Uncertainties Surrounding Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex.

Background: Madariaga virus (MADV), a member of the eastern equine encephalitis virus (EEEV) complex, circulates in Latin America and exhibits distinct evolutionary and ecological features compared to the North American EEEV. While published data have shed light on MADV ecology, several key aspects remain unknown. Methods: In this study, we compiled data on virus isolation, vector competence, and animal serology collected over six decades in Latin America to identify critical knowledge gaps on MADV transmission and ecology. Results: Specific vertebrate animals serving as amplifying hosts and the mosquito species acting as enzootic and epizootic vectors have not yet been identified. Other aspects that remain unclear are the virus current geographic distribution, the role of equines as hosts in epizootic cycles, and the full impact of MADV on human health in endemic regions. Conclusions: The numerous knowledge gaps surrounding MADV, its widespread distribution in Latin America, and its potential to cause severe disease in animals and humans emphasize the urgent need for increased research efforts, heightened awareness, and intensified surveillance towards this potential emerging threat.

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.

Safety and immunogenicity of a novel trivalent recombinant MVA-based equine encephalitis virus vaccine: A Phase 1 clinical trial.

BACKGROUND: Three encephalitic alphaviruses-western, eastern, and Venezuelan equine encephalitis virus (WEEV, EEEV and VEEV)-can cause severe disease and have the potential to be used as biological weapons. There are no approved vaccines for human use. A novel multivalent MVA-BN-WEV vaccine encodes the envelope surface proteins of the 3 viruses and is thereby potentially able to protect against them all, as previously demonstrated in animal models. This first-in-human study assessed the safety, tolerability, and immunogenicity of MVA-BN-WEV vaccine in healthy adult participants. METHODS: Forty-five participants were enrolled into 3 dose groups (1 × 10E7 Inf.U, 1 × 10E8 Inf.U, and 2 × 10E8 Inf.U), received 2 doses 4 weeks apart, and were then monitored for 6 months. RESULTS: The safety profile of MVA-BN-WEV was acceptable at all administered doses, with incidence of local solicited AEs increased with increasing dose and no other clinically meaningful differences between dose groups. One SAE (Grade 2 pleural effusion) was reported in the lowest dose group and assessed as possibly related. No AEs resulted in death or led to withdrawal from the second vaccination or from the trial. The most common local solicited AE was injection site pain, and general solicited AEs were headache, fatigue, and myalgia. MVA-BN-WEV induced humoral immune responses; WEEV-, EEEV- and VEEV-specific neutralizing antibody responses peaked 2 weeks following the second vaccination, and the magnitude of these responses increased with dose escalation. The highest dose resulted in seroconversion of all (100 %) participants for WEEV and VEEV and 92.9 % for EEEV, 2 weeks following second vaccination, and durability was observed for 6 months. MVA-BN-WEV induced cellular immune responses to VEEV E1 and E2 (EEEV and WEEV not tested) and a dose effect for peptide pool E2. CONCLUSION: The study demonstrated that MVA-BN-WEV is well tolerated, induces immune responses, and is suitable for further development. CLINICAL TRIAL REGISTRY NUMBER: NCT04131595.

Comprehensive Assessment of Inactivation Methods for Madariaga Virus.

The Eastern Equine Encephalitis Virus (EEEV) is an emerging public health threat, with the number of reported cases in the US increasing in recent years. EEEV is a BSL3 pathogen, and the North American strain is a US Federal Select Agent (SA). These restrictions make experiments with EEEV difficult to perform, as high-tech equipment is often unavailable in BSL3 spaces and due to concerns about generating aerosols during manipulations. Therefore, a range of inactivation methods suitable for different downstream analysis methods are essential for advancing research on EEEV. We used heat, chemical, and ultraviolet (UV)-based methods for the inactivation of infected cells and supernatants infected with the non-select agent Madariaga virus (MADV). Although the MADV and EEEV strains are genetically distinct, differing by 8-11% at the amino acid level, they are expected to be similarly susceptible to various inactivation methods. We determined the following to be effective methods of inactivation: heat, TRIzol LS, 4% PFA, 10% formalin, and UV radiation for infected supernatants; TRIzol, 2.5% SDS with BME, 0.2% NP40, 4% PFA, and 10% formalin for infected cells. Our results have the potential to expand the types and complexity of experiments and analyses performed by EEEV researchers.

Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

Commonly Reported Mosquito-Borne Viruses in the United States: A Primer for Pharmacists.

