Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity.

Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

Association of THBS3 with Glycoprotein D Promotes Pseudorabies Virus Attachment, Fusion, and Entry.

Pseudorabies virus (PRV) is a neurotropic virus causing obvious neurological disorders and reproductive failure in pigs. PRV entry into target cells is a complex multistep process initiated by interacting viral envelope glycoproteins with cellular receptors. In the current study, we found that thrombospondin 3 (THBS3) plays an important role in PRV entry into target cells, indicating that THBS3 is a new PRV coreceptor. To confirm this hypothesis, the knockdown of THBS3 in several permissive cells inhibited PRV primary infection, and overexpression of THBS3 in PK15 cells promoted PRV infection. CRISPR-Cas9 knockout markedly reduced PRV infection in PK15 cells. Antibodies against THBS3 blocked PRV infection in naturally permissive target cells. Moreover, soluble THBS3 protein neutralized the infectivity of PRV. Mechanistically, THBS3 interacted with the PRV gD via its N and C termini to facilitate PRV binding in permissive and nonpermissive cells. Also, in the absence of Nectin-1, THBS3 promoted cell-to-cell fusion mediated by virus glycoproteins. While THBS3 alone could not increase virus entry, overexpression of it in the presence of Nectin-1 promoted virus entry into CHO-K1 cells. Our results have identified THBS3 as a critical player in PRV binding and subsequent membrane fusion and entry. IMPORTANCE Herpesvirus entry occurs through a cascade of virus-cell interactions, and multiple surface glycoproteins play a role in virus binding and entry during the virus invasion process. Early studies showed that attachment to cells by PRV, as well as other alphaherpesviruses, is mediated by interactions between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). However, gD may also be involved in virus binding in an HSPG-independent manner. To date, the respective cellular receptors are still unknown. In this report, we identified a host molecule, THBS3, involved in gD-mediated PRV binding and subsequent membrane fusion and entry, which increases our understanding of the initial events in alpha herpesvirus infections.

Pseudorabies virus kinase UL13 phosphorylates H2AX to foster viral replication.

The DNA damage response (DDR) pathway is critical for maintaining genomic integrity and sustaining organismal development. Viruses can either utilize or circumvent the DDR to facilitate their replication. Pseudorabies virus (PRV) infection was shown to induce apoptosis via stimulating DDR. However, the underlying mechanisms have not been fully explored to date. This study showed that PRV infection robustly activates the ATM and DNA-PK signaling pathways shortly after infection. However, inhibition of ATM, but not DNA-PK, could dampen PRV replication in cells. Importantly, we found that PRV-encoded serine/threonine kinase UL13 interacts with and subsequently phosphorylates H2AX. Furthermore, we found that UL13 deletion largely attenuates PRV neuroinvasiveness and virulence in vivo. In addtion, we showed that UL13 contributes to H2AX phosphorylation upon PRV infection both in vitro and in vivo, but does not affect ATM phosphorylation. Finally, we showed that knockdown of H2AX reduces PRV replication, while this reduction can be further enhanced by deletion of UL13. Taken together, we conclude that PRV-encoded kinase UL13 regulates DNA damage marker γH2AX and UL13-mediated H2AX phosphorylation plays a pivotal role in efficient PRV replication and progeny production.

Pseudorabies Virus Infection Triggers NF-κB Activation via the DNA Damage Response but Actively Inhibits NF-κB-Dependent Gene Expression.

