Western Equine Encephalitis Virus in Birds

Overview and Taxonomy of Western Equine Encephalitis Virus in Avian Reservoirs

Western Equine Encephalitis Virus (WEEV) occupies a distinctive and epidemiologically critical position within the genus Alphavirus, family Togaviridae, representing one of the most historically significant arboviral pathogens of the Americas. Taxonomically, WEEV is classified within the Western Equine Encephalitis (WEE) antigenic complex, a group of closely related alphaviruses that also includes Eastern Equine Encephalitis Virus (EEEV) and Venezuelan Equine Encephalitis Virus (VEEV), among others. This complex is characterized by serological cross-reactivity and shared ecological traits, most notably the utilization of avian species as primary amplification hosts in enzootic cycles [15]. The WEEV virion is a spherical, enveloped particle approximately 65–70 nm in diameter, housing a single-stranded, positive-sense RNA genome of roughly 11.5 kb. The genomic architecture encodes four nonstructural proteins (nsP1–nsP4) essential for viral replication and three major structural proteins: the capsid (C), and two surface glycoproteins, E1 and E2, which form trimeric spikes mediating receptor attachment and membrane fusion [1, 8, 15]. Critically, WEEV is not a monotypic entity; extensive genomic and phylogenetic analyses have resolved multiple distinct lineages. Contemporary phylogenetic studies, particularly those arising from the 2023–2024 South American epizootic, have identified at least three major lineages: a historically recognized North American lineage (Lineage A), a South American lineage (Lineage B), and a novel, emerging lineage designated Lineage C, which has been responsible for the recent fatal equine cases in Brazil, Uruguay, and Argentina [12, 14]. This genetic diversity is not merely a taxonomic curiosity; it underpins significant variation in virulence, vector competence, and receptor tropism, factors that directly shape the virus’s behavior within avian reservoir populations.

The role of birds as the principal vertebrate reservoirs for WEEV is a foundational tenet of its enzootic transmission cycle, a paradigm established through pioneering field and experimental studies spanning over seven decades. Unlike the mammalian incidental hosts (humans and equids), which are considered dead-end hosts due to insufficient viremia to infect feeding mosquitoes, numerous avian species develop high-titer, prolonged viremias that are exquisitely tailored to infect the primary mosquito vector, Culex tarsalis, and other ornithophilic Culex spp. [3, 11, 15]. This ecological specialization has profound consequences: avian reservoir competence directly dictates the intensity of viral amplification and the consequent risk of spillover into human and equine populations. Critically, the relationship between WEEV and its avian hosts is not a simple binary of infection versus resistance; it represents a complex, co-evolutionary dynamic shaped by differential susceptibility, variable duration of viremia, and the emergence of chronic infections.

Molecularly, the avian host tropism of WEEV is now being understood at an unprecedented structural resolution, revealing a sophisticated mechanism of receptor recognition that distinguishes pathogenic from attenuated strains. For decades, the very-low-density lipoprotein receptor (VLDLR) and its family members were recognized as entry mediators for WEEV; however, this paradigm has been fundamentally refined by the recent discovery that protocadherin 10 (PCDH10) serves as a more universal and physiologically relevant receptor for WEEV entry into neuronal cells [1, 8, 10]. Cryo-electron microscopy studies have elucidated the precise atomic interactions: the extracellular cadherin repeat 1 (EC1) of PCDH10 inserts into a cleft formed between adjacent E2-E1 heterodimers within a single trimeric spike on the viral surface [8, 10]. Astonishingly, this receptor usage exhibits a species-specific shift that is directly relevant to avian reservoirs. The nonpathogenic, mosquito-adapted strain Imperial-181 has lost the ability to bind to mammalian PCDH10 due to a critical mutation (E2Q153L), yet it retains the capacity to bind avian PCDH10 [10]. This differential binding is contingent upon a single amino acid residue at position 89 of avian PCDH10 (arginine), which is absent in the mammalian ortholog [10]. This elegant structural finding provides a molecular explanation for the presumed attenuation of Imperial-181 in mammals while preserving its competence for replication in avian hosts, thereby maintaining its enzootic fitness. Concurrently, virulent epidemic strains like McMillan have retained the ability to engage multiple receptors, including VLDLR and ApoER2, in a strain-dependent manner, with the E2 V265F mutation conferring VLDLR-binding capability that expands mammalian tropism and neuroinvasiveness [8, 9]. Therefore, the taxonomic and pathogenic diversity of WEEV is inextricably linked to its receptor usage, with avian-adapted strains representing a distinct ecological subset that underpins the enzootic cycle.

Experimental infection studies have systematically catalogued the differential host competence of various avian species, providing a quantitative framework for understanding reservoir dynamics. In comprehensive inoculation studies across 27 bird species from California, 11 of 20 species inoculated with WEEV were deemed competent hosts, defined as sustaining a viremia of ≥2 log₁₀ PFU/0.1 mL, the established threshold for infecting Culex tarsalis [3]. Crucially, this study revealed that species with the highest field antibody prevalence were not necessarily the most competent experimental hosts, challenging assumptions based solely on serosurveillance. The house finch (Haemorhous mexicanus), a common peridomestic species, consistently demonstrated high viremia titers and robust antibody responses, while mourning doves (Zenaida macroura) exhibited a more nuanced pattern, with some individuals harboring infectious virus for extended periods [3, 5]. The landmark work of Hammon, Reeves, and Sather in the early 1950s using wild birds from Kern County, California, demonstrated that viremia could reach titers of 10⁻⁶ and persist for 3–4 days following subcutaneous inoculation [4]. This level of viremia is more than sufficient to infect feeding mosquitoes and underscores the remarkable amplification potential within avian communities. Notably, the same study found that St. Louis encephalitis virus (SLEV) produced significantly lower viremias in the same avian species [4], highlighting the uniquely high replication capacity of WEEV in birds. Immunosuppression experiments, using agents like cyclophosphamide and dexamethasone, have further elucidated the host-pathogen interplay, showing that immunocompromised states can dramatically elevate and prolong viremia, although this effect was more pronounced for SLEV than for WEEV [5, 6]. These findings suggest that natural variations in avian immune status, possibly due to stress, co-infection, or nutritional deficits, could create "superspreader" individuals that disproportionately contribute to viral amplification.

The phenomenon of chronic WEEV infection in birds remains one of the most intriguing and controversial aspects of its enzootic biology, with direct implications for viral overwintering and geographic dispersal. Overwintering, the persistence of WEEV during periods of low vector activity, has long been a puzzle in arbovirology, given the lack of vertical transmission in Culex tarsalis and the short lifespan of adult mosquitoes. Experimental evidence has demonstrated that a small proportion of infected birds can harbor viral RNA for extended periods exceeding six weeks post-inoculation, and infectious virus has been recovered from mourning doves and other species after blind passage in mosquito cells [3, 5]. In the studies by Reisen et al., six birds (one house finch, three mourning doves, one Brewer’s sparrow, and one white-crowned sparrow) contained WEEV RNA detected by RT-PCR at necropsy more than six weeks post-inoculation [3]. However, attempts to induce viral recrudescence through immunosuppression with cyclophosphamide the following spring were unsuccessful in mourning doves, and no viral RNA was detected in sera, spleen, lung, or kidney tissues at necropsy [5]. These results suggest that while chronic infection can occur, it may be a rare event and not a reliable mechanism for population-level persistence. Nevertheless, the potential for chronically infected birds to serve as long-distance dispersal agents is profound, and the recent re-emergence of WEEV in South America during 2023–2024, after nearly 40 years of apparent silence, has been epidemiologically linked to migratory bird stopovers, strongly implicating avian transport in the introduction of the novel Lineage C into new geographic areas [2, 13]. This event underscores that the taxonomic classification of WEEV must be considered within a dynamic phylogeographic context, where avian movement patterns are the principal drivers of lineage distribution and emergence.

Evolutionary analyses of WEEV over the past century have revealed a pattern of enigmatic decline followed by re-emergence that is intimately tied to its avian reservoir hosts. During the mid-to-late 20th century, WEEV activity in North America experienced a dramatic and puzzling reduction in both enzootic circulation and equine/human disease incidence. Genomic studies investigating this "submergence" have uncovered evidence of positive selection acting upon specific amino acid residues during this period, particularly in the structural polyprotein [16]. Contrary to the hypothesis that genetic drift or fitness reductions were responsible for the decline, these positively selected mutations were found to confer a fitness advantage in the enzootic host-vector system (house sparrows and Culex tarsalis), while having no measurable effect on mammalian virulence in hamster models [16]. This remarkable finding indicates that the decline in WEEV activity was not due to viral attenuation in birds or mosquitoes, but rather to unidentified ecological factors, such as changes in land use, vector populations, or herd immunity in avian communities, that interrupted the amplification cycle. Importantly, residues associated with mammalian virulence were likely eliminated from the population through negative selection or genetic drift, not because they were detrimental to enzootic transmission [16]. This evolutionary trajectory has important taxonomic implications: the WEEV lineages circulating today in South America may represent a distinct ecotype that has re-optimized for avian amplification while retaining the capacity to cause severe neurological disease in spillover hosts, as tragically evidenced by 217 human cases and 12 fatalities in the 2023–2024 outbreak [2, 12].

