Equine Herpesvirus 1

Overview and Taxonomy of Equine Herpesvirus 1

Equine herpesvirus 1 (EHV-1) is a highly prevalent, globally distributed, and economically devastating pathogen of equids, classified within the family Herpesviridae, subfamily Alphaherpesvirinae, and genus Varicellovirus [1, 3, 17]. This taxonomic placement aligns EHV-1 with other significant alphaherpesviruses, including human herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), reflecting a shared evolutionary ancestry and fundamental biological properties such as neurotropism, latency, and the capacity for reactivation [12, 19]. The virion structure is characteristic of the herpesvirus family, comprising a double-stranded DNA genome encased within an icosahedral capsid, a proteinaceous tegument layer, and a lipid envelope studded with at least twelve distinct glycoproteins that mediate viral entry, cell-to-cell spread, and immune evasion [15, 20]. The linear genome of EHV-1 is approximately 150 kilobase pairs in length and is organized into unique long (UL) and unique short (US) regions, each flanked by inverted repeat sequences, a genomic architecture that facilitates recombination and genetic diversity [13, 15, 16]. The World Organisation for Animal Health (WOAH) recognizes EHV-1 as a notifiable pathogen due to its capacity to cause severe outbreaks of abortion and neurological disease, underscoring its significance to international equine health and trade [3, 5].

The historical recognition of EHV-1 as a distinct viral entity dates to the mid-20th century, when it was first identified as the causative agent of equine rhinopneumonitis and viral abortion [21, 22]. Early experimental inoculations by Jackson and Kendrick in 1971 definitively established the link between EHV-1 infection and paralytic neurological disease, a syndrome now termed equine herpesvirus myeloencephalopathy (EHM) [21, 22]. These seminal studies demonstrated that pregnant mares inoculated with the virus developed a spectrum of neurological signs, from mild ataxia to severe recumbency, with histopathological examination revealing a primary vasculitis of the central nervous system (CNS) [21, 22]. This work laid the foundation for understanding EHV-1 as a pathogen capable of causing not only respiratory and reproductive disease but also life-threatening neurological complications. Subsequent molecular characterization, beginning with restriction endonuclease fingerprinting in the 1980s, revealed the existence of two major genomic subtypes, subtype 1 (predominantly associated with abortion) and subtype 2 (more frequently isolated from respiratory cases), providing the first evidence of genetic heterogeneity within the species [23, 24]. These early studies by Allen and colleagues, analyzing over 300 field isolates, demonstrated that while subtype 1 isolates exhibited remarkable genetic stability over decades, subtype 2 isolates displayed greater diversity, suggesting different evolutionary pressures and epidemiological dynamics [23, 24].

The modern taxonomic understanding of EHV-1 has been refined through comprehensive genomic sequencing and phylogenetic analyses. The species is now recognized to encompass a continuum of genetic variants rather than discrete pathotypes, although a single nucleotide polymorphism (SNP) in open reading frame 30 (ORF30), encoding the catalytic subunit of the viral DNA polymerase, has garnered significant attention. This SNP results in a non-synonymous substitution of asparagine (N) to aspartic acid (D) at amino acid position 752 (N752D), which was initially proposed as a marker for "neuropathogenic" strains [11, 13]. The D752 genotype has been statistically associated with an increased risk of neurological disease and hypervirulence, with one Irish study estimating that the odds of neurological disease were 27 times greater for infections with the D752 genotype compared to the N752 genotype [11]. However, a critical and evolving consensus, supported by the 2024 ACVIM consensus statement and multiple large-scale epidemiological investigations, now emphasizes that this genotype is neither necessary nor sufficient for the development of EHM [1, 3, 9]. Pusterla et al. (2020) demonstrated in a cohort of 65 EHM-diagnosed horses that the frequency of the D752 and N752 genotypes was not significantly different, and clinical outcomes, including severity of ataxia, fever, and mortality, were comparable between the two groups, with the notable exception of a higher incidence of urinary incontinence in D752-infected horses [9]. Furthermore, the major 2021 Valencia outbreak, which involved over 750 horses and resulted in widespread neurological disease and multiple fatalities, was caused by a virus harboring the A2254 (N752) genotype, definitively proving that non-neuropathogenic strains can trigger severe EHM outbreaks [2, 3, 6, 8]. This outbreak strain also carried a unique SNP (A713G) in ORF11, which has been proposed as a novel epidemiological marker for tracking the spread of this particular lineage [8].

The genomic diversity of EHV-1 extends well beyond the ORF30 locus. Multi-locus typing and whole-genome sequencing have revealed the existence of at least 13 distinct UL clades circulating globally, with evidence of inter-clade recombination, as well as recombination with the closely related equine herpesvirus 4 (EHV-4) and equine herpesvirus 8 (EHV-8) [13, 16]. Bryant et al. (2018) sequenced 78 EHV-1 strains isolated over 35 years and demonstrated that while most neurological isolates clustered within a few clades, abortion isolates were distributed across nine clades, indicating that abortigenic potential is a widespread property of the species [13]. Importantly, this study also identified three neurological isolates from linked outbreaks that lacked the D752 substitution but possessed a different, previously unrecognized polymorphism in ORF30, suggesting alternative genetic determinants of neurovirulence [13]. The attenuated vaccine strain Kentucky A (KyA) has been fully sequenced and shown to harbor large deletions in genes encoding glycoproteins I and E (gI/gE) and gp2, as well as multiple point mutations, which collectively contribute to its attenuation and high replication titers in cell culture [15]. These genomic features highlight the plasticity of the EHV-1 genome and the potential for rapid evolution under selective pressure from host immunity or vaccination.

Epidemiologically, EHV-1 is considered endemic in horse populations worldwide, with seroprevalence studies demonstrating widespread exposure. In northern Morocco, a type-specific ELISA revealed that 12.8% of unvaccinated and 21.8% of vaccinated horses were seropositive for EHV-1, while all horses tested were positive for EHV-4, indicating near-universal exposure to the latter [7]. In Australia, a 25-year retrospective study of 600 aborted equine fetuses detected EHV-1 DNA in 3% of cases, confirming its role as a persistent cause of infectious abortion [10]. The virus is maintained in populations through the establishment of lifelong latency, primarily in the trigeminal ganglion and lymphoid tissues, with periodic reactivation triggered by stress, transport, immunosuppression, or concurrent disease [3, 17, 19]. This latent reservoir poses a continuous challenge for disease control, as clinically healthy horses can intermittently shed virus without overt signs, facilitating silent transmission within and between premises [4]. The 2021 Valencia outbreak exemplifies this risk: the virus was likely introduced by a latently infected horse attending the show-jumping competition, and the delay in diagnosis and implementation of biosecurity measures allowed rapid spread among the 752 horses present, with subsequent geographic dissemination across Europe [2, 3, 6]. Risk factor analysis from this outbreak identified male sex (stallions and geldings were six times more likely to become infected), age greater than nine years, and housing in poorly ventilated central areas of stabling tents as significant predictors of EHM development [2].

The host range of EHV-1 is not restricted to domestic horses. Natural infection and disease have been documented in donkeys, mules, and a wide array of non-equid species, including cattle, camelids, cervids, and even ursids and felids, as assessed under the European Animal Health Law framework [5, 14]. In Ethiopia, a severe EHM outbreak affecting horses, mules, and donkeys revealed that donkeys experienced particularly fulminant disease, with sudden death without premonitory signs of paralysis, and 98.9% of isolates from affected equids carried the D752 genotype [14]. Additionally, the closely related equine herpesvirus 9 (EHV-9), originally isolated from Thomson’s gazelles, has been shown to cause neurological disease in experimentally infected horses, further blurring the taxonomic and pathogenic boundaries within the equid alphaherpesvirus group [18]. The ability of EHV-1 to cross species barriers and cause severe disease in novel hosts underscores its potential as an emerging pathogen and highlights the need for vigilant surveillance at the human-animal interface, particularly given the economic importance of equids in agriculture, sport, and transportation. The Food and Agriculture Organization (FAO) and WOAH have both emphasized the need for improved diagnostic capacity and biosecurity protocols to mitigate the impact of EHV-1 on global equine health and trade [3, 5].

Molecular Pathogenesis of Equine Herpesvirus 1

The molecular pathogenesis of Equine Herpesvirus 1 (EHV-1) represents a sophisticated and multi-stage process that begins at the mucosal surface and culminates in systemic dissemination, immune evasion, and severe endotheliopathy. The virus has evolved a remarkable array of strategies to subvert host defenses, exploit immune cells for transport, and establish latency, all of which contribute to its status as one of the most consequential pathogens in equine medicine.

Initial Entry and Respiratory Epithelial Infection

The molecular cascade of EHV-1 infection commences with the inhalation of aerosolized virus and its deposition on the respiratory epithelium. A seminal discovery in the understanding of EHV-1 entry was the identification of equine major histocompatibility complex (MHC) class I molecules as functional entry receptors [32]. Unlike herpes simplex virus, which utilizes specific glycoprotein receptors, EHV-1 glycoprotein D (gD) binds directly to equine MHC class I molecules on the surface of target cells [32]. This interaction is highly specific; exogenous expression of equine MHC class I heavy chain genes in non-permissive murine cells confers susceptibility to infection, and antibodies directed against gD or equine MHC class I can block viral entry [32]. The efficiency of this entry mechanism is further refined at the molecular level: a single amino acid residue at position 173 within the α2 domain of the MHC class I molecule is critical for determining receptor function. Specifically, the presence of alanine at this position is permissive for viral entry, while glutamine or threonine residues abrogate receptor activity [35]. This precise molecular requirement may explain the strict host range of EHV-1 and its predilection for equine endothelial cells and leukocytes, which abundantly express the appropriate MHC class I isoforms.

Once internalized, the virus undergoes uncoating and begins replication within the nuclei of respiratory epithelial cells. Studies using ex vivo tissue explants have demonstrated that EHV-1 replicates in a plaque-wise manner within the nasal mucosa, with plaques visible as early as 24 hours post-inoculation [29]. The virus exhibits a clear tropism for respiratory epithelium, with significantly larger plaques formed in nasal mucosa compared to vaginal mucosa, reflecting an evolutionary adaptation to the respiratory route of entry [29]. Critically, viral replication does not directly breach the basement membrane via cell-to-cell spread; instead, EHV-1 subverts monocytic cells (CD172a+ cells) that infiltrate the infected epithelium, hijacking these cells to invade the underlying lamina propria [29].

Orchestration of Leukocyte Recruitment and Early Immune Modulation

The transition from a localized respiratory infection to systemic dissemination is a highly regulated process that relies on the virus's ability to manipulate the host chemokine response. Research has revealed a striking dichotomy between neuropathogenic and abortigenic EHV-1 strains in their capacity to recruit immune cells to the upper respiratory tract (URT). Neurovirulent EHV-1 strains enhance the expression and bioactivity of the chemokines CXCL9 and CXCL10 in primary respiratory epithelial cells, resulting in the efficient recruitment of both monocytic CD172a+ cells and T lymphocytes [28]. Conversely, abortigenic strains temper monocyte migration, and this difference appears to be mediated, at least in part, by the viral glycoprotein gp2. Deletion of gp2 from a neuropathogenic strain paradoxically increased the recruitment of monocytic and T cells to epithelial cell supernatants, revealing that gp2 normally functions to temper leukocyte migration [28]. This suggests a sophisticated viral orchestration: neuropathogenic strains may selectively recruit the very cells that will later serve as vehicles for systemic dissemination, while abortigenic strains may limit early inflammation to favor localized replication in the reproductive tract.

Concurrent with this chemokine manipulation, EHV-1 mounts a formidable assault on the host's innate antiviral defenses, particularly the type I interferon (IFN) system. In equine endothelial cells (EECs), infection with a neuropathogenic strain results in a transient induction of IFN-β followed by a rapid and profound shutdown [31]. This inhibition operates at multiple levels of the IFN signaling cascade. The virus significantly reduces the mRNA expression of Toll-like receptor 3 (TLR3) and TLR4 as early as 6 hours post-infection (hpi), effectively dampening the sensitization phase of the IFN response [31]. Downstream of receptor engagement, EHV-1 reduces mRNA levels of interferon regulatory factor 7 (IRF7) and IRF9, and decreases the cellular expression of tyrosine kinase 2 (TYK2) [31]. The most critical attack, however, occurs at the level of signal transducer and activator of transcription 2 (STAT2). EHV-1 blocks the phosphorylation and nuclear translocation of STAT2 in response to exogenous IFN, with this inhibition being dependent on viral late gene expression [31]. This strategic blockade of the JAK-STAT pathway effectively renders infected cells blind to the antiviral effects of interferon, creating a permissive environment for viral replication and spread.

Cell-Associated Viremia: The T Lymphocyte as a Trojan Horse

The hallmark of EHV-1 pathogenesis is the establishment of a cell-associated viremia, which is critical for viral transport from the respiratory tract to secondary target organs, including the pregnant uterus and the central nervous system. While early studies identified peripheral blood mononuclear cells (PBMCs) as the primary carriers, the molecular details of this relationship have been elucidated in remarkable detail. EHV-1 is predominantly a T-lymphotropic virus; studies using infective center assays and mitogen stimulation demonstrated that T lymphocytes, rather than B cells or monocytes, harbor the highest viral load during peak viremia [34]. More recent work has confirmed that activated T lymphocytes are significantly more susceptible to infection than their quiescent counterparts, and that CD4+ T cells are infected with greater efficiency than CD8+ T cells, particularly those derived from blood versus lymph nodes [12].

The virus has evolved a suite of immune evasion mechanisms that allow it to persist within T lymphocytes despite the presence of neutralizing antibodies. Upon infection of T cells, all classes of viral proteins are expressed early, yet the expression of viral glycoproteins on the cell surface is restricted [12]. This "stealth" phenotype prevents immune recognition and destruction by antibody-dependent cellular cytotoxicity or complement. Furthermore, the release of progeny virions from infected T lymphocytes is severely hampered, leading to an accumulation of viral nucleocapsids within the T cell nucleus [12]. This intracellular retention is not a defect but a deliberate strategy: the virus maintains a high concentration of infectious material within the cell, ready for transfer upon contact with target endothelial cells.

