Equine Herpesvirus 4: Respiratory Disease Reference

Overview and Taxonomy of Equine Herpesvirus 4: Respiratory Disease Reference

Equine herpesvirus 4 (EHV-4), historically designated as equine rhinopneumonitis virus type 2, represents one of the most ubiquitous and economically significant viral pathogens affecting the global equid population. As a member of the Alphaherpesvirinae subfamily within the Herpesviridae family, EHV-4 is a double-stranded DNA virus that has co-evolved with its equine hosts over millennia, establishing a delicate balance between viral persistence and host immune surveillance [4]. This virus is the primary etiological agent of contagious upper respiratory tract disease in horses worldwide, a condition clinically referred to as equine rhinopneumonitis, and is responsible for substantial morbidity, disruption to training and competition schedules, and significant economic losses across the equine industry [1, 13]. Understanding the taxonomic position, genomic architecture, and biological characteristics of EHV-4 is fundamental to comprehending its pathogenesis, epidemiology, and the challenges associated with disease control and prevention.

Taxonomic Classification and Phylogenetic Position

EHV-4 is classified within the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. This taxonomic assignment places it in close phylogenetic proximity to other significant equine and non-equine alphaherpesviruses, including equine herpesvirus 1 (EHV-1), equine herpesvirus 8 (EHV-8), equine herpesvirus 9 (EHV-9), bovine herpesvirus 1 (BoHV-1), and the human pathogen varicella-zoster virus (VZV) [2, 18]. Among the equid alphaherpesviruses, EHV-4 is most closely related to EHV-1, sharing approximately 80–85% genomic homology, yet the two viruses exhibit distinct epidemiological patterns, host cell tropisms, and clinical manifestations [12, 24]. This genetic similarity has historically complicated diagnostic differentiation, necessitating the development of type-specific molecular assays, including the recently validated multiplex real-time PCR assay targeting the melanocortin 1 receptor (MC1R) endogenous control, which can reliably discriminate between EHV-1 and EHV-4 with analytical sensitivity as low as four copies per reaction [18].

Phylogenetic analyses based on complete genome sequencing and partial gene sequencing, particularly of the glycoprotein B (gB) gene and the DNA polymerase gene, have revealed that EHV-4 field isolates cluster into two major subclades [4, 19]. Bayesian phylogenetic reconstruction using ancient viral genomes recovered from archaeological horse remains dated to approximately 3,900 years before present has provided a minimal time estimate for EHV-4 diversification, pushing the evolutionary origin of these subclades back to nearly a thousand years ago, substantially earlier than previous estimates based solely on modern sequence data [4]. This ancient genome, recovered from a horse associated with the Sintashta spoke-wheeled chariot culture in the Southeastern Urals, represents the oldest known EHV-4 sequence and underscores the long-standing association between this virus and domesticated horses [4]. Contemporary phylogenetic studies have further demonstrated that EHV-4 strains circulating in different geographic regions, including Europe, Australia, Japan, Africa, and the Middle East, exhibit considerable genetic diversity, with evidence of widespread natural recombination among field isolates [12, 13, 17]. This recombination, a hallmark of alphaherpesvirus evolution, contributes to the generation of genomic diversity and may have implications for virulence, antigenicity, and vaccine efficacy [12].

Genomic Architecture and Genetic Diversity

The EHV-4 genome is a linear double-stranded DNA molecule of approximately 144–150 kilobase pairs (kbp), organized into a unique long (UL) region and a unique short (US) region, each flanked by inverted repeat sequences [13, 19]. The complete genome sequence of the reference strain NS80567, along with several Japanese and Australian isolates, has been determined, revealing a coding capacity for over 76 open reading frames (ORFs), many of which are conserved among alphaherpesviruses [19]. Comparative genomic analyses have identified that the EHV-4 genome encodes several genes with known or putative roles in viral replication, immune evasion, and pathogenesis. Notably, ORF1 and ORF2 are present in EHV-4 but absent or truncated in many EHV-1 strains, and these gene products have been implicated in host range determination and virulence [23]. Conversely, deletion of ORF2 in the neuropathogenic EHV-1 strain Ab4 resulted in reduced virulence and nasal shedding, highlighting the potential functional importance of this genomic region across the equid alphaherpesviruses [14, 23].

Restriction endonuclease fingerprinting and whole-genome sequencing have demonstrated substantial genomic diversity among EHV-4 field isolates, with polymorphisms distributed throughout the genome [13, 17]. Restriction fragment length polymorphism (RFLP) analysis of 23 Japanese isolates using BamHI, BglII, EcoRI, SacI, and SalI revealed distinct digestion patterns, including mobility shifts and loss or gain of fragments, providing a molecular epidemiological tool for tracing outbreak strains [17]. This genetic variability is further exemplified by the observation that ORF24 and ORF71, which contain variable repeat sequences, exhibit size polymorphisms that correlate with epizootiological relatedness, allowing differentiation between independent outbreak strains and those sharing a common source [17]. The full genome sequences of EHV-4 isolates from an outbreak in Germany confirmed that, despite the virus being genetically stable overall, different RFLP profiles and genome sequences among contemporaneous isolates suggested the involvement of multiple sources of infection, likely through independent reactivation events from latency [13].

Antigenic Structure and Serological Classification

EHV-4 is antigenically related to but distinct from EHV-1, a feature that has necessitated the development of type-specific serological assays. The virus encodes a panel of structural glycoproteins embedded in the viral envelope, including glycoprotein B (gB), gC, gD, gE, gG, gH, and gL, which serve as major targets for the host humoral immune response [18, 24]. Among these, gB and gD are highly immunogenic and have been used extensively for the molecular detection and phylogenetic characterization of EHV-4 field strains [2, 24]. The gG gene, which encodes a secreted glycoprotein unique to alphaherpesviruses, exhibits sufficient sequence divergence between EHV-1 and EHV-4 to serve as a reliable target for type-specific PCR and serological discrimination [18].

Seroprevalence studies conducted across diverse geographic regions consistently demonstrate that EHV-4 is more prevalent than EHV-1 in most equid populations. In a molecular survey of equids in Morocco, 27% of 154 samples tested positive for EHV-4 by PCR, compared to only 1.94% for EHV-1, underscoring the endemic nature of EHV-4 circulation [2]. Similarly, a study of large-scale donkey farms in the Liaocheng area of China reported that 5.22% of 230 donkeys were seropositive for EHV-4 alone, with an additional 4.78% seropositive for both EHV-1 and EHV-4, bringing the total exposure rate to approximately 10% [15]. In the Arabian horse population in Egypt, molecular detection revealed that 30% of 80 samples were positive for EHV-4, confirming active viral circulation in this region as well [24]. These findings, together with seroprevalence data from Europe, North America, Australia, and Asia, establish that EHV-4 is a universally distributed pathogen that infects a substantial proportion of the global equid population, with many animals experiencing subclinical or mild infections that contribute to the maintenance of the virus in the environment [1, 4, 7].

Host Range and Species Tropism

While the horse (Equus caballus) is considered the natural reservoir and primary host for EHV-4, the virus has been documented to infect other equid species, including donkeys (Equus asinus), mules, and possibly zebras. The first reported outbreak of EHV-4-associated respiratory disease in donkeys occurred on an ecological donkey milk farm in Romania, where 37 of 300 jennies developed severe upper respiratory tract disease, with concomitant late abortions and neonatal deaths [3]. Pathological examination revealed acute, diffuse necrotizing bronchointerstitial pneumonia with intranuclear viral inclusion bodies, and RT-PCR confirmed the presence of EHV-4 [3]. This outbreak, which resulted in nine fatalities and three neurological cases, demonstrated that EHV-4 can cause severe disease in donkeys, challenging the traditional view that EHV-4 is exclusively a pathogen of horses. Subsequent epidemiological investigations in Ethiopia have detected EHV-4 in donkeys with respiratory signs, although at lower prevalence compared to horses, suggesting that species-specific differences in susceptibility may exist [5].

The detection of EHV-4 in a variety of equid hosts underscores the importance of considering cross-species transmission in the epidemiology of this virus. However, EHV-4 is not recognized as a zoonotic pathogen, and there is no evidence of transmission to humans. The World Organisation for Animal Health (WOAH) does not list EHV-4 as a notifiable disease, although its close relative EHV-1 is subject to international surveillance and control measures due to its potential to cause abortion storms and neurologic disease [16, 21]. Nonetheless, the economic impact of EHV-4-related respiratory disease on the equine industry is substantial, with outbreaks leading to training interruptions, cancellation of competitions, and veterinary treatment costs [1, 8, 10].

Epidemiological Significance and Global Distribution

EHV-4 is considered endemic in virtually all countries where horses are maintained, with seroprevalence rates typically ranging from 60% to 80% in adult horse populations [4]. The virus is transmitted primarily through the respiratory route via direct nose-to-nose contact, aerosolized respiratory secretions, or indirect contact through contaminated fomites, including feed buckets, water troughs, grooming equipment, and human handlers [1, 20]. The incubation period is typically 2 to 10 days, during which infected horses shed large quantities of virus in nasal secretions, often before the onset of clinical signs, facilitating rapid spread within susceptible populations [1, 13].

Outbreaks of EHV-4 respiratory disease are commonly observed in congregate settings such as boarding stables, breeding farms, training facilities, and equestrian events, where the mixing of horses from diverse geographic origins and immune statuses occurs [1, 13, 20]. A comprehensive analysis of an outbreak at the Large Animal Teaching Hospital in Copenhagen, Denmark, illustrated the dynamics of EHV-4 transmission within a hospital setting, where the index case was traced to a single horse that introduced the virus, followed by secondary spread over a seven-week period [1]. The outbreak strain was characterized by full genome sequencing and found to be more closely related to Australian and Japanese strains than to other European strains, highlighting the global movement of EHV-4 variants through horse transport [1]. Environmental surveillance using pooled stall sponge sampling at a multi-week equestrian show during winter months detected EHV-4 in 1.37% of 948 pooled samples from 53 barns, confirming that the virus can be silently shed by clinically healthy horses and accumulate in the environment, particularly during colder months when ventilation is reduced [20].

Seasonal trends in EHV-4 infection have been observed, with a higher incidence in late autumn, winter, and early spring, likely due to a combination of factors including increased crowding, reduced ventilation in stables, and stress associated with weaning, transportation, and changes in management practices [7, 13, 20]. In a training facility for Thoroughbred yearlings in Japan, monitoring over several years revealed that pyretic horses infected with EHV-4 occurred most frequently between August and September, coinciding with the arrival and mixing of new yearlings [7]. The implementation of an earlier vaccination program significantly reduced infection rates in this population, from 6.8–10.9‰ in 2018–2021 to 1.2–3.8‰ in 2021–2023, demonstrating the potential for targeted intervention to mitigate endemic disease [7].

Clinical Manifestations and Respiratory Disease

EHV-4 is primarily associated with acute upper respiratory tract disease, clinically characterized by pyrexia (often reaching 39–41°C), serous to mucopurulent nasal discharge, mandibular lymphadenopathy, cough, and increased lung sounds on auscultation [1, 13, 24]. The disease is most severe in young horses, particularly weanlings and yearlings, in whom primary infection can result in substantial morbidity [7, 13]. In adult horses with pre-existing immunity, infections are often subclinical or mild, although reactivation of latent virus under conditions of stress can lead to recrudescent disease with viral shedding [1, 13]. The clinical signs observed during the Danish outbreak included pyrexia, nasal discharge, mandibular lymphadenopathy, and increased lung sounds, with the majority of infected horses recovering without complications within one to two weeks [1].

