Pseudorabies Virus

Taxonomy and Genomic Organization of Pseudorabies Virus

Taxonomic Classification and Nomenclature

Pseudorabies virus (PRV), the etiological agent of Aujeszky’s disease (AD), occupies a well-defined position within the Herpesviridae family. It is classified under the subfamily Alphaherpesvirinae, genus Varicellovirus, and is officially designated as Suid alphaherpesvirus 1 (SuHV-1) by the International Committee on Taxonomy of Viruses (ICTV) [1, 12]. This taxonomic assignment places PRV in close evolutionary proximity to other significant alphaherpesviruses, including Human alphaherpesvirus 1 (herpes simplex virus type 1, HSV-1), Human alphaherpesvirus 3 (varicella-zoster virus, VZV), and Bovine alphaherpesvirus 1 (BoHV-1) [5, 7, 10]. The genus Varicellovirus is distinguished by its members’ capacity to establish latency in sensory ganglia and cause a range of clinical manifestations from mild mucocutaneous lesions to severe neurological disease in their respective hosts.

Phylogenetic analyses based on complete genomic sequences and individual glycoprotein genes have consistently resolved PRV isolates into two major clades. Clade 1 comprises primarily classical, historically circulating strains, including the prototypical vaccine strain Bartha-K61 and early European isolates. Clade 2, further subdivided into clade 2.1 and clade 2.2, encompasses the more recently emerged variant strains that have dominated the epidemiological landscape since the early 2010s [20, 26]. Clade 2.2 represents the currently predominant genotype worldwide and is most frequently associated with cross-species transmission events, including human infections [20]. This phylogenetic bifurcation reflects substantial genetic divergence driven by ongoing recombination, point mutations, and selective pressures from vaccination programs, particularly in China where the Bartha-K61 vaccine was widely deployed before the emergence of vaccine-escape variants [3, 14, 19].

The taxonomic distinctiveness of PRV is further underscored by its broad host tropism, which is exceptional among the alphaherpesviruses. While the natural host is the domestic pig (Sus scrofa domesticus) and wild boar, PRV can productively infect a remarkably wide range of mammals, including cattle, sheep, goats, dogs, cats, rodents, rabbits, and, as increasingly documented, humans [1, 5, 8, 11]. This capacity to overcome species barriers is a hallmark of PRV biology and is intimately linked to the structural and functional properties of its genomic repertoire, particularly the envelope glycoproteins that mediate entry into host cells.

Genome Architecture and Physical Organization

The PRV genome is a linear, double-stranded DNA molecule approximately 145 kilobase pairs (kbp) in length, with a G+C content of approximately 73.5%, among the highest of any herpesvirus [1, 2, 31]. The genome is organized into two unique regions: a long unique segment (UL) of approximately 110 kbp and a short unique segment (US) of approximately 9 kbp. These unique regions are flanked by inverted repeat sequences. The UL region is bracketed by the terminal repeat long (TRL) and internal repeat long (IRL) elements, while the US region is flanked by the internal repeat short (IRS) and terminal repeat short (TRS) elements. This arrangement, denoted as TRL–UL–IRL–IRS–US–TRS, creates a genome structure that can invert the US region relative to UL, generating four possible genomic isomers, all of which are encapsidated at equimolar ratios [1, 31].

The genome encodes an estimated 70 to 100 distinct proteins, including both structural and non-structural components [2, 27]. Early genomic annotation efforts, based on sequence homology with HSV-1 and functional studies, identified approximately 72 open reading frames (ORFs) [34]. However, subsequent high-depth transcriptomic analyses employing Illumina, PacBio, and Oxford Nanopore sequencing platforms have substantially revised this estimate, revealing a far more complex transcriptional landscape than previously appreciated [27, 34, 35].

Moldován et al. (2018) utilized a multi-platform sequencing approach (Illumina HiScanSQ, PacBio RS-II, and Oxford Nanopore MinION) to identify 19 previously unknown putative protein-coding genes, all of which are 5′-truncated forms of longer annotated PRV genes [27]. Additionally, 19 non-coding RNAs were detected, including 5′- and 3′-truncated transcripts without in-frame ORFs, antisense RNAs, and RNA molecules originating from genomic regions previously thought to be transcriptionally silent [27]. In silico analysis further identified 145 upstream ORFs (uORFs), many located on longer 5′ isoforms of transcripts, suggesting a previously unrecognized layer of translational regulation [27].

The lytic replication origins constitute critical cis-acting elements within the genome. PRV possesses two origins of lytic replication: OriL, located within the UL region between the UL21 and UL22 genes, and OriS, present in the inverted repeat sequences flanking US [34, 35]. OriL is a 130–140 bp palindromic sequence that serves as the binding site for the origin-binding protein (UL9). Notably, transcriptomic studies have identified a highly abundant polyadenylated non-coding RNA, designated CTO-S (coterminal transcript short), encoded in close proximity to OriL, whose transcription is fully dependent on the viral transactivator IE180 [35]. This observation suggests a functional interplay between transcription and replication machineries at this genomic locus, potentially influencing the regulation of DNA synthesis [35].

Capsid, Tegument, and Envelope Architecture at the Molecular Level

The virion architecture of PRV conforms to the canonical herpesvirus structural paradigm: an electron-dense DNA core enclosed within an icosahedral capsid, surrounded by a proteinaceous tegument layer, and finally enveloped by a lipid bilayer studded with viral glycoproteins [1, 6].

Capsid Structure: The PRV capsid, approximately 125 nm in diameter, is composed of 162 capsomers arranged in an icosahedral lattice with T=16 triangulation number. Cryo-electron microscopy (cryo-EM) structures of the PRV A-capsid (empty capsid) and C-capsid (DNA-containing capsid) have been resolved to near-atomic resolution (approximately 3.5–4.0 Å), providing unprecedented detail of capsid assembly and organization [6]. The major capsid protein, UL19 (VP5), forms both hexons and pentons, while the triplex proteins UL38 (VP19C) and UL18 (VP23) stabilize intercapsomer connections. A unique feature revealed in the PRV C-capsid is the decoration of the portal complex, a dodecameric ring of UL6 protein located at a single capsid vertex, with capsid-associated tegument complexes (CATCs) [6]. This structural detail, which is highly reminiscent of but not identical to that observed in HSV-1, provides a molecular interface for tegument attachment and uncoating during viral entry.

Tegument Organization: The tegument is a structurally dynamic protein layer that bridges the capsid and envelope. It contains at least 15 distinct viral proteins, including the inner tegument proteins (e.g., UL36/VP1/2, UL37), the outer tegument proteins (e.g., UL11, UL16, UL21, UL46, UL47, UL48, UL49), and the US3 kinase [10, 22]. The tegument is not simply a passive scaffold; it plays active roles in capsid transport, nuclear targeting, modulation of host antiviral responses, and virion morphogenesis. For instance, the inner tegument protein pUL21 interacts with the cytoplasmic dynein light chain Roadblock-1 via its carboxyl terminus, facilitating retrograde axonal transport of the capsid along microtubules, an essential step for neuroinvasion [22]. The tegument also contains the UL13 protein, a serine/threonine kinase that functions as an antagonist of the cGAS-STING signaling axis. UL13 recruits the cellular E3 ubiquitin ligase RNF5 to promote K27-/K29-linked ubiquitination and proteasomal degradation of STING, thereby suppressing type I interferon production [4].

Envelope Glycoproteins: The viral envelope is derived from host cell membranes and contains at least 11 distinct glycoproteins, many of which are essential for attachment, entry, cell-to-cell spread, and immune evasion. The core fusion machinery comprises four essential glycoproteins: gB (the fusion protein), gH/gL (the regulatory heterodimer), and gD (the receptor-binding protein) [23]. The interaction between gD and cellular receptors, primarily nectin-1, but also nectin-2 and heparan sulfate proteoglycans, triggers a conformational cascade that activates gB-mediated membrane fusion [23, 28]. The crystal structure of PRV gD in both its free and nectin-1-bound states, solved by Li et al. (2017), reveals a V-set immunoglobulin-like fold with a unique hydrophobic loop insertion that mediates receptor engagement [28]. Notably, the membrane-proximal region of gD negatively regulates receptor binding; removal of this region significantly increases affinity for nectin-1, suggesting an autoinhibitory mechanism [28].

Beyond the core fusion machinery, several other glycoproteins contribute to virulence and pathogenesis. Glycoprotein E (gE) and glycoprotein I (gI) form a heterodimeric Fc receptor that facilitates cell-to-cell spread and immune evasion by binding the Fc region of host antibodies [2, 30]. Glycoprotein C (gC) mediates initial attachment to heparan sulfate proteoglycans on the cell surface, while glycoprotein N (gN) and glycoprotein M (gM) are involved in virion maturation and egress [23]. The major immunodominant glycoprotein gB, which elicits potent neutralizing antibody responses, is a class III fusion protein that undergoes extensive conformational rearrangement during membrane fusion. Structural studies have identified two classes of protective antibodies targeting gB: complement-dependent neutralizing antibodies that bind the crown region (domain IV), and a complement-independent, broadly neutralizing antibody (1H1) that binds the base of domain I near the fusion loops [29]. This structural and immunological characterization has direct implications for subunit vaccine design.

Genomic Variation, Recombination, and Evolution

The PRV genome is characterized by a relatively high degree of genetic plasticity compared to other alphaherpesviruses such as HSV-1 and VZV [5]. This plasticity is manifested through point mutations, insertions, deletions, and, most significantly, recombination events that occur both between different PRV genotypes and between vaccine and wild-type strains [3, 14, 19, 20]. The emergence of variant PRV strains in China since 2011, exemplified by isolates such as JSY7, JSY13, FJ62, and hSD-1/2019, exemplifies the evolutionary dynamism of this virus [8, 14, 19, 31].