The World Organisation for Animal Health (WOAH) and the World Health Organization (WHO) classify WEEV as a notifiable pathogen of significant public health and economic consequence, and the avian reservoir component is central to surveillance and control strategies. Serosurveillance of wild bird populations has historically been a cornerstone of arbovirus monitoring programs, with hemagglutination-inhibition (HI) and complement fixation-inhibition (CFI) tests being deployed extensively across the Americas. In the Atlantic Forest region of Brazil, a 13-year surveillance effort testing nearly 40,000 wild birds found that only 0.7% exhibited monotypic HI antibody responses for WEEV, with positive serology distributed across 66 species and 22 families, predominantly in unforested habitats and agricultural areas [7]. This low seroprevalence belies a high force of infection in certain species and highlights the challenge of detecting enzootic circulation before spillover events. Molecular diagnostic tools, including real-time PCR and multiplex amplicon sequencing, have now revolutionized the ability to detect WEEV in avian tissues and swabs, but official protocols have been compromised by primer-probe mismatches against contemporary South American lineages, necessitating continuous genomic surveillance to maintain diagnostic accuracy [2]. The integration of avian telemetry, viral phylogenetics, and ecological niche modeling, informed by climate change projections that predict shifts in high-risk areas toward northern South America and the Pantanal, is now essential for predicting and mitigating future epizootics [13]. In summary, the taxonomy of WEEV cannot be divorced from its avian reservoir biology; the virus’s genetic lineages, receptor specificity, evolutionary trajectory, and outbreak potential are all profoundly shaped by its interactions with the diverse and mobile avian communities that sustain it.

Molecular Pathogenesis of Western Equine Encephalitis Virus: Protocadherin-10 and Neuronal Entry Mechanisms in Avian and Mammalian Hosts

The molecular pathogenesis of Western equine encephalitis virus (WEEV) is fundamentally defined by its capacity to invade the central nervous system (CNS) and establish a productive infection within neurons. For decades, the identity of the cellular receptors mediating this neurotropism remained one of the most elusive questions in alphavirology. The recent identification of Protocadherin-10 (PCDH10) as a high-affinity neuronal receptor for WEEV has revolutionized our understanding of viral entry, tropism, and the differential pathogenesis observed between avian reservoir hosts and mammalian incidental hosts [1, 10]. This discovery, coupled with the structural elucidation of receptor engagement, provides a molecular framework for explaining the virus’s pronounced neurovirulence, its host range, and the evolutionary pressures that have shaped its receptor usage over time.

The Discovery and Structural Basis of PCDH10 as a WEEV Receptor

The seminal work by Yang et al. (2024) employed a comprehensive overexpression screen of 6,133 membrane-associated proteins in HEK293T cells, utilizing GFP-tagged pseudotyped viruses bearing the WEEV E2/E1 glycoproteins (SINV-WEEV and VSV-ΔG-WEEV). This unbiased approach identified PCDH10 as a potent mediator of viral entry, dramatically increasing infection rates for both pseudotypes [1]. PCDH10 is a member of the cadherin superfamily, characterized by its extracellular cadherin (EC) repeats, and is known to be enriched at neuronal synapses, playing critical roles in neural development and synaptic plasticity. Its expression pattern, highly concentrated within the CNS, immediately provided a compelling explanation for WEEV’s pronounced neurotropism.

Subsequent cryo-electron microscopy (cryo-EM) studies have provided atomic-level resolution of the WEEV-PCDH10 interaction. Liang et al. (2025) determined the structure of WEEV virus-like particles (VLPs) in complex with the first two extracellular cadherin repeats (EC1-EC2) of human PCDH10 at a remarkable 2.99 Å resolution [10]. The structural data reveal a highly specific binding mechanism: the EC1 domain of PCDH10 inserts deeply into a cleft formed by two adjacent E2-E1 heterodimers within a single trimeric spike on the viral surface. Crucially, the EC2 domain makes no direct contact with the viral envelope, indicating that the high-affinity interaction is mediated entirely by the N-terminal EC1 domain [10]. This binding mode is distinct from that of other alphavirus-receptor interactions, underscoring the unique evolutionary adaptation of WEEV to this neuronal protein. The residues on the viral E2 glycoprotein involved in this interaction are highly conserved among pathogenic WEEV strains but are absent in closely related alphaviruses, confirming the specificity of this receptor-ligand pair [8, 10].

Differential Receptor Usage: PCDH10, VLDLR, and the Molecular Basis of Host Tropism

A critical aspect of WEEV pathogenesis is its differential behavior in avian versus mammalian hosts. Birds serve as the primary amplifying reservoir, typically developing a high-titer viremia sufficient to infect feeding mosquitoes, yet they rarely exhibit overt neurological disease [3, 4]. In contrast, humans and equids are considered dead-end or incidental hosts, where infection frequently leads to severe, often fatal encephalitis [11, 18]. The molecular basis for this dichotomy is now being elucidated through the lens of receptor usage and structural compatibility.

While PCDH10 is a general receptor for WEEV, the virus also utilizes members of the low-density lipoprotein receptor (LDLR) family, including VLDLR and ApoER2, as alternative entry mediators [1, 8, 9]. However, receptor usage is not static across all WEEV strains. A landmark finding by Liang et al. (2025) demonstrated that nonpathogenic WEEV strains, such as Imperial-181, have evolved to lose the ability to bind mammalian PCDH10 while retaining affinity for avian PCDH10 [10]. This functional shift is governed by a single amino acid polymorphism at position 153 of the E2 glycoprotein. The pathogenic strain 71V-1658 possesses a glutamine at this position (E2-Q153), which is critical for PCDH10 binding. The nonpathogenic Imperial-181 strain carries a leucine substitution (E2-Q153L), which abrogates binding to human PCDH10 but not to its avian ortholog [10].

This finding has profound implications for understanding WEEV ecology and pathogenesis. The ability to bind avian PCDH10 allows the virus to efficiently infect and replicate in its natural reservoir hosts, birds, without causing significant neuropathology. The structural basis for this species-specificity lies in a single residue difference in the avian PCDH10 receptor itself. An arginine at position 89 (R89) on avian PCDH10 is essential for its interaction with the Imperial-181 strain, a residue that is not conserved in the mammalian ortholog [10]. This elegant co-evolutionary adaptation ensures that the virus can maintain its enzootic cycle in birds while limiting its pathogenic potential in this critical host population.

Conversely, the acquisition of mutations that permit binding to mammalian PCDH10, such as the E2-Q153L reversion or the E2-V265F mutation that enables VLDLR binding, confers the ability to invade the mammalian CNS with devastating consequences [8-10]. The virulent McMillan strain, for example, engages VLDLR through multiple LA repeats (LA1-2, LA2-3, etc.), which bind in a cleft between E2-E1 heterodimers, a mode distinct from other alphaviruses [8]. This suggests that pathogenic WEEV strains have evolved a multi-receptor strategy to enhance entry into mammalian neurons, where PCDH10 and VLDLR are both highly expressed.

Neuronal Entry and the Pathogenesis of Encephalitis

The molecular entry of WEEV into neurons is the initiating event in a cascade that culminates in severe encephalitis. Following a mosquito bite, the virus replicates locally in the skin and muscle, establishing a primary viremia. In birds, this viremia is robust and prolonged, often lasting 3–4 days with titers sufficient to infect feeding vectors [4]. In mammals, the viremia is typically lower and shorter-lived, but the virus is rapidly shuttled to the CNS. The mechanism of neuroinvasion is thought to involve direct infection of endothelial cells of the blood-brain barrier (BBB), or via a "Trojan horse" mechanism where infected leukocytes carry the virus across the BBB. Once within the CNS parenchyma, the expression of PCDH10 on neuronal cell surfaces provides a high-affinity docking site for the virus.

The cryo-EM structures reveal that PCDH10 engagement triggers viral internalization, likely via clathrin-mediated endocytosis, a common entry pathway for alphaviruses. The low pH of the endosome then catalyzes a conformational change in the E1 glycoprotein, leading to fusion of the viral and endosomal membranes and release of the viral genome into the cytoplasm [8, 10]. The specificity of this interaction is underscored by the fact that PCDH10 is not merely a tethering factor but a true entry receptor that facilitates the entire internalization process.

Once inside neurons, WEEV replication is rapid and cytopathic. The virus hijacks the host cell machinery, leading to profound neuronal dysfunction and death. Histopathological examination of infected brains, whether from experimental mouse models or naturally infected horses, reveals severe, bilateral lesions in rostroventral regions of the encephalon, particularly the olfactory bulb, piriform cortex, and thalamic regions [17, 19]. Viral antigen and RNA are detected predominantly within neuronal bodies and processes, with associated gliosis and inflammatory cell infiltration [19]. The pronounced involvement of the olfactory bulb in aerosol challenge models suggests that the olfactory nerve may serve as a direct route of neuroinvasion, bypassing the BBB entirely [17]. This is particularly relevant for understanding the high case fatality rate (up to 40%) associated with aerosol exposure, as the virus can rapidly access the CNS without requiring a systemic viremia [17].

Implications for Viral Evolution and Re-Emergence

The molecular understanding of WEEV receptor usage provides critical insights into the virus’s puzzling epidemiology. WEEV activity declined dramatically in North America during the late 20th century, a phenomenon that has been attributed to ecological factors rather than a loss of viral fitness [16]. Genomic analyses have revealed that during this period of decline, WEEV underwent positive selection for mutations that enhanced fitness in the enzootic host (birds) and vector (Culex tarsalis), while residues associated with mammalian virulence were likely eliminated by negative selection or genetic drift [16]. This suggests that the virus was under selective pressure to maintain its enzootic cycle, even at the cost of reduced virulence in incidental mammalian hosts.