The nuclear factor kappa B (NF-κB) pathway is known to integrate signaling associated with very diverse intra- and extracellular stressors, including virus infections, and triggers a powerful (proinflammatory) response through the expression of NF-κB-regulated genes. Typically, the NF-κB pathway collects and transduces threatening signals at the cell surface or in the cytoplasm leading to nuclear import of activated NF-κB transcription factors. In the current work, we demonstrate that the swine alphaherpesvirus pseudorabies virus (PRV) induces a peculiar mode of NF-κB activation known as "inside-out" NF-κB activation. We show that PRV triggers the DNA damage response (DDR) and that this DDR response drives NF-κB activation since inhibition of the nuclear ataxia telangiectasia-mutated (ATM) kinase, a chief controller of DDR, abolished PRV-induced NF-κB activation. Initiation of the DDR-NF-κB signaling axis requires viral protein synthesis but occurs before active viral genome replication. In addition, the initiation of the DDR-NF-κB signaling axis is followed by a virus-induced complete shutoff of NF-κB-dependent gene expression that depends on viral DNA replication. In summary, the results presented in this study reveal that PRV infection triggers a noncanonical DDR-NF-κB activation signaling axis and that the virus actively inhibits the (potentially antiviral) consequences of this pathway, by inhibiting NF-κB-dependent gene expression. IMPORTANCE The NF-κB signaling pathway plays a critical role in coordination of innate immune responses that are of vital importance in the control of infections. The current report generates new insights into the interaction of the alphaherpesvirus pseudorabies virus (PRV) with the NF-κB pathway, as they reveal that (i) PRV infection leads to NF-κB activation via a peculiar "inside-out" nucleus-to-cytoplasm signal that is triggered via the DNA damage response (DDR), (ii) the DDR-NF-κB signaling axis requires expression of viral proteins but is initiated before active PRV replication, and (iii) late viral factor(s) allow PRV to actively and efficiently inhibit NF-κB-dependent (proinflammatory) gene expression. These data suggest that activation of the DDR-NF-κB during PRV infection is host driven and that its potential antiviral consequences are actively inhibited by the virus.

A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.

Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC50 = 2.514 µg/ml and 4.297 µg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, 316QPAEPFP322, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.

Pseudorabies Virus Inhibits Type I and Type III Interferon-Induced Signaling via Proteasomal Degradation of Janus Kinases.

Both type I and III interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to trigger the expression of antiviral IFN-stimulated genes (ISGs). We report that the porcine alphaherpesvirus pseudorabies virus (PRV) triggers proteasomal degradation of the key Janus kinases Jak1 and to a lesser extent Tyk2, thereby inhibiting both type I and III IFN-induced STAT1 phosphorylation and suppressing IFN-induced expression of ISGs. UV-inactivated PRV did not interfere with IFN signaling. In addition, deletion of the EP0 gene from the PRV genome or inhibition of viral genome replication did not affect PRV-induced inhibition of IFN signaling. To our knowledge, this is the first report describing Janus kinase degradation by alphaherpesviruses. These findings thus reveal a novel alphaherpesvirus evasion mechanism of type I and type III IFNs. IMPORTANCE Type I and III interferons (IFNs) trigger signaling via Janus kinases that phosphorylate and activate signal transducer and activator of transcription (STAT) transcription factors, leading to the expression of antiviral interferon-stimulated genes (ISGs) that result in an antiviral state of host cells. Viruses have evolved various mechanisms to evade this response. Our results indicate that an alphaherpesvirus, the porcine pseudorabies virus (PRV), inhibits both type I and III IFN signaling pathways by triggering proteasome-dependent degradation of the key Janus kinases Jak1 and Tyk2 and consequent inhibition of STAT1 phosphorylation and suppression of ISG expression. Moreover, we found that this inhibition is not caused by incoming virions and does not depend on expression of the viral EP0 protein or viral true late proteins. These data for the first time address alphaherpesvirus evasion of type III IFN-mediated signaling and reveal a previously uncharacterized alphaherpesvirus mechanism of IFN evasion via proteasomal degradation of Janus kinases.

CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo.

Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

ATP-dependent activation of NLRP3 inflammasome in primary murine macrophages infected by pseudorabies virus.