Recombination analysis has provided direct evidence for natural interclade recombination. The JSY13 strain, isolated from a Bartha-K61-vaccinated swine farm, was shown to be a recombinant between the minor parental genotype I (Bartha-like) and the major parental genotype II (JSY7-like) [14]. Similarly, the FJ62 strain isolated in Sichuan province exhibited a mosaic genome: its gB gene was 100% identical to the wild boar-derived MY-1 strain (genotype I), while its gC, gD, and gE genes clustered with contemporary Chinese porcine isolates (genotype II) [19]. Such recombinant strains often display intermediate virulence phenotypes, as demonstrated by the moderate pathogenicity of JSY13 compared to the highly virulent JSY7 [14]. The detection of recombination between live attenuated vaccine strains and field viruses raises significant concerns for vaccination strategies, as vaccine-derived genetic material can be incorporated into circulating virulent strains, potentially generating novel pathogens with altered antigenicity and virulence [3, 14].

Whole-genome sequencing of variant strains has identified specific genetic markers associated with increased pathogenicity and immune escape. The gE protein of Chinese variant strains, including JSY7, JSY13, and HN1201, contains a characteristic aspartate insertion at position 57 (Asp57) in the extracellular domain, a signature that is absent in classical strains and the Bartha-K61 vaccine [14, 31, 32]. Additional mutations in gB, gC, and gD have been correlated with enhanced virulence, altered cell tropism, and reduced neutralization by vaccine-induced antibodies [20, 26]. This genetic divergence may explain the failure of the Bartha-K61 vaccine to provide complete protection against variant strains in the field [1, 3, 24].

The evolutionary dynamics of PRV are further shaped by selection pressures exerted by host immune responses. Analysis of glycoprotein sequences across diverse PRV strains has identified positively selected amino acid sites in gB, gC, gD, and gE that are predicted to be associated with adaptation to new hosts and immune evasion [20]. The global population size of clade 2.2 (variant PRV) has increased markedly since 2011, with an effective reproduction number greater than 1 from 2011 to 2016, indicating sustained transmission and expansion [20]. This expansion underscores the ongoing risk of cross-species transmission and the need for continuous genomic surveillance.

Transcriptomic Complexity and Non-Coding RNA Repertoire

The PRV transcriptome is far more complex than initially appreciated. High-resolution RNA-sequencing studies have revealed that nearly the entire viral genome is transcribed during lytic infection, with the exception of loci within the large internal and terminal repeats and several small intergenic repetitive sequences [34]. The transcription program is temporally regulated, with immediate-early (IE), early (E), and late (L) kinetic classes, governed by the sole immediate-early transactivator IE180 [15]. IE180 is a multifunctional protein that activates transcription from all viral promoters and is essential for lytic replication [34].

Alternative polyadenylation is a prominent feature of PRV gene expression, with single-base resolution mapping revealing alternative polyadenylation sites in numerous genes [34]. The ep0 gene, which encodes the early protein 0 (EPO), a key transactivator and virulence determinant, undergoes alternative splicing, generating transcript isoforms with distinct 3′ untranslated regions (UTRs) [34]. This splicing event may influence mRNA stability, translational efficiency, or subcellular localization.

The discovery of 19 non-coding RNAs (ncRNAs) and 50 distinct transcript length isoforms has fundamentally altered our understanding of PRV gene regulation [27]. Among these ncRNAs, the CTO-S transcript is particularly noteworthy. This highly abundant polyadenylated ncRNA is expressed from a locus between UL21 and UL22, its transcription is strictly dependent on IE180, and it overlaps the OriL replication origin [35]. CTO-S is not a microRNA precursor, and its functional role remains under investigation. However, its close genomic proximity to a replication origin and its temporal expression pattern suggest a potential role in modulating DNA replication or chromatin structure at the origin [35].

Additionally, two novel transcripts, CTO-L (a long readthrough product of UL21) and a short transcript originating from a previously uncharacterized region, were identified as overlapping the replication origin [27]. The presence of stable polyadenylated transcripts spanning replication origins raises the possibility of regulatory crosstalk between the transcription and replication machineries, a phenomenon observed in other herpesviruses but not previously documented for PRV [34, 35].

Genomic Basis for Vaccine Vector Design and Genetic Manipulation

A significant practical implication of PRV genomic organization is its utility as a viral vector for vaccine development. PRV possesses extensive non-essential regions that can accommodate foreign genes without compromising viral replication [2]. The capacity to delete virulence-associated genes, such as gE, gI, TK (thymidine kinase), and US3, while retaining immunogenicity has enabled the construction of safe, attenuated recombinant strains [16, 21, 24, 25]. For example, the gE/gI deletion serves as a DIVA (Differentiating Infected from Vaccinated Animals) marker, allowing serological discrimination between vaccinated and naturally infected animals [2, 17, 24].

The PRV vector platform has been exploited to express immunogens from other swine pathogens, including classical swine fever virus (CSFV) E2 protein and African swine fever virus (ASFV) CD2v protein, demonstrating the feasibility of multivalent vaccines [16, 21]. The insertion site for foreign genes is typically the intergenic region between gG and gD, which is transcriptionally active but non-essential for replication [21]. Advances in genome editing technologies, particularly CRISPR/Cas9-mediated homologous recombination, have dramatically accelerated the construction of recombinant PRV BACs (bacterial artificial chromosomes), achieving recombination efficiencies as high as 86% [33]. These tools enable rapid, precise manipulation of the PRV genome for basic research and translational applications.

The genetic stability of recombinant PRV strains is a critical consideration for vaccine safety. Serial passaging studies have demonstrated that foreign genes inserted into the gG-gD intergenic region are stably maintained for at least 20 passages in cell culture [16]. However, the potential for recombination with wild-type strains, as documented for the Bartha-K61 vaccine, necessitates careful biosafety assessment [14]. The discovery of natural recombinants between vaccine and field strains underscores the importance of monitoring for genomic reversion and the emergence of novel chimeric viruses in vaccinated populations [3, 14].

Implications of Genomic Variation for Zoonotic Potential

The recent recognition of PRV as a zoonotic pathogen capable of causing severe encephalitis in humans has intensified interest in the genomic determinants of cross-species transmission [5, 8, 9, 13, 18]. Since 2017, at least 25 human cases of PRV infection have been reported in China, predominantly in individuals with occupational exposure to swine (e.g., butchers, pork vendors, farmers) [5]. The first human PRV isolate, hSD-1/2019, was obtained from the cerebrospinal fluid of an encephalitis patient and was found to be genetically closest to contemporary Chinese PRV variant strains [8]. Phylogenetic analysis placed hSD-1/2019 within clade 2.2, the same lineage associated with the 2011 swine outbreak [8, 20].

Comparative genomic analysis of human-derived PRV isolates and swine-derived variant strains has not identified a specific "zoonotic" signature; rather, human infections appear to be caused by the same genetically heterogeneous variant strains circulating in pig populations [8]. This suggests that

Molecular Pathogenesis of Pseudorabies Virus Infection

Molecular Determinants of Viral Entry and Host Tropism

The initiation of pseudorabies virus (PRV) infection is governed by a sophisticated, multi-step entry process that determines both host range and tissue tropism. PRV, classified within the Alphaherpesvirinae subfamily, genus Varicellovirus, employs a core fusion machinery conserved across the Herpesviridae family, consisting of glycoprotein B (gB), the heterodimeric gH/gL complex, and the receptor-binding glycoprotein D (gD) [1, 23]. Unlike the prototypic human herpes simplex viruses (HSV-1/2), which exhibit stringent dependence on all four glycoproteins for membrane fusion, PRV displays unique mechanistic flexibility; notably, PRV can mediate cell-cell fusion in the absence of gD, a phenomenon not observed with HSV, highlighting fundamental differences in the activation thresholds of their respective fusion machineries [23].

The molecular basis of host cell recognition is primarily dictated by the interaction between gD and cellular receptors, among which nectin-1 is the most effective and widely utilized across multiple alphaherpesviruses [28]. High-resolution structural studies of PRV gD in both its free and nectin-1-bound states have revealed a three-dimensional fold homologous to its HSV counterpart, yet with distinct interface residues that mediate receptor engagement [28]. PRV gD exhibits comparable binding affinities for both swine and human nectin-1, a finding of profound significance given the documented zoonotic potential of PRV variant strains [8, 9, 28]. The structural analysis further demonstrated that removal of membrane-proximal residues from PRV gD paradoxically increases its affinity for nectin-1, suggesting a conformational masking mechanism that regulates receptor accessibility [28]. The fusion process itself is executed by gB, the bona fide fusogen, which contains fusion loops within domain I that insert into the target membrane [29]. Importantly, monoclonal antibody studies have identified two distinct classes of neutralizing antibodies against gB: complement-dependent antibodies targeting the crown region (domain IV) and a rare complement-independent antibody (1H1) that binds the fusion loop-containing domain I, directly neutralizing the virus by interfering with membrane fusion [29]. This structural vulnerability at the base of gB represents a critical target for vaccine design and therapeutic intervention.

Once fusion is accomplished, the viral capsid and associated tegument proteins are delivered into the cytoplasm, where the journey to the nucleus commences. The tegument is a structurally complex layer residing between the capsid and envelope, composed of at least 20 distinct proteins that orchestrate immediate-early events in infection [1, 6].