The transfer mechanism itself is a masterful piece of viral biology. When an infected T lymphocyte encounters an endothelial cell of the endometrium or central nervous system, a late viral protein(s) orchestrates the formation of a specialized junction known as the "viral synapse" [12]. This process involves T cell polarization, the formation of actin-rich projections, and the anterograde dynein-mediated transport of viral progeny toward the cell-cell interface. The transfer of virus occurs directly from the T cell to the endothelial cell, bypassing the extracellular space and thus evading neutralizing antibodies [12]. Transcriptomic analysis of PBMCs during peak viremia has further revealed a complex interplay of host responses, including the upregulation of interferon defense responses, chemokine signaling, cell adhesion pathways, and coagulation cascades, all of which contribute to the vascular pathology that defines the later stages of disease [25].

The Molecular Basis of Neuropathogenicity: The ORF30 Polymorphism

No discussion of EHV-1 pathogenesis is complete without addressing the controversial role of the single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30). An A-to-G substitution at nucleotide 2254 results in a change from asparagine (N) to aspartic acid (D) at amino acid position 752 (N752D) within the catalytic subunit of the viral DNA polymerase [11]. This mutation has been statistically associated with an increased incidence of neurological disease, leading to the classification of "neuropathogenic" (D752) and "non-neuropathogenic" (N752) strains.

The epidemiological evidence supporting the significance of this mutation is substantial. In a 28-year study of 272 Irish isolates, viruses harboring the G2254/D752 genotype had a 27-fold greater association with neurological disease compared to those with the A2254/N752 genotype [11]. Similarly, a study of EHM outbreaks in Ethiopia found that 90 out of 91 clinically affected equids (98.9%) harbored the D752 genotype, including horses, mules, and donkeys [14]. However, the relationship is not absolute. Multiple studies have demonstrated that N752 strains can and do cause neurological disease, albeit at a lower frequency. A retrospective analysis of 65 EHM cases in the United States found no significant difference in the frequency of D752 versus N752 genotypes among affected horses, nor did the genotype significantly impact the overall clinical outcome or severity of ataxia [9]. The one notable exception was urinary incontinence, which was significantly more common in horses infected with D752 strains [9].

This paradox suggests that while the ORF30 polymorphism is a significant molecular marker, it is not the sole determinant of neuropathogenicity. The outbreak at the CES Valencia Spring Tour in 2021 provides a compelling case in point. The virus responsible for this large-scale neurological outbreak was genotyped as A2254 (N752), the so-called "non-neuropathogenic" variant [2, 3, 8]. Genomic sequencing of five isolates from this outbreak revealed a mutation in open reading frame 11 (ORF11; A713G) that was not present in 249 other EHV-1 sequences, suggesting that other viral genes can compensate for or modulate the neuropathogenic potential [8]. Furthermore, whole-genome sequencing of 78 strains over 35 years has revealed up to 13 distinct viral clades circulating in the UK, with neurological isolates grouping into five different clades, indicating that neuropathogenicity is polygenic [13].

Endothelium as the Battleground: Vasculitis, Thrombosis, and Immunopathology

The final stage of EHV-1 pathogenesis is defined by infection of the endothelial lining of small blood vessels, leading to vasculitis, thrombosis, and ischemic necrosis of downstream tissues. This is the common pathway for both abortion and neurological disease, with the specific target organ (pregnant uterus versus CNS) determining the clinical manifestation. The histopathological hallmark of EHM is a lymphohistiocytic vasculitis, characterized by infiltration of mononuclear cells into the vessel wall, endothelial swelling, fibrinoid necrosis, and thrombosis [21, 27]. In experimental infections with the neuropathogenic Ab4 strain, horses that developed EHM exhibited frank vasculitis not only in the spinal cord but also in the lungs, endometrium, and eyes, underscoring the systemic nature of the vascular pathology [27].

The molecular events that trigger this vasculitic response are a complex interplay of direct viral cytopathology and immune-mediated damage. Transcriptomic profiling of PBMCs from horses that develop EHM compared to those that do not has identified several critical pathways that distinguish the "EHM phenotype" [33]. Horses that develop EHM show an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1, and a skewing of the immune response toward a T-helper 2 (Th2) phenotype [33]. Coagulation pathways are also dysregulated, which likely contributes to the thrombotic component of the disease [33].

The role of the host immune response, particularly in aged horses, cannot be overstated. While EHM occurs in only ~10% of infected horses overall, the incidence increases dramatically to >70% in mares over 20 years of age [30]. Using an "old mare model" of EHM, researchers have identified a distinct immunological signature associated with susceptibility. Non-EHM horses (typically young horses) show an early and robust upregulation of type I interferon (IFN-α) in nasal secretions, with upregulation of IRF7, IRF9, IL-1β, CXCL10, and TBET in blood, followed by an IFN-γ response during viremia [30]. This profile is indicative of a strong, timely cellular immune response. In stark contrast, EHM horses (aged mares) show low IFN-α levels in nasal secretions, and peak levels of IRF7, IRF9, CXCL10, and TGF-β coincide with viremia rather than preceding it [30]. Most significantly, EHM horses exhibit significantly higher levels of the anti-inflammatory cytokine IL-10 in nasal secretions, PBMCs, and cerebrospinal fluid, along with higher serum IgG3/5 antibody titers. This suggests that a shift toward a regulatory or Th2-type immune response, rather than a protective type 1 response, is a key risk factor for EHM [30]. This immunophenotype likely allows for higher and more prolonged cell-associated viremia, which is known to be a prerequisite for CNS endothelial infection.

The culmination of this process in the CNS is a fulminant neuroinflammatory response. In a mouse model of EHV-1 encephalitis, infection with neurovirulent strains leads to severe infiltration of monocytes and CD8+ T cells into the brain, driven by a complex chemokine storm involving CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9, and CXCL10 [26]. Pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-12β, and TNF are abundant, and Toll-like receptors (TLR2, TLR3, TLR9) are upregulated [26]. Strikingly, no expression of IFN-γ or IL-12α was detected, reinforcing the concept that a failure of the type 1 immune response is a central feature of neuropathogenesis [26]. The activated innate immune mechanisms, rather than controlling the virus, contribute directly to the extensive neuropathology. This immune-mediated damage, rather than direct viral lysis of neurons, is believed to be the primary driver of the clinical signs of ataxia, paralysis, and urinary incontinence that characterize equine herpesvirus myeloencephalopathy.

Epidemiology and Risk Factors for EHV-1 Infection and EHM

Equine herpesvirus-1 (EHV-1) is an exceedingly prevalent pathogen distributed across the globe, and its epidemiological profile is characterized by endemic circulation within virtually all managed equid populations, punctuated by episodic outbreaks of severe disease, most notably equine herpesvirus myeloencephalopathy (EHM) and abortion storms. Understanding the complex interplay of viral, host, and environmental factors that govern the transmission, reactivation, and clinical expression of EHV-1 is paramount for the design of effective control strategies. This section provides an exhaustive analysis of the epidemiological landscape and the multifactorial risk determinants for EHV-1 infection and its most devastating sequela, EHM.

Global Distribution and Seroprevalence

EHV-1 is considered endemic in equine populations across Europe, North America, and beyond, with serological surveys consistently demonstrating widespread exposure. The virus is not subject to eradication programs, and its ubiquitous nature is underpinned by its capacity for latency and reactivation. The European Food Safety Authority (EFSA) has formally assessed EHV-1 infection within the framework of the Animal Health Law, concluding that it can be considered eligible for Union intervention, although with a low certainty for categorization as a high-consequence disease requiring immediate eradication (Category A) [3, 5]. This assessment reflects the reality that while EHV-1 is endemic and economically significant, it is not subject to the same stringent stamping-out policies as, for example, foot-and-mouth disease. The World Organisation for Animal Health (WOAH) includes EHV-1 as a listed disease due to its socioeconomic impact and trade implications, underscoring its global relevance.

Seroprevalence rates vary significantly by region, management system, and vaccination history. In a recent survey of horse populations in northern Morocco, a region where the virus is endemic, type-specific ELISA testing revealed that 12.8% of unvaccinated horses and 21.8% of vaccinated horses were seropositive for EHV-1 [7]. Notably, the same study found that 100% of sampled horses were seropositive for the closely related EHV-4, highlighting the near-universal exposure to this alphavirus. The lower seroprevalence for EHV-1 compared to EHV-4 aligns with the general understanding that EHV-4 is even more widespread and primarily causes mild respiratory disease, whereas EHV-1 carries a greater risk for systemic spread and severe outcomes. Importantly, the fact that a proportion of vaccinated horses remained seropositive, yet with low neutralizing antibody titers, points to the limitations of current vaccination protocols in inducing robust, long-lasting humoral immunity [7]. The presence of seropositive animals in both vaccinated and unvaccinated groups underscores the endemic nature of the infection and the continuous circulation of the virus even in well-managed herds.

In contrast to the high seroprevalence in adult populations, the detection of EHV-1 as a cause of abortion in specific regions can be surprisingly low. A 25-year retrospective study in Australia using qPCR on aborted fetal tissues found a prevalence of EHV-1 DNA of only 3% [10]. Similarly, a study in Brazil reported a low occurrence of EHV-1 as a cause of abortion and perinatal mortality, with only 2 of 105 fetuses testing positive by PCR [39]. These data suggest that while EHV-1 is a primary infectious cause of abortion, the proportion of abortions attributable to this virus relative to other causes (e.g., bacterial placentitis, non-infectious factors) can be highly variable and may be overestimated in some clinical reports. The discrepancy between high seroprevalence and relatively low abortion prevalence in some populations indicates that many infections are subclinical or result only in mild respiratory disease, and that specific host or viral factors are required for the more severe manifestations.

Transmission Dynamics and Viral Shedding

The primary route of EHV-1 transmission is direct or indirect contact with infectious nasal secretions from acutely infected horses. Following a short incubation period of 2–10 days, infected horses shed large quantities of virus in nasal discharge, typically for 7–10 days, although shedding can be prolonged in some individuals [1, 40]. The virus is highly contagious and can be transmitted via fomites (e.g., shared water buckets, grooming equipment, tack, and human hands), aerosols over short distances, and potentially through contaminated feed and bedding. The 2021 outbreak in Valencia, Spain, which involved over 750 horses at a show-jumping competition, provided a stark illustration of the explosive potential of this virus when introduced into a high-density, stressed population. The outbreak rapidly spread within the tented stabling complex, leading to cancellation of the event, lockdown of the facility, and subsequent geographic dissemination of the virus across Europe, likely due to a delay in diagnosis and late application of biosecurity measures [2, 3, 6, 8]. Genomic sequencing of the outbreak strain revealed it was closely related to strains circulating in Belgium and the United Kingdom, suggesting a common source or a chain of transmission linked to the international movement of horses [6].

A critical and often underappreciated aspect of EHV-1 transmission is the role of subclinically shedding horses. A study following a multi-county EHM outbreak in California found that EHV-1 DNA was detected by qPCR in nasal swabs from clinically healthy sport horses during mandatory quarantine periods [4]. This "silent" shedding represents a significant risk for outbreak propagation, as seemingly healthy animals can introduce the virus into new populations. The ACVIM consensus statement underscores that latency is established in lymphoid tissues and the trigeminal ganglion, and that reactivation can occur spontaneously or be triggered by stress (e.g., transport, weaning, intense competition, corticosteroid administration), leading to renewed shedding and transmission without overt clinical signs in the carrier [1, 40].

Recent research has also identified urine as a potential, albeit secondary, source of EHV-1 shedding. During two EHM outbreaks in Spain, viral DNA was detected in urine samples from 11 of 18 hospitalized horses, and the duration of DNA detection in urine was longer than in buffy coat samples and comparable to that in nasal swabs [37]. Although the concentration of viral DNA in urine was lower than in nasal swabs, this finding raises the possibility of environmental contamination and transmission through urine-soaked bedding, particularly in stabling environments. The clinical relevance of urinary shedding for transmission remains to be fully determined, but it warrants consideration in biosecurity protocols.

Viral Strain Diversity and the Neuropathogenicity Debate

Epidemiological investigations into EHV-1 have long sought to identify viral genetic determinants that predict disease outcome. The most widely studied polymorphism is a single nucleotide substitution in the DNA polymerase gene (ORF30) resulting in an amino acid change from asparagine (N) to aspartic acid (D) at position 752 (N752D). This change, found in so-called "neuropathogenic" strains (D752), has been statistically associated with an increased risk of neurological disease. Early studies, including a comprehensive analysis of 272 Irish isolates from 238 outbreaks over 28 years, found that the association between the D752 genotype and neurological disease was highly significant, with the odds of neurological disease in horses infected with D752 strains estimated to be 27 times greater than those infected with N752 strains [11]. Furthermore, that study linked the D752 genotype with "hypervirulence," defined as outbreaks involving multiple abortion or neurological cases [11]. In Ethiopia, a study of EHM outbreaks in horses, donkeys, and mules found that 98.9% of clinically affected animals harbored the D752 genotype, while the N752 variant was found in only a single donkey [14]. This near-perfect association in an outbreak setting strongly suggests that this genotype is a major contributor to EHM in that region.

However, the binary classification of strains as "neuropathic" or "non-neuropathic" based solely on the ORF30 polymorphism is now recognized as an oversimplification. Subsequent large-scale studies have fundamentally challenged this paradigm. A study of 65 horses diagnosed with EHM at a diagnostic laboratory in the United States, the largest case series of its kind, found that there was no significant difference in the frequency of the D752 versus N752 genotype among EHM-affected horses [9]. Furthermore, aside from an increased frequency of urinary incontinence in D752-infected animals, there were no significant differences in the severity of ataxia, fever, lethargy, or overall outcome between the two genotype groups [9]. This finding has been corroborated by the 2024 ACVIM consensus statement, which explicitly states that the neuropathogenic potential of an EHV-1 strain cannot be predicted based on the ORF30 genotype alone [1]. The EFSA assessment also concluded that the respiratory, reproductive, and neurological signs are not strain-specific and that the virus responsible for the 2021 Valencia outbreak (genotype A2254, corresponding to N752) did not possess the "classical" neuropathogenic marker [3].

This apparent contradiction, that a mutation associated with neurological disease in some studies is not predictive in others, highlights the importance of other viral and host factors. Whole-genome sequencing of EHV-1 isolates has revealed a high degree of diversity. Analysis of 78 strains from the UK over 35 years identified up to 13 distinct clades circulating in the UK, with neurological isolates grouping into 5 clades and the majority possessing the N752D substitution, although some neurological isolates from linked outbreaks had a different polymorphism [13]. This suggests that multiple genetic pathways can lead to the neurological phenotype. Furthermore, the 2021 European outbreak strain contained a unique mutation (A713G in ORF11) not found in over 250 other sequenced isolates, providing a new potential marker for epidemiological surveillance [8]. Recombination between different EHV-1 clades, and even between EHV-1 and EHV-4 or EHV-8, has also been detected, further contributing to genetic diversity and the potential emergence of novel strains with altered virulence [13, 16]. Thus, the molecular epidemiology of EHV-1 is a dynamic landscape, and reliance on a single genetic marker for risk assessment is inadequate.