Pseudorabies virus (PRV), an alphaherpesvirus, causes respiratory and reproductive diseases in pigs and severe nervous symptom in other susceptible hosts. Previous studies showed that PRV infection induced a systemic inflammatory response in mice, indicating that pro-inflammatory cytokines participated in viral neuropathy in mice. The pro-inflammatory cytokine IL-1ß is a key mediator of the inflammatory response and plays an important role in host-response to pathogens. However, the secretion of IL-1ß and its relationship with inflammasome activation during PRV infection remains poorly understood. In this study, we found that PRV infection caused significant secretion of several pro-inflammatory cytokines in macrophages and promoted IL-1ß secretion in an ATP-dependent manner. Furthermore, the expression of IL-1ß can be induced by only PRV infection and depended on NF-κB pathway activation, while the subsequent secretion of IL-1ß was mediated by ATP-induced P2 × 7R activation, loss of intracellular K+, and the subsequent NLRP3 inflammasome activation. By using a mouse infection model, we also found that ATP exacerbated clinical signs and death of mice infected by PRV in a NLRP3-dependent manner. These results indicate that ATP facilitates activation of NLRP3 inflammasome and enhances the pathogenicity of PRV in mice during its acute infection.

Untargeted LC-MS based metabolomic profiling of iPAMs to investigate lipid metabolic pathways alternations induced by different Pseudorabies virus strains.

Owing to viral recombination, interspecies transmission, and evolution, variant pseudorabies virus (PRV) strains exhibit different biological characteristics and pathogenicity. To improve the understanding of common and specific metabolic changes that occur upon infection by different PRV strains, we herein describe the comprehensive analysis of metabolites of PRV vaccine strain (Bartha K61), classical strain (EA) and variant strain (HNX) infection in immortalized porcine alveolar macrophage cells. Compared with uninfected cells, cells infected with Bartha K61, EA and HNX had 246, 225, and 272 differing metabolites, respectively. In the three types of PRV-strain-infected cells, lipids and lipid-like molecules accounted for over 50 % of the altered metabolites. As these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of the host metabolism. We analyzed the potential relationship between virus replication and the virus-mediated host metabolism. Our study resulted in the first reconstruction of the major lipid metabolic pathways involved in PRV infection, including those of glycerophospholipids, sphingolipids, glycerolipids, and fatty acyls. In addition, the metabolic perturbations caused by different PRV strain infections are consistent across many species, however, our results also revealed many specific metabolic alterations during HNX infection, such as the enrichment of phosphatidylinositol and 15R-PGE2 methyl ester 15-acetate, and the diminishment of phosphatidylethanolamine, phosphatidic acid, and ceramides. These strain-specific altered metabolites may be linked to the unique biological characteristics and pathogenicity of the HNX strain.

Emergence of a novel pathogenic recombinant virus from Bartha vaccine and variant pseudorabies virus in China.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has resulted in substantial economic losses in the swine industry worldwide. Previous reports have shown that the PRV variant is responsible for the Pseudorabies outbreaks in Bartha-K61-vaccinated farms in China. However, there is limited information about the evolution of recombination of the PRV variant. Here, we isolated two PRV variants from a Bartha-K61-vaccinated swine farm, named them the JSY7 and JYS13 strains, analysed their complete genomic sequences and evaluated pathogenicity. As results, the JSY7 and JSY13 strains showed different cytopathic effects and plaque sizes. The JSY7 and JSY13 strains had the same Aspartate insertions in the gE protein as other PRV variants. The JSY7 and JSY13 strains were clustered into the same clade based on a genomic phylogenetic analysis. However, the JSY7 strain was relatively close to recent PRV isolates in China, while the JSY13 strain was more closely related to earlier PRV isolates. Interestingly, the gC gene phylogenetic tree showed that the JSY7 strain belonged to genotype II lineage 3, while the JSY13 strain belonged to genotype I and is the same branch with the Bartha strain. Furthermore, the PRV variants were relatively distant from the Bartha strain in the phylogenetic analysis of the gB, gC and gD genes. Importantly, a recombination analysis showed that the JSY13 strain might be a natural recombinant between the minor parental genotype I Bartha strain and the major parental genotype II JSY7 strain. Finally, we also found that the JSY13 strain showed a moderate virulence compared to the JSY7 strain in mice. Taken together, our data provide direct evidence for genomic recombination of PRV in nature, which may play an important role in the evolution and virulence of PRV. This discovery suggests that live PRV vaccine can act as genetic donors for genomic recombination.

The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation.

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.

Typical gene expression profile of pseudorabies virus reactivation from latency in swine trigeminal ganglion.

