Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals for human use against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthritogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus, and Western equine encephalitis virus. 4'-Fluorouridine (4'-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4'-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4'-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4'-FlU in a mouse model. Although mutations against 4'-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4'-FlU for the treatment of alphavirus encephalitis.IMPORTANCEVenezuelan equine encephalitis virus (VEEV) is a mosquito-spread virus that can cause encephalitis in people and animals. There are no FDA-approved countermeasures to treat VEEV infections in humans. 4'-Fluorouridine (4'-FlU) is currently being developed to treat multiple viral infections, including VEEV. A major problem with antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we treated infected mice with 4'-FlU and measured how well the compound inhibited virus replication and prevented severe disease. In addition, we identified mutations in VEEV's polymerase that confer reduced susceptibility to 4'-FlU. We then assessed if viruses encoding for these mutations were still pathogenic. Although VEEV can develop mild resistance to 4'-FlU in vitro, administration of 4'-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4'-FlU. These results suggest that 4'-FlU has strong potential as a future treatment for alphavirus infections.

Equine meningoencephalomyelitis is an important cause of morbidity and mortality and is associated with a wide variety of infectious etiologies. Because of the lack of large retrospective studies, the prevalence and incidence of these diseases are unknown. Here we describe 171 cases of meningoencephalomyelitis in horses submitted to the Section of Anatomic Pathology at the New York State Animal Health Diagnostic Center (Cornell University, Ithaca, NY, USA) from 1996-2023. Neuroinflammatory disease was identified in 5.4% of submitted horses with a wide breed, age, and sex distribution. A parasitic cause was identified in 32 (19%) cases, with protozoa in 18 (11%) cases and metazoa in 14 (8%) cases. A viral cause was identified in 31 (18%) cases, corresponding to infection by equid alphaherpesvirus 1 (EqAHV1; 12 of 31, 39%), eastern equine encephalitis virus (10 of 31; 32%), West Nile virus (5 of 31; 16%), and rabies virus (4 of 31; 13%), followed by 14 bacterial (8%) cases and 7 fungal (4%) cases. Of the remaining 87 of 171 (51%) cases, 20 (23%) had some histologic features, although not conclusive, of protozoal disease, and 8 (9%) of EqAHV1 infection. However, 59 (68%) cases did not have any neuropathologic changes that would support a definitive diagnosis. Although we found the expected causes of equine meningoencephalomyelitis in our study, the large number of cases with unknown etiologic diagnoses highlights the challenges of definitively proving causes of neuroinflammation in the horse and supports the need for improved ante- and postmortem testing.

Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for large-scale outbreaks in tropical regions. Its RNA replication depends on the assembly of functional replication complexes using the P123 and P1234 polyprotein precursors and their cleavage products, the nonstructural proteins (nsP1–nsP4). To dissect this process, we developed a trans-complementation assay using either plasmid-based expression or tetracycline-inducible stable cell lines expressing individual nsPs to rescue the activities of defective replicases. CHIKV nsP1, as well as nsP1 from closely related alphaviruses such as Ross River virus, successfully complemented CHIKV replicases carrying RNA capping-deficient mutations in nsP1. However, no complementation was observed for a replicase with an nsP1 mutation that completely disrupted membrane association. CHIKV and Eastern equine encephalitis virus (EEEV) nsP4 formed functional replication complexes with matching P123, as well as with P123 from most of alphaviruses. Genomes of CHIKV and EEEV lacking the nsP4 region remained infectious in cells expressing the corresponding nsP4 and could be propagated under these conditions. CHIKV replicase containing a mutation in the protease active site of nsP2 was also rescued by transient expression of wild-type nsP2. In contrast, replicases with mutations in the active site of the NTPase/RTPase/helicase domain of nsP2, or in nsP3 affecting phosphorylation or ADP-ribose binding/hydrolysis, could not be complemented. These results reveal key functional interdependencies among CHIKV nonstructural proteins. The inducible cell lines and trans-complementation platform for CHIKV and EEEV lacking nsP4 represent valuable tools for generating conditionally infectious virus systems and for facilitating high-throughput antiviral and neutralizing antibody screening under lower biosafety conditions.

