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Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Pseudorabies (PR), a highly contagious disease caused by the pseudorabies virus (PRV), represents a significant threat to the global swine industry. Despite the success of developed countries in controlling the PRV epidemic through swine pseudorabies eradication programs, wild boars, as a potential source of infection, still require sustained attention and effective control measures. Concurrently, there has been considerable global attention directed towards cases of PRV infection in humans. In consideration of the aforementioned factors, this paper presents a comprehensive review of recent developments in the PRV genome, epidemiology, vaccine research, and molecular detection methods. The epidemiology section presents an analysis of the transmission routes, susceptible animal groups, and geographic distribution of PRV, as well as an examination of the trend of the epidemic in recent years. In the field of vaccine research, the current development of genetically engineered vaccines is emphasized, and the immunogenicity and safety of vaccines are discussed. Moreover, the molecular detection techniques utilized to identify PRV, including immunological methods, nucleic acid detection methods, biosensors, and so forth, are presented in a systematic manner. Finally, this paper presents a comprehensive discussion of the current status of PRV-related research and offers insights into future directions, with the aim of providing a foundation for the scientific prevention and control of PRV. [ABSTRACT FROM AUTHOR]

Background: Pseudorabies (PR) caused by the re-emerging of pseudorabies virus (PRV) variant has outbroken among PRV vaccine immunized swine in many pig farms, which has caused serious social and economic consequences since the end of 2011. The PRV UL49.5 protein can inactivate the transporter associated with antigen processing (TAP), thereby downregulating the cell surface expression of swine leukocyte antigen class I (SLA-I) to evade host immune surveillance. Methods: In this study, based on the PRV ΔgE/TK strain, PRV ΔgE/TK/UL49.5 triple gene deletion strain was constructed through homologous recombination and deletion of the PRV UL49.5 gene by the Cre-LoxP system. Its growth curve and effect on SLA-I transcription level were determined. Preliminary studies were carried out on serum neutralizing antibody levels, IFN-γ and IL-4 cytokines levels in mice immunized with PRV ΔgE/TK/UL49.5, and the viral load and challenge protection in mice tissues after challenge. Results: The growth characteristics of PRV ΔgE/TK/UL49.5 strain were similar to those of PRV ΔgE/TK strain. The level of SLA-I was returned to normal after the deletion of PRV UL49.5 gene. The immunization of PRV ΔgE/TK/UL49.5 did not affect the weight gain of mice. Immunized mice could induce high levels of serum neutralization antibodies and immune cytokines, including IFN-γ and IL-4, which could provide complete protection against virulent PRV challenge. No obvious pathological damage was observed in lung, brain and trigeminal ganglion of mice immunized with PRV ΔgE/TK/UL49.5, and the tissue viral load was the lowest. Conclusions: PRV ΔgE/TK/UL49.5 strain can induce enhanced immunogenicity and had the potential to be used as a candidate strain. [ABSTRACT FROM AUTHOR]

Alpha-ketoglutarate-dependent dioxygenase, also known as fat mass and obesity-associated protein (FTO), is an RNA demethylase that mediates the demethylation of N6,2-O-dimethyladenosine (m6Am) and N6-methyladenosine (m6A). Both m6Am and m6A are prevalent modifications in mRNA and affect different aspects of transcript biology, including splicing, nuclear export, translation efficiency, and degradation. The role of FTO during (herpes) virus infection remains largely unexplored. In this study, we show that the UL13 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) triggers phosphorylation of FTO. In primary epithelial cells, depletion of FTO leads to increased expression of antiviral interferon-stimulated genes (ISGs) and UL13 triggers FTO-dependent suppression of ISG expression. Although PRV infection suppresses m6Am levels in host small nuclear RNA, this is independent of UL13. The current data highlight FTO as an important regulator of antiviral ISG expression and suggest that UL13-mediated phosphorylation of FTO may serve as a previously unrecognized viral strategy to suppress the antiviral interferon response. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is a herpes virus, also known as Aujeszky's disease virus (ADV), which can cause a highly infectious disease pseudorabies (PR) in a variety of mammals. In the past, it has been debated whether PRV can infect humans, but more and more cases of PRV infection have been reported since 2017. The illness has claimed many victims and left survivors with serious sequelae. This indicates that humans may ignore the zoonotic ability of PRV. This review aims to summarize the pathology and pathogenesis of PRV and speculate on how it infects humans. This paper provides a comprehensive overview of the progression of PRV, including its virology characteristics, genomic organization, and genetic evolution. It also synthesises the existing literature on PRV infection in humans, and analyses the factors contributing to PRV zoonosis. Finally, the pathogenesis of PRV-infected pigs and other mammals was summarized, and the pathogenesis of PRV-infected humans was speculated. [ABSTRACT FROM AUTHOR]