Tegument-Dependent Intracellular Transport and Neuroinvasion

The alphaherpesvirus lifecycle is distinguished by its ability to infect peripheral neurons and establish latency, a process that demands efficient retrograde axonal transport of viral capsids from axon terminals to the neuronal cell body. PRV has evolved a sophisticated mechanism to hijack the host microtubule motor machinery, primarily dynein. While the tegument protein VP1/2 has long been recognized as a dynein adaptor, the inner tegument protein pUL21 has emerged as a critical facilitator of neuroinvasion [22]. pUL21 physically interacts with the cytoplasmic dynein light chain Roadblock-1, an interaction that is essential for the retrograde transport of PRV capsids in neuronal cells [22]. Deletion or mutation of the carboxyl terminus of pUL21, which is the domain responsible for Roadblock-1 binding, severely impairs retrograde axonal transport in vitro and attenuates neuroinvasion in vivo [22]. This finding underscores the exquisite specialization of PRV tegument proteins for neuronal trafficking, a process that distinguishes pathogenic alphaherpesviruses from less neuroinvasive viruses.

The capsid itself, once transported to the nuclear pore, releases the viral genome into the nucleus for replication. Near-atomic resolution structures of the PRV A-capsid (empty) and C-capsid (DNA-containing) have elucidated the intricate architecture of the capsid shell and the portal complex [6]. The C-capsid portal is decorated with capsid-associated tegument complexes (CATCs), which are thought to link the capsid to the nuclear pore and facilitate genome ejection [6]. The structural homology between PRV and other herpesviruses, including human pathogens, positions PRV as a valuable model for studying herpesvirus assembly and nuclear egress, while also providing a structural platform for developing oncolytic or therapeutic agents [6].

Subversion of Host Antiviral Innate Immunity

PRV devotes a substantial portion of its coding capacity to counteracting the host antiviral response, particularly the type I interferon (IFN-I) system. The virus has evolved a multi-pronged strategy to target each node of the cGAS-STING, RIG-I, and PKR signaling pathways, ensuring efficient replication despite robust innate immune activation [4, 7, 36, 38].

Targeting the cGAS-STING Axis: The tegument protein UL13 functions as a potent antagonist of STING-mediated signaling. UL13 directly interacts with the cyclic dinucleotide (CDN) domain of STING and recruits the host E3 ubiquitin ligase RING-finger protein 5 (RNF5) [4]. This recruitment promotes K27-/K29-linked ubiquitination of STING, targeting it for proteasomal degradation [4]. The functional consequence is a profound suppression of IFN-β production and downstream interferon-stimulated gene (ISG) expression. A PRV mutant lacking UL13 (ΔUL13) is significantly impaired in its ability to antagonize STING, leading to enhanced type I IFN responses and marked attenuation of pathogenicity in a murine model [4]. This mechanism illustrates a novel immune evasion strategy: the virus does not merely block signaling but actively degrades a central adaptor protein.

Suppressing RIG-I and OASL Pathways: The PRV UL24 protein counteracts the antiviral activity of oligoadenylate synthetase-like (OASL) protein. OASL enhances RIG-I-mediated IFN induction in response to DNA viruses like PRV [36]. PRV infection downregulates OASL expression at both mRNA and protein levels. UL24 specifically impairs RIG-I signaling, thereby inhibiting the transcription of IFN and ISGs [36]. Moreover, UL24 reduces RIG-I-induced expression of endogenous OASL in an IRF3-dependent manner, effectively neutralizing this arm of the antiviral response [36].

Inhibition of Stress Granule Formation and Translational Arrest: Viruses often trigger cellular stress responses that lead to the formation of stress granules (SGs), which sequester translationally stalled mRNAs and promote antiviral signaling. PRV infection paradoxically activates the eIF2α kinases PKR and PERK, yet the virus efficiently blocks SG formation by dephosphorylating eIF2α [38]. This dephosphorylation occurs early in infection and is independent of the cellular GADD34/PP1 phosphatase complex, suggesting that PRV encodes its own phosphatase or recruits an alternative host phosphatase [38]. Pharmacological inhibition of PP1 with salubrinal partially restores eIF2α phosphorylation and suppresses viral replication, confirming the functional importance of this evasion strategy [38].

Modulation of Autophagy: Autophagy serves as an intrinsic antiviral defense mechanism, and PRV has evolved to subvert it. PRV infection induces autophagy during the early stages, prior to significant viral protein expression, but as viral proteins accumulate, autophagy is suppressed [42]. The conserved alphaherpesvirus US3 tegument protein is responsible for this suppression, acting through activation of the AKT/mTOR signaling pathway [42]. Inhibition of autophagy increases infectious PRV titers, indicating that the autophagic pathway indeed restricts PRV replication and that its suppression is critical for viral fitness [42].

Systemic Inflammatory Response and the "Mad Itch": In non-natural hosts such as rodents, cattle, and dogs, PRV infection precipitates a characteristic and lethal neuropathic pruritus known as the "mad itch" [10, 41]. This phenomenon is not merely a consequence of viral replication but is driven by a maladaptive systemic inflammatory response. Infection of mice with the virulent PRV-Becker strain induces massive neutrophil infiltration at the inoculation site and dorsal root ganglia (DRGs), accompanied by elevated plasma levels of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), Gro-1, and monocyte chemoattractant protein-1 [41]. Crucially, the attenuated vaccine strain PRV-Bartha also replicates in the peripheral nervous system and spreads to the brain, yet it fails to induce this specific inflammatory cytokine storm, and infected mice do not develop pruritus [41]. This indicates that virulence is not solely determined by replicative capacity or neuroinvasiveness but by the ability to trigger a specific, lethal inflammatory cascade. The identification of IL-6 and G-CSF as central mediators of PRV-induced neuroinflammation provides a mechanistic link between the virus and the neuropathic itch, a symptom that has baffled researchers for over a century [10, 41].

Molecular Basis of Neuropathogenesis and Latency

PRV is a highly neurotropic virus. In its natural host, the pig, initial replication occurs in mucosal epithelium, after which the virus invades the peripheral nervous system (PNS) and can establish latent infection in sensory ganglia [1, 10]. In non-natural hosts, PRV progresses rapidly from the PNS to the central nervous system (CNS), causing acute, invariably fatal encephalomyelitis [1, 7, 10]. The molecular determinants of this differential pathogenesis are only partially understood but involve specific viral genes and host factors.

Viral Genes Governing Neurovirulence: Systematic knockout studies using CRISPR/Cas9 technology have identified the thymidine kinase (TK) and glycoprotein M (gM) as critical pathogenicity determinants in the context of current Chinese variant strains [30]. Deletion of either TK or gM dramatically attenuates virulence in mice, while deletion of other genes traditionally considered virulence factors, such as gE, gI, Us2, Us3, Us9, gG, gN, and EP0, does not significantly reduce virulence in this model [30]. This finding challenges earlier assumptions and suggests that the molecular basis of virulence is strain-dependent and may have shifted with the emergence of variant PRV strains in China. TK is essential for viral nucleotide metabolism in non-dividing cells such as neurons, and its deletion cripples the virus's ability to replicate in the CNS. gM, a membrane protein involved in virion morphogenesis and cell-to-cell spread, appears to play a non-redundant role in neuroinvasion [30].

Genomic Variation and Recombination-Driven Pathogenesis: The emergence of highly pathogenic PRV variant strains in China since 2011 has been linked to extensive genomic recombination [3, 14, 19, 20]. Phylogenetic analyses reveal that PRV can be divided into two main clades, with frequent interclade and intraclade recombination events driving genetic diversity [20]. Notably, recombinant strains have been isolated from vaccinated herds, demonstrating that live attenuated vaccine strains (e.g., Bartha-K61) can serve as genetic donors for recombination with circulating field strains, potentially generating novel recombinants with altered virulence and immune evasion profiles [14]. One such recombinant, the JSY13 strain, was identified as a natural hybrid between the Bartha vaccine strain (genotype I) and the virulent JSY7 variant (genotype II), exhibiting moderate virulence compared to its parental strains [14]. These recombination events are not merely academic; they have direct implications for vaccine efficacy and disease control, as the antigenic composition of recombinants may diverge sufficiently to evade vaccine-induced immunity.

Zoonotic Potential and Human Pathogenesis: The isolation of a PRV strain (hSD-1/2019) from the cerebrospinal fluid of a human encephalitis patient in China provided definitive evidence that PRV can cross the species barrier and cause severe neurological disease in humans [8]. This isolate is phylogenetically closest to Chinese pig variant strains and exhibits high pathogenicity in experimentally infected pigs, inducing acute neurological symptoms [8]. Human cases, although rare, have been characterized by acute encephalitis, bilateral necrotizing retinitis, endophthalmitis, and high mortality or severe sequelae [8, 9, 13, 18, 37, 39, 40]. The portal of entry in humans is likely via direct contact with infected pigs or contaminated materials, and the virus gains access to the CNS via the trigeminal or olfactory nerves [11, 13]. The molecular mechanisms enabling PRV to infect human neurons are consistent with the utilization of nectin-1, which is conserved across species, and the capacity of PRV gD to bind human nectin-1 with high affinity [28]. The World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) recognize PRV as a significant pathogen of swine, and the accumulating evidence of zoonotic transmission underscores the need for continued surveillance and a One Health approach to PRV control [1, 3, 5, 8].