An alphavirus vaccine development utilizing RNA replication-defective strategy.

ABSTRACTMost human pathogens are zoonotic, transmitted from vertebrate hosts to humans. However, it is still unclear how the topology of host co‐occurrence networks may contribute to disease transmission. To address this uncertainty, we examined the host co‐occurrence networks of 22 zoonotic pathogens from six continents (70 networks). First, we distinguished two major gradients of variability in host network topology—size (numbers of nodes and edges) and connectance/modularity. Larger networks with high connectance but low modularity have a greater potential for zoonotic disease transmission. These networks encompassed the hosts of 10 pathogens that cause emerging, re‐emerging, and/or genetically diversifying diseases: St. Louis encephalitis virus, influenza A virus, West Nile virus, Toxoplasma gondii, Eastern equine encephalitis virus, Avian orthoavulavirus 1, Japanese encephalitis virus, Usutu virus, Sindbis virus, and Coxiella burnetii. Second, we identified the top 87 hosts with the most connections to other hosts across networks, for example, Columba livia (rock pigeon), Passer domesticus (house sparrow), Hirundo rustica (barn swallow), Sturnus vulgaris (European starling), Anas platyrhynchos (mallard), and Gallinula chloropus (common moorhen). These species were highly connected in 7–27 networks of 2–11 pathogens. Notably, 50 of the 87 hosts were migratory, urban, or semi‐urban, highlighting the risk of zoonotic spread in developed areas.

We present an updated checklist of mosquito species in New York State, integrating historical records, modern surveillance, and recent literature. A total of 68 species across 10 genera are documented, including invasive taxa (Aedes albopictus, Ae. japonicus) and historically rare species (Orthopodomyia alba, Or. signifera). This synthesis emphasizes species of public and veterinary significance, such as Culex pipiens and Culiseta melanura, vectors of West Nile and eastern equine encephalitis viruses. Analysis of more than 3 million specimens collected since 1999 confirms the absence of Ae. aegypti, historically introduced but unable to persist in New York. Urbanization, container-breeding ecology, climate change, and global commerce continue to shape mosquito distributions and pathogen risk. This checklist bridges historic and current data, providing a reliable reference for surveillance, vector control, and future research.

Toward a more detailed understanding of the Eastern equine encephalitis virus (EEEV) cycle, individual blood-fed Culiseta melanura (Coquillett) mosquitoes were collected at an enzootic focus in New York State. Blooded females were tested for EEEV by multiplex real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR. Host bloodmeals were identified using DNA amplification and sequencing of the cytochrome B gene. In 2018, 577 individual mosquitoes were tested. Virus was detected in the bodies of 16, of which two had the virus in their legs. In 2022, 606 individual mosquitoes were tested, and the virus was detected in the bodies of six, of which one had the virus in its legs. Virus in the legs suggested a disseminated infection. The qRT-PCR cycle threshold (Ct) values for individual bodies ranged from 17.6 to 38.3, and for legs, 22.8 to 28.1. Host sources for 17 of the 22 were: Passeriformes, Bombycillidae, cedar waxwings (Bombycilla cedrorum); Icteridae, red-winged blackbird (Agelaius phoeniceus); Turdidae, American robin (Turdus migratorius), veery (Catharus fuscescens), and wood thrush (Hylocichla mustelina); Vireonidae, yellow-throated vireo (Vireo flavifrons); Passerellidae, song sparrow (Melospiza melodia), and field sparrow (Spizella pusilla); Parulidae, American redstart (Setophaga ruticilla); and Gruiformes, Rallidae, American coot (Fulica americana). In both years, red-winged blackbirds were a source of blood for Cs. melanura in the earliest 2 d after EEEV was detected. Annually, the earliest detection of the virus was in blooded-gravid mosquitoes before non-blooded mosquitoes in 15 of 17 yr. These results support the thesis that birds contribute to the introduction and re-emergence of EEEV to this enzootic focus.