Background: Pseudorabies virus (PRV), porcine parvovirus (PPV) and porcine circovirus 3 (PCV3) are common in swine farms in China. Single infection or co-infection with PRV, PPV and/or PCV3 was difficult to distinguish between their clinical symptoms and pathological changes. Therefore, a quick and accurate detection method is needed for epidemiological surveillance, disease management, import and export control. Methods: In the present study, we established a multiplex real-time PCR assay based on SYBR Green I for the simultaneous detection of PRV, PPV and PCV3 genomes. Results: PRV, PPV and PCV3 were distinguished in the same sample by their different melting temperatures (Tm), with melting peaks at 90 °C for PRV, 84 °C for PPV and 80 °C for PCV3, respectively, and other non-targeted swine pathogens did not exhibit specific melting peaks. The assay showed a high degree of linearity (R2≧0.995), and the detection limits were 4.76 copies/μL for PRV, 3.67 copies/μL for PPV, 3.07 copies/μL for PCV3 and 1.87 × 102 copies/μL for the three mixed plasmids, respectively. In this research, 81 clinical samples from pig farms in nine different regions of Guangdong Province were used to evaluate this new method. The detection rate of the multiplex real-time PCR assay was higher than that of the conventional PCR assay. Conclusions: This multiplex real-time PCR assay could be used as a diagnostic tool that is rapid, sensitive and reliable for the detection of co-infection of PRV, PPV and PCV3 as well as for molecular epidemiological surveillance. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV), causing Aujeszky's disease in swine, has important economic impact on the pig industry in China and even poses a threat to public health. Although this disease has been controlled by vaccination with PRV live attenuated vaccines (LAVs), the potency of PRV LAVs in inducing cellular immunity has not been well characterized. In this study, using PRV Bartha K61 strain (BK61), the most-used PRV LAVs, as a model, we re-examined the cellular immune response elicited by the BK61 in mice and pigs by multicolor flow cytometry. We found that phenotypic activation of T cells, NK cells and B cells was hardly detected after vaccination. However, antigen-specific IFN-γ-producing CD4 T cells rather than CD8 T cells were dominantly detected but at low frequency upon restimulation with live BK61 virus. These BK61-specific CD4 T cells are also able to simultaneously produce TNF-α and IL-2, showing characteristics of multifunctional T cells. However, BK61-specific CD4 T cells showed weak secondary response upon challenge with PRV DX strain. Further vaccination with PRV-infected dendritic cells (DCs) transiently increased the percentage of IFN-γ-positive CD4 and CD8 T cells but eventually restored to low frequency and did not improve the protective efficacy of BK61 against challenge, suggesting that PRV BK61 induced a relatively weak cellular immunity that could not be overcome by the DC vaccination. Similar immune responses were also observed following vaccination with another PRV LAV HD/c in mice and pigs, suggesting that this may be an intrinsic drawback of PRV LAVs in inducing cellular immunity. Our results demonstrated that PRV LAVs elicited a CD4 Th1-biased weak cellular immunity which is implicative for the development of PRV-vectored vaccine. [ABSTRACT FROM AUTHOR]

Herpesviruses carry an assortment of proteins in the interstitial space between the capsid and membrane envelope, collectively referred to as the tegument. Upon virion fusion with a cell, envelope integrity is disrupted, and many tegument constituents disperse into the cytosol to carry out individual effector functions, while others direct transport of the capsid to the nucleus. To gain insight into the tegument dynamics that occur with disruption of envelope integrity, we used a combination of single-particle fluorescence and biochemical approaches that leveraged the previously established use of n-ethylmaleimide to inhibit virion dynamics. We document that the large tegument protein (pUL36), which is stably bound to the capsid surface at its C-terminus, is also conditionally bound to the capsid via its N-terminal deubiquitinase (DUB) domain. The DUB is released, while remaining tethered to the capsid by the pUL36 C-terminus, by a mechanism dependent on reactive cysteines. Mutation of these cysteines locks the DUB in a capsid bound state and suppresses enzymatic activity. IMPORTANCE Neuroinvasive alphaherpesviruses, such as herpes simplex virus and pseudorabies virus, cause a broad range of diseases in humans and other animals. Novel strategies to interfere with the virion structural rearrangements required for infectivity could prove valuable to treat infections, yet critical aspects of the virion architecture and its metastability remain poorly defined. In this study, we document that the pUL36 tegument protein exhibits conditional capsid binding in its N-terminal deubiquitinase domain that regulates enzymatic activity during infection. [ABSTRACT FROM AUTHOR]

The type I interferon signaling pathway constitutes a pivotal component of the innate immune response, encompassing the cGAS/STING and JAK/STAT pathways. Drugs that affect the body's innate immune response could potentially be used as broad-spectrum antivirals. In this study, the antiviral activities of 25 flavo noids against pseudorabies virus (PRV) were tested in PK-15 cells. Eight active flavo noids were identified, with IC50 values ranging from 23.24 to 323.09 µM. Subsequently, the regulatory effects of these flavonoids on the cGAS/STING pathway in PRV-infected cells were investigated. It was found that Myricetin significantly increased the transcriptional levels of cGAS, STING, IRF3, and IFN-ß, which had been reduced by PRV infection. The regulation of the type I in terferon signaling pathways by myricetin following PRV infection was further investigated through the production of cGAMP and the assessment of transcriptional and protein levels of pivotal genes and proteins. To confirm the activation of the innate immune response, a dual luciferase gene reporter study found that the expression of the IFN-ß promoter in the myricetin-treated group was significantly elevated in a cellular model of type I interferon signaling pathway, and the contents of IFN-ß were also significant ly higher than those observed in the infected-untreated group in a PRV-infected mice model. Moreover, the transcriptional and protein levels of key genes and proteins in cell and mouse models exhibited analogous outcomes to those observed in PRV-infected cells. These findings suggest that myricetin can effectively activate the type I interferon signaling pathway, thereby enhancing the innate immune response during PRV infection. IMPORTANCE PRV, belonging to the Herpesviridae family, is an easily overlooked zoonotic pathogen that can threaten human health. The immunoprotective efficacy of conventional vaccines is significantly reduced due to the continuous mutation of the PRV genome, which constantly generates new viral strains. Therefore, there is a need to develop potent therapeutic drugs. PRV is capable of evading the host's natural immunity by suppressing the host's type I interferon signaling pathway, and the search for drugs that activate natural immunity can induce the body to produce type I IFN interferon and exert antiviral effects. Accordingly, the present study sought to identify active compounds from flavonoids that modulate the type I IFN interferon signaling pathway and thus inhibit the proliferation of PRV, which provides a new idea for the development of anti-PRV drugs from flavonoids that modulate the type I IFN interferon signaling pathway to enhance the body's antiviral immunity. [ABSTRACT FROM AUTHOR]

