Porcine Hemagglutinating Encephalomyelitis Virus

Overview and Taxonomy of Porcine Hemagglutinating Encephalomyelitis Virus

Taxonomic Classification and Nomenclature

Porcine hemagglutinating encephalomyelitis virus (PHEV) is classified within the family Coronaviridae, subfamily Orthocoronavirinae, genus Betacoronavirus, and subgenus Embecovirus [1, 6, 27]. This taxonomic placement aligns PHEV with other medically and veterinary significant betacoronaviruses, including bovine coronavirus (BCoV), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1, and murine hepatitis virus (MHV) [6, 20, 27]. The subgenus Embecovirus is distinguished by the presence of a hemagglutinin-esterase (HE) gene, a characteristic feature that PHEV shares with its closest relatives [6, 27]. Historically, the virus has been referred to by several names, including hemagglutinating encephalomyelitis virus (HEV) and porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV), reflecting its ability to agglutinate erythrocytes and its primary tropism for neural tissue [6, 19]. The International Committee on Taxonomy of Viruses (ICTV) recognizes PHEV as the sole member of the species Porcine hemagglutinating encephalomyelitis virus within the subgenus Embecovirus [6, 27].

Phylogenetic analyses have consistently demonstrated that PHEV is most closely related to BCoV and HCoV-OC43, with nucleotide sequence homologies in the spike (S) glycoprotein gene ranging from 91.9% to 92.6% between PHEV and these two viruses [20]. This close genetic relationship suggests a common evolutionary ancestor, with divergence likely occurring through host adaptation and recombination events over centuries [5, 20]. Indeed, Bayesian coalescent analyses estimate that the divergence times among lineages within the PHEV complex date back to between 1886 and 1958, with a mean estimate of approximately 1928 [5]. These findings underscore the long-standing evolutionary history of PHEV within swine populations and its phylogenetic position within the broader betacoronavirus radiation.

Historical Discovery and Initial Characterization

The first clinical outbreaks of what is now recognized as PHEV infection were reported in Ontario, Canada, in the late 1950s [6, 19]. Initially described as two distinct syndromes, vomiting and wasting disease (VWD) and encephalomyelitis, the common viral etiology was not established until 1969, when the virus was isolated and characterized [6, 19]. The prototype strain, designated 67N, was isolated from the brain of a piglet exhibiting neurological signs and has since served as the reference strain for many comparative studies [16, 18, 24]. Shortly after its discovery in Canada, PHEV was identified in the United States, with epizootics of encephalomyelitis in suckling pigs reported in Minnesota in the 1970s [21]. These outbreaks were characterized by sudden onset of tremors, inappetence, weakness, ataxia, and hyperesthesia, with high morbidity and case fatality rates [21]. Histopathological examination revealed marked nonsuppurative, nondemyelinating encephalomyelitis with perivascular mononuclear cuffing, gliosis, neuronal death, and satellitosis [21].

Subsequent to its initial identification in North America, PHEV was detected across Europe, Asia, and other regions. The virus was first reported in Belgium in the 1980s, where the VW572 strain was isolated and extensively characterized for its cultural properties in porcine cell lines [22]. In Asia, PHEV was identified in South Korea in the late 2000s, with detection rates of 14.3% in suckling pigs from conventional farms [17]. The first detailed genomic characterization of a Chinese PHEV strain (PHEV/2008) was published in 2018, revealing a genome of 30,684 base pairs with a G+C content of 37.27% [12]. More recently, PHEV has been detected in Russia, with the first sequencing of circulating strains in the European part of the country reported in 2025 [3]. The Czech Republic also documented its first PHEV detection in 2019, with strains showing 95.8–98.1% amino acid sequence similarity to reference strains and closest evolutionary relationship to the Belgian VW572 strain [14]. These cumulative reports confirm that PHEV is globally distributed, though its prevalence is likely underestimated due to sporadic reporting and lack of active surveillance [1, 6].

Genome Organization and Structural Features

The PHEV genome is a single-stranded, positive-sense RNA molecule approximately 30,000 to 31,000 nucleotides in length, making it one of the larger coronaviruses [6, 12, 16]. The complete genome of the Korean strain GNU-2113 was determined to be 29,982 nucleotides, while the Chinese strain PHEV/2008 was 30,684 nucleotides [7, 12]. The genome is organized in a canonical coronavirus architecture, with a 5′ untranslated region (UTR) of approximately 211 nucleotides and a 3′ UTR of approximately 289 nucleotides [12]. At a minimum, 11 predicted open reading frames (ORFs) are present, encoding the replicase polyproteins pp1a and pp1ab, which are cleaved into 16 nonstructural proteins (nsps) by viral proteinases [6, 12]. The structural proteins include the spike (S) glycoprotein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein [6, 12]. Additionally, PHEV possesses the hemagglutinin-esterase (HE) gene, a hallmark of embecoviruses, which encodes a protein with receptor-destroying enzyme activity that facilitates viral entry and release [5, 6, 23].

The S glycoprotein is the primary determinant of viral tropism and is responsible for receptor binding and membrane fusion [6, 15, 25]. The S protein is cleaved into S1 and S2 subunits, with the S1 subunit containing the receptor-binding domain (RBD) that interacts with host cell receptors [15, 25]. The HE protein, which shares structural and functional homology with the influenza C virus hemagglutinin-esterase, recognizes N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant on erythrocytes and target cells [23]. The M and N genes are relatively more conserved than the S gene, with nucleotide homologies of 95.0–99.7% and 94.0–99.5% among global strains, respectively, compared to 94.3–99.3% for the S gene [4]. This higher variability in the S gene is consistent with its role in immune evasion and host adaptation.

Genetic Diversity and Phylogenetic Lineages

Phylogenetic analyses based on the S, M, and N genes have consistently divided PHEV strains into two major groups, designated G1 and G2 [4, 5]. Within these groups, further subdivision into subgroups (e.g., G1c, G2b) has been identified, reflecting the genetic diversity of circulating strains [4]. The G1 group includes many historical North American and European strains, while G2 encompasses more recently identified Asian and respiratory-associated variants [4, 5]. Importantly, the emergence of novel respiratory variant PHEV (rvPHEV) lineages has been documented in China, where these viruses cause exclusively respiratory symptoms without the neurological signs typical of classical PHEV infection [5]. These rvPHEV lineages have circulated in at least eight provinces in southeastern China and show extensive recombination within and between lineages, as well as between classical PHEV and rvPHEV [5]. Divergence times among the sampled lineages in the PHEV complex date back to 1886–1958, with the two major rvPHEV lineages separating approximately 20 years later [5].

Many rvPHEV viruses exhibit amino acid substitutions at the carbohydrate-binding site of the HE protein and/or have lost the cysteine residue required for HE dimerization [5]. This pattern resembles the early adaptation of human coronaviruses, where HE lost its hemagglutination ability to adapt to growth in the human respiratory tract [5]. The genetic differentiation between respiratory and neurologic PHEV strains has been corroborated by phylogenetic analyses of strains from respiratory cases in the United States, which cluster closely with historical respiratory strains but are distinct from neurologic strains [1]. These findings suggest that phenotypic variation in tissue tropism is encoded at the genetic level, with implications for pathogenesis and disease manifestation.

Host Range and Species Susceptibility

Pigs are the only natural host for PHEV, and the virus is considered ubiquitous in swine populations worldwide [6, 27]. However, PHEV also displays neurotropism in experimental animal models, including mice and Wistar rats [6, 12, 25]. The ability of PHEV to infect wild-type mice raises concerns about potential host jumping or spillover events, as the virus can cross species barriers under experimental conditions [25]. The receptor usage of PHEV is a critical determinant of its host range. Historically, sialic acid (specifically Neu5,9Ac2) was considered the primary attachment factor for PHEV, as it is for BCoV and influenza C virus [23]. However, recent studies have identified additional protein receptors that mediate PHEV entry. Dipeptidyl peptidase 4 (DPP4) has been shown to interact with the PHEV spike RBD and is required for efficient infection in porcine and murine cells [25]. The DPP4 residues N229 and N321 participate in RBD binding via their linked carbohydrate moieties, and removal of these N-glycosylations enhances the RBD-DPP4 interaction and viral invasion, suggesting that glycosylation acts as a shielding mechanism that restricts cross-species transmission [25].

More recently, dipeptidase 1 (DPEP1) has been identified as a functional receptor for PHEV, with cryo-electron microscopy revealing that the PHEV spike trimer samples both open and closed conformations at steady state, unlike other embecoviruses [28]. The RBD of the PHEV spike shares no detectable sequence homology with those of closely related viruses, yet the elements involved in receptor binding are conserved across embecoviruses, indicating a striking versatility of the RBD to accommodate highly variable sequences that confer novel receptor specificities [28]. Additionally, cell-surface glycans such as sialic acid and heparan sulfate act as attachment factors that facilitate viral entry, with the expression of heparan sulfate proteoglycans (HSPGs) being regulated by PHEV RNA replication [10]. The tetraspanin CD81 has also been implicated as a receptor for the PHEV-VW572 isolate, mediating entry into neuronal cells [2]. These multifaceted receptor interactions highlight the complexity of PHEV entry and its potential for host range expansion.

Epidemiological Significance and Global Distribution

PHEV is highly prevalent and circulates subclinically in most swine herds worldwide [6, 9]. Serological studies using validated enzyme-linked immunosorbent assays (ELISAs) have demonstrated that PHEV infection is endemic in commercial swine populations. In the United States, a large-scale serosurvey of breeding females from 104 farms across 19 states revealed an overall seroprevalence of 53.35%, with a herd seroprevalence of 96.15% [9]. Similarly, in free-range Iberian pigs in central-western Spain, PHEV antibodies were detected in 68.0% of animals, with lower prevalence (22.6%) in wild boars, consistent with endemic exposure in domestic pigs and sporadic circulation in wildlife [26]. In China, a comprehensive surveillance study in Guangxi province from 2021 to 2024 found a PHEV positivity rate of 2.81% by RT-qPCR in tissue samples and nasopharyngeal swabs, with higher rates in swabs (3.98%) than in tissues (2.05%) [4]. These data underscore the widespread nature of PHEV infection, even in the absence of clinical disease.

The World Organisation for Animal Health (WOAH) recognizes PHEV as a pathogen of economic importance, particularly in herds with high gilt replacement rates, specific pathogen-free animals, and gnotobiotic swine [6, 29]. Clinical disease is typically observed only in piglets under 4 weeks of age, with morbidity and mortality rates approaching 100% in naive herds [6, 21]. The age-dependent susceptibility is attributed to the waning of maternal antibodies, which provide passive immunity to nursing piglets [6]. In older pigs, PHEV infection is subclinical, characterized by active viral replication and shedding followed by a robust humoral and cell-mediated immune response that results in viral clearance [11]. The virus is shed in nasal secretions and feces, with detection in oral fluids up to 28 days post-inoculation in grower pigs [11]. No viremia is detected, indicating that PHEV spreads via neural routes rather than hematogenously [8, 11].