Mosquito-borne diseases are a public health concern. Pharmacists are often a patient's first stop for health information and may be asked questions regarding transmission, symptoms, and treatment of mosquito borne viruses (MBVs). The objective of this paper is to review transmission, geographic location, symptoms, diagnosis and treatment of MBVs. We discuss the following viruses with cases in the US in recent years: Dengue, West Nile, Chikungunya, LaCrosse Encephalitis, Eastern Equine Encephalitis Virus, and Zika. Prevention, including vaccines, and the impact of climate change are also discussed.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host-pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.

Mapping Eastern (EEE) and Venezuelan Equine Encephalitides (VEE) among Equines Using Geographical Information Systems, Colombia, 2008-2019.

INTRODUCTION: Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) viruses are zoonotic pathogens affecting humans, particularly equines. These neuroarboviruses compromise the central nervous system and can be fatal in different hosts. Both have significantly influenced Colombia; however, few studies analyse its behaviour, and none develop maps using geographic information systems to characterise it. OBJECTIVE: To describe the temporal-spatial distribution of those viruses in Colombia between 2008 and 2019. METHODS: Retrospective cross-sectional descriptive study, based on weekly reports by municipalities of the ICA, of the surveillance of both arboviruses in equines, in Colombia, from 2008 to 2019. The data were converted into databases in Microsoft Access 365®, and multiple epidemiological maps were generated with the Kosmo RC1®3.0 software coupled to shape files of all municipalities in the country. RESULTS: In the study period, 96 cases of EEE and 70 of VEE were reported, with 58% of EEE cases occurring in 2016 and 20% of EEV cases in 2013. The most affected municipalities for EEE corresponded to the department of Casanare: Yopal (20), Aguazul (16), and Tauramena (10). In total, 40 municipalities in the country reported ≥1 case of EEE. CONCLUSIONS: The maps allow a quick appreciation of groups of neighbouring municipalities in different departments (1° political division) and regions of the country affected by those viruses, which helps consider the expansion of the disease associated with mobility and transport of equines between other municipalities, also including international borders, such as is the case with Venezuela. In that country, especially for EEV, municipalities in the department of Cesar are bordering and at risk for that arboviral infection. there is a high risk of equine encephalitis outbreaks, especially for VEE. This poses a risk also, for municipalities in the department of Cesar, bordering with Venezuela.

West Nile Virus and Eastern Equine Encephalitis Virus High Probability Habitat Identification for the Selection of Sentinel Chicken Surveillance Sites in Florida.

To mitigate the effects of West Nile virus (WNV) and eastern equine encephalitis virus (EEEV), the state of Florida conducts a serosurveillance program that uses sentinel chickens operated by mosquito control programs at numerous locations throughout the state. Coop locations were initially established to detect St. Louis encephalitis virus (SLEV), and coop placement was determined based on the location of human SLEV infections that occurred between 1959 and 1977. Since the introduction of WNV into Florida in 2001, WNV has surpassed SLEV as the primary arbovirus in Florida. Identifying high probability locations for WNV and EEEV transmission and relocating coops to areas of higher arbovirus activity would improve the sensitivity of the sentinel chicken surveillance program. Using 2 existing models, this study conducted an overlay analysis to identify areas with high probability habitats for both WNV and EEEV activity. This analysis identified approximately 7,800 km2 (about 4.5% of the state) as high probability habitat for supporting both WNV and EEEV transmission. Mosquito control programs can use the map resulting from this analysis to improve their sentinel chicken surveillance programs, increase the probability of virus detection, reduce operational costs, and allow for a faster, targeted response to virus detection.

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

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.

Ecology and Epidemiology of Eastern Equine Encephalitis Virus in the Northeastern United States: An Historical Perspective.

In the current review, we examine the regional history, ecology, and epidemiology of eastern equine encephalitis virus (EEEV) to investigate the major drivers of disease outbreaks in the northeastern United States. EEEV was first recognized as a public health threat during an outbreak in eastern Massachusetts in 1938, but historical evidence for equine epizootics date back to the 1800s. Since then, sporadic disease outbreaks have reoccurred in the Northeast with increasing frequency and northward expansion of human cases during the last 20 yr. Culiseta melanura (Coquillett) (Diptera: Culicidae) serves as the main enzootic vector that drives EEEV transmission among wild birds, but this mosquito species will occasionally feed on mammals. Several species have been implicated as bridge vectors to horses and humans, with Coquilletstidia perturbans (Walker) as a leading suspect based on its opportunistic feeding behavior, vector competence, and high infection rates during recent disease outbreaks. A diversity of bird species are reservoir competent, exposed to EEEV, and serve as hosts for Cs. melanura, with a few species, including the wood thrush (Hlocichia mustelina) and the American robin (Turdus migratorius), contributing disproportionately to virus transmission based on available evidence. The major factors responsible for the sustained resurgence of EEEV are considered and may be linked to regional landscape and climate changes that support higher mosquito densities and more intense virus transmission.