The recent re-emergence of WEEV in South America (2023–2024), causing significant outbreaks in Argentina, Uruguay, and Brazil, underscores the virus’s continued threat [2, 12, 19]. Genomic characterization of these outbreak strains has identified a novel lineage (Lineage C) that is phylogenetically distinct from North American strains [12, 14]. The receptor usage profile of these emerging strains remains a critical area of investigation. Given that the outbreak originated in Argentina and spread via migratory bird movements [2, 13], it is plausible that these strains retain high affinity for avian PCDH10, enabling efficient amplification in bird populations. However, their ability to cause severe neurological disease in horses and humans suggests that they also possess the molecular determinants necessary for engaging mammalian PCDH10 and/or VLDLR [19].

The structural biology of the WEEV-receptor interaction also opens new avenues for therapeutic intervention. The highly conserved binding interface between the E2 glycoprotein and PCDH10 EC1 represents an attractive target for small-molecule inhibitors or monoclonal antibodies that could block viral entry. The development of such entry inhibitors is a priority, given the lack of approved vaccines or antivirals for WEEV [1, 11, 20]. Furthermore, the trivalent MVA-based vaccine platform, which encodes the envelope polyproteins of WEEV, EEEV, and VEEV, has shown complete protection in mouse aerosol challenge models, highlighting the potential for immunological targeting of the viral glycoproteins [21].

In summary, the molecular pathogenesis of WEEV is inextricably linked to its ability to exploit PCDH10 for neuronal entry. The differential binding affinity of WEEV strains for avian versus mammalian PCDH10 provides a molecular explanation for the distinct clinical outcomes observed in reservoir and incidental hosts. This receptor-mediated tropism, combined with the virus’s capacity to engage alternative receptors like VLDLR, underpins its neurovirulence and its potential for re-emergence. As WEEV continues to circulate and evolve, a deep understanding of these molecular mechanisms is essential for predicting future outbreak risk and developing effective countermeasures.

Epidemiology of Western Equine Encephalitis Virus in Birds: Outbreak Dynamics, Transmission Cycles, and Geographic Expansion

Western equine encephalitis virus (WEEV) persists in nature through an intricate enzootic cycle that relies almost exclusively on avian reservoir hosts and ornithophilic mosquito vectors, principally Culex tarsalis in North America and related Culex species in South America. Birds are not merely passive carriers; they are the amplification engines that drive the virus’s geographic expansion and determine the magnitude of epizootic spillover into equids and humans. Understanding the epidemiology of WEEV in birds requires a synthesis of historical outbreak patterns, experimental host-competence data, vector–host interactions, and the molecular evolutionary forces that shape contemporary viral lineages. The recent re‑emergence of WEEV in South America during 2023–2024, after a nearly 40‑year intermission, has reinvigorated research into these dynamics and underscores the critical role of migratory birds in translocating the virus across vast distances [2, 12, 19].

Transmission Cycles: Enzootic Maintenance and Epizootic Amplification

The classic transmission cycle of WEEV involves a continuous, year‑round enzootic system in which Culex tarsalis mosquitoes acquire the virus by feeding on viremic passerine birds, become persistently infected, and subsequently transmit the virus to new avian hosts. A viremia of at least 2 log₁₀ plaque‑forming units per 0.1 mL of blood is required to infect feeding Cx. tarsalis [3]. Experimental inoculations of 27 bird species from California’s San Joaquin and Coachella valleys revealed that 11 of 20 species tested met or exceeded this threshold and were therefore considered competent reservoir hosts; house finches, house sparrows, mourning doves, and Brewer’s sparrows consistently produced high‑titer viremias lasting 3–4 days [3, 4]. Hammon and colleagues demonstrated in the early 1950s that viremia titers could reach 10⁻⁶ in wild birds, with detectable virus appearing within 24 hours of inoculation and persisting for up to four days [4]. Antibody responses developed within 15–29 days, conferring lifelong immunity that protects against subsequent infection, though a small proportion of birds (e.g., mourning doves and white‑crowned sparrows) may harbor viral RNA for weeks without shedding infectious virus [3, 5].

Importantly, WEEV does not rely solely on the Culex–passerine dyad. During periods of high vector abundance, bridge vectors such as Aedes species may become involved, facilitating spillover from the enzootic cycle into accidental hosts (horses and humans) [15, 18]. However, the primary determinant of outbreak magnitude remains the density of competent avian reservoirs and the rate of infectious mosquito bites. The World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) have historically listed WEEV as a notifiable disease for equids, recognizing that avian amplification precedes equine epizootics by weeks [12, 19]. In South America, the 2023–2024 outbreak involved 2,548 equine cases and 217 human cases (12 fatal), all following a surge in Cx. tarsalis populations and seroconversions in sentinel birds [12].

Host Competence and the Role of Immunosuppression

Not all bird species contribute equally to WEEV amplification. Host competence is a composite measure of viremia magnitude, duration, and vector susceptibility. In a landmark California field study, only 11 of 20 species had a host competence index ≥1, meaning they sustained viremias above the transmission threshold for at least one day [3]. Interestingly, species with high field antibody prevalence (e.g., Western scrub‑jays) were not necessarily the most competent hosts, suggesting that exposure history and ecological exposure risk can decouple seroprevalence from reservoir efficiency [3]. Mourning doves, for example, develop poor viremias for St. Louis encephalitis virus but are moderately competent for WEEV; however, experimental immunosuppression with cyclophosphamide significantly augmented WEEV viremia in house finches, although it did not increase the frequency of chronic infection [5, 6]. These data reinforce the concept that stress, nutritional status, and co‑infections may temporarily elevate an individual bird’s infectiousness, but long‑term persistence is rare. The U.S. Centers for Disease Control and Prevention (CDC) has long recognized that chronic infections in birds are unlikely to serve as overwintering mechanisms, making the virus dependent on continuous transmission during warmer months.

A novel and underappreciated component of the enzootic cycle involves reptiles. DNA barcoding of blood meals from Culex mosquitoes in Florida and Arizona revealed that lizards (e.g., Anolis and Sceloporus species) constituted up to 60% of Cx. nigripalpus and Cx. tarsalis blood meals in southern regions [22]. Whether lizards can sustain WEEV viremia and infect mosquitoes remains poorly studied; preliminary work suggests they may act as dilution hosts for flaviviruses, but their role in alphavirus transmission is ambiguous. Given the extensive feeding upon lizards by key WEEV vectors, further investigation is urgently needed.

Geographic Expansion and Molecular Epidemiology

WEEV historically caused large, recurring summer outbreaks in the western United States and Canada, with periodic incursions into the Midwest. After the 1980s, enzootic circulation declined dramatically, and disease in humans and horses virtually disappeared [16]. Bergren et al. (2020) used phylogenetics to demonstrate that during this “submergence” period, six positively selected amino acid residues appeared in the viral genome, and competition experiments in house sparrows and Cx. tarsalis showed that these mutations conferred a fitness advantage in the enzootic cycle [16]. Paradoxically, this adaptation to birds and mosquitoes did not increase mammalian virulence, in fact, residues linked to virulence in hamsters were apparently lost through negative selection. The authors concluded that ecological factors (e.g., changes in irrigation, vector control, and land use) rather than viral fitness explains the North American decline [16]. Conversely, the 2023–2024 South American outbreak, centered in Argentina, Uruguay, and Brazil, has been traced to a novel lineage (proposed as lineage C) that shares a common ancestor with an Argentine strain from 1958 [2, 12, 14]. Phylogenomic and epidemiological evidence strongly implicates migratory birds as the vehicle for this expansion. The outbreak likely originated in Argentina and then spread northeast into Uruguay and the Brazilian state of Rio Grande do Sul along known migration flyways of the Southern Cone [2, 13].

Climate change models, using Maxent and climatic variables such as thermal seasonality and annual rainfall, forecast a poleward and eastward expansion of WEEV risk areas in South America, with Paraguay, northern Brazil, and the Pantanal becoming highly suitable [13]. The coincidence of the outbreak with migratory stopover sites reinforces the risk that birds will seed WEEV into previously unaffected regions. These projections are of grave concern to the CDC and PAHO, as WEEV is a category B select agent and a potential biothreat due to its aerosol infectivity [17, 21]. Currently, there are no licensed human vaccines; however, equine vaccines are available and are critical for outbreak control, as recommended by WOAH during the 2023–2024 epizootic [12, 19].

Viral Receptor Tropism and Implications for Avian–Mammalian Host Shifts

Recent structural biology advances have illuminated the molecular basis of WEEV host range. PCDH10, a neuronal cadherin, was identified as a universal receptor for all WEEV strains, while VLDLR and ApoER2 serve as alternative receptors for some strains [1, 8-10]. Crucially, nonpathogenic strains (e.g., Imperial‑181) have lost the ability to bind mammalian PCDH10 but retain strong affinity for avian PCDH10 [10]. A single amino acid change at position 153 (Q153L) in the E2 glycoprotein can restore mammalian receptor binding, indicating a low barrier to a host‑shift that could enhance zoonotic potential [10]. Additionally, a single polymorphism (E181K or E81K) in E2 determines whether a strain can use VLDLR for entry [9]. These findings suggest that the avian reservoir exerts selective pressure that maintains compatibility with avian receptors while tolerating loss of mammalian receptor usage, a trade‑off that may have contributed to the decades‑long quiescence of WEEV in North America. The re‑emerging South American strains, now undergoing rapid evolution, must be closely monitored for any mutations that could expand their mammalian tropism.

