and hemophagocytic lymphohistiocytosis in a 5-month-old infant.

Eastern equine encephalitis virus infection is a rare sporadic central nervous system infection transmitted by a mosquito vector. Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease associated with the inability of an overactive immune system to effectively respond to infections. Many viruses are known to trigger primary, as well as secondary, HLH. We report a pediatric case of eastern equine encephalitis virus-associated HLH which caused severe neurologic injury and death.

Isolation of eastern equine encephalitis virus from Aedes albopictus in Florida.

Fourteen strains of eastern equine encephalitis (EEE) virus were isolated from Aedes albopictus mosquitoes collected in Polk County, Florida. These are the first isolations of an arbovirus of proven public health and veterinary importance from naturally infected Ae. albopictus in the United States since established populations of this introduced mosquito were first discovered in 1985. The widespread distribution of Ae. albopictus in Florida and in other areas of the United States where EEE is endemic raises concern that this species may become an epizootic and epidemic vector of EEE virus.