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Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne New World alphaviruses that cause encephalitis in equids and humans. These viruses can cause severe disease and death, as well as long-term severe neurological symptoms in survivors. Despite the pathogenesis and weaponization of these viruses, there are no approved therapeutics for treating infection.In this review, we describe the molecular pathogenesis of these viruses, discuss host-pathogen interactions needed for viral replication, and highlight new avenues for drug development with a focus on host-targeted approaches.Current approaches have yielded some promising therapeutics, but additional emphasis should be placed on advanced development of existing small molecules and pursuit of pan-encephalitic alphavirus drugs. More research should be conducted on EEEV and WEEV, given their high lethality rates.

Complexes or assemblages of culicid species and subspecies from the southern region of Latin America are difficult to differentiate morphologically (Harbach 2012, Laurito et al. 2017). These complexes include taxa with differences in their vectorial capacity and therefore in their epidemiological importance (Dujardin and Schofield 2004). However, morphological variation within the species and superposition of diagnostic characters based on classical structures often do not allow for a correct identification. The aim of this work was to evaluate, through geometric morphometry, differences in the wing shape of female mosquitoes belonging to the Pipiens Assemblage (Cx. pipiens and Cx. quinquefasciatus) of Argentina. Fil: Garzón, Maximiliano Javier. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Grupo de Estudio de Mosquitos; Argentina Fil: Grech, Marta Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigación Esquel de Montaña y Estepa Patagóica. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Esquel. Centro de Investigación Esquel de Montaña y Estepa Patagónica; Argentina Fil: Lizuain, Arturo Andrés. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Centro Nacional de Diagnóstico e Investigaciones Endemo-epidémicas; Argentina Fil: Schweigmann, Nicolas Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.

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.

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Western equine encephalitis (WEE) was once prevalent and routinely isolated from mosquitoes in Colorado; however, isolations of Western equine encephalitis virus (WEEV) have not been reported from mosquito pools since the early 1990s. The objective of the present study was to test pools of Culex tarsalis (Coquillett) mosquitoes sampled from Weld County, CO, in 2016 for evidence of WEEV infection. Over 7,000 mosquitoes were tested, but none were positive for WEEV RNA. These data indicate that WEEV either was not circulating enzootically in Northern Colorado, was very rare, and would require much more extensive mosquito sampling to detect, or was heterogeneously distributed spatially and temporally and happened to not be present in the area sampled during 2016. Even though the reported incidence of WEE remains null, screening for WEEV viral RNA in mosquito vectors offers forewarning toward the detection and prevention of future outbreaks.

The three encephalitic alphaviruses, western, eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV) are potential biothreat agents due to high infectivity through aerosol exposure, ease of production in large amounts, and relative stability in the environment. Currently, there is no licensed vaccine for human use to these three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this study, the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform was used to construct and produce three monovalent recombinant MVA-BN-based encephalitic alphavirus vaccines, MVA-BN-W, MVA-BN-E, and MVA-BN-V. Additionally, a MVA-BN-based construct was designed to produce antigens against all three alphaviruses, the trivalent vaccine MVA-BN-WEV. The protective efficacy of these vaccines was evaluated in vivo. Female BALB/c mice were immunized with two doses of each monovalent MVA-BN-based alphavirus vaccine, a mixture of the three monovalent vaccines, MVA-BN-W + E + V, or the trivalent vaccine MVA-BN-WEV at a four-week interval. Two weeks after the booster immunization, the mice were instilled intranasally with 5 × 103 to 1 × 104 plaque forming units of WEEV, EEEV, or VEEV. All mice immunized with monovalent vaccines survived the respective virus challenge without any signs of illness or weight loss, while all the control mice died. The triple mixture of vaccines or the trivalent vaccine also provided 90 to 100% protection to the mice against WEEV and VEEV challenges, and 60% to 90% protection against EEEV challenge. These data suggest that each monovalent MVA-BN-W, MVA-BN-E, and MVA-BN-V is a potential vaccine candidate against respective encephalitic alphavirus and the three monovalent vaccines can be given in a mixture (MVA-BN-W + E + V) or the trivalent vaccine MVA-BN-WEV can serve as a true multivalent vaccine without significantly reducing efficacy against WEEV and VEEV despite slightly reduced efficacy against EEEV challenge.

Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α–synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of ⍺-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of ⍺-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.

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.

VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses., Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century., Author summary Equally important to understanding how arboviruses emerge is understanding the conditions in which they experience reductions in activity. Western equine encephalitis virus (WEEV) provides a unique case study due to its reduction in equine and human incidence and wildlife transmission activity during the second half of the twentieth century. Despite those reductions, we identified six amino acid substitutions that appeared to increase fitness in avian hosts and/or mosquito vectors. We also found no effect of these mutations on mammalian virulence. Our results suggest that ecological factors likely explain the reduction in WEEV activity, even in the face of adaptive evolution.

RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus. IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 7 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites., Author summary Eastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in hematopoietic cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3’ untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3’ UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo. These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.

Eastern, Venezuelan and western equine encephalitis viruses (EEEV, VEEV, and WEEV) are mosquito-borne viruses that cause substantial disease in humans and other vertebrates. Vaccines are limited and current treatment options have not proven successful. In this report, we vaccinated outbred mice with lipid-antigen-nucleic acid-complexes (LANACs) containing VEEV E1 + WEEV E1 antigen and characterized protective efficacy against lethal EEEV, VEEV, and WEEV challenge. Vaccination resulted in complete protection against EEEV, VEEV, and WEEV in CD-1 mice. Measurements of bioluminescence and plaque reduction neutralization tests (PRNTs) indicate that LANAC VEEV E1 + WEEV E1 vaccination is sterilizing against VEEV and WEEV challenge; whereas immunity to EEEV is not sterilizing. Passive transfer of rabbit VEEV E1 + WEEV E1 immune serum to naive mice extended the mean time to death (MTD) of EEEV challenged mice and provided significant protection from lethal VEEV and WEEV challenge.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus ; Togaviridae ) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed ( Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that is vectored by mosquitos and cycled in rodents. It can cause disease in equines and humans characterized by a febrile illness that may progress into encephalitis. Like the capsid protein of other viruses, VEEV capsid is an abundant structural protein that binds to the viral RNA and interacts with the membrane-bound glycoproteins. It also has protease activity, allowing cleavage of itself from the growing structural polypeptide during translation. However, VEEV capsid protein has additional nonstructural roles within the host cell functioning as the primary virulence factor for VEEV. VEEV capsid inhibits host transcription and blocks nuclear import in mammalian cells, at least partially due to its complexing with the host CRM1 and importin α/β1 nuclear transport proteins. VEEV capsid also shuttles between the nucleus and cytoplasm and is susceptible to inhibitors of nuclear trafficking, making it a promising antiviral target. Herein, the role of VEEV capsid in viral replication and pathogenesis will be discussed including a comparison to proteins of other alphaviruses.

Western equine encephalitis virus (WEEV) naturally cycles between mosquitos and birds or rodents, with a case fatality rate of up to 15% in humans during epizootic outbreaks. There are no medical countermeasures to treat WEEV infection, and accidental aerosol exposure increases the case fatality rate up to 40%. Understanding the pathogenesis of infection is required to develop and assess medical countermeasures. This study describes the clinical and pathological findings of mice infected with WEEV by the aerosol route, and use as a model for WEEV infection in humans. Balb/c mice were infected by the aerosol route with a dose range of high-virulence WEEV strain Fleming to establish the median lethal dose (MLD). The disease course was acute, culminating in severe clinical signs, neuroinvasion, and dose-dependent mortality. Further groups of mice were exposed by the aerosol route, periodically sacrificed, and tissues excised for histopathological examination and virology. Viral titres peaked four days post-challenge in the brain and lungs, corresponding with severe bilateral lesions in rostroventral regions of the encephalon, especially in the olfactory bulb and piriform cortex. Recapitulation of the most serious clinical presentations of human WEEV disease in mice may prove a useful tool in the evaluation of medical countermeasures.

