Equine Encephalitis Virus Research Articles

Peculiarities of Circulation of Venezuelan Equine Encephalomyelitis Virus (Scientific Review)

Reviewed are some peculiarities of circulation of Venezuelan equine encephalomyelitis virus (VEE), the enzootic strains of which play an increasingly important part as etiological agents of human infections nowadays. The majority of VEE cases among the residents of the endemic regions within the inter-epidemic period are caused by the ID subtype of VEE that includes 6 genotypes. Isolated from humans have been the genotypes Columbia/Venezuela, Panama/Peru, and Peru/Bolivia, ID subtype of VEE. Possibility of emergence of new VEE outbreaks is associated with ID subtype mutation into the IC subtype, which is assumed to be a potential threat as etiological agent of the disease at present.

Immuno-Chromatographic Wicking Assay for the Rapid Detection of Dengue Viral Antigens in Mosquitoes (Diptera: Culicidae)

There is a threat for dengue virus (DENV) reemergence in many regions of the world, particularly in areas where the DENV vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), are readily available. However, there are currently no accurate and reliable diagnostic methods to provide critical, real-time information for early detection of DENV within the vector populations to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect DENV in a pool of female Aedes mosquitoes infected with any of the four viral serotypes. The DENV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 30 min. It was highly specific and did not cross-react with samples spiked with West Nile, yellow fever, Japanese encephalitis, Rift Valley fever, chikungunya, Venezuelan equine encephalomyelitis, Ross River, LaCrosse, or Caraparu viruses. The DENV assay can provide real-time critical information on the presence of DENV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

Leukocyte Response to Eastern Equine Encephalomyelitis Virus in a Wild Passerine Bird

Leukocyte counts are frequently used to assess the immunologic status of animals; however, few studies have directly looked at the predictive value of leukocyte counts and an animal's ability to respond to an infection with a pathogen. Understanding how an animal's leukocyte profile is altered by an active infection can assist with interpretation of leukocyte profiles in animals for which infection status is not known. In this study we examine the leukocyte counts of gray catbirds (Dumetella carolinensis) infected with eastern equine encephalomyelitis virus (EEEV). Blood smears were collected from infected catbirds on -4, 2, 5, and 14 days postinoculation (dpi) with EEEV, and from a corresponding uninfected control group, to monitor leukocyte counts. Although we found that preinfection leukocyte counts were not a reliable predictive of a catbird's viremia, we did find that infected catbirds exhibited significant hematologic changes in response to EEEV infection. We observed a significant drop in all subpopulations of leukocytes (i.e., lymphocytes, monocytes, and granulocytes) following infection. Lymphocytes and granulocytes still had not recovered to preinfection levels at 14 dpi. Uninfected catbirds also exhibited statistically significant changes in leukocyte counts, but this was due to a slight increase at 14 dpi and was not considered biologically relevant. Studies such as this can provide important information for field ecoimmunologists that use leukocyte counts to assess immunocompetence in free-living animals.

Novel alphavirus-based vaccine induces strong immune responses against prostate-specific antigen (PSA) and mediates tumor cell elimination in a mouse model of prostate cancer

Prostate cancer is the second most frequently diagnosed cancer in men worldwide and sixth leading cancer-related cause of death in males. Treatments for recurrent prostate cancer are only rarely curative, and the majority of patients eventually demonstrate progressive disease. Animal and clinical studies suggested that immunological approaches can be useful for prostate cancer treatment and prevention. The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate-specific differentiation antigens is an attractive concept. Prostate specific antigen (PSA) is a serine protease of the glandular kallikrein gene family with highly restricted expression on prostate tumor cells and normal prostate. Previous attempts to develop PSA-based vaccines for prostate cancer primarily using poxvirus and adenovirus vectors resulted in promising clinical and immune responses. However, these approaches had limitations due to pre-existing antibody responses to viral vectors and insufficient safety. Previously we have constructed novel non-replicative alphavirus vector platform derived from live attenuated human TC-83 IND vaccine against Venezuelan equine encephalitis (VEE) virus infection. In earlier studies, vaccines based on alphaviruses demonstrated efficiency in targeted gene delivery to DC and induction of protective CTL immunity against several human viral pathogens and breast cancer in a rodent animal model. In this study we tested novel TC-83 based PSA-encoding alphavirus vaccine (vVLP-PSA) against prostate cancer using transgenic mouse model with established neonatal tolerance to human PSA. Preventive vaccination using vVLP-PSA demonstrated induction of strong PSA-specific CD8+ T-cell response as detected by IFNγ ELISPOT assay and intracellular cytokine staining. Potent expansion of PSA-specific CD8+ effector T-cells was found in peripheral blood and spleens of vaccinated mice using PSA peptide-loaded MHC I dextramers. vVLP-PSA vaccination also induced PSA-specific antibody production in immunized mice. Injection of TRAMP tumor cells expressing PSA into vaccinated mice resulted in elevated infiltration of tumor site with CD8+ T-cells and rapid elimination of PSA-expressing tumor cells during first several weeks after tumor challenge. Tumor survival analysis revealed tendency for delayed tumor growth in vVLP-PSA immunized mice. Altogether, our data demonstrate that TC83-based vaccine encoding PSA induces strong PSA-specific CTL response and mediates efficient elimination of antigen-expressing prostate tumor cells promptly after tumor challenge.

