Equine Encephalitis Virus Research Articles

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.

Safety and immunogenicity of an inactivated eastern equine encephalitis virus vaccine

BackgroundEastern equine encephalitis virus (EEEV) is a mosquito borne alphavirus spread primarily in Atlantic and Gulf Coast regions of the United States. EEEV is the causative agent of a devastating meningoencephalitis syndrome, with approximately 30% mortality and significant morbidity. There is no licensed human vaccine against EEEV. An inactivated EEEV vaccine has been offered under investigational new drug (IND) protocols at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. MethodsHealthy at-risk laboratory personnel received inactivated PE-6 strain EEEV (TSI-GSD 104) vaccine under two separate IND protocols. Protocol FY 99–11 (2002–2008) had a primary series consisting of doses on day 0, 7, and 28. Protocol FY 06–31 (2008–2016) utilized a primary series with doses on day 0 and 28, and month 6. Participants with an inadequate immune response, plaque reduction neutralization test with 80% cut-off (PRNT80) titer < 40, received booster vaccination. Volunteers with prior EEEV vaccination were eligible to enroll for booster doses based on annual titer evaluation. ResultsThe FY06-31 dosing schema resulted in significantly greater post-primary series immune response (PRNT80 ≥ 40) rates (84% vs 54%) and geometric mean titers (184.1 vs 39.4). The FY 06–31 dosing schema also resulted in significantly greater cumulative annual immune response rates from 1 to up to 7 years post vaccination (75% vs 59%) and geometric mean of titers (60.1 vs 43.0). The majority of probably or definitely related adverse events were mild and local; there were no probably or definitely related serious adverse events. ConclusionsInactivated PE-6 EEEV vaccine is safe and immunogenic in at-risk laboratory personnel. A prolonged primary series, with month 6 dose, significantly improved vaccine immunogenicity both post-primary series and longitudinally on annual titers. Despite decades of safe use under IND, full licensure is not planned due to manufacturing constraints, and ongoing development of alternatives.

A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge.

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.

49. Clinical Characteristics of the 2019 Eastern Equine Encephalitis Virus Outbreak in Michigan

BackgroundEastern Equine Encephalitis Virus (EEEV) is a mosquito-borne alphavirus responsible for unpredictable outbreaks of severe neurologic disease in humans. While the vast majority of human EEEV infections are either asymptomatic or clinically nonspecific, a minority of patients develops neuroinvasive disease (EEE), which is a devastating illness with a mortality of at least 30%. No treatments are known to be effective. EEEV infection is relatively rare in the United States, with an annual average nationwide incidence of 7 cases between 2009 and 2018. However, 2019 was an exceptionally active year for human EEEV disease, yielding 38 nationwide confirmed cases, including 10 in Michigan, comprising the state’s largest outbreak to date.MethodsEEE cases were identified by a regional network of physicians. Cases were defined by presentation with clinical symptoms of encephalitis, and by identification of EEEV IgM antibodies or RNA in cerebrospinal fluid (CSF), or EEEV-specific IgM in serum as confirmed by plaque reduction neutralization test. Radiographic images were evaluated and clinical data abstracted through chart review and clinical follow-up where possible.ResultsRecords from 7 patients were identified and reviewed. The median age was 64, with a male predominance, and all presented in August. Notably, commercial arboviral CSF serology was uniformly negative on the initial CSF sample, and diagnosis was not made until a mean of 23 days (range: 12–38 days) after presentation. Testing in public health laboratories yielded the diagnosis in 5 out of 7 cases. Imaging findings were heterogeneous, but most patients exhibited abnormal findings in the thalamus and/or basal ganglia, and one patient displayed prominent pons and midbrain abnormalities. 4 patients died, while 2 patients survived with severe neurologic sequelae, and 1 patient recovered without sequelae. One patient underwent a limited postmortem examination, which revealed diffuse meningoencephalitis and focal vascular necrosis.ConclusionEEE is a frequently fatal condition whose diagnosis is often delayed, and for which no effective treatments are known. Improved diagnostics are needed to facilitate further clinical studies of EEE and encourage the development of potential therapies.Disclosures All Authors: No reported disclosures