Host Risk Factors: Age, Sex, and Immune Competence

Perhaps the most critical determinants of EHM risk are host-specific factors, with age emerging as the most robust and clinically relevant predictor. Multiple observational and experimental studies have demonstrated a dramatically increased incidence of EHM in older horses, particularly aged mares. A landmark experimental model used to study this phenomenon involved infecting old mares ( >20 years of age) with the neuropathogenic strain EHV-1 Ab4. In these studies, EHM developed in 100% of old mares, often leading to severe, recumbent disease requiring euthanasia, whereas only 1 of 9 young horses developed mild ataxia [30, 33]. This striking difference is not explained by a higher viral load in the nasal passages; in fact, aged mares shed significantly less virus from the nose than young horses. Instead, they developed a more profound cell-associated viremia, coinciding with a single peak of fever, and a dysregulated immune response [30].

Transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from these aged mares revealed that EHM-susceptible animals exhibited a distinct immunophenotype prior to and during infection. Key features included an upregulation of IL-6, a dysregulation of T-cell activation pathways (e.g., AP-1 signaling), and a skewing of the adaptive response towards a T-helper 2 (Th2) phenotype, characterized by increased IL-10 production and a lack of robust, early type-I interferon (IFN) and IFN-γ responses [30, 33]. In contrast, non-EHM horses mounted a rapid and effective innate response, with early upregulation of IFN-α in nasal secretions and IRF7/IRF9, IL-1β, CXCL10, and TBET in the blood [30]. This failure of the aged immune system to mount a timely, cell-mediated antiviral response is believed to permit unrestricted viral replication in endothelial cells of the central nervous system, leading to the vasculitis and thrombosis that are the pathogenic hallmarks of EHM [26, 27, 38]. The histopathological changes in EHM are not a direct viral cytolytic effect on neurons but rather an immune-mediated vasculopathy, with lymphohistiocytic infiltration and fibrinoid necrosis of small arteries and veins in the CNS [21, 22, 27]. This vasculitis leads to ischemia, edema, and secondary neurological deficits. The findings from the old mare model strongly suggest that immunosenescence, the age-related decline in immune function, is a primary risk factor for EHM.

Sex appears to be another important risk factor, although the data are complex and context-dependent. The Valencia outbreak study reported that stallions and geldings were six times more likely to develop clinical EHV-1 infection (fever and/or other signs) compared to mares [2]. This finding is intriguing and may relate to differences in stress physiology, management (e.g., stabling of males in more central, high-traffic areas), or sex hormone influences on immune function. However, when it comes to severe EHM requiring hospitalization and resulting in death in that same outbreak, male sex was not a significant predictor; the risk was instead associated with age >9 years [2]. Conversely, the Ethiopian outbreak study of working equids found that females (63.7%) were more frequently affected with EHM than males [14], and the experimental old mare model specifically selects aged females because of their high susceptibility [30, 33]. It appears that while male horses may be more prone to becoming infected and showing clinical signs of respiratory disease, the progression to EHM is more strongly linked to age and female status in certain populations. The underlying mechanisms for this sex-based difference in EHM risk are poorly understood but may involve hormonal modulation of endothelial cell susceptibility or immune regulation. Pregnant mares are also uniquely at risk for both abortion and EHM, historically recognized since the early experimental studies of EHV-1 where non-pregnant animals did not develop neurological disease while pregnant mares did [22].

Breed predisposition has also been reported. In the US study of 65 EHM cases, Quarter Horses and Saddlebreds were overrepresented, and Warmbloods underrepresented, among horses infected with the D752 genotype compared to the N752 genotype [9]. This suggests that certain breeds may be more commonly infected with specific viral genotypes, possibly due to management practices or genetic susceptibility to infection with particular strains.

Environmental and Management Risk Factors

The explosive nature of EHM outbreaks is inextricably linked to environmental and management factors that facilitate viral transmission and increase host susceptibility. The 2021 Valencia outbreak provides a textbook case. The event was a high-intensity show-jumping competition, which inherently involves stress from transport, unfamiliar environment, intense physical exertion, and social mixing. Horses were housed in a large, temporary tented structure, which likely had suboptimal ventilation and high horse density. The study from this outbreak identified that horses located in the middle of the tent were significantly more likely to develop EHM compared to those at the periphery [2]. This finding directly implicates poor air circulation and higher concentrations of aerosolized virus in the central, less ventilated areas as a key risk factor. The authors emphasized the "crucial role of stable design, position, and ventilation in EHV outbreaks" [2].

Other critical management risk factors include:

• High population density and frequent horse movements: Shows, sales, training facilities, and veterinary hospitals are high-risk environments. The mixing of horses from different origins, some of whom may be latently infected and reactivating, creates a perfect storm for transmission.
• Stressful events: Long-distance transport, weaning, intensive training, and concurrent illness are well-documented triggers for reactivation of latent virus and for increasing susceptibility to primary infection [1, 40].
• Delayed diagnosis and biosecurity: The EFSA report on the Valencia outbreak noted that the wide geographic spread of the virus was possibly due to a delay in diagnosis and the late application of biosecurity measures [3]. Once a horse develops fever or neurological signs, immediate isolation and testing are essential. The use of quarantine and qPCR testing of clinically healthy horses, as implemented after the California outbreak, proved highly successful at preventing further EHM cases and allowing safe return to competition [4].
• Vaccination status and history: While vaccination against EHV-1 is widely practiced, its ability to prevent EHM is limited. Systematic reviews and the ACVIM consensus statement have concluded that the evidence for successful vaccination against EHM is limited, and no vaccine label carries a claim for protection against the neurological form [1, 36]. However, vaccination may reduce nasal shedding and viremia, thereby potentially decreasing transmission and the severity of disease. The field study in Morocco showed that vaccinated horses had higher seroprevalence but low neutralizing antibody titers [7], and the immunological studies indicate that current vaccines induce a more restricted IgG isotype response than natural infection and may not effectively promote the cytotoxic T-lymphocyte (CTL) responses thought to be critical for controlling EHM [19, 41]. Thus, a history of vaccination should not be interpreted as providing reliable protection against EHM, but it remains a recommended component of a comprehensive prevention program [1].

Co-Infections and Other Pathogens

The epidemiology of EHV-1 is complicated by the frequent presence of other respiratory pathogens. The California study of clinically healthy show horses found that EHV-1 was circulating alongside EHV-4, equine influenza virus, equine rhinitis B virus, Streptococcus equi subsp. equi, and gammaherpesviruses (EHV-2 and EHV-5) [4]. Co-infections can exacerbate clinical disease, complicate diagnosis, and alter transmission dynamics. For instance, infection with EHV-2 has been shown to reactivate EHV-1 from latency, and the presence of multiple viruses may overwhelm the host's immune defenses, increasing the risk of severe outcomes. The transcriptomic study of PBMCs from EHV-1-infected horses also detected transcripts of EHV-2 and EHV-5, suggesting that co-infection is common and may influence the host response [25].

Summary of Key Epidemiological Features

• Endemic Presence: EHV-1 is ubiquitous in equine populations worldwide, with seroprevalence varying but indicating widespread exposure.
• Transmission: Primarily via direct contact with nasal secretions; also via fomites, aerosols, and potentially urine. Subclinical shedders play a critical role in silent transmission.
• Latency and Reactivation: The virus establishes lifelong latency, with periodic reactivation triggered by stress, leading to renewed shedding and disease.
• Viral Genetics: The ORF30 polymorphism (N752D) is associated with increased risk of EHM in some populations but is neither necessary nor sufficient for the development of neurological disease. The viral genome is diverse, with multiple clades circulating and

Clinical Manifestations: Respiratory, Reproductive, and Neurologic Forms

Equine herpesvirus-1 (EHV-1) infection manifests as a spectrum of clinical syndromes that range from subclinical to rapidly fatal, reflecting the virus’s sophisticated capacity to exploit host cellular machinery and evade immune surveillance. The clinical expression of disease is governed by a complex interplay between viral strain characteristics, host immune status, age, sex, environmental stressors, and pregnancy state. The three principal clinical forms, respiratory, reproductive, and neurologic, are not mutually exclusive; they frequently overlap within outbreaks and even within individual animals, underscoring the systemic nature of EHV-1 pathogenesis. Understanding the nuanced presentation of each form is critical for early recognition, effective outbreak management, and the implementation of targeted therapeutic interventions.

Respiratory Form: The Portal of Entry and Primary Replication Site

The respiratory form represents the most common and often the initial clinical manifestation of EHV-1 infection. Following inhalation of aerosolized virus or direct contact with contaminated fomites, EHV-1 establishes primary replication within the epithelial cells of the upper respiratory tract, particularly the nasal mucosa and nasopharynx [1, 17]. This initial replication phase is typically subclinical or associated with mild, self-limiting disease in immunocompetent adult horses, but it can be more pronounced in young, immunologically naïve animals, particularly weanlings and yearlings [1, 30].

Clinically, the respiratory form is characterized by a biphasic fever pattern, with the first temperature spike occurring 24–48 hours post-infection, coinciding with viral replication in the respiratory epithelium, and a second spike approximately 4–6 days later, which correlates with the onset of cell-associated viremia [1, 30]. Fever often exceeds 39.5°C (103°F) and may persist for 2–4 days. Accompanying signs include serous to mucopurulent nasal discharge, conjunctivitis, submandibular lymphadenopathy, lethargy, anorexia, and a dry, harsh cough [1, 17]. In many cases, the respiratory signs are indistinguishable from those caused by equine herpesvirus-4 (EHV-4), equine influenza virus, or Streptococcus equi subsp. equi, necessitating laboratory confirmation for definitive diagnosis [1, 4].

The severity of respiratory disease is influenced by viral dose, strain virulence, and host factors. Experimental infections with the neuropathogenic strain Ab4 in young horses consistently produce marked respiratory signs, including a pronounced biphasic fever, profuse nasal shedding, and significant lethargy [30]. In contrast, older mares infected with the same strain may exhibit minimal to no respiratory signs, yet paradoxically develop severe neurologic disease [30, 33]. This dichotomy highlights the critical role of age-related immune senescence in shaping clinical outcome. The respiratory epithelium is not merely a passive barrier; it actively orchestrates the early immune response. EHV-1 infection of primary equine respiratory epithelial cells (ERECs) induces the expression of chemokines such as CXCL9 and CXCL10, which recruit monocytic cells and T lymphocytes to the site of infection [28]. Neuropathogenic strains have been shown to elicit a more robust chemokine response than abortigenic strains, potentially facilitating more efficient leukocyte recruitment and subsequent viral dissemination [28].

Viral shedding from the respiratory tract is a critical epidemiological feature. Nasal shedding of EHV-1 typically begins 1–2 days post-infection, peaks around day 4–6, and can persist for 14–21 days [1, 43]. The magnitude and duration of shedding are highly variable; some horses may shed virus for up to 28 days [1]. Importantly, clinically healthy horses can actively shed EHV-1, serving as silent reservoirs for transmission. A study following a multi-county EHM outbreak in California found that a significant proportion of clinically normal sport horses were shedding EHV-1, underscoring the risk of silent transmission at equine gatherings [4]. The World Organisation for Animal Health (WOAH) recognizes the economic and welfare impact of EHV-1, and its guidelines emphasize the importance of quarantine and PCR testing of apparently healthy horses to prevent undetected spread during outbreaks [4].

Reproductive Form: Abortion, Neonatal Disease, and Emerging Urogenital Manifestations

The reproductive form of EHV-1 infection is one of the most economically devastating consequences of the disease, causing significant losses to the equine breeding industry worldwide. EHV-1 is a primary infectious cause of abortion in mares, typically occurring in the last trimester of pregnancy, between 7 and 11 months of gestation [1, 10, 11]. Abortion can occur sporadically or in “abortion storms,” where multiple mares on a single premises abort within a short timeframe [1, 11]. The pathogenesis of EHV-1 abortion is a consequence of the virus’s ability to establish cell-associated viremia. Following replication in the respiratory tract, EHV-1 infects peripheral blood mononuclear cells (PBMCs), primarily T lymphocytes and monocytes, and is transported to the pregnant uterus [12, 34]. The virus then infects the endothelial cells of the endometrial microvasculature, leading to vasculitis, thrombosis, and ischemic necrosis of the endometrium [1, 27]. This vascular damage compromises the maternal-fetal interface, allowing the virus to cross into the fetal compartment. Fetal infection results in widespread viral replication in fetal tissues, particularly the liver, lung, spleen, and thymus, leading to fetal death and expulsion [1, 39].

The hallmark of EHV-1 abortion is that the mare typically shows no prodromal signs of illness; abortion is often sudden and unexpected [1]. The fetus is usually expelled fresh, with minimal autolysis, and may appear grossly normal. However, histopathological examination of fetal tissues reveals characteristic lesions, including multifocal hepatic necrosis, intranuclear eosinophilic inclusion bodies in hepatocytes and bronchial epithelial cells, and interstitial pneumonia [39]. The placenta may show no gross lesions, but microscopic examination can reveal vasculitis and thrombosis of chorionic villi [1].

The neuropathogenic potential of the virus is not a prerequisite for abortigenic capacity. While early studies suggested a strong association between the D752 (neuropathogenic) genotype and neurologic disease, and the N752 (non-neuropathogenic) genotype with abortion, subsequent large-scale epidemiological studies have demonstrated that both genotypes can cause abortion [9, 11, 13]. A 28-year retrospective study of EHV-1 isolates in Ireland found that the majority of abortion outbreaks were caused by viruses with the N752 genotype, but a statistically significant association between the D752 genotype and “hypervirulence” (defined as outbreaks involving multiple abortion or neurological cases) was observed [11]. Similarly, in Poland, 3.1% of EHV-1-associated abortions were caused by the neuropathogenic genotype, indicating its presence in the reproductive disease spectrum [45]. The 2021 Valencia outbreak, which was caused by a virus lacking the classical D752 neuropathogenic marker (A2254 genotype), resulted in both respiratory and neurological disease but was not associated with widespread abortion, suggesting that other viral genetic determinants influence tissue tropism and clinical outcome [2, 3, 6, 8].