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses., Highlights • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2- b ]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950–957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535–3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222–9241, 2013, http://dx.doi.org/10.1021/jm401330r ). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 μM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae , Bunyaviridae , Picornaviridae , and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535–3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950–957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222–9241, 2013, http://dx.doi.org/10.1021/jm401330r ). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

Vaccinations against the encephalitic alphaviruses (western, eastern, and Venezuelan equine encephalitis virus) are of significant interest to biological defense, public health, and agricultural communities alike. Although vaccines licensed for veterinary applications are used in the Western Hemisphere and attenuated or inactivated viruses have been used under Investigational New Drug status to protect at-risk personnel, there are currently no licensed vaccines for use in humans. Here, we will discuss the need for a trivalent vaccine that can protect humans against all three viruses, recent progress to such a vaccine, and a strategy to continue development to Food and Drug Administration licensure.

Western equine encephalitis virus (WEEV) is an arbovirus from the genus Alphavirus , family Togaviridae , which circulates in North America between birds and mosquitoes, occasionally causing disease in humans and equids. In recent decades, human infection has decreased dramatically; the last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed the genomic sequences of 33 low-passage-number strains with diverse geographic and temporal distributions and performed comprehensive phylogenetic analyses. Our results indicated that WEEV is a highly conserved alphavirus with only approximately 5% divergence in its most variable genes. We confirmed the presence of the previously determined group A and B lineages and further resolved group B into three sublineages. We also observed an increase in relative genetic diversity during the mid-20th century, which correlates with the emergence and cocirculation of several group B sublineages. The estimated WEEV population size dropped in the 1990s, with only the group B3 lineage being sampled in the past 20 years. Structural mapping showed that the majority of substitutions in the envelope glycoproteins occurred at the E2-E2 interface. We hypothesize that an event occurred in the mid-20th century that resulted in the increased genetic diversity of WEEV in North America, followed by genetic constriction due to either competitive displacement by the B3 sublineage or stochastic events resulting from a population decline. IMPORTANCE Western equine encephalitis virus (WEEV) has caused several epidemics that resulted in the deaths of thousands of humans and hundreds of thousands of equids during the past century. During recent decades, human infection decreased drastically and the virus has not been found in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed 33 complete genome sequences and conducted comprehensive phylogenetic analyses. We confirmed the presence of two major lineages, one of which diverged into three sublineages. Currently, only one of those sublineages is found circulating in nature. Understanding the evolution of WEEV over the past century provides a unique opportunity to observe an arbovirus that is in decline and to better understand what factors can cause said decline.

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5x10(4) TCID 50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69-3A2 antibody is a promising candidate for future passive vaccine development.

Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine.

Cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (IRF-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. To investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using the neurotropic alphavirus western equine encephalitis virus (WEEV). We found that WEEV activated IRF-3-mediated neuronal innate immune pathways in a replication-dependent manner, and abrogation of IRF-3 function enhanced virus-mediated injury by WEEV and the unrelated flavivirus St. Louis encephalitis virus. Furthermore, IRF-3-dependent neuronal protection from virus-mediated cytopathology occurred independently of autocrine or paracrine type I interferon activity. Despite being partially controlled by IRF-3-dependent signals, WEEV also disrupted antiviral responses by inhibiting pattern recognition receptor pathways. This antagonist activity was mapped to the WEEV capsid gene, which disrupted signal transduction downstream of IRF-3 activation and was independent of capsid-mediated inhibition of host macromolecular synthesis. Overall, these results indicate that innate immune pathways have important cytoprotective activity in neurons and contribute to limiting injury associated with infection by neurotropic arboviruses.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