Host Range, Transmission, and Global Epidemiology

Host Range: The Suid-Centric Paradigm and Its Exceptions

Pseudorabies virus (PRV), the etiological agent of Aujeszky’s disease, exhibits a remarkably broad host range in vitro and in vivo, yet its ecological and evolutionary success is fundamentally anchored to a single reservoir: members of the family Suidae. Pigs, including domestic swine (Sus scrofa domesticus) and wild boar (Sus scrofa), are recognized as the only natural hosts in which PRV can complete its full replication cycle, establish lifelong latency, and be efficiently transmitted to naïve conspecifics [1, 3, 7]. In swine, infection is often subclinical in adult animals or manifests as respiratory distress in growing pigs, reproductive failure in pregnant sows, and fatal neurological disease in neonatal piglets [1]. This differential pathogenicity is a hallmark of the host-virus adaptation: PRV has co-evolved with suids to permit viral persistence without invariably causing rapid host death, a strategy that ensures continued transmission.

In stark contrast, infection in non-natural hosts, which includes a staggering diversity of mammals such as cattle, sheep, goats, dogs, cats, rabbits, rodents, and even mink, is almost uniformly fatal [1, 3, 7, 45]. The clinical signature in these species is a severe, rapidly progressive neurological syndrome characterized by intense pruritus, known colloquially as the “mad itch,” followed by profound central nervous system (CNS) dysfunction, coma, and death [10, 41]. The pathophysiological basis for this dichotomy lies in the differential neuroinvasive capacity and host immune response. In non-natural hosts, PRV invades the peripheral nervous system (PNS) with extraordinary efficiency, traveling via retrograde axonal transport to the CNS, where it triggers a fulminant, necrotizing encephalitis [7, 22]. Critically, the host inflammatory response in these animals, driven by a cytokine storm involving interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), is itself a major contributor to mortality, rather than direct viral cytopathology alone [41]. This systemic inflammatory response syndrome (SIRS) is not observed in swine, further underscoring the species-specific nature of PRV pathogenesis.

The host range extends to endangered wildlife, raising significant conservation concerns. Documented fatal infections in the Iberian lynx (Lynx pardinus) in Spain and the Florida panther highlight the threat PRV poses to already vulnerable populations [46]. In these cases, transmission likely occurs through predation on infected wild boar or feral swine, creating a sylvatic cycle that complicates eradication efforts. Avian species are generally considered resistant, although experimental infections have been reported; the virus is not considered a significant pathogen in birds under natural conditions [3].

Zoonotic Potential: From Neglected Pathogen to Confirmed Human Threat

For over a century, PRV was considered a strict veterinary pathogen with no public health significance. This paradigm was shattered definitively in 2017 with the first molecularly confirmed cases of human PRV encephalitis in China [8, 9, 39]. Since then, a growing body of evidence has established PRV as an emerging zoonotic agent, capable of causing severe, life-threatening disease in humans [5, 11]. As of 2022, at least 25 human cases have been reported in China, with the actual number likely higher due to underdiagnosis [5]. The clinical presentation in humans is dominated by acute encephalitis, often with a rapid onset of fever, headache, seizures, and altered consciousness [8, 13, 37]. A particularly striking feature is the frequent involvement of the eyes, manifesting as bilateral acute retinal necrosis (ARN) or endophthalmitis, which can lead to permanent vision loss even in survivors [18, 39, 40]. Respiratory failure requiring mechanical ventilation is common, and the mortality rate is substantial [8, 13].

The causative agent in these human cases has been molecularly characterized. In a landmark study, Liu et al. (2020) successfully isolated a live PRV strain, designated hSD-1/2019, from the cerebrospinal fluid (CSF) of a patient with acute encephalitis [8]. This represented the first-ever isolation of PRV from a human patient. Phylogenetic analysis demonstrated that hSD-1/2019 is genetically closest to the variant PRV strains currently circulating in Chinese swine herds, and experimental infection of pigs with this human isolate reproduced the severe neurological disease characteristic of the variant strains [8]. This finding provides unequivocal evidence for direct pig-to-human transmission and confirms that the variant strains, which emerged in 2011, possess an enhanced zoonotic potential compared to classical PRV strains [3, 8].

All reported human cases share a common epidemiological thread: direct, high-intensity exposure to pigs or their bodily fluids. The vast majority of patients have been swine farmers, butchers, slaughterhouse workers, or pork vendors [9, 13, 37]. The portal of entry is hypothesized to be through broken skin or mucous membranes following contact with contaminated material, such as infected blood, placental tissues, or respiratory secretions [39]. Ocular exposure, perhaps through splashes, may explain the high incidence of retinal necrosis [18, 40]. Importantly, there is no evidence of human-to-human transmission, indicating that humans are dead-end hosts. The World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) have not issued specific travel advisories, but the World Organisation for Animal Health (WOAH) recognizes the potential public health implications and emphasizes the need for robust surveillance in swine populations to mitigate zoonotic risk. The Food and Agriculture Organization (FAO) also highlights the economic and food security risks posed by PRV to the global pig industry.

Transmission Dynamics: Routes, Reservoirs, and Shedding

PRV transmission is governed by a combination of direct and indirect routes, with the respiratory and oronasal pathways being paramount. In swine, the primary mode of transmission is via direct contact between infected and susceptible pigs. The virus is shed in high titers in nasal secretions, saliva, and ocular fluids during the acute phase of infection [1, 3]. Aerosolized virus can also travel short distances within confinement facilities, making fomite-mediated spread a significant concern. The virus is relatively stable in the environment, particularly in cool, moist conditions, and can persist in contaminated feed, water, bedding, and equipment for days to weeks, facilitating indirect transmission [1].

A critical feature of PRV biology is its ability to establish lifelong latency in the trigeminal ganglia and other sensory neurons of the PNS [7, 10]. Latently infected pigs are clinically normal but can periodically reactivate the virus, especially under stress (e.g., transport, farrowing, co-infection with other pathogens). Reactivation leads to renewed viral shedding in nasal secretions, often without overt clinical signs, creating a cryptic source of infection that perpetuates the cycle within herds [1]. This latency mechanism is the single greatest obstacle to eradication in endemic regions.

Venereal transmission is also well-documented. Infected boars can shed PRV in semen, and artificial insemination with contaminated semen has been implicated in outbreaks [1]. Vertical transmission from sow to fetus is a major cause of reproductive disease, leading to abortion, stillbirth, and mummified fetuses [1, 44]. The detection of PRV in wild boar fetuses confirms that intrauterine infection occurs in this reservoir as well, further entrenching the virus in the sylvatic cycle [44].

Wild boar and feral swine serve as the primary wildlife reservoir for PRV. In many European countries and parts of the United States, seroprevalence in wild boar populations can exceed 50%, providing a persistent source of virus that can spill over into domestic swine operations, particularly in areas with poor biosecurity [20, 44]. The virus can also be transmitted from wild boar to other wildlife through predation or scavenging, as evidenced by fatal infections in lynx and other carnivores [46]. This sylvatic cycle is a major impediment to regional eradication, as wildlife populations are largely unmanaged and inaccessible to vaccination campaigns.

Global Epidemiology: A Tale of Two Eras

The global epidemiology of PRV can be divided into two distinct eras: the pre-variant era (before 2011) and the post-variant era (2011–present). During the latter half of the 20th century, the widespread deployment of effective marker vaccines, particularly the Bartha-K61 strain, led to the successful eradication of PRV from commercial swine herds in many developed nations, including the United States, much of Western Europe, New Zealand, and Canada [1, 3]. These countries now maintain PRV-free status, a testament to the efficacy of comprehensive vaccination programs combined with test-and-removal strategies. However, the virus remains endemic in feral swine populations in these regions, posing a constant threat of re-introduction to domestic herds [5, 44].

The situation in Asia, and particularly in China, is dramatically different. China is the world’s largest producer and consumer of pork, with an immense, densely concentrated swine population. PRV was first reported in China in the 1950s, and the Bartha-K61 vaccine was widely used for decades with apparent success [43]. However, in late 2011, a devastating outbreak of pseudorabies swept through Bartha-K61-vaccinated pig farms across multiple provinces in China [1, 3, 26, 43]. This outbreak was caused by the emergence of highly pathogenic PRV variant strains (classified as clade 2.2) that were antigenically and genetically distinct from the classical strains used in the vaccine [3, 20, 26].

These variant strains, such as HN1201, JS-2012, and FJ-2012, possess several key genetic differences compared to classical strains, including specific amino acid substitutions in the major glycoproteins gB, gC, gD, and gE [20, 26, 31]. These changes are thought to be responsible for the increased virulence and, critically, the ability to partially evade immunity induced by the Bartha-K61 vaccine [3, 24, 26]. The variant strains cause more severe clinical disease in pigs of all ages, with higher mortality rates in growing pigs and more pronounced reproductive failure in sows [32]. Epidemiological surveys conducted between 2012 and 2017 across 27 Chinese provinces revealed an average PRV-gE (wild-type virus) positive rate of 8.27% in clinical samples, with rates fluctuating between 5.57% and 12.19% annually [26]. This indicates sustained, widespread circulation of the variant virus in the Chinese swine population.

The emergence of the variant strains has not been confined to China. Phylogenetic analyses have shown that clade 2.2 (the variant clade) is now the most prevalent genotype globally and is the clade most frequently associated with cross-species transmission events, including those to humans [20]. The effective reproduction number (Rₑ) for this clade was estimated to be >1 from 2011 to 2016, indicating that the virus was actively spreading and not under control [20]. Recombination events between different PRV genotypes, including recombination between the Bartha vaccine strain and circulating variant strains, have been documented in the field [14, 19]. This natural recombination generates novel chimeric viruses with unpredictable virulence and antigenic profiles, further complicating control efforts and driving the ongoing evolution of the virus [5, 14].