Alphaviruses are transmitted by Aedes mosquitoes and cause large-scale epidemics worldwide. Chikungunya virus (CHIKV) infection can cause febrile seizures known as chikungunya fever (CHIKF), which ultimately leads to severe joint pain and myalgia. While a vaccine has recently been introduced against CHIKV, at present, no anti-viral drug is available. CHIKV, like other alphaviruses, has a short 6K protein capable of forming an ion channel. Blocking this ion channel with drugs can therefore serve as a potential way to curtail CHIKV infection. To that end, we screened a repurposed drug library using three bacteria-based channel assays to detect blockers against 6K viroporin, yielding several hits. Interestingly, several of the blockers were able to inhibit the 6K protein from the similar Eastern equine encephalitis virus (EEEV), while others were not, pointing to structural specificity which may be explained by modeling studies. In conclusion, our study provides a starting point for developing a new route to potentially inhibit CHIKV.

Venezuelan (VEEV), Eastern (EEEV), and Western (WEEV) equine encephalitis viruses are mosquito-transmitted alphaviruses in the family Togaviridae with the potential to cause fatal neuroinvasive disease in humans. These viruses can also be infectious when aerosolized and, thus, are potential biothreat agents. Human infection can progress rapidly to encephalitis, with fatality rates of 1 to 10% in symptomatic VEEV and WEEV infections and 30 to 70% in symptomatic EEEV infections. Currently, there are no antiviral agents or vaccines approved for encephalitic alphaviruses. An investigational live-attenuated VEEV vaccine was generated more than 40 years ago but is highly reactogenic, poorly immunogenic, and causes disease in up to 20% of recipients. Formalin-inactivated vaccines for EEEV and WEEV are also poorly immunogenic and no longer available. Here, we developed a trivalent vaccine against VEEV, EEEV, and WEEV using a combination of attenuated chimeric Sindbis-VEEV/EEEV/WEEV viruses to streamline production and an H2O2 inactivation treatment for enhanced safety and virion surface epitope preservation. The vaccines were adjuvanted with alum and tested for immunogenicity and protection in mouse and nonhuman primate models of lethal, aerosolized alphavirus infection. Two doses conferred complete protection in mice, and a similar regimen showed substantial or complete protection in nonhuman primates when administered at low and high doses, respectively. These results suggest that this inactivated vaccine is effective against the three encephalitogenic alphaviruses and may meet the need to counter the public health threat and biothreat posed by these viruses.

ABSTRACT The New World alphaviruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), and Venezuelan Equine Encephalitis Virus (VEEV), are known to cause neurological diseases that pose a significant threat to public health concerns and bioterrorism preparedness challenges due to their potential for aerosol transmission. Currently, no FDA-approved vaccines or antiviral drugs are available for humans, although ongoing studies are exploring potential solutions. Most vaccine evaluation methods rely on live virus models, which require handling in biosafety level 3 (BSL-3) facilities. In this study, we constructed pseudoviruses for NW alphaviruses using the vesicular stomatitis virus (VSV) system by expressing the glycoproteins E3-E2-E1 on the surface of the VSV vector. In vitro cell infection experiments revealed that the pseudovirus titres of EEEV and VEEV were comparatively higher. Bioluminescence imaging in a mouse model was used to assess infection in vivo. When injected into the brain, this was the main site of infection for NW alphavirus-based pseudoviruses. When administered via the tail vein, the pseudovirus primarily infected the spleen, while intraperitoneally injected pseudoviruses mainly infected the intestines and thymus. Furthermore, we systematically evaluated the correlation between neutralizing antibody titres induced by DNA immunization and the protection against homologous virus strains. This study establishes a safe, convenient, and efficient system for evaluating the protective effects of vaccines against NW alphaviruses, which can be operated in a BSL-2 facility.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus that can cause severe encephalitis in humans and horses with a high case fatality rate. There are no licensed EEEV vaccines or therapeutics for human use, warranting the need to better understand the human immune response against EEEV. Here we present a cryo-EM reconstruction of the chimeric virus, Sindbis (SINV)/EEEV, in complex with a potently neutralizing and efficacious intact human IgG1 antibody in a mouse model of infection and disease. This antibody requires bivalency to recognize a quaternary epitope on the E2 glycoprotein and cross-links two virus spikes across the icosahedral two-fold axis through a unique binding mode. Kinetic analysis of the binding interaction provides insights into this distinguishing feature. Mechanistically, the antibody inhibits viral entry into cells through blockade of receptor binding and early fusion events but does not block egress, thereby, exclusively targeting an epitope found on intact virions. The discovery of the quaternary epitope and unique binding mode recognized by this antibody together advance our understanding of the complexity of antibody-antigen interactions and can aid in vaccine design to elicit recognition of distinct epitopes of clinically relevant alphaviruses.