Neonatal disease is another manifestation of the reproductive form. Foals infected in utero may be born alive but are often weak, febrile, and succumb to severe interstitial pneumonia and multi-organ failure within the first few days of life [1, 42]. These foals are a significant source of viral shedding and can serve as amplifiers of infection within a breeding farm.

An emerging and less well-characterized urogenital manifestation is equine idiopathic haemorrhagic cystitis (EIHC), which has been associated with EHV-1 infection. A case report described a gelding presenting with stranguria, pollakiuria, and haematuria, with cystoscopic findings of bladder mucosal ulceration and hemorrhage [44]. Histopathology revealed chronic active ulcerative cystitis with neutrophilic, lymphoplasmacytic, and eosinophilic infiltrates, and PCR testing of bladder mucosa was positive for EHV-1 [44]. While the exact role of EHV-1 in EIHC remains to be fully elucidated, this case highlights the potential for EHV-1 to cause disease beyond the classical triad of respiratory, reproductive, and neurologic syndromes. Furthermore, experimental infection studies have demonstrated that EHV-1 can be detected in the reproductive organs of all male horses, causing interstitial lymphoplasmacytic and histiocytic orchitis, raising concerns about potential long-term effects on fertility and the possibility of venereal transmission [27]. The detection of EHV-1 DNA in urine samples from naturally infected horses during EHM outbreaks further supports the potential for urogenital involvement and suggests that urine may serve as an additional, albeit likely minor, route of viral excretion [37].

Neurologic Form: Equine Herpesvirus Myeloencephalopathy (EHM)

Equine herpesvirus myeloencephalopathy (EHM) is the most severe and feared manifestation of EHV-1 infection, representing a true medical emergency. Although EHM occurs in only approximately 10–14% of infected horses, its high case-fatality rate, prolonged recovery times, and potential for large-scale outbreaks at equine events make it a disease of paramount concern to the equine industry [1, 17, 38, 40]. EHM is not a direct viral infection of neurons but rather an immunopathological disease driven by endothelial cell infection and subsequent vasculitis in the central nervous system (CNS) [1, 22, 38, 40].

The pathogenesis of EHM is a multi-step process. Following primary respiratory infection and the establishment of cell-associated viremia, EHV-1-infected PBMCs, particularly T lymphocytes, travel to the CNS [12, 34]. The virus is then transferred to the endothelial cells of the spinal cord and brain microvasculature via a process of viral synapse formation, a sophisticated mechanism that allows direct cell-to-cell transfer of virus while evading neutralizing antibodies [12]. Infection of CNS endothelial cells triggers a cascade of events: viral replication within the endothelium leads to endothelial cell damage, activation of the coagulation cascade, and a robust local inflammatory response [22, 26, 38]. This results in vasculitis, thrombosis, and perivascular cuffing with mononuclear cells, leading to ischemic and hemorrhagic necrosis of the surrounding neural tissue [22, 27, 40]. The resulting neuropathology is primarily vascular in origin, explaining the often asymmetric and multifocal nature of the clinical signs.

The clinical presentation of EHM is highly variable, ranging from mild ataxia to recumbency and death. The hallmark of EHM is the acute onset of symmetrical or asymmetrical ataxia, typically affecting the hindlimbs more severely than the forelimbs [1, 9, 40]. Affected horses may exhibit a “bunny-hopping” gait, toe dragging, and a wide-based stance. As the disease progresses, horses may become unable to rise, leading to recumbency, which carries a grave prognosis. Other common neurologic signs include urinary incontinence (often with a distended bladder and dribbling urine), tail hypotonia, and perineal hypalgesia [1, 9, 40]. Cranial nerve deficits are less common but can include facial nerve paralysis, dysphagia, and vestibular signs. A key clinical feature is that fever and respiratory signs may precede the onset of neurologic signs by several days, but in many cases, particularly in older horses, neurologic dysfunction is the first and only clinical sign observed [30, 33].

The risk of developing EHM is influenced by a constellation of host and viral factors. Age is the single most significant host risk factor. While EHM can occur in horses of any age, the incidence increases dramatically in older horses, particularly mares over 20 years of age, in whom the risk can exceed 70% [30, 33, 38]. This age-related susceptibility is attributed to immunosenescence, with older horses exhibiting a dysregulated immune response characterized by a delayed and diminished type I interferon response, a shift towards a T-helper 2 (Th2) and regulatory T-cell phenotype, and an upregulation of pro-inflammatory and pro-coagulant pathways [30, 33]. Sex also plays a role; a large outbreak study found that stallions and geldings were six times more likely to develop EHV-1 infection compared to mares, although the risk of EHM specifically was not significantly different between sexes [2]. However, once infected, older mares are disproportionately represented among EHM cases [30]. Breed may also influence susceptibility, with Quarter Horses and Saddlebreds overrepresented in some studies of EHM caused by the D752 genotype [9].

Environmental and management factors are critical. The 2021 Valencia outbreak provided compelling evidence that stable design and ventilation are major determinants of EHM risk. Horses housed in the middle of a large tent structure, where ventilation was poorest, were significantly more likely to develop EHM compared to those housed at the periphery [2]. This finding underscores the importance of airborne transmission and the role of high viral loads in enclosed, poorly ventilated spaces. Stressors such as long-distance transport, weaning, and concurrent illness are well-established triggers for reactivation of latent EHV-1 and subsequent clinical disease [1, 17].

The role of viral genotype in EHM has been a subject of intense debate. The discovery of a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30), resulting in an asparagine-to-aspartic acid substitution at amino acid 752 (N752D), was initially hailed as a definitive marker for “neuropathogenic” strains [11, 13]. Epidemiological studies confirmed a strong statistical association between the D752 genotype and outbreaks of EHM [11]. However, it has become increasingly clear that this marker is neither necessary nor sufficient for the development of EHM. Numerous outbreaks of EHM have been caused by viruses with the N752 (non-neuropathogenic) genotype, including the large 2021 Valencia outbreak [2, 3, 6, 8, 9]. A comprehensive study of 65 EHM cases found no significant difference in the frequency of the D752 versus N752 genotype among affected horses, and with the exception of a higher incidence of urinary incontinence in D752-infected horses, there was no difference in clinical disease severity or outcome [9]. These findings have led to a paradigm shift: the N752D substitution is a risk factor for, but not a determinant of, neuropathogenicity. Other viral genetic determinants, including mutations in ORF11 (A713G) identified in the Valencia outbreak, likely contribute to neurotropism and virulence [8]. The current consensus, supported by the European Food Safety Authority (EFSA) and WOAH, is that EHV-1 strains should not be strictly categorized as “neuropathogenic” or “non-neuropathogenic” based solely on the ORF30 genotype, as all strains possess the potential to cause EHM under the right host and environmental conditions [3, 5].

The immune response to EHV-1 in the CNS is a double-edged sword. While a robust and timely type I interferon (IFN) response, particularly IFN-α, is associated with protection from EHM, a delayed or dysregulated response correlates with severe disease [30, 31]. Horses that develop EHM exhibit low levels of IFN-α in nasal secretions and a delayed upregulation of interferon regulatory factors (IRF7, IRF9) in the blood [30]. In contrast, horses that resist EHM show an early and robust IFN-α response [30]. Furthermore, EHM is associated with an upregulation of IL-10, a regulatory cytokine, and a shift towards IgG3/5 antibody isotypes, indicative of a Th2-biased response [30]. The virus itself actively subverts the host interferon response. EHV-1 infection of equine endothelial cells has been shown to inhibit the type I IFN signaling pathway by reducing the expression of Toll-like receptors (TLR3 and TLR4), blocking the phosphorylation and nuclear translocation of STAT2, and downregulating IRF7 and IRF9 [31]. This viral interference with innate immunity creates a permissive environment for viral replication and spread within the CNS. The resulting immunopathology, driven by the influx of CD8+ T cells and the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF, contributes to the extensive neuropathology observed in EHM [26, 33].

Diagnostic Approaches: Molecular, Serological, and Virological Methods

The accurate and timely diagnosis of Equine Herpesvirus 1 (EHV-1) infection is a cornerstone of effective outbreak management, clinical decision-making, and epidemiological surveillance. Given the virus’s capacity for rapid dissemination, latency, and the severe consequences of equine herpesvirus myeloencephalopathy (EHM) and abortion, diagnostic approaches must be both sensitive and specific. The diagnostic landscape for EHV-1 has evolved considerably, moving from classical virological techniques to highly sensitive molecular assays, while serological methods remain indispensable for retrospective diagnosis and seroprevalence studies. The 2024 ACVIM consensus statement [1] and a subsequent systematic review on diagnostic testing [1] have provided critical frameworks for evaluating these methodologies, emphasizing that no single test is sufficient for all clinical scenarios. The choice of diagnostic approach must be guided by the stage of infection, the clinical presentation, the sample type, and the specific objective, whether it be detecting acute infection, confirming EHM, monitoring viral shedding, or conducting large-scale surveillance.

Molecular Diagnostic Methods: The Gold Standard for Acute Infection

Polymerase chain reaction (PCR)-based techniques, particularly quantitative real-time PCR (qPCR), have become the diagnostic modality of choice for the detection of EHV-1 nucleic acid. The ACVIM consensus statement [1] and the European Food Safety Authority (EFSA) assessment [3] both affirm that PCR performed on appropriate samples is the most sensitive and rapid method for confirming active infection. The primary advantage of qPCR lies in its ability to detect viral DNA directly from clinical specimens without the need for viable virus, making it ideal for samples that may have been compromised during transport or storage. Furthermore, qPCR provides quantitative data on viral load, which has been shown to correlate with clinical severity and the risk of developing EHM [42, 43]. A systematic review by Soboll-Hussey et al. [42] established a clear relationship between the magnitude and duration of cell-associated viremia, as measured by qPCR in peripheral blood, and the incidence of both abortion and EHM. This quantitative capacity allows clinicians to stratify risk and monitor the efficacy of antiviral interventions.

Sample Selection and Diagnostic Windows. The selection of appropriate biological samples is paramount for maximizing diagnostic sensitivity. For the detection of acute respiratory infection, nasal swabs (NS) are the specimen of choice, as viral shedding from the respiratory epithelium is most pronounced during the first 7–10 days post-infection [43]. However, for the diagnosis of EHM and abortion, the detection of cell-associated viremia is critical. Whole blood, specifically the buffy coat (BC) fraction, is the preferred sample for detecting viremia, as EHV-1 establishes a cell-associated infection in peripheral blood mononuclear cells (PBMCs), particularly T lymphocytes [12, 34]. The seminal work by Scott et al. [34] demonstrated that T lymphocytes are the primary leukocyte subpopulation harboring EHV-1 during viremia, a finding that has been confirmed by modern molecular studies [12]. The duration of viremia is typically shorter than nasal shedding, often peaking around day 4–6 post-infection and becoming undetectable by day 14 [34, 43]. Therefore, timing of blood collection is crucial.

A novel and potentially valuable sample type has emerged from recent outbreak investigations: urine. Álvarez et al. [37] demonstrated that EHV-1 DNA can be detected in urine samples from naturally infected horses during EHM outbreaks. Notably, viral DNA was detectable in urine for a longer duration and at slightly higher concentrations compared to buffy coat samples, although with lower concentrations than in nasal swabs [37]. This finding suggests that urine may serve as a complementary, non-invasive sample for extending the diagnostic window for viremia, particularly in horses where blood sampling is challenging or when late-stage infection is suspected. The pathophysiological basis for urinary shedding is likely related to the vasculitis and endothelial damage characteristic of EHM, which can affect the renal and urinary tract vasculature, as evidenced by cases of EHV-1-associated haemorrhagic cystitis [44].

Genotyping and Molecular Epidemiology. Beyond simple detection, molecular methods enable genotyping of EHV-1 strains, which has profound implications for understanding pathogenesis and outbreak tracing. The most well-characterized genetic marker is the single nucleotide polymorphism (SNP) at position 2254 in open reading frame 30 (ORF30), which encodes the viral DNA polymerase. This SNP results in an amino acid substitution from asparagine (N) to aspartic acid (D) at codon 752 (N752D). Historically, the D752 genotype was termed the “neuropathogenic” marker due to its statistical association with EHM outbreaks [11, 13, 14]. However, this association is not absolute. The 2024 ACVIM consensus [1] and the EFSA assessment [3] both emphasize that the N752D polymorphism is not a definitive determinant of neurovirulence. Pusterla et al. [9] found no significant difference in the frequency of the D752 genotype among 65 horses with EHM, and importantly, the non-neuropathogenic N752 genotype was also frequently isolated from neurological cases. Similarly, the large 2021 Valencia outbreak was caused by a strain with the A2254 (N752) genotype, yet it produced a high incidence of EHM [2, 6, 8]. This indicates that host factors, viral load, and other viral genetic determinants are critical contributors to neuropathogenesis [30, 33, 38].

Despite this nuance, genotyping remains a valuable epidemiological tool. Multi-locus sequence typing, particularly sequencing of the ORF68 gene, has been used to group EHV-1 isolates into geographical clades and track viral spread [11, 14]. Garvey et al. [11] demonstrated that ORF68 sequencing, combined with multi-locus analysis of 26 ORFs, could differentiate at least 10 distinct clades circulating in Ireland over 28 years, enabling the tracing of virus between premises. More recently, whole-genome sequencing has provided unprecedented resolution. The 2021 Valencia outbreak was linked to a strain carrying a novel SNP (A713G) in ORF11, which was not present in 249 other global isolates, providing a specific molecular marker for that outbreak [8]. Furthermore, genomic analysis of the Valencia strains revealed their close genetic relationship to viruses from Belgium and the UK, suggesting a common source of infection at the international equestrian event [6]. These advanced molecular techniques, while not yet routine in clinical practice, are indispensable for global surveillance and understanding viral evolution, including the detection of recombination events between EHV-1 and other equine herpesviruses [13, 16].

Serological Diagnostic Methods: Retrospective and Population-Level Insights

Serological assays detect the host’s humoral immune response to EHV-1 infection and are primarily used for retrospective diagnosis, seroprevalence studies, and vaccine response monitoring. The most widely used serological tests are the virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA). The VNT is considered the reference standard for detecting neutralizing antibodies, which are primarily directed against viral glycoproteins such as gB, gC, and gD [20]. However, the VNT has limitations, including the requirement for cell culture facilities, a turnaround time of 2–3 days, and the inability to differentiate between antibodies induced by EHV-1 and the closely related EHV-4 due to cross-reactivity [7, 46]. This cross-reactivity is a significant confounder in endemic populations where both viruses circulate.