Pseudorabies virus (PRV) establishes a lifelong latent infection in swine trigeminal ganglion (TG) following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Muscle injection combined with intravenous deliver of the synthetic corticosteroid dexamethasone (DEX) consistently induces reactivation from latency in pigs. In this study, PRV-free piglets were infected with PRV. Viral shedding in nasal and ocular swabs demonstrated that PRV infection entered the latent period. The anti-PRV antibody was detected by enzyme-linked immunosorbent assay and the serum neutralization test, which suggested that the PRV could establish latent infection in the presence of humoral immunity. Immunohistochemistry and viral genome detection of TG neurons suggested that PRV was reactivated from latency. Viral gene expressions of IE180, EP0, VP16, and LLT-intron were readily detected at 3-h post-DEX treatment, but gB, a γ1 gene, was not detectable. The differentially expressed phosphorylated proteins of TG neurons were analyzed by ITRAQ coupled with LC-MS/MS, and p-EIF2S2 differentially expression was confirmed by western blot assay. Taken together, our study provides the evidence that typical gene expression in PRV reactivation from latency in TG is disordered compared with known lytic infection in epithelial cells.

Swine Promyelocytic Leukemia Isoform II Inhibits Pseudorabies Virus Infection by Suppressing Viral Gene Transcription in Promyelocytic Leukemia Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.

Pseudorabies virus encephalitis in humans: a case series study.

Pseudorabies virus (PRV) is known to cause severe encephalitis in juvenile pigs and various non-native hosts; recent evidences suggest that PRV might cause encephalitis in humans. In a multicenter cohort study in China, next-generation sequencing of cerebrospinal fluid (CSF) was performed to detect pathogens in all patients with clinically suspected central nervous system infections. This study involved all the patients whose CSF samples were positive for PRV-DNA; their clinical features were evaluated, and species-specific PCR and serological tests were sequentially applied for validation. Among the 472 patients tested from June 1, 2016, to December 1, 2018, six were positive for PRV-DNA, which were partially validated by PCR and serological tests. Additionally, we retrospectively examined another case with similar clinical and neuroimaging appearance and detected the presence of PRV-DNA. These patients had similar clinical manifestations, including a rapid progression of panencephalitis, and similar neuroimaging features of symmetric lesions in the basal ganglia and bilateral hemispheres. Six of the patients were engaged in occupations connected with swine production. PRV infection should be suspected in patients with rapidly progressive panencephalitis and characteristic neuroimaging features, especially with exposure to swine.

Detection of a gE-deleted Pseudorabies virus strain in an Italian red fox.

This study describes an Aujeszky's disease case in an adult male red fox found in an urban area in Central Italy, that exhibited a fatal infection with neurological lesions, but neither itching nor skin lesions. Diagnostic examinations included histology, and parasitological, bacteriological and virological analyses. Detection of parasitic enteric pathogens, bacteria, E. coli, Leptospira spp., rabies, canine distemper virus, parvovirus, hepatitis E virus and pseudorabies virus (PrV) was performed. Results showed the presence of a gE-deleted PrVthat was closely related to the NIA-3 strain but differed from the PrV strains currently circulating in wild boars and domestic pigs in Italy. All the results led to the conclusion that the fox suffered from Aujeszky's disease caused by a gE-deleted PrV strain closely related to a vaccine strain. The epidemiological link between the PrV vaccine strain and fox infection remains unclear. It could involve vaccinated pigs as a primary source of infection by direct or indirect contact with the red fox or less likely it could be related to improper use of the vaccine in the fox.

Effects of gE/gI deletions on the miRNA expression of PRV-infected PK-15 cells.