The emergence of respiratory PHEV variants has expanded the clinical spectrum of PHEV-associated disease. In 2015, acute outbreaks of influenza-like illness in exhibition swine in Michigan, USA, were attributed to PHEV, with testing ruling out influenza A virus [13]. Subsequent retrospective studies confirmed PHEV in 7.62% of cases with necrotizing bronchitis or bronchiolitis, and the virus was detected via in-situ hybridization in respiratory epithelium [1]. Co-infection with other respiratory pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) suggests that PHEV may contribute to the porcine respiratory disease complex (PRDC) [1]. These findings have prompted the inclusion of PHEV in the differential diagnosis for respiratory disease in nursery pigs, particularly when neurological signs are absent [1, 5]. The genetic and phenotypic diversity of PHEV, coupled with its global distribution and potential for cross-species transmission, underscores the need for continued surveillance and research into this understudied betacoronavirus.

Molecular Pathogenesis and Neurotropism of PHEV

Porcine hemagglutinating encephalomyelitis virus (PHEV) stands as the sole known neurotropic coronavirus endemic to swine, representing a singular model for understanding betacoronavirus invasion of the central nervous system (CNS) [4, 6]. The molecular pathogenesis of PHEV is a multifaceted orchestration of host cell subversion, beginning at the mucosal surface and culminating in severe, often fatal, neurological disease. This journey involves a sophisticated interplay of viral structural proteins with host receptors, the exploitation of endocytic and exocytic pathways, the manipulation of cellular stress responses and autophagy, and a robust, yet ultimately subverted, innate immune response. While traditionally defined by its neurotropism, contemporary research has unveiled significant genetic and phenotypic plasticity, with the emergence of respiratory variants that challenge the classical paradigm of PHEV pathogenesis [1, 5]. This section provides an exhaustive analysis of the molecular mechanisms underpinning PHEV’s neurotropic lifecycle, from initial host engagement to the establishment of persistent infection within the CNS, while also contextualizing the evolutionary divergence that permits alternate tissue tropisms.

Initial Attachment and Receptor Engagement: A Multimodal Strategy

The initial steps of PHEV infection are characterized by a complex, multi-receptor engagement strategy that dictates both tissue tropism and host range. Unlike many coronaviruses that rely on a single primary protein receptor, PHEV appears to utilize a combination of glycoconjugates and proteinaceous receptors for attachment and entry. The S1 subunit of the viral spike (S) protein is the primary determinant of host-cell recognition. Early work established that PHEV agglutinates erythrocytes by binding to N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface [23], a receptor-destroying enzyme (RDE) activity also found in its hemagglutinin-esterase (HE) protein. This binding to sialic acids serves as a critical attachment factor, particularly in the upper respiratory tract. It has been demonstrated that cell-surface glycans, including sialic acid (SA) and heparan sulfate (HS), act as critical interacting factors for PHEV, with removal of these moieties from neuroblastoma (N2a) cells significantly inhibiting viral infection [10]. This suggests that initial tethering to the glycocalyx is an essential prerequisite for subsequent high-affinity receptor binding.

The identity of the protein receptor(s) mediating PHEV entry has been a subject of intense investigation, revealing a remarkable degree of isolate-specific and functional diversity. The neural cell adhesion molecule (NCAM) was identified as an early candidate, with the S1 domain between residues 291-548 shown to interact with NCAM, suggesting this region as a potential receptor-binding domain (RBD) [15]. More recent and groundbreaking research has identified dipeptidyl peptidase 4 (DPP4) as a functional receptor for PHEV [25]. Strikingly, porcine and murine DPP4 bind the viral spike RBD with high affinity, and the glycosylation state of DPP4, particularly at residues N229 and N321, acts as a critical species barrier. Removal of these N-glycans enhances RBD-DPP4 interaction and viral invasion, indicating that PHEV has evolved to tolerate differences in DPP4 ortholog glycosylation as a key mechanism for its ability to infect both pigs and mice, a potential host-jump event [25]. This work was extended by the discovery that dipeptidase 1 (DPEP1) also serves as a functional receptor for PHEV [28]. Cryo-electron microscopy revealed that the PHEV spike trimer samples open and closed conformations, and the RBD binds to DPEP1 via conserved structural elements, showcasing the versatility of the embecovirus RBD to accommodate variable sequences and confer novel receptor specificities [28]. This usage of DPEP1 may be particularly important for infection of renal and respiratory epithelia. Furthermore, significant isolate-specific differences exist. While the PHEV-VW572 strain efficiently uses the tetraspanin CD81 receptor for entry into neuronal cells, the Gent/PS412 isolate does not, highlighting that receptor tropism is a key variable in PHEV neuropathogenesis [2]. This complexity is further augmented by the role of integrins; PHEV binds to integrin α5β1, initiating a signaling cascade that is crucial for viral entry and cytoskeletal rearrangement [35, 41]. Collectively, PHEV employs a "barcoded" entry strategy, where the initial attachment factor (sialic acid) is followed by engagement of one or more protein receptors (NCAM, DPP4, DPEP1, CD81, integrins) likely depending on the viral strain and target cell type, a redundancy that broadens its tissue tropism.

Cellular Entry and Intracellular Trafficking: Hijacking the Endocytic Machinery

Following attachment, PHEV entry into neural cells is mediated by a dynamin-dependent, clathrin-mediated endocytosis (CME) pathway [36]. A comprehensive study employing pharmacological inhibitors, RNA interference, and dominant-negative mutants confirmed that PHEV entry into Neuro-2a cells requires clathrin, the adaptor protein Eps15, and a low-pH environment, characteristic of a classical CME mechanism [36]. This process is also critically dependent on membrane cholesterol and the small GTPases Rab5 and Rab7, which orchestrate early and late endosomal trafficking, respectively [36]. The virus exploits the host’s endosomal network for uncoating and genome release, a process that is exquisitely sensitive to endosomal acidification.

The invasion of PHEV is not a passive event; it actively manipulates the host cytoskeleton. Binding to integrin α5β1 activates the focal adhesion kinase (FAK) pathway, which in turn triggers the Rac1/Cdc42-PAK-LIMK-cofilin signaling axis [35]. This leads to a biphasic remodeling of the actin cytoskeleton: an initial polymerization event that facilitates viral uptake, followed by a later depolymerization phase that may aid in intracellular transport [35]. This cytoskeletal rearrangement is essential for the virus to overcome the physical barrier of the cortical actin network beneath the plasma membrane. The endocytic pathway is also critical for the establishment of long-distance infection within neurons. The virus co-opts the NGF/TrkA signaling endosome system for retrograde transport. The viral infection suppresses Unc51-like kinase 1 (Ulk1), a master regulator of this process, leading to aberrant Rab5 activation and premature degradation of activated pTrkA, thereby disrupting retrograde neurotrophic signaling and contributing to neurodegeneration [37]. This molecular sabotage explains the progressive neurite outgrowth failure observed in PHEV-infected neurons.

Non-Lytic Egress and Intercellular Spread: The Exosomal Highway

A defining feature of PHEV neuropathogenesis is its non-lytic mode of egress, which allows for stealthy dissemination within the CNS without immediately destroying host neurons. PHEV, particularly the neurovirulent VW572 strain, exploits the multivesicular body (MVB) pathway for viral exit. It has been shown that PHEV-VW572, but not Gent/PS412, uses MVB-derived exosomes for viral egress [2, 32]. Complete virions are packaged into intracellular vesicles and released as "PHEV-exosome" hybrids near the plasma membrane [2, 32]. This exosomal transport serves a dual purpose: it protects the virus from immune surveillance and facilitates the transfer of viral components (proteins and RNA) to nonpermissive bystander cells, including microglia, thereby amplifying infection and spreading neuroinflammation without direct cell-to-cell contact [32].

The final step of this egress pathway is intimately tied to the lysosome. In a paradigm distinct from other betacoronaviruses like SARS-CoV-2, which require deacidified lysosomes for release, PHEV actively induces lysosomal acidification to promote its egress [42]. This is driven by the host protein progranulin (PGRN). PHEV infection enhances PGRN trafficking to the lysosome, where it recruits vacuolar-type ATPase (V-ATPase), intensifying acidification and triggering Arl8b-dependent lysosomal exocytosis [42]. This process is not merely a byproduct of infection but is a required step for efficient viral release and neural dissemination; PGRN knockout mice are resistant to PHEV infection [42]. This unique reliance on lysosomal acidification for egress underscores the exquisite co-evolution of PHEV with its host cell's vesicular trafficking machinery.

Subversion of Host Cell Homeostasis: Autophagy, ER Stress, and Apoptosis

PHEV exerts profound effects on host cell homeostasis, intricately manipulating the autophagy-lysosome system, the unfolded protein response (UPR), and programmed cell death to create an environment conducive to its replication. Infection triggers a non-canonical form of autophagy that is independent of the canonical initiator Ulk1 but dependent on AMPK-driven BECN1 phosphorylation [34, 38]. This leads to the formation of autophagosomes, which paradoxically act as a scaffold for viral replication complexes. However, PHEV disrupts the completion of autophagy at a late stage by blocking the fusion of autophagosomes with lysosomes, as evidenced by the lack of p62 degradation [38]. This "atypical" autophagy provides the virus with replication membranes while preventing its own degradation. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, is also manipulated in a complex manner: PHEV infection suppresses overall TFEB expression while paradoxically activating its nuclear translocation by inhibiting MTORC1, ensuring the lysosomal machinery is primed for viral egress [33].

Concurrently, PHEV replication in the endoplasmic reticulum (ER) induces massive ER stress, activating all three branches of the UPR: PERK, IRE1, and ATF6 [31]. The PERK/eIF2α pathway, in particular, is a double-edged sword. While it phosphorylates eIF2α to attenuate global protein translation (a host defense), PHEV likely encodes mechanisms to selectively translate its own genome. The phosphorylated eIF2α also promotes the formation of stress granules (SGs), which are RNA-protein aggregates that sequester translation factors [31]. In an intriguing finding, PHEV-induced SGs actually inhibit viral replication, suggesting this UPR branch acts as a potent innate restriction factor, whereas the autophagy arm of the UPR is co-opted for viral benefit [31]. Ultimately, this pervasive cellular stress drives the cell toward apoptosis. PHEV induces apoptosis in infected cells via a caspase-dependent pathway, involving both the death receptor (caspase-8) and mitochondrial (caspase-9) pathways, converging on effector caspase-3 [40]. While this cell death is detrimental to the host, it may facilitate viral release and the subsequent infection of neighboring cells, contributing to the neuropathology.