Following reactivation of a latent alphaherpesvirus infection, viral particles are assembled in neuronal cell bodies, trafficked anterogradely within axons to nerve termini, and spread to adjacent epithelial cells. The virally encoded membrane proteins US9p and the glycoprotein heterodimer gE/gI of pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) play critical roles in anterograde spread, likely as a tripartite gE/gI-US9p complex. Two kinesin motors, kinesin-1 and kinesin-3, are implicated in the egress of these viruses, but how gE/gI-US9p coordinates their activities is poorly understood. Here, we report that PRV, in addition to associating with the kinesin-3 motor KIF1A, recruits the neuronal kinesin-1 isoforms KIF5A and KIF5C, but not the broadly expressed isoform KIF5B, during egress from differentiated CAD neurons. Similarly, in the axons of dorsal root ganglia (DRG)-derived sensory neurons, PRV colocalized with KIF5C but not KIF5B. In differentiated CAD cells, the association of KIF1A with egressing PRV was dependent upon US9p, whereas the recruitment of KIF5 isoforms required gE/gI. Consistent with these findings, the number of PRV particles trafficking within CAD neurites and the axons of DRG neurons increased when kinesin-1 motor activity was upregulated by hyperacetylating microtubules using trichostatin A (TSA) or tubacin, and this enhanced trafficking depended upon the presence of gE/gI. We propose that, following its recruitment by US9p, KIF1A delivers PRV particles to a location where KIF5 motors are subsequently added by a gE/gI-dependent mechanism. KIF5A/C isoforms then serve to traffic viral particles along axons, resulting in characteristic recrudescent infection. IMPORTANCE Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors. [ABSTRACT FROM AUTHOR]

Abstract The primary cause of viral encephalitis (VE) is invasion of the central nervous system (CNS) by the virus, which leads to neuroinflammation and poses a significant threat to global public health. Microglia, as CNS-resident macrophages, play a crucial role in neuroinflammation and are often identified as the preferred target for the prevention or treatment of VE. In this study, we used pseudorabies virus (PRV)-induced VE in mice and pigs as a model to investigate the regulation of microglial responses during viral encephalitis and explored the mechanism of microglial activation. Cellular experiments revealed that microglial activation was accompanied by cell migration, characteristic morphological changes, phagocytosis, inflammatory cytokine production, and antigen presentation. Transcriptome analysis revealed that genes related to inflammation in PRV-infected BV2 cells were significantly enriched. The expression of the NOD1 gene in BV2 cells was significantly increased during PRV infection, after which NOD1 in BV2 cells was silenced by siRNA and overexpressed via a plasmid. NOD1 was found to be involved in the secretion of cytokines in BV2 cells by regulating the MAPK/NF-κB signalling pathway. Mouse and pig experiments have shown that NOD1 is involved in the secretion of cytokines by microglia by regulating the MAPK/NF-κB signalling pathway during PRV infection.

Abstract Pseudorabies virus (PRV) can establish lifelong latent infection in peripheral nervous ganglion, and persistent infections in peripheral blood lymphocytes. Establishing an infection in the lymphocytes does not only enable the PRV to escape host immune surveillance but pass through the placental barrier, leading to fetal death and abortion. Due to the pathogenicity of the PRV, it poses a huge challenge in its prevention and control. The PRV escapes host immunity through downregulation of swine leukocyte antigen class I (SLA I) molecules on infected cells. However, data on the molecular mechanisms of the SLA I suppression remains scant. Here, in order to verify the effect of candidate proteins PRV pUL44 and pUS6 on PRV immune escape related molecules SLA I and peptide loading complex (PLC), we detected the expression of SLA I and PLC components after expressing PRV pUL44 and pUS6. The effects of pUS6 and pUL44 on SLA I and PLC were analyzed by qRT-PCR and Western blot at mRNA and protein level, respectively. Cells expressing pUS6 or pUL44 genes showed a significantly suppressed expression of surface and total SLA I molecules. In addition, unlike UL44, the US6 gene was shown to downregulate the transporter associated with antigen processing 1 (TAP1), TAP2 and Tapasin molecules. The results show that PRV pUS6 may participate in virus immune escape by directly regulating the SLA I, TAP dimer and Tapasin molecules, thus blocking the transportation of TAP-bound peptides to the ER to bind SLA I molecules. We provide a theoretical basis on the mechanism of TAP mediated immune escape by the PRV.

The primary cause of viral encephalitis (VE) is invasion of the central nervous system (CNS) by the virus, which leads to neuroinflammation and poses a significant threat to global public health. Microglia, as CNS-resident macrophages, play a crucial role in neuroinflammation and are often identified as the preferred target for the prevention or treatment of VE. In this study, we used pseudorabies virus (PRV)-induced VE in mice and pigs as a model to investigate the regulation of microglial responses during viral encephalitis and explored the mechanism of microglial activation. Cellular experiments revealed that microglial activation was accompanied by cell migration, characteristic morphological changes, phagocytosis, inflammatory cytokine production, and antigen presentation. Transcriptome analysis revealed that genes related to inflammation in PRV-infected BV2 cells were significantly enriched. The expression of the NOD1 gene in BV2 cells was significantly increased during PRV infection, after which NOD1 in BV2 cells was silenced by siRNA and overexpressed via a plasmid. NOD1 was found to be involved in the secretion of cytokines in BV2 cells by regulating the MAPK/NF-κB signalling pathway. Mouse and pig experiments have shown that NOD1 is involved in the secretion of cytokines by microglia by regulating the MAPK/NF-κB signalling pathway during PRV infection. [ABSTRACT FROM AUTHOR]