Natural products as leads to potential mosquitocides

Mosquitoes are the crucial vectors for a number of mosquito-borne infectious diseases i.e. dengue, yellow fever, chikungunya, malaria, Rift Valley fever, elephantiasis, Japanese Encephalitis, and Murray Valley encephalitis etc. Besides, they also transmit numerous arboviruses (arthropod-borne viruses) for example West Nile virus, Saint Louis encephalitis virus, Eastern equine encephalomyelitis virus, Everglades virus, Highlands J virus, and La Crosse Encephalitis virus. The emergence of widespread insecticide resistance and the potential environmental issues associated with some synthetic insecticides (such as DDT) has indicated that additional approaches to control the proliferation of mosquito population would be an urgent priority research. The present review highlights some natural product mosquitocides that are target-specific, biodegradable, environmentally safe, and botanicals in origin.

Serologic Responses of West Nile Virus Seronegative Mature Horses to West Nile Virus Vaccines

A 42-day study was conducted to assess the impact of three West Nile virus vaccines given either as separate injections or incorporated with their counterpart equine encephalitis and tetanus vaccines on serological responses under field use conditions. Two hundred forty mature, West Nile virus seronegative (<4) horses were followed serologically pre- and postprimary and secondary vaccination with six different vaccination programs, all including West Nile virus antigens. Forty horses were unvaccinated sentinel horses. All vaccines stimulated both a primary and secondary (booster) response to vaccination that was significantly higher than that of seronegative controls. However, inclusion of West Nile virus with equine encephalitis viruses and tetanus toxoid in vaccines had a significant detrimental impact on West Nile virus serum neutralization antibody production to both the primary and secondary vaccinations.

Risk of Exposure to Eastern Equine Encephalomyelitis Virus Increases with the Density of Northern Cardinals

For a variety of infectious diseases, the richness of the community of potential host species has emerged as an important factor in pathogen transmission, whereby a higher richness of host species is associated with a lowered disease risk. The proposed mechanism driving this pattern is an increased likelihood in species-rich communities that infectious individuals will encounter dead-end hosts. Mosquito-borne pathogen systems potentially are exceptions to such “dilution effects” because mosquitoes vary their rates of use of vertebrate host species as bloodmeal sources relative to host availabilities. Such preferences may violate basic assumptions underlying the hypothesis of a dilution effect in pathogen systems. Here, we describe development of a model to predict exposure risk of sentinel chickens to eastern equine encephalitis virus (EEEV) in Walton County, Florida between 2009 and 2010 using avian species richness as well as densities of individual host species potentially important to EEEV transmission as candidate predictor variables. We found the highest support for the model that included the density of northern cardinals, a highly preferred host of mosquito vectors of EEEV, as a predictor variable. The highest-ranking model also included Culiseta melanura abundance as a predictor variable. These results suggest that mosquito preferences for vertebrate hosts influence pathogen transmission.