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily of Herpesviridae. PRV-induced pseudorabies is a highly contagious disease that has caused huge economic losses to the global swine industry. The PRV gE/gI gene deletion vaccine strain (Fa ΔgE/gI strain) constructed from the PRV Fa wild-type strain was shown to have a protective effect against infection. However, the interaction between PRV gE/gI genes and host miRNA needs further exploration, and little is known about the regulatory mechanisms of non-coding RNAs during PRV infection. miRNAs play a key regulatory role in viral infection and immune responses, so we analyzed the differential expression of miRNAs induced by the PRV Fa ΔgE/gI strain and Fa wild-type strain in the PK15 cell line. High-throughput sequencing reads were aligned to known Sus scrofa pre-miRNAs in the miRBase database. Target genes of differentially expressed miRNAs were predicted using the miRGen 3.0 database, then filtered miRNA target genes were subjected to Gene Ontology (GO) analysis and Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis. Stem-loop quantitative real-time PCR was performed to confirm the accuracy of high-throughput sequencing data. In total, 387, 472, and 490 annotated and novel mature miRNAs were identified from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells, respectively. Five PRV-encoded miRNAs were also identified. GO analysis showed that target genes of differentially expressed miRNAs in PRV Fa ΔgE/gI strain-infected and Fa wild-type strain-infected PK-15 cells were mainly involved in biological regulation and metabolic processes. STRING analysis showed that immune-related target genes of differentially expressed miRNAs in the Toll-like receptor signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-κB signaling pathway, and transforming growth factor-ß signaling pathway were interrelated. This is the first report of the small RNA transcriptome in PRV mutant wild-type strain-infected and Fa ΔgE/gI strain-infected porcine cell lines. Our findings will contribute to the prevention and treatment of PRV mutant strains.

Prevalence of Porcine Pseudorabies Virus and Its Coinfection Rate in Heilongjiang Province in China from 2013 to 2018.

Pseudorabies (PR) is a highly contagious disease affecting a wide range of animals, which annually causes great economic losses in China. In this study, a total number of 18,815 serum samples and 1,589 tissue samples were collected from 311 intensive pig farms (≥350 sows) located in eight cities in Heilongjiang province, and tested by ELISA and PCR. Overall, the serum positive rates of gE and gB protein were 16.3% and 84.5%, respectively. The PCR-positive rate of PR virus (PRV) in tissue samples was 17.8%. The coinfection rates of PRV with porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and classical swine fever virus (CSFV) were also measured. The rate of PRV coinfected with PRRSV was 36.0% followed by 12.9% with PCV2 and 1.8% with CSFV, respectively. These results clearly demonstrate PRV prevalence and its coinfection rate in Heilongjiang province, indicating high PR endemic in pig farms in this region. This study provides data for further epidemiological investigations and a reference for developing PRV control strategies in this region and in China.

From Severe Herpes Zoster to Rare Suid Herpesvirus Encephalitis: A New Twist of the Varicellovirus Genus Infection in Patients with Kidney Diseases.

Both the herpes zoster virus and suid herpesvirus type 1 (SuHV-1) belong to the Varicellovirus genus of the α-herpesviridae subfamily. They may cause opportunistic infections especially in patients with kidney diseases, varying from latent illness to overt lethality. Under these circumstances, impaired renal function is both the culprit for and victim of the infection. However, fulminant eruption of severe skin herpes zoster in lupus nephritis (LN) patients under prolonged immunosuppressive therapy is rare and even more rarely seen is the SuHV-1 encephalitis in human. Facing the evolution of these rare infections, we hence chose to review the clinical pathogenicity of these two viruses which were cognate in origin but distinct in virulence. As such, we began with the first of the two above viral diseases and proceeded with peculiar renal involvement, unique clinical symptoms and pertinent lethal risk. Of importance, LN was used to exemplify the reciprocally detrimental interactions between impaired renal function and suppressed immune response. Then in a manner similar to the gradient overlay, SuHV-1 encephalitis was discussed focusing on its neurotropic features, specific MRI findings and exclusive test of high throughput sequencing. Our report highlighted novel presentations of the Varicellovirus genus infection by providing a productive multidisciplinary communication with pointed disclosure of the renal involvement. It may therefore be of great medical relevance and educational value for clinicians, especially the unseasoned ones, to foresee and manage similar cases in susceptible patients.

Pseudorabies Virus Infection of Epithelial Cells Leads to Persistent but Aberrant Activation of the NF-κB Pathway, Inhibiting Hallmark NF-κB-Induced Proinflammatory Gene Expression.

The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.