Naturally circulating strains of eastern equine encephalitis virus (EEEV) bind heparan sulfate (HS) receptors and this interaction has been linked to neurovirulence. Previous studies associated EEEV-HS interactions with three positively charged amino acid clusters on the E2 glycoprotein. One of these sites has recently been reported to be critical for binding EEEV to the very-low-density lipoprotein receptor (VLDLR), an EEEV receptor protein. The proteins apolipoprotein E receptor 2 (ApoER2) isoforms 1 and 2, and LDLR have also been shown to function as EEEV receptors. Herein, we investigate the individual contribution of each HS interaction site to EEEV HS- and protein receptor-dependent infection in vitro and EEEV replication in animals. We show that each site contributes to both EEEV-HS and EEEV-protein receptor interactions, providing evidence that altering these interactions can affect disease in mice and eliminate mosquito infectivity. Thus, multiple HS-binding sites exist in EEEV E2, and these sites overlap functionally with protein receptor interaction sites, with each type of interaction contributing to tissue infectivity and disease phenotypes.

Broad spectrum antiviral BDGR-164 provides protection against lethal neurotropic alphavirus infection in mice.

Ebola virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Chikungunya virus, Monkeypox virus, Eastern equine encephalitis virus, Tick-borne encephalitis virus, and Venezuelan equine encephalitis virus can cause severe infections and diseases, such as hemorrhagic fever, encephalitis, meningitis, and chikungunya fever. These viruses pose significant threats to public health due to their high infectivity and mortality rates, and they could potentially be used as bioterrorism agents. Early detection is the most critical step for effective prevention and control of infection. However, there is no sensitive nucleic acid detection method for the nine high-threat viral agents tested simultaneously. In this study, we developed multiplex probe amplification (MPA) with Melting Curve PCR method, named MPA-nine-viruses, which overcame the limitation of the available fluorescence channel number and realized simultaneous detection of nine high-threat viral agents in a single reaction, with high sensitivity and specificity for the targets.

Raptors may serve as both intermediate and definitive hosts for Sarcocystis spp. Past research has documented fatal encephalitis in raptors caused by various Sarcocystis spp., whereas other surveys have found a high prevalence of tissue cysts without evidence of disease. Little is known about the prevalence of Sarcocystis in raptors in the eastern USA. The aim of this study was to determine the occurrence of tissue cysts and histopathologic changes associated with Sarcocystis spp. infection in raptors in eastern Tennessee. Tissues of 33 raptors from Tennessee, USA, were assessed with histopathologic examination. Cysts consistent with Sarcocystis spp. were present in the heart, skeletal, or tracheal muscle of 11 (33%) raptors, without any associated inflammation. Tissues from histopathologic-positive raptors were then tested with PCR targeting of the 18S rRNA gene of Sarcocystis. Sequence analysis of PCR products revealed that six raptors had sequences most similar to Sarcocystis falcatula, and two had sequences most similar to Sarcocystis halieti. In addition, one S. falcatula-positive Bald Eagle (Haliaeetus leucocephalus) had lymphoplasmacytic meningoencephalitis and was positive via immunohistochemistry for eastern equine encephalitis virus. Our study supports findings in other geographic regions that raptors commonly serve as hosts for Sarcocystis spp. without evidence of associated disease.