While EHV-4 is most commonly associated with respiratory disease, its potential to cause reproductive and neurological disease has been documented, although these manifestations are less frequent than with EHV-1. EHV-4 has been sporadically isolated from cases of abortion and neonatal death, and an outbreak in a Romanian donkey farm reported ten concurrent late abortions and three neurological cases, suggesting that certain strains or host conditions may predispose to more severe outcomes [3]. However, the association between EHV-4 and neurological disease remains controversial, and the virus is not considered a primary cause of equine herpesvirus myeloencephalopathy (EHM) [6, 11]. The biological basis for the differential pathogenicity between EHV-4 and EHV-1 is multifactorial and includes differences in the ability to establish cell-associated viremia, infect endothelial cells, and disseminate to secondary target organs such as the pregnant uterus and spinal cord [22]. These host-pathogen interactions are profoundly influenced by the immune status of the host, the viral strain involved, and environmental stressors [9, 13], elements that will be explored in greater depth in subsequent sections of this reference.

Molecular Pathogenesis of Equine Herpesvirus 4

Equine herpesvirus 4 (EHV-4), a member of the Alphaherpesvirinae subfamily, is a ubiquitous and highly successful pathogen of equids, primarily associated with acute upper respiratory tract disease, clinically termed equine rhinopneumonitis. While often considered less virulent than its close relative, equine herpesvirus 1 (EHV-1), EHV-4 is a significant cause of morbidity in the global horse population, with seroprevalence rates reaching 60-80% in some regions [4]. The molecular pathogenesis of EHV-4 is a complex, multi-stage process that begins at the mucosal surface of the upper respiratory tract (URT) and involves sophisticated mechanisms of viral entry, replication, immune evasion, and the establishment of lifelong latency. Understanding these molecular events is critical for developing effective vaccines and antiviral strategies, as the virus remains a persistent threat to equine health and the equine industry worldwide.

Initial Infection and Viral Entry at the Respiratory Epithelium

The pathogenesis of EHV-4 is initiated upon inhalation of infectious virions shed in respiratory secretions from acutely infected or reactivated latently infected horses [1, 13]. The primary site of viral entry is the mucosal epithelium of the upper respiratory tract, including the nasal passages, pharynx, and tonsillar tissue [22]. The virus must first overcome the physical and immunological barriers of the respiratory mucosa. The apical surface of the equine respiratory epithelium presents a formidable challenge, as the primary receptors for alphaherpesvirus entry, such as nectin-1 and nectin-2, are typically located on the basolateral surface of epithelial cells [29]. To circumvent this, EHV-4, like EHV-1, exploits disruptions in epithelial integrity. Environmental factors, such as exposure to pollens, mycotoxins, or bacterial toxins (e.g., α-hemolysin from Staphylococcus aureus and adenylate cyclase toxin from Bordetella bronchiseptica), can compromise tight junction integrity, allowing the virus to access its basolateral receptors [29]. This highlights a critical interplay between environmental and microbial co-factors in predisposing the respiratory tract to EHV-4 infection.

Once access to the basolateral receptor is achieved, viral entry is mediated by the coordinated action of multiple viral glycoproteins. The attachment glycoprotein gC binds to cell surface heparan sulfate proteoglycans, concentrating the virus on the cell surface. This is followed by the core fusion machinery, composed of gB, gH, and gL, which catalyzes the fusion of the viral envelope with the host cell membrane, releasing the nucleocapsid into the cytoplasm [22]. The virus then traffics to the nucleus, where viral DNA replication and transcription of immediate-early (IE) and early (E) genes commence, hijacking the host cellular machinery for viral propagation.

Viral Replication, Cytopathic Effect, and Host Cell Shutoff

Following entry, EHV-4 undergoes a rapid lytic replication cycle within the epithelial cells of the URT. The cytopathic effect (CPE) is characterized by cell rounding, detachment, and the formation of syncytia (multinucleated giant cells) [3]. Histopathological examination of infected respiratory tissues reveals acute, diffuse necrotizing bronchointerstitial pneumonia with occasional intraepithelial intranuclear viral inclusion bodies, which are hallmark features of active alphaherpesvirus replication [3]. The virus efficiently replicates to high titers, leading to extensive destruction of the respiratory epithelium. This epithelial damage is the direct cause of the primary clinical signs observed during EHV-4 infection, including pyrexia, serous to mucopurulent nasal discharge, and mandibular lymphadenopathy [1, 13].

A key aspect of EHV-4 pathogenesis is its ability to modulate the host cell environment to favor viral replication. Like other alphaherpesviruses, EHV-4 encodes factors that contribute to host cell shutoff, a process that degrades host mRNA and inhibits host protein synthesis, thereby freeing up cellular resources for viral production and dampening the host’s antiviral response. While specific studies on EHV-4 host shutoff are limited, the virus shares significant genetic homology with EHV-1, which encodes the virion host shutoff (vhs) protein (UL41). It is highly probable that EHV-4 employs a similar mechanism to suppress the expression of interferon (IFN) and other antiviral genes early in infection [22]. The rapid replication and cell lysis in the URT are essential for the virus to achieve high titers for shedding and transmission to new hosts.

Cell-Associated Viremia and Dissemination

A critical divergence in the pathogenesis of EHV-4 compared to EHV-1 lies in its capacity to establish a robust cell-associated viremia. While EHV-1 is renowned for its ability to infect peripheral blood mononuclear cells (PBMCs), particularly CD172a+ monocytic cells, and disseminate to the endothelium of the pregnant uterus and central nervous system (CNS), EHV-4 is far less efficient in this regard [22, 36]. This fundamental difference is the primary molecular explanation for why EHV-4 is rarely associated with abortion or equine herpesvirus myeloencephalopathy (EHM), which are devastating sequelae of EHV-1 infection [3, 6, 34].

The molecular basis for this restricted dissemination is an area of active investigation. The initial steps are similar: after replicating in the epithelium, EHV-4 infects CD172a+ monocytic cells that are recruited to the site of infection. Chemokines such as CCL2 and CCL5, secreted by infected epithelial cells, play a crucial role in attracting these target cells to the lamina propria [36]. However, the efficiency of this process appears to be strain-dependent and generally lower for EHV-4. Once inside the monocytic cells, EHV-4 must successfully evade intracellular innate immune sensors and complete its replication cycle to produce infectious progeny that can be transported via the lymphatics and bloodstream. The virus’s ability to suppress type I interferon (IFN-α/β) responses within these cells is a key determinant of its success in establishing viremia [9, 27]. EHV-4 appears to be more susceptible to these innate immune restrictions within PBMCs compared to EHV-1, resulting in a lower magnitude and shorter duration of cell-associated viremia. This restricted viremia is the primary reason EHV-4 is predominantly a respiratory pathogen, as it fails to efficiently reach and infect the endothelial cells of the spinal cord or placenta, which are the pathological targets for EHM and abortion, respectively [11, 30, 31].

Immune Evasion Strategies at the Mucosal Surface

To establish a productive infection, EHV-4 must counteract the host’s innate and adaptive immune defenses at the respiratory mucosa. The virus has evolved several sophisticated immune evasion strategies, many of which are shared with EHV-1. A primary target is the type I interferon (IFN) pathway. Upon viral entry, pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and cytosolic DNA sensors detect viral components and trigger a signaling cascade leading to the production of IFN-α/β. This, in turn, induces the expression of hundreds of interferon-stimulated genes (ISGs) that establish an antiviral state [9, 27, 35]. EHV-4, like other alphaherpesviruses, encodes proteins that interfere with this pathway at multiple levels. For instance, the viral IE protein (ICP0) and other early proteins can inhibit the activation of interferon regulatory factors (IRFs) and suppress the JAK-STAT signaling pathway, thereby blunting the induction of ISGs [22].

The role of mucosal antibodies, particularly IgG4/7 and IgG1 isotypes, is paramount in controlling EHV-4 infection at the point of entry. Studies on EHV-1 have demonstrated that pre-existing mucosal IgG4/7 antibodies can neutralize the virus, preventing its attachment and entry into epithelial cells [14, 25, 28]. In immune horses, these antibodies rapidly neutralize the virus, leading to incomplete viral replication and a lack of detectable viral shedding [27, 28]. The induction of these protective mucosal antibodies is a key goal of vaccination. Intramuscular vaccination with inactivated EHV-1/4 vaccines has been shown to boost mucosal IgG4/7 levels, which correlates with protection from clinical disease and reduced viral shedding [25]. Conversely, in non-immune horses, the absence of these neutralizing antibodies allows for unimpeded viral replication, leading to high viral loads, pronounced clinical signs, and the induction of a strong type I IFN response [27, 28]. The virus also appears to modulate the B-cell response, potentially skewing it away from a protective IgG4/7 response in naïve animals.

Latency and Reactivation: The Molecular Basis for Persistence

A defining feature of all alphaherpesviruses, including EHV-4, is their ability to establish lifelong latency in the host, a state of reversible non-productive infection [16, 18, 33]. Following the resolution of primary respiratory disease, the virus enters a latent state in sensory neurons of the trigeminal ganglia (TG) and in cells of the respiratory-associated lymphoid tissue (RALT) [33]. During latency, the viral genome persists as a circular episome in the nucleus of the infected cell, with a highly restricted pattern of gene expression. Only a specific set of latency-associated transcripts (LATs) are produced, while the lytic gene cascade is silenced. The molecular mechanisms governing the establishment, maintenance, and reactivation of EHV-4 latency are complex and involve epigenetic regulation of the viral genome, including histone modifications and DNA methylation.

Reactivation from latency is a stochastic event that can be triggered by various stress factors, including transportation, weaning, co-infection with other pathogens, corticosteroid administration, and the physiological stress of competition or parturition [13, 16]. Upon reactivation, the latent viral genome is re-activated, and the lytic replication cycle resumes. This leads to the production of infectious virus, which is then shed in nasal secretions, often in the absence of overt clinical signs [32, 37]. This silent shedding is a critical epidemiological feature, as it allows the virus to circulate undetected within a population, serving as a source of infection for naïve animals [20, 32]. The ability of EHV-4 to reactivate and be shed by clinically normal horses underscores the challenge of controlling its spread, as quarantine and testing protocols may fail to identify these silent shedders [32]. The high seroprevalence of EHV-4 globally is a direct consequence of this efficient latency-reactivation cycle, which ensures the virus’s persistence within the equine population [4, 15].

Genetic Diversity and Recombination

The molecular pathogenesis of EHV-4 is further shaped by its genomic plasticity. While EHV-4 is considered genetically stable, field isolates exhibit a degree of genomic diversity that can influence virulence and tissue tropism [13, 17, 19]. Restriction fragment length polymorphism (RFLP) analysis and full-genome sequencing have revealed that EHV-4 isolates can be grouped into distinct clades, and this diversity is particularly evident in genes encoding surface glycoproteins and proteins involved in immune evasion [12, 17, 19]. Crucially, EHV-4 demonstrates a much higher propensity for natural recombination than EHV-1 [12, 26]. This widespread recombination, detected through high-throughput sequencing of field isolates, allows for the rapid exchange of genetic material between different circulating strains [12]. This process can generate novel viral variants with altered pathogenic potential, such as increased ability to replicate in the URT, enhanced immune evasion capabilities, or even, in rare cases, an expanded tissue tropism. The ability to recombine is a powerful evolutionary tool that allows EHV-4 to adapt to changing host immune pressures and environmental conditions, contributing to its continued success as a pathogen. The recombination events are not limited to EHV-4 strains alone; bioinformatic analyses have provided evidence of inter-species recombination between EHV-1 and EHV-4, and between EHV-1 and EHV-8, further complicating the evolutionary landscape of these viruses [26].

