Western Equine Encephalitis Research Articles

Outbreak of Western Equine Encephalitis Virus Infection Associated with Neurological Disease in Horses Following a Nearly 40-Year Intermission Period in Argentina.

Western equine encephalitis virus (WEEV) is a mosquito-borne arbovirus (genus Alphavirus, family Togaviridae) that has re-emerged in South America in late 2023, causing severe disease in both horses and humans after a nearly 40-year intermission period. We here describe the virological, serological, pathological, and molecular features of WEEV infection in horses during the 2023-2024 outbreak in Argentina. WEEV-infected horses developed neurological signs with mild to severe encephalitis associated with minimal to abundant WEEV-infected cells, as demonstrated by WEEV-specific in situ hybridization. The distribution of viral RNA was multifocal, with predominance within neuronal bodies, neuronal processes, and glial cells in the medulla oblongata and thalamic regions. Phylogenetic analysis of partial nsP4 sequences from three viral isolates obtained from three different provinces of Argentina support grouping with other temporally current WEEV strains from Uruguay and Brazil under a recently proposed novel lineage.

PCDH10 is a neuronal receptor for western equine encephalitis virus

PCDH10 is a neuronal receptor for western equine encephalitis virus

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

Serum amyloid A increases following routine vaccination of healthy adult horses.

To measure the effect of routine vaccination on serum amyloid A (SAA) concentration in apparently healthy horses. We hypothesized that routine vaccination would increase SAA in healthy horses. 21 apparently healthy client-owned horses and 15 Kansas State University College of Veterinary Medicine-owned horses. In experiment 1 (n = 8 horses), a blinded, randomized, prospective, crossover study was performed. Horses were either vaccinated (rabies, tetanus, West Nile, Eastern and Western equine encephalomyelitis, equine herpesvirus-1/-4, influenza) or administered saline, and SAA was measured at 6, 12, and 24 hours and daily until day 10 with a commercial lateral-flow immunoassay. In experiment 2 (n = 28 horses), a prospective, observational study measured SAA after vaccination at 12 and 24 hours and daily until day 10. A linear mixed-effect model with repeated measures over time blocked by horse tested the effect of treatment on SAA. A repeated-measures correlation tested the correlation between SAA and temperature. Over time, vaccinated horses had increased model-adjusted SAA compared to unvaccinated horses without clinical evidence of adverse reaction (P < .01). In experiment 1, the model-adjusted SAA after vaccination peaked on day 2 (median, 1,872 µg/mL; IQR, 1,220.8 to 2,402.5 µg/mL) and returned to normal (< 20 µg/mL) by day 9 (median, 6 µg/mL; IQR, 0.8 to 23.5 µg/mL) after vaccination. In experiment 2, vaccinated horses had increased SAA over time; temperature and SAA were not correlated (P = .78). Results of this study indicated that routine vaccination results in increased SAA concentration and provided evidence for a period of convalescence following vaccination. Measuring SAA for 10 days following vaccination cannot be used as an indicator of illness.

Western equine encephalomyelitis in Argentina after 35 years: The importance of an early and specific diagnosis

Western equine encephalomyelitis in Argentina after 35 years: The importance of an early and specific diagnosis

Outbreak of Western Equine Encephalitis in humans in Argentina and Uruguay during late 2023 to early 2024

Outbreak of Western Equine Encephalitis in humans in Argentina and Uruguay during late 2023 to early 2024

Shifts in receptors during submergence of an encephalitic arbovirus

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1–3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2–E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

Syrian Hamsters Model Does Not Reflect Human-like Disease after Aerosol Exposure to Encephalitic Alphaviruses.

Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies. Each year, there are a limited number of human cases of encephalitic alphaviruses; thus, licensure of a vaccine or therapeutic would require approval under the FDA animal rule. Approval under the FDA animal rule requires the disease observed in the animal model to recapitulate what is observed in humans. Currently, initial testing of vaccines and therapeutics is performed in the mouse model. Unfortunately, alphavirus disease manifestations in a mouse do not faithfully recapitulate human disease; the VEEV mouse model is lethal whereas in humans VEEV is rarely lethal. In an effort to identify a more appropriate small animal model, we evaluated hamsters in an aerosol exposure model of encephalitic alphavirus infection. The pathology, lethality, and viremia observed in the infected hamsters was inconsistent with what is observed in NHP models and humans. These data suggest that hamsters are not an appropriate model for encephalitic alphaviruses to test vaccines or potential antiviral therapies.

MVA-based vaccines are protective against lethal eastern equine encephalitis virus aerosol challenge in cynomolgus macaques.