Diagnostic and Surveillance Challenges

Surveillance of WEEV in birds relies on serology (hemagglutination‑inhibition, plaque‑reduction neutralization, and complement fixation‑inhibition tests) and molecular detection [7, 23]. A recent genomic study of the Uruguayan outbreak discovered that widely used real‑time PCR primers and probes contain mismatches to the circulating lineage, leading to potential false negatives in official diagnostic protocols [2]. This highlights the need for regular updating of molecular assays as new lineages emerge. The CDC and national reference laboratories must incorporate sequence data from ongoing outbreaks to maintain diagnostic sensitivity.

In summary, the epidemiology of WEEV in birds is a dynamic interplay between host competence, vector behavior, viral evolution, and environmental change. Migratory birds are both the primary reservoirs and the most efficient long‑distance disseminators of the virus. The unprecedented re‑emergence of WEEV in South America after a 40‑year hiatus, coupled with climate‑driven range expansion, demands enhanced avian surveillance and a rapid, coordinated international response. The virus’s remarkable ability to shift receptor usage while maintaining enzootic fitness in birds underscores the need for continued molecular surveillance and the development of pan‑alphavirus vaccines and therapeutics.

Diagnostic Approaches for Western Equine Encephalitis Virus in Avian Samples: PCR Assays and Genomic Characterization

The accurate and timely diagnosis of Western equine encephalitis virus (WEEV) in avian samples is a cornerstone of effective surveillance, outbreak response, and a fundamental prerequisite for understanding the complex enzootic transmission cycles that sustain this pathogen. Birds, as the primary vertebrate reservoir hosts, are not merely incidental casualties of WEEV infection; they are the engines of viral amplification and geographic dissemination. Consequently, diagnostic approaches applied to avian samples must be exquisitely sensitive, capable of detecting low-titer viremias that may be transient, and robust enough to handle the inherent variability of field-collected samples. The modern diagnostic landscape for WEEV in birds is dominated by molecular techniques, particularly reverse transcription-polymerase chain reaction (RT-PCR) and its quantitative variants (RT-qPCR), which have largely supplanted traditional virus isolation for primary screening due to their speed, sensitivity, and capacity for high-throughput analysis. However, the integration of genomic characterization, primarily through next-generation sequencing (NGS), has elevated our diagnostic capacity from mere detection to a sophisticated understanding of viral evolution, emergence, and transmission dynamics. This section provides an exhaustive analysis of these molecular diagnostic approaches, their biological underpinnings, and their critical role in the context of the recent and dramatic re-emergence of WEEV in South America.

The Foundational Role of RT-PCR and RT-qPCR in Avian Surveillance

The detection of WEEV RNA in avian tissues and blood represents the most direct and reliable method for confirming active infection. The gold-standard molecular approach has historically been conventional RT-PCR, which amplifies a specific region of the viral genome, followed by gel electrophoresis for amplicon visualization. However, the field has decisively shifted toward real-time, or quantitative, RT-PCR (RT-qPCR). This technique offers several critical advantages: it provides quantitative data on viral load (viral RNA copies per unit of sample), it is performed in a closed-tube system that minimizes the risk of cross-contamination, and it offers significantly greater sensitivity and a broader dynamic range of detection.

The biological rationale for employing these assays in avian samples is deeply rooted in the pathogenesis of WEEV in its reservoir hosts. As demonstrated by seminal experimental infection studies, birds typically develop a robust but transient viremia following inoculation. Hammon et al. [4] established that viremia in wild birds can be detected within 24 hours, reaching titers as high as (10^{-6}) (a 1:1,000,000 dilution of serum still infectious) and lasting for only 3 to 4 days. This narrow window of detectability necessitates a diagnostic tool of extreme sensitivity. RT-qPCR, with its ability to detect as few as 10-100 RNA copies per reaction, is ideally suited for this task. Furthermore, the work of Reisen et al. [3] demonstrated that host competence, a measure of a bird species’ ability to transmit the virus to a feeding mosquito, is defined by a viremia threshold of (\geq 2 \log_{10}) plaque-forming units per 0.1 ml. RT-qPCR can not only confirm infection but can also quantify the viremia, allowing researchers to infer the potential epidemiological significance of an infected bird. A bird with a high viral RNA load is far more likely to be a competent amplifier of the virus than one with a low or undetectable load.

The design of primers and probes for these assays is a matter of paramount importance. The target region is most often a highly conserved segment of the viral genome, such as the (nsP4) gene (encoding the RNA-dependent RNA polymerase) or the (E1) envelope glycoprotein gene. The recent 2023-2024 South American outbreak has provided a stark lesson in the consequences of primer-template mismatch. Tomás et al. [2] performed genomic characterization of Uruguayan strains and discovered "mispairing in real-time PCR primers and probes that may affect official diagnostic protocols." This finding is of critical concern for organizations like the World Organisation for Animal Health (WOAH) and the World Health Organization (WHO), which rely on standardized diagnostic protocols for global surveillance. A single nucleotide polymorphism (SNP) in the primer binding site of a newly emerged viral lineage can lead to false-negative results, undermining surveillance efforts and delaying outbreak response. This underscores the necessity of continuously updating molecular diagnostic assays based on circulating viral sequences, a task that is now intimately linked with genomic surveillance.

Genomic Characterization: From Detection to Evolutionary Insight

While PCR assays provide a binary (positive/negative) or quantitative answer, genomic characterization through NGS provides a narrative. It tells the story of where the virus came from, how it is spreading, and whether it is evolving in ways that could alter its pathogenicity, transmissibility, or receptor tropism. The application of NGS to avian samples is a powerful, albeit more complex and costly, diagnostic and research tool.

The process typically begins with RNA extraction from the avian sample (blood, serum, or tissue homogenate). For WEEV, which has a single-stranded, positive-sense RNA genome of approximately 11.5 kb, the RNA is often enriched for viral sequences. This can be achieved through ribosomal RNA depletion or, more specifically, through a multiplex PCR approach that amplifies the entire viral genome in a series of overlapping amplicons. Tomás et al. [2] successfully employed a "novel multiplex PCR assay combined with next-generation Illumina sequencing" to obtain complete genomes from 15 Uruguayan strains. This approach is particularly valuable for field samples where the viral load may be low, as it specifically amplifies the target viral RNA, increasing the depth of sequencing coverage.

The resulting genomic data is then subjected to rigorous bioinformatic analysis. Phylogenetic reconstruction, as performed by Campos et al. [12] and Vissani et al. [19], allows for the classification of new isolates into distinct lineages. The identification of a "novel WEEV lineage" (proposed as lineage C) from fatal equine cases in Brazil [12, 14] is a direct result of such genomic characterization. This lineage was found to be distinct from historical North American strains and from other South American strains, suggesting a unique evolutionary trajectory. Furthermore, phylogenetic analysis of partial (nsP4) sequences from Argentine isolates confirmed their grouping with contemporaneous strains from Uruguay and Brazil [19], providing molecular evidence for a single, large-scale outbreak spreading across national borders.

The epidemiological implications of this genomic data are profound. By combining phylogenetic trees with epidemiological data (e.g., dates of onset, geographic locations), researchers can infer transmission pathways. Tomás et al. [2] concluded that "phylogenetic and epidemiological data suggest that the outbreak originated in Argentina and spread to Uruguay and Brazil, likely by movements of infected birds." This is a powerful demonstration of how genomic characterization of avian samples can be used to track the spatial and temporal dynamics of an outbreak. The role of migratory birds as long-distance dispersal agents is a well-established concept in arbovirology [13], and genomic data provides the definitive evidence to support these epidemiological models.

Advanced Applications: Detecting Chronic Infections and Receptor Tropism

Beyond acute diagnosis and outbreak tracking, molecular diagnostics are being applied to answer more nuanced questions about WEEV biology in avian hosts. One long-standing hypothesis is that birds may serve as overwintering reservoirs for WEEV, maintaining the virus in a temperate climate when mosquito vectors are inactive. This would require the establishment of a chronic or persistent infection. Early experimental work provided tantalizing evidence for this. Reisen et al. [3] detected viral RNA by RT-PCR in the tissues of several bird species (house finches, mourning doves, sparrows) more than six weeks post-inoculation, and in a few cases, infectious virus was recovered after blind passage. However, subsequent studies, such as those on mourning doves by Reisen et al. [5], failed to detect viral RNA in sera, spleen, lung, or kidney tissues at necropsy, and immunosuppression did not induce a relapse. This suggests that while chronic infection may occur in some individuals, it is not a universal or robust phenomenon. The use of highly sensitive RT-qPCR on a wide range of tissues (not just blood) is critical to resolving this question, as the virus may be sequestered in specific organs at very low levels.

Another frontier in WEEV diagnostics is the genotyping of specific viral determinants of host range and virulence. The recent discovery of protocadherin 10 (PCDH10) as a major neuronal receptor for WEEV [1, 8, 10] has opened new avenues for understanding viral tropism. Critically, it has been shown that nonpathogenic WEEV strains, such as Imperial-181, have lost the ability to bind to mammalian PCDH10 but retain affinity for avian PCDH10 [10]. A single amino acid change in the E2 glycoprotein (E2Q153L) was identified as the key determinant of this receptor switch [10]. Diagnostic assays can now be designed to specifically screen for this or other receptor-binding mutations. For example, a targeted RT-PCR or a sequencing panel could be used to rapidly determine whether a WEEV strain isolated from a bird carries the "avian-tropic" or "mammalian-tropic" signature. This information is of immense value for risk assessment: the detection of a strain with high affinity for mammalian PCDH10 or VLDLR [8, 9] in an avian population would signal a heightened risk of spillover into equine and human populations.