Epidemiology and Transmission Dynamics of Equine Herpesvirus 4

Global Distribution and Endemic Status

Equine herpesvirus 4 (EHV-4) is globally enzootic, representing one of the most prevalent viral pathogens affecting equids worldwide. The virus establishes a persistent presence across diverse geographic regions, with seroprevalence estimates ranging from 60–80% in domestic horse populations [4]. This remarkable ubiquity underscores the virus's evolutionary success and its profound co-adaptation with equine hosts over millennia. A landmark paleovirological study by Lebrasseur et al. recovered an ancient EHV-4 genome with 4.2X average depth-of-coverage from an Early Bronze Age archaeological specimen excavated in the Southeastern Urals, dating to approximately 3,900 years before present [4]. Bayesian phylogenetic reconstructions from this ancient genome provide a minimal time estimate for EHV-4 diversification to approximately 4,000 years ago, coinciding temporally with the dissemination of modern domestic horses across the Central Asian steppes alongside Sintashta spoke-wheeled chariots [4]. This analysis fundamentally revises previous diversification timelines, which had placed the emergence of the two major EHV-4 subclades in the 16th century based solely on modern sequence data; the updated estimates push this divergence to nearly a millennium ago [4]. Such deep evolutionary history explains the intricate host–pathogen equilibrium that characterizes contemporary EHV-4 epidemiology.

Contemporary molecular surveys consistently confirm widespread viral circulation. In Morocco, a study evaluating 154 equid samples detected EHV-4 in 27% of specimens, in stark contrast to the 1.94% positivity rate for EHV-1 [2]. Similarly, investigations of Arabian horse populations in northern Egypt between 2021 and 2022 revealed that 30% of 80 samples tested positive for EHV-4, with mixed EHV-1/EHV-4 infections identified in five animals [24]. The virus has been documented in diverse equid populations including horses, donkeys, and their hybrids, with species-specific susceptibility patterns emerging as an important epidemiological consideration [3, 5]. In Ethiopia, detection rates for EHV-4 were substantially lower than those for gammaherpesviruses, with 8.1% of 160 equids with respiratory disease testing positive for EHV-4, while no EHV-4 was detected among 111 clinically healthy controls [40]. This finding underscores the strong association between EHV-4 detection and clinical respiratory disease, differentiating it from the gammaherpesviruses EHV-2 and EHV-5 which are frequently identified in asymptomatic carriers [39, 40].

Transmission Mechanisms and Viral Shedding Dynamics

Transmission of EHV-4 occurs primarily through direct nose-to-nose contact via respiratory secretions, with fomites and contaminated equipment serving as secondary routes of dissemination [1, 10]. The virus initially infects the epithelial cells of the upper respiratory tract mucosa, where it undergoes rapid local replication [28]. Infected horses shed infectious virus in nasal secretions, with shedding patterns characterized by a relatively brief but intense period of contagiousness. During a documented outbreak at the Large Animal Teaching Hospital, University of Copenhagen, nine horses tested EHV-4 positive over approximately seven weeks, with detailed clinical registrations revealing pyrexia, nasal discharge, mandibular lymphadenopathy, and increased lung sounds upon auscultation as the predominant clinical signs [1]. Notably, a tenth horse (Eq10) tested positive almost three weeks after the last outbreak-associated horse, and partial genome sequencing confirmed infection with a distinct wild-type strain, demonstrating that the hospital's biosecurity measures successfully eliminated the original outbreak strain despite continued viral circulation in the broader equine population [1].

The duration and magnitude of viral shedding are modulated by host immune status, age, and prior exposure history. Horses with pre-existing mucosal immunity, particularly those possessing EHV-1/4-specific IgG4/7 antibodies at the upper respiratory tract mucosa, exhibit significantly reduced viral replication and shedding [25, 28]. Experimental studies using immune horses demonstrated that pre-existing mucosal IgG1 and IgG4/7 antibodies neutralize incoming virus, preventing complete viral replication and thereby abrogating infectious shedding [28]. Transcriptomic profiling revealed that immune horses rapidly upregulate B-cell pathway genes upon challenge, with antileukoproteinase (SLPI) expression increasing 2.1-fold within 24 hours, while non-immune horses mount a robust type I interferon response characterized by IFIT2 and IFIT3 upregulation coincident with active viral replication [27]. This dichotomy in innate immune activation patterns has profound implications for transmission dynamics, as immune horses do not shed infectious virus and thus constitute dead-end hosts in the transmission chain.

Reservoir Hosts, Latency, and Reactivation

The capacity for lifelong latency, a hallmark of all alphaherpesviruses, is central to EHV-4's epidemiological persistence. Following primary infection, the virus establishes latency in the trigeminal ganglion, respiratory-associated lymphoid tissue (RALT), and potentially peripheral blood mononuclear cells, though the precise distribution of EHV-4 latency has been less extensively characterized than that of EHV-1 [11, 33]. Latent infections undergo periodic reactivation, typically triggered by physiological stressors including weaning, transportation, intensive training, concurrent infections, parturition, or adverse environmental conditions such as seasonal transitions and temperature extremes [13, 15]. Reactivation results in renewed viral shedding, often without overt clinical signs, creating a continuous source of infectious virus that perpetuates endemic circulation.

The role of donkeys as reservoir hosts warrants particular attention. A landmark outbreak investigation on an ecological donkey milk farm in Romania documented the first EHV-4-associated respiratory disease in donkeys from that country [3]. Among 300 jennies, 37 initially presented with severe upper respiratory tract disease, with ten concomitant late abortions or neonatal deaths and three neurological cases, resulting in nine fatalities [3]. EHV-4 was confirmed by real-time PCR, and acute, diffuse necrotizing bronchointerstitial pneumonia with intraepithelial intranuclear viral inclusion bodies was identified histopathologically [3]. Similarly, serological surveys of large-scale donkey farms in Liaocheng, China revealed that 62.96% of 27 farms tested seropositive for EHV, with 5.22% of 230 donkeys positive for EHV-4 antibodies alone and 4.78% positive for both EHV-1 and EHV-4 [15]. The seropositivity rate was significantly higher in donkeys aged 1–4 years compared to those under one year, and significantly higher in fall and winter compared to spring and summer, consistent with stress-associated reactivation patterns [15].

Outbreak Patterns and Risk Factors

Outbreaks of EHV-4 typically occur when susceptible animals are congregated under conditions of physiological stress. A large outbreak at a Standardbred horse-breeding farm in northern Germany in 2017 exemplifies this dynamic: of 84 horses, 76 were tested and 41 were affected, including 20 foals, 10 stallions, and 11 mares [13]. Epidemiological investigation revealed that stress caused by seasonal changes, management practices, routine equestrian activities, and exercise contributed as multifactorial causes for disease outbreak [13]. Importantly, 73% of infected mares and their corresponding foals shed virus simultaneously, illustrating the close contact transmission dynamics within maternal–offspring pairs [13]. Restriction fragment length polymorphism (RFLP) analysis of four EHV-4 isolates obtained during this outbreak revealed distinct profiles in each isolate, suggesting the involvement of multiple sources of infection, either through simultaneous introduction of different strains or reactivation of latent viruses with distinct genomic characteristics [13].

Age constitutes a critical risk factor, with young animals, particularly foals and yearlings, exhibiting heightened susceptibility to primary infection and clinical disease. In a study of Thoroughbred yearlings at a training facility in Japan, pyretic horses infected with EHV-1/4 occurred most frequently between August and September across three consecutive years (2018–2021), with nine confirmed EHV-4 cases and five cases involving both viruses [7]. The implementation of an updated vaccination program that commenced earlier, before all yearlings had arrived, resulted in infection rates declining from 6.8–10.9‰ in baseline years to 1.2–1.7‰ in the final year of observation [7]. This intervention demonstrated that strategic vaccination timing can disrupt transmission cycles even within an endemic setting.

Co-infections with other respiratory pathogens modulate EHV-4 transmission dynamics. In Ethiopian equids, concurrent infections with EHV-1 and EHV-2 (31%), EHV-1 and EHV-5 (17%), and all three viruses (13%) were documented in animals with respiratory disease [5]. Furthermore, bacterial infections with Staphylococcus aureus or Bordetella bronchiseptica can predispose the respiratory epithelium to enhanced EHV-4 replication. Mechanistic studies using equine respiratory mucosal explants demonstrated that the bacterial exotoxins α-hemolysin (Hla) and adenylate cyclase toxin (ACT) decrease epithelial thickness, induce detachment of epithelial cells, and cause partial loss of cilia, with these morphological changes correlating with increased EHV-4 replication in the epithelium [29]. Such synergistic interactions between viral and bacterial pathogens likely amplify transmission efficiency during polymicrobial outbreaks.

Spatial and Temporal Transmission Dynamics

Environmental transmission of EHV-4, while less efficient than direct contact, contributes to viral persistence in congregate settings. Environmental surveillance at a multi-week equestrian show during winter months detected EHV-4 in 1.37% of 948 pooled stall sponge samples, with detection frequency increasing during colder months compared to spring and summer [20]. This seasonal variation aligns with serological observations from donkey farms in China, where positivity rates were significantly higher in fall and winter [15]. The World Organisation for Animal Health (WOAH) recognizes that while EHV-4 is not a WOAH-listed disease, its impact on equine health and international movement warrants vigilant surveillance within the framework of regional control programs.

The Danish hospital outbreak provided unique insights into within-facility transmission patterns. Through detailed mapping of horse locations and movements combined with serial qPCR testing and antibody serology, the most likely "patient zero" (Eq1) was identified [1]. The outbreak strain's complete genome sequence revealed closer phylogenetic relatedness to Australian and Japanese EHV-4 strains than to other European isolates, though this observation likely reflects the limited sequence data available from European isolates rather than true intercontinental transmission [1]. Crucially, the study demonstrated that a different wild-type strain infected a horse nearly three weeks after the last outbreak case, confirming that stringent biosecurity, including testing and movement restrictions, can successfully interrupt transmission chains even in hospital environments where susceptible and infected horses commingle.

Evolutionary Dynamics and Strain Diversity

EHV-4 exhibits substantial genomic diversity that facilitates rapid adaptation and complicates epidemiological tracking. Restriction endonuclease analysis of 23 Japanese EHV-4 field isolates revealed distinct differences including mobility shifts of fragments and loss/gain of restriction sites [17]. Two genes containing repeat sequences, ORFs 24 and 71, showed size variation among epizootiologically unrelated isolates, while related isolates maintained identical fragment sizes, providing a molecular epidemiological tool for outbreak tracing [17].

Whole-genome sequencing of 14 Australian EHV-4 isolates and subsequent bioinformatic analysis provided compelling evidence of widespread natural recombination among field isolates, in stark contrast to EHV-1, where only a single potential recombination event was detected among 22 isolates [12]. This fundamental biological difference has profound epidemiological implications: recombination allows EHV-4 to generate genetic diversity more rapidly than point mutation alone, potentially facilitating immune evasion, altering tissue tropism, and modulating virulence. The demonstration of recombination between EHV-1 and EHV-4, and between EHV-1 and EHV-8, further emphasizes the dynamic nature of equid alphaherpesvirus evolution [12, 26].

Genome sequences of eight Japanese EHV-4 isolates, including two from aborted fetuses and five from horses with respiratory disease, clustered into two phylogenetic groups that did not correlate with pathogenicity [19]. Comparison of predicted amino acid sequences failed to identify any genes specifically associated with abortion, suggesting that host factors, viral load, or stochastic events, rather than fixed genetic determinants, govern the infrequent occurrence of abortion following EHV-4 infection [19]. Similarly, partial genome sequencing of the Danish outbreak strain revealed that the hospitalized horses were infected with a wild-type EHV-4 strain more closely related to Australian and Japanese isolates than to European strains, though this may reflect sequencing bias rather than genuine geographic structuring [1]. The complete genome sequence obtained from this outbreak contributes to the growing database necessary for robust phylogeographic analyses.