Immune Evasion: The Nucleocapsid Protein as a Master Immunosuppressor

To establish a persistent infection within the immunologically privileged CNS, PHEV must effectively subvert the innate antiviral response. The virus achieves this through a highly targeted strategy centered on its nucleocapsid (N) protein. The N protein exerts a dual-pronged attack on the RIG-I-like receptor (RLR) signaling pathway. First, it directly binds to the caspase activation and recruitment domain (CARD) of RIG-I via its C-terminal domain (CTD), competitively blocking TRIM25-mediated K63-linked ubiquitination, which is essential for RIG-I activation [30]. Second, the N protein concurrently disrupts the function of IRF3, the master transcription factor for early interferon (IFN) induction, by blocking its homodimerization, phosphorylation, and nuclear translocation [30]. This simultaneous sabotage of the sensor (RIG-I) and the effector (IRF3) creates an "immune-permissive window" during early infection. Consequently, the virus relies on a delayed, less-efficient IRF7-driven IFN response (>12 hours post-infection), which is inadequate to control the initial burst of viral replication [30]. This strategy is distinct from the nonstructural protein-mediated evasion seen in other coronaviruses, highlighting the N protein as a central virulence factor evolved for neurotropism.

The innate response is not entirely silenced, however. PHEV infection of the respiratory epithelium triggers a robust interferon-stimulated gene (ISG) response, including RSAD2, MX1, and ISG15, along with chemokines like CCL5 and CXCL10 [39, 43]. This leads to astrogliosis, a hallmark of viral encephalitis. Astrocytes are not directly infected by PHEV; rather, they are activated by IFN-β secreted from infected neurons [43]. This astrocyte activation, while a hallmark of neuroinflammation, may also contribute to pathology and is a target for therapeutic intervention.

Genetic Determinants of Neurotropism and the Emergence of Respiratory Variants

The canonical PHEV pathotype is defined by its neurotropism. Infection typically begins in the upper respiratory tract (turbinate and tonsils), after which the virus invades the peripheral nervous system (PNS) via sensory nerve endings and travels retrogradely to the CNS, establishing infection in the brainstem, cerebellum, and cerebrum [6, 8]. This neuroinvasion is facilitated by the virus's ability to use axonal transport pathways [37]. However, a paradigm-shifting discovery has been the identification of novel respiratory variant PHEV (rvPHEV) lineages that cause exclusively respiratory symptoms without neurological signs [5]. These variants have circulated in at least eight provinces in southeastern China [5]. Phylogenetic and recombination analyses show that these rvPHEV lineages are the result of extensive recombination, both within and between classical PHEV and the novel lineages, with divergence times dating back to approximately 1928 [5].

Crucially, rvPHEV strains possess specific genetic signatures that likely underpin this altered tropism. Many of these viruses show amino acid substitutions at the carbohydrate-binding site of the hemagglutinin esterase (HE) protein or have lost the cysteine required for HE dimerization [5]. This loss of HE function is a striking parallel to the adaptation of human coronavirus OC43, where HE lost its hemagglutination ability to adapt to growth in the human respiratory tract. Furthermore, the receptor-binding profile of these strains likely differs. The genetic differentiation of respiratory and neurologic PHEV strains has been phylogenetically confirmed, with respiratory strains clustering separately from neurologic ones [1]. This suggests that a limited number of critical mutations, particularly in the S and HE genes, can fundamentally shift the tissue tropism of PHEV from the CNS to the respiratory tract. This plasticity means that PHEV should now be considered a significant primary respiratory pathogen in swine, and a contributor to the porcine respiratory disease complex (PRDC), as evidenced by its detection in pigs with influenza-like illness and necrotizing bronchitis [1, 13]. The high genetic diversity and complex evolutionary trajectory of PHEV, particularly in regions like Southern China [4], underscore the virus’s potential to generate novel pathotypes with unpredictable consequences for swine health and potential zoonotic risk, as also recognized by the World Organisation for Animal Health (WOAH) in considerations of emerging coronaviruses.

Emerging Respiratory Pathogenesis and Role in Porcine Respiratory Disease Complex

The traditional conceptualization of porcine hemagglutinating encephalomyelitis virus (PHEV) as a strictly neurotropic pathogen has undergone a fundamental paradigm shift in recent years. While PHEV has been historically recognized for its capacity to induce vomiting and wasting disease (VWD) and encephalomyelitis in neonatal piglets, accumulating evidence now positions this betacoronavirus as a significant, albeit underappreciated, contributor to respiratory pathology in swine [1, 5, 6]. This emerging respiratory phenotype, characterized by influenza-like illness and its integration into the multifactorial porcine respiratory disease complex (PRDC), represents one of the most consequential developments in contemporary PHEV research.

Historical Context and the Sentinel Respiratory Outbreak

The first definitive link between PHEV and respiratory disease was established during acute outbreaks of influenza-like illness in exhibition swine at agricultural fairs in Michigan, USA, during 2015 [13]. These outbreaks, initially suspected to involve influenza A virus due to the clinical presentation of coughing, sneezing, nasal discharge, and lethargy, were systematically ruled out for influenza and subsequently identified as PHEV-positive through molecular diagnostics [13]. This sentinel event catalyzed a re-evaluation of PHEV’s pathogenic repertoire, prompting investigators to question whether respiratory tropism represented an aberrant, sporadic occurrence or a more widespread, previously unrecognized aspect of PHEV biology.

Subsequent retrospective and prospective investigations have confirmed that the Michigan outbreak was not an isolated phenomenon. A comprehensive diagnostic investigation by Arunsiripate et al. (2025) demonstrated PHEV positivity via quantitative PCR (qPCR) in 83.33% of pigs examined specifically for respiratory disease, with histopathological lesions including necrotizing bronchitis and bronchiolitis [1]. Critically, in-situ hybridization (ISH) confirmed the presence of PHEV mRNA within respiratory epithelial cells, providing direct molecular evidence of viral replication in the respiratory tract rather than mere contamination or passive carriage [1]. Immunohistochemical analysis further revealed significant macrophage infiltration in affected lung tissue, indicating an active inflammatory response to viral infection [1].

Genetic Basis for Respiratory Tropism: The Emergence of Respiratory Variant PHEV

The genetic underpinnings of PHEV’s respiratory phenotype have been elucidated through large-scale phylogenetic and genomic analyses. He et al. (2023) conducted a landmark retrospective epidemiological study of swine populations in China, identifying novel lineages of PHEV complex coronaviruses that cause exclusively respiratory symptoms with no signs of the neurological symptoms typically associated with classical PHEV infection [5]. Through extensive surveillance spanning at least eight provinces in southeastern China, these investigators demonstrated that the novel respiratory variant PHEV (rvPHEV) lineages have circulated widely, suggesting that the respiratory phenotype is not a rare aberration but a stable, transmissible viral characteristic [5].

Phylogenetic and recombination analyses of twenty-four complete genomes identified two major viral lineages causing respiratory symptoms, with extensive recombination occurring within them, between them, and between classical PHEV and the novel rvPHEV lineages [5]. Divergence time estimates indicate that the two major rvPHEV lineages separated approximately 20 years after the initial divergence of the PHEV virus complex, which dates back to 1886–1958 (mean estimate 1928) [5]. This temporal framework suggests that respiratory-adapted PHEV strains have been circulating for decades, potentially evading detection due to the historical focus on neurological disease.

The molecular basis for this phenotypic shift appears to involve critical modifications in the hemagglutinin esterase (HE) protein. Many rvPHEV viruses exhibit amino acid substitutions at the carbohydrate-binding site of HE and/or have lost the cysteine residue required for HE dimerization [5]. This observation is particularly striking because it mirrors the early adaptation of human coronaviruses, where HE lost its hemagglutination ability to adapt to growth in the human respiratory tract [5]. The convergent evolution between rvPHEV and human coronaviruses underscores the selective pressure exerted by the respiratory environment and highlights the potential for PHEV to serve as a model for understanding coronavirus host adaptation.

Further supporting the genetic distinction between respiratory and neurologic strains, phylogenetic analysis by Arunsiripate et al. (2025) demonstrated that PHEV strains recovered from respiratory cases cluster closely with historical respiratory strains but remain distinct from neurologic strains [1]. This genetic differentiation suggests that specific viral determinants, likely residing in the spike (S) glycoprotein and HE genes, confer differential tissue tropism. The S gene, in particular, exhibits the highest genetic variability among PHEV structural proteins, with nucleotide homology ranging from 94.3% to 99.3% among circulating strains in Guangxi province, China, compared to the more conserved M and N genes [4]. This hypervariability of the S gene, which encodes the primary receptor-binding and fusion machinery, is consistent with its role in mediating host cell entry and tissue tropism [4, 16].

Receptor Usage and Cellular Entry in the Respiratory Tract

The molecular mechanisms by which PHEV gains entry into respiratory epithelial cells have been progressively elucidated, revealing a complex interplay of multiple receptors and attachment factors. Historically, PHEV was known to utilize N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for hemagglutination, a property shared with bovine coronavirus and influenza C virus [23]. However, recent investigations have dramatically expanded our understanding of PHEV receptor usage.

Dipeptidyl peptidase 4 (DPP4) has been identified as a candidate binding target of the PHEV spike protein, with both porcine and murine DPP4 demonstrating high affinity for the viral spike receptor-binding domain (RBD) [25]. Structural studies reveal that the PHEV spike protein binds to the outer surface of blades IV and V of the DPP4 β-propeller domain, with DPP4 residues N229 and N321 participating in RBD binding via their linked carbohydrate entities [25]. Intriguingly, removal of these N-glycosylations profoundly enhanced the RBD-DPP4 interaction and viral invasion, suggesting that these glycans act as shielding mechanisms that modulate receptor accessibility and potentially influence species barriers [25].

In a parallel and potentially convergent finding, dipeptidase 1 (DPEP1) has been identified as a functional receptor for PHEV, with cryo-electron microscopy revealing that PHEV, unlike other embecoviruses, samples open and closed conformations of its spike trimer at steady state [28]. The X-ray structure of the RBD/DPEP1 complex demonstrated that the elements involved in receptor binding are conserved across embecoviruses, revealing a striking versatility of the RBD to accommodate highly variable sequences that confer novel receptor specificities [28]. This receptor plasticity may underpin PHEV’s ability to infect multiple tissue types, including the respiratory epithelium.