Type-Specific ELISAs and IgG Isotyping. To overcome the cross-reactivity issue, type-specific ELISAs have been developed. These assays often utilize recombinant glycoprotein G (gG) from EHV-1 and EHV-4, as gG is a type-specific antigen that elicits a divergent antibody response [7, 46]. Fuentealba et al. [46] developed an agar gel immunodiffusion (AGID) test using recombinant EHV-1 gD, which demonstrated 100% specificity and 99.5% sensitivity compared to VNT, offering a simpler and more rapid alternative for serological screening. However, even type-specific ELISAs have limitations in differentiating recent infection from past exposure or vaccination, as antibodies can persist for months to years.

The characterization of IgG sub-isotypes has emerged as a more refined serological approach. Goodman et al. [41] demonstrated that EHV-1-specific IgG4/7 levels strongly correlate with virus neutralization titers, while IgG1/3 levels can distinguish naturally infected horses from those only vaccinated. Horses that had survived a neurological outbreak had significantly higher IgG1/3 and neutralizing antibody titers compared to vaccinated-only horses [41]. This differential IgG isotype profile reflects the more robust and diverse antigenic stimulation provided by natural infection versus the restricted response induced by modified-live virus (MLV) vaccines. More recent work by Giessler et al. [30] has linked specific IgG sub-isotype profiles to clinical outcome. In their experimental model, horses that developed EHM had significantly higher serum IgG3/5 antibody titers compared to horses that did not develop neurological signs. This suggests that the quality, not just the quantity, of the antibody response may be a correlate of protection or risk for EHM. High-throughput IgG isotyping, as proposed by Goodman et al. [41], could therefore be a valuable tool for screening vaccine candidates and identifying horses at increased risk for severe disease.

Paired Serology for Confirming Recent Infection. For clinical diagnosis, a single serological result is of limited value due to the high seroprevalence of EHV-1 in many populations [7]. The EFSA assessment [3] and the ACVIM consensus [1] recommend the use of paired serum samples collected 2–4 weeks apart to demonstrate a four-fold or greater rise in antibody titer, which is indicative of recent infection or reactivation. This approach is particularly useful in retrospective investigations of abortion storms or EHM outbreaks where acute-phase samples were not collected for PCR. However, seroconversion may be delayed or absent in cases of primary infection in young foals or in immunocompromised animals. Furthermore, in horses with high pre-existing antibody titers from prior exposure or vaccination, a significant rise may not be detectable, leading to false-negative interpretations.

Virological Methods: Historical Cornerstone and Contemporary Utility

Classical virological techniques, including virus isolation (VI) in cell culture and immunohistochemistry (IHC), remain relevant in specific diagnostic contexts, although they have been largely supplanted by molecular methods for routine diagnosis. Virus isolation involves inoculating clinical samples (e.g., nasal swabs, buffy coat, or tissue homogenates) onto permissive equine cell lines, such as equine dermal (E. Derm) or equine kidney cells, and observing for cytopathic effect (CPE) [2, 39]. The primary advantage of VI is that it confirms the presence of infectious virus, which is critical for understanding transmission risk. However, VI is labor-intensive, time-consuming (often requiring 3–7 days), and less sensitive than qPCR, as it requires viable virus that can replicate in culture. The 2021 Valencia outbreak successfully isolated EHV-1 from clinical samples, confirming the presence of replication-competent virus in the outbreak setting [2].

Histopathology and Immunohistochemistry. In post-mortem diagnosis, particularly for abortion cases, histopathological examination and IHC are invaluable. The characteristic lesions of EHV-1 abortion include multifocal necrosis, eosinophilic intranuclear inclusion bodies in hepatocytes and bronchial epithelium, and vasculitis [27, 39]. IHC, using monoclonal or polyclonal antibodies against EHV-1 antigens (e.g., gB or gC), can confirm the presence of viral antigen within these lesions, providing a definitive diagnosis [27, 39]. A retrospective study in Brazil found that a combination of PCR, VI, histopathology, and IHC increased the diagnostic yield for EHV-1 abortion compared to any single method alone [39]. In cases of EHM, IHC on central nervous system (CNS) tissue can demonstrate viral antigen within endothelial cells of the spinal cord and brain, confirming the role of vasculitis in the pathogenesis [27]. However, IHC is less sensitive than qPCR, particularly in tissues with low viral loads or when autolysis has degraded antigens.

The Role of Diagnostic Testing in Outbreak Management. The practical application of these diagnostic methods is best illustrated by their use during outbreaks. The 2021 Valencia outbreak serves as a critical case study. The outbreak was initially identified through qPCR testing of nasal swabs from febrile horses, which allowed for rapid confirmation of EHV-1 [2]. Subsequent genotyping of the ORF30 region revealed the A2254 (non-neuropathic) genotype, yet the outbreak still resulted in a 40% incidence of neurological signs among infected horses [2]. This underscores the point that reliance on the “neuropathogenic” genotype for risk assessment is insufficient. The outbreak was controlled through a strategy of mass qPCR testing of all horses on the premises, quarantine of positive animals, and movement restrictions [2, 4]. A similar strategy was employed in California following a multi-county EHM outbreak, where qPCR testing of clinically healthy horses during a mandatory quarantine period was successful at identifying silent shedders and preventing further EHM cases [4]. These field experiences highlight that qPCR, despite its limitations in predicting neurovirulence, is the most effective tool for identifying infected animals, breaking the chain of transmission, and enabling a safe return to competition. The detection of EHV-1 DNA in urine, as demonstrated by Álvarez et al. [37], may further enhance outbreak surveillance by providing an additional, non-invasive sample matrix for screening large populations.

Vaccination Strategies and Immunoprophylaxis

The development and deployment of effective vaccination strategies against equine herpesvirus-1 (EHV-1) represent one of the most formidable challenges in contemporary equine infectious disease medicine. Despite decades of research and the commercial availability of several vaccine formulations, the consensus among leading veterinary authorities, including the American College of Veterinary Internal Medicine (ACVIM) and the European Food Safety Authority (EFSA), is that currently available vaccines provide, at best, incomplete protection against the full spectrum of EHV-1 disease, particularly the most devastating manifestations: equine herpesvirus myeloencephalopathy (EHM) and abortion [1, 3]. This reality is underscored by the 2024 ACVIM consensus statement, which explicitly notes that evidence for successful vaccination against EHV-1 infection is limited, and that improvements in experimental design and reporting are critically needed [1]. The systematic review underpinning this statement, conducted by Osterrieder et al. (2023), performed a comprehensive meta-analysis of vaccination studies and concluded that while some vaccines may reduce nasal shedding and viremia, the protection afforded against EHM is inconsistent and often marginal [36]. This section provides an exhaustive analysis of the biological, immunological, and practical dimensions of EHV-1 vaccination, dissecting the mechanisms of vaccine-induced immunity, the limitations of current products, and the strategic imperatives for future immunoprophylaxis.

The Immunological Basis for Vaccination: Correlates of Protection and Pathogenesis

To understand why vaccination against EHV-1 remains suboptimal, one must first appreciate the sophisticated immune evasion strategies employed by the virus. EHV-1 is an alphaherpesvirus that has co-evolved with its equid hosts for millennia, developing a repertoire of mechanisms to subvert both innate and adaptive immunity. Following intranasal infection, the virus replicates in the respiratory epithelium, where it must contend with the host's initial interferon (IFN) response. Critically, EHV-1 has been shown to actively inhibit the type I IFN signaling pathway. Oladunni et al. (2019) demonstrated that EHV-1 targets both the sensitization and induction steps of the type I IFN response in equine endothelial cells (EECs), significantly reducing Toll-like receptor 3 (TLR3) and TLR4 mRNA expression, blocking the phosphorylation and nuclear translocation of signal transducer and activator of transcription 2 (STAT2), and thereby crippling the antiviral state [31]. This strategic inhibition of innate immunity creates a window for the virus to establish a cell-associated viremia, which is the central pathogenic event leading to secondary disease.

The cell-associated viremia is not a passive process; it is an active, virus-driven exploitation of the host's own immune cells. EHV-1 infects peripheral blood mononuclear cells (PBMCs), with a particular tropism for T lymphocytes. Poelaert et al. (2019) elegantly elucidated the molecular choreography of this process, showing that activated T lymphocytes, especially CD4+ cells, become efficiently infected despite the presence of neutralizing antibodies [12]. The virus restricts the expression of viral glycoproteins on the surface of infected T cells, preventing immune recognition, and hampers the release of progeny virions, leading to an accumulation of nucleocapsids in the T cell nucleus. When these infected T lymphocytes contact endothelial cells of the endometrium or central nervous system (CNS), late viral proteins orchestrate the formation of a virological synapse, facilitating direct cell-to-cell transfer of viral progeny [12]. This mechanism allows EHV-1 to reach its target organs, the pregnant uterus and the CNS, while largely evading humoral immunity. This explains why neutralizing antibodies, while effective at reducing cell-free virus in the respiratory tract, are insufficient to prevent the cell-associated viremia that seeds EHM and abortion.

The immune correlates of protection against EHM are therefore more complex than simple antibody titers. The work of Giessler et al. (2024), using an experimental "old mare model" that reliably induces EHM, has provided crucial insights. They demonstrated that protection from EHM is associated with a timely induction of type I IFN (IFN-α) in nasal secretions and an early upregulation of interferon regulatory factors (IRF7/IRF9), pro-inflammatory cytokines (IL-1β), and chemokines (CXCL10) in the blood, followed by a robust IFN-γ response during viremia [30]. In contrast, horses that developed EHM exhibited low nasal IFN-α levels, a dysregulated cytokine response, and significantly higher levels of the regulatory cytokine IL-10 in nasal secretions, PBMCs, and cerebrospinal fluid (CSF). Furthermore, EHM-susceptible horses showed a skewed IgG sub-isotype response, with higher titers of IgG3/5, which are associated with a T-helper 2 (Th2)-biased or regulatory immunophenotype [30]. These findings align with transcriptomic analyses by Zarski et al. (2021), who found that PBMCs from EHM-affected horses showed an upregulation of IL-6, a dysregulation of T-cell activation through AP-1, and a skewing towards a Th2 response, while non-EHM horses mounted a more effective cellular immune response [33]. Collectively, these data indicate that an effective vaccine must not only induce neutralizing antibodies but also, and more importantly, prime a robust, early, and appropriately polarized cell-mediated immune response, particularly a CD4+ and CD8+ T-cell response capable of eliminating infected cells before they can establish the cell-associated viremia.

Commercially Available Vaccines: Composition, Claims, and Evidence of Efficacy

The global market for EHV-1 vaccines includes both modified-live virus (MLV) and inactivated (killed) products, often formulated in combination with equine herpesvirus-4 (EHV-4) and, in some cases, equine influenza virus. The ACVIM consensus statement and the systematic review by Osterrieder et al. (2023) provide a sobering assessment of their field efficacy [1, 36]. No commercially available vaccine carries a label claim for protection against EHM, and the evidence for prevention of abortion is variable. The systematic review, which included a meta-analysis of multiple experimental challenge studies, found that while some vaccines could reduce the magnitude and duration of nasal shedding and, to a lesser extent, cell-associated viremia, the effect on clinical disease, particularly neurological signs, was inconsistent and often statistically non-significant [36].

The immunological limitations of current vaccines are multifaceted. First, the humoral response induced by vaccination is often narrow in scope. Goodman et al. (2011) compared the IgG isotype profiles of horses vaccinated with a commercial MLV vaccine to those of horses that had been naturally infected or exposed during an outbreak. They found that the MLV vaccine induced a more restricted IgG isotype response (primarily IgG4/7) compared to the diverse IgG1/3 and IgG4/7 responses seen in naturally infected horses [41]. This suggests that current vaccines may not adequately stimulate the full repertoire of antibody effector functions, including complement fixation and opsonization, which are mediated by IgG1/3. Second, the induction of cell-mediated immunity, particularly cytotoxic T lymphocytes (CTLs), is suboptimal. The EHV-1-specific IFN-γ-producing CD4+ T-cell numbers were significantly higher in outbreak-exposed horses compared to vaccinated-only horses, indicating that natural infection primes a more robust T-cell memory than current vaccines [41]. This is a critical deficit, given that CTL activity is considered a primary correlate of protection against EHV-1, as it is the only immune mechanism capable of eliminating virus-infected cells that are the vehicle for viremia [19].

Furthermore, the phenomenon of immune interference and the virus's ability to establish latency complicate vaccination strategies. EHV-1 establishes lifelong latency in the trigeminal ganglion and lymphoid tissues, and reactivation can occur during periods of stress, immunosuppression, or following corticosteroid administration [1, 3, 47]. Vaccination does not prevent the establishment of latency, nor does it reliably prevent reactivation. A study by Brini et al. (2021) in Moroccan horse populations highlighted the practical challenges of field vaccination. They found that 21.8% of horses vaccinated with a monovalent EHV-1 vaccine were seropositive by type-specific ELISA, but the virus neutralization test (VNT) revealed low mean antibody titers (1:49 for EHV-1), suggesting that the vaccine-induced antibody response was weak and potentially short-lived [7]. This underscores the need for rigorous vaccination schedules and booster protocols, as well as the potential for vaccine failure in the face of high challenge doses or immunosuppressed individuals.

Strategic Considerations for Vaccination Programs

Given the limitations of current vaccines, vaccination strategies must be integrated into a comprehensive biosecurity and management framework. The ACVIM consensus statement and EFSA scientific opinions emphasize that vaccination should be promoted as part of a multi-layered control program, but it cannot be relied upon as a standalone measure [1, 3]. The primary goals of vaccination are to reduce the severity of respiratory disease, decrease the magnitude and duration of nasal shedding (thereby reducing transmission), and, ideally, lower the risk of viremia and subsequent secondary disease.

Vaccination of Broodmares: The prevention of EHV-1 abortion is a primary target for vaccination in breeding operations. Inactivated vaccines are commonly used in pregnant mares, typically administered at 5, 7, and 9 months of gestation. The rationale is to boost humoral immunity and provide passive transfer of antibodies to the foal via colostrum. However, the evidence base for this practice is not robust. The systematic review by Osterrieder et al. (2023) found that while some studies showed a reduction in abortion rates in vaccinated mares, the quality of evidence was low, and many studies lacked appropriate control groups [36]. The ACVIM consensus statement notes that vaccination may reduce the risk of abortion but does not eliminate it, and that strict biosecurity, including isolation of pregnant mares from new arrivals and horses returning from events, is paramount [1]. The 2021 Valencia outbreak, which involved a show-jumping competition and resulted in widespread dissemination of the virus, tragically illustrated how quickly EHV-1 can spread through a naive or under-vaccinated population, leading to EHM cases and deaths [2, 6, 8].