The global distribution of PRV is thus highly heterogeneous. While the disease is controlled or eradicated in the commercial sectors of North America, Western Europe, and Oceania, it remains a major, uncontrolled epizootic threat across much of Asia, Eastern Europe, and parts of South America. The virus is also endemic in wild boar populations across Eurasia and North America, ensuring its long-term persistence. The re-emergence of PRV in China since 2011, coupled with the confirmed zoonotic potential of the variant strains, has fundamentally altered the global risk assessment for this pathogen. The WOAH classifies PRV as a notifiable disease, and its re-emergence underscores the critical need for continued surveillance, the development of next-generation vaccines effective against variant strains, and stringent biosecurity measures to prevent spillover from wildlife and spillback into human populations.

Clinical Manifestations and Pathological Features in Susceptible Hosts

The clinical trajectory and pathological landscape of pseudorabies virus (PRV) infection are profoundly dictated by host species, age, viral strain virulence, and route of inoculation. As a member of the Alphaherpesvirinae subfamily, PRV exhibits a remarkably broad host range, infecting most mammals except for higher-order primates, though the latter caveat has been challenged by emerging human case reports. Critically, the clinical outcome bifurcates sharply between the natural host, swine, and all other susceptible mammals. In swine, infection can present as a spectrum from subclinical to fatal, depending on age and immune status, whereas in non-natural hosts, including cattle, dogs, cats, sheep, goats, rodents, and, as increasingly documented, humans, infection is almost invariably acute, neurotropic, and rapidly fatal, often characterized by the pathognomonic “mad itch” (pruritus infesta) [1, 5, 10]. The World Organisation for Animal Health (WOAH, formerly OIE) classifies pseudorabies as a notifiable disease, underscoring its transboundary economic impact, particularly within the global swine industry. This section provides a granular, systems-level dissection of the clinical manifestations and correlative pathological features across susceptible hosts, drawing upon experimental infections and natural outbreak investigations.

1. Clinical and Pathological Features in Swine (the Natural Host)

In swine, the sole species in which PRV can establish a productive, lifelong infection with latency, clinical expression is a function of age, immunological naivety, and viral variant pathogenicity. The emergence of highly pathogenic variant strains since 2011, particularly in China, has recalibrated the expected clinical picture [1, 3, 32].

1.1 Neonatal and Suckling Piglets (Generally < 2–4 weeks of age)

This cohort is the most severely affected, presenting with a peracute to acute neurological syndrome. The incubation period is typically 24–48 hours post-infection. Initial signs include profound depression, anorexia, and pyrexia (>41°C), rapidly escalating to classic central nervous system (CNS) signs: incoordination, ataxia, opisthotonos, paddling movements, nystagmus, and generalized tremors. Death frequently occurs within 24–48 hours of onset, with mortality approaching 100% in naïve herds [1, 31, 32]. Pathologically, gross lesions may be minimal or absent, but microscopic examination reveals a severe, non-suppurative meningoencephalitis and ganglioneuritis. Perivascular cuffing by mononuclear cells (lymphocytes, plasma cells, macrophages), neuronal necrosis, neuronophagia, and gliosis are prominent throughout the brain, particularly the brainstem, pons, and cerebrum [31]. The olfactory bulb is consistently involved, reflecting the primary neuroinvasive route. Viral antigen is demonstrable within neurons and glial cells via immunohistochemistry. Importantly, these piglets lack a mature immune response, allowing unchecked viral replication and rapid centrifugal spread into the CNS without the modulating effects of adaptive immunity [1, 7].

1.2 Weaner and Growing-Finishing Pigs (4 weeks – 6 months)

Clinical disease in this age group is dominated by severe respiratory distress, reflecting a shift in viral tropism from purely neurological to the respiratory epithelium. This is due to the maturation of the immune system, which partially restricts CNS invasion but does not prevent lytic replication in the upper and lower respiratory tract. Affected pigs exhibit high fever, anorexia, coughing, sneezing, labored breathing (dyspnea), and nasal discharge [1, 24]. The respiratory phase can be compounded by secondary bacterial infections, particularly Pasteurella multocida and Mycoplasma hyopneumoniae, leading to fibrinous bronchopneumonia. Neurological signs are less frequent but may include dullness, circling, and head pressing. Mortality is lower than in neonates but can range from 10–50%, depending on the strain and management. Pathologically, the hallmark is a severe necrotizing rhinitis and tonsillitis, followed by a diffuse interstitial pneumonia [24, 31]. Lungs are heavy, edematous, and fail to collapse, with histological features of alveolar septal thickening, necrosis of bronchiolar epithelium, and mononuclear infiltration. The brain may show mild to moderate non-suppurative encephalitis, particularly in the trigeminal ganglia and brainstem, though clinical correlates are often absent [32].

1.3 Adult Sows and Boars (Reproductive Manifestations)

In sexually mature swine, PRV infection is a leading cause of reproductive failure. The clinical presentation is highly dependent on the stage of gestation. Infection of pregnant sows, particularly in the first and second trimesters, leads to transplacental infection of fetuses, resulting in embryonic death, resorption, and returning to estrus. Later gestation infections (third trimester) produce late-term abortion, stillbirths, mummified fetuses, and the birth of weak, trembling piglets that die within hours [1, 43, 44]. The clinical syndrome is often summarized by the acronym SMEDI (Stillbirth, Mummification, Embryonic Death, Infertility). Sows themselves may show only transient pyrexia and anorexia, but the herd-level impact is devastating. Pathologically, aborted fetuses may show autolysis, and the placenta reveals areas of necrosis and vasculitis. Viral antigen is detectable in fetal tissues, including the brain, lung, and liver, confirming vertical transmission [44]. In boars, infection can cause orchitis, epididymitis, and shedding of virus in semen, contributing to venereal transmission.

2. Clinical and Pathological Features in Non-Natural Hosts: The “Mad Itch” Syndrome and Fulminant Neuropathy

In all species other than swine, PRV typically causes a rapidly ascending, highly lethal neurological disease. The cardinal and most distinctive clinical sign is an intense, uncontrollable neuropathic pruritus, from which the disease derives its common name, “Aujeszky’s disease” or “mad itch” [10, 41]. This symptom is virtually never observed in swine.

2.1 Rodents (Mice, Rats) and Rabbits (Experimental Models)

These species serve as critical models for understanding PRV neuropathogenesis. Following peripheral inoculation (e.g., footpad, snout), the virus enters sensory nerve endings and undergoes retrograde axonal transport to the dorsal root ganglia (DRG) and spinal cord, then ascends to the brainstem. Initial signs (24–48 hours) include hyperesthesia and self-directed pruritus, with mice incessantly biting and scratching the inoculation site, leading to exoriation and mutilation [41]. This behavior is not a result of a local inflammatory response at the skin, but rather a reflection of viral replication in DRG neurons, where the virus induces a massive, specific host inflammatory response. Laval et al. (2018) demonstrated that infection with the virulent PRV-Becker strain in mice leads to a systemic inflammatory response syndrome (SIRS) characterized by markedly elevated plasma levels of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), along with chemokines such as Gro-1 [41]. This systemic inflammation, rather than direct viral cytolysis alone, is the primary driver of acute mortality. Histologically, the footpad and affected DRG show necrosis and massive neutrophil infiltration [41]. The brain shows severe, non-suppurative encephalitis, with prominent perivascular cuffing, gliosis, and hemorrhage, particularly in the brainstem and thalamus. Attenuated strains like Bartha fail to induce this inflammatory cascade and are non-lethal [41].

2.2 Carnivores: Dogs, Cats, and Fur-Bearing Animals

Dogs and cats are exquisitely susceptible and infection is almost invariably fatal. Clinical onset is acute, with initial signs of restlessness, hypersalivation, dysphagia (due to pharyngeal paralysis), vomiting, and a hoarse bark or meow. The “mad itch” is intensely characteristic: the animal vigorously licks, bites, or rubs a focal area, usually the face or flank, often until the skin is raw. This pruritus is followed by rapidly progressive neurological signs, ataxia, circling, head pressing, seizures, and coma, culminating in death within 24–48 hours [10, 48] . In dogs, Zhang et al. (2015) documented a unique pathological signature: systemic hemorrhage and congestion were the most marked macroscopic findings, including petechiae and ecchymoses on the endocardium and epicardium, pulmonary hemorrhage, and incomplete splenic contraction. Microscopically, nonsuppurative ganglioneuritis, brainstem encephalitis, and myocardial necrosis with exudation were prominent. Importantly, the respiratory distress often observed in dogs was attributed not to primary pneumonitis, but to cardiogenic lesions from viral myocarditis. Viral antigen was confined to the brainstem and peripheral ganglia, highlighting the selective neurotropic targeting [48].

In fur-bearing animals like minks, outbreaks are devastating. A documented outbreak in Shandong Province, China, caused by a vaccine-resistant PRV variant, resulted in 87.2% mortality (3,522/4,028 minks). Clinical signs included anorexia, hypersalivation, and hindlimb paralysis [45]. Pathological findings are consistent with acute encephalitis.