Effects of seasonality and land use on the diversity, relative abundance, and distribution of mosquitoes on St. Kitts, West Indies

BackgroundMosquito surveys that collect local data on mosquito species’ abundances provide baseline data to help understand potential host-pathogen-mosquito relationships, predict disease transmission, and target mosquito control efforts.MethodsWe conducted an adult mosquito survey from November 2017 to March 2019 on St. Kitts, using Biogents Sentinel 2 traps, set monthly and run for 48-h intervals. We collected mosquitoes from a total of 30 sites distributed across agricultural, mangrove, rainforest, scrub and urban land covers. We investigated spatial variation in mosquito species richness across the island using a hierarchical Bayesian multi-species occupancy model. We developed a mixed effects negative binomial regression model to predict the effects of spatial variation in land cover, and seasonal variation in precipitation on observed counts of the most abundant mosquito species observed.ResultsThere was high variation among sites in mosquito community structure, and variation in site level richness that correlated with scrub forest, agricultural, and urban land covers. The four most abundant species were Aedes taeniorhynchus, Culex quinquefasciatus, Aedes aegpyti and Deinocerites magnus, and their relative abundance varied with season and land cover. Aedes aegypti was the most commonly occurring mosquito on the island, with a 90% probability of occurring at between 24 and 30 (median = 26) sites. Mangroves yielded the most mosquitoes, with Ae. taeniorhynchus, Cx. quinquefasciatus and De. magnus predominating. Psorophora pygmaea and Toxorhynchites guadeloupensis were only captured in scrub habitat. Capture rates in rainforests were low. Our count models also suggested the extent to which monthly average precipitation influenced counts varied according to species.ConclusionsThere is high seasonality in mosquito abundances, and land cover influences the diversity, distribution, and relative abundance of species on St. Kitts. Further, human-adapted mosquito species (e.g. Ae. aegypti and Cx. quinquefasciatus) that are known vectors for many human relevant pathogens (e.g. chikungunya, dengue and Zika viruses in the case of Ae. aegypti; West Nile, Spondweni, Oropouche virus, and equine encephalitic viruses in the case of Cx. quinqefasciatus) are the most wide-spread (across land covers) and the least responsive to seasonal variation in precipitation.

Insect-specific viruses and arboviruses in adult male culicids from Midwestern Brazil.

Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilhéus (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.

Effect of Environment and Elevation on Seasonal Prevalence of Culicoides in West Bengal

Background: Culicoides, popularly known as ‘biting midges’ is a group of vector insects causing transmission of pathogens to livestock and other closely related mammals leading to animal health deterioration. They are among the smallest and most abundant hematophagous vectors of Blue tongue virus (BTV), Epizootic haemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine encephalitis virus (EEV), protozoans, filarial nematodes throughout the world. They are known to spread livestock diseases in India in the states of Andhra Pradesh, Assam, Gujarat, Haryana, Himachal Pradesh, Jammu and Kashmir, Jharkhand, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Nagaland, Punjab, Rajasthan, Sikkim, Tamil Nadu, Tripura, Uttar Pradesh and West Bengal. 30 Culicoides vectors are reported worldwide, 13 of them from India, of which 11 are found in West Bengal. In the Indian subcontinent, biting midges live throughout the year, with two distinct peaks - one in the monsoon (August-September) and another peak during post-monsoon or beginning of winter (November) and there is a steady decrease during the summer months (May). Adults thrive best at low-temperature range (19-32oC) and rainfall plays an important role in Culicoides breeding and life-cycle. But a detailed account on the ecology, breeding sites, biology and bionomics of immature and adults have been poorly understood with very scanty literature. The present research is aimed to provide an exhaustive detail on the ecological behavior of Culicoides and their prevalence pattern based on the effect of meteorological parameter and elevation across different regions of West Bengal. Methods: Insect samples were collected using sweep nets in the early morning, afternoon and just before sunset for a period of four years from January 2014 to December 2017 in six agro-climatic regions of West Bengal. After collection, they were transferred in 70% ethanol, kept in a cool place, sorted and different parts of the specimen were mounted on glass slides in the phenol-balsam mixture and identified through Leica DFC 295 binocular light microscope. Ecological graph analysis were generated using software PAST 3.0 and MS-EXCEL 2016. Maps on sampling sites was generated using software ARC GIS 10.5). Result: Five Culicoides species-Culicoides oxystoma, C. actoni, C. peregrinus,C. orientalis and C. anophelis were collected. The results show highest prevalence in post-monsoon months at the coastal-saline region, at an of elevation 5-15m with ideal temperature of 14-35oC and rainfall of 1100-2000mm in West Bengal, with the highest dominance of Culicoides oxystoma throughout the year.