Having recently characterized a CD-1 outbred mouse model of pathogenesis for Western equine encephalitis virus, we examined the possible protective effects of cationic liposome-DNA complexes (CLDCs) against encephalitic arboviral infection. In this investigation, mice were pre-treated, co-treated, or post-treated with CLDC then challenged with a subcutaneous or aerosol dose of the highly virulent WEEV-McMillan strain, lethal in mice 4-5 days after inoculation. Pre-treatment with CLDCs provided a significant protective effect in mice, which was reflected in significantly increased survival rates. Further, in some instances a therapeutic effect of CLDC administration up to 12h after WEEV challenge was observed. Mice treated with CLDC had significantly increased serum IFN-gamma, TNF-alpha, and IL-12, suggesting a strong Th1-biased antiviral activation of the innate immune system. In virus-infected animals, large increases in production of IFN-gamma, TNF-alpha, MCP-1, IL-12, and IL-10 in the brain were observed by 72h after infection, consistent with neuroinvasion and viral replication in the CNS. These results indicate that strong non-specific activation of innate immunity with an immune therapeutic such as CLDC is capable of eliciting significant protective immunity against a rapidly lethal strain of WEEV and suggest a possible prophylactic option for exposed individuals.

The encephalitides caused by Venezuelan (VEEV), eastern (EEEV), and western (WEEV) equine encephalitis viruses are important natural diseases of horses and humans and potential agents of biowarfare or bioterrorism. No licensed vaccines or specific therapies exist to prevent or treat human infections with VEEV, EEEV, or WEEV. Well-characterized animal models are needed to support the development of such medical countermeasures under the United States Food and Drug Administration’s “Animal Rule.” This review focuses on the pathological features and pathogenetic mechanisms of these alphaviral encephalitides in animal models, with an emphasis on aerosol infections. Infection of mice, nonhuman primates, and other species with VEEV, EEEV, and WEEV causes encephalitis and often death. There is great variability in the specific manifestations of disease in the different models, however. Many aspects of the disease in animal models and in humans remain to be characterized using modern methods. Especially needed is a better understanding of the fundamental mechanisms involved in 3 key phases of the pathogenesis of alphavirus encephalitis. These are the early extraneural phase, the process of neuroinvasion itself, and virus and host factors related to neurovirulence. A greater understanding of these aspects could provide avenues for the development of medical countermeasures and better establish suitable animal models of alphavirus encephalitis for testing them under the Animal Rule.

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed ≤2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 103 p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 103 p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

We developed two types of chimeric Sindbis virus (SINV)/western equine encephalitis virus (WEEV) alphaviruses to investigate their potential use as live virus vaccines against WEE. The first-generation vaccine candidate, SIN/CO92, was derived from structural protein genes of WEEV strain CO92-1356, and two second-generation candidates were derived from WEEV strain McMillan. For both first- and second-generation vaccine candidates, the nonstructural protein genes were derived from SINV strain AR339. Second-generation vaccine candidates SIN/SIN/McM and SIN/EEE/McM included the envelope glycoprotein genes from WEEV strain McMillan; however, the amino-terminal half of the capsid, which encodes the RNA-binding domain, was derived from either SINV or eastern equine encephalitis virus (EEEV) strain FL93-939. All chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in 6-week-old mice. Vaccinated mice developed little or no detectable disease and showed little or no evidence of challenge virus replication; however, all developed high titers of neutralizing antibodies. Upon intranasal challenge with high doses of virulent WEEV strains, mice vaccinated with >or=10(5)PFU of SIN/CO92 or >or=10(4)PFU of SIN/SIN/McM or SIN/EEE/McM were completely protected from disease. These findings support the potential use of these live-attenuated vaccine candidates as safe and effective vaccines against WEE.