MVA-based monovalent eastern equine encephalitis virus (MVA-BN-EEEV) and multivalent western, eastern, and Venezuelan equine encephalitis virus (MVA-BN-WEV) vaccines were evaluated in the cynomolgus macaque aerosol model of EEEV infection. Macaques vaccinated with two doses of 5 × 108 infectious units of the MVA-BN-EEEV or MVA-BN-WEV vaccine by the intramuscular route rapidly developed robust levels of neutralizing antibodies to EEEV that persisted at high levels until challenge at day 84 via small particle aerosol delivery with a target inhaled dose of 107 PFU of EEEV FL93-939. Robust protection was observed, with 7/8 animals receiving MVA-BN-EEEV and 100% (8/8) animals receiving MVA-BN-WEV surviving while only 2/8 mock vaccinated controls survived lethal challenge. Complete protection from viremia was afforded by both vaccines, with near complete protection from vRNA loads in tissues and any pathologic evidence of central nervous system damage. Overall, the results indicate both vaccines are effective in eliciting an immune response that is consistent with protection from aerosolized EEEV-induced disease.

The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses

Members of the low-density lipoprotein receptor (LDLR) family, including LDLRAD3, VLDLR, and ApoER2, were recently described as entry factors for different alphaviruses. However, based on studies with gene edited cells and knockout mice, blockade or abrogation of these receptors does not fully inhibit alphavirus infection, indicating the existence of additional uncharacterized entry factors. Here, we perform a CRISPR-Cas9 genome-wide loss-of-function screen in mouse neuronal cells with a chimeric alphavirus expressing the Eastern equine encephalitis virus (EEEV) structural proteins and identify LDLR as a candidate receptor. Expression of LDLR on the surface of neuronal or non-neuronal cells facilitates binding and infection of EEEV, Western equine encephalitis virus, and Semliki Forest virus. Domain mapping and binding studies reveal a low-affinity interaction with LA domain 3 (LA3) that can be enhanced by concatenation of LA3 repeats. Soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in cell culture and in mice. Our results establish LDLR as a low-affinity receptor for multiple alphaviruses and highlight a possible path for developing inhibitors that could mitigate infection and disease.

Exploring the Intricacies of Encephalitis in Mosquito-Borne Diseases through Vector-Host-Pathogen Interactions: A Critical Review

Environmental changes, medication resistance and sociodemographic shifts have all contributed to a dramatic increase in vector-borne diseases in the last 40 years, impacting both people and domestic animals. Pandemics like the Dengue fever epidemic that hit Bangladesh in 2023 show how devastating these illnesses may be on a global scale. The authors of this work stress the significance of comprehending vector-host-pathogen pathways via their examination of arboviruses in Asia. The ecology and biology of Culex, Culiseta and Aedes species in connection to Dengue Virus, Japanese Encephalitis (JE), Eastern Equine Encephalitis (EEE) and Western Equine Encephalitis (WEE) are explored in a thorough literature review that utilizes MeSH terminology. Beginning with the Japanese Encephalitis Virus (JEV), this article examines the virus’s transmission from insects to vertebrates and, inadvertently, to humans as a disease. We then go on to dengue encephalitis, breaking its intricate pathophysiology into parts. This includes aspects like immune-mediated consequences, systemic problems and direct invasion. The Aedes mosquito is a key player in the human-mosquito-human cycle that transmits Dengue Virus (DENV) and the extrinsic incubation time impacts outbreaks. Continuing to focus on the neuroinvasive effects on horses and people, we have Eastern Equine Encephalopathy (EEE). The interaction between birds and Culiseta melanura mosquitoes highlights the transmission of the enzootic cycle. At last, we look at Western Equine Encephalitis (WEE), which WEEV causes and how it affects both horses and people. Importantly, Culex species, which include mosquitoes and birds, serve as vectors in the enzootic cycle. Factors including climate change and international travel are included in the study’s conclusion, which emphasizes the significance of continuing research to monitor and reduce the worldwide effect of these arboviruses.

Vertebrate-class-specific binding modes of the alphavirus receptor MXRA8

MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups invitro and invivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.

Severe Neurologic Disease in a Horse Caused by Tick-Borne Encephalitis Virus, Austria, 2021.

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses.

Hemp (Cannabis sativa cv. Kompolti) essential oil and its nanoemulsion: Prospects for insecticide development and impact on non-target microcrustaceans