Madariaga Virus

The area affected by the disease caused by Madariaga virus has been expanding recently. Given the geographic location of the endemic regions that overlap with tourist destinations, as well as the confirmed possibility of importing the disease, we cannot rule out the risk of appearance of this disease in Russia. The aim of the study was to analyse the properties of Madariaga virus (Alphavirus genus, eastern equine encephalomyelitis virus complex) and some epidemiological and epizootological characteristics of the virus-associated disease which manifests itself in diffuse inflammation of brain and spinal cord. Based on both ecological and molecular genetic characteristics of Madariaga virus, it is classified as an individual virus which is part of the eastern equine encephalomyelitis virus complex. There is evidence that this pathogen can cause epizootic outbreaks in horses, infect other types of mammals (rats, bats), as well as, potentially, birds and reptiles. The reservoir of infection is the short-tailed cane mouse (Zygodontomys brevicauda). Human cases have also been described, and the potential route of infection was identified as transmission via mosquito bites. Vectors of pathogen transmission are mosquitoes of the Culex, Aedes, and Psorophora genera. Serological studies carried out in Panama revealed the presence of antibodies to Madariaga virus in 2–5% of the examined patients, which indicates the existence of asymptomatic infection along with clinically apparent infection. Phylogenetic analysis of strains isolated from infected people showed that the strains belong to lineage III of the eastern equine encephalomyelitis virus subtype common in Central and South America. The paper summarises the results of assessment of potential risk factors of Madariaga virus infection in endemic regions that was performed using the Akaike information criterion. Risk groups in endemic regions are represented by farm workers and fishermen. The results of the studies show that the area affected by the disease is expanding, and the strains that belong to genetic lineage III of the eastern equine encephalomyelitis virus pose the greatest epidemic risk.

Barcoding of the Genus Culicoides (Diptera: Ceratopogonidae) in Austria-An Update of the Species Inventory Including the First Records of Three Species in Austria.

Ceratopogonidae are small nematoceran Diptera with a worldwide distribution, consisting of more than 5400 described species, divided into 125 genera. The genus Culicoides is known to comprise hematophagous vectors of medical and veterinary importance. Diseases transmitted by Culicoides spp. Such as African horse sickness virus, Bluetongue virus, equine encephalitis virus (Reoviridae) and Schmallenberg virus (Bunyaviridae) affect large parts of Europe and are strongly linked to the spread and abundance of its vectors. However, Culicoides surveillance measures are not implemented regularly nor in the whole of Austria. In this study, 142 morphologically identified individuals were chosen for molecular analyses (barcoding) of the mitochondrial cytochrome c oxidase subunit I gene (mt COI). Molecular analyses mostly supported previous morphologic identification. Mismatches between results of molecular and morphologic analysis revealed three new Culicoides species in Austria, Culicoides gornostaevae Mirzaeva, 1984, which is a member of the Obsoletus group, C. griseidorsum Kieffer, 1918 and C. pallidicornis Kieffer, 1919 as well as possible cryptic species. We present here the first Austrian barcodes of the mt COI region of 26 Culicoides species and conclude that barcoding is a reliable tool with which to support morphologic analysis, especially with regard to the difficult to identify females of the medically and economically important genus Culicoides.

Mosquito Iridescent Virus: New Records from Nature and Infections Using Strelkovimermis spiculatus (Mermithidae) as a Vector Under Laboratory Conditions.