Ecology of Eastern Equine Encephalitis Virus in the Southeastern United States: Incriminating Vector and Host Species Responsible for Virus Amplification, Persistence, and Dispersal.

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) is a mosquito-borne pathogen found in eastern North America that causes severe disease in humans and horses. The mosquito Culiseta melanura (Coquillett) (Diptera: Culicidae) is the primary enzootic vector of EEEV throughout eastern North America while several mosquito species belonging to diverse genera serve as bridge vectors. The ecology of EEEV differs between northern and southern foci, with respect to phenology of outbreaks, important vertebrate hosts, and bridge vector species. Active transmission is limited to roughly half of the year in northern foci (New York, New Hampshire, Massachusetts, Connecticut), while year-round transmission occurs in the southeastern region (particularly Florida). Multiple phylogenetic analyses indicate that EEEV strains circulating in northern foci are likely transported from southern foci by migrating birds. Bird species that overwinter or migrate through Florida, are bitten by Cs. melanura in late spring, and arrive at northern breeding grounds in May are the most likely candidates to disperse EEEV northward. Available data indicate that common yellowthroat and green heron satisfy these criteria and could serve as virus dispersers. Understanding the factors that drive the phenology of Cs. melanura reproduction in the south and the timing of avian migration from southern foci could provide insight into how confluence of these biological phenomena shapes outbreaks of EEE throughout its range. This information could be used to develop models predicting the likelihood of outbreaks in a given year, allowing vector control districts to more efficiently marshal resources necessary to protect their stakeholders.

The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat.

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito-transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into the fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on single-particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

Fatal Human Infection with Evidence of Intrahost Variation of Eastern Equine Encephalitis Virus, Alabama, USA, 2019.

Eastern equine encephalitis virus (EEEV) is an arbovirus in the family Togaviridae, genus Alphavirus, found in North America and associated with freshwater/hardwood swamps in the Atlantic, Gulf Coast, and Great Lakes regions. EEEV disease in humans is rare but causes substantial illness and death. To investigate the molecular epidemiology and microevolution of EEEV from a fatal case in Alabama, USA, in 2019, we used next-generation sequencing of serum and cerebrospinal fluid (CSF). Phylogenetic inference indicated that the infecting strain may be closely related to isolates from Florida detected during 2010-2014, suggesting potential seeding from Florida. EEEV detected in serum displayed a higher degree of variability with more single-nucleotide variants than that detected in the CSF. These data refine our knowledge of EEEV molecular epidemiologic dynamics in the Gulf Coast region and demonstrate potential quasispecies bottlenecking within the central nervous system of a human host.

PERK Is Critical for Alphavirus Nonstructural Protein Translation.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

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.

Designing multivalent immunogens for alphavirus vaccine optimization.

There is a pressing need for vaccines against mosquito-borne alphaviruses such as Venezualen and eastern equine encephalitis viruses (VEEV, EEEV). We demonstrate an approach to vaccine development based on physicochemical properties (PCP) of amino acids to design a PCP-consensus sequence of the epitope-rich B domain of the VEEV major antigenic E2 protein. The consensus "spike" domain was incorporated into a live-attenuated VEEV vaccine candidate (ZPC/IRESv1). Mice inoculated with either ZPC/IRESv1 or the same virus containing the consensus E2 protein fragment (VEEVconE2) were protected against lethal challenge with VEEV strains ZPC-738 and 3908, and Mucambo virus (MUCV, related to VEEV), and had comparable neutralizing antibody titers against each virus. Both vaccines induced partial protection against Madariaga virus (MADV), a close relative of EEEV, lowering mortality from 60% to 20%. Thus PCP-consensus sequences can be integrated into a replicating virus that could, with further optimization, provide a broad-spectrum vaccine against encephalitic alphaviruses.