Innate cell-autonomous antiviral responses are essential first lines of defense against central nervous system infections but may also contribute to neuropathogenesis. We investigated the relationships between innate immunity and neuronal differentiation using an in vitro culture system with human cell lines to analyze cellular responses to the neurotropic alphavirus western equine encephalitis virus. Human neuronal cells displayed a maturation-dependent reduction in virus-induced cytopathology that was independent of autocrine interferon α or β activity. In addition, maturation was associated with enhanced responsiveness to exogenous stimuli, such that differentiated neurons required five- to ten-fold less type I interferon to suppress viral replication or virus-induced cytopathology compared to immature cells, although this enhanced responsiveness extended to only a subset of unique type I interferons. These results demonstrate that maturation-dependent changes in human neuronal cells may be key determinants in the innate immune response to infections with neurotropic alphaviruses.

Western equine encephalitis virus (WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNalpha) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNalpha provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNalpha delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection.

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.

Song sparrows, Melopiza melodia, inoculated subcutaneously with either western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV) or West Nile virus (family Flaviviridae, genus Flavivirus, WNV) developed elevated viremias, and they were considered to be competent experimental hosts for both viruses. However, birds that ingested from three to 20 mosquitoes containing comparable amounts of either WEEV or WNV failed to become infected, indicating limited oral susceptibility. Comparatively few field-collected birds had antibodies against either WEEV or WNV, indicating that this species was infrequently bitten by infectious mosquitoes in nature and probably was of limited importance in viral amplification.

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of

House finches, Carpodacus mexicanus, were experimentally infected with high and standard doses of western equine encephalomyelitis virus (WEEV) or St. Louis encephalitis virus (SLEV) to determine whether high doses would produce an elevated viremia response and a high frequency of chronic infections. Finches inoculated with approximately100,000 plaque forming units (PFU) of WEEV or SLEV produced viremia and antibody responses similar to those in finches inoculated with approximately 100 PFU of WEEV or 1000 PFU of SLEV, the approximate quantities of virus expectorated by blood-feeding Culex tarsalis Coquillett. Infected finches were held through winter and then necropsied. Only one finch inoculated with the high dose of SLEV developed a chronic infection. Our data indicated that elevated infectious doses of virus may not increase the viremia level or the frequency of chronic infection in house finches.

After-hatching and hatching year, mourning doves were infected by inoculation with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses; some birds in each group also were treated with the immunosuppressant cyclophosphamide before and during infection. Cyclophosphamide treatment significantly increased the WEE viremia but did not alter the antibody response. In contrast, cyclophosphamide-treated and -untreated doves did not develop a detectable SLE viremia but became antibody positive. Antibody peaked at 10 wk after inoculation for both viruses and remained detectable in most birds throughout the 26-wk study. When treated with cyclophosphamide the following spring, birds did not relapse and develop a detectable viremia. Previously infected birds were protected when challenged with conspecific virus (i.e., none produced a detectable viremia), but there was no anamnestic antibody response to reinfection. In agreement with our failure to detect relapses, all birds were negative for viral RNA when sera, spleen, lung, and kidney tissues were tested by reverse transcriptase-polymerase chain reaction after necropsy. Our results indicated that adult mourning doves were an incompetent host for SLE virus and probably do not serve as a suitable overwintering or dispersal host for either WEE and SLE viruses.

Chimeric cDNA clones, pMWE1000 and pMWE2000, differing by five nucleotides at their 5′ termini, were constructed of the 5′ two-thirds of the western equine encephalitis (WEE) virus genome (encoding nonstructural proteins) and the 3′ one-third of the eastern equine encephalitis (EEE) virus genome (encoding structural proteins). The WEE virus sequences were derived from full-length cDNA clones, pWE1000 and pWE2000, which were isogenic except for five nucleotide differences at their 5′ termini and were responsible for significant differences in mouse virulence. Each cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length RNA. Transfection of BHK-21 cells with the chimeric RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 105 PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900–7909).

While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.