Iridoviridae is a DNA virus family that affects both vertebrates and invertebrates. Immature aquatic stages of many dipteran species infected with iridovirus have been found in different places worldwide. The most represented genera of the Culicidae family are Aedes and Psorophora. To date, sixteen species of Aedes naturally infected with iridoviruses have been reported. Moreover, there are four records for the genus Psorophora, one for Culiseta, and two for Culex. In this paper, we report two new mosquito species as natural hosts of iridoviridae in Argentina: Aedes albifasciatus (Macquart) and Culex dolosus (Lynch Arribalzaga). We also analyzed the ability of a Cx. pipiens-Invertebrate Iridescent Virus to replicate in vivo in the larval stage of two mosquito species, Culex apicinus Philippi and Ae. aegypti (L.) using Strelkovimermis spiculatus as a vector, under laboratory conditions. Although Ae. aegypti is the most recognized mosquito vector of important arboviruses responsible for emergent diseases, Cx. apicinus and Ae. albifasciatus may also be implicated in enzootic or epizootic cycles of virus transmission, such as the St. Louis Encephalitis virus and the Western Equine Encephalomyelitis virus.

Electrocardiography Abnormalities in Macaques after Infection with Encephalitic Alphaviruses.

Eastern (EEEV) and Venezuelan (VEEV) equine encephalitis viruses (EEVs) are related, (+) ssRNA arboviruses that can cause severe, sometimes fatal, encephalitis in humans. EEVs are highly infectious when aerosolized, raising concerns for potential use as biological weapons. No licensed medical countermeasures exist; given the severity/rarity of natural EEV infections, efficacy studies require animal models. Cynomolgus macaques exposed to EEV aerosols develop fever, encephalitis, and other clinical signs similar to humans. Fever is nonspecific for encephalitis in macaques. Electrocardiography (ECG) metrics may predict onset, severity, or outcome of EEV-attributable disease. Macaques were implanted with thermometry/ECG radiotransmitters and exposed to aerosolized EEV. Data was collected continuously, and repeated-measures ANOVA and frequency-spectrum analyses identified differences between courses of illness and between pre-exposure and post-exposure states. EEEV-infected macaques manifested widened QRS-intervals in severely ill subjects post-exposure. Moreover, QT-intervals and RR-intervals decreased during the febrile period. VEEV-infected macaques suffered decreased QT-intervals and RR-intervals with fever onset. Frequency-spectrum analyses revealed differences in the fundamental frequencies of multiple metrics in the post-exposure and febrile periods compared to baseline and confirmed circadian dysfunction. Heart rate variability (HRV) analyses revealed diminished variability post-exposure. These analyses support using ECG data alongside fever and clinical laboratory findings for evaluating medical countermeasure efficacy.

2773. Safety and Immunogenicity Study of Eastern Equine Encephalitis Vaccine

BackgroundEastern equine encephalitis virus (EEEV) is an alphavirus with a high mortality rate and serious neurological sequelae in infected persons making this virus an important human pathogen.MethodsFollowing written informed consent, eligible subjects received two priming doses of EEEV vaccine, inactivated, TSI-GSD 104, 0.5 mL subcutaneously on days 0 and 28 days followed by a mandatory booster, 0.1 mL intradermal, at 6 months. Serum samples were collected pre-vaccination, days 21–35 following dose 2, as well as before and 21–35 days after dose 3. Sera with a Plaque Reduction Neutralization Test80 titer (PRNT80) ≥ 1:40 were considered responders with adequate titers for the purpose of biocontainment suite entry.ResultsSixty-seven (67) subjects were enrolled in this study to receive the primary vaccination series. All 67 subjects received at least 1 primary vaccination; 66 completed the 2 primary doses; 58 completed the 2 primary doses and the 6-month dose. Of these, 38 (56.7%) reported one or more adverse events. Fatigue was reported in 13 (19.4%), headache in 9 (13.4%), upper respiratory tract infection in 6 (9.0%), nausea in 5 (7.5%), pyrexia in 5 (7.5%), oropharyngeal pain in 4 (6.0%), myalgia in 4 (6.0%), injection site pain in 7 (10.4%), injection site hematoma in 4 (6.0%) and injection site erythema in 3 (4.5%) subjects. Adverse events were mostly mild or moderate and transient. PRNT80 titers ≥ 1:40 was observed in 39/65 (60%) subjects who received both primary doses of EEEV vaccine compared with 48/57 (84%) subjects who completed the 2-dose primary series and the 6-month dose and also had blood drawn for titer. Females had a higher response rate (61.5%) at the pre 6-month boost titer than did males (34.3%) (p = 0.0231). Similarly, the pre 6-month boost geometric mean titer (GMT) for females was 35.5 vs. 21.9 for males (p = 0.0231). The post 6-month boost GMT for females was 146.7 and 181.5 for males (P = 0.13).ConclusionInactivated Eastern Equine Encephalitis Virus vaccine, TSI-GSD 104, Lot 2-1-89 appears to be safe and immunogenic. This Phase 2 vaccine study supports a priming dose schedule of Days 0 and 28 and 6-month. The 6 month dose is anamnestic improving the overall response rate and level of antibody for this primary dosing schedule.Disclosures All authors: No reported disclosures.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from −80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at −20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.