Beyond protein receptors, cell-surface glycans, including sialic acid (SA) and heparan sulfate (HS), act as critical attachment factors for PHEV [10]. Glycan depletion assays demonstrate that removing SA or HS from target cells inhibits PHEV infection, while soluble sugar monomers competitively bind PHEV and affect viral infectivity [10]. The expression of heparan sulfate proteoglycans (HSPGs), including syndecans and glypicans, is regulated by PHEV RNA replication, suggesting a dynamic interplay between the virus and the host cell glycocalyx [10]. Furthermore, the tetraspanin CD81 has been identified as a receptor for the PHEV-VW572 isolate, mediating entry into neuronal cells, though the relevance of this receptor for respiratory epithelial cells remains to be determined [2].

Primary Replication in the Respiratory Tract: Evidence from In Vivo and Ex Vivo Models

The respiratory tract has been definitively established as the primary site of PHEV replication, preceding neuroinvasion in classical infections and serving as the sole site of replication in respiratory variant strains. Comprehensive characterization of PHEV infection in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs by Mora-Díaz et al. (2021) demonstrated that the highest concentration of virus was detected in turbinate and trachea at 5 days post-inoculation, followed by tonsils, lungs, and tracheobronchial lymph nodes [8]. Viral shedding in nasal secretions was detected from 1 to 10 days post-inoculation, confirming active replication in the upper respiratory tract [8].

The development of an ex vivo air-liquid interface porcine respiratory epithelial cell culture (ALI-PREC) system has provided unprecedented insights into PHEV’s interaction with the respiratory epithelium. This system, which closely resembles the epithelial lining of the tracheobronchial region, demonstrated that PHEV replicates actively in ALI-PRECs, causing cytopathic changes and disruption of ciliated columnar epithelia [8]. Transcriptome analysis of PHEV-infected ALI-PRECs revealed downregulation of cilia-associated genes, including CILK1, DNAH11, LRRC-23, -49, and -51, and the acidic sialomucin CD164L2, providing a molecular explanation for the impairment of mucociliary clearance observed during infection [39].

The innate immune response to PHEV in the respiratory epithelium is characterized by a robust interferon response. Transcriptome analysis demonstrated significant upregulation of interferon-stimulated genes, including RSAD2, MX1, IFIT, and ISG15, as well as chemokine genes CCL5 and CXCL10 [39]. This antiviral signaling cascade, while intended to restrict viral replication, also contributes to the inflammatory milieu that characterizes PHEV-associated respiratory disease. The activation of the PERK/PKR-eIF2α pathway in response to PHEV-induced endoplasmic reticulum stress represents an additional host defense mechanism, attenuating global protein translation and facilitating stress granule formation to restrict viral replication [31].

Immune Evasion Mechanisms Facilitating Respiratory Infection

PHEV has evolved sophisticated strategies to subvert the host innate immune response, facilitating its replication in the respiratory tract and subsequent dissemination. The viral nucleocapsid (N) protein has emerged as a central immunosuppressor, targeting the RIG-I-MAVS signaling pathway to delay and attenuate the interferon response [30]. Mechanistically, the N protein directly engages RIG-I’s caspase activation and recruitment domain (CARD) via its C-terminal domain, competitively blocking TRIM25-mediated K63-linked ubiquitination and silencing RIG-I activation [30]. Concurrently, the N protein disrupts IRF3 activation by interfering with homodimerization, phosphorylation, and nuclear translocation, abrogating its function as the dominant early antiviral mediator [30].

This dual-pronged attack on the innate immune system creates an immune-permissive window during which PHEV can replicate unchecked. The virus activates RIG-I-MAVS signaling but hijacks this pathway to induce a delayed IRF7-dependent interferon response (>12 hours post-infection), permitting substantial viral replication before the host can mount an effective antiviral state [30]. The inadequate IRF7-driven interferon induction (less than 3-fold at the mRNA level) fails to compensate for IRF3 inactivation, further compromising the host’s ability to control infection [30].

In the context of respiratory infection, this immune evasion strategy may have particular significance. The respiratory epithelium is a frontline barrier that relies heavily on rapid interferon signaling to contain viral infections. By delaying and attenuating this response, PHEV can establish a foothold in the respiratory tract before adaptive immune mechanisms are mobilized. This may explain the prolonged viral shedding observed in nasal secretions (up to 28 days post-inoculation in grower pigs) and the ability of PHEV to persist in swine populations despite widespread seropositivity [9, 11].

Integration into the Porcine Respiratory Disease Complex

The recognition of PHEV as a respiratory pathogen has necessitated its inclusion in the differential diagnosis of PRDC, a multifactorial disease syndrome involving interactions between viral and bacterial pathogens, environmental factors, and host susceptibility. PRDC is characterized by reduced growth performance, cough, dyspnea, and mortality, with significant economic implications for the swine industry. The World Organisation for Animal Health (WOAH) recognizes the importance of respiratory pathogens in swine health, and the inclusion of PHEV in diagnostic panels represents an evolving understanding of PRDC etiology.

Epidemiological investigations have demonstrated that PHEV frequently co-occurs with other respiratory pathogens, suggesting synergistic or additive effects in disease pathogenesis. Arunsiripate et al. (2025) documented PHEV co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), a cornerstone pathogen in PRDC, in clinical cases [1]. The retrospective component of their study identified PHEV in 7.62% of cases with necrotizing bronchitis or bronchiolitis, indicating that PHEV is not merely an incidental finding but a significant contributor to respiratory pathology [1].

The development of multiplex molecular diagnostic tools has facilitated the detection of PHEV alongside other respiratory pathogens. Sunaga et al. (2019) developed a one-run real-time PCR detection system (Dempo-PCR) capable of screening 17 porcine respiratory pathogens, including PHEV, in a single assay [44]. Similarly, triplex and quadruplex RT-PCR assays have been developed for the simultaneous detection of PHEV with other neurotropic and respiratory viruses, including pseudorabies virus, classical swine fever virus, and Japanese encephalitis virus [45, 47]. These diagnostic advances enable comprehensive characterization of the respiratory pathogen landscape and facilitate the identification of PHEV’s role in polymicrobial infections.

The detection of PHEV in nasopharyngeal swabs from clinically affected pigs underscores its relevance to respiratory disease surveillance. Shi et al. (2024) reported a positivity rate of 3.98% in nasopharyngeal swabs compared to 2.05% in tissue samples from Guangxi province, China, suggesting that non-invasive sampling methods can effectively detect PHEV in respiratory secretions [4]. This finding has practical implications for herd-level monitoring and biosecurity assessment.

Epidemiological Significance and Subclinical Circulation

The epidemiological landscape of PHEV respiratory infection is characterized by widespread subclinical circulation punctuated by acute outbreaks. Serological surveys have demonstrated that PHEV is endemic in swine herds worldwide, with a seroprevalence of 53.35% in U.S. sow herds and 96.15% herd-level seroprevalence [9]. In free-range Iberian pigs from Spain, PHEV seroprevalence reached 68.0%, with lower but detectable levels (22.6%) in wild boars, suggesting spillover from domestic populations [26]. The detection of PHEV in wild boars across Europe, including Italy, Spain, Germany, and Poland, raises concerns about wildlife reservoirs and the potential for viral maintenance in sylvatic cycles [29].

The subclinical nature of PHEV infection in older pigs, as demonstrated by Mora-Díaz et al. (2020), who found that 7-week-old pigs developed no clinical signs despite active viral replication and shedding for up to 28 days, complicates efforts to assess the true burden of respiratory disease attributable to PHEV [11]. This subclinical shedding, characterized by detection of virus in oral fluid and feces without viremia, allows PHEV to circulate undetected within herds, potentially contributing to PRDC through immunosuppression or synergistic interactions with other pathogens [11].

The economic impact of PHEV-associated respiratory disease, while difficult to quantify precisely, is likely substantial. The high mortality rates in neonatal pigs during classical outbreaks are well-documented, but the insidious effects of subclinical respiratory infection on growth performance, feed conversion efficiency, and susceptibility to secondary bacterial infections may represent a greater cumulative economic burden [6, 46]. The Food and Agriculture Organization (FAO) has emphasized the importance of understanding the full pathogenic spectrum of swine coronaviruses to inform disease control strategies and mitigate economic losses.

Comparative Pathology and Interspecies Considerations

The emergence of respiratory PHEV strains raises important questions about viral evolution and the potential for interspecies transmission. PHEV is known to infect not only pigs but also mice and Wistar rats, and the identification of DPP4 as a receptor that mediates host range expansion suggests that PHEV may have the capacity to cross species barriers [6, 25]. The observation that PHEV tolerance to DPP4 orthologs is a putative determinant of cross-species transmission highlights the need for surveillance at the human-animal interface [25].

The genetic and functional similarities between PHEV and human betacoronaviruses, including OC43 and HKU1, as well as SARS-CoV-2, have led to the proposal that PHEV may serve as a surrogate model for studying betacoronavirus neuropathogenesis and respiratory adaptation [2, 32]. The convergent evolution of HE protein modifications in rvPHEV and human coronaviruses suggests that the selective pressures shaping coronavirus evolution in respiratory tracts may be conserved across species [5]. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have emphasized the importance of understanding coronavirus ecology in animal reservoirs to predict and prevent future zoonotic emergence.

The recent identification of PHEV in Russia, with the first sequencing of Russian strains and phylogenetic analysis revealing their relationship to European isolates, underscores the global distribution of this virus and the need for coordinated international surveillance efforts [3]. The genetic diversity observed among PHEV strains, including the identification of novel genotypes in South Korea with large unique deletions in nonstructural protein 3 and NS2, suggests that the virus continues to evolve under endemic pressure, potentially giving rise to variants with altered tissue tropism or pathogenic potential [7].

Global Epidemiology and Risk Factors for PHEV Infection

Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus within the subgenus Embecovirus, has been recognized as a significant pathogen in swine populations worldwide since its first identification in Canada in the late 1950s [1, 3, 20]. Despite its early discovery and global circulation, PHEV remains one of the least researched porcine coronaviruses, with much of its epidemiology and risk factor landscape still poorly characterized [1, 3-73]. This section aims to provide a comprehensive, evidence-based synthesis of the global distribution, transmission dynamics, host range, and key risk factors influencing PHEV infection and disease manifestation, drawing exclusively from the curated source set provided.

1. Global Distribution and Seroprevalence

PHEV is considered endemic in most pig-producing countries, with serological surveys confirming its widespread subclinical circulation [1, 3-73]. Early reports from Canada described outbreaks of vomiting and wasting disease (VWD) and encephalomyelitis in suckling piglets, with the virus isolated and characterized as HEV (later PHEV) [1, 20]. Subsequent studies in the United States confirmed the presence of PHEV in swine herds, often associated with respiratory or neurological signs, though many infections remained subclinical [1, 3-73].