Seroprevalence and Population Immunity

Serological surveys provide critical insights into population-level immunity and transmission intensity. In Turkey, development of an in-house ELISA enabled detection of EHV-1/4 antibodies in horse populations, revealing widespread exposure [38]. On large-scale donkey farms in Liaocheng, China, 62.96% of farms demonstrated seropositivity for EHV, with positive rates varying by county, Dong'e County exhibited the highest proportion at 21.28% [15]. Age-specific seroprevalence patterns showed significantly higher positivity in donkeys aged 1–4 years compared to those under one year, consistent with waning maternal antibody and subsequent natural exposure [15].

The humoral immune response to EHV-4 is dominated by IgG4/7 isotypes, which play a crucial role in mucosal protection. Intramuscular vaccination with inactivated EHV-1/4 vaccine in previously exposed horses elicited significant increases in serum IgG4/7 that persisted throughout the study period, with mucosal antibodies increasing after five out of six vaccine injections [25]. This IgG4/7 response correlated with protection from viral replication at the upper respiratory tract, as demonstrated in experimental EHV-1 challenge studies [14, 28]. Notably, horses with robust pre-existing mucosal IgG4/7 did not mount a type I interferon response upon challenge, suggesting that neutralizing antibodies at the mucosal surface prevent viral entry and subsequent innate immune activation [27, 28]. The half-life and durability of these protective antibodies influence herd immunity thresholds and the optimal timing of booster vaccinations.

Transmission in Special Populations: Donkeys and Mules

The epidemiology of EHV-4 in donkeys and mules has historically been understudied relative to horses, but emerging evidence indicates these species play important roles in viral maintenance and transmission. In Ethiopia, EHV-1 was detected in a significantly higher proportion of donkeys (76%) compared to horses (51.5%), while horses were fourteen times more likely to be positive for EHV-2 [5]. Species-specific susceptibility to EHVs likely reflects differences in host immune responses, receptor expression patterns, or management practices. The Romanian outbreak in an ecological donkey farm documented not only respiratory disease but also abortion and neurological signs, expanding the recognized clinical spectrum of EHV-4 in donkeys [3]. The detection of EHV-4-specific antibodies in 15 of 28 sampled animals (53.6%) during this outbreak indicates that seroconversion rates can be high in naive populations during acute outbreaks [3].

Genetic characterization of

Clinical Manifestations of Equine Herpesvirus 4 Respiratory Disease

Equine herpesvirus 4 (EHV-4) is the quintessential etiological agent of equine rhinopneumonitis, a globally endemic respiratory syndrome that affects equids of all ages, though its clinical expression is profoundly modulated by host immune status, age, and environmental stressors. While EHV-4 shares a close phylogenetic relationship with its more notorious counterpart, EHV-1, the clinical manifestations of EHV-4 infection are overwhelmingly confined to the respiratory tract, with a markedly lower propensity for systemic dissemination and the devastating sequelae of abortion and myeloencephalopathy [1, 6, 13]. This fundamental distinction in clinical behavior underscores a divergence in pathogenetic mechanisms, driven by differences in cellular tropism and immune evasion strategies. The clinical picture of EHV-4 respiratory disease is therefore best understood as a spectrum ranging from inapparent or subclinical infection to overt, sometimes severe, upper and lower respiratory tract inflammation, with the most pronounced signs typically observed in weanlings, yearlings, and young adult horses exposed to the virus for the first time or under conditions of immunological stress [1, 13, 40].

The Typical Acute Respiratory Syndrome

The hallmark clinical presentation of acute EHV-4 infection is an afebrile to moderately febrile respiratory illness with an incubation period of approximately 2 to 10 days following viral entry into the upper respiratory tract (URT) epithelium [8, 22]. The initial febrile response is often biphasic, with an early temperature spike coinciding with viral replication in the nasal mucosa, followed by a second, often more pronounced, pyrexic episode as the virus engages secondary lymphoid tissues and the host mounts a systemic inflammatory response [1, 13]. In a meticulously documented outbreak within a Danish equine teaching hospital, the most consistently recorded clinical signs included pyrexia (frequently exceeding 38.5°C), serous to mucopurulent nasal discharge, mandibular lymphadenopathy, and increased lung sounds upon thoracic auscultation [1]. These findings are echoed in outbreak investigations from Germany, where 41 of 84 horses on a Standardbred breeding farm exhibited respiratory illness characterized by bilateral nasal discharge, coughing, and submandibular lymph node enlargement [13]. The nasal discharge, initially thin and serous, often progresses to a thicker, mucopurulent consistency within 48–72 hours as secondary bacterial colonization, often by opportunistic pathogens such as Streptococcus equi subsp. zooepidemicus, complicates the primary viral insult [1, 8, 44]. Ocular involvement, manifesting as conjunctivitis and epiphora, is reported in a subset of cases, reflecting the ability of EHV-4 to infect the conjunctival epithelium [1, 13].

Pyrexia and Systemic Signs

The febrile response is a cardinal feature of acute EHV-4 respiratory disease. In the Danish outbreak, pyrexia was documented as the most frequent initial sign, often preceding other clinical abnormalities by 12–24 hours [1]. The magnitude and duration of fever are generally less severe than that observed with EHV-1 infection, and it typically resolves within 3–5 days in uncomplicated cases [13, 23]. However, in immunologically naive foals or animals under concurrent stress, the fever may be more sustained and pronounced [3, 13]. Associated systemic signs include lethargy, partial to complete anorexia, and a transient depression in overall demeanor. These systemic effects are driven by the release of proinflammatory cytokines, including interleukin-1β, tumor necrosis factor-α, and interleukin-6, which act on the hypothalamus to elevate the thermoregulatory set point [22, 42].

Nasal Discharge and Ocular Involvement

The evolution of the nasal discharge provides a valuable clinical indicator of disease progression. In the earliest phase of infection, the discharge is profuse, watery, and bilateral, reflecting the exudation of serous fluid from inflamed nasal mucosa [1, 13]. As the infection advances, the discharge becomes mucoid and, in the presence of secondary bacterial infection, purulent. This transition is often accompanied by a worsening of respiratory effort and the development of a moist, productive cough [1, 13, 40]. Conjunctivitis, characterized by hyperemia of the conjunctival membranes and serous ocular discharge, is reported in a significant minority of cases, particularly in foals [13]. The presence of ocular signs should prompt consideration of EHV-1 as a differential, as this manifestation is more common with that virus, but it is nonetheless recognized in EHV-4 infections [13].

Mandibular Lymphadenopathy

Enlargement and palpation of the submandibular lymph nodes is a highly consistent finding in EHV-4 respiratory disease [1, 13]. The nodes are typically firm, mildly to moderately enlarged, and may be slightly painful upon palpation. This lymphadenopathy reflects the active replication of the virus within the local lymphoid tissues, particularly the respiratory-associated lymphoid tissue (RALT), which is a critical initial site for viral propagation and immune priming [22, 36]. The enlargement is symmetrical and bilateral, and it generally resolves over the course of 7–14 days as the adaptive immune response clears the infection.

Respiratory Auscultation Findings

Auscultation of the thorax frequently reveals increased bronchovesicular sounds, particularly over the cranioventral lung fields [1]. In more severe cases, wheezes and crackles may be appreciated, indicative of lower airway involvement and the development of bronchitis or bronchopneumonia [1, 3]. While EHV-4 is primarily an upper respiratory pathogen, the virus can extend into the lower airways, especially in young foals or animals with compromised respiratory defenses, leading to a diffuse bronchointerstitial pneumonia [3, 13]. In a comprehensive study of Australian horses, the detection of EHV-4 by qPCR was associated with clinical signs of mild respiratory disease, though the overall prevalence of EHV-4 in the study population was low (1.2% of 407 horses) [39]. This reinforces the concept that while EHV-4 can cause overt illness, a substantial proportion of infections may be subclinical or result in only minor clinical signs.

Age-Related and Population-Specific Clinical Patterns

The clinical expression of EHV-4 respiratory disease is strikingly age-dependent. In foals under six months of age, maternally derived antibodies provide a degree of protection, though this wanes substantially by weaning age [13]. Consequently, the peak incidence of clinical disease occurs in weanlings and yearlings, a period coinciding with the loss of passive immunity and the stress of commingling, transport, and training [7, 13]. In a Japanese study of Thoroughbred yearlings in training, the incidence of pyretic episodes attributable to EHV-4 infection peaked between August and September, precisely when these young animals were being introduced to the training facility [7]. The clinical signs in this cohort were dominated by fever and mild respiratory signs, and the implementation of an earlier vaccination program significantly reduced the infection rate, underscoring the role of immune maturity in moderating clinical severity [7].

In adult horses, EHV-4 infection is often subclinical or associated with very mild, transient respiratory signs. However, in a Tibetan donkey farm in Romania, an EHV-4 outbreak manifested with severe upper respiratory tract disease in 37 of 300 jennies, with ten concomitant late abortions and neonatal deaths, and three neurological cases [3]. This report is exceptional, as EHV-4 is rarely associated with abortion or neurological disease, and it suggests that in certain populations, particularly donkeys, which may have a different baseline immune status, the virus may behave with enhanced virulence [3, 19, 34]. The study confirmed the diagnosis by RT-PCR and histology, demonstrating acute, diffuse necrotizing bronchointerstitial pneumonia with intranuclear inclusion bodies in the affected donkeys [3]. This case illustrates that while the classic clinical picture of EHV-4 is a mild upper respiratory infection, severe disease can occur, especially in naïve populations or under conditions of high viral challenge.

Subclinical and Latent Infection

A critical aspect of the clinical epidemiology of EHV-4 is the prevalence of subclinical infection. The virus is endemic in equine populations worldwide, with seroprevalence rates often exceeding 60% [4, 15]. The vast majority of infections result in either no observable clinical signs or a very mild, self-limiting malaise that goes undetected. In clinically healthy sport horses monitored after an EHV-1 outbreak in California, EHV-4 was detected at low but stable frequencies in nasal swabs from animals with no outward signs of illness [32]. Similarly, in environmental stall sampling at a multi-week equestrian show, EHV-4 was detected in 1.37% of pooled sponge samples, indicating that the virus is shed into the environment by apparently healthy carriers [20]. This phenomenon of subclinical shedding is a central feature of EHV-4 epidemiology, as it allows the virus to persist and spread undetected within a herd, with outbreaks often triggered only when the virus is introduced to a group of susceptible naive animals or when reactivation from latency occurs due to stress [13, 16, 37].

The establishment of latency is a hallmark of all alphaherpesviruses, and EHV-4 is no exception. After primary infection, the virus becomes latent within the trigeminal ganglia and, potentially, in respiratory-associated lymphoid tissue [22, 33]. Reactivation of latent virus can be precipitated by a variety of stressors, including transport, weaning, corticosteroid administration, intercurrent illness, and the physical exhaustion associated with training or competition [13, 37, 45]. When reactivation occurs, the animal may again shed virus in nasal secretions, often without any overt clinical signs, thereby perpetuating the cycle of transmission. This phenomenon was elegantly documented in the German outbreak, where epidemiological investigation suggested that stress from seasonal changes, management practices, and routine equestrian activities acted as a multifactorial trigger for reactivation and subsequent dissemination of EHV-4 among in-housed mares and foals [13].

Complications and Atypical Manifestations

While respiratory disease is the hallmark of EHV-4 infection, a small proportion of cases may develop complications. The most notable of these is secondary bacterial pneumonia, which can transform a mild viral illness into a life-threatening bronchopneumonia, particularly in foals and immunocompromised adults [3, 8]. The aspiration of purulent material and the breaching of the mucociliary clearance barrier by the virus predispose the lower airways to colonization by opportunistic bacteria, most commonly Streptococcus equi subsp. zooepidemicus and Bordetella bronchiseptica [29, 44]. Co-infections with other respiratory viruses, such as EHV-1, EHV-2, and EHV-5, are also well-documented and may result in a more severe or prolonged clinical course [39, 40, 43]. In Ethiopian equids, concurrent infections with EHV-4 and EHV-2 were detected in a significant proportion of animals with respiratory signs, though the precise contribution of each virus to the overall clinical picture remains difficult to disentangle [5, 40].