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.

Eastern equine encephalitis virus evades induction of the host immune response through miR-142-3p restriction of myeloid cell replication

Abstract Eastern equine encephalitis virus (EEEV) is an alphavirus that causes the most virulent mosquito-borne disease in North America. We have previously determined that a myeloid cell-specific miRNA, miR-142-3p, binds to the 3′ non-translated region of EEEV suppressing virus replication and the induction of systemic IFN-α/β, promoting severe disease. Deletion of the miR-142-3p binding sites (virus 11337) rescues virus replication in myeloid cells, induces systemic IFN-α/β production, and leads to virus attenuation in vivo. We hypothesized that miR-142-3p restriction of EEEV replication in myeloid cells suppresses the adaptive immune response. Infection of C57BL6 mice with the myeloid tropic 11337 virus lead to increased cellularity in the popliteal lymph node (PLN) within 12 hours, predominantly comprised of CD11b+ F4/80+ Ly-6C+ inflammatory macrophages. Recruitment of inflammatory macrophages was significantly delayed in the non-myeloid tropic WT infected mice. NK cells and neutrophils were also recruited to the PLN after 11337 infection but not WT infection. In addition, 11337 infection of myeloid cells lead to increased cytokine and chemokine mRNA levels in the PLN compared to WT infection. In the spleen, 11337 infection lead to an increased frequency and number of epitope-specific CD8+ T cells compared to WT infection; however, the frequency was similar in the PLN. On day 6, epitope-specific CD8+ T cells expressed both KLRG1+ and CD127+during a WT infection while 11337 infection lead to the generation of short-lived effector cells (KLRG1+ CD127−). These results demonstrate that miR-142-3p restriction of EEEV replication in myeloid cells limits the induction of the host immune response and the generation of effector CD8+ T cells.

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

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

An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding.

ABSTRACTAlphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.

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

BackgroundEastern 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.ResultsOur 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.ConclusionsThese 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.

Environmental Drivers of Seasonal Patterns of Host Utilization by Culiseta melanura (Diptera: Culicidae) in Florida.

Field studies were carried out in four Florida counties to investigate winter and spring ecology of host use by Culiseta melanura (Coquillet), the primary vector of eastern equine encephalomyelitis virus (EEEV) in North America. Bloodmeal analysis by PCR was used to identify 233 host bloodmeals, which mainly originated from birds (78.5%) and reptiles (17.2%), primarily Anolis spp. lizards. Across counties, the percentage of bloodmeals from reptiles (7-37% depending upon county) increased with increasing day length and temperature in the spring. Multiple logistic regression revealed that differences in reptile host use across collection sites were largely explained by differences in average day length and temperature on the day of collection, and is probably owing to environment-driven behavioral patterns of ectothermic animals. Although past studies have demonstrated reptile biting by epizootic vectors of EEEV, including Culex (Melanoconion) spp., this is the first study to demonstrate widespread and common feeding upon ectothermic hosts by Cs. melanura. This work suggests that reptiles, particularly anole lizards, play a role in the ecology of EEEV in Florida either as amplifying hosts or as noncompetent hosts which dilute vector feedings thereby suppressing transmission. Detailed laboratory studies investigating impacts of environmental variables (temperature and photoperiod) on EEEV competence of anoles are needed to assess whether these animals support virus amplification.