Simple Summary: The pseudorabies virus (PRV) remains a devastating pathogen in swine, significantly impacting animal health and causing substantial losses in the breeding industry. By summarizing the latest advancements, including innate immunity, metabolic pathways, autophagic processes, protein degradation systems, and others, this work underscores the pivotal role of various cellular factors and pathways in shaping the PRV–host dynamic. Additionally, the work highlights viral sophisticated escape mechanisms that enable it to evade host defenses for self-replication. We hope that this work can provide a profound understanding of PRV–host interactions, laying the groundwork for the development of innovative therapeutic strategies to combat this virus. The pseudorabies virus (PRV), also known as suid alphaherpesvirus 1 (SuAHV-1), has garnered significant attention due to its broad host range and the economic losses it incurs in the swine industry. This review aims to provide a comprehensive understanding of the intricate virus–host interactions during PRV infection, focusing on the evasion strategies of the virus against the host responses. We also summarize the mechanisms by which PRV manipulates the host cell machinery to facilitate its replication and spread, while simultaneously evading detection and clearance by the immune system. Furthermore, we discuss the latest advancements, such as metabolic, autophagic, and apoptotic pathways in studying these interactions, highlighting the role of various cellular factors and pathways in elucidating virus–host dynamics. By integrating these insights, the article aims to provide a comprehensive overview of the molecular mechanisms underlying PRV pathogenesis and host response, paving the way for the development of novel therapeutic strategies against this virus. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is the pathogenic agent of pseudorabies, causing serious reproductive failure in swine. However, it is still unknown whether PRV uterine inoculation impairs blastocyst implantation. In the present study, a PRV infection mouse model was developed. Pregnant mice were inoculated with either 104 or 105 50% tissue culture infectious dose (TCID50) units of PRV on gestation day 2 (GD2). Viral DNA was detected in tissues by PCR and/or in situ hybridization. Histopathological change and expression of proinflammatory cytokines in uterus were analyzed by H.E. staining and qPCR, respectively. Apoptosis was also investigated by TUNEL assay, and the expression of apoptosis-related proteins including Bax and Bcl-2 was detected by Western blot. The results showed that intrauterine exposure of PRV on GD2 reduced the number of embryo implantation site. Abundant viral DNA was detected in spinal marrow and brain, and small amounts of PRV genomes were detected in embryo implantation site, ovary as well as thymus. Considerable inflammatory cells infiltrating in the endometrium, with high levels of pro-inflammatory cytokines of interleukin (IL)-6, IL-1β and tumor necrosis factor- α mRNA after infection. In addition, PRV infection promoted apoptosis in stroma and endothelium of the mouse endometrium. Collectively, intrauterine inoculation of PRV during early pregnancy causes cytokine release syndrome and apoptosis in endometrium, which impairs mouse embryo implantation. [ABSTRACT FROM AUTHOR]

Since 2011, the emergence of Pseudorabies virus (PRV) variants has led to significant vaccine failures, resulting in severe economic losses in China’s swine industry. Conventional PRV vaccines have shown limited efficacy against these emergent variants, underscoring the urgent need for novel immunization strategies. This study aimed to develop and evaluate a novel recombinant PRV vaccine candidate with improved safety and immunogenicity profiles. Utilizing the homology-directed repair (HDR)-CRISPR/Cas9 system, we generated a recombinant PRV strain, designated PRV SX-10ΔgI/gE/TK/UL24, with deletions in the gI, gE, TK, and UL24 genes. In vitro analyses demonstrated that the recombinant virus exhibited similar replication kinetics and growth curves comparable to the parental strain. The immunological properties of the recombinant PRV were assessed in murine and porcine models. All animals inoculated with PRV SX-10ΔgI/gE/TK/UL24 survived without exhibiting significant clinical signs or pathological alterations. Immunological assays revealed that PRV SX-10ΔgI/gE/TK/UL24 elicited significantly higher levels of gB-specific antibodies, neutralizing antibodies, and cytokines (including IFN-γ, IL-2, and IL-4) compared to both the Bartha-K61 and PRV SX-10ΔgI/gE/TK strains. Notably, both murine and porcine subjects immunized with PRV SX-10ΔgI/gE/TK/UL24 demonstrated enhanced protection against challenges with the variant PRV SX-10 strain, compared to other vaccine strains. These findings suggest that PRV SX10ΔgI/gE/TK/UL24 represents a promising PRV vaccine candidate strain, offering valuable insights for the prevention and control of PRV in clinical applications. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) can establish lifelong latent infection in peripheral nervous ganglion, and persistent infections in peripheral blood lymphocytes. Establishing an infection in the lymphocytes does not only enable the PRV to escape host immune surveillance but pass through the placental barrier, leading to fetal death and abortion. Due to the pathogenicity of the PRV, it poses a huge challenge in its prevention and control. The PRV escapes host immunity through downregulation of swine leukocyte antigen class I (SLA I) molecules on infected cells. However, data on the molecular mechanisms of the SLA I suppression remains scant. Here, in order to verify the effect of candidate proteins PRV pUL44 and pUS6 on PRV immune escape related molecules SLA I and peptide loading complex (PLC), we detected the expression of SLA I and PLC components after expressing PRV pUL44 and pUS6. The effects of pUS6 and pUL44 on SLA I and PLC were analyzed by qRT-PCR and Western blot at mRNA and protein level, respectively. Cells expressing pUS6 or pUL44 genes showed a significantly suppressed expression of surface and total SLA I molecules. In addition, unlike UL44, the US6 gene was shown to downregulate the transporter associated with antigen processing 1 (TAP1), TAP2 and Tapasin molecules. The results show that PRV pUS6 may participate in virus immune escape by directly regulating the SLA I, TAP dimer and Tapasin molecules, thus blocking the transportation of TAP-bound peptides to the ER to bind SLA I molecules. We provide a theoretical basis on the mechanism of TAP mediated immune escape by the PRV. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) exhibits a complex interplay of host-pathogen interactions, primarily by modulating host cell death pathways to optimize its replication and spread in Neuro-2a cells. Using high-throughput RNA sequencing, we identified 2,382 upregulated differentially expressed genes (DEGs) and 3,998 downregulated DEGs, indicating a intricate interaction between viral pathogenesis and host cellular responses. This research offers valuable insights into the molecular processes involved in PRV infection, highlighting the substantial inhibition of crucial cell death pathways in Neuro-2a cells, including necroptosis, pyroptosis, autophagy, ferroptosis, and cuproptosis. Cells infected with PRV exhibit decreased expression of genes critical in these pathways, potentially as a mechanism to avoid host immune reactions and ensure cell survival to support ongoing viral replication. This extensive inhibition of apoptosis and metabolic alterations highlights the sophisticated tactics utilized by PRV, enhancing our comprehension of herpesvirus biology and the feasibility of creating specific antiviral treatments. This research contributes to our understanding of how viruses manipulate host cell death and presents potential opportunities for therapeutic interventions to disrupt the virus's lifecycle. [ABSTRACT FROM AUTHOR]