Vaccination for EHM Prevention: This remains the most contentious and challenging area. No vaccine has been proven to prevent EHM in a controlled, statistically powered study. The ACVIM consensus statement is unequivocal: "Evidence for successful vaccination against... EHV-1 infection was limited" [1]. The pathogenesis of EHM is intimately linked to the cell-associated viremia and the subsequent immunopathology in the CNS. The virus's ability to infect T lymphocytes and travel within them to the CNS endothelium, where it triggers a vasculitis and thrombosis, is a process that is largely resistant to antibody-mediated neutralization [12, 30]. The host immune response itself contributes to the pathology; the pro-inflammatory cytokine storm and the infiltration of CD8+ T cells and monocytes into the CNS, as demonstrated in mouse models, can exacerbate neurological damage [26]. Therefore, an ideal vaccine would need to induce a sterilizing immunity at the respiratory mucosa or, failing that, a rapid and potent CTL response that eliminates infected cells before they can establish a significant viremia. Current vaccines do not achieve this.

Vaccination Schedules and Booster Protocols: The optimal vaccination schedule varies by product and risk profile. For horses at high risk of exposure, such as those that travel frequently to shows, events, or breeding farms, more frequent boosters (every 3-6 months) are often recommended. The ACVIM statement suggests that for horses in high-risk environments, vaccination every 6 months with an inactivated vaccine may be considered, although the evidence for this practice is based on expert opinion rather than controlled trials [1]. It is crucial to note that vaccination should not be performed on horses that are already incubating the disease or showing clinical signs, as it may exacerbate the condition. Furthermore, the use of MLV vaccines in pregnant mares is generally contraindicated due to the risk of vaccine-induced abortion, although some products have specific label claims for use in pregnant mares.

Future Directions: The Quest for a Protective Vaccine

The development of a next-generation EHV-1 vaccine that provides robust, durable protection against EHM and abortion is a major research priority. The insights from recent pathogenesis studies point towards several promising avenues. A successful vaccine must induce a strong, early, and appropriately polarized immune response. The work of Giessler et al. (2024) suggests that shifting the immune response away from a regulatory/Th2 phenotype and towards a Th1/cytotoxic phenotype is critical [30]. This could be achieved through the use of novel adjuvants that selectively stimulate Toll-like receptors (TLRs) to promote IFN-γ and CTL induction. The identification of equine MHC class I molecules as the entry receptor for EHV-1, as demonstrated by Sasaki et al. (2011), also opens the door for novel vaccine designs that target the virus-receptor interaction [32, 35].

Subunit vaccines targeting key viral glycoproteins, such as glycoprotein D (gD), the primary attachment protein, and glycoprotein B (gB), which mediates fusion, are under investigation. The use of recombinant gD in diagnostic tests has already been established [46], and its potential as a vaccine antigen is being explored. However, the virus's ability to evade immunity through cell-to-cell spread and its exploitation of T cells as Trojan horses suggests that a multi-antigen approach, incorporating both envelope glycoproteins and internal proteins (such as the immediate-early protein or tegument proteins) that are targets for CTL, may be necessary. The deletion of specific virulence genes, as seen in the attenuated KyA strain (which lacks gI, gE, and full-length gp2), provides a blueprint for developing safer and more immunogenic MLV vaccines [15]. However, the risk of recombination with field strains, although considered low for EHV-1 compared to EHV-4 [16], must be carefully evaluated.

In conclusion, the current state of EHV-1 vaccination is characterized by a significant gap between clinical need and product capability. While vaccination remains a cornerstone of equine health management and can contribute to reducing the burden of respiratory disease and viral shedding, it does not provide reliable protection against the most severe outcomes, EHM and abortion. The path forward requires a paradigm shift in vaccine design, moving from a focus on humoral immunity to the induction of robust, cell-mediated immunity that can intercept the virus at the critical juncture of cell-associated viremia. Until such a vaccine is developed and validated, the equine industry must rely on a rigorous, multi-faceted approach that combines strategic vaccination with strict biosecurity, early detection through PCR testing, and effective outbreak management protocols, as recommended by the World Organisation for Animal Health (WOAH) and the ACVIM [1, 3].

Pharmacologic Interventions and Treatment Protocols

The management of equine herpesvirus-1 (EHV-1) infection, particularly its most devastating manifestation, equine herpesvirus myeloencephalopathy (EHM), remains one of the most challenging and contentious areas in equine internal medicine. The therapeutic landscape is characterized by a critical scarcity of robust, randomized, placebo-controlled clinical trials. The updated ACVIM consensus statement [1] and its underpinning systematic review on pharmacologic interventions [48] have solidified this assessment, concluding that evidence for effective pharmaceutical treatment is severely limited. Consequently, current protocols are largely extrapolated from in vitro data, experimental infection models in horses and mice, anecdotal field experience, and principles borrowed from the management of other human and veterinary herpesviruses. The overarching therapeutic goals are threefold: (1) inhibition of viral replication to reduce viremia and shedding, (2) modulation of the aberrant and often destructive host inflammatory response, and (3) provision of aggressive supportive care to manage neurological deficits and secondary complications. An understanding of the complex immunopathogenesis, where viral infection of endothelial cells triggers a CD8+ T-cell and monocytic vasculitis and thrombosis in the central nervous system (CNS) [22, 26], is fundamental to rationalizing these interventions.

Antiviral Agents: Nucleoside Analogues and Beyond

The cornerstone of antiviral strategy against EHV-1 is the use of nucleoside analogues, which function by interfering with viral DNA polymerase activity. Acyclovir, a deoxyguanosine analogue commonly used against human alphaherpesviruses, has been the most extensively studied in horses, yet its clinical utility is profoundly constrained by its pharmacokinetic profile. Oral bioavailability of acyclovir in horses is exceptionally poor, typically less than 3%, necessitating intravenous administration or the use of its prodrug, valacyclovir. Valacyclovir is metabolized rapidly into acyclovir, achieving higher plasma concentrations after oral dosing. However, even with valacyclovir (e.g., 40 mg/kg PO q8h), plasma acyclovir concentrations often fail to consistently exceed the in vitro 50% inhibitory concentration (IC50) for EHV-1, which varies widely among strains [1, 48]. The systematic review by Goehring et al. [48] found no evidence from controlled trials that either acyclovir or valacyclovir alters the course of EHM, reduces viremia duration, or improves survival rates. Their use is further complicated by potential nephrotoxicity, particularly with intravenous acyclovir in dehydrated animals, and the significant cost of prolonged therapy. Despite this lack of definitive proof, valacyclovir is frequently deployed during outbreaks, especially in high-value horses or those with early signs of viremia, under the premise that a partial antiviral effect is better than none. The application is often prophylactic in exposed, but not yet clinically ill, horses.

Ganciclovir, a more potent nucleoside analogue with greater activity against cytomegalovirus and other beta- and alphaherpesviruses, has shown superior in vitro efficacy against EHV-1 compared to acyclovir. Its ophthalmic formulation (ganciclovir gel) is used for EHV-1-associated ocular disease, such as keratitis or uveitis, which can occur secondary to viral-induced vasculitis [27]. The use of systemic ganciclovir is rare due to its cost, requirement for intravenous administration, and significant potential for bone marrow suppression and neutropenia. Cidofovir, another antiviral with a longer duration of action, remains largely experimental in equine practice. In summary, the efficacy of current antiviral agents is widely regarded as marginal at best, and they are considered a supportive gesture rather than a definitive curative therapy. The failure of these drugs may be partially explained by the cell-associated nature of the viremia. EHV-1 exploits T lymphocytes and monocytic cells to disseminate, shielding the virus within these cells from antiviral agents and neutralizing antibodies [12, 34]. Once the virus has reached the CNS endothelium and initiated vasculitis, the inflammatory cascade, rather than active viral replication, becomes the primary driver of pathology, rendering antiviral therapy of limited benefit at that stage.

Anti-Inflammatory and Immunomodulatory Therapy: Managing the Host Response

Given the central role of immunopathology in EHM, the use of corticosteroids is perhaps the most debated pharmacologic intervention. The inflammatory lesions of EHM, lymphohistiocytic vasculitis, thrombosis, and perivascular cuffing in the CNS, are driven by a dysregulated host immune response [22, 26, 38]. Although theoretically appealing to suppress this inflammation, the use of corticosteroids carries the significant risk of promoting viral replication by suppressing the type I interferon response and cytotoxic T-cell activity [19, 30]. The ACVIM consensus statement [1] recommends that corticosteroids, such as dexamethasone or prednisolone, be reserved for rapidly deteriorating neurological cases where the risk of inflammatory damage outweighs the risk of enhanced viral replication. There are no controlled trials, however, demonstrating a survival benefit. In contrast, aggressive use of non-steroidal anti-inflammatory drugs (NSAIDs), most commonly flunixin meglumine or phenylbutazone, is a mainstay of therapy for controlling fever, endotoxemia (due to secondary bacterial translocation from gut stasis), and the generalized systemic inflammatory response. These agents are indicated for pyrexia and to improve mentation, but their efficacy in directly mitigating CNS inflammation is limited. Supportive care often includes DMSO (dimethyl sulfoxide), administered intravenously or via nasogastric tube, for its purported free-radical scavenging, anti-inflammatory, and mild diuretic effects to reduce CNS edema. The evidence for DMSO is almost entirely anecdotal and derived from spinal cord injury models in non-equine species.

The emerging understanding of the immune correlates of protection and risk for EHM has opened discussions on more targeted immunomodulation. Research by Giessler et al. [30] and Zarski et al. [33] has identified that horses developing EHM exhibit a dysfunctional immune response characterized by a delayed and suppressed type I interferon (IFN-α/β) response, coupled with a shift toward a regulatory or TH-2-type immunity, marked by elevated IL-10 and IgG3/5 antibody isotypes. In contrast, protected horses mount a robust, early IFN-α response and a functional CD4+ and CD8+ T-cell response [30]. This suggests that recombinant equine interferon (IFN-α) , administered intranasally or systemically early in the course of infection, might be a logical therapeutic. While some commercial IFN-α products exist, their use is off-label for EHV-1 and robust trial data are lacking. EHV-1, however, actively counteracts the host interferon response by inhibiting the JAK-STAT signaling pathway, specifically by blocking STAT2 phosphorylation and nuclear translocation [31], which may render exogenous IFN-α therapy less effective than anticipated. Another immunomodulatory strategy involves the use of plasma transfusions from hyperimmunized horses or horses that have recovered from EHV-1 infection. This passive administration of neutralizing antibodies may help reduce cell-free viremia and shedding [19], but it is expensive, logistically challenging, and carries its own risks (e.g., transfusion reactions). Non-specific immunostimulants, such as equine biological response modifiers (e.g., Propionibacterium acnes extract), have been used anecdotally but lack any supportive evidence.

Supportive Care and Management of Complications

The high mortality and morbidity associated with EHM are often attributable to secondary complications rather than primary viral infection. Aggressive supportive care is therefore paramount and represents the therapeutic area with the most measurable impact on outcome. Urinary incontinence is a common and severe complication, significantly associated with the neuropathogenic D752 genotype [9]. An areflexic bladder leads to urinary retention, overflow incontinence, and a high risk of ascending bacterial cystitis or pyelonephritis. Management requires regular bladder catheterization (indwelling or intermittent) under strict aseptic technique. Monitoring for hematuria and pollakiuria is critical, as EHV-1-associated hemorrhagic cystitis has been reported [44]. Antimicrobial therapy should be instituted based on urine culture and sensitivity when urinary tract infection is confirmed. Gastrointestinal stasis (ileus) is another critical issue, often resulting from autonomic dysfunction and recumbency. Horses with EHM that become recumbent require intensive nursing, including body slings (if used judiciously to avoid aspiration), frequent turning to prevent pressure sores and myopathy, and enteral or parenteral nutritional support. Aspiration pneumonia is a leading cause of death in recumbent horses due to dysphagia. Therefore, feeding must be carefully managed, often with a soft, slurried diet, and horses should be positioned with their heads elevated.

Vasculitis and thrombosis are the pathological hallmarks of EHM. While no drug is approved for this specific indication, low-molecular-weight heparin (e.g., dalteparin or enoxaparin) or aspirin have been used empirically to inhibit platelet aggregation and thrombus formation. Their efficacy is unproven, and the risk of hemorrhage, particularly in the CNS, must be considered. Anticoagulant therapy is generally not recommended unless there is clear evidence of consumptive coagulopathy (disseminated intravascular coagulation, DIC). Finally, the World Organisation for Animal Health (WOAH) and many national veterinary authorities mandate strict biosecurity and movement restrictions during outbreaks, which are arguably more effective than any single pharmacologic agent in limiting the spread of this economically critical pathogen [1, 3]. The management of an EHV-1 outbreak hinges on early detection via qPCR on nasal swabs and buffy coat samples [2, 37, 43], immediate isolation of affected and exposed horses, and strict implementation of hygiene protocols. The treatment of affected horses, particularly those with EHM, remains a heroic but often futile effort, underscoring the critical importance of preventative vaccination and biosecurity as the primary pillars of control.

Outbreak Management, Biosecurity, and Prevention Strategies

The management of Equine Herpesvirus-1 (EHV-1) outbreaks, the implementation of rigorous biosecurity protocols, and the deployment of effective prevention strategies constitute a triad of non-negotiable interventions for safeguarding equine populations against this pervasive and economically devastating pathogen. The multifaceted nature of EHV-1, its capacity for latency, its diverse clinical manifestations ranging from mild respiratory disease to fatal myeloencephalopathy (EHM) and abortion storms, and its ability to spread rapidly through both direct and indirect contact, demands a sophisticated, multi-layered approach that integrates epidemiological principles, diagnostic precision, and behavioral management. The cornerstone of any robust EHV-1 prevention strategy lies in acknowledging that absolute eradication is unattainable in endemic regions, and thus, the operational goal shifts to minimizing viral transmission, reducing the magnitude and duration of viremia, and preventing the severe sequelae of infection [1, 3]. This section provides an exhaustive analysis of the contemporary evidence underpinning outbreak control, biosecurity, and prevention, drawing from the most recent consensus statements, outbreak investigations, and systematic reviews.