EEEH!? A case of eastern equine encephalitis in a dog

Eastern equine encephalitis virus (EEEV) is an arthropod-borne, positive-sense RNA alphavirus posing a substantial threat to public health. Unlike similar viruses such as SARS-CoV-2, EEEV replicates efficiently in neurons, producing progeny viral particles as soon as 3–4 hours post-infection. EEEV infection, which can cause severe encephalitis with a human mortality rate surpassing 30%, has no licensed, targeted therapies, leaving patients to rely on supportive care. Although the general characteristics of EEEV infection within the host cell are well-studied, it remains unclear how these interactions lead to rapid production of progeny viral particles, limiting development of antiviral therapies. Here, we present a novel rule-based model that describes attachment, entry, uncoating, replication, assembly, and export of both infectious virions and virus-like particles within mammalian cells. Additionally, it quantitatively characterizes host ribosome activity in EEEV replication via a model parameter defining ribosome density on viral RNA. To calibrate the model, we performed experiments to quantify viral RNA, protein, and infectious particle production during acute infection. We used Bayesian inference to calibrate the model, discovering in the process that an additional constraint was required to ensure consistency with previous experimental observations of a high ratio between the amounts of full-length positive-sense viral genome and negative-sense template strand. Overall, the model recapitulates the experimental data and predicts that EEEV rapidly concentrates host ribosomes densely on viral RNA. Dense packing of host ribosomes was determined to be critical to establishing the characteristic positive to negative RNA strand ratio because of its role in governing the kinetics of transcription. Sensitivity analysis identified viral transcription as the critical step for infectious particle production, making it a potential target for future therapeutic development.

Eastern equine encephalitis virus (EEEV) is an arthropod-borne, positive-sense RNA alphavirus posing a substantial threat to public health. Unlike similar viruses such as SARS-CoV-2, EEEV replicates efficiently in neurons, producing progeny viral particles as soon as 3-4 hours post-infection. EEEV infection, which can cause severe encephalitis with a human mortality rate surpassing 30%, has no licensed, targeted therapies, leaving patients to rely on supportive care. Although the general characteristics of EEEV infection within the host cell are well-studied, it remains unclear how these interactions lead to rapid production of progeny viral particles, limiting development of antiviral therapies. Here, we present a novel rule-based model that describes attachment, entry, uncoating, replication, assembly, and export of both infectious virions and virus-like particles within mammalian cells. Additionally, it quantitatively characterizes host ribosome activity in EEEV replication via a model parameter defining ribosome density on viral RNA. To calibrate the model, we performed experiments to quantify viral RNA, protein, and infectious particle production during acute infection. We used Bayesian inference to calibrate the model, discovering in the process that an additional constraint was required to ensure consistency with previous experimental observations of a high ratio between the amounts of full-length positive-sense viral genome and negative-sense template strand. Overall, the model recapitulates the experimental data and predicts that EEEV rapidly concentrates host ribosomes densely on viral RNA. Dense packing of host ribosomes was determined to be critical to establishing the characteristic positive to negative RNA strand ratio because of its role in governing the kinetics of transcription. Sensitivity analysis identified viral transcription as the critical step for infectious particle production, making it a potential target for future therapeutic development.

Eastern equine encephalomyelitis virus (EEEV) is endemic in Michigan, showing an upsurge in human cases and in infections of white-tailed deer, horses, and other animals in the past decade (2010-2020). However, blood-host associations of the enzootic mosquito vector Culiseta melanura in the Great Lakes region are poorly known compared with other better-studied regions. Vertebrate sources of blood meals of Cs. melanura collected from resting boxes were determined through sequencing of the mitochondrial cytochrome B gene generated from polymerase chain reaction. Thirty-six unique avian species were detected in the samples, and 42% of the blood meals originated from only two species (American robin and northern cardinal). This result shows that although the Cs. melanura population investigated here used a wide range of avian hosts, American robin and northern cardinal are the main hosts in southwestern Michigan.

Optimization of a panel of behavioral tests for use in containment using a golden Syrian hamster model.