In China, PHEV has been detected in multiple provinces, with strains such as PHEV-JLsp09 and PHEV-CC14 isolated from clinical outbreaks in Jilin and Guangxi, respectively [1, 3-73]. A large-scale surveillance study in Guangxi during 2021–2024 tested 6,986 tissue and swab samples, finding a PHEV positivity rate of 2.81% (196/6,986), with higher rates in nasopharyngeal swabs (3.98%) than in tissue samples (2.05%) [4]. This study also revealed that PHEV strains from Guangxi clustered into subgroups G1 and G2, with complex evolutionary trajectories and high genetic diversity [4]. Similarly, a retrospective study in South Korea detected PHEV in 22 of 239 samples (9.2%) from 17 farms, with higher prevalence in suckling pigs (14.3%) than in weaners (6.5%) and growers (7%) [17]. In the Czech Republic, a 2019 survey found PHEV in 7.9% of nasal swabs from pigs of various ages, with strains showing 95.8–98.1% amino acid identity to reference strains [14]. In Spain, seroprevalence studies using an indirect ELISA based on the S1 protein revealed that PHEV antibodies were present in 68% of Iberian pigs and 22.6% of wild boars from central-western regions, indicating endemic circulation in free-range systems [5, 66]. These findings collectively underscore that PHEV is highly prevalent and circulates subclinically in most swine herds worldwide, with sporadic clinical outbreaks often limited to neonatal piglets [1, 3-73].

2. Transmission Dynamics and Host Range

PHEV is primarily a neurotropic coronavirus that invades the central nervous system (CNS) via peripheral nerves, causing encephalomyelitis in susceptible piglets [1, 3-73]. However, recent evidence has also demonstrated a respiratory tropism, with PHEV detected in the upper respiratory tract and causing mucociliary disruption, interferon responses, and chemokine induction in air-liquid interface porcine respiratory epithelial cell cultures [39]. This dual tropism suggests that PHEV may initially replicate in the respiratory epithelium before gaining access to the CNS via neural pathways [1, 20, 39].

Transmission occurs primarily through direct contact between pigs, via nasal secretions and feces, with viral shedding detected in oral fluid (1–28 days post-inoculation) and feces (1–10 days) in experimentally infected grower pigs [8]. The virus can also be shed in nasal secretions and feces from subclinically infected carriers, contributing to endemic circulation within herds [1, 3-73]. Importantly, PHEV does not produce viremia, and viral RNA is detected only in tissues except liver, with highest concentrations in turbinate, trachea, tonsils, lungs, and tracheobronchial lymph nodes at early time points [8]. This pattern aligns with a respiratory-to-neural invasion pathway.

The host range of PHEV is largely restricted to pigs, but experimental infections have demonstrated neurotropism in mice and rats, suggesting potential cross-species transmission [1, 3-73]. Wild boars have been identified as seropositive for PHEV antibodies in Spain, with prevalence reaching 22.6% in some regions, indicating that free-ranging populations may serve as reservoirs or spillover hosts [5, 66]. Furthermore, the virus has been shown to utilize dipeptidyl peptidase 4 (DPP4) as a functional receptor, with the spike protein’s receptor-binding domain (RBD) binding to DPP4 orthologs across species, including human DPP4 [28]. This finding raises concerns about potential host jumps, though the current zoonotic risk remains low due to the virus’s strong neurotropism in pigs and limited replication in human cells [28, 72]. Nonetheless, cross-neutralization studies have revealed that human sera collected after SARS-CoV-2 infection can neutralize PHEV and bovine coronavirus (BCV) in vitro, suggesting prior exposure or immune memory to related betacoronaviruses [72]. This highlights the need for continued surveillance at wildlife-livestock interfaces to monitor spillover events [5, 66, 72].

3. Risk Factors for Infection and Clinical Disease

Age is the most critical determinant of clinical disease outcome. PHEV causes fatal encephalomyelitis primarily in piglets under 4 weeks of age, with morbidity and mortality rates approaching 100% in naive herds [1, 3-73]. Older pigs (>4 weeks) typically experience subclinical or mild respiratory infections, with no neurological signs [1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

Diagnostic Approaches: Histopathology, Molecular Detection, and Serology

The accurate and timely diagnosis of porcine hemagglutinating encephalomyelitis virus (PHEV) infection necessitates a multifaceted approach that integrates classical histopathological evaluation with advanced molecular and serological techniques. Given the virus's capacity to induce distinct clinical syndromes, ranging from classical neurological disease and vomiting and wasting disease (VWD) to recently identified respiratory presentations, a comprehensive diagnostic algorithm is essential for both clinical management and epidemiological surveillance [1, 5, 6]. The diagnostic landscape for PHEV has evolved considerably, transitioning from historical reliance on virus isolation and hemagglutination to highly sensitive and specific molecular assays and robust serological platforms that enable population-level monitoring.

Histopathological Examination and In Situ Detection

Histopathological analysis remains a cornerstone for the presumptive diagnosis of PHEV infection, particularly in cases presenting with neurological signs. The hallmark lesion in the central nervous system (CNS) is a non-suppurative encephalomyelitis, characterized by lymphoplasmacytic perivascular cuffing, diffuse gliosis, neuronal degeneration and necrosis, and neuronophagia [1, 21, 57]. These pathological changes are most pronounced in the gray matter of the brainstem, cerebrum, and spinal cord, reflecting the virus's profound neurotropism [6, 12, 21]. In acutely infected piglets, gross pathological findings may include meningeal hyperemia and hemorrhage, with histopathological examination revealing satellitosis and the formation of glial nodules [57, 67]. The severity of neuronal injury correlates with clinical outcome, as extensive neuronal loss underpins the high mortality observed in neonatal pigs [6, 57].

Recent investigations have substantially expanded the histopathological spectrum associated with PHEV, demonstrating that the virus can induce significant respiratory pathology. In a landmark study examining PHEV as a potential respiratory pathogen, Arunsiripate et al. (2025) documented necrotizing bronchitis and bronchiolitis in pigs with clinical respiratory disease, with PHEV confirmed by quantitative PCR (qPCR) in 83.33% of examined cases [1]. These pulmonary lesions were accompanied by prominent macrophage infiltration into the alveolar spaces and interstitium, as demonstrated by immunohistochemical (IHC) analysis [1]. This finding is particularly significant as it establishes PHEV as a contributor to the porcine respiratory disease complex (PRDC), often in co-infection with other pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) [1]. The retrospective component of this study identified PHEV in 7.62% of cases presenting with necrotizing bronchitis or bronchiolitis, underscoring the virus's underappreciated role in respiratory disease [1].

The application of in situ hybridization (ISH) has proven instrumental in definitively localizing PHEV within specific cell types in affected tissues. Using ISH, Arunsiripate et al. (2025) confirmed the presence of PHEV mRNA within respiratory epithelial cells of the bronchial and bronchiolar lining, providing direct evidence of viral replication at this site [1]. Similarly, immunohistochemical staining using antibodies directed against the PHEV nucleocapsid (N) protein has been successfully employed to demonstrate viral antigen in neurons of the cerebral cortex in both naturally infected piglets and experimentally inoculated mice [57, 59, 67]. These in situ techniques not only confirm infection but also provide critical insights into viral tropism and pathogenesis. For instance, the observation that PHEV antigen is localized to neurons in the CNS supports the hypothesis that the virus utilizes axonal transport along neural circuits to invade the brain following peripheral replication [6, 12].

The utilization of ex vivo models, such as air-liquid interface porcine respiratory epithelial cell cultures (ALI-PRECs), has further refined our understanding of PHEV-induced histopathology at the cellular level. Mora-Díaz et al. (2021) demonstrated that PHEV replication in ALI-PRECs leads to cytopathic changes, including disruption of the ciliated columnar epithelium and loss of ciliary function, confirming the upper respiratory tract as a primary site of infection [8]. Transcriptome analysis of these cultures revealed downregulation of cilia-associated genes, including CILK1, DNAH11, and multiple leucine-rich repeat-containing proteins (LRRC-23, -49, -51), providing a molecular basis for the observed mucociliary dysfunction [39]. These ex vivo findings are histopathologically relevant, as they recapitulate the in vivo scenario where respiratory epithelial damage facilitates viral spread and secondary bacterial colonization.

Beyond the CNS and respiratory tract, PHEV infection induces pathological changes in other organ systems. In experimentally infected cesarean-derived, colostrum-deprived (CDCD) piglets, gastritis characterized by lymphoplasmacytic infiltration with perivasculitis and neuritis involving gastric ganglia degeneration was a prominent finding [8]. This gastric pathology likely contributes to the vomiting and wasting syndrome characteristic of PHEV infection. Ultrastructural examination by transmission electron microscopy (TEM) has revealed coronavirus-like particles within homogenized brain tissue suspensions and has been instrumental in characterizing the virus's entry and egress mechanisms. Notably, TEM studies have demonstrated that PHEV virions are packaged within multivesicular body (MVB)-derived exosomes, which serve as vehicles for intercellular transmission, particularly in neuronal cells [2, 32]. This exosomal pathway represents a sophisticated mechanism for viral dissemination that may evade host immune recognition [32, 42].

Molecular Detection: From Conventional RT-PCR to Advanced Multiplex Platforms

Molecular detection methods have become the gold standard for PHEV diagnosis due to their unparalleled sensitivity, specificity, and rapid turnaround time [6, 27]. Reverse transcription PCR (RT-PCR) and its real-time quantitative variant (RT-qPCR) are the most widely employed techniques, targeting various regions of the PHEV genome including the nucleocapsid (N) gene, spike (S) glycoprotein gene, and polymerase gene [18, 47, 60]. The N gene is particularly favored as a target due to its relative conservation among PHEV strains, ensuring broad detection across genetic lineages [4, 45, 47]. Early work by Sekiguchi et al. (2004) established nested RT-PCR targeting the spike protein gene as a highly sensitive method, capable of detecting PHEV viral RNA in samples where virus isolation in cell culture failed [18]. This nested format, while sensitive, has largely been supplanted by real-time methods that offer quantitative data and reduced risk of cross-contamination.

The development of multiplex real-time RT-PCR assays represents a significant advancement, allowing for the simultaneous detection and differentiation of PHEV from other swine pathogens that cause similar clinical signs. This is particularly important given the overlapping neurological and respiratory presentations caused by viruses such as porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) [45, 47]. Hu et al. (2023) developed a quadruplex qRT-PCR targeting the PHEV N gene, PRV gB gene, CSFV 5'UTR, and JEV NS1 gene, achieving a limit of detection (LOD) of 15 copies/μL for each pathogen with no cross-reactivity against other common swine viruses [47]. Applying this assay to 1,977 clinical samples from Guangxi, China, revealed PHEV positivity rates of 1.57%, effectively demonstrating the virus's circulation in the region [47]. Similarly, a triplex assay for PHEV, PRV, and CSFV has been validated, with high specificity and a detection limit comparable to singleplex assays [45].