Foundational Principles of EHV-1 Outbreak Prevention and Control

The biological complexity of EHV-1 dictates the strategic imperatives for its control. The virus’s ability to establish lifelong latency in trigeminal ganglia and lymphoid tissues, with subsequent reactivation under immunosuppressive stress (e.g., transport, weaning, corticosteroid administration, concurrent illness), means that a clinically healthy horse can suddenly become a potent source of viral shedding [1, 3, 17]. This latent reservoir is the primary impediment to eradication and necessitates a paradigm of continuous vigilance. Epidemiological analyses have irrevocably demonstrated that the most significant outbreaks, such as the 2021 Valencia Spring Tour outbreak, are often precipitated by the congregation of horses from diverse geographic origins at high-density events, where stress, mixing, and inadequate ventilation create a perfect storm for viral amplification and dissemination [2, 6]. The Valencia outbreak, which involved over 750 horses and resulted in the lockdown of the venue, highlighted that the virus causing the outbreak was genetically related to strains circulating in Belgium and the United Kingdom, underscoring the role of international movement in viral spread [6]. Critically, the strain responsible did not possess the classical D752 neuropathogenic marker (ORF30), yet it caused significant neurological disease and mortality, reinforcing the contemporary understanding that EHM is not exclusively a property of a single viral genotype but is heavily influenced by host factors (age, sex, immune status) and environmental context [2, 3, 8, 9]. Thus, prevention cannot rely solely on strain surveillance; it must be holistic.

The World Organisation for Animal Health (WOHA) and the Food and Agriculture Organization (FAO) have long championed the principles of compartmentalization and biosecurity for managing transboundary animal diseases, and these principles are directly applicable to EHV-1 given its endemic status and the high value of the equine industry. A successful prevention program integrates pre-emptive management (biosecurity and vaccination), rapid detection and confirmation (diagnostic surveillance), and aggressive containment (movement control and isolation). The recent European Food Safety Authority (EFSA) assessment, conducted within the framework of the Animal Health Law (Regulation (EU) No 2016/429), categorized EHV-1 infection as a disease with a high probability of fulfilling criteria for categories D (prevention and control measures) and E (surveillance), but with negligible probability for category A (immediate eradication) [5]. This classification is pragmatic: it acknowledges that while EHV-1 is too widespread to eradicate, robust control measures can significantly mitigate its impact.

Pre-Event Biosecurity: The Bedrock of Prevention

Biosecurity for EHV-1 must be conceptualized as a continuous process, not a reactive protocol triggered by an outbreak. The primary goal is to prevent the introduction of the virus onto a premises (bio-exclusion) and, if introduction occurs, to prevent its spread within the premises (bio-containment). The risk of introduction is primarily mediated by the movement of horses, but fomites, including shared tack, water buckets, feed, grooming equipment, trailers, and even human clothing and hands, represent a highly significant and often underestimated vector [1, 3].

Quarantine and Health Monitoring: The most critical biosecurity measure is the strict quarantine of all new arrivals. The ACVIM consensus statement emphasizes that an isolation period of 21 to 28 days is essential, as this exceeds the typical incubation period and window for peak viral shedding [1, 3]. During this quarantine, horses should be housed in a physically separate facility (ideally a different airspace) and attended to last in the daily routine. A comprehensive health monitoring protocol must be implemented, including twice-daily temperature checks. Fever (≥38.6°C or 101.5°F) is often the earliest and most sensitive indicator of EHV-1 infection, often preceding the onset of respiratory signs or neurological deficits by 24 to 48 hours [2, 40]. Any pyrexic horse should be immediately isolated from the quarantine group pending diagnostic investigation.

Ventilation and Stable Design: The Valencia outbreak data provided a stark lesson in the critical role of stable design. Logistic regression analysis revealed that horses housed in the middle of a large tent structure, where ventilation was poorest and aerosol transmission was potentiated, were significantly more likely to develop EHM [2]. This finding is biologically plausible given the high concentration of virus shed in nasal secretions (up to 10⁶ PFU/mL) and the ability of the virus to survive in the environment for at least 7-14 days on surfaces and in aerosols [3]. Consequently, stabling should prioritize cross-ventilation, minimize shared airspace, and maintain physical barriers (solid walls rather than bars) between stalls to reduce direct contact and fomite transmission. Show organizers and boarding stable managers should design facilities to create distinct air handling zones.

Vaccination as a Biosecurity Tool: Vaccination against EHV-1 is widely practiced, but its role in biosecurity must be understood with nuance. The 2023 ACVIM consensus statement, underpinned by a systematic review and meta-analysis, concluded that currently available vaccines (both inactivated and modified-live virus) reduce the incidence and severity of respiratory disease, and may reduce the magnitude and duration of nasal shedding post-challenge [1, 36]. However, no vaccine currently licensed provides reliable, sterilizing immunity against infection, nor does any vaccine have a label claim for protection against EHM. The meta-analysis found no significant evidence that vaccination prevented the development of EHM or abortion under field conditions [36]. This does not render vaccination useless; rather, it must be employed as a risk-mitigation tool. Vaccination reduces the viral load shed by an infected horse, thereby lowering the environmental contamination pressure and the likelihood of transmission to cohorts. A strategic vaccination protocol, typically administering vaccines at 6-month intervals, with the final dose given 2-4 weeks prior to high-risk events (shows, sales, breeding), is recommended to prime the immune system and potentially reduce the duration of viremia [1]. However, reliance on vaccination alone is a dangerous fallacy. The seroprevalence studies from Morocco, for example, showed that vaccinated horses had low neutralizing antibody titers, and a significant proportion (21.8%) remained seropositive for EHV-1 yet were still susceptible to infection, indicating that field vaccination protocols may be suboptimal [7]. Furthermore, the immune response to vaccination differs qualitatively from that to natural infection; vaccination primarily induces a Th2-type humoral response with limited cytotoxic T lymphocyte (CTL) activity, whereas protective immunity against cell-associated viremia requires robust Th1-type cell-mediated immunity and early interferon-gamma (IFN-γ) production [30, 41]. This immunological gap explains why vaccines fail to prevent the most severe consequences of infection.

Proactive Surveillance and Diagnostic Strategies

Early detection is the single most effective intervention for curtailing an EHV-1 outbreak. The gold standard for diagnosing acute infection is quantitative polymerase chain reaction (qPCR) on nasal swabs and whole blood (buffy coat) [1, 43]. The sensitivity and rapid turnaround time (hours) of qPCR make it indispensable for outbreak management. A strategic sampling protocol should be implemented for any horse exhibiting pyrexia, respiratory signs, or neurologic ataxia. Importantly, during an outbreak, testing of in-contact, clinically healthy horses is equally critical. The study following the 2022 California EHM outbreaks demonstrated that a significant number of clinically healthy, show-going horses were shedding EHV-1 (as well as other respiratory pathogens like EHV-4 and Streptococcus equi), acting as silent vectors facilitating widespread transmission [4]. The mandatory qPCR testing and quarantine period imposed by the United States Equestrian Federation (USEF) were successful in preventing further outbreaks subsequent to the initial incident [4].

Novel Diagnostic Frontiers : Urine Sampling: Recent research has revealed an additional diagnostic dimension. A study investigating two EHM outbreaks in Spain in 2021 and 2023 detected EHV-1 DNA in urine samples from a majority of infected horses [37]. Notably, the viral DNA was detected in urine for a longer duration than in buffy coat samples, with slightly higher concentrations in some cases. While the clinical relevance of this finding for transmission remains to be fully elucidated (urine is unlikely to be a major route of environmental contamination compared to nasal secretions), urine sampling offers a potentially valuable complementary diagnostic specimen, particularly for horses where blood or nasal swabs are difficult to obtain or when the timing of sampling is suboptimal [37]. This finding expands the diagnostic toolkit available to clinicians during an outbreak.

Molecular Epidemiology and Strain Tracking: During a large outbreak, molecular characterization of the viral strain can provide critical epidemiological insights. Sequencing of the ORF68 gene or whole-genome sequencing of isolates allows for phylogenetic tracing to identify the source of an outbreak and track chains of transmission [6, 11]. The Irish study analyzing isolates over 28 years demonstrated that multi-locus typing could differentiate strains and corroborate epidemiological links between premises [11]. The identification of a unique A713G marker in the 2021 European outbreak strain provided a specific tool to trace the spread of that particular clade [8]. In a practical outbreak setting, saving extracted DNA or viral isolates for future sequencing can inform control strategies and legal or insurance-related investigations.

Serology : A Retrospective Tool: While PCR is essential for real-time diagnosis, serological testing (paired serum neutralization or type-specific ELISA) has a role in retrospective confirmation of infection, particularly in horses that have recovered or in vaccinated populations where PCR may be negative [1, 7, 46]. A four-fold or greater rise in antibody titer between acute and convalescent serum (taken 14-21 days apart) confirms recent infection. However, serology cannot distinguish between vaccine-induced antibodies and those from natural infection [41].

Containment and Movement Management During an Outbreak

Once an EHV-1 infection is confirmed or strongly suspected, the immediate priority shifts to containment. The 2021 Valencia outbreak is a textbook case of the consequences of delayed containment: the initial diagnosis was not made until horses had dispersed from the central venue, leading to widespread geographic dissemination of the virus across Europe [3, 6]. The EFSA report explicitly noted that "the outbreak reported in Valencia was followed by wide geographic spread of the virus possibly due to a delay in diagnosis and late application of biosecurity measures" [3].

Immediate Isolation and Movement Ban: The index case and all exposed cohorts must be immediately isolated in a designated "hot zone" with dedicated personnel, equipment, and footbaths. A complete moratorium on horse movement onto and off the affected premises must be enforced. The consensus recommendation is that no horses should be allowed to leave the premises until at least 21 days have passed since the detection of the last clinical case, provided that no new cases or fevers occur [1, 3]. This 21-day clock often feels economically devastating to show barns or training stables, but it is a scientifically justified period based on the maximum duration of viremia and viral shedding observed in experimental and field studies [40, 42, 43].

Risk Stratification and Zoning: Not all horses on a premises face equal risk. The premises should be geographically and operationally zoned into areas of high, medium, and low risk. The "clean" zone (unexposed, healthy horses) should be managed with strict barrier nursing to prevent breach. All affected or suspect horses are in the "dirty" zone. Personnel movement should be unidirectional from clean to dirty, and rigorous hand hygiene and footbath disinfection (using agents effective against enveloped viruses, such as accelerated hydrogen peroxide, 2% chlorhexidine, or quaternary ammonium compounds) are mandatory between zones [1].

Symptomatic and Supportive Care as a Control Measure: While treatment is discussed in detail elsewhere, it is relevant here that aggressive supportive care of EHM cases, including anti-inflammatory therapy, bladder management, and prevention of secondary infections, also serves a biosecurity function by reducing the duration of severe clinical signs and the associated high viral load in the environment [1, 48]. Horses with severe ataxia and urinary incontinence are particularly problematic, as urine may contain viral DNA [37], and the inability to maintain sternal recumbency increases the risk of pressure sores and secondary infections that prolong viral shedding.

Termination of the Outbreak: The official end of an outbreak is declared when no new cases (no fever, no neurologic signs, and negative PCR on nasal swabs and blood) have occurred for a period at least twice the maximum incubation period, typically 14-21 days after the resolution of the last clinical case. Prior to lifting the travel ban, a final round of surveillance PCR testing on all in-contact horses is recommended, even if they appear clinically normal, to identify persistent subclinical shedders [1, 4].

Environmental and Vector Considerations

EHV-1 is an enveloped virus, which renders it relatively susceptible to desiccation and common disinfectants compared to non-enveloped viruses. However, it can persist for up to 7 days on wood, metal, and fabric surfaces, and for even longer in organic matter (manure, hay, feed) at low temperatures and high humidity [3]. Disinfection protocols must therefore include a pre-cleaning step to remove organic load, followed by application of an appropriate disinfectant. Contaminated bedding must be removed and properly composted or incinerated.

Role of Latent Infection and the "Trojan Horse": Arguably the most challenging aspect of outbreak management is the presence of latently infected horses. A horse that appears clinically recovered and tests negative on nasal swab and blood PCR may still harbor reactivatable virus in its trigeminal ganglia and lymphoid tissues [1, 12]. Any significant stressor, a long trailer ride, a new environment, concurrent infection, or even administration of corticosteroids, can trigger reactivation and a new episode of shedding. This biological reality means that quarantine protocols must be absolute, and that even "clean" horses from an unaffected premises should be treated with caution if they have a history of prior EHV-1 exposure. The molecular details of how the virus "bridles" T lymphocytes to reach target organs, evading neutralizing antibodies, underscore the sophistication of the pathogen and the limitations of immune-based control strategies [12].

Future Directions in Prevention and Control

The current evidence base, meticulously reviewed by the ACVIM panel, reveals significant gaps in our armamentarium [1]. The failure of existing vaccines to reliably prevent EHM is the most pressing challenge. Future prevention strategies are likely to pivot towards:

1. Correlates of Protection: Identifying specific immune markers that predict protection from EHM, such as early IFN-γ responses, robust CD8+ CTL activity, and balanced Th1/Th2 profiles, will guide next-generation vaccine design [30, 33]. The identification of an "at-risk immunophenotype" in aged mares (high IL-10, TGF-β, and IgG3/5) provides a target for vaccine modulation [30].
2. Therapeutic Vaccines and Immunomodulators: Given the limitations of prophylactic vaccination, research into immunomodulators (e.g., recombinant IFN-α, TLR agonists) that can be administered at the time of an outbreak to boost innate immunity may prove valuable [31, 48]. The antivirals valacyclovir and ganciclovir have shown promise in reducing viremia and severity of disease, but evidence is currently limited to small experimental and clinical studies, and widespread use is hampered by cost and lack of licensed efficacy data [48].
3. Genomic Surveillance: The establishment of a global genomic surveillance network for EHV-1, similar to that used for influenza, would allow real-time tracking of emerging variants and recombination events, enabling more rapid risk assessment and targeted biosecurity measures [6, 11, 13].

In summary, effective management of EHV-1 requires a zero-tolerance approach to biosecurity breaches, combined with the intellectual honesty to acknowledge that current vaccines and treatments are imperfect. The foundation of prevention is meticulous management: rigorous quarantine, optimal ventilation, stress reduction, and immediate diagnostic testing of any febrile horse. The future of control lies in a deeper immunological understanding of protection and the development of interventions that specifically target the cell-associated viremia and endothelial infection that underpin the most devastating outcomes of this infection.

