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.

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.

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.

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.

First Detection of Madariaga virus in Mosquitoes Collected in a Wild Environment of Northeastern Argentina.

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.

Emergence of Madariaga virus as a cause of acute febrile illness in children, Haiti, 2015-2016.

Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America.

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.

Eastern equine encephalitis in puppies in Michigan and New York states.

Eastern equine encephalitis virus (EEEV) is classified as a select agent and is capable of causing mortality in humans and a number of veterinary species. Herein, we describe 3 cases of EEE in puppies in Michigan and New York. Two puppies were euthanized following an acute history of seizures and obtundation. A littermate of one of these puppies died 2 wk earlier following a history of anorexia and fever. All 3 puppies lacked significant gross anatomic lesions at autopsy and tested negative for rabies virus. In all 3 puppies, histologic examination revealed necrotizing, neutrophilic, lymphoplasmacytic meningoencephalitis with strong positive immunohistochemical labeling for EEEV antigen in neurons and glial cells. The diagnosis of EEE was confirmed by PCR in one puppy and by plaque reduction neutralization testing in the other 2 dogs. EEE is rare in dogs, and has only been reported in puppies. The initial clinical signs of EEE in puppies are typically nonspecific, including anorexia, fever, and diarrhea, but rapidly progress to severe neurologic disease characterized by seizures and recumbency. Although rare, EEE should be considered as a differential diagnosis for neurologic disease in puppies, especially after more common diseases, such as canine distemper, rabies, and toxoplasmosis have been ruled out.

Evaluation of Partially Submerged Sticky Traps On Lake Spillways For Adult Black Fly (Diptera: Simuliidae) Surveillance and Arbovirus Detection.

Sentinel surveillance systems demonstrate an improved ability to supplement monitoring data and anticipate arbovirus outbreaks (i.e., sentinel avian species). Management complications can arise during unpredictable or unseasonal disease detections, especially in rural areas where resident distribution is patchy. Using spillways near residential lake communities as static surveillance locations, we tested a novel partially submerged sticky trapping technique and screened wild populations of adult female black flies (Diptera: Simuliidae) for West Nile virus (WNV) and eastern equine encephalitis virus (EEEV). Trap site selection criteria considered the density of immature black fly colonization on spillway surfaces and the number of positive detections of arboviral targets in nearby Culex mosquito populations. On average (±standard error), sticky traps captured 134 (±33) adult black flies over a 24-h period, with 1 trap capturing as many as 735 individuals. Although we detected positive cases of WNV from 20 Culex mosquito trapping sites within 16 km (approx. flight radius) of the selected lake spillways, mixed pools of adult female Simulium vittatum complex and Simulium decorum were all negative for both arboviruses. This study yielded an application for partially submerged sticky traps to collect adult female black flies. Its potential uses for monitoring the infection rates of more well-documented Simulium parasites are discussed.

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

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

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.

Isolation and Characterization of Madariaga Virus from a Horse in Paraíba State, Brazil.

Madariaga virus (MADV), the new species designation for the South American isolates of eastern equine encephalitis virus (EEEV), is genetically divergent and substantially different in ecology and pathogenesis from North American EEEV strains. We isolated and characterized a MADV isolate obtained from a horse in Brazil. Our results support previous phylogenetic studies showing there are three genetically distinct MADV lineages. The MADV isolate from Paraíba State belongs to the South American lineage III and is closely related to Peruvian, Colombian and Venezuelan isolates.

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

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

Evaluation of the Honey-Card Technique for Detection of Transmission of Arboviruses in Florida and Comparison With Sentinel Chicken Seroconversion.

Zoonotic mosquito-borne viruses, such as the West Nile virus (WNV) and eastern equine encephalitis virus (EEEV), are major public health threats in the United States. Early detection of virus transmission and targeted vector management are critical to protect humans against these pathogens. Sentinel chickens and pool screening of mosquitoes, the most widely used methods of arbovirus early detection, have technical time-lags that compromise their early-detection value. The exploitation of sugar-feeding by trapped mosquitoes for arbovirus surveillance may represent a viable alternative to other methods. Here we compared effectiveness of sugar-impregnated nucleic-acid preserving substrates (SIPS) and sentinel chicken program for detecting WNV, EEEV, and St. Louis encephalitis virus in gravid traps, CO2-baited light traps, and resting traps at 10 locations in two Florida counties. In St. Johns County, comparable numbers of EEEV detections were made by SIPS traps (18) and sentinel chickens (22), but fewer WNV detections were made using SIPS (1) than sentinel chickens (13). In Volusia County, seven arbovirus detections were made via the sentinel chicken program (one EEEV and six WNV), whereas only one arbovirus detection (WNV) was made using SIPS. CO2-baited light traps captured >90% of total mosquitoes, yet yielded <30% of arbovirus detections. Resting traps and gravid traps captured a fraction of total mosquitoes, yet yielded roughly equivalent numbers of arbovirus detections, as did light traps. Challenges to successful deployment of SIPS include optimization of traps for collecting all vector species, increasing sugar-feeding rates of trapped vectors, and developing tractable methods for arbovirus detection.

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.

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

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

MRI findings in eastern equine encephalitis: the "parenthesis" sign.

Two patients with eastern equine encephalitis (EEE) presented to a tertiary referral center. Both subjects' brain magnetic resonance imaging showed T2/FLAIR (fluid-attenuated inversion recovery) hyperintensities including linear areas of hyperintensity in the external and internal capsules with sparing of the lentiform nuclei. Single case reports of imaging findings in EEE exist with nonspecific patterns of abnormality. We propose that this "( ) parentheses sign" on T2 or FLAIR imaging may distinguish EEE from other processes.

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

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