The most sensitive molecular approach currently available is digital PCR (dPCR), which provides absolute quantification of target nucleic acids without reliance on standard curves. Shi et al. (2024) developed a triplex crystal digital RT-PCR (cdRT-PCR) for PHEV, PRV, and CSFV, achieving LODs of 4.812, 4.047, and 5.243 copies/reaction, respectively, approximately 50-fold more sensitive than conventional multiplex RT-qPCR [45]. This extraordinary sensitivity is particularly valuable for detecting low-level viral shedding during subclinical infections or in samples with degraded RNA. The cdRT-PCR assay demonstrated excellent repeatability, with intra-assay coefficients of variation (CVs) of 0.73–1.87% and inter-assay CVs of 0.57–2.95%, and when applied to 1,367 clinical samples, showed a 98.98% coincidence rate with reference multiplex RT-qPCR [45]. Such digital platforms are poised to become the reference standard for PHEV detection in research and surveillance contexts.

For high-throughput screening in the context of porcine respiratory disease complex, broad-spectrum real-time PCR systems have been developed. Sunaga et al. (2019) described a "Dempo-PCR" system capable of detecting 17 different porcine respiratory pathogens in a single run, including PHEV [44]. This TaqMan-based assay employs novel primer-probe sets and demonstrates high sensitivity for all targets, enabling comprehensive etiological investigation of respiratory disease outbreaks [44]. Such multiplex capabilities are essential given the frequent observation of PHEV co-infections with PRRSV, swine influenza virus, and bacterial pathogens, as documented by Arunsiripate et al. (2025) [1] and Rho et al. (2010) [17].

Large-scale epidemiological surveillance using RT-qPCR has revealed the widespread circulation of PHEV. In a comprehensive study spanning 2021-2024 in Guangxi province, China, Shi et al. (2024) tested 6,986 clinical samples using a quadruplex RT-qPCR, finding an overall PHEV positivity rate of 2.81% (196/6,986) [4]. Notably, positivity rates were higher in nasopharyngeal swabs (3.98%) compared to tissue samples (2.05%), supporting the role of respiratory shedding in viral transmission [4]. This study further demonstrated that PHEV strains circulating in southern China exhibit high genetic diversity, with phylogenetic analysis of the S, M, and N genes revealing the presence of both G1 and G2 lineages, and evidence of recombination and mutation [4]. Similarly, a study in the Czech Republic detected PHEV in 7.9% of nasal swabs using RT-PCR, with phylogenetic analysis showing that Czech strains clustered most closely with the Belgian VW572 strain [14]. These molecular surveillance efforts underscore the necessity of continuously monitoring circulating strains to ensure diagnostic assays remain effective against evolving viral genotypes.

The isolation of PHEV in cell culture, while less commonly employed for primary diagnosis, remains a valuable adjunct for obtaining viral isolates for characterization and research. PHEV can be propagated in primary and secondary pig-derived cell lines, including primary pig kidney (PPK), primary pig testicle (PPT), secondary pig thyroid (SPTh), and continuous cell lines such as PK-15, SK-K, and ST [22, 24, 63]. SPTh and PPK cells have been identified as the most susceptible for virus cultivation and quantitation, with cytopathic effect (CPE) characterized by syncytium formation, cell rounding, and detachment from the monolayer typically observed within 18-72 hours post-inoculation [22, 63]. Andries and Pensaert (1980) established that for virus isolation from clinical specimens, blind passage is recommended if the hemagglutination (HA) test remains negative at 7 days post-inoculation [22]. The adaptation of PHEV to cell culture, particularly the mouse-adapted 67N strain in SK-K cells, has facilitated the development of plaque assay systems for viral quantitation and neutralization assays [24].

Serological Diagnosis: ELISA Platforms and Hemagglutination Inhibition

Serological assays are critical for determining population-level exposure, monitoring immune status, and evaluating vaccine efficacy. The development of reliable serological tools for PHEV has lagged behind that for other swine coronaviruses, but significant progress has been made in recent years [6, 9]. The virus neutralization (VN) test has historically been considered the gold standard for detecting PHEV-specific antibodies, demonstrating high specificity [53, 65]. However, VN assays are labor-intensive, require cell culture facilities, and take several days to complete, making them impractical for large-scale surveillance [9].

The hemagglutination inhibition (HI) test has also been used, leveraging the ability of PHEV to agglutinate erythrocytes from chickens, mice, rats, and other species via its hemagglutinin-esterase (HE) protein's interaction with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) receptors [6, 23]. However, concerns regarding specificity have been raised, as sera from several animal species, including mice, rabbits, and humans, exhibit HI activity without corresponding VN activity, suggesting that HI may detect non-neutralizing antibodies or cross-reactive factors [65]. Sasaki et al. (2003) demonstrated that VN tests using FS-L3 cells cultured without serum provided a more reliable measure of PHEV infection than HI, as VN activity was detected exclusively in sera from pigs, rats, cows, and dogs, species known to be susceptible to PHEV infection [65].

The development of enzyme-linked immunosorbent assays (ELISAs) has revolutionized PHEV serology, enabling high-throughput, cost-effective testing. The most extensively validated assay is the S1-based indirect IgG ELISA developed by Mora-Díaz et al. (2020) [9]. This assay utilizes the S1 subunit of the PHEV spike protein as the coating antigen and has undergone rigorous validation using known-status serum samples from pigs experimentally inoculated with PHEV or other swine coronaviruses. Using receiver operating characteristic (ROC) analysis, a sample-to-positive (S/P) cutoff of ≥0.6 was established, yielding both high sensitivity and specificity, with no cross-reactivity observed against porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) [9]. Application of this ELISA to a large-scale serosurvey of 2,756 serum samples from breeding females across 104 U.S. sow farms in 19 states revealed an overall PHEV seroprevalence of 53.35%, with herd-level seroprevalence reaching 96.15% [9]. These data underscore that PHEV is endemic and highly prevalent in U.S. swine herds, despite the absence of clinical disease in most operations.

A complementary indirect ELISA using soluble antigen extracted from PHEV-infected FS-L3 cells has been developed by Trang et al. (2020) [53]. This assay, employing RBS buffer containing 0.2% NP-40 for antigen extraction, demonstrated excellent diagnostic performance when compared to VN tests on 150 porcine serum samples. ROC analysis yielded an area under the curve (AUC) of 0.996, with a sensitivity of 95.35% and specificity of 96.88% at the optimal cutoff [53]. Correlation between the indirect ELISA and VN test was strong (R = 0.850), with near-perfect agreement (kappa value = 0.932), indicating that this ELISA can reliably substitute for VN testing in sero-epidemiological studies [53].

The utility of serological testing extends beyond research to practical herd management. Given that no commercial PHEV vaccines are currently available, neonatal protection depends entirely on lactogenic immunity derived from immune dams [6, 9]. Serological profiling of gilts and sows allows producers to assess the risk of PHEV-associated disease in their offspring. In herds where maternal immunity is low, management strategies such as promoting natural virus circulation through early exposure of gilts may be implemented to boost herd immunity and prevent clinical outbreaks in vulnerable neonates [6].

Recent serosurveillance studies have documented PHEV exposure in diverse swine populations. Encinas et al. (2026) reported antibodies to PHEV in 68.0% of Iberian pigs and 22.6% of wild boars in central-western Spain, indicating endemic circulation in free-range production systems and sporadic spillover into wildlife [26]. Cross-reactivity studies have demonstrated that human sera from both pre-pandemic and SARS-CoV-2 convalescent individuals can neutralize bovine coronavirus (BCV) and immunostain PHEV-infected cells, suggesting that exposure to endemic human betacoronaviruses (e.g., OC43) elicits antibodies that cross-react with related animal coronaviruses [72]. This finding has implications for interpreting serological data in wildlife and livestock populations and highlights the potential for cross-species transmission events.

Rapid diagnostic tests have been developed to enable on-site detection of PHEV antigen. Chen et al. (2012) described an immunochromatographic strip assay utilizing colloidal gold-labeled monoclonal antibody (MAb) 4D4 as the detection reagent and MAb 1E2 as the capture antibody on the test line [62]. This strip could detect PHEV with a hemagglutination unit of 2 within 10 minutes, showing 100% specificity and 97.78% sensitivity relative to RT-PCR, with excellent agreement (kappa = 0.976) [62]. The strips demonstrated remarkable stability, retaining sensitivity and specificity after storage at room temperature for 6 months or 4°C for 12 months [62]. Such point-of-care tests are invaluable for rapid outbreak response and surveillance in field settings where laboratory infrastructure is limited.

Metabolic and Antiviral Interventions Targeting PHEV Replication

The development of effective therapeutic strategies against porcine hemagglutinating encephalomyelitis virus (PHEV) represents a critical unmet need in swine medicine, given the virus’s capacity to cause devastating neurological disease in neonatal piglets, its emerging role in the porcine respiratory disease complex (PRDC), and the complete absence of licensed vaccines or specific antiviral drugs [6, 27, 46]. PHEV, a neurotropic betacoronavirus within the subgenus Embecovirus, establishes infection primarily in the upper respiratory tract before disseminating via peripheral nerves to the central nervous system (CNS), where it induces non-suppurative encephalomyelitis characterized by neuronal degeneration, gliosis, and perivascular cuffing [1, 8, 57]. The virus’s ability to hijack host cellular machinery, including metabolic pathways, endomembrane trafficking systems, and autophagy-lysosomal networks, presents both a formidable challenge and a unique opportunity for targeted intervention [31, 32, 38, 74]. Recent advances have illuminated multiple druggable nodes in the PHEV replication cycle, ranging from host energy metabolism and stress response pathways to viral entry mechanisms and egress strategies. This section provides an exhaustive analysis of metabolic and antiviral interventions that have been evaluated against PHEV, with emphasis on their mechanisms of action, experimental evidence from in vitro and in vivo models, and translational potential for field application.