Spatial epidemiology of eastern equine encephalitis in Florida

BackgroundEastern Equine Encephalitis virus (EEEV) is an alphavirus with high pathogenicity in both humans and horses. Florida continues to have the highest occurrence of human cases in the USA, with four fatalities recorded in 2010. Unlike other states, Florida supports year-round EEEV transmission. This research uses GIS to examine spatial patterns of documented horse cases during 2005–2010 in order to understand the relationships between habitat and transmission intensity of EEEV in Florida.MethodsCumulative incidence rates of EEE in horses were calculated for each county. Two cluster analyses were performed using density-based spatial clustering of applications with noise (DBSCAN). The first analysis was based on regional clustering while the second focused on local clustering. Ecological associations of EEEV were examined using compositional analysis and Euclidean distance analysis to determine if the proportion or proximity of certain habitats played a role in transmission.ResultsThe DBSCAN algorithm identified five distinct regional spatial clusters that contained 360 of the 438 horse cases. The local clustering resulted in 18 separate clusters containing 105 of the 438 cases. Both the compositional analysis and Euclidean distance analysis indicated that the top five habitats positively associated with horse cases were rural residential areas, crop and pastureland, upland hardwood forests, vegetated non-forested wetlands, and tree plantations.ConclusionsThis study demonstrates that in Florida tree plantations are a focus for epizootic transmission of EEEV. It appears both the abundance and proximity of tree plantations are factors associated with increased risk of EEE in horses and therefore humans. This association helps to explain why there is are spatially distinct differences in the amount of EEE horse cases across Florida.

Detection of Eastern Equine Encephalomyelitis Virus RNA in North American Snakes

The role of non-avian vertebrates in the ecology of eastern equine encephalomyelitis virus (EEEV) is unresolved, but mounting evidence supports a potential role for snakes in the EEEV transmission cycle, especially as over-wintering hosts. To determine rates of exposure and infection, we examined serum samples from wild snakes at a focus of EEEV in Alabama for viral RNA using quantitative reverse transcription polymerase chain reaction. Two species of vipers, the copperhead (Agkistrodon contortrix) and the cottonmouth (Agkistrodon piscivorus), were found to be positive for EEEV RNA using this assay. Prevalence of EEEV RNA was more frequent in seropositive snakes than seronegative snakes. Positivity for the quantitative reverse transcription polymerase chain reaction in cottonmouths peaked in April and September. Body size and sex ratios were not significantly different between infected and uninfected snakes. These results support the hypothesis that snakes are involved in the ecology of EEEV in North America, possibly as over-wintering hosts for the virus.

Eastern Equine Encephalitis in Tennessee: 2002–2008

Human and equine outbreaks caused by eastern equine encephalomyelitis virus (EEEV) typically occur in North America adjacent to coastal wetlands associated with the presence of Culiseta melanura (Coquillet) mosquitoes. Eastern equine encephalomyelitis (EEE) is an emerging disease in Tennessee, as the first records of equine disease began in 2002. In 2006 we trapped and tested mosquitoes for EEEV at hardwood swamps in western Tennessee that were at the epicenter of a multi-equine outbreak in fall of 2005. Additionally, the Tennessee Valley Authority tested mosquito pools collected in Tennessee swamps from 2000 to 2007 for the presence of arboviruses. Two pools of EEEV positive Culex erraticus (Dyer and Knab) mosquitoes were found (one each in 2003 and 2004) in a county adjacent to where the 2005 outbreak occurred. In 2008, another EEE outbreak involving multiple horses occurred in West Tennessee. A brain specimen was collected from a horse during this outbreak and the first isolate of EEEV from Tennessee was obtained. In total, 74,531 mosquitoes collected from 2000 to 2008 were tested via polymerase chain reaction and VecTest for EEEV. The traditional enzootic vector, Cs. melanura, was found in low numbers at all collection sites. Cx. erraticus, however, was consistently found in high numbers and was the only mosquito species in which EEEV was detected. We suggest that EEE transmission may be maintained by Cx. erraticus in a nontraditional cycle. We discuss the importance of a nontraditional cycle from the perspective of EEEV adaptation and emergence.

State-of-the-Art Therapeutic Medical Countermeasures for Viral Threat Agents

In recent years, there has been an increase in the perceived threat of biological agents being used against civilian populations. This has prompted an urgent need for the development and procurement of medical countermeasures (MCMs) against highly pathogenic viruses that can prevent morbidity and mortality from infections caused by these agents. To date, antiviral drug development has been largely focused on clinically prevalent chronic infections due to their commercial viability. This has left a huge gap in the drug development path for acute infections of biodefense importance. In this review, we discuss the antiviral research and development initiatives focusing specifically on poxviruses, filoviruses, and equine encephalitis viruses (EEV). We discuss the benefits and technical challenges in the current development strategies and the hurdles in the licensure path for MCMs against these highly pathogenic viruses under the FDA Animal Rule, and we provide recommendations for the path forward.