In conclusion, the diagnostic approach to WEEV in avian samples has evolved into a multi-layered, high-resolution discipline. RT-qPCR remains the workhorse for rapid, sensitive, and quantitative detection of acute infections, forming the backbone of surveillance systems recommended by agencies like the CDC and WOAH. However, the true power of modern diagnostics lies in the integration of genomic characterization. NGS not only confirms the presence of the virus but also provides the evolutionary context necessary to track its spread, identify emerging lineages, and even predict its potential for cross-species transmission. As WEEV continues its dramatic re-emergence in the Americas, the continued refinement and application of these molecular tools to avian samples will be indispensable for protecting both animal and public health.

Susceptibility and Lethality of Western Equine Encephalitis Virus in Avian Species: Experimental and Field Evidence

The maintenance and amplification of Western equine encephalitis virus (WEEV) within avian populations constitutes a cornerstone of its enzootic transmission cycle, a paradigm recognized since the virus’s initial characterization in the western United States. Avian species serve as the primary reservoir hosts, a role predicated on their unique virological and ecological interactions with the virus. Unlike the severe, often lethal neuroinvasive disease observed in humans and equids, WEEV infection in most avian species is characterized by a robust, high-titer viremia without overt clinical illness or significant mortality. This asymptomatic carrier state, however, belies a complex interplay of susceptibility, host competence, and viral kinetics that is exquisitely tuned to the ecology of the primary mosquito vector, Culex tarsalis. Understanding the nuances of avian susceptibility and lethality is not merely an academic exercise; it is the foundational knowledge required for predicting viral amplification, outbreak risk, and the potential for geographic spread, particularly in the context of the 2023–2024 re-emergence of WEEV across South America [2, 12, 13].

Experimental Elucidation of Avian Host Competence

The most comprehensive body of experimental evidence on avian susceptibility stems from decades of work by Reisen and colleagues, who systematically evaluated the response of numerous passerine and columbiform species to WEEV inoculation. In a landmark study involving 27 species from California, 133 of 164 birds (81.1%) inoculated subcutaneously with sympatric WEEV strains developed a detectable viremia [3]. This high infection rate underscores the intrinsic susceptibility of the avian host. However, the critical metric for ecological significance is not merely the presence of viremia but its magnitude and duration. The threshold for infecting a feeding Culex tarsalis mosquito is approximately ≥2 log₁₀ plaque-forming units per 0.1 mL of blood. Using this metric, Reisen et al. [3] calculated a host competence index, defined as the average number of days a species’ viremia remained at or above this infectious threshold. Of the 20 species tested with WEEV, 11 were deemed competent hosts, including the house finch (Haemorhous mexicanus), house sparrow (Passer domesticus), and several species of sparrows and blackbirds. This experimental framework directly demonstrates that susceptibility is not uniform; it is a graded continuum where some species are highly efficient amplifiers while others, despite becoming infected, contribute minimally to enzootic transmission.

The temporal dynamics of viremia are equally critical. Foundational work by Hammon, Reeves, and Sather [4] demonstrated that viremia in experimentally infected wild birds appears rapidly, often within 24 hours post-inoculation, peaks at titers as high as 10⁻⁶ (as measured by the dilution of serum that still produced infection), and persists for a relatively brief period of 3 to 4 days. This narrow window of high-titer viremia perfectly synchronizes with the feeding cycle of the mosquito vector, maximizing the probability of transmission. The fact that birds develop neutralizing antibodies within 15 to 29 days, which confer lifelong protection against re-infection, explains why epizootic waves are often seasonal and linked to the recruitment of naïve juvenile birds into the population [4, 5]. Critically, this work established that wild birds are significantly more important than domestic fowl as amplification hosts, given the higher viremias they achieve, a conclusion that remains a central tenet of WEEV epidemiology [4].

Species-Specific Differences and the Role of Immunosuppression

Within the broad category of “avian hosts,” significant variation exists in both susceptibility and the potential for lethality. While WEEV is generally considered non-lethal for birds, experimental infections have documented mortality events that provide crucial insights into the host-virus interface. In the Reisen et al. [3] study, lethal infections were not the norm, but they did occur in six birds: one house finch, three mourning doves (Zenaida macroura), one Brewer’s sparrow, and one white-crowned sparrow. The presence of viral RNA detected by RT-PCR in these birds at necropsy more than six weeks post-inoculation suggests that mortality may be associated with the establishment of a persistent, rather than a fully cleared, infection. This is supported by the recovery of infectious WEEV from three mourning doves after blind passage in mosquito cells, a finding that points to the existence of a chronic carrier state in some individuals [3]. The mourning dove, in particular, has been a subject of intense scrutiny. Separate experiments demonstrated that while after-hatching year and hatching-year mourning doves readily produce WEEV-specific antibodies, they do not develop a detectable viremia when infected with St. Louis encephalitis virus, but are competent for WEEV [5]. This stark difference in susceptibility between closely related arboviruses highlights the exquisite specificity of the WEEV-avian interaction.

The role of the host’s physiological state, specifically immune competence, profoundly alters susceptibility and disease outcome. Experimental immunosuppression using cyclophosphamide or the corticosteroid dexamethasone in house finches resulted in a marked enhancement of both the amplitude and duration of WEEV viremia [6]. Cyclophosphamide treatment in mourning doves similarly significantly increased WEEV viremia without altering the antibody response, indicating that the immunosuppression primarily affected the cell-mediated arms of the immune system responsible for controlling viral replication [5]. These findings are of immense epidemiological significance. They suggest that periods of stress, such as migration, extreme weather events, or concurrent infections, could transiently transform a “subclinical” infection into a state of hyper-viremia, dramatically increasing the bird’s infectivity for mosquitoes. Furthermore, the fact that immunosuppression did not lead to the reactivation of a chronic infection (relapse) in doves treated with cyclophosphamide the following spring argues against a major role for persistent infection as an overwintering mechanism, at least for this species [5].

Field Evidence, Serosurveillance, and the 2023–2024 South American Outbreak

Experimental data are powerfully complemented by field serosurveys, which document the real-world prevalence of WEEV infection across diverse avian populations. Long-term surveillance in the Atlantic Forest region of São Paulo, Brazil, from 1978 to 1990, screened nearly 40,000 wild birds for hemagglutination-inhibiting (HI) antibodies. Only 0.7% (269 birds) showed a monotypic reaction for WEEV, but these positive birds were distributed across 66 species and 22 families, revealing a remarkably broad host range [7]. This low overall seroprevalence is consistent with a virus that causes an acute, transient infection followed by lifelong immunity; the proportion of seropositive birds in a population at any given time reflects the recency and intensity of local viral transmission. The study emphasized that positive birds were concentrated in unforested habitats, particularly near agricultural fields, linking WEEV activity to human-modified landscapes [7]. This pattern aligns with the ecology of Culex tarsalis, which thrives in agricultural drainage and irrigation systems.

The most dramatic field evidence for the role of birds in WEEV dispersal and evolution comes from the explosive 2023–2024 outbreak in South America. After a nearly 40-year intermission period, WEEV re-emerged in Argentina, rapidly spreading to Uruguay and Brazil [2, 12, 14, 19]. Genomic analysis of the newly isolated strains has been pivotal. Phylogenetic studies revealed that the Uruguayan and Brazilian outbreak strains share a common evolutionary origin with an Argentine strain from 1958, forming a distinct lineage (proposed as Lineage C) separated from North American strains [2, 12]. The authors of these studies explicitly conclude that the rapid geographic expansion of the outbreak was “likely by movements of infected birds” [2]. This conclusion is supported by the work of Lorenz et al. [13], whose environmental modeling identified that high-risk areas for WEEV in South America coincide with migratory bird stopover sites. The timing of the outbreak (November 2023–April 2024) aligns perfectly with the austral summer, a period of peak mosquito activity and the fledging of juvenile, immunologically naïve birds, providing a large cohort of susceptible amplifying hosts.

Molecular Determinants of Avian Tropism and Pathogenesis

The cellular and molecular basis for the stark difference in disease outcome between avian and mammalian hosts is now being unraveled at the atomic level. The discovery of Protocadherin 10 (PCDH10) as a primary neuronal receptor for WEEV has provided a critical piece of this puzzle [1]. PCDH10 is a neuronal-enriched transmembrane protein, and its utilization by WEEV explains the virus’s potent neurotropism in mammals. Crucially, structural biology studies have revealed that WEEV strains exhibit differential receptor tropism. Liang et al. [10] demonstrated that nonpathogenic WEEV strains, such as Imperial-181, have evolved to lose the ability to bind to mammalian PCDH10 while retaining the ability to bind to avian PCDH10. This switch is mediated by a single amino acid polymorphism, E2Q153L, which restores binding to avian PCDH10 [10]. The structural basis for this difference is that residue 89 on avian PCDH10 is an arginine, which is essential for interaction with the Imperial-181 strain [10]. Similarly, the very-low-density lipoprotein receptor (VLDLR), another receptor for WEEV, shows strain-specific utilization. A single mutation (V265F) in the E2 glycoprotein can allow a non-VLDLR-binding strain to gain this receptor-binding ability [8, 9].