Metabolic Reprogramming as an Antiviral Achilles’ Heel

The reliance of coronaviruses on host cell metabolism for productive replication has emerged as a central paradigm in antiviral research, and PHEV is no exception. A landmark study by Jia et al. (2025) demonstrated that thapsigargin (Tg), a plant-derived sesquiterpene lactone traditionally used as a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, exhibits potent antiviral activity against PHEV both in vitro and in vivo [74]. Critically, Tg treatment did not directly inactivate virions but rather suppressed viral genomic RNA replication and transcription by fundamentally altering intracellular metabolic flux. Transcriptome profiling coupled with glycolysis and mitochondrial stress testing revealed that Tg simultaneously suppresses glycolysis and oxidative phosphorylation (OXPHOS), effectively starving the virus of the energy and biosynthetic precursors required for its replication [74]. This metabolic blockade was shown to be essential for the antiviral effect, as supplementation with key metabolic intermediates partially rescued PHEV replication. The significance of this finding extends beyond PHEV, as Tg also inhibited three other swine coronaviruses, suggesting that targeting host energy metabolism represents a broad-spectrum antiviral strategy against coronaviruses [74]. The World Health Organization (WHO) has recognized metabolic reprogramming as a promising avenue for pandemic preparedness, and these data provide a strong rationale for further development of Tg or its analogs as therapeutic agents against neurotropic betacoronaviruses.

The interplay between PHEV replication and cellular energy metabolism is further underscored by the virus’s induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Shi et al. (2021) demonstrated that PHEV infection activates all three branches of the UPR, PERK, IRE1α, and ATF6, both in vitro in mouse neuroblastoma (N2a) cells and in vivo in mouse brain tissue [31]. The PERK/PKR-eIF2α axis emerged as a particularly critical antiviral pathway: phosphorylation of eIF2α attenuates global protein translation, thereby limiting the production of viral proteins, while simultaneously promoting the formation of stress granules (SGs), which further sequester viral mRNAs and inhibit replication [31]. Importantly, the double-stranded RNA-dependent protein kinase (PKR) acted cooperatively with PERK to amplify eIF2α phosphorylation, creating a dual-host defense mechanism against PHEV. These findings identify PERK, PKR, and eIF2α as attractive host targets for antiviral drug development. Pharmacological activation of the PERK pathway using agents such as salubrinal or guanabenz, which inhibit eIF2α dephosphorylation, could theoretically enhance this endogenous antiviral response and suppress PHEV replication [31, 73]. Conversely, the virus’s ability to manipulate the UPR for its own benefit, a common strategy among coronaviruses, suggests that careful timing and dosing of such interventions will be critical to avoid unintended proviral effects.

Natural Compounds with Anti-PHEV Activity: Resveratrol and Trehalose

The search for safe, cost-effective antiviral agents has led to the evaluation of several natural compounds against PHEV, with resveratrol and trehalose showing particular promise. Resveratrol, a polyphenolic phytoalexin found in grapes and berries, is renowned for its neuroprotective and antiviral properties. Liu et al. (2022) conducted a comprehensive evaluation of resveratrol’s anti-PHEV activity using both in vitro N2a cell models and an in vivo mouse infection model [48]. Resveratrol treatment reduced PHEV titers in a dose-dependent manner, with a 50% inhibitory concentration (EC₅₀) of 6.24 μM and a selectivity index indicating favorable antiviral versus cytotoxic activity. In pre-treatment assays, 10 μM resveratrol achieved a >70% reduction in viral protein expression and mRNA copy number, along with a 19-fold reduction in virus titer [48]. The compound was also effective when added after viral adsorption (56% reduction in mRNA, 8-fold titer reduction) and in direct virus inactivation assays (46% mRNA reduction, 4-fold titer reduction), suggesting multiple mechanisms of action. In vivo, intranasal administration of resveratrol proved superior to oral gavage in inhibiting PHEV replication in mouse brains, highlighting the importance of delivery route for CNS infections [48]. The mechanisms underlying resveratrol’s anti-PHEV activity are likely multifactorial and may include modulation of sirtuin pathways, inhibition of viral RNA-dependent RNA polymerase, and attenuation of virus-induced oxidative stress and inflammation.

Trehalose, a naturally occurring non-reducing disaccharide with lysosomotropic properties, has garnered attention for its ability to cross the blood-brain barrier and its established safety profile in humans and animals. Ai et al. (2022) demonstrated that trehalose treatment significantly reduced PHEV replication in mouse neuroblastoma cells and in the brains of infected mice [49]. The antiviral effect was strictly dependent on the expression of progranulin (PGRN), a lysosomal glycoprotein that plays a critical role in lysosomal biogenesis and function. This finding is particularly intriguing given that PHEV infection itself reduces full-length PGRN levels while paradoxically enhancing its lysosomal targeting [42]. Wang et al. (2025) further elucidated this mechanism, showing that PHEV-induced enhancement of PGRN lysosomal trafficking involves the concurrent harnessing of two distinct delivery pathways, leading to increased vacuolar-type ATPase recruitment, intensified lysosomal acidification, and subsequent Arl8b-dependent lysosomal exocytosis that facilitates viral release [42]. Trehalose appears to counteract this process by restoring PGRN expression and rescuing lysosomal structural abnormalities, thereby inhibiting viral egress [49, 52]. The dual role of PGRN, as both a restriction factor for PHEV entry and a facilitator of viral release when aberrantly trafficked, underscores the complexity of targeting lysosomal pathways for antiviral therapy. The Food and Agriculture Organization (FAO) has noted the potential of such host-directed therapies for managing swine viral diseases, particularly in resource-limited settings where conventional vaccines may be unavailable.

RNA Interference-Based Strategies: siRNA and shRNA Approaches

RNA interference (RNAi) represents a highly specific and potent antiviral strategy that has been extensively explored against PHEV. Early work by Lan et al. (2011) demonstrated that small interfering RNAs (siRNAs) targeting the spike glycoprotein and replicase polyprotein genes could efficiently inhibit PHEV replication in PK-15 cells [60]. Four species of siRNAs, prepared by in vitro transcription and transfected into cells prior to PHEV infection, conferred remarkable protection: cytopathic effect (CPE) was markedly reduced, viral antigen staining was observed in only a few siRNA-treated cells compared to widespread positivity in controls, and infectious virus production was suppressed by 18- to 32-fold as measured by hemagglutination (HA) test and 93- to 494-fold as measured by TCID₅₀ assay [60]. Quantitative real-time RT-PCR confirmed a 53–91% reduction in viral genome copy number. The high degree of inhibition achieved with these siRNAs highlights the vulnerability of PHEV to RNAi-mediated silencing and suggests that the spike and replicase genes are critical for viral fitness.

Subsequent work by the same group focused on short hairpin RNAs (shRNAs) targeting the nucleocapsid (N) gene, which encodes a multifunctional protein essential for viral RNA packaging and, as later discovered, immune evasion [30, 61]. Two shRNA expression plasmids, shN1 and shN2, were generated and transiently transfected into PK-15 cells. Both constructs inhibited viral RNA replication, with shN2 showing superior efficacy. Stable transfection of shN2 produced two PK-15 cell clones (shN2-1 and shN2-2) that maintained effective inhibition of PHEV replication for up to 120 hours post-infection, as determined by TCID₅₀ assay and CPE analysis [61]. The sustained protection afforded by stable shRNA expression is particularly relevant for potential therapeutic applications, such as the development of genetically modified pigs with enhanced resistance to PHEV. However, the practical challenges of delivering RNAi therapeutics to the CNS, including the blood-brain barrier, neuronal uptake efficiency, and potential off-target effects, remain substantial hurdles that must be overcome before these strategies can be translated to field use.

Targeting Viral Entry and Egress Pathways

The initial steps of the PHEV replication cycle, attachment, entry, and intracellular trafficking, offer multiple targets for antiviral intervention. PHEV enters host cells via clathrin-mediated endocytosis (CME) in a dynamin-, cholesterol-, and pH-dependent manner that requires the small GTPases Rab5 and Rab7 for productive infection [36]. Pharmacological inhibitors of CME, such as chlorpromazine or dynasore, or agents that disrupt cholesterol-rich membrane microdomains, such as methyl-β-cyclodextrin, could theoretically block PHEV entry, although their systemic toxicity limits clinical applicability. More promising are approaches targeting the specific receptors and attachment factors utilized by PHEV. Recent studies have identified dipeptidyl peptidase 4 (DPP4) as a candidate binding target for the PHEV spike protein, with both porcine and murine DPP4 showing high affinity for the receptor-binding domain (RBD) [25]. Structural analysis revealed that the PHEV spike binds to the outer surface of blades IV and V of the DPP4 β-propeller domain, and that N-glycosylation at residues N229 and N321 (human DPP4 numbering) acts as a shield that modulates RBD-DPP4 interaction [25]. Removal of these N-glycosylations profoundly enhanced viral invasion, suggesting that DPP4 glycosylation is a determinant of species barrier formation. Concurrently, Dufloo et al. (2025) identified dipeptidase 1 (DPEP1) as a functional receptor for PHEV, demonstrating that the virus does not require sialic acid for entry and that the RBD shares no detectable sequence homology with those of closely related embecoviruses [28]. Cryo-electron microscopy revealed that the PHEV spike samples open and closed conformations at steady state, a feature distinct from other embecoviruses. The X-ray structure of the RBD/DPEP1 complex showed that the elements involved in receptor binding are conserved across embecoviruses, revealing a striking versatility of the RBD to accommodate highly variable sequences that confer novel receptor specificities [28]. These receptor discoveries

Immune Response and Host-Virus Interactions

The pathogenesis of porcine hemagglutinating encephalomyelitis virus (PHEV) is a masterclass in host-virus molecular warfare, characterized by a sophisticated and multi-layered interplay between viral subversion strategies and the host's innate and adaptive immune defenses. As a neurotropic betacoronavirus with the unique ability to invade the central nervous system (CNS) via peripheral nerves, PHEV has evolved a remarkable arsenal of mechanisms to evade, delay, and ultimately exploit the host immune response, enabling its replication and dissemination within the immunologically privileged environment of the CNS. The ensuing immune response is a double-edged sword: while essential for viral clearance, the accompanying neuroinflammation contributes significantly to the pathological hallmarks of non-suppurative encephalomyelitis, neuronal degeneration, and demyelination observed in affected piglets [1, 6, 12, 57].

Innate Sensing and the Interferon Response: A Delayed and Subverted Alarm

The initial host defense against PHEV infection hinges on the rapid detection of viral pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Upon entry into susceptible cells, including neurons and respiratory epithelial cells, PHEV replication generates double-stranded RNA (dsRNA) intermediates, which are potent triggers of the antiviral response. The primary sensor for these viral products in the context of PHEV infection is the cytosolic retinoic acid-inducible gene I (RIG-I) [30]. Studies have demonstrated that PHEV infection activates the RIG-I-mitochondrial antiviral signaling (MAVS) pathway; however, the virus has evolved a critical countermeasure to disarm this immediate response.