The kidneys are essential organs that help maintain homeostasis, and their function is regulated by the neural system. Despite the anatomical multi-synaptic connection between the central autonomic nuclei and the kidneys, it remains unclear whether there are any variations in neural connections between the nervous systems and the renal cortex and medulla in male and female mice. Here, we used the pseudorabies virus to map the central innervation network of the renal cortex and medulla in both sexes. The data revealed that specific brain regions displayed either a contralateral-bias or ipsilateral-bias pattern while kidney-innervating neurons distributed symmetrically in the midbrain and hindbrain. Sex differences were observed in the distribution of neurons connected to the left kidney, as well as those connected to the renal cortex and medulla. Our findings provide a comprehensive understanding of the brain-kidney network in both males and females and may help shed light on gender differences in kidney function and disease susceptibility in humans. [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudorabies virus (PRV) is an ideal model for mechanistic investigations into α-herpesvirus. The neurotropism and latent infection of PRV have been extensively studied. Apart from neurological symptoms, diarrhea caused by PRV infection is also an essential cause of mortality in newborn and weaned piglets. However, little research has been done on PRV invasion of the gut. To fill this gap, a nasal drip PRV-infection mouse model was developed, consisting of three groups: the challenged group (Group A), the immunization-challenged group (Group B), and a mock group (Group C). The results showed that immunization with PRV XJ delgE/gI/TK successfully prevented intestinal damage caused by PRV drop-nose infection. Subsequently, intestines were collected for transcriptional analysis. Differentially expressed genes analysis revealed that PRV XJ delgE/gI/TK was effective in reducing the organismal intestinal transcriptional activity caused by PRV. The Group A vs Group C and Group A vs Group B had similar Kyoto Encyclopedia of Genes and Genomes (KEGG)-enriched signaling pathways and the differentially expressed genes were primarily enriched in pathways, such as cell adhesion molecules, focal adhesion kinase, and actin cytoskeleton regulation. Notably, transcriptome analysis indicated that genes associated with the focal adhesion kinase (FAK) signaling pathway (ECM-ITGA/ITGB-p-FAK) were significantly more highly expressed in Group A than in Group B and Group C. The results of quantitative real-time PCR (RT-qPCR) and western blotting were consistent with KEGG analysis. Therefore, we hypothesized that PRV promotes self-infection through activation of the ECM-ITGA/ITGB-p-FAK signaling pathway and that PRV XJ delgE/gI/TK immunization could attenuate the intestinal damage caused by PRV by inhibiting the activation of this pathway.IMPORTANCEPseudorabies virus (PRV) poses a significant threat to the swine industry and public health due to its ability to infect multiple species, including humans, leading to substantial economic losses and potential health risks. This study addresses a critical gap in understanding the impact of PRV infection on the gut, which has been less explored compared to its neurological effects. By developing a drip-nose PRV-infection mouse model, the research indicated that PRV might promote self-infection through activation of the ECM-ITGA/ITGB-p-FAK signaling pathway, and PRV XJ delgE/gI/TK immunization effectively prevents intestinal damage by significantly reducing the expression of genes in the ECM-ITGA/ITGB-p-FAK signaling pathway. The research has important implications for the swine industry and public health by contributing to the development of better vaccines and treatments, ultimately helping to control PRV and prevent its cross-species transmission.

Abstract Pseudorabies have caused enormous economic losses in China’s pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China.

Abstract Serine/threonine kinase receptor-associated protein (STRAP) serves as a scaffold protein and is engaged in a variety of cellular activities, although its importance in antiviral innate immunity is unknown. We discovered that STRAP works as an interferon (IFN)-inducible positive regulator, facilitating type I IFN signaling during pseudorabies virus infection. Mechanistically, STRAP interacts with TBK1 to activate type I IFN signaling. Both the CT and WD40 7 − 6 domains contribute to the function of STRAP. Furthermore, TBK1 competes with PRV-UL50 for binding to STRAP, and STRAP impedes the degradation of TBK1 mediated by PRV-UL50, thereby increasing the interaction between STRAP and TBK1. Overall, these findings reveal a previously unrecognized role for STRAP in innate antiviral immune responses during PRV infection. STRAP could be a potential therapeutic target for viral infectious diseases.

Abstract Background Pseudorabies is an infection of domestic and wild pigs that has occasionally been reported in dogs with fatal encephalitis. Hunting dogs are predisposed to pseudorabies exposure due to incorrect practices (administration of raw infected meat) or close contact with infected wild boars. This study described an outbreak of pseudorabies in two hunting dogs in the Campania region, southern Italy. Case presentation Two hunting dogs were hospitalized after a hunting trip, with fever, itching, and self-inflicted lesions. Laboratory tests showed mild anemia and marked leukocytosis. Despite conservative therapy, both animals died 48 h after the presentation of symptoms. One of the carcasses was sent to the Department of Veterinary Medicine and Animal Production in Naples to confirm the suspicion of pseudorabies. DNA was extracted from different matrices and used as a template for real-time PCR to detect PRV. Several samples (brain, cerebellum, brainstem, lung, and liver) tested positive. Subsequent sequence analyses of glycoprotein E from DNA extracted from the brain stem revealed a sequence similarity to those described in previous cases of pseudorabies in dogs in Italy, France and Belgium. One month after the outbreak, blood samples were collected from 42 dogs belonging to the same hunting team and from 245 dogs (cohort population) living in the Campania region. All samples were tested with two commercial ELISAs to detect seroconversion against glycoproteins B and E. A seroprevalence of 19% was observed in the hunting team affected by the outbreak, while only 0.8% was observed in the regional dog population. Conclusions The data reported in this study demonstrate potential exposure to PRV by dead-end hosts, particularly hunting dogs. The sequencing results indicated the homogeneity of PRV strains circulating in the different Italian regions.