Abortion and neonatal death are exceedingly rare complications of EHV-4 infection, in stark contrast to EHV-1 [3, 19]. The Romanian donkey outbreak, however, stands as a stark exception, with ten abortions and neonatal deaths occurring concurrently with the respiratory outbreak [3]. This observation is supported by molecular characterization of EHV-4 isolates from aborted fetuses in Japan, where whole genome sequencing failed to identify any specific genes that could reliably predict abortigenic potential [19]. It appears that EHV-4 lacks the ability to efficiently establish cell-associated viremia, a prerequisite for transplacental transmission, under most circumstances [22]. Nevertheless, the virus can, under certain ill-defined host or environmental conditions, cross the placental barrier and cause fetal death, though this is the exception rather than the rule.

Equine herpesvirus myeloencephalopathy (EHM) has been rarely attributed to EHV-4, and the association remains controversial [6, 13]. A few reports describe neurological signs, primarily ataxia and recumbency, in association with EHV-4 infection, but the causality is difficult to confirm, and the clinical presentation appears distinct from the severe, often necrotizing vasculitis that characterizes EHV-1-induced EHM [3, 41, 46]. In the Romanian donkey outbreak, three neurological cases were reported, but the precise etiology of the neurological signs was not fully elucidated, and EHV-1 co-infection or secondary factors could not be excluded [3]. The consensus within the field is that EHV-4 is a respiratory pathogen of primary importance, and while it may sporadically be implicated in other disease syndromes, its clinical identity remains firmly anchored in the upper respiratory tract.

Diagnostic Methods for Equine Herpesvirus 4

The accurate and timely diagnosis of Equine Herpesvirus 4 (EHV-4) infection is paramount for effective outbreak management, implementation of biosecurity protocols, and understanding the epidemiological dynamics of this ubiquitous pathogen. Given that EHV-4 is endemic in global equine populations, with seroprevalence rates often exceeding 60-80% [4], diagnostic methods must be capable of differentiating acute infection from latent carriage, distinguishing EHV-4 from its closely related alphaherpesvirus counterpart EHV-1, and providing results with sufficient speed to inform clinical decision-making. The diagnostic armamentarium for EHV-4 has evolved considerably, moving from traditional virus isolation and serological profiling to highly sensitive molecular techniques that now represent the gold standard for detection and characterization.

Molecular Detection: Quantitative PCR and Multiplex Assays

The cornerstone of contemporary EHV-4 diagnosis is the polymerase chain reaction (PCR), particularly real-time quantitative PCR (qPCR). This methodology offers unparalleled sensitivity, specificity, and rapid turnaround time, making it indispensable for both clinical diagnosis and epidemiological surveillance. The development of a multiplex real-time PCR assay (EHV1-4MP) represents a significant advancement, enabling the simultaneous detection and differentiation of EHV-1 and EHV-4 in a single reaction [18]. This assay, which incorporates an endogenous internal control targeting the equine melanocortin 1 receptor (MC1R) gene, demonstrates analytical sensitivity of approximately four copies for EHV-4 per reaction and exhibits 100% concordance with singleplex assays when applied to clinical specimens including upper respiratory swabs, washes, blood, placenta, lung, and brain tissue [18]. Critically, this multiplex assay has been engineered to exclude closely related equid herpesviruses such as EHV-3, EHV-8, and EHV-9, addressing a significant specificity limitation of earlier PCR designs [18].

The selection of appropriate clinical specimens is critical for maximizing diagnostic yield. Nasal swabs and nasopharyngeal swabs are the specimens of choice for detecting active respiratory shedding, as EHV-4 replicates extensively in the upper respiratory tract epithelium [1, 13]. During an outbreak investigation at the Large Animal Teaching Hospital in Denmark, systematic qPCR testing of nasal swabs from hospitalized horses successfully identified nine EHV-4-positive cases over a seven-week period, demonstrating the utility of this approach for tracking viral transmission within a facility [1]. Whole blood samples can also be submitted for PCR analysis, although the detection of EHV-4 DNA in blood is less common than for EHV-1, as EHV-4 is less frequently associated with cell-associated viremia [49]. For post-mortem diagnosis, particularly in cases of abortion or neonatal death, tissues such as lung, liver, spleen, and placenta should be collected for molecular testing [3, 51].

The timing of sample collection relative to the onset of clinical signs profoundly influences diagnostic sensitivity. Viral shedding in nasal secretions typically peaks during the acute febrile phase, and samples collected within the first 24-72 hours of pyrexia and nasal discharge yield the highest probability of detection [1, 13]. In a study of an EHV-4 outbreak in Germany, qPCR confirmed viral involvement in all 41 affected horses, with foals and their corresponding mares often shedding virus concurrently, underscoring the importance of testing both dam and offspring during outbreak investigations [13]. It is worth noting that PCR assays can detect viral DNA even in the absence of infectious virus, as nucleic acid may persist after viable virus has been cleared by the host immune response. Therefore, positive PCR results should be interpreted in conjunction with clinical findings and, where possible, serological data.

Virus Isolation and Traditional Virological Techniques

While molecular methods have largely supplanted virus isolation for routine diagnosis, the isolation of EHV-4 in cell culture remains an important tool for obtaining viral isolates for genetic characterization, antiviral susceptibility testing, and vaccine development. EHV-4 can be propagated in a variety of equine cell lines, including equine embryonic lung cells, equine dermal cells, and primary equine kidney cells [13, 17]. The virus typically produces characteristic cytopathic effects (CPE) within 2-5 days post-inoculation, including cell rounding, detachment, and syncytia formation [13]. However, virus isolation is labor-intensive, requires specialized laboratory facilities, and has lower sensitivity compared to PCR, particularly when samples have been improperly stored or transported. In a Moroccan study, virus isolation was unsuccessful for all EHV-4-positive samples despite successful molecular detection, highlighting the challenges associated with this technique [2]. Similarly, in a Brazilian investigation of equine abortion, only one of two PCR-positive fetuses yielded a positive virus isolation result [51].

Restriction fragment length polymorphism (RFLP) analysis of isolated viral DNA has historically been employed for epidemiological strain typing. Digestion of EHV-4 genomic DNA with restriction endonucleases such as BamHI, BglII, EcoRI, SacI, and SalI reveals distinct fragment patterns that can differentiate epizootiologically unrelated isolates [13, 17]. In the German outbreak study, four EHV-4 isolates from different animals exhibited distinct RFLP profiles, suggesting the involvement of multiple viral strains or reactivation from latency rather than a single point-source introduction [13]. While RFLP analysis has largely been superseded by whole-genome sequencing for high-resolution typing, it remains a useful, cost-effective tool for preliminary strain discrimination in resource-limited settings.

Serological Diagnostics: ELISA and Virus Neutralization

Serological testing provides retrospective evidence of EHV-4 infection and is valuable for assessing population-level exposure, vaccine responses, and confirming recent infection through demonstration of seroconversion. The virus neutralization test (VNT) has long been considered a reference standard for detecting EHV-4-specific antibodies. This assay measures the ability of serum antibodies to neutralize infectious virus in cell culture and provides a functional assessment of humoral immunity [13, 48]. However, VNT is labor-intensive, requires live virus and cell culture facilities, and can be hampered by the presence of non-specific inhibitors in equine serum. Furthermore, cross-reactivity between EHV-1 and EHV-4 antibodies is a well-recognized limitation, as the two viruses share significant antigenic homology, particularly in glycoprotein B and other structural proteins [24, 38].

Enzyme-linked immunosorbent assays (ELISAs) have been developed to address these limitations and offer higher throughput, standardization, and the ability to differentiate between EHV-1 and EHV-4 infections. Type-specific ELISAs, often based on peptide antigens derived from unique regions of the viral glycoprotein G (gG), have demonstrated excellent discriminatory power [7, 13]. An in-house ELISA developed for serological detection of EHV-1/4 antibodies in Turkish horses showed good correlation with reference tests and provided valuable data on the seroprevalence of these viruses in the local equine population [38]. Indirect ELISAs have also been successfully applied to detect EHV-4-specific antibodies in donkeys, as demonstrated during an outbreak on an ecological donkey milk farm in Romania, where 15 of 28 symptomatic animals were seropositive [3].

The interpretation of serological results requires careful consideration of the timing of sample collection. A single positive serological result indicates prior exposure or vaccination but does not confirm active infection. Definitive diagnosis of recent infection requires demonstration of a four-fold or greater rise in antibody titers between acute and convalescent serum samples collected 2-4 weeks apart [1, 13]. In the Danish outbreak investigation, pre- and post-infection sera were tested for EHV-4 antibodies, and the observed seroconversion patterns helped confirm the timing of infection and identify the likely index case [1]. It is important to note that maternally derived antibodies in foals can confound serological interpretation for several months after birth, and vaccination histories must be carefully documented to avoid misinterpretation.

Recent research has highlighted the importance of mucosal antibody responses, particularly IgG4/7 isotypes, in protection against EHV-1 and EHV-4 infection at the upper respiratory tract [14, 25, 28]. While these assays are not yet widely available for routine clinical diagnosis, they represent a promising frontier for evaluating vaccine efficacy and individual protective immunity. Studies have shown that intramuscular vaccination with inactivated EHV-1/4 vaccines can induce mucosal IgG4/7 antibodies in previously exposed horses, and these antibodies correlate with reduced viral shedding and protection from clinical disease [25, 28]. The development of standardized assays for measuring mucosal antibody responses could revolutionize our approach to diagnosing immune status and predicting susceptibility to infection.

Genomic Characterization and Phylogenetic Analysis

The application of next-generation sequencing (NGS) and whole-genome sequencing has transformed our understanding of EHV-4 molecular epidemiology and evolution. Complete genome sequences of EHV-4 isolates from diverse geographic regions, including Japan, Australia, Germany, and Denmark, have revealed a genetically stable virus with evidence of widespread natural recombination [12, 13, 19]. Phylogenetic analyses have identified two major subclades of EHV-4, with estimates suggesting diversification occurred approximately 1,000 years ago, coinciding with the spread of modern domestic horses across the Central Asian steppes [4]. This evolutionary history is considerably older than previously thought and underscores the long-standing co-adaptation between EHV-4 and its equine host.

Partial genome sequencing, particularly of the glycoprotein B (gB) gene and the DNA polymerase gene, is frequently employed for phylogenetic characterization and molecular epidemiological studies [2, 24]. In a study of Arabian horses in Egypt, sequencing of the gB gene revealed high similarity between local EHV-4 strains and reference sequences from GenBank, confirming the genetic homogeneity of this virus across geographic regions [24]. Similarly, analysis of EHV-4 isolates from Morocco demonstrated that the circulating strains clustered with global reference sequences, providing no evidence for geographically distinct lineages [2].

The ability to track viral transmission through genomic analysis was elegantly demonstrated during the Danish outbreak investigation. Partial genome sequencing revealed that a horse testing positive nearly three weeks after the last outbreak case was infected by a different wild-type EHV-4 strain, indicating that the hospital had successfully eliminated the original outbreak strain through testing and enhanced biosecurity measures [1]. This finding highlights the power of genomic epidemiology for distinguishing between ongoing transmission and independent introduction events. Furthermore, the complete genome sequence of the outbreak strain showed closer relatedness to Australian and Japanese strains than to other European isolates, suggesting long-distance viral dispersal and underscoring the need for more comprehensive global sequence data [1].