Winter Biology of Wetland Mosquitoes at a Focus of Eastern Equine Encephalomyelitis Virus Transmission in Alabama, USA

At temperate latitudes, vectors and pathogens must possess biological mechanisms for coping with cold temperatures and surviving from one transmission season to the next. Mosquitoes that overwinter in the adult stage have been proposed as winter maintenance hosts for certain arboviruses. In the cases of West Nile virus (family Flaviviridae, genus Flavivirus) and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus), discovery of infected overwintering females lends support to this hypothesis, but for other arboviruses, in particular Eastern equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, EEEV), overwintering of the virus in mosquito hosts as not been demonstrated. In the current study, we collected overwintering mosquitoes from a focus of EEEV transmission in the southeastern United States to determine whether mosquitoes serve as winter maintenance hosts for EEEV and to document overwintering biologies of suspected vectors. No virus was detected via reverse transcription-polymerase chain reaction of > 500 female mosquitoes collected during three winters. Investigation into the winter biologies indicated that Anopheles punctipennis (Say), Culex erraticus (Dyar & Knab), Culex peccator Dyar & Knab, and Uranotaenia sapphirina (Osten Sacken) overwinter as females. Females of these species were collected from hollow trees and emergence traps placed over ground holes. Southern magnolia, Magnolia grandiflora L., trees were preferred overwintering sites of culicine mosquitoes. Emergence from underground overwintering sites peaked in mid-March, when air temperatures reached 18-22 degrees C, and the first blood-engorged females of Cx. erraticus and Cx. peccator were collected during this same period. Blood-fed Culex territans Walker females were collected as early as mid-February. This work provides insight into the overwintering biologies of suspected virus vectors at a site of active EEEV transmission and provides limited evidence against the hypothesis that EEEV persists through intertransmission periods in overwintering mosquitoes.

Inhibition of Dengue Virus by Targeting Viral NS4B Protein

We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 μM; and 50% cytotoxic concentration [CC(50)], >40 μM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis-P104L-abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.

Use of Sindbis/Eastern Equine Encephalitis Chimeric Viruses in Plaque Reduction Neutralization Tests for Arboviral Disease Diagnostics

Eastern equine encephalitis virus (EEEV) is a highly virulent, mosquito-borne alphavirus that causes severe and often fatal neurological disease in humans and horses in eastern North American, the Caribbean, and Mexico and throughout Central and South America. EEEV infection is diagnosed serologically by anti-EEEV-specific IgM detection, with confirmation by the plaque reduction neutralization test (PRNT), which is highly specific for alphaviruses. Live virus is used in the PRNT procedure, which currently requires biosafety level 3 containment facilities and select agent security in the case of EEEV. These requirements restrict the ability of public health laboratories to conduct PRNTs. Sindbis virus (SINV)/EEEV recombinant constructs have been engineered to express the immunogenic structural proteins from 2 wild-type EEEV strains in an attenuated form. These SINV/EEEVs, which are not classified as select agents, were evaluated as alternative diagnostic reagents in a PRNT using human, equine, and murine sera. The results indicate that the chimeric viruses exhibit specificity comparable to that of wild-type EEEV, with only a slight reduction in sensitivity. Considering their benefits in increased safety and reduced regulatory requirements, these chimeric viruses should be highly useful in diagnostic laboratories throughout the Americas.

Test of Recrudescence Hypothesis for Overwintering of Eastern Equine Encephalomyelitis Virus in Gray Catbirds

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) epizootics are infrequent, but they can lead to high mortality in infected horses and humans. Despite the importance of EEEV to human and animal health, little is known about how the virus overwinters and reinitiates transmission each spring, particularly in temperate regions where infected adult mosquitoes are unlikely to survive through the winter. One hypothesis to explain the mechanism by which this virus persists from year to year is the spring recrudescence of latent virus in avian reservoir hosts. In this study, we tested the recrudescence hypothesis with gray catbirds (Dumatella carolinensis) captured in northern Ohio (July-August 2007). Birds were experimentally infected with EEEV on 1 October 2007. In January 2008, they were then exposed to exogenous testosterone and/or extended photoperiod to initiate reactivation of latent EEEV infection. All birds became viremic with EEEV, with mean viremia of 6.0 log10 plaque-forming units/ml serum occurring at 1 d postinoculation. One male in the testosterone, long-day treatment group had EEEV viral RNA in a cloacal swab collected on 18 January 2008. Otherwise, no other catbirds exhibited reactivated infections in cloacal swabs or blood. Antibody titers fluctuated over the course of the study, with lowest titers observed in January 2008, which corresponded with the lowest mean weight of the birds. No EEEV viral RNA was detected in the blood, kidney, spleen, brain, liver, and lower intestine upon necropsy at 19 wk postinfection.

