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.

Evaluation of a Methoprene Aerial Application for the Control of Culiseta melanura (Diptera: Culicidae) in Wetland Larval Habitats

Eastern equine encephalitis virus (EEEV) is an arbovirus endemic to the eastern United States. Human cases are rare but can be serious. The primary enzootic vector is Culiseta melanura (Coquillett) (Diptera: Culicidae), an ornithophagic mosquito. We conducted an aerial application of a granular methoprene formulation in Hockomock Swamp (Massachusetts), which represents a focus of EEEV transmission. Water collected from inside and outside Cs. melanura crypts was evaluated in bioassays of early fourth instar Cs. melanura larvae using treated and untreated water. Adult eclosion rates were 36% significantly lower in treated compared with untreated water (P < 0.05). Eclosion rates for water collected from inside crypts were significantly higher (62%) than rates from outside crypts (30%) (P < 0.05), indicating higher efficacy outside crypts. We tested whether reduced methoprene efficacy inside the crypts was due to reduced chemical penetration into this habitat. Chemical water analyses confirmed that methoprene concentrations were lower inside the crypts (0.1 ± 0.05 ppb) compared to water from outside crypts (1.79 ± 0.41 ppb). The susceptibility of Cs. melanura to methoprene was also determined to allow for comparison against concentrations observed in water collected from the field (LC-95: 1.95 ± 0.5 ppb). Overall, methoprene-treated water prevented mosquito development for up to 4 wk, but with a reduction in efficacy between 4- and 6-wk post-application. Our results suggest that aerial methoprene applications can effectively treat open water in wetlands but may not provide efficacious control of Cs. melanura due to an inability to penetrate larval habitats.

Distribución potencial de Culiseta melanura (Coquillett) (Diptera: Culicidae) en América

Culiseta melanura es un mosquito importante debido a su papel en el ciclo de transmisión del virus de la Encefalitis Equina del Este. Se distribuye desde el sur de Quebec a través de Maine y el área de los Grandes Lagos hasta el sur de Florida y el Este de Texas, y en el noreste de México. En este estudio determinamos la distribución potencial de Cs. melanura en el continente americano y la importancia de las variables bioclimáticas en la conformación de dicha distribución. Para representar la distribución potencial de Cs. melanura se utilizó un modelo de máxima entropía (MaxEnt, versión 3.4.1). Se utilizó un total de 80 registros geográficos obtenidos de GBIF y literatura pertinente. Los datos de clima se obtuvieron de 19 capas climáticas disponibles en la base de datos WorldClim versión 2.0. El área bajo la curva del modelo generado tuvo un valor de 0.98, lo cual indica que es un modelo con buena confiabilidad. Los resultados mostraron áreas de idoneidad de hábitat en la costa sur de Alaska, costa este y oeste de Canadá, centro de Estados Unidos, Colombia, Ecuador, Perú, sur de Chile, sur de Brasil, costa este de Argentina y Uruguay. Esta información puede ser útil en los programas de vigilancia entomológica para el monitoreo de Cs. melanura en países como Estados Unidos y Canadá donde actualmente esta especie está presente.

Field Analysis of Biological Factors Associated With Sites at High and Low to Moderate Risk for Eastern Equine Encephalitis Virus Winter Activity in Florida

Eastern equine encephalitis virus (EEEV) is the most pathogenic arbovirus endemic to the United States. Studies have demonstrated Florida’s role as a regional reservoir for the virus and its ability to support year-round transmission. Previous research has developed risk index models for mapping locations most at risk for EEEV transmission. We compared vector abundance, vector feeding behavior, potential host species, and fauna presence at high versus low–moderate risk sites during the winter and spring. Predicted high-risk sites had a significantly greater abundance of mosquitoes overall, including Culiseta melanura (Coquillett) (Diptera: Culicidae), the primary enzootic vector of EEEV. Twenty host species were identified from Cs. melanura bloodmeals, with the majority taken from avian species. Culiseta melanura largely fed upon the Northern Cardinal (Cardinalis cardinalis (Passeriformes: Cardinalidae)), which accounted for 20–24.4% of the bloodmeals obtained from this species in years 1 and 2, respectively. One EEEV-positive mosquito pool (Cs. melanura) and nine EEEV seropositive sentinel chickens were confirmed during winter-spring collections from high-risk sites; no seropositive chickens nor mosquito pools were found at the low–moderate risk sites. These results suggest that high-risk sites for EEEV activity are characterized by habitats that support populations of Cs. melanura and which may also provide ample opportunities to feed upon Northern Cardinals. The overall low level of mosquito populations during the winter also suggests that control of Cs. melanura populations in winter at high-risk sites may prove effective in reducing EEEV transmission during the peak summer season.