Emerging Diagnostic Technologies and Future Directions

The diagnostic landscape for EHV-4 continues to evolve with the development of novel technologies and approaches. Metagenomic next-generation sequencing (mNGS) offers the potential for unbiased detection of all pathogens present in a clinical sample, including unexpected or novel viruses [50]. While currently too expensive and technically demanding for routine use, mNGS has proven valuable for investigating complex respiratory disease outbreaks where co-infections with multiple pathogens are suspected. Transcriptomic profiling of host gene expression in nasal secretions or peripheral blood mononuclear cells represents another frontier, with studies identifying early innate immune signatures, such as upregulation of interferon-stimulated genes and antileukoproteinase (SLPI), that correlate with protection from EHV-1 infection [27, 47]. These host-response biomarkers could potentially be developed into rapid diagnostic tests that identify infected animals before viral shedding reaches detectable levels.

Point-of-care testing technologies, including isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), hold promise for field-deployable EHV-4 diagnosis. These assays can be performed with minimal equipment, provide results in under an hour, and could facilitate real-time biosecurity decisions during outbreaks. However, rigorous validation studies comparing their performance to established qPCR assays are needed before widespread implementation can be recommended.

The World Organisation for Animal Health (WOAH) provides guidelines for the diagnosis of equine herpesvirus infections, emphasizing the importance of using validated methods and appropriate quality assurance measures. For international movement of horses and participation in equestrian events, many jurisdictions require negative PCR results for EHV-1 and EHV-4 within a specified timeframe prior to travel [32]. The development of standardized, internationally recognized diagnostic protocols is essential for facilitating safe horse movement while minimizing the risk of disease introduction into naive populations. As our understanding of EHV-4 pathogenesis and immune responses deepens, diagnostic methods will continue to advance, providing ever more powerful tools for controlling this ubiquitous and economically significant pathogen.

Outbreak Management and Biosecurity Measures

The management of an Equine Herpesvirus 4 (EHV-4) outbreak presents a formidable challenge to equine practitioners, facility managers, and regulatory authorities, demanding a coordinated, multi-pronged strategy that integrates rapid diagnostics, rigorous biosecurity protocols, strategic vaccination, and comprehensive epidemiological investigation. The global endemicity of EHV-4, coupled with its propensity for latency and reactivation, necessitates a proactive and scientifically grounded approach to curtail viral dissemination and mitigate clinical disease. The following analysis delineates the critical components of outbreak management and biosecurity, drawing upon contemporary outbreak investigations and experimental evidence to provide an authoritative framework for containment.

Early Detection and Diagnostic Confirmation

The cornerstone of effective outbreak management is the rapid and accurate identification of the causative agent. In the context of a suspected EHV-4 outbreak, the deployment of molecular diagnostics, particularly quantitative polymerase chain reaction (qPCR), is non-negotiable. The development and validation of a multiplex real-time PCR assay (EHV1-4MP) that simultaneously detects EHV-1 and EHV-4, while including an endogenous internal control targeting the melanocortin 1 receptor (MC1R) gene, has demonstrated exceptional analytical sensitivity, approximately two copies for EHV-1 and four copies for EHV-4 per reaction [18]. This assay’s ability to differentiate EHV-4 from closely related equid herpesviruses such as EHV-3, EHV-8, and EHV-9 [18] is crucial for accurate diagnosis, as misidentification can lead to inappropriate biocontainment measures.

The utility of qPCR in outbreak settings is powerfully illustrated by the detailed investigation of an EHV-4 outbreak at the Large Animal Teaching Hospital, University of Copenhagen, Denmark [1]. Nasal swabs obtained throughout the seven-week outbreak were tested by qPCR, allowing for the temporal mapping of viral shedding and the identification of the most likely “patient zero” (Eq1) based on location, viral detection, and antibody responses [1]. This study further demonstrated the value of partial and full genome sequencing. Sequencing revealed that a tenth horse (Eq10), which tested positive nearly three weeks after the last outbreak horse, was infected by a different wild-type EHV-4 strain, proving that the hospital’s biosecurity measures and testing protocols had successfully eliminated the initial outbreak strain [1]. This case underscores a critical principle: sequencing should not be viewed as a retrospective research tool alone but as an active epidemiological instrument capable of confirming the cessation of transmission and detecting the introduction of new viral strains.

Furthermore, the clinical presentation must guide sampling strategy. Nasal secretions are the primary sample type for detecting active respiratory shedding, as viremia is not a consistent feature of EHV-4 infection, unlike EHV-1. Serological analysis of paired acute and convalescent sera can confirm recent infection, particularly in cases where viral load has waned by the time of sampling [1, 38]. In the German outbreak on a Standardbred horse-breeding farm, a combination of virus isolation, qPCR, and serological follow-up using virus neutralization tests and peptide-specific ELISAs was employed to confirm EHV-4 involvement in all 41 affected horses [13]. This multi-modal diagnostic approach provides the highest level of confidence in outbreak confirmation.

Immediate Quarantine and Movement Controls

Upon suspicion or confirmation of EHV-4, the immediate implementation of stringent movement controls is the single most critical biosecurity measure. The 2024 ACVIM consensus statement on EHV-1, which shares considerable epidemiological relevance with EHV-4 given their related transmission dynamics, provides a framework that can be adapted for EHV-4 [11]. Following European Food Safety Authority (EFSA) guidance for EHV-1, no movements of horses should be allowed from an affected facility until at least 21 days after the detection of the last clinical case [16]. This period accounts for the typical duration of viral shedding and the incubation period.

The rapid dissemination of EHV-4 in congregate settings was tragically demonstrated in a fulminant outbreak of equine herpesvirus myeloencephalopathy (EHM) in a population of aged working equids. Although this outbreak was caused by EHV-1, the biosecurity failures are directly instructive for EHV-4 management. The outbreak was predisposed by the grouping of 33 unvaccinated animals in two large pens with shared water and feed troughs. Within the first week, 42.4% of the animals developed neurological deficits, and the attack rate was exceptionally high [41]. The shared resources, water troughs, feed bins, and communal pens, served as efficient fomites, allowing the virus to propagate rapidly through a naïve population [41]. For EHV-4, which primarily causes respiratory disease, the risk of aerosol and fomite transmission is similarly high, especially in enclosed barns with poor ventilation.

Quarantine zones must be clearly demarcated. Infected horses should be isolated in a dedicated isolation unit, ideally with separate air handling, dedicated equipment (stainless steel buckets, halters, lead ropes), and dedicated personnel who do not interact with susceptible horses. Strict protocols for entering and exiting the isolation area must be enforced, including the use of disposable gloves, boots, and coveralls. The Danish outbreak study highlighted the importance of reinforcing biosecurity measures, including the segregation of horses based on qPCR status and the meticulous cleaning of stalls and equipment, which ultimately allowed the hospital to clear the outbreak [1]. The strategy of quarantine and EHV-1 qPCR testing of clinically healthy horses following multi-county EHM outbreaks in California was also successful at eliminating additional outbreaks and facilitating a safe return to competition, with no reported respiratory disease outbreaks at subsequent shows [32]. This demonstrates that active surveillance of healthy in-contact animals is a vital component of outbreak containment.

Zonal Biosecurity and Environmental Decontamination

Establishing a zonal biosecurity system is essential for preventing the spread of EHV-4 within and beyond an affected facility. The facility should be divided into clearly defined “clean” and “dirty” zones. The infected zone (dirty) encompasses the isolation area and any paths used to access it. A transition zone (buffer) should be established where personnel change footwear and clothing. The clean zone houses all susceptible, unexposed horses. All movement of horses, feed, bedding, and equipment must be unidirectional from clean to dirty zones, with no reverse movement permitted.

EHV-4, like all alphaherpesviruses, is enveloped and is therefore susceptible to most common disinfectants, including accelerated hydrogen peroxide, potassium peroxymonosulfate, and 10% bleach solutions. The virus can survive on fomites for extended periods under favorable environmental conditions. Environmental sampling at a multi-week equestrian show during winter months detected EHV-4 in 1.37% of pooled stall sponges, alongside higher frequencies of S. zooepidemicus (28.69%) and EHV-2 (14.45%) [20]. Although the frequency of EHV-4 detection was low compared to other pathogens, its presence in the environment confirms that contaminated stalls can serve as a reservoir for infection, particularly during colder months when viral survival is prolonged [20]. Therefore, after removal of infected horses, stalls must be thoroughly cleaned of organic material (manure, bedding, feed) before disinfection. Cationic detergents and formalin-based disinfectants are also effective. All equipment, grooming kits, tack, feed tubs, water buckets, should be dedicated to the isolation unit and disinfected or discarded after the outbreak.

Vaccination as a Prophylactic and Outbreak Intervention Tool

Vaccination remains a cornerstone of EHV-4 prevention, though its role in the acute management of an ongoing outbreak is nuanced. The updated vaccination program implemented in a training facility for Thoroughbred yearlings in Japan provides compelling evidence for the efficacy of prophylactic vaccination in reducing infection rates. By revising the schedule to administer a live EHV-1 vaccine (Equi N Tect ERP) as early as possible after arrival, followed by a second dose two months later, the investigators observed a dramatic reduction in the infection rate for EHV-1/4. In the pre-intervention period (2018–2021), infection rates peaked between August and September at 6.8‰–10.9‰; under the new program (2021–2023), rates fell to 2.6‰–3.8‰ in the first year and 1.2‰–1.7‰ in the second year (P < 0.05) [7]. This study underscores the principle that vaccination timing and coverage are critical: closing the “immunity gap” for early-arriving yearlings prevented the establishment of a viral reservoir within the training population.

Research into the immunological mechanisms of protection has identified mucosal antibodies, particularly IgG4/7, as key correlates of protection at the site of viral entry. Intramuscular vaccination with a commercial inactivated EHV-1/4 vaccine in previously exposed horses significantly increased EHV-1-specific mucosal antibodies in the upper respiratory tract, dominated by the IgG4/7 isotype [25]. Furthermore, pre-existing mucosal IgG1 and IgG4/7 antibodies have been shown to neutralize EHV-1 and prevent viral replication at the epithelium of the upper respiratory tract, thereby blocking the progression of infection [14, 28]. These findings have profound implications for outbreak management: booster vaccination of at-risk horses in the face of an outbreak may not provide immediate protection (due to the lag time required for antibody production), but it can raise herd immunity and reduce the severity and duration of shedding in recently exposed animals [7, 25].

However, vaccination is not a panacea. A systematic review and meta-analysis on EHV-1 vaccination concluded that evidence for successful vaccination against the neurological form is limited [52], and existing vaccines do not provide sterilizing immunity. The 2024 ACVIM consensus statement similarly noted that improvements in experimental design and reporting are needed in future vaccine studies [11]. For EHV-4, which is predominantly a respiratory pathogen, the goal of vaccination is to reduce clinical disease severity and viral shedding, not necessarily to prevent infection entirely. During an outbreak, emergency vaccination of unaffected cohorts may be considered, but it must be combined with strict quarantine and testing to be effective.

Stress Reduction and Management of Latency

A critical but often overlooked aspect of EHV-4 outbreak management is the role of stress in triggering viral reactivation from latency. The German outbreak on a Standardbred horse-breeding farm explicitly identified “stress caused by seasonal changes, management practices, routine equestrian activities, and exercises” as multifactorial contributors to the disease outbreak [13]. The investigators concluded that the different restriction fragment length polymorphism profiles and genome sequences of the isolated viruses suggested the involvement of more than one animal as a source of infection, likely due to reactivation from a latent state [13]. This observation is supported by the global prevalence of EHV-4, with seroprevalence rates of 60-80% in the horse population [4], indicating that the vast majority of horses are latently infected.