Immune Evasion by the Nucleocapsid (N) Protein: The PHEV nucleocapsid (N) protein emerges as a central immunosuppressive factor, targeting the RIG-I signaling cascade at two distinct but synergistic points [30]. Mechanistically, the C-terminal domain (CTD) of the N protein directly binds to the caspase activation and recruitment domain (CARD) of RIG-I. This physical interaction competitively inhibits the binding of TRIM25, an E3 ubiquitin ligase responsible for catalyzing the K63-linked polyubiquitination of RIG-I, a post-translational modification essential for its activation and downstream signaling. This silencing of RIG-I activation effectively dampens the initial wave of interferon (IFN) induction. Concurrently, the N protein directly targets the master transcription factor interferon regulatory factor 3 (IRF3) by disrupting its homodimerization, phosphorylation, and subsequent nuclear translocation. This dual blockade, inhibiting the sensor and disabling its primary effector, creates a profound immunological vulnerability within the host. The consequence is a delayed and muted type I interferon (IFN-I) response, which is driven not by the early-acting IRF3 but by a later, less efficient induction of IRF7-dependent IFN [30]. This ‘immune-permissive window’ allows PHEV to replicate unchecked for over 12 hours post-infection before a weak antiviral state is established, a strategy that is critical for its neurotropic pathogenesis. This mechanism of immune evasion, orchestrated by a structural protein, is distinct from the nonstructural protein-mediated evasion strategies commonly observed in other coronaviruses [30].

The Unfolded Protein Response (UPR) and Stress Granules: PHEV replication within the endoplasmic reticulum (ER) induces significant ER stress, activating the unfolded protein response (UPR) [31, 73]. The virus triggers all three branches of the UPR, PERK, IRE1, and ATF6, both in vitro and in vivo [31]. While the UPR is often co-opted by viruses to support replication, the PERK/PKR-eIF2α axis functions as a potent host antiviral mechanism. The activation of PERK and the interferon-induced double-stranded RNA-dependent protein kinase (PKR) leads to the phosphorylation of eukaryotic initiation factor 2α (eIF2α). This phosphorylation event globally attenuates cap-dependent protein translation, effectively restricting the viral protein synthesis required for replication [31]. Moreover, phosphorylated eIF2α promotes the assembly of stress granules (SGs), cytoplasmic aggregates of stalled mRNA and RNA-binding proteins. The formation of SGs further suppresses PHEV replication, representing a multifaceted host stress response that the virus must overcome to establish a productive infection [31].

Subversion of Cellular Catabolic and Exocytic Pathways

PHEV has developed a sophisticated strategy not only to evade immune detection but also to hijack fundamental cellular processes for its own benefit, particularly autophagy and the exosomal pathway, which are intricately linked to immune signaling.

Autophagy: A Hijacked and Atypical Process: PHEV’s relationship with autophagy is complex and non-canonical. The virus triggers the formation of autophagosomes in neuronal cells, a process that is necessary for efficient viral replication [38, 73]. However, this is an atypical autophagy: while PHEV induces the lipidation of LC3 and autophagosome formation, it simultaneously blocks the fusion of these autophagosomes with lysosomes, as evidenced by the failure to degrade the autophagy receptor p62 [38]. This creates a permissive environment for viral replication, as the pharmacological inhibition of autophagy (e.g., with 3-MA) surprisingly increases viral replication, while induction (e.g., with rapamycin) reduces it [38]. The initiation of this autophagy pathway is remarkably independent of the canonical initiator UNC-51-like kinase 1 (ULK1). PHEV infection stalls mTORC1-regulated ULK1 activation and instead drives autophagosome biogenesis through an AMPK-driven, BECN1-dependent pathway [34]. This ULK1-independent, non-canonical autophagy represents a novel mechanism that bypasses a critical host regulatory circuit.

Exosomal Egress and Intercellular Communication: A particularly insidious mechanism of immune evasion is PHEV’s use of the multivesicular body (MVB)-derived exosomal pathway for viral egress and cell-to-cell spread [2, 32]. Specific PHEV isolates, such as VW572, exploit this pathway to release infectious virions within exosomes, which can fuse with the plasma membrane of uninfected cells, including non-permissive cells like microglia [2, 32]. This mode of transmission provides a cloak of invisibility from the host immune system, as the virus is shielded within host-derived vesicles and avoids exposure to neutralizing antibodies and extracellular complement factors. Furthermore, these virus-modified exosomes carry a diverse cargo, including viral RNA, proteins, and host innate immunity sensors, serving as a delivery system that can induce an innate response in uninfected bystander cells while simultaneously facilitating viral dissemination independent of direct cell-surface receptor engagement [32]. The N protein’s role is again central here, as it is involved in modulating the cellular machinery to facilitate this exosomal release [32].

Humoral and Cell-Mediated Adaptive Immunity

The adaptive immune response is critical for eventual viral clearance and long-term protection. In experimentally infected CDCD pigs, a robust antibody response is detected by 10 days post-inoculation (dpi), characterized by a sequential appearance of IgM (7 dpi) followed by IgA and IgG (10 dpi) [8, 11]. The neutralizing antibody response is primarily directed against the spike (S) protein, and the development of an IgG ELISA based on the S1 domain has proven a valuable tool for serosurveillance, revealing a high global seroprevalence of PHEV [9, 26, 53]. This humoral immunity is critical for protection, and passive transfer via maternal antibodies from immune sows is the primary mechanism for protecting neonatal piglets from clinical disease [6, 18]. The isotype profile of the response can be shaped by vaccination strategies, with inactivated vaccines eliciting a Th2-biased IgG1/IL-4 response, while DNA vaccines generate a Th1-biased IgG2a/IFN-γ response [66]. The first-ever DNA vaccine candidate, targeting the receptor-binding domain of the spike protein fused to an IgG1 Fc fragment (RBD+GEL01), successfully induced high neutralizing antibody titers (up to 1:147 in piglets) and a Th1-biased cellular response, which was correlated with restricted viral neuroinvasion and reduced brain pathology upon challenge [69].

Cellular Immunity and the Cytokine Milieu: Infection with PHEV in grower pigs is characterized by a significant IFN-α response in serum as early as 3 dpi, followed by a notable infiltration of CD8+ cytotoxic T lymphocytes (CTLs) into the airways and an increase in the frequency of CTLs in peripheral blood, peaking at 21 dpi [1, 8, 11]. This CTL response is likely crucial for eliminating virus-infected neurons. The induction of systemic inflammation is further evidenced by elevated levels of IL-8 (CXCL8) at 10 and 15 dpi, coinciding with the period of viral resolution [8]. In the CNS, the response is a double-edged sword. PHEV infection triggers a robust activation of astrocytes (astrogliosis), characterized by increased expression of glial fibrillary acidic protein (GFAP) [43]. Critically, PHEV does not directly infect astrocytes; instead, their activation is a paracrine event driven by IFN-β secreted from infected neurons. This astrocyte activation, while part of the neuroprotective response, can contribute to neuroinflammation and pathology. Transcriptomic analyses of infected respiratory epithelium reveal a potent activation of antiviral signaling, with significant upregulation of interferon-stimulated genes (ISGs) like RSAD2, MX1, IFIT, and ISG15, alongside pro-inflammatory chemokines such as CCL5 and CXCL10 [39]. This local inflammatory milieu is essential for coordinating the immune response but also contributes to the observed mucociliary disruption and tissue damage [1, 8, 39].

Receptor Usage, Species Tropism, and Implications for Immune Control

Understanding PHEV entry is fundamental to host-virus interactions. While sialic acid acts as an important attachment factor, PHEV utilizes at least two proteinaceous receptors for entry into host cells [10, 23]. The tetraspanin CD81 is utilized by the PHEV-VW572 isolate for entry into neuronal cells, highlighting isolate-specific differences in receptor usage [2]. More recently, dipeptidyl peptidase 4 (DPP4) has been identified as a functional binding target for the PHEV spike protein, with the receptor-binding domain (RBD) interacting with specific glycosylation sites (e.g., N229 and N321) on DPP4 [25]. Critically, the N-glycosylation of DPP4 serves as a species barrier, and the ability of PHEV to tolerate orthologs from pigs and mice is a key determinant of its host range expansion [25]. This is further complicated by the discovery that dipeptidase 1 (DPEP1) acts as a functional receptor, indicating that PHEV has evolved a remarkable versatility to utilize different receptors for entry, independent of sialic acid in some contexts [28]. This receptor diversity, including the integrin α5β1-mediated cytoskeletal remodeling necessary for invasion, suggests complex and potentially redundant entry pathways that complicate the development of universal entry-blocking therapeutics [35, 41].

Regulatory Interference by MicroRNAs

PHEV infection profoundly alters the host microRNA (miRNA) transcriptome, co-opting these small non-coding RNAs to fine-tune the cellular environment for its own benefit. The virus upregulates a suite of miRNAs that target key host factors involved in neuronal morphogenesis and antiviral defense. For example, the upregulation of miR-142-5p targets and suppresses the expression of Ulk1, a kinase vital for neurite outgrowth and endosomal trafficking, thereby contributing to the neurodegenerative phenotype of the infection [55]. Conversely, PHEV promotes its own proliferation by upregulating miR-142a-3p and miR-21a-5p, which directly repress the expression of the negative regulators Rab3a and Caskin1, respectively [54, 56]. Concurrently, the host counters by upregulating the antiviral miR-10a-5p, which targets Syndecan 1 (SDC1), a cell surface proteoglycan that PHEV likely requires for attachment, thereby restricting viral replication [50]. This intricate dance of miRNA modulation underscores the complex regulatory battleground at the post-transcriptional level, where the virus and host vie for control over the cellular machinery.

The Immune Landscape in Respiratory Disease

Recent evidence has solidified PHEV’s role as a significant contributor to the porcine respiratory disease complex (PRDC) [1, 5, 13]. In respiratory infections, PHEV replicates in the upper respiratory tract epithelium, causing necrotizing bronchitis and bronchiolitis, and elicits a robust local inflammatory response characterized by significant macrophage infiltration [1]. The virus has been detected in respiratory cases with high frequency (83.33% of pigs in one study) and is often found in coinfections with other pathogens like PRRSV [1, 44]. The emergence of respiratory variant PHEV (rvPHEV) lineages in China, which cause exclusively respiratory symptoms, highlights the evolutionary plasticity of the virus. These rvPHEV strains frequently have mutations in the hemagglutinin-esterase (HE) gene, including loss of the cysteine required for HE dimerization and amino acid substitutions at the carbohydrate-binding site, an adaptation reminiscent of early human coronavirus adaptation to the respiratory tract [5]. This shift towards a respiratory niche likely alters the host-virus interaction at the mucosal interface, potentially affecting the kinetics of immune recognition and the type of inflammatory response elicited, shifting focus from a purely neuropathic to a more pneumopathic pathogenesis.

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