The Crab Hole Mosquito Blues

Venezuelan equine encephalomyelitis (VEE) epizoodemics were reported at 6–10-year intervals in northern South America beginning in the 1920s. In 1937, epizootic VEE virus was isolated from infected horse brain and shown as distinct from the North American equine encephalomyelitis viruses. Subsequently, epizootic and sylvatic strains were isolated in distinct ecosystems; isolates were characterized serologically as epizootic subtype I, variants A/B and C; or sylvatic (enzootic) subtype I, variants D, E, and F, and subtypes II, III, and IV. In 1969, variant I-A/B virus was transported from a major outbreak in northern South America to the borders of El Salvador, Guatemala, and Honduras. This musical poem describes the history and ecology of VEE viruses and the epidemiology of an unprecedented 1969 movement of VEE viruses from South America to equids and humans in Central America from Costa Rica to Guatemala and Belize and in Mexico and the United States that continued until 1972.

Infection dynamics of western equine encephalomyelitis virus (Togaviridae: Alphavirus) in four strains of Culex tarsalis (Diptera: Culicidae): an immunocytochemical study.

BACKGROUND: Vector competence describes the efficiency with which vector arthropods become infected with and transmit pathogens and depends on interactions between pathogen and arthropod genetics as well as environmental factors. For arbovirus transmission, the female mosquito ingests viremic blood, the virus infects and replicates in midgut cells, escapes from the midgut, and disseminates to other tissues, including the salivary glands. Virus-laden saliva is then injected into a new host. For transmission to occur, the virus must overcome several "barriers", including barriers to midgut infection and/or escape and salivary infection and/or escape. By examining the spatial/temporal infection dynamics of Culex tarsalis strains infected with western equine encephalomyelitis virus (WEEV), we identified tissue tropisms and potential tissue barriers, and evaluated the effects of viral dose and time postingestion. METHODS: Using immunostained paraffin sections, WEEV antigens were tracked in four Cx. tarsalis strains: two recently colonized California field strains - Coachella Valley, Riverside County (COAV) and Kern National Wildlife Refuge (KNWR); and two laboratory strains selected for WEEV susceptibility (high viremia producer, HVP), and WEEV resistance (WR). RESULTS AND CONCLUSIONS: Tissues susceptible to WEEV infection included midgut epithelium, neural ganglia, trachea, chorionated eggs, and salivary glands. Neuroendocrine cells in the retrocerebral complex were occasionally infected, indicating the potential for behavioral effects. The HVP and COAV strains vigorously supported viral growth, whereas the WR and KNWR strains were less competent. Consistent with earlier studies, WEEV resistance appeared to be related to a dose-dependent midgut infection barrier, and a midgut escape barrier. The midgut escape barrier was not dependent upon the ingested viral dose. Consistent with midgut infection modulation, disseminated infections were less common in the WR and KNWR strains than in the HVP and COAV strains. Once the virus disseminated from the midgut, all strains were able to develop salivary gland infections. The possible roles of observed pathology will be discussed in a subsequent paper.

Variation in Western Equine Encephalomyelitis Viral Strain Growth in Mammalian, Avian, and Mosquito Cells Fails to Explain Temporal Changes in Enzootic and Epidemic Activity in California

The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20-30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.

Dissemination of western equine encephalomyelitis virus in the potential vector, Culex pipiens pallens

Two western equine encephalomyelitis virus (WEEV) strains have been isolated in China. Our previous studies have verified that the mosquito Culex pipiens pallens Coquillett (Diptera: Culicidae) infected with WEEV was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. In this study, we observed the distribution of WEEV antigen in orally-infected Cx. p. pallens by immunohistochemistry in order to better understand the initial infection, dissemination, and transmission of WEEV in the potential vector. Orally-infected WEEV dissemination varied within the different tissues of Cx. p. pallens, with virus antigen consistently observed in the salivary glands, foregut, midgut epithelial cells, Malpighian tubules, hindgut, and ovarian follicles of some individuals after various days of extrinsic incubation. We suggest that Cx. p. pallens, the potential vector of WEEV, has the ability to harbor the virus through the alimentary system, and the midgut epithelial cell may be the initial site of WEEV replication after ingestion of a viremic blood meal.

REVIEW PAPER

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.