Therefore, outbreak management must include an assessment of stressors: recent transport, weaning, overcrowding, poor ventilation, concurrent disease, or intense training schedules. Mitigation strategies should include providing optimal nutrition, ensuring adequate ventilation in barns, reducing stocking density, and temporarily suspending rigorous exercise or competition schedules for affected and at-risk horses. Management practices that minimize immunosuppression are not merely supportive care; they are a direct intervention against the primary mechanism of viral re-emergence [13].

Communication and Epidemiological Tracing

Finally, effective outbreak management demands transparent and rapid communication among veterinarians, facility managers, horse owners, and regulatory authorities. The World Organisation for Animal Health (WOAH) provides guidelines for the control of equine diseases, and while EHV-4 is not a WOAH-listed disease (unlike EHV-1 in some contexts), reporting to national animal health authorities is advisable for epidemiological surveillance. The utility of molecular epidemiology in tracing transmission pathways cannot be overstated. In the Danish study, genome sequencing not only distinguished the outbreak strain from a subsequent introduction but also revealed that the outbreak strain was more closely related to Australian and Japanese strains than to other European strains [1]. This finding highlights that international movement of horses can introduce novel strains to naïve populations, and genomic surveillance can track these introductions.

During an outbreak, a centralized log should be maintained documenting the location, clinical status, qPCR results, and movement history of every horse on the premises. This log facilitates the identification of high-risk contacts and the tracing of potential spread to other facilities. The Spanish EHV-1 outbreak in Valencia (2021), which spread to nine other European countries and Qatar, was tracked using a specific single nucleotide polymorphism (A713G) in ORF11, allowing for the confirmation of epidemiological links [53]. A similar approach should be applied to EHV-4 outbreaks, leveraging the genetic diversity observed among field isolates [12, 17] to support targeted control measures and limit further dissemination.

Viral Genomics and Strain Diversity of EHV-4

Equid herpesvirus 4 (EHV-4) is a member of the Alphaherpesvirinae subfamily, genus Varicellovirus, and is genetically distinct from its close relative, equine herpesvirus 1 (EHV-1), despite sharing a common ancestor and a high degree of genomic synteny. The EHV-4 genome is a linear double-stranded DNA molecule of approximately 144–150 kilobase pairs (kbp), organized into a unique long (UL) region and a unique short (US) region, the latter flanked by internal and terminal repeat sequences. This genomic architecture is characteristic of the alphaherpesviruses and underpins the virus's capacity for latency, reactivation, and recombination. The complete genome sequence of the EHV-4 reference strain NS80567, along with a growing number of field isolates from diverse geographic regions, has provided a foundational framework for understanding the molecular epidemiology, evolutionary dynamics, and strain-specific virulence determinants of this globally endemic pathogen [1, 13, 19].

Genomic Architecture and Evolutionary Timescale

The evolutionary history of EHV-4 has been dramatically revised by the recovery of an ancient viral genome from archaeological horse remains. Bayesian phylogenetic analysis of a 4.2X coverage EHV-4 genome obtained from a ~3,900-year-old specimen excavated in the Southeastern Urals provided a minimal time estimate for EHV-4 diversification to approximately 4,000 years before present [4]. This temporal calibration places the emergence of EHV-4 in the context of the Sintashta culture and the spread of spoke-wheeled chariotry across the Central Asian steppes, a period of intense horse domestication and long-distance movement. Critically, this ancient genome analysis revised the previously estimated diversification time of the two major EHV-4 subclades from the 16th century, based solely on modern sequence data, to nearly a thousand years ago, demonstrating that the virus has co-evolved with its equine host for millennia [4]. This deep evolutionary history is reflected in the genomic stability of EHV-4, yet also in the subtle but significant genetic variation that has accumulated across geographically distinct lineages.

Evidence for Widespread Natural Recombination

One of the most striking features of EHV-4 genomics is the pervasive role of natural recombination in shaping its population structure. High-throughput sequencing of complete genomes from Australian and New Zealand isolates revealed widespread recombination events across the EHV-4 genome, in stark contrast to the near-absence of such events among EHV-1 isolates analyzed in the same study [12]. This fundamental difference in recombination frequency between the two equine alphaherpesviruses has profound implications for their respective evolutionary trajectories. For EHV-4, recombination serves as a powerful mechanism for generating genetic diversity, potentially allowing the virus to escape immune pressure, alter tissue tropism, or modulate virulence. The recombination events detected were not confined to a single genomic region but were distributed across the UL and US segments, suggesting that co-infection of a single host with multiple EHV-4 strains is a common occurrence in nature [12]. This finding is corroborated by outbreak investigations where multiple distinct EHV-4 strains have been identified within a single facility, as observed in a Danish hospital outbreak where a second, unrelated wild-type strain (Eq10) was detected after the index outbreak strain had been contained [1]. The capacity for recombination also complicates phylogenetic analyses and vaccine design, as circulating strains may represent mosaics of ancestral lineages rather than simple clonal lineages.

Restriction Fragment Length Polymorphism and Genomic Diversity

Before the advent of widespread whole-genome sequencing, restriction fragment length polymorphism (RFLP) analysis provided the first insights into EHV-4 genomic diversity. Early work on 23 Japanese EHV-4 field isolates demonstrated that restriction endonuclease digestion patterns (using BamHI, BglII, EcoRI, SacI, and SalI) exhibited distinct differences, including mobility shifts of fragments and loss or gain of restriction sites [17]. This RFLP-based diversity was particularly evident in genes containing repeat sequences, such as ORF24 and ORF71, where the size of amplified fragments varied among epizootiologically unrelated isolates but remained conserved among isolates from the same outbreak [17]. These repeat regions, likely prone to slipped-strand mispairing during replication, serve as hypervariable markers that can be exploited for molecular epidemiology. Subsequent full-genome sequencing of seven Japanese EHV-4 isolates, including two from aborted fetuses and five from horses with respiratory disease, confirmed that the isolates cluster into two major phylogenetic groups, but critically, this clustering did not correlate with pathogenicity (i.e., abortion versus respiratory disease) [19]. Comparative analysis of predicted amino acid sequences across all open reading frames failed to identify any single gene or mutation consistently associated with the abortigenic phenotype, suggesting that EHV-4-induced abortion may result from host-specific factors, viral load, or synergistic interactions between multiple viral genes rather than a discrete "abortigenic marker" [19].

Contemporary Outbreak Strains and Global Phylogenetic Relationships

The application of next-generation sequencing to contemporary EHV-4 outbreaks has revealed a complex global phylogeography. The complete genome sequence of an EHV-4 strain responsible for a major respiratory outbreak at a Standardbred breeding farm in northern Germany in 2017 was determined by de novo assembly, yielding a ~144 kbp genome [13]. Despite the involvement of multiple animals as sources of infection, as evidenced by different RFLP profiles among four isolated viruses, the overall genome was noted to be genetically stable, with the observed diversity attributed to reactivation of latent virus from different individuals rather than rapid de novo mutation [13]. Similarly, the full genome of the outbreak strain from the 2022 Danish hospital outbreak was sequenced and found to be more closely related to Australian and Japanese EHV-4 strains than to other European strains, a finding that underscores the limited sequence data available from Europe and the potential for long-distance viral transport via the international movement of horses [1]. This observation aligns with the broader understanding that EHV-4 is endemic globally, with seroprevalence rates of 60–80% in many populations, and that strain circulation is not strictly geographically confined [4, 15]. Molecular characterization of EHV-4 strains from Egypt, based on partial glycoprotein B (gB) gene sequencing, showed high similarity to both local and foreign reference strains, confirming the global homogeneity of this gene while also highlighting the utility of gB sequencing for species-level identification [24].

Strain Diversity in Non-Equine Hosts and Diagnostic Implications

While EHV-4 is primarily a pathogen of horses, its host range extends to other equids, including donkeys and mules. An outbreak of EHV-4-associated respiratory disease in an ecological donkey milk farm in Romania, confirmed by RT-PCR, demonstrated that donkeys are susceptible to clinical infection with EHV-4, presenting with severe upper respiratory tract disease, late abortions, and neurological signs [3]. Serological surveys in large-scale donkey farms in China have further confirmed the circulation of EHV-4 in asinine populations, with 5.22% of donkeys testing positive for EHV-4 antibodies alone and 4.78% positive for both EHV-1 and EHV-4 [15]. The genetic characterization of EHV-4 strains from donkeys is still in its infancy, but these findings emphasize the need for species-specific surveillance. Importantly, the development of highly specific multiplex real-time PCR assays, such as the EHV1-4MP assay, has improved diagnostic accuracy by enabling the simultaneous detection of EHV-1 and EHV-4 while excluding closely related viruses like EHV-3, EHV-8, and EHV-9 [18]. This molecular precision is essential for distinguishing EHV-4 from other equid herpesviruses that may cause similar clinical signs, particularly in regions where multiple EHV species co-circulate [2, 5, 40].

Implications for Vaccine Development and Antiviral Strategies

The genomic diversity of EHV-4, particularly the evidence for recombination and the lack of a single virulence determinant, poses challenges for vaccine development. Current commercial vaccines are often bivalent, targeting both EHV-1 and EHV-4, and rely on inactivated whole-virus preparations that may not reflect the antigenic diversity of circulating field strains [7, 25]. The observation that EHV-4 strains from different geographic regions can be more closely related to each other than to local strains suggests that a globally effective vaccine may be feasible, but the potential for recombination to generate novel antigenic variants remains a concern [1, 12]. Furthermore, the identification of decitabine as a potent inhibitor of EHV-4 replication in vitro, acting through the upregulation of interferon-stimulated genes, opens new avenues for antiviral therapy that are informed by viral genomics [6]. Understanding the genetic basis of drug susceptibility or resistance will be critical as these compounds move toward clinical application. The World Organisation for Animal Health (WOAH) recognizes EHV-1 and EHV-4 as significant pathogens of equids, and genomic surveillance is increasingly recognized as a tool for monitoring the emergence of strains with altered pathogenicity or vaccine escape potential.

Immune Response and Vaccination Strategies for EHV-4

The immune response to equine herpesvirus type 4 (EHV-4) is a complex, multi-layered process that begins at the mucosal surface of the upper respiratory tract (URT) and involves both innate and adaptive arms of the equine immune system. Understanding this response is critical not only for elucidating the pathogenesis of EHV-4-induced respiratory disease but also for the rational design of effective vaccination strategies. While EHV-4 is often considered less virulent than its close relative, equine herpesvirus type 1 (EHV-1), it remains a significant cause of endemic respiratory disease in the global equine population, with seroprevalence rates estimated between 60-80% [4]. The virus is enzootic in most countries, and outbreaks are frequently precipitated by stress, management practices, and the gathering of horses at events [1, 13]. The immune mechanisms that control EHV-4 infection share many features with those described for EHV-1, but distinct epidemiological and genetic characteristics of EHV-4, including its propensity for widespread natural recombination [12], necessitate a focused examination of its immunobiology and the specific challenges it presents for vaccine development.

Innate Immune Recognition and the Mucosal Barrier

The initial encounter between EHV-4 and the host occurs at the respiratory epithelium. The innate immune system provides the first line of defense, and its efficacy is a major determinant of whether infection will be contained or will progress to more severe disease. The equine respiratory tract has evolved multiple antiviral barriers, which can be compromised by environmental factors such as pollen, mycotoxins, and bacterial toxins, thereby increasing susceptibility to alphaherpesvirus infection [29]. Upon viral entry, pattern recognition receptors (PRRs) on epithelial cells and resident immune cells recognize pathogen-associated molecular patterns (PAMPs), triggering a signaling cascade that leads to the production of type I interferons (IFN-α/β) and a suite of pro-inflammatory cytokines and chemokines.