In response to the 1984 St. Louis encephalitis (SLE) epidemic in the Los Angeles Basin of southern California (USA), an investigative program was initiated to evaluate the interactive components of the SLE virus transmission cycle. From 1987 through 1996 (10 yr), 52,589 birds were bled and their sera tested for SLE and western equine encephalomyelitis (WEE) virus antibodies by the hemagglutination inhibition (HAI) test. Eighty-three percent of the birds tested were house finches (Carpodacus mexicanus) (48.7%) and house sparrows (Passer domesticus) (34.6%); 1.1% of these birds were positive for SLE antibodies. Prevalence of WEE antibodies was negligible. The analysis of 5,481 sera from rock doves (Columbia livia) yielded 3.6% SLE positives and 0.4% WEE positives. Collection sites were maintained as study sites when identified as positive bird, mosquito, and SLE virus activity localities; others were abandoned. Serial serum samples from 7,749 banded house sparrows and 9,428 banded house finches from these selected sites demonstrated year-round SLE virus transmission. One location exhibited significant numbers of house finches undergoing annual SLE seroconversion and a number of seroconversion-reversion-reconversion sequences suggesting either viral reinfection from mosquitoes or recrudescence by latent virus. A proportion of both bird species also lived for longer than 1 yr, thus, increasing the possibility of virus carry-over from autumn to spring. Assessment of concurrently collected mosquitoes indicated no correlative association between mosquito populations and SLE seroconversion and reconversion. European house sparrows introduced in the 1800's may have provided a supplemental link to the existing SLE virus enzootic cycle involving endemic house finches. Meteorological factors are reviewed as possible important correlates of SLE epidemics. The house finch/house sparrow serosurveillance system is also evaluated for use as an "Early Warning" indicator of SLE virus activity.

The mechanism for long-term maintenance of western equine encephalomyelitis (WEE) virus in California was investigated by studying genetic variation in the E2 portion of the genome of 55 strains of WEE virus isolated since 1938 from different locations in California. Four major lineages were evident: virus strains isolated from the Central Valley since 1993 and Los Angeles in 1991 formed lineage A; southern California strains isolated since 1978 and isolates from the Central Valley from 1978 to 1987 formed lineage B; northern California isolates from 1968 to 1971 formed lineage C; and early isolates from 1938 to 1961 formed a fourth lineage, D. The separation of strains from north and south of the Tehachapi and San Bernardino Mountains (i.e., the Central Valley and southern California, respectively) since 1991 indicates that there has been little recent movement of virus between the two regions and recent strains from these two locations appear to be evolving independently. However, within the Central Valley and within southern California, virus appears to circulate freely, perhaps by movement of birds or mosquito vectors. Although the current virus lineage in the Central Valley may have been introduced from an unknown source in 1991, introduction and establishment of new viral genotypes from outside California do not seem to occur regularly. It appears most likely that virus is maintained in separate geographic areas of California through local persistence in enzootic foci.

The incidence of Western Equine Encephalitis (WEE) in humans and equids peaked during the mid-20th century and has declined to fewer than 1–2 human cases annually during the past 20 years. Using the mouse model, changes in WEE virus (WEEV) virulence were investigated as a potential explanation for the decline in the number of cases. Evaluation of 10 WEEV strains representing a variety of isolation locations, hosts, and all decades from the 1940's to the 1990's yielded no evidence of a decline in virulence. These results suggest that ecological factors affecting human and equine exposure should be investigated to explain the decline in WEE.

Neurotropic alphaviruses, which include western equine encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. We previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted Sindbis virus infection in mice. We describe here further development of this series, resulting in 10-fold improvement in potency in a WEEV replicon assay and up to 40-fold increases in half-lives in mouse liver microsomes. Using a rhodamine123 uptake assay in MDR1-MDCKII cells, we were able to identify structural modifications that markedly reduce recognition by P-glycoprotein, the key efflux transporter at the blood-brain barrier. In a preliminary mouse PK study, we were able to demonstrate that two new analogues could achieve higher and/or longer plasma drug exposures than our previous lead and that one compound achieved measurable drug levels in the brain.

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90–100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.