Serine/threonine kinase receptor-associated protein (STRAP) serves as a scaffold protein and is engaged in a variety of cellular activities, although its importance in antiviral innate immunity is unknown. We discovered that STRAP works as an interferon (IFN)-inducible positive regulator, facilitating type I IFN signaling during pseudorabies virus infection. Mechanistically, STRAP interacts with TBK1 to activate type I IFN signaling. Both the CT and WD40 7 − 6 domains contribute to the function of STRAP. Furthermore, TBK1 competes with PRV-UL50 for binding to STRAP, and STRAP impedes the degradation of TBK1 mediated by PRV-UL50, thereby increasing the interaction between STRAP and TBK1. Overall, these findings reveal a previously unrecognized role for STRAP in innate antiviral immune responses during PRV infection. STRAP could be a potential therapeutic target for viral infectious diseases. [ABSTRACT FROM AUTHOR]

Pseudorabies have caused enormous economic losses in China's pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China. [ABSTRACT FROM AUTHOR]

Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines. [ABSTRACT FROM AUTHOR]

To analyze a case of acute retinal necrosis (ARN) associated with pseudorabies virus (PRV) infection and discusses the clinical characteristics of PRV-induced ARN (PRV-ARN). Case report and literature review of ocular features in PRV-ARN. A 52-year-old female diagnosed with encephalitis presented with bilateral vision loss, mild anterior uveitis, vitreous opacity, occlusive retinal vasculitis, and retinal detachment in her left eye. The result of metagenomic next-generation sequencing (mNGS) indicated that both cerebrospinal fluids and vitreous fluid tested positive for PRV. PRV, a zoonosis, can infect both humans and mammals. Patients affected with PRV may experience severe encephalitis and oculopathy, and the infection has been associated with high mortality and disability. ARN is the most common ocular disease, which develops rapidly following encephalitis and is characterized by five figures: bilateral onset, rapid progression, severe visual impairment, poor response to systemic antiviral drugs, and an unfavorable prognosis. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) disrupts coordination between the bladder and the external urinary sphincter (EUS), leading to transient or permanent voiding impairment, which is more severe in males. Male versus female differences in spinal circuits related to the EUS as well as post‐SCI rewiring are essential for understanding of sex‐/gender‐specific impairments and possible recovery mechanisms. To quantitatively assess differences between EUS circuits in males versus females and in spinal intact (SI) versus SCI animals, we retrogradely traced and counted EUS‐related neurons. In transgenic ChAT‐GFP mice, motoneurons (MNs), interneurons (INs), and propriospinal neurons (PPNs) were retrogradely trans‐synaptically traced with PRV614‐red fluorescent protein (RFP) injected into EUS. EUS‐MNs in dorsolateral nucleus (DLN) were separated from other GFP+ MNs by tracing them with FluoroGold (FG). We found two morphologically distinct cell types in DLN: FG+ spindle‐shaped bipolar (SB‐MNs) and FG− rounded multipolar (RM‐MNs) cholinergic cells. Number of MNs of both types in males was twice as large as in females. SCI caused a partial loss of MNs in all spinal nuclei. After SCI, males showed a fourfold rise in the number of RFP‐labeled cells in retro‐DLN (RDLN) innervating hind limbs. This suggests (a) an existence of direct synaptic interactions between spinal nuclei and (b) a post‐SCI increase of non‐specific inputs to EUS‐MNs from other motor nuclei. Number of INs and PPNs deferred between males and females: In SI males, the numbers of INs and PPNs were ∼10 times larger than in SI females. SCI caused a twofold decrease of INs and PPNs in males but not in females. [ABSTRACT FROM AUTHOR]

Importance: As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear. Objective: This study examined the function of EP0 to provide a direction for its in-depth analysis. Methods: In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells. Results: This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization. Conclusions and Relevance: These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV), causing Aujeszky’s disease in swine, has important economic impact on the pig industry in China and even poses a threat to public health. Although this disease has been controlled by vaccination with PRV live attenuated vaccines (LAVs), the potency of PRV LAVs in inducing cellular immunity has not been well characterized. In this study, using PRV Bartha K61 strain (BK61), the most-used PRV LAVs, as a model, we re-examined the cellular immune response elicited by the BK61 in mice and pigs by multicolor flow cytometry. We found that phenotypic activation of T cells, NK cells and B cells was hardly detected after vaccination. However, antigen-specific IFN-γ-producing CD4 T cells rather than CD8 T cells were dominantly detected but at low frequency upon restimulation with live BK61 virus. These BK61-specific CD4 T cells are also able to simultaneously produce TNF-α and IL-2, showing characteristics of multifunctional T cells. However, BK61-specific CD4 T cells showed weak secondary response upon challenge with PRV DX strain. Further vaccination with PRV-infected dendritic cells (DCs) transiently increased the percentage of IFN-γ-positive CD4 and CD8 T cells but eventually restored to low frequency and did not improve the protective efficacy of BK61 against challenge, suggesting that PRV BK61 induced a relatively weak cellular immunity that could not be overcome by the DC vaccination. Similar immune responses were also observed following vaccination with another PRV LAV HD/c in mice and pigs, suggesting that this may be an intrinsic drawback of PRV LAVs in inducing cellular immunity. Our results demonstrated that PRV LAVs elicited a CD4 Th1-biased weak cellular immunity which is implicative for the development of PRV-vectored vaccine.

ABSTRACT The interaction between Z-DNA binding protein 1 (ZBP1) and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been uncovered in several viral infections. However, the role of this molecular pathway during infection with the alpha-herpesvirus pseudorabies virus (PRV) remains largely elusive. Here, we report that during PRV infection, ZBP1-mediated NLRP3 inflammasome activation is inhibited by the viral tegument protein VP22, thereby facilitating viral infection. Through a combination of RNA sequencing and genetic studies, we demonstrate that PRV VP22 functions as a virus-encoded virulence factor by evading the inhibitory effects of ZBP1 on virus infection. Importantly, the replication and pathogenicity of a recombinant PRV lacking VP22 are significantly increased in ZBP1-deficient cells and mice. Mechanistically, PRV VP22 interacts with ZBP1, impeding the recruitment of receptor-interacting protein kinase 3 and Caspase-8, thereby inhibiting NLRP3 activation. Furthermore, we show that the N-terminal 1–50 amino acid domain of VP22 dominantly destabilizes ZBP1-mediated function. Taken together, these findings identify a functional link between PRV infection and ZBP1-mediated NLRP3 inflammatory response, providing novel insights into the pathogenesis of PRV and other herpesviruses.IMPORTANCEZ-DNA binding protein 1 (ZBP1) functions as a pivotal innate immune sensor that regulates inflammatory cell death during viral infections. However, its role in pseudorabies virus (PRV) infection remains unknown. Here, we demonstrate that ZBP1 serves as a restrictive factor by triggering the activation of the NLR family pyrin domain-containing 3 inflammasome, a process counteracted by PRV-encoded protein VP22. Furthermore, VP22 interferes with the interaction between ZBP1 and receptor-interacting protein kinase 3/Caspase-8, particularly through its N-terminal 1–50 amino acids. Importantly, deficiency in ZBP1 enhances the replication and virulence of recombinant viruses lacking VP22 or its N-terminal 1–50 amino acids. These findings reveal how PRV escapes ZBP1-mediated inflammatory responses during infection, potentially informing the rational design of therapeutic interventions.