In non-immune horses, a robust type I interferon response is a hallmark of acute EHV-1 infection, and by extension, is critical for EHV-4. Transcriptomic profiling of nasopharyngeal samples from EHV-1-infected horses has demonstrated a significant upregulation of interferon-induced proteins with tetratricopeptide repeats (IFIT2 and IFIT3) and the secretion of nasal IFN-α, which correlates with active viral replication [27]. This response is essential for establishing an antiviral state in neighboring cells and activating natural killer (NK) cells. However, EHV-4, like other alphaherpesviruses, has evolved sophisticated immune evasion strategies to subvert this response. The virus can modulate the expression of interferons, cytokines, and chemokines at the site of infection, thereby dampening the host's ability to control early replication [22, 54]. The recruitment of CD172a+ monocytic cells to the site of infection, driven by chemokines such as CCL2 and CCL5, is a critical step in the pathogenesis of EHV-1, as these cells can become infected and facilitate viral dissemination from the epithelium to the lamina propria and ultimately to the bloodstream [36]. It is highly plausible that similar mechanisms are at play during EHV-4 infection, given the shared pathogenic pathway of these two alphaherpesviruses.

A key finding from recent research is the role of antileukoproteinase (SLPI) in the innate immune defense of immune horses. In horses with pre-existing immunity, an EHV-1 challenge did not induce a type I IFN response or IFIT expression. Instead, a rapid increase in SLPI gene expression and protein secretion was observed within 24 hours [27]. SLPI is a potent serine protease inhibitor with known anti-inflammatory and antimicrobial properties. Its rapid induction in immune horses suggests that it may be part of a highly efficient, non-inflammatory innate mechanism that prevents viral entry or replication at the mucosal surface, thereby obviating the need for a more disruptive inflammatory cascade. This finding highlights a fundamental difference in the innate immune response between naive and immune animals and points to novel targets for vaccine-induced immunity.

The Adaptive Humoral Immune Response: The Central Role of Mucosal IgG4/7

The adaptive immune response, particularly the humoral arm, is paramount for long-term protection against EHV-4. While systemic antibodies are important, the most critical effectors are those present at the portal of entry, the mucosa of the URT. Pre-existing mucosal immunity, especially the presence of EHV-specific IgG4/7 antibodies, has been shown to correlate strongly with protection from clinical disease, reduced viral shedding, and the prevention of cell-associated viremia [14, 25, 28]. This is a cornerstone concept for understanding immunity to equine alphaherpesviruses.

The equine immunoglobulin G (IgG) system is complex, with seven distinct sub-isotypes (IgG1-IgG7). Among these, IgG4/7 and IgG1 have been identified as the primary neutralizing antibodies against EHV-1. Mechanistic studies have demonstrated that purified mucosal IgG1 and IgG4/7 can effectively neutralize EHV-1, while other isotypes such as IgG3/5, IgG6, and IgA do not possess this neutralizing capability [28]. In immune horses, a rapid anamnestic B-cell response occurs upon re-exposure, leading to a swift increase in intranasal IgG4/7 antibodies. This rapid response effectively neutralizes the incoming virus, preventing it from establishing a productive infection in the epithelium. Consequently, viral replication is incomplete, viral copy numbers remain low or undetectable, and infectious virus is not shed [14, 28]. This state of "sterilizing immunity" at the mucosal surface is the gold standard for protection.

In contrast, non-immune horses lack these pre-existing neutralizing mucosal antibodies. Upon primary infection, they experience a period of high viral replication and shedding before a de novo adaptive response can be mounted. This response is characterized by a delayed and less robust production of mucosal antibodies, allowing the virus to replicate to high titers, cause clinical signs (pyrexia, nasal discharge, lymphadenopathy), and establish cell-associated viremia [1, 28]. The induction of serum IgG1 is an early marker of infection, but it is the later, sustained production of IgG4/7 that correlates with eventual recovery and long-term immunity [23]. The importance of the IgG4/7 isotype is further underscored by studies showing that horses fully protected from EHV-1 challenge had high pre-existing levels of these antibodies, while partially protected or susceptible horses had lower levels [14].

Cellular Immunity and the Role of T-Cells

While humoral immunity is critical for preventing infection at the mucosal surface, cellular immunity, mediated by T-cells, is essential for controlling and clearing infection once it has been established, particularly in the context of cell-associated viremia. The cell-mediated immune (CMI) response is directed against virus-infected cells and is crucial for eliminating the virus from the host and for long-term control of latency.

Following EHV-1 infection, there is an increase in IFN-γ secretion by peripheral blood mononuclear cells (PBMCs) upon ex vivo re-stimulation with the virus, coinciding with the period of viremia [23]. IFN-γ is a key cytokine produced by CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ Th1 cells, and it plays a vital role in activating macrophages and enhancing the antiviral state. Studies using a mouse model have shown that intranasal treatment with CpG-B oligodeoxynucleotides, which are potent inducers of Th1-type immunity, protected mice from a lethal EHV-1 challenge. This protection was associated with a massive upregulation of IFN-γ and interferon-stimulated genes (ISGs) in the lungs, suggesting that IFN-γ is a major factor in the protective immune response [35].

The development of EHM in EHV-1 infection is associated with a dysregulated immune response. Horses that develop EHM show a delayed or weak type I IFN response, an upregulation of regulatory cytokines like IL-10, and a skewing of the immune response towards a Th2-type or regulatory phenotype [9, 47]. Transcriptomic analysis of PBMCs from EHM-affected horses revealed an upregulation of IL-6, dysregulation of T-cell activation, and a Th2-skewed response, contrasting with the robust cellular and Th1-type immunity seen in horses that only develop mild respiratory disease [47]. This suggests that a strong, early, and balanced CMI response, characterized by IFN-γ production, is critical for preventing severe outcomes. For EHV-4, which rarely causes neurological disease, the immune response is generally more effective at containing the virus to the respiratory tract. However, the fundamental principles of CMI, the need for IFN-γ-producing T-cells to control viremia and limit viral spread, are equally applicable.

Vaccination Strategies: Current Approaches and Future Directions

Vaccination against EHV-4 is a cornerstone of respiratory disease control, but the development of a universally protective vaccine has been challenging due to the virus's ability to establish latency, its immune evasion mechanisms, and the complex nature of protective immunity. Current vaccination strategies can be broadly categorized into inactivated (killed) vaccines, modified-live virus (MLV) vaccines, and novel experimental approaches.

Inactivated Vaccines: Commercially available inactivated bivalent EHV-1/4 vaccines are widely used. These vaccines are considered safe, as they cannot revert to virulence. Their primary mechanism of action is the induction of systemic humoral immunity. A landmark study by Wagner et al. (2025) demonstrated that intramuscular (i.m.) vaccination with an inactivated EHV-1/4 vaccine could significantly boost mucosal antibodies (mucAbs) in the URT of previously exposed horses [25]. The study showed that after i.m. vaccination, serum IgG4/7 levels increased significantly and remained high. Critically, mucAbs in the nasal passages, dominated by the IgG4/7 isotype, also increased after most vaccine injections. This finding is of paramount importance, as it provides evidence that systemic i.m. vaccination can effectively boost the key antibody isotype at the site of viral entry, challenging the long-held assumption that mucosal immunity can only be achieved through intranasal vaccination. The study also noted a transient increase in serum IgG1 after the first vaccination, which is typical of a primary response to an inactivated antigen [25]. Other studies have explored optimizing the inactivation process, for example, using a combination of formaldehyde and binary ethylenimine (BEI) to create a more potent inactivated vaccine that provides protective antibody titers for up to six months [48].

Modified-Live Virus (MLV) Vaccines: MLV vaccines are designed to induce a more comprehensive immune response, including both humoral and cellular arms, by mimicking a natural infection without causing severe disease. A live EHV-1 vaccine (Equi N Tect ERP) has been used in Japan to successfully reduce the incidence of EHV-1 and EHV-4 infections in Thoroughbred yearlings. By implementing an earlier vaccination schedule, researchers observed a significant reduction in the rate of pyretic horses and overall infection rates, demonstrating the field efficacy of an MLV strategy when applied with optimized timing [7].

A highly promising area of research involves the genetic manipulation of EHV-1 to create safer and more immunogenic vaccine candidates. The deletion of specific virulence genes, such as ORF2, from the neuropathogenic EHV-1 strain Ab4 has yielded a mutant virus (Ab4ΔORF2) that is attenuated yet retains strong immunogenicity. Vaccination with Ab4ΔORF2 resulted in reduced fever and nasal virus shedding compared to the parent virus, while inducing a similar adaptive immune response, including high levels of EHV-1-specific IgG4/7 antibodies [23]. In a subsequent challenge study, horses previously infected with Ab4ΔORF2 were fully or partially protected from challenge with the virulent parent virus, with protection correlating with pre-existing mucosal IgG4/7 antibodies and a rapid anamnestic response [14]. This approach of deleting immune evasion or virulence genes represents a rational strategy for developing next-generation EHV-1/4 vaccines.

Novel and Adjunctive Strategies: Several innovative approaches are being explored to enhance protection against equine herpesviruses. One strategy involves using a live-attenuated equine influenza vaccine (Flu Avert® I.N.) to stimulate innate immunity in the respiratory tract. In vitro studies on primary equine respiratory epithelial cells (ERECs) showed that treatment with Flu Avert modulated chemokine expression (IL-8, CCL2, CXCL9) and reduced EHV-1 replication, suggesting that this intranasal vaccine could provide non-specific, short-term protection against EHV-1 during periods of high risk [54]. Another approach is concurrent vaccination, where an EIV vaccine and an inactivated EHV vaccine are administered together. This strategy was shown to significantly enhance EIV-specific IFN-γ production without compromising humoral responses, indicating a potential benefit for boosting cellular immunity [56].

The use of antiviral compounds as a prophylactic or therapeutic adjunct is also under investigation. A screening of 42 antiviral compounds against EHV-4 in vitro identified decitabine as a highly effective inhibitor. Importantly, transcriptomic analysis revealed that decitabine's mechanism of action involves the upregulation of genes implicated in the interferon response, suggesting it enhances the innate antiviral pathway rather than directly targeting a viral enzyme [6]. This immunomodulatory approach could be a valuable tool in managing outbreaks.

Challenges and Future Perspectives

Despite the availability of vaccines, EHV-4 remains endemic, and outbreaks continue to occur. Several factors contribute to this challenge. First, the virus's ability to establish lifelong latency and reactivate under stress means that vaccinated horses can still become shedders and serve as a source of infection [13, 16]. Second, the genetic diversity of EHV-4, driven by widespread natural recombination [12], may lead to antigenic variation that could reduce the efficacy of vaccines derived from older or geographically distinct strains. Third, while current vaccines can reduce clinical signs and viral shedding, they often do not provide complete "sterilizing immunity" at the mucosal surface, allowing for subclinical infection and transmission [32].

The World Organisation for Animal Health (WOAH) recognizes the significant economic impact of equine herpesviruses and emphasizes the importance of biosecurity, surveillance, and vaccination for disease control. Future vaccine development must focus on inducing durable, high-titer mucosal IgG4/7 antibodies and robust T-cell memory, particularly IFN-γ-producing cells. The identification of specific correlates of protection, such as the ratio of mucosal IgG4/7 to other isotypes or the frequency of EHV-4-specific CTLs, will be crucial for evaluating new vaccine candidates. Furthermore, a deeper understanding of the host genetic factors that influence susceptibility to severe disease, as explored in genome-wide association studies for EHM [55], could pave the way for personalized vaccination strategies. Ultimately, an integrated approach combining optimized vaccination protocols, rigorous biosecurity, stress reduction, and potentially immunomodulatory therapies will be required to achieve better control of EHV-4 in the global equine population.

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