2.3 Ruminants: Cattle, Sheep, and Goats

In cattle, PRV infection presents as a peracute, febrile illness with severe pruritus, often localized to the flank, perineum, or neck. Affected animals rub violently against fences or objects, leading to extensive skin abrasions. The disease rapidly progresses to muscle tremors, incoordination, recumbency, opisthotonos, and convulsions. Death occurs within 24–72 hours. The isolation of a variant PRV strain (e.g., from Eastern China) causing acute bovine death confirmed that these novel strains pose a significant threat to the cattle industry [47]. Pathologically, the brain reveals a severe non-suppurative encephalitis with widespread neuronal necrosis and demyelination. Interestingly, the trigeminal nerve and ganglia are heavily infected, consistent with the pathogenesis observed in other species [47].

2.4 Wild and Endangered Felids (Lynx, Panthers)

PRV is a significant threat to endangered species. The first reported case in an Iberian lynx (Lynx pardinus) in Spain demonstrated that wild felids are susceptible. The lynx presented with alopecia at the ventral neck, indicative of the pruritus, and bloody gastrointestinal contents. Histopathological examination revealed a moderate non-suppurative meningoencephalitis with diffuse demyelination, necrotizing gastritis, and enteritis. Viral antigen was detected in neurons, glial cells, tonsil, and gastric epithelium, confirming multiorgan neurotropic spread [46]. This case underscores the risk of spillover from suid reservoirs into vulnerable wildlife populations.

3. Emerging Zoonotic Manifestations: Human PRV Encephalitis and Ocular Disease

Historically considered a veterinary pathogen, PRV has been increasingly recognized as a zoonotic agent capable of causing severe, often devastating, neurological and ocular disease in humans. Since 2017, over 25 cases have been reported in China, with a likely higher incidence undiagnosed [5, 8, 11]. The World Health Organization (WHO) has not yet formally listed PRV as a priority zoonotic pathogen, but the accumulating evidence demands heightened surveillance.

3.1 Clinical Presentation of Human Encephalitis

All reported human cases have involved direct occupational exposure to swine or swine by-products (e.g., slaughterhouse workers, pork vendors, veterinarians, and farmers). The incubation period is estimated at 3–7 days. The clinical onset is acute, with high-grade fever, severe headache, and rapid progression to altered consciousness, seizures (including tonic-clonic), and coma [8, 9, 13, 37]. Respiratory failure requiring mechanical ventilation is a common and critical complication, reported in up to 80% of patients [13]. The clinical syndrome is indistinguishable from other causes of viral encephalitis, including herpes simplex virus (HSV). Diagnosis is routinely confirmed via metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF), which reveals PRV-specific nucleotide sequences [8, 9, 37]. CSF analysis typically shows a mild lymphocytic pleocytosis, elevated protein, and normal glucose, findings consistent with viral meningitis. The first-ever human PRV isolate, hSD-1/2019, was obtained from a CSF sample and phylogenetically clustered with Chinese variant PRV strains circulating in pigs, providing definitive proof of zoonotic spillover [8]. Serological testing reveals positive PRV-specific antibodies (anti-gB, anti-gE) in both CSF and serum [8].

3.2 Radiological and Pathological Correlates in Humans

Brain magnetic resonance imaging (MRI) reveals a characteristic pattern of multifocal, bilateral inflammatory lesions with a predilection for the limbic system. The most consistently reported abnormalities are hyperintensities on T2-weighted and FLAIR sequences involving the bilateral temporal lobes and insular cortex [9, 13]. Extension into the frontal lobes, basal ganglia, caudate nucleus, and thalamus is also documented [37]. This neuroimaging pattern is reminiscent of HSV encephalitis but is distinct in its more extensive involvement of deep gray matter structures.

3.3 Ocular Manifestations: Acute Retinal Necrosis and Endophthalmitis

A uniquely severe and visually devastating complication of human PRV infection is bilateral acute retinal necrosis (ARN) and endophthalmitis [18, 39, 40]. This manifests weeks to months after the acute encephalitic phase, as the patient recovers consciousness. Presenting symptoms include rapid, painless, bilateral vision loss. Ophthalmoscopic examination reveals severe vitreous haze, necrotizing retinitis with a “tattered fishnet” appearance, and multiple retinal holes leading to retinal detachment [39, 40]. Next-generation sequencing of vitreous humor has confirmed the presence of PRV genomic material, indicating that the virus has a tropism for retinal tissue [18, 39, 40]. Histopathological examination of vitreous specimens is not typically performed, but the clinical picture suggests a direct viral cytopathic effect on retinal neurons, leading to rapid tissue necrosis

Advanced Diagnostic Approaches for Pseudorabies Virus

The accurate and timely diagnosis of pseudorabies virus (PRV) infection is a cornerstone of effective disease surveillance, control, and eradication programs. The clinical presentation of Aujeszky’s disease (AD) can be highly variable, ranging from reproductive failure in sows and fatal neurological disease in piglets to subclinical respiratory infections in growing pigs, making differential diagnosis from other swine pathogens essential [1, 43]. Furthermore, the emergence of highly pathogenic PRV variant strains since 2011, which have demonstrated the capacity to overcome immunity conferred by classical vaccines like Bartha-K61, has necessitated the development of advanced diagnostic tools capable of differentiating infected from vaccinated animals (DIVA) and detecting novel viral genotypes [3, 14, 26]. The diagnostic landscape for PRV has evolved dramatically, moving from classical virological and serological methods to a sophisticated arsenal of molecular, genomic, and high-throughput sequencing technologies. This section provides an exhaustive analysis of these advanced diagnostic approaches, detailing their underlying principles, specific applications, and critical roles in managing the ongoing threat of PRV to global swine production and, increasingly, to public health.

Molecular Detection and Genomic Characterization

The advent of polymerase chain reaction (PCR) and its derivatives has revolutionized the detection of PRV, offering unparalleled sensitivity, specificity, and speed compared to traditional virus isolation. These molecular methods are now the gold standard for confirming acute infections, screening subclinically infected herds, and conducting large-scale epidemiological surveys.

Real-Time Quantitative PCR (qPCR) and Conventional PCR

Conventional PCR targeting conserved regions of the PRV genome, such as the glycoprotein B (gB), gD, or gE genes, has been widely employed for routine diagnosis and is a standard method for detecting viral DNA in clinical samples including tonsils, lymph nodes, brain tissue, and nasal swabs [17, 44, 46]. The gE gene is of particular importance because it is absent in most commercially available gE-deleted marker vaccines, allowing for the differentiation of wild-type infection from vaccination [17, 26]. Real-time quantitative PCR (qPCR) has largely supplanted conventional PCR due to its ability to provide quantitative viral load data, which is crucial for assessing the severity of infection, monitoring viral shedding, and evaluating the efficacy of antiviral interventions or vaccines [39, 49, 50]. A study by Sun et al. (2018) utilized qPCR targeting the gE gene to screen over 16,000 clinical samples across 27 provinces in China, establishing a robust epidemiological baseline and demonstrating the sustained circulation of wild-type PRV even in vaccinated populations [26]. The analytical sensitivity of these assays is exceptionally high; for instance, a real-time recombinase-aided amplification (RAA) assay, an isothermal alternative to qPCR, achieved a detection limit of three 50% tissue culture infectious doses (TCID₅₀) per reaction, comparable to the most sensitive qPCR protocols [49]. This level of sensitivity is critical for detecting low-level viral shedding or latent infections, which are key to understanding transmission dynamics.

Isothermal Amplification Technologies: Recombinase-Aided Amplification (RAA)

While qPCR remains the laboratory standard, its reliance on sophisticated thermal cyclers limits its application in resource-limited settings or for on-farm, point-of-care diagnostics. Isothermal amplification technologies, such as recombinase-aided amplification (RAA), address this critical gap. The real-time RAA assay developed by Tu et al. (2021) is a paradigm of this approach. This method amplifies specific DNA targets at a constant low temperature (typically 37-42°C) using a recombinase, single-stranded DNA binding proteins, and a strand-displacing polymerase. The assay, designed against the conserved gE gene, demonstrated 100% specificity for wild-type PRV, showing no cross-reactivity with other major porcine viruses or gE-deleted vaccine strains [49]. Its diagnostic accordance rate with qPCR was an impressive 97.57% when testing 206 clinical tissue samples [49]. A particularly innovative feature of this assay is that the amplified products can be visualized under a portable blue light instrument, eliminating the need for expensive detection equipment. This makes real-time RAA an exceptionally powerful tool for rapid, on-site screening during outbreak investigations, enabling immediate implementation of biosecurity measures without the delays associated with sample transport to centralized laboratories.

Next-Generation Sequencing (NGS) and Metagenomics

Perhaps the most transformative advancement in PRV diagnostics has been the application of next-generation sequencing (NGS), particularly metagenomic NGS (mNGS). This unbiased, hypothesis-free approach has been instrumental in confirming PRV as a zoonotic pathogen capable of causing severe human encephalitis, a discovery that classical diagnostic methods had failed to make for decades [8, 9, 13, 37]. In human cases presenting with acute encephalitis of unknown etiology, mNGS of cerebrospinal fluid (CSF) has consistently identified PRV-specific nucleotide sequences, providing the definitive etiological diagnosis [8, 9, 13]. For example, Liu et al. (2020) used mNGS to detect PRV sequences (7-6198 reads) in the CSF of four encephalitis patients, leading to the first-ever isolation of a PRV strain (hSD-1/2019) from a human case [8]. Similarly, NGS of vitreous humor has confirmed PRV as the cause of bilateral acute retinal necrosis and endophthalmitis in patients with occupational exposure to swine, demonstrating the virus’s ability to invade ocular tissues [18, 39, 40].