ABSTRACT Pseudorabies virus (PRV), a member of the alpha-herpesviruses, can infect both the nervous and reproductive systems of pigs, causing neonatal mortality and reproductive failure in sows, which incurs substantial economic losses. Neurotropism is a common characteristic of various viruses, allowing them to cross the blood-brain barrier and access the central nervous system. However, the precise mechanisms by which PRV affects the blood-brain barrier are not well understood. To investigate the mechanism of PRV’s interaction with the blood-brain barrier and its engagement with the PI3K/Akt signaling pathway during infection, an in vitro monolayer cell model of the blood-brain barrier was established. Our research found that PRV activates Matrix metallopeptidase 2 (MMP2), which degrades Zonula occludens-1 (ZO-1) and consequently enhances the permeability of the blood-brain barrier. PRV infection elevated the transcriptional levels of tissue inhibitor of metalloproteinases 1 (TIMP1) and inhibited its degradation through the ubiquitin-proteasome pathway, leading to higher intracellular concentrations of TIMP1 protein. TIMP1 regulates apoptosis and inhibits PRV replication in mouse brain microvascular endothelial cells through the PI3K/Akt/mTOR signaling pathway. In summary, our study delineates the mechanism through which PRV compromises the blood-brain barrier and provides insights into the host’s antiviral defense mechanisms post-infection.IMPORTANCEPRV, known for its neurotropic properties, is capable of inducing severe neuronal damage. Our study discovered that following PRV infection, the expression of MMP2 was upregulated, leading to the degradation of ZO-1. Furthermore, upon PRV infection in the host, the promoter of TIMP1 is significantly activated, resulting in a significant increase in TIMP1 protein levels. This upregulation of TIMP1 inhibits the proliferation of PRV through the PI3K/Akt signaling pathway. This study elucidated the mechanism through which PRV, including the PRV XJ delgE/gI/TK strains, compromises the blood-brain barrier and identifies the antiviral response characterized by the activation of the PI3K/Akt signaling pathway within infected host cells. These findings provide potential therapeutic targets for the clinical management and treatment of PRV.

Pseudorabies virus (PRV) poses a significant threat to the global swine industry, characterized by high morbidity and a range of sequelae in infected pigs. Mitochondria serve as a crucial platform for innate immunity, playing a pivotal role in a wide array of antiviral responses. In our recent study, we revealed that PRV infection induces mitochondrial disruption, which in turn triggers PINK1/PARKIN-mediated mitophagy. We also show that this process leads to the degradation of the mitochondrial antiviral signaling protein (MAVS) and the inhibition of antiviral interferon production and signaling, ultimately facilitating viral replication.

Pseudorabies virus (PRV) is the pathogenic agent of pseudorabies, causing serious reproductive failure in swine. However, it is still unknown whether PRV uterine inoculation impairs blastocyst implantation. In the present study, a PRV infection mouse model was developed. Pregnant mice were inoculated with either 104 or 105 50% tissue culture infectious dose (TCID50) units of PRV on gestation day 2 (GD2). Viral DNA was detected in tissues by PCR and/or in situ hybridization. Histopathological change and expression of proinflammatory cytokines in uterus were analyzed by H.E. staining and qPCR, respectively. Apoptosis was also investigated by TUNEL assay, and the expression of apoptosis-related proteins including Bax and Bcl-2 was detected by Western blot. The results showed that intrauterine exposure of PRV on GD2 reduced the number of embryo implantation site. Abundant viral DNA was detected in spinal marrow and brain, and small amounts of PRV genomes were detected in embryo implantation site, ovary as well as thymus. Considerable inflammatory cells infiltrating in the endometrium, with high levels of pro-inflammatory cytokines of interleukin (IL)-6, IL-1β and tumor necrosis factor-α mRNA after infection. In addition, PRV infection promoted apoptosis in stroma and endothelium of the mouse endometrium. Collectively, intrauterine inoculation of PRV during early pregnancy causes cytokine release syndrome and apoptosis in endometrium, which impairs mouse embryo implantation.

Background: Pseudorabies virus (PRV), responsible for pseudorabies, is one of the most important pathogen in swine industry. PRV infection suppresses interferon-ß (IFN-ß) and tumor necrosis factor- (TNF-) expression, which consequently results in an immune escape in the host. Methods: In this study, distribution of peripheral blood T and B cell sub-populations was analyzed by flow cytometry after infecting mice with 1105 TCID50 PRV. Result: We found that the levels of CD3+ T, CD4+ T and CD8+ T lymphocyte in infection group were significantly decreased at 24 hpi (p<0.01) and remained at a low level until 72 hpi compared to the control group. Interestingly, the levels of CD19+ T lymphocyte were significantly lower after 24 hpi (p<0.01) and lasted until 48 hpi, then increased sharply and then dropped at 96 hpi. Collectively, these results indicated that PRV infection could play a negative role in adaptive immunity in mice by decreasing the levels of B and T lymphocytes. [ABSTRACT FROM AUTHOR]

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The pseudorabies virus (PRV) is identified as a double-helical DNA virus responsible for causing Aujeszky's disease, which results in considerable economic impacts globally. The enzyme tryptophanyl-tRNA synthetase 2 (WARS2), a mitochondrial protein involved in protein synthesis, is recognized for its broad expression and vital role in the translation process. The findings of our study showed an increase in both mRNA and protein levels of WARS2 following PRV infection in both cell cultures and animal models. Suppressing WARS2 expression via RNA interference in PK-15 cells led to a reduction in PRV infection rates, whereas enhancing WARS2 expression resulted in increased infection rates. Furthermore, the activation of WARS2 in response to PRV was found to be reliant on the cGAS/STING/TBK1/IRF3 signaling pathway and the interferon-alpha receptor-1, highlighting its regulation via the type I interferon signaling pathway. Further analysis revealed that reducing WARS2 levels hindered PRV's ability to promote protein and lipid synthesis. Our research provides novel evidence that WARS2 facilitates PRV infection through its management of protein and lipid levels, presenting new avenues for developing preventative and therapeutic measures against PRV infections. [ABSTRACT FROM AUTHOR]