This exquisite molecular adaptation provides a mechanistic explanation for the virus’s ability to productively infect birds without causing the neuropathology seen in mammals. In birds, WEEV appears to have evolved to use receptors that permit replication in tissues that support high-titer viremia (e.g., lymphoid tissues, vascular endothelium) without efficiently entering the central nervous system. The virus’s envelope glycoproteins, which mediate receptor binding and membrane fusion, are the key determinants of this host range. Bergren et al. [16] used reverse genetics and competition fitness assays to demonstrate that mutations that became fixed in WEEV during its late 20th-century decline in North America were under positive selection for increased fitness in enzootic hosts (house sparrows and Culex tarsalis). Importantly, these same mutations had no effect on virulence in a mammalian (hamster) model. This suggests that the selective pressure driving WEEV evolution is primarily exerted within its avian-mosquito transmission cycle, and that mammalian virulence is a secondary, perhaps even an accidental, byproduct of this adaptation.

Lethality and the Enzootic Fitness Advantage

The fundamental observation that WEEV infection is rarely lethal in its avian reservoir hosts is not a sign of viral weakness but a hallmark of an exquisitely co-evolved relationship. A virus that kills its reservoir host rapidly reduces its own opportunity for transmission. The hallmark of a successful enzootic arbovirus is the ability to generate a high-titer, sustained viremia without causing significant morbidity or mortality. The WOAH (World Organisation for Animal Health) and the WHO (World Health Organization) classify WEEV as a serious zoonotic pathogen, but for birds, it is primarily a silent amplifier. The cases of mortality reported in experimental settings, such as the mourning doves and finches in Reisen’s studies, likely represent a breakdown of this delicate balance, perhaps due to host age, nutritional stress, or concurrent infections [3, 6].

Furthermore, the concept of “lethality” must be considered in an evolutionary context. The experimental evolution work by Bergren et al. [16] showed that the WEEV strains that became dominant during a period of enzootic decline in North America were actually fit for the avian host. The decline in human and equine cases was not due to the virus losing fitness, but rather to ecological changes, perhaps shifts in land use, vector control, or mosquito behavior, that reduced spillover from the competent avian reservoir. The re-emergence in South America in 2023–2024 serves as a stark reminder that the virus remains in an enzootic state within wild bird populations, capable of explosive amplification when ecological conditions favor its mosquito vector [11, 13]. The detection of antibodies in Brazilian birds decades ago and the recent genomic linkage of current outbreak strains to a 1958 Argentine isolate confirm the long-term persistence of WEEV within South American avian communities, likely in a cryptic, subclinical cycle [7, 12, 24]. Thus, the susceptibility of birds to WEEV is not just a biological fact; it is the engine that drives the global epidemiology of this re-emerging threat.

Evolutionary Phylogeny and Phylogeography of Western Equine Encephalitis Virus: Insights from South American Outbreaks and Avian Reservoir Movements

Historical Context and the Enigma of WEEV Emergence

Western equine encephalitis virus (WEEV) presents a singularly compelling case study in arboviral evolution and phylogeography, characterized by dramatic shifts in geographic distribution, host tropism, and epidemic potential. For much of the 20th century, WEEV was a dominant cause of arboviral encephalitis in the western United States and Canada, with large-scale epizootics involving equids and sporadic human cases that carried a case fatality rate of up to 15% [17, 18]. However, beginning in the late 20th century, WEEV underwent a remarkable and poorly understood decline in North America, with enzootic circulation and clinical disease virtually disappearing [16]. This "submergence" of WEEV, as it has been termed, was initially hypothesized to result from genetic drift, fitness reductions, or ecological shifts. Yet, rigorous evolutionary analyses have challenged this narrative. Bergren et al. (2020) demonstrated that during the period of decline, six amino acid residues in the WEEV genome were under positive selection, not genetic drift, and that these mutations conferred a fitness advantage in enzootic hosts (house sparrows) and vectors (Culex tarsalis) [16]. This finding is profound: it suggests that WEEV did not become less fit for its natural transmission cycle; rather, it adapted more efficiently to birds and mosquitoes. The decline in North American human and equine cases is therefore more likely attributable to ecological factors, such as changes in land use, vector control, or avian host population dynamics, than to viral attenuation [16]. This evolutionary backdrop sets the stage for understanding the virus's dramatic re-emergence in South America, a continent where WEEV has historically circulated at lower, enzootic levels but has now erupted in a major epizootic event.

Phylogenetic Architecture: The Emergence of a Novel South American Lineage

The 2023–2024 WEEV outbreak in South America represents the most significant resurgence of this pathogen in nearly four decades, with over 2,500 equine cases and 217 human cases reported across Argentina, Uruguay, and Brazil [2, 12]. Genomic characterization of strains from this outbreak has fundamentally reshaped our understanding of WEEV phylogeny. Prior to this event, WEEV was broadly classified into two major lineages: Lineage A (North American strains) and Lineage B (South American strains). However, comprehensive sequencing of viruses from fatal equine cases in Rio Grande do Sul, Brazil, and from Uruguayan outbreaks has led to the proposal of a third, distinct lineage, Lineage C [12, 14]. This novel lineage is phylogenetically distinct from both historical North American isolates and previously characterized South American strains, including an Argentine strain from 1958 that clusters with the current outbreak viruses [2].

The phylogenetic analyses reveal a complex evolutionary history. The 2023–2024 South American strains share a common ancestor with the 1958 Argentine strain, indicating a long-term, cryptic circulation of this lineage within the continent [2]. Crucially, these South American Lineage C viruses are genetically distinct from the North American Lineage A strains that caused the historical epidemics in the United States and Canada [2, 12]. This genetic divergence is not merely a matter of geographic isolation; it reflects fundamental differences in receptor usage and host adaptation. Recent structural biology studies have shown that WEEV entry into host cells is mediated by two principal receptors: the newly identified protocadherin 10 (PCDH10), a neuronal-enriched protein, and the very-low-density lipoprotein receptor (VLDLR) [1, 8-10]. Critically, the receptor tropism of WEEV is strain-dependent and has shifted over evolutionary time. Nonpathogenic strains, such as Imperial-181, have lost the ability to bind mammalian PCDH10 while retaining affinity for avian PCDH10, a finding that explains their attenuated phenotype in mammals [10]. In contrast, virulent epidemic strains engage both PCDH10 and VLDLR [10]. The South American Lineage C viruses, given their high pathogenicity in horses and humans, likely possess a receptor-binding profile that enables efficient entry into mammalian neurons, a hypothesis that warrants direct experimental validation.

Phylogeographic Dynamics: Avian Reservoirs as Drivers of Continental Spread

The phylogeographic reconstruction of the 2023–2024 outbreak provides compelling evidence for the role of avian reservoirs in the rapid, long-distance dispersal of WEEV. Epidemiological and genomic data converge to indicate that the outbreak originated in Argentina and subsequently spread southward and eastward into Uruguay and Brazil [2]. The temporal and spatial patterns of case detection, coupled with the high genetic similarity of viral isolates across these countries, are inconsistent with a model of slow, vector-borne diffusion. Instead, the data strongly implicate the movement of infected migratory birds as the primary mechanism of viral transport [2, 13].

This hypothesis is biologically plausible and supported by decades of experimental and field data. WEEV is maintained in an enzootic cycle between ornithophilic Culex mosquitoes (primarily Cx. tarsalis in North America and Cx. quinquefasciatus and Cx. nigripalpus in South America) and a wide range of passerine and columbiform birds [3, 4, 22]. Experimental infections have demonstrated that numerous avian species, including house finches, mourning doves, and sparrows, develop viremias of sufficient magnitude (≥2 log10 PFU/0.1 mL) to infect feeding mosquitoes [3]. Critically, some birds, such as mourning doves and house finches, can harbor viral RNA for weeks post-inoculation, and in rare cases, infectious virus can be recovered from tissues long after the acute viremia has resolved [3, 5]. This capacity for prolonged infection, potentially reactivated by immunosuppression, provides a mechanism for virus persistence and transport over vast distances [6].

The 2023–2024 outbreak coincided with the migratory stopover periods of several Neotropical bird species that traverse the Southern Cone of South America [13]. The Atlantic Flyway, which connects breeding grounds in Patagonia with wintering areas in northern Argentina, Uruguay, and southern Brazil, is a well-documented route for avian-borne arboviruses. The rapid appearance of WEEV cases in Uruguay and Brazil within weeks of the initial Argentine outbreaks is entirely consistent with the flight speeds and migration patterns of competent avian hosts such as the White-rumped Sandpiper (Calidris fuscicollis) or the Swainson's Thrush (Catharus ustulatus). Furthermore, serosurveys conducted in the Atlantic Forest region of São Paulo, Brazil, between 1978 and 1990 detected hemagglutination-inhibition antibodies against WEEV in 66 species of wild birds, confirming that South American avifauna has been exposed to this virus for decades and likely serves as a long-term reservoir [7].

Evolutionary Drivers of Receptor Tropism and Host Switching

The evolutionary trajectory of WEEV is inextricably linked to its interactions with avian and mammalian hosts at the molecular level. The identification of PCDH10 as a general, high-affinity receptor for WEEV has revolutionized our understanding of viral neurotropism and host range [1, 10]. PCDH10 is a member of the cadherin superfamily and is highly expressed on neuronal surfaces, explaining the pronounced neuroinvasiveness of WEEV [1]. Cryo-electron microscopy structures have revealed that the PCDH10 EC1 domain inserts into a cleft formed by adjacent E2-E1 glycoprotein heterodimers on the viral spike [8, 10]. This binding interface is highly conserved among WEEV strains, but a single amino acid substitution at position 153 of the E2 glycoprotein (Q153L) is sufficient to ablate binding to mammalian PCDH10 while preserving binding to avian PCDH10 [10]. This mutation is present in the nonpathogenic Imperial-181 strain, providing a structural explanation for its attenuation in mammals.