Beyond human diagnostics, NGS is a powerful tool for characterizing the genomic evolution of PRV field strains. Whole-genome sequencing of isolates from outbreaks has revealed the molecular basis for the increased virulence of variant strains and the emergence of natural recombinants. For instance, sequencing of the JSY7 and JSY13 strains isolated from a vaccinated farm provided direct evidence of natural recombination between a Bartha-K61 vaccine-like strain (genotype I) and a circulating variant strain (genotype II), resulting in a novel recombinant with intermediate virulence [14]. Such detailed genomic characterization is essential for understanding viral evolution, tracking the spread of specific clades (e.g., the globally dominant Clade 2.2), and identifying amino acid changes in key glycoproteins (gB, gC, gD, gE) that may be associated with immune escape or adaptation to new hosts, including humans [3, 20, 26].

Transcriptome and Epigenetic Profiling

Advanced sequencing platforms have also been leveraged to create a comprehensive map of the PRV transcriptome, revealing a complexity far beyond the previously annotated 72 coding sequences. Using a multi-platform approach combining Illumina short-read, PacBio RS-II, and Oxford Nanopore long-read sequencing, researchers have identified 19 previously unknown putative protein-coding genes (all 5′ truncated forms of known genes), 19 non-coding RNAs, and over 50 distinct transcript length isoforms [27]. This high-resolution transcriptome map, which includes the discovery of a highly abundant short non-coding RNA (CTO-S) near the origin of lytic replication (OriL), provides a detailed genetic blueprint for future functional studies and the identification of novel diagnostic targets [27, 34, 35]. The detection of these novel RNA species, including antisense transcripts and those overlapping replication origins, opens new avenues for developing diagnostic assays that target not just DNA but specific viral RNA signatures, potentially differentiating between lytic and latent infections.

Serological and Immunological Assays

Serological testing remains a mainstay for herd-level surveillance, monitoring vaccine efficacy, and confirming PRV-free status for international trade, as recommended by the World Organisation for Animal Health (WOAH). The key to modern serological diagnostics is the DIVA principle, which relies on detecting antibodies against viral proteins that are absent in marker vaccines.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is the most widely used serological method due to its high throughput, objectivity, and suitability for automation. Two primary types of ELISA are employed: one targeting antibodies against glycoprotein B (gB), which is present in both wild-type virus and all vaccines, indicating exposure to PRV; and another targeting glycoprotein E (gE), which is deleted in the most common marker vaccines (e.g., Bartha-K61). A positive gB ELISA result indicates the animal has been exposed to PRV (either through infection or vaccination), while a positive gE ELISA result specifically indicates infection with a wild-type, field strain [17]. Large-scale epidemiological studies rely heavily on this approach. For instance, Ma et al. (2020) tested over 16,000 serum samples from 362 pig farms in Shandong, China, and found that while 91.5% were gB-positive (indicating high vaccine coverage or exposure), 52.7% were gE-positive, revealing a high prevalence of wild-type virus circulation [17]. This data is critical for assessing the failure of current vaccination strategies and guiding the development of new, more effective vaccines derived from local variant strains [24, 25].

Virus Neutralization Test (VNT)

The virus neutralization test (VNT) is the gold standard for assessing functional antibody titers and is often used to confirm ELISA results or for international trade requirements. It measures the ability of serum antibodies to neutralize live virus in cell culture, providing a direct correlate of protective immunity. Studies evaluating vaccine efficacy, such as those comparing the Bartha-K61 vaccine against variant PRV challenges, have used VNT to quantify neutralizing antibody responses [24]. While highly specific, VNT is labor-intensive, time-consuming (requiring several days), and requires cell culture facilities, limiting its use in high-throughput surveillance.

Advanced Immunoassays: Monoclonal Antibody Epitope Mapping

At the cutting edge of immunological diagnostics is the detailed characterization of B-cell epitopes using monoclonal antibodies (mAbs). Li et al. (2017) isolated a panel of 15 mAbs against the PRV glycoprotein B (gB) and used X-ray crystallography to map their epitopes. They identified two functionally distinct classes of neutralizing antibodies: one class (complement-dependent) binds to the crown region (domain IV) of gB, while a single, exceptionally potent mAb (1H1) binds to the base of domain I, near the fusion loops, and neutralizes the virus directly by interfering with membrane fusion [29]. This high-resolution structural and functional mapping of protective epitopes has profound implications for designing next-generation subunit vaccines and developing epitope-based serological assays that can distinguish between responses to vaccination and natural infection with unprecedented precision.

Advanced Virological and Microscopic Techniques

While molecular and serological methods dominate modern diagnostics, classical virological techniques remain essential for isolating live virus for characterization, vaccine development, and research.

Virus Isolation and Electron Microscopy

Virus isolation in permissive cell lines (e.g., Vero, PK-15, ST cells) is the definitive method for confirming the presence of infectious virus. The characteristic cytopathic effect (CPE), plaque formation, cell rounding, and syncytia formation, is used for initial identification [14, 16]. This method is crucial for obtaining viral isolates for whole-genome sequencing, pathogenicity studies, and vaccine seed stock. Transmission electron microscopy (TEM) provides direct visualization of the virus. The isolation of the first human PRV strain, hSD-1/2019, was confirmed by TEM, which revealed typical herpesvirus morphology, an icosahedral nucleocapsid surrounded by a tegument layer and a lipid envelope with glycoprotein spikes, indistinguishable from swine PRV isolates [8]. This visual confirmation was critical in definitively establishing the zoonotic link.

Immunohistochemistry (IHC) and In Situ Hybridization

Immunohistochemistry (IHC) is a powerful technique for visualizing the spatial distribution of PRV antigens within tissues, providing critical insights into pathogenesis. By using specific antibodies against PRV proteins (e.g., gB or gE), IHC can pinpoint viral antigen in specific cell types, such as neurons in the brainstem and peripheral ganglia, or in epithelial cells of the tonsils and gastric mucosa [46, 48]. This technique has been instrumental in demonstrating the neurotropic nature of PRV, showing viral invasion of the central nervous system (CNS) in both natural and experimental infections [48]. In situ hybridization (ISH), which uses labeled DNA or RNA probes to detect viral nucleic acids, offers a complementary approach, particularly for detecting latent virus or when antigen expression is low.

Diagnostic Challenges and Future Directions

Despite these advances, significant diagnostic challenges remain. The high genetic diversity of PRV, driven by frequent recombination and mutation, can lead to false-negative results in PCR assays if primer or probe binding sites are not conserved across all circulating strains [14, 20]. The emergence of novel recombinants, such as those between vaccine and field strains, further complicates the molecular landscape [14]. Furthermore, the ability of PRV to establish lifelong latency in the peripheral nervous system (PNS) of pigs means that a negative PCR result from a nasal swab or tonsil scraping does not rule out the presence of a latent infection that can reactivate under stress [10]. The development of diagnostic tools capable of detecting latent viral genomes or transcripts (e.g., latency-associated transcripts, LATs) is a critical unmet need [15].

Future diagnostic approaches will likely integrate multi-omics data. For example, combining whole-genome sequencing of isolates with transcriptomic profiling of the host response could identify specific biomarkers of infection or reactivation. The development of rapid, multiplexed, point-of-care devices that can simultaneously detect PRV alongside other major swine pathogens (e.g., African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus) would be a game-changer for field diagnostics [17, 21]. Given the increasing evidence of PRV as a zoonotic threat, with the World Health Organization (WHO) and other public health bodies taking note, the development of standardized, validated diagnostic protocols for use in both veterinary and human clinical laboratories is paramount. The continued refinement of CRISPR-based diagnostic platforms, which offer rapid, specific, and field-deployable detection, holds immense promise for the next generation of PRV surveillance and control.

Vaccination Strategies and Novel Therapeutic Interventions

The control and eventual eradication of pseudorabies (Aujeszky’s disease) represent a formidable challenge in veterinary virology, underscored by the virus’s ability to establish latency, its broad host range, and the continuous emergence of immune-evasive variant strains. The cornerstone of global PRV management has historically been vaccination, a strategy that has evolved from classical inactivated and attenuated vaccines to sophisticated, rationally designed recombinant and vectored platforms. Concurrently, the limitations of vaccination, particularly against emerging variants and in non-porcine hosts, have spurred intensive investigation into novel therapeutic interventions, ranging from small-molecule antivirals and natural products to immunomodulatory agents and gene-editing technologies. This section provides an exhaustive analysis of the current landscape of vaccination strategies and the burgeoning field of novel therapeutics for PRV, integrating molecular mechanisms, epidemiological context, and translational potential.

The Evolution and Limitations of Classical Vaccination

The global effort to control PRV has been historically dominated by the use of modified live virus (MLV) vaccines, with the Bartha-K61 strain serving as the archetypal example. This attenuated strain, characterized by deletions in the glycoprotein E (gE) and glycoprotein I (gI) genes, among others, has been instrumental in reducing clinical disease and viral shedding in swine populations for decades [1, 3]. The success of the Bartha-K61 vaccine is predicated on its ability to induce robust humoral and cellular immune responses while allowing for serological differentiation between vaccinated and naturally infected animals (DIVA strategy), primarily through the detection of antibodies against the deleted gE protein [1]. However, the re-emergence of highly pathogenic PRV variant strains in China since 2011 has starkly revealed the limitations of this classical approach. These variants, which are genetically and antigenically distinct from the Bartha-K61 strain, have been shown to cause severe disease and mortality even in vaccinated herds, indicating a significant breach in vaccine-induced protective immunity [1, 3, 43]. Phylogenetic analyses have demonstrated that the circulating variant strains belong to a distinct clade (Clade 2.2) with substantial genetic divergence from the vaccine strain, particularly in key immunogenic glycoproteins such as gB, gC, and gD [20, 26]. This antigenic drift, coupled with the potential for recombination between vaccine strains and field viruses, as evidenced by the isolation of natural recombinants like the JSY13 strain, which contains genetic material from both the Bartha vaccine and a virulent variant, has raised critical concerns about the long-term sustainability of relying on a single, decades-old vaccine backbone [14, 19]. The World Organisation for Animal Health (WOAH) recognizes the profound economic impact of PRV, and the failure of established vaccines to control these new variants has necessitated a paradigm shift in vaccine design.