Introduction: Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput. Methods: This study was designed to produce monoclonal antibodies targeting PRV through immunization ofmice using the eukaryotic systemto express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips. Results and discussion: The strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336×103 copies/µL (the number of viral genome copies permicroliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality. [ABSTRACT FROM AUTHOR]

In recent years, pseudorabies virus (PRV) variants have resulted in an epidemic in swine herds and huge economic losses in China. Therefore, it is essential to develop an efficacious vaccine against the spread of PRV variants. Here, the triple-gene-deletion virus and the triple-gene-deletion plus gC virus were constructed by homologous recombination (HR). And then, their growth capacity, proliferation ability, and immune efficacy were evaluated. The results showed that the growth kinetics of the recombinant viruses were similar to those of the parental strain PRV-AH. Compared with the triple-gene-deletion virus group, the more dominant level of neutralizing antibody (NA) can be induced in the triple-gene-deletion plus gC virus group with the same 106.0 TCID50 dose after 4 and 6 weeks post-initial immunization (PII) (p < 0.0001). In addition, the antibody titers in mice immunized with the triple-gene-deletion plus gC virus were significantly higher than those immunized with triple-gene deletion virus with the same 105.0 TCID50 dose after 6 weeks PII (p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 105.0 TCID50, the level of NA was close to that in the triple-gene deletion virus group with 106.0 TCID50 at 6 weeks PII. Meanwhile, the cytokines IL-4 and IFN-γ in sera were tested by enzyme-linked immunosorbent assay (ELISA) in each group. The highest level of IL-4 or IFN-γ was also elicited in the triple-gene deletion plus gC virus group at a dose of 106.0 TCID50. After challenge with PRV-AH, the survival rates of the triple-gene deletion plus gC virus immunized groups were higher than those of other groups. In immunized groups with 105.0 TCID50, the survival rate shows a significant difference between the triple-gene deletion plus gC virus group (75%, 6/8) and the triple-gene deletion virus group (12.5%, 1/8). In general, the immune efficacy of the PRV TK/gI/gE-deleted virus can be increased with additional gC insertion in mice, which has potential for developing an attenuated vaccine candidate for PRV control. [ABSTRACT FROM AUTHOR]

Abstract Background Pseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro. Methods Primary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting. Results Virus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection. Conclusions The data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

In 2022, a significant PRV outbreak in a southwestern China pig farm led to a high incidence of sow abortion. A serological analysis using gE antigen-based ELISA revealed a high prevalence (69.30%) of PRV gE antibodies among the affected pigs, with a significant variation across different pig populations (1.11–76.12%). We collected additional 5552 pig serum samples and 580 pig cerebrospinal fluid (CSF) samples from various pig farms in Southwest China between 2022 and 2024. The seropositive rates for PRV gE antibodies ranged from 2.36% and 8.65% in the serum samples, while the positive detection rates for the PRV gE gene in the cerebrospinal fluid samples, as determined by PCR, were between 1.06% and 2.36%. The PCR analysis and sequencing of the PRV gB, gC, gE, and TK genes from eight randomly selected samples identified two distinct strains, CQ1 and CQ2. CQ1’s gC gene showed similarity to the vaccine strain Bartha, while the other genes aligned with Chinese classical strains, suggesting its potential genetic recombination. CQ2 aligned with the Chinese classical strain SC. Although the overall PRV infection in Southwest China’s pig farms is relatively low, occasional outbreaks with high positivity rates are observed. These findings highlight the necessity for increased surveillance and stringent control measures to safeguard the swine industry.

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Abstract Background Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood. Methods To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing. Results The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed. Conclusions Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.

Abstract The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.

Abstract Background Oncolytic viruses are now well recognized as potential immunotherapeutic agents against cancer. However, the first FDA-approved oncolytic herpes simplex virus 1 (HSV-1), T-VEC, showed limited benefits in some patients in clinical trials. Thus, the identification of novel oncolytic viruses that can strengthen oncolytic virus therapy is warranted. Here, we identified a live-attenuated swine pseudorabies virus (PRV-LAV) as a promising oncolytic agent with broad-spectrum antitumor activity in vitro and in vivo. Methods PRV cytotoxicity against tumor cells and normal cells was tested in vitro using a CCK8 cell viability assay. A cell kinase inhibitor library was used to screen for key targets that affect the proliferation of PRV-LAV. The potential therapeutic efficacy of PRV-LAV was tested against syngeneic tumors in immunocompetent mice, and against subcutaneous xenografts of human cancer cell lines in nude mice. Cytometry by time of flight (CyTOF) and flow cytometry were used to uncover the immunological mechanism of PRV-LAV treatment in regulating the tumor immune microenvironment. Results Through various tumor-specific analyses, we show that PRV-LAV infects cancer cells via the NRP1/EGFR signaling pathway, which is commonly overexpressed in cancer. Further, we show that PRV-LAV kills cancer cells by inducing endoplasmic reticulum (ER) stress. Moreover, PRV-LAV is responsible for reprogramming the tumor microenvironment from immunologically naïve (“cold”) to inflamed (“hot”), thereby increasing immune cell infiltration and restoring CD8+ T cell function against cancer. When delivered in combination with immune checkpoint inhibitors (ICIs), the anti-tumor response is augmented, suggestive of synergistic activity. Conclusions PRV-LAV can infect cancer cells via NRP1/EGFR signaling and induce cancer cells apoptosis via ER stress. PRV-LAV treatment also restores CD8+ T cell function against cancer. The combination of PRV-LAV and immune checkpoint inhibitors has a significant synergistic effect. Overall, these findings point to PRV-LAV as a serious potential candidate for the treatment of NRP1/EGFR pathway-associated tumors.