Collection and DNA Detection of Dirofilaria immitis (Rhabditida Onchocercidae), Using a Novel Primer Set, in Wild-Caught Mosquitoes From Gainesville, FL

Dirofilaria immitis, the causative agent of dog heartworm disease, is an important cause of canine morbidity and mortality, expensive to treat, and severe infections are often fatal. Much is known about the pathogen in the canine host, yet little is known on the basic ecology of the nematode in the mosquito vector. Thus, to evaluate the effectiveness of collection techniques on ability to capture dog heartworm-infected mosquitoes (Diptera Culicidae), we conducted a field study spanning 111 wk. Four methods were used: two aspirators types, sweep netting, and a CDC trap. All sites had canines present in either residential yards (n = 4) or dog kennel facilities (n = 3). Collected mosquitoes were sorted by site, trap, species, and date, then pooled into groups of up to 25 individuals. Mosquito head and thorax pools were extracted for DNA, that was screened using currently available protocols. These protocols were found unreliable; thus, we developed a novel qPCR primer and probe set. Using this method, the original samples were re-assayed and provided 494 positive pools. Approximately 10% of positive samples were confirmed by Sanger sequencing. Twenty-two mosquito species tested positive for dog heartworm DNA, including a new association with Wyeomyia mitchellii (Theobald). Although Aedes atlanticus (Dyar and Knab), Anopheles crucians Wiedemann, and Culiseta melanura (Coquillett) composed nearly 36% of the total collection, these species represented 42% of the qPCR positive pools. Infection rates within commonly collected mosquitoes ranged up to 2.5%, with more rarely collected species ranging up to 14%. The CDC trap was the most effective collection method at trapping infected mosquitoes.

Seasonal Changes of Host Use by Culiseta melanura (Diptera: Culicidae) in Central Florida

The mosquito Culiseta melanura (Coquillett) is the primary enzootic vector of eastern equine encephalitis virus (EEEV), a zoonotic Alphavirus endemic to eastern North America. In its northern range, Cs. melanura is considered a strict avian biter, transmitting EEEV among susceptible birds in a cycle of enzootic amplification. In its southern range, however, Cs. melanura is more general in host use, feeding heavily upon birds but also reptiles and mammals. The goal of this study was to better understand how host use of Cs. melanura changes throughout the year in Florida, where year-round EEEV transmission is observed. Mosquitoes were sampled in 2018 from nine sites across three central Florida counties. In total, 213 Cs. melanura bloodmeals were identified by PCR consisting of 39 species of birds, reptiles, and mammals. Avian bloodmeals were prominent throughout the year (range = 30–85%), and songbirds were a large portion of identified bloodmeals (37.1%). Reptiles surpassed birds only in spring (April–June), and brown anole (Anolis sagrei Duméril and Bibron, 1837 [Reptilia: Dactyloidae]) was the most commonly detected single host species (22.1% overall). Mammalian bloodmeals were mainly observed in summer, with humans being the most fed on mammal (12.7% overall). This study reveals that in southern foci of EEEV transmission, Cs. melanura host use varies throughout the year with reptiles providing the majority of blood meals in spring (51.3%), and birds are fed on more than other host groups during all other seasons (50.6–70.1%). In addition, feeding on mammals increases during summer months, which may implicate Cs. melanura in epizootic transmission in Florida.

Cases of Eastern equine encephalitis in humans associated with Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes with the virus in New York State from 1971 to 2012 by analysis of aggregated published data

From 1971 to 2012, in New York State, years with human Eastern equine encephalitis (EEE) were more strongly associated with the presence of Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes infected with the EEE virus (Fisher's exact test, one-sided P = 0.005, 0.03, 0.03) than with Culiseta morsitans, Aedes vexans, Culex pipiens-restuans, Anopheles quadrimaculatus or Anopheles punctipennis (P = 0.05, 0.40, 0.33, 1.00, 1.00). The estimated relative risk of a case in a year in which the virus was detected vs. not detected was 14.67 for Ae. canadensis, 6.38 for Cq. perturbans and 5.50 for Cs. morsitans. In all 5 years with a case, Cs. melanura with the virus was detected. In no year was there a case in the absence of Cs. melanura with the virus. There were 18 years with no case in the presence of Cs. melanura with the virus. Such observations may identify the time of increased risk, and when the methods may be used to prevent or reduce exposure to vector mosquito species in this geographic region.