Formulation development and non-target effect studies are two crucial steps for the registration of new botanical insecticides. Previous reports put in evidence the potential of hemp (Cannabis sativa L.) essential oil (EO) as a new candidate ingredient for developing green insecticides. However, the validation of its target and non-target effects after proper encapsulation is missing so far. In the current work, we investigated the insecticidal efficacy of the EO obtained from the hemp variety ‘Kompolti’, and we optimized its formulation development into a nanoemulsion (NE). The latter was assayed against larvae of Culex quinquefasciatus Say, a well-known vector of Wuchereria bancrofti Cobbold, avian malaria, and several arboviruses, such as Western equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, and Zika virus. Considering the percentage of encapsulated EO (7.5% w/w), the developed NE showed mortality on Cx. quinquefasciatus larvae, in terms of LC50(90) values [72.2 (207.2) ppm], like those of pure EO [50.8(142.3) ppm]. Non-target toxicity experiments showed limited toxicity on the aquatic microcrustacean Daphnia magna Straus (i.e., mortality <16% after 48 h of exposure to the EO-based NE at the LC90 estimated on mosquito larvae). Considering that the LC50 values detected in this study are lower than those belonging to many EOs, and that the application at sublethal doses caused mortality between 39.1% and 40.8% as well as a reduction in mosquito fertility resulting in a reduction in natality of 40.4% and 45.1% for the EO and EO-based NE, respectively, both products may be considered as potential environmentally friendly and sustainable insecticides.

Using fruit flies to delve into mosquito insecticide resistance

With more than 3,000 species and an almost ubiquitous presence, the economic importance of mosquitoes cannot be overemphasized. Anopheles mosquitoes are vectors for infectious diseases such as malaria – an endemic disease in tropical and sub-tropical regions of the world that infects more than 200 million people worldwide and causes over 400,000 deaths annually, with most casualties being infants or inhabitants of sub-Saharan Africa. The Aedes aegytpi and Culex quinquefasciatus species of mosquitoes are also vectors for arboviruses such as chikungunya virus, dengue virus, western equine encephalitis virus, and Zika virus. Consequently, insecticides are frequently used to stem the population of mosquitoes. Nevertheless, mosquito insecticide resistance has emerged as a major problem that has contributed to numerous failed eradication campaigns for the aforementioned diseases. In this mini-review, we expound on how fruit flies (Drosophila melanogaster) could be a complementary model system for studying mosquito insecticide resistance, with the ultimate goal of confirming any promising leads in mosquitoes.

Vaccine elicitation and structural basis for antibody protection against alphaviruses

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect invivo against a broad range of alphaviruses.

Isolation of Flaviviruses and Alphaviruses with Encephalitogenic Potential Diagnosed by Evandro Chagas Institute (Pará, Brazil) in the Period of 1954-2022: Six Decades of Discoveries.

Viruses with encephalitogenic potential can cause neurological conditions of clinical and epidemiological importance, such as Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Dengue virus, Zika virus, Chikungunya virus, Mayaro virus and West Nile virus. The objective of the present study was to determine the number of arboviruses with neuroinvasive potential isolated in Brazil that corresponds to the collection of viral samples belonging to the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute (SAARB/IEC) of the Laboratory Network of National Reference for Arbovirus Diagnosis from 1954 to 2022. In the analyzed period, a total of 1,347 arbovirus samples with encephalitogenic potential were isolated from mice; 5,065 human samples were isolated exclusively by cell culture; and 676 viruses were isolated from mosquitoes. The emergence of new arboviruses may be responsible for diseases still unknown to humans, making the Amazon region a hotspot for infectious diseases due to its fauna and flora species characteristics. The detection of circulating arboviruses with the potential to cause neuroinvasive diseases is constant, which justifies the continuation of active epidemiological surveillance work that offers adequate support to the public health system regarding the virological diagnosis of circulating arboviruses in Brazil.

Evaluation of an equine multi-antigen rabies combination vaccine in pregnant mares

Mares are commonly vaccinated during pregnancy, especially in the late third trimester, with multiple vaccine products containing a variety of antigens. Currently, a new core antigens vaccine containing rabies virus, tetanus toxoid, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus, and West Nile virus with and without the inclusion of Venezuelan equine encephalomyelitis virus is used in later term mares. Use of this vaccine has not been evaluated for safety in pregnant broodmares. We determined the safety of this vaccine in pregnant mares as it pertains to live foaling rate and per cycle pregnancy rate. Findings indicated that the use of a new multi-antigen rabies combination vaccine had no impact on live foaling rate nor per cycle pregnancy rates.

Sequelae and Animal Modeling of Encephalitic Alphavirus Infections.

Eastern (EEEV), Venezuelan (VEEV), and western equine encephalitis viruses (WEEV) are members of the genus Alphavirus, family Togaviridae. Typically spread by mosquitoes, EEEV, VEEV, and WEEV induce febrile illness that may develop into more severe encephalitic disease, resulting in myriad severe neurologic sequelae for which there are no vaccines or therapeutics. Here, we summarize the clinical neurologic findings and sequelae induced by these three encephalitic viruses and describe the various animal models available to study them. We emphasize the crucial need for the development of advanced animal modeling combined with the use of telemetry, behavioral testing, and neuroimaging to facilitate a detailed mechanistic understanding of these encephalitic signs and sequelae. Through the use of these systems, much-needed therapeutics and vaccines can be developed.