Next-Generation Recombinant and Marker Vaccines

In response to the inadequacy of classical vaccines, a new generation of rationally designed vaccines has been developed, leveraging advanced molecular biology techniques to enhance immunogenicity, safety, and DIVA compatibility. A primary strategy involves the deletion of additional virulence-associated genes from the PRV genome to create more attenuated and safer vaccine backbones. The thymidine kinase (TK) gene, a classic virulence determinant for alphaherpesviruses, has been a primary target. Studies using CRISPR/Cas9-mediated gene editing have confirmed that deletion of the TK gene, along with glycoprotein M (gM), significantly reduces PRV virulence in mouse models, providing a rational basis for constructing safer live-attenuated vaccines [30]. Consequently, triple-gene-deleted vaccines (e.g., gE-/gI-/TK-) derived from circulating variant strains have been developed and evaluated. In direct comparative trials, a prototype gE-/gI-/TK- vaccine derived from the variant XJ5 strain demonstrated equivalent protective efficacy to a high-dose Bartha-K61 vaccine against sublethal challenge with the homologous variant, highlighting that a vaccine based on a contemporary strain can be as effective as the gold-standard vaccine when administered at an appropriate dose [24]. However, the critical advantage of these newer constructs lies in their enhanced safety profile and the potential for broader protection against heterologous variants.

Another innovative approach involves the substitution of key immunogenic genes from variant strains into the backbone of the attenuated Bartha-K61 vaccine. For instance, a chimeric vaccine, PRV B-gD&gCS, was constructed by replacing the gD and gC genes of Bartha-K61 with those from the Chinese variant strain AH02LA using bacterial artificial chromosome (BAC) technology [25]. This strategy aims to preserve the proven safety and growth characteristics of the Bartha backbone while updating the antigenic repertoire to match circulating field strains. Remarkably, this chimeric vaccine not only provided complete clinical protection against lethal challenge but also demonstrated a superior ability to reduce or even completely prevent virus shedding post-challenge compared to the parental Bartha-K61 vaccine [25]. This finding is of paramount importance for eradication programs, as reducing shedding is critical for interrupting transmission chains. Furthermore, the development of self-assembling nanoparticle vaccines represents a quantum leap in subunit vaccine design. By displaying the PRV glycoprotein D (gD) on the surface of lumazine synthase (LS) 60-meric protein scaffolds via a covalent SpyTag003/SpyCatcher003 coupling system, researchers have created a highly immunogenic platform [51]. This LS-gD nanoparticle, when formulated with a potent adjuvant (ISA 201VG), elicited robust humoral and cellular immune responses in both mice and piglets, providing effective protection against PRV challenge and eliminating pathological symptoms in the brain and lungs [51]. This technology offers a safer, non-replicating alternative to live vaccines, with the potential for rapid adaptation to new variants by simply swapping the target antigen.

PRV as a Viral Vector for Multivalent Vaccines

The unique biology of PRV, particularly its large double-stranded DNA genome with numerous non-essential regions, makes it an exceptionally versatile viral vector for developing multivalent vaccines against co-infecting swine pathogens [2]. The capacity of PRV to accommodate large foreign gene inserts and induce long-lasting immunity (over 4 months) has been exploited to construct recombinant PRV strains expressing protective antigens from other devastating viruses. A landmark example is the development of a recombinant PRV strain (JS-2012-ΔgE/gI-E2) that expresses the E2 glycoprotein of classical swine fever virus (CSFV) [21]. This bivalent vaccine, based on a gE/gI-deleted variant PRV backbone, was shown to provide complete protection against lethal challenges with both virulent PRV and CSFV in a single shot [21]. This approach not only simplifies vaccination schedules but also addresses the challenge of differentiating infected from vaccinated animals (DIVA) for both diseases, as the vaccine lacks PRV gE and the CSFV E2 marker can be distinguished from other CSFV proteins. Similarly, the utility of PRV as a vector extends to African swine fever virus (ASFV), a global threat for which no commercial vaccine exists. A recombinant PRV with deletions in gE, gI, and TK was engineered to express the ASFV CD2v protein [16]. This construct was safe, immunogenic, and provided 100% protection against a virulent PRV challenge in mice, while also inducing ASFV-specific antibodies and cellular immune responses [16]. These studies underscore the immense potential of PRV-vectored vaccines to combat multiple swine diseases simultaneously, a strategy that could revolutionize disease control in intensive farming systems.

Novel Therapeutic Interventions: Antivirals and Natural Products

While vaccination remains the primary prophylactic tool, the lack of effective antiviral drugs for treating PRV infections, especially in acute cases or in non-porcine hosts (including humans), has driven the search for novel therapeutic agents. The identification of host-virus interaction points has revealed several druggable targets. For instance, the PRV tegument protein UL13 has been shown to recruit the E3 ubiquitin ligase RNF5 to degrade STING, a central adaptor in the cGAS-STING DNA-sensing pathway, thereby inhibiting type I interferon production [4]. Conversely, the host protein OASL exerts an antiviral effect by enhancing RIG-I-mediated interferon signaling, a pathway that is antagonized by the PRV UL24 protein [36]. These intricate immune evasion mechanisms provide potential targets for therapeutic intervention aimed at restoring or enhancing innate antiviral responses.

A diverse array of natural products has demonstrated significant anti-PRV activity in vitro and in vivo. Quercetin, a flavonoid, exhibited a potent inhibitory effect against multiple PRV strains (including the virulent HNX strain) with a high selectivity index (229) and a low IC50 (2.618 μM). Mechanistically, quercetin appears to interact with the viral gD protein, thereby inhibiting viral adsorption [52]. In a mouse model, quercetin injection protected against lethal PRV challenge, reducing brain viral loads and mortality [52]. Kaempferol, another flavonoid, improved survival rates in PRV-infected mice (22.22%) compared to acyclovir (16.67%) and dramatically reduced viral gene copies in the brain by over 700-fold [15]. Its mechanism involves inhibiting the transcription of the immediate-early gene IE180 and the latency-associated transcript (LAT), suggesting a potential role in disrupting both lytic replication and latency establishment [15]. Resveratrol, a stilbenoid, has also shown promise, reducing mortality and improving growth performance in PRV-infected piglets. Its antiviral mechanism is linked to the inhibition of IκB kinase (IKK) activation, thereby blocking NF-κB signaling and subsequent viral gene expression and inflammation [50, 57]. Other natural compounds, such as germacrone from Rhizoma Curcuma and polysaccharides from Radix isatidis, have also demonstrated anti-PRV activity, primarily by inhibiting early stages of the viral replication cycle [53, 56]. Furthermore, antimicrobial peptides like piscidin 1 have shown potent, dose-dependent virucidal activity by directly interacting with virus particles and protecting cells from PRV-induced apoptosis, with in vivo efficacy in reducing mouse mortality [54].

Repurposed Drugs and Gene-Editing Therapies

The repurposing of existing pharmaceuticals offers a rapid and cost-effective pathway to therapeutic development. Ivermectin, a well-known antiparasitic agent, has been identified as a specific inhibitor of importin α/β-dependent nuclear transport. Since PRV DNA replication occurs in the nucleus, ivermectin was tested and found to inhibit PRV replication in a dose-dependent manner by blocking the nuclear localization of the viral DNA polymerase accessory subunit UL42 [55]. In a mouse model, ivermectin treatment increased survival rates and alleviated clinical signs and brain lesions [55]. This finding is particularly significant given ivermectin’s established safety profile and widespread availability, making it a potential candidate for field use. Another repurposed drug, chloroquine, an antimalarial agent, has also shown anti-PRV activity, as demonstrated using a recombinant PRV expressing firefly luciferase for high-throughput screening [59].

The advent of gene-editing technologies, particularly CRISPR/Cas9, has opened unprecedented avenues for both vaccine development and direct antiviral therapy. The technology has been used to rapidly construct recombinant PRV strains with targeted gene deletions, such as the simultaneous substitution of virulence genes, which can then be excised using Cre/Lox systems for enhanced safety [58]. This approach has yielded vaccine candidates with proven protective efficacy in pigs [58]. Moreover, CRISPR/Cas9 has been employed to generate bacterial artificial chromosomes (BACs) of PRV with unprecedented efficiency (up to 86%), facilitating the rapid manipulation of the viral genome for functional studies and vaccine design [33]. Beyond vaccine construction, CRISPR/Cas9 can be directly applied as an antiviral strategy. By designing single guide RNAs (sgRNAs) that target essential PRV genes, the system can cleave the viral genome, thereby inhibiting replication. A recombinant PRV expressing firefly luciferase was used to screen and identify highly effective sgRNAs that potently inhibited PRV replication, demonstrating the feasibility of this approach for developing sequence-specific antiviral therapies [59]. This strategy holds promise for treating acute PRV infections or for use in eliminating latent viral reservoirs, a concept that is being explored for other herpesviruses.

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