Concurrently, WEEV has evolved the capacity to utilize VLDLR and ApoER2 as alternative receptors, particularly in virulent strains [8, 9]. The binding mode for VLDLR is distinct from that of other alphaviruses; the LA1-2 repeats of VLDLR insert into the same E2-E1 cleft as PCDH10, but the interaction is stabilized by additional contacts with the E2 B domain [8]. A single polymorphism in the E2 glycoprotein (e.g., V265F or E181K) can determine whether a WEEV strain can engage VLDLR [8, 9]. This plasticity in receptor usage has profound implications for host switching. The South American Lineage C viruses, which are highly pathogenic in equids and humans, likely possess an E2 genotype that permits efficient engagement of both PCDH10 and VLDLR in mammalian cells. This dual-receptor tropism may enhance viral entry into a broader range of cell types, including neurons and glial cells, and may facilitate the rapid neuroinvasion observed in fatal equine cases [19].

Implications for Surveillance and Pandemic Preparedness

The re-emergence of WEEV in South America, after a 40-year intermission, underscores the inadequacy of current surveillance paradigms and the critical need for a One Health approach that integrates virology, ornithology, and climatology. The World Organisation for Animal Health (WOAH) and the World Health Organization (WHO) have historically classified WEEV as a notifiable pathogen, but surveillance in South America has been sporadic. The 2023–2024 outbreak revealed significant gaps in diagnostic capacity; genomic analysis of Uruguayan strains identified mispairing in real-time PCR primers and probes used in official protocols, potentially leading to false-negative results [2]. This is a critical failure that must be urgently addressed.

Furthermore, climate change is projected to expand the geographic range of WEEV transmission. Ecological niche modeling using Maxent has identified thermal seasonality and annual rainfall as the key environmental determinants of WEEV occurrence [13]. Under future climate scenarios, high-risk areas are predicted to shift, with Paraguay, Venezuela, Colombia, and extensive regions of Brazil (including the Northeast, Midwest, and Pantanal biomes) becoming increasingly suitable for WEEV transmission [13]. These regions are also major stopover sites for migratory birds, creating a perfect storm for viral amplification and spillover. The CDC and PAHO have issued alerts regarding this potential expansion, but proactive surveillance in avian populations, particularly in migratory stopover sites, remains woefully underfunded.

The Enigma of Viral Persistence and Overwintering

One of the most enduring mysteries in WEEV ecology is the mechanism by which the virus persists through temperate winters when mosquito activity ceases. The experimental data on avian chronic infection are equivocal. While some studies have detected viral RNA in tissues of house finches and mourning doves weeks after inoculation, infectious virus was only rarely recovered, and immunosuppression with cyclophosphamide did not reliably induce relapse [3, 5, 6]. This suggests that true chronic, recrudescent infections in birds are uncommon and may not be the primary overwintering mechanism. Alternative hypotheses include vertical transmission in mosquitoes, persistence in reptile hosts (lizards have been shown to constitute a significant proportion of Culex blood meals in the southern US), or reintroduction each spring by migratory birds from tropical regions where transmission is continuous [22, 25]. The 2023–2024 South American outbreak, which occurred during the austral summer, may have been fueled by a combination of these factors, with the initial introduction from a tropical reservoir followed by rapid amplification along migratory flyways. Understanding these mechanisms is not merely an academic exercise; it is essential for predicting the timing and location of future outbreaks and for designing effective control strategies, including the strategic vaccination of equids as recommended by WOAH.

Host-Virus Interactions and Reservoir Competence: Factors Influencing Western Equine Encephalitis Virus Persistence in Bird Populations

The perpetuation of Western equine encephalitis virus (WEEV) within its natural transmission cycle hinges upon a complex interplay between the virus and its avian reservoir hosts. Unlike accidental or dead-end hosts such as humans and equids, birds must support a viremia of sufficient magnitude and duration to infect feeding mosquito vectors, thereby completing the enzootic cycle. However, the simple presence of a viremia is insufficient to explain long-term persistence; the capacity for a host to contribute to viral maintenance across seasons and geographical regions, its reservoir competence, is governed by a suite of molecular, immunological, and ecological factors. Understanding these factors is critical for predicting outbreak risk and designing surveillance strategies, particularly in the context of the virus's recent re-emergence in South America after decades of relative quiescence [2, 12, 19]. The avian host is not merely a passive vessel for viral replication but an active participant in a dynamic evolutionary and ecological relationship that dictates WEEV's spatial and temporal distribution.

Quantitative and Qualitative Aspects of Reservoir Competence

The foundational characteristic of a competent avian reservoir is the ability to generate a viremia that surpasses the infection threshold for the primary mosquito vector, Culex tarsalis. Experimental infections have demonstrated that this competence is highly variable across bird species. Reisen and colleagues (2003) established a critical benchmark, defining host competence as the average number of days a bird maintains a viremia ≥2 log10 plaque-forming units per 0.1 ml, the minimum required to infect Cx. tarsalis [3]. In their comprehensive study of 20 California bird species, eleven were classified as competent hosts for WEEV, while others, such as mourning doves, consistently fell below this threshold [3, 5]. Early work by Hammon, Reeves, and Sather (1951) corroborated this species-specific variability, noting that many wild birds developed readily detectable viremias within 24 hours of infection, peaking at titers up to 10⁻⁶, but that the duration and peak titer were not uniform across taxa [4]. The viremia profile is not solely a function of the host species; it is also shaped by the viral strain itself. The classic work by Bergren et al. (2020) demonstrated that positively selected mutations in the WEEV genome, which became fixed in the population during the virus's mid-20th-century decline, actually enhanced replicative fitness in house sparrows (Passer domesticus), a key enzootic host [16]. This finding challenges the simplistic notion that declining disease incidence equates to diminished viral fitness in the primary reservoir, suggesting instead that ecological or vector-related factors were the primary drivers of the observed "submergence" [16]. Thus, reservoir competence is a quantitative trait determined by the intersection of host genetics, viral genetics, and the physiological state of the avian host.

Molecular Determinants of Viral Entry and Host Specificity

At the molecular level, the interaction between the WEEV envelope glycoprotein and host cell receptors is a primary determinant of host range and tissue tropism. The identification of protocadherin 10 (PCDH10) as a general, high-affinity receptor for WEEV represents a paradigm shift in understanding viral entry [1]. Structural analyses have revealed that the EC1 ectodomain of PCDH10 inserts into a cleft formed by adjacent E2-E1 heterodimers on the viral spike, a binding mechanism unique to WEEV among alphaviruses [8, 10]. Crucially, the specificity of this interaction governs host susceptibility. While pathogenic strains like 71V-1658 bind both human and avian PCDH10, non-pathogenic strains such as Imperial-181 have evolved to lose affinity for mammalian PCDH10 while retaining the ability to bind the avian ortholog [10]. This receptor tropism shift is mediated by a single amino acid polymorphism at residue 153 of the E2 glycoprotein; the E2Q153L mutation in Imperial-181 restores its ability to engage avian PCDH10 but not the human form [10]. Furthermore, an arginine residue at position 89 on the avian PCDH10 protein is essential for this interaction, providing a molecular basis for the virus's preferential amplification in avian populations [10]. Alongside PCDH10, WEEV can also utilize the very-low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) for entry, particularly in mammalian systems [1, 8, 9]. However, a single polymorphism in E2 (e.g., V265F or E181K) can determine whether a strain can engage VLDLR [8, 9]. This receptor plasticity allows WEEV to maintain a broad host range while also permitting the emergence of strains that are highly adapted to avian hosts, thereby ensuring efficient replication in reservoirs without necessarily posing a threat to accidental hosts. The structural basis of this differential receptor usage highlights the fine molecular tuning that underpins the virus's enzootic maintenance.

The Role of Chronic Infection and Immunosuppression in Viral Persistence

A critical factor for the overwintering and long-distance dispersal of WEEV is the potential for prolonged or chronic infection in avian hosts. The question of whether birds can harbor infectious virus for weeks or months, thereby bridging gaps in mosquito activity, has been a central focus of investigation. Experimental evidence from antibody surveys and viral RNA detection has proven instructive. An early study by Ferreira et al. (1994) detected hemagglutination-inhibiting antibodies for WEEV in 0.7% of nearly 40,000 wild birds in Brazil, indicating widespread but generally low-level historical exposure [7]. More directly, Reisen et al. (2003) used RT-PCR to detect WEEV RNA in tissues of six birds (one house finch, three mourning doves, one Brewer’s sparrow, and one white-crowned sparrow) at necropsy more than six weeks post-inoculation [3]. However, infectious virus could only be recovered from the three mourning doves after blind passage in mosquito cells, suggesting that the viral RNA load in many birds was either too low for direct isolation or represented non-infectious viral remnants [3].

Subsequent work by Reisen et al. (2004) specifically addressed the overwintering potential of mourning doves (Zenaida macroura) for WEEV. Even when birds were treated with the potent immunosuppressant cyclophosphamide, which significantly amplified their acute viremia, they did not experience a detectable relapse of viremia the following spring [5]. Similarly, the authors could not detect viral RNA in serum, spleen, lung, or kidney tissues at necropsy 26 weeks post-infection [5]. This strongly suggests that mourning doves, despite being a common host, do not serve as a suitable overwintering reservoir for WEEV. In contrast, a parallel study in house finches (Haemorhous mexicanus) found that immunosuppression with cyclophosphamide or dexamethasone increased the frequency of chronic infection for the flavivirus St. Louis encephalitis virus (SLEV) but not for WEEV [6]. These findings indicate a fundamental difference in the persistence biology of these two co-circulating arboviruses. The failure of immunosuppression to induce chronic WEEV infection or relapse in these experimental systems implies that WEEV persistence in temperate regions may rely more heavily on other mechanisms, such as continuous low-level transmission in subtropical zones, vertical transmission in vectors, or introduction via migratory birds, rather than long-term cryptic infection in individual birds.