References

[1] Lunn DP, Burgess B, Dorman DC, Goehring L, Gross P, Osterrieder K, et al.. Updated ACVIM consensus statement on equine herpesvirus‐1. Journal of Veterinary Internal Medicine. 2024. DOI: https://doi.org/10.1111/jvim.17047

[2] Couroucé A, Normand C, Tessier C, Pomares R, Thevenot J, Marcillaud-Pitel C, et al.. Equine herpesvirus-1 outbreak during a show-jumping competition: a clinical and epidemiological study.. Journal of Equine Veterinary Science. 2023. DOI: https://doi.org/10.1016/j.jevs.2023.104869

[3] Carvelli A, Nielsen S, Paillot R, Broglia A, Kohnle L. Clinical impact, diagnosis and control of Equine Herpesvirus‐1 infection in Europe. EFSA journal. European Food Safety Authority. 2022. DOI: https://doi.org/10.2903/j.efsa.2022.7230

[4] Wilcox A, Barnum S, Wademan C, Corbin R, Escobar E, Hodzic E, et al.. Frequency of Detection of Respiratory Pathogens in Clinically Healthy Show Horses Following a Multi-County Outbreak of Equine Herpesvirus-1 Myeloencephalopathy in California. Pathogens. 2022. DOI: https://doi.org/10.3390/pathogens11101161

[5] Nielsen S, Álvarez J, Bicout D, Calistri P, Canali E, Drewe J, et al.. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Equine Herpesvirus‐1. EFSA journal. European Food Safety Authority. 2022. DOI: https://doi.org/10.2903/j.efsa.2022.7036

[6] Vereecke N, Carnet F, Pronost S, Vanschandevijl K, Theuns S, Nauwynck H. Genome Sequences of Equine Herpesvirus 1 Strains from a European Outbreak of Neurological Disorders Linked to a Horse Gathering in Valencia, Spain, in 2021. Microbiology Resource Announcements. 2021. DOI: https://doi.org/10.1128/MRA.00333-21

[7] Brini ZE, Fihri OF, Paillot R, Lotfi C, Amraoui F, Ouadi HE, et al.. Seroprevalence of Equine Herpesvirus 1 (EHV-1) and Equine Herpesvirus 4 (EHV-4) in the Northern Moroccan Horse Populations. Animals. 2021. DOI: https://doi.org/10.3390/ani11102851

[8] Sutton G, Normand C, Carnet F, Couroucé A, Garvey M, Castagnet S, et al.. Equine Herpesvirus 1 Variant and New Marker for Epidemiologic Surveillance, Europe, 2021. Emerging Infectious Diseases. 2021. DOI: https://doi.org/10.3201/eid2710.210704

[9] Pusterla N, Hatch K, Crossley B, Wademan C, Barnum S, Flynn K. Equine herpesvirus-1 genotype did not significantly affect clinical signs and disease outcome in 65 horses diagnosed with equine herpesvirus-1 myeloencephalopathy.. The Veterinary Journal. 2020. DOI: https://doi.org/10.1016/j.tvjl.2019.105407

[10] Akter R, Legione A, Sansom FM, El-Hage C, Hartley C, Gilkerson J, et al.. Detection of Coxiella burnetii and equine herpesvirus 1, but not Leptospira spp. or Toxoplasma gondii, in cases of equine abortion in Australia - a 25 year retrospective study. PLoS ONE. 2020. DOI: https://doi.org/10.1371/journal.pone.0233100

[11] Garvey M, Lyons R, Hector R, Walsh C, Arkins S, Cullinane A. Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017. Pathogens. 2019. DOI: https://doi.org/10.3390/pathogens8010007

[12] Poelaert KC, Cleemput JV, Laval K, Favoreel H, Couck L, Broeck WVd, et al.. Equine Herpesvirus 1 Bridles T Lymphocytes To Reach Its Target Organs. Journal of Virology. 2019. DOI: https://doi.org/10.1128/JVI.02098-18

[13] Bryant N, Wilkie G, Russell C, Compston L, Grafham D, Clissold L, et al.. Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks. Transboundary and Emerging Diseases. 2018. DOI: https://doi.org/10.1111/tbed.12809

[14] Negussie H, Demissie DG, Tessema TS, Nauwynck H. Equine Herpesvirus‐1 Myeloencephalopathy, an Emerging Threat of Working Equids in Ethiopia. Transboundary and Emerging Diseases. 2017. DOI: https://doi.org/10.1111/tbed.12377

[15] Shakya AK, O’callaghan D, Kim S. Comparative Genomic Sequencing and Pathogenic Properties of Equine Herpesvirus 1 KyA and RacL11. Frontiers in Veterinary Science. 2017. DOI: https://doi.org/10.3389/fvets.2017.00211

[16] Vaz P, Horsington J, Hartley C, Browning GF, Ficorilli N, Studdert M, et al.. Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.. Journal of General Virology. 2016. DOI: https://doi.org/10.1099/jgv.0.000378

[17] Afify AF, Hassanien RT, Naggar RFE, Rohaim M, Munir M. Unmasking the Ongoing Challenge of Equid Herpesvirus- 1 (EHV-1): A Comprehensive Review.. Microbial Pathogenesis. 2024. DOI: https://doi.org/10.1016/j.micpath.2024.106755

[18] Taniguchi A, Fukushi H, Matsumura T, Yanai T, Masegi T, Hirai K. Pathogenicity of a new neurotropic equine herpesvirus 9 (gazelle herpesvirus 1) in horses.. Journal of Veterinary Medical Science. 2000. DOI: https://doi.org/10.1292/JVMS.62.215

[19] Rusli N, Mat K, Harun H. A review: interactions of equine herpesvirus-1 with immune system and equine lymphocyte.. Open Journal of Veterinary Medicine. 2014. DOI: https://doi.org/10.4236/OJVM.2014.412036

[20] Mahmoud HYAH, Andoh K, Hattori S, Terada Y, Noguchi K, Shimoda H, et al.. Characterization of Glycoproteins in Equine Herpesvirus-1. Journal of Veterinary Medical Science. 2013. DOI: https://doi.org/10.1292/jvms.13-0168

[21] Jackson T, Kendrick JW. Paralysis of horses associated with equine herpesvirus 1 infection.. Journal of the American Veterinary Medical Association. 1971. DOI: https://doi.org/10.2460/javma.1971.158.08.1351

[22] Jackson T, Osburn BI, Cordy DR, Kendrick JW. Equine herpesvirus 1 infection of horses: studies on the experimentally induced neurologic disease.. American Journal of Veterinary Research. 1977. DOI: https://doi.org/10.2460/ajvr.1977.38.06.709

[23] Allen GP, Yeargan M, Lw T, Jt B, Wh M. Molecular epizootiologic studies of equine herpesvirus-1 infections by restriction endonuclease fingerprinting of viral DNA.. American Journal of Veterinary Research. 1983. DOI: https://doi.org/10.2460/ajvr.1983.44.02.263

[24] Allen GP, Yeargan M, Turtinen LW, Bryans J. A new field strain of equine abortion virus (equine herpesvirus-1) among Kentucky horses.. American Journal of Veterinary Research. 1985. DOI: https://doi.org/10.2460/ajvr.1985.46.01.138

[25] Zarski L, Weber P, Lee Y, Hussey GS. Transcriptomic Profiling of Equine and Viral Genes in Peripheral Blood Mononuclear Cells in Horses during Equine Herpesvirus 1 Infection. Pathogens. 2021. DOI: https://doi.org/10.3390/pathogens10010043

[26] Mesquita L, Costa RC, Zanatto DA, Bruhn F, Mesquita LLR, Lara M, et al.. Equine herpesvirus 1 elicits a strong pro-inflammatory response in the brain of mice.. Journal of General Virology. 2021. DOI: https://doi.org/10.1099/jgv.0.001556

[27] Holz C, Sledge D, Kiupel M, Nelli R, Goehring L, Hussey GS. Histopathologic Findings Following Experimental Equine Herpesvirus 1 Infection of Horses. Frontiers in Veterinary Science. 2019. DOI: https://doi.org/10.3389/fvets.2019.00059

[28] Poelaert KC, Cleemput JV, Laval K, Xie J, Favoreel H, Nauwynck H. Equine herpesvirus 1 infection orchestrates the expression of chemokines in equine respiratory epithelial cells.. Journal of General Virology. 2019. DOI: https://doi.org/10.1099/jgv.0.001317

[29] Negussie H, Li Y, Tessema TS, Nauwynck H. Replication characteristics of equine herpesvirus 1 and equine herpesvirus 3: comparative analysis using ex vivo tissue cultures. Veterinary Research. 2016. DOI: https://doi.org/10.1186/s13567-016-0305-5

[30] Giessler K, Goehring L, Jacob S, Davis A, Esser M, Lee Y, et al.. Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses. Journal of General Virology. 2024. DOI: https://doi.org/10.1099/jgv.0.001987

[31] Oladunni FS, Sarkar S, Reedy S, Balasuriya UBR, Horohov D, Chambers T. Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells. Journal of Virology. 2019. DOI: https://doi.org/10.1128/JVI.01342-19

[32] Sasaki M, Hasebe R, Makino Y, Suzuki T, Fukushi H, Okamoto M, et al.. Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D. Genes to Cells. 2011. DOI: https://doi.org/10.1111/j.1365-2443.2011.01491.x

[33] Zarski L, Giessler K, Jacob S, Weber P, Mccauley A, Lee Y, et al.. Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses. Viruses. 2021. DOI: https://doi.org/10.3390/v13030356

[34] Jc S, Sk D, Ac M. In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies.. American Journal of Veterinary Research. 1983. DOI: https://doi.org/10.2460/ajvr.1983.44.07.1344

[35] Sasaki M, Kim E, Igarashi M, Ito K, Hasebe R, Fukushi H, et al.. Single Amino Acid Residue in the A2 Domain of Major Histocompatibility Complex Class I Is Involved in the Efficiency of Equine Herpesvirus-1 Entry*. Journal of Biological Chemistry. 2011. DOI: https://doi.org/10.1074/jbc.M111.251751

[36] Osterrieder K, Dorman DC, Burgess B, Goehring L, Gross P, Neinast C, et al.. Vaccination for the prevention of equine herpesvirus‐1 disease in domesticated horses: A systematic review and meta‐analysis. Journal of Veterinary Internal Medicine. 2023. DOI: https://doi.org/10.1111/jvim.16895

[37] Álvarez AV, Jose-Cunilleras E, Dorrego-Rodriguez A, Santiago-Llorente I, Cuesta-Torrado Mdl, Troya-Portillo L, et al.. Detection of equine herpesvirus-1 (EHV-1) in urine samples during outbreaks of equine herpesvirus myeloencephalopathy.. Equine Veterinary Journal. 2023. DOI: https://doi.org/10.1111/evj.14007

[38] Hussey G, Giessler K. Contribution of the immune response to the pathogenesis of equine herpesvirus-1 (EHV-1): Are there immune correlates that predict increased risk or protection from EHV-1 myeloencephalopathy?. The Veterinary Journal. 2022. DOI: https://doi.org/10.1016/j.tvjl.2022.105827

[39] Silva AAd, Cunha EM, Lara M, Villalobos E, Nassar A, Mori E, et al.. Low occurrence of equine herpesvirus 1 (EHV-1) as cause of abortion and perinatal mortality in Brazil. Arquivos do Instituto Biológico. 2018. DOI: https://doi.org/10.1590/1808-1657000852017

[40] Pusterla N, Hussey G. Equine herpesvirus 1 myeloencephalopathy.. The Veterinary clinics of North America. Equine practice. 2014. DOI: https://doi.org/10.1016/j.cveq.2014.08.006

[41] Goodman L, Wimer CL, Dubovi E, Gold C, Wagner B. Immunological Correlates of Vaccination and Infection for Equine Herpesvirus 1. Clinical and Vaccine Immunology. 2011. DOI: https://doi.org/10.1128/CVI.05522-11

[42] Soboll-Hussey G, Dorman DC, Burgess B, Goehring L, Gross P, Neinast C, et al.. Relationship between equine herpesvirus‐1 viremia and abortion or equine herpesvirus myeloencephalopathy in domesticated horses: A systematic review. Journal of Veterinary Internal Medicine. 2023. DOI: https://doi.org/10.1111/jvim.16948

[43] Pusterla N, Dorman DC, Burgess B, Goehring L, Gross M, Osterrieder K, et al.. Viremia and nasal shedding for the diagnosis of equine herpesvirus‐1 infection in domesticated horses. Journal of Veterinary Internal Medicine. 2023. DOI: https://doi.org/10.1111/jvim.16958

[44] Easther R, Manthorpe E, Woolford L, Kawarizadeh A, Hemmatzadeh F, Agne G. Eosinophilic Inflammation and Equine Herpesvirus-1 associated with Haemorrhagic Cystitis in a Horse. Case Report.. Journal of Equine Veterinary Science. 2022. DOI: https://doi.org/10.1016/j.jevs.2022.104161

[45] Stasiak K, Rola J, Ploszay G, Socha W, Żmudziński J. Detection of the neuropathogenic variant of equine herpesvirus 1 associated with abortions in mares in Poland. BMC Veterinary Research. 2015. DOI: https://doi.org/10.1186/s12917-015-0416-7

[46] Fuentealba N, Sguazza G, Scrochi M, Bravi M, Zanuzzi C, Corva S, et al.. Production of equine herpesvirus 1 recombinant glycoprotein D and development of an agar gel immunodiffusion test for serological diagnosis.. Journal of Virological Methods. 2014. DOI: https://doi.org/10.1016/j.jviromet.2014.02.025

[47] Bond SL, Workentine M, Hundt J, Gilkerson J, Léguillette R. Effects of nebulized dexamethasone on the respiratory microbiota and mycobiota and relative equine herpesvirus‐1, 2, 4, 5 in an equine model of asthma. Journal of Veterinary Internal Medicine. 2019. DOI: https://doi.org/10.1111/jvim.15671

[48] Goehring L, Dorman DC, Osterrieder K, Burgess B, Dougherty KA, Gross P, et al.. Pharmacologic interventions for the treatment of equine herpesvirus‐1 in domesticated horses: A systematic review. Journal of Veterinary Internal Medicine. 2024. DOI: https://doi.org/10.1111/jvim.17016