The Ecological and Evolutionary Context of Avian Persistence

The persistence of WEEV is not solely a product of individual host-virus interactions but is profoundly influenced by the ecological landscape and evolutionary pressures acting on the virus. The recent 2023–2024 outbreak in South America, which followed nearly four decades of apparent silence, provides a powerful case study [2, 12, 19]. Genomic and epidemiological data strongly suggest that the outbreak originated in Argentina and spread to Uruguay and Brazil, likely facilitated by the movements of infected migratory birds [2, 12, 13]. Lorenz et al. (2024) modeled the distribution of WEEV in South America and found that environmental variables, particularly thermal seasonality and annual rainfall, are key determinants of virus occurrence [13]. Their models predict that climate change will shift high-risk areas, potentially expanding the range of WEEV into regions such as the Pantanal and parts of northern Brazil, precisely where migratory bird stopovers are concentrated [13]. This underscores the dynamic relationship between avian migration patterns, climate-driven changes in vector distribution, and the re-emergence of WEEV.

The evolutionary pressures that shape WEEV's adaptation to its avian hosts are also evident from genomic surveillance. The 2023–2024 South American outbreak has been linked to a novel lineage (proposed as lineage C), which is genetically distinct from historical North American strains [2, 12]. Phylogenetic analysis of partial nsP4 and structural gene sequences from horses in Argentina, Uruguay, and Brazil confirms that these contemporary strains share a common evolutionary origin with a 1958 Argentine strain, suggesting a long-term, low-level circulation of a genetically stable viral lineage in South America [2, 19]. This continuity, despite decades without significant outbreaks, points to the existence of a robust enzootic cycle in bird populations that is capable of maintaining the virus at a low, undetected level. As articulated by Gonzalez and Vincent (2019), understanding these maintenance cycles is essential for anticipating the emergence of arboviral threats [26]. The WEEV example demonstrates that viral persistence in birds is a consequence of both molecular adaptation, such as the fine-tuning of receptor usage [10] and positive selection for enzootic fitness [16], and ecological contingencies, including the movement of birds across vast distances and the capacity of the virus to exploit environmental windows of increased vector-host contact. The vector's own internal barriers, such as salivary gland escape barriers in Cx. tarsalis [25], further modulate transmission efficiency, creating a complex network of factors that collectively determine the success and spatiotemporal dynamics of WEEV perpetuation in avian populations.

References

[1] Yang Y, Zhao L, Li Z, Wang S, Xu Z, Wang Y. PCDH10 is a neuronal receptor for western equine encephalitis virus. Cell Research. 2024. DOI: https://doi.org/10.1038/s41422-024-01031-1

[2] Tomás G, Marandino A, Rodríguez S, Wallau G, Dezordi F, Oliveira ALSd, et al.. Diagnosis and genomic characterization of the largest western equine encephalitis virus outbreak in Uruguay during 2023–2024. npj Viruses. 2024. DOI: https://doi.org/10.1038/s44298-024-00078-6

[3] Reisen W, Reisen W, Chiles R, Martinez V, Fang Y, Green E. Experimental Infection of California Birds with Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses. Journal of medical entomology. 2003. DOI: https://doi.org/10.1603/0022-2585-40.6.968

[4] Hammon W, Reeves W, Sather G. Western equine and St. Louis encephalitis viruses in the blood of experimentally infected wild birds and epidemiological implications of findings.. Journal of Immunology. 1951. DOI: https://doi.org/10.4049/jimmunol.67.4.357

[5] Reisen W, Chiles R, Martinez V, Fang Y, Green E. Encephalitis Virus Persistence in California Birds: Experimental Infections in Mourning Doves (Zenaidura macroura). Journal of medical entomology. 2004. DOI: https://doi.org/10.1603/0022-2585-41.3.462

[6] Reisen W, Chiles R, Green E, Fang Y, Mahmood F, Martinez V, et al.. Effects of immunosuppression on encephalitis virus infection in the house finch, Carpodacus mexicanus. Journal of medical entomology. 2003. DOI: https://doi.org/10.1603/0022-2585-40.2.206

[7] Ferreira IB, Pereira LE, Rocco I, Marti A, Souza Ld, Iversson LB. Surveillance of arbovirus infections in the Atlantic Forest Region, State of São Paulo, Brazil. I. Detection of hemagglutination-inhibiting antibodies in wild birds between 1978 and 1990.. Revista do Instituto de Medicina Tropical de São Paulo. 1994. DOI: https://doi.org/10.1590/S0036-46651994000300011

[8] Ma B, Cao Z, Ding W, Zhang X, Xiang Y, Cao D. Structural basis for the recognition of two different types of receptors by Western equine encephalitis virus.. Cell Reports. 2025. DOI: https://doi.org/10.1016/j.celrep.2025.115724

[9] Liang S, Xu Z, Liu X, Yang Y, Zhao L, Hu C, et al.. Structural insights into VLDLR recognition by western equine encephalitis virus. Nature Communications. 2025. DOI: https://doi.org/10.1038/s41467-025-66330-6

[10] Liang S, Yang Y, Liu Y, Xu Z, Hou J, Li D, et al.. Structural basis for engagement of Western Equine Encephalitis Virus with the PCDH10 receptor. Nature Communications. 2025. DOI: https://doi.org/10.1038/s41467-025-61659-4

[11] Wang L, Zheng R, Li Z, Zhang L. Western equine encephalitis virus: A comprehensive review of epidemics, transmission, hosts, and strategies for mitigation. Virulence. 2025. DOI: https://doi.org/10.1080/21505594.2025.2580162

[12] Campos A, Franco AC, Godinho F, Huff R, Candido DS, Cardoso JdC, et al.. Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023–2024. Emerging Infectious Diseases. 2024. DOI: https://doi.org/10.3201/eid3009.240530

[13] Lorenz C, Azevedo TSd, Chiaravalloti-Neto F. Effects of climate change on the occurrence and distribution of Western equine encephalitis virus in South America.. Public Health. 2024. DOI: https://doi.org/10.1016/j.puhe.2024.12.031

[14] Campos A, Franco AC, Godinho F, Huff R, Cardoso J, Morais P, et al.. Molecular epidemiology of Western equine encephalitis virus in Brazil, 2023–2024. medRxiv. 2024. DOI: https://doi.org/10.1101/2024.04.15.24305848

[15] . western equine encephalitis virus. CABI Compendium. 2022. DOI: https://doi.org/10.1079/cabicompendium.60874

[16] Bergren NA, Haller SL, Rossi S, Seymour R, Huang JH, Miller AL, et al.. “Submergence” of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions. PLoS Pathogens. 2020. DOI: https://doi.org/10.1371/journal.ppat.1008102

[17] Phelps A, O’Brien L, Eastaugh LS, Davies C, Lever M, Ennis J, et al.. Susceptibility and Lethality of Western Equine Encephalitis Virus in Balb/c Mice When Infected by the Aerosol Route. Viruses. 2017. DOI: https://doi.org/10.3390/v9070163

[18] Simon LV, Coffey R, Fischer M. Western equine encephalitis. Definitions. 2020. DOI: https://doi.org/10.32388/tk7iot

[19] Vissani MA, Alamos F, Tordoya MS, Minatel L, Schammas J, Santos MDD, et al.. Outbreak of Western Equine Encephalitis Virus Infection Associated with Neurological Disease in Horses Following a Nearly 40-Year Intermission Period in Argentina. bioRxiv. 2024. DOI: https://doi.org/10.3390/v16101594

[20] Ma J, Wang H, Zheng X, Wu H, Yang S, Xia X. Western equine encephalitis virus virus‐like particles from an insect cell‐baculovirus system elicit the strong immune responses in mice. Biotechnology Journal. 2021. DOI: https://doi.org/10.1002/biot.202100008

[21] Henning LN, Endt K, Steigerwald R, Anderson M, Volkmann A. A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge. Frontiers in Immunology. 2021. DOI: https://doi.org/10.3389/fimmu.2020.598847

[22] Reeves LE, Burkett-Cadena N. Lizards Are Important Hosts for Zoonotic Flavivirus Vectors, Subgenus Culex, in the Southern USA. Frontiers in Tropical Diseases. 2022. DOI: https://doi.org/10.3389/fitd.2022.842523

[23] Mj T, Mccammon. Detection of arbovirus antibodies in avian sera by the complement fixation-inhibition test.. American Journal of Veterinary Research. 1979. DOI: https://doi.org/10.2460/ajvr.1979.40.02.299

[24] Vasconcelos P, Rosa JF, Rosa ATD, Dégallier N, Pinheiro F, Filho GCS. Epidemiologia das encefalites por arbovírus na amazônia brasileira. Revista Do Instituto De Medicina Tropical De Sao Paulo. 1991. DOI: https://doi.org/10.1590/S0036-46651991000600007

[25] Stauft C, Phillips A, Wang TT, Olson K. Identification of salivary gland escape barriers to western equine encephalitis virus in the natural vector, Culex tarsalis. bioRxiv. 2022. DOI: https://doi.org/10.1101/2022.01.11.475797

[26] Gonzalez J, Vincent T. Moving Arbovirology in a Changing World. . 2019. DOI: https://doi.org/10.33696/casereports.1.004