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Pseudorabies virus (PRV), an α-herpesvirus, induces immunosuppression and can lead to severe neurological diseases. N-methyl-D-aspartate receptor (NMDAR), an important excitatory nerve receptor in the central nervous system, is linked to various nervous system pathologies. The link between NMDAR and PRV-induced neurological diseases has not been studied. In vivo studies revealed that PRV infection triggers a reduction in hippocampal NMDAR expression, mediated by inflammatory processes. Extensive hippocampal neuronal degeneration was found in mice on the 6th day by hematoxylin-eosin staining, which was strongly correlated with increased NMDAR protein expression. In vitro studies utilizing the CCK-8 assay demonstrated that treatment with an NMDAR antagonist significantly heightened the cytotoxic effects of PRV on T lymphocytes. Notably, NMDAR inhibition did not affect the replication ability of PRV. However, it facilitated the accumulation of pro-inflammatory cytokines in PRV-infected T cells and enhanced the transcription of the CD25 gene through the secretion of interleukin-2 (IL-2), consequently exacerbating immunosuppression. In this study, we found that NMDAR has functional activity in T lymphocytes and is crucial for the inflammatory and immune responses triggered by PRV infection. These discoveries highlight the significant role of NMDAR in PRV-induced neurological disease pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pseudorabies virus (PRV) infection causes systemic inflammatory responses and inflammatory damages in infected animals, which are associated with the activation of inflammasome and pyroptosis in infected tissues. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas, the deficiency of viral UL4 was able to reduce ASC-dependent inflammasome activation and the occurrences of pyroptosis. Mechanistically, the 132-145 aa of UL4 permitted its translocation from the nucleus to the cytoplasm to interact with cytoplasmic ASC to promote the activation of NLRP3 and AIM2 inflammasome. Further research showed that UL4 promoted the phosphorylation levels of SYK and JNK to enhance the ASC phosphorylation, which led to the increase of ASC oligomerization, thus promoting the activation of NLRP3 and AIM2 inflammasome and enhanced GSDMD-mediated pyroptosis. In vivo experiments further showed that PRV UL4 (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) infection did not lead to a significant decrease in viral titers at 12 h. p. i, but it induced lower levels of IL-1β, IL-18, and GSDMD-NT, which led to an alleviated inflammatory infiltration and pathological damage in the lungs and brains, and a lower death rate compared with wild-type PRV strain infection. Taken together, our findings unravel that UL4 is an important viral regulator to manipulate the inflammasome signaling and pyroptosis of host cells to promote the pathogenicity of PRV, which might be further exploited as a new target for live attenuated vaccines or therapeutic strategies against pseudorabies in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells. Pseudorabies virus (PRV) is a significant veterinary pathogen in pigs, causing neurological sequalae that ultimately lead to the animal's demise. PRV is known to trigger apoptotic cell death during the late stages of infection. The virion host shutdown protein (VHS) encoded by UL41 plays a crucial role in the PRV infection process. In this study, we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3, thereby inhibiting the translocation and phosphorylation of IRF3. Notably, mutating the conserved amino acid sites (E192, D194, and D195) in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV, suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection. These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response., Competing Interests: Conflict of interest All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Pseudorabies virus (PRV), an alphaherpesvirus, is a neglected zoonotic pathogen. Dectin-1 sensing of β-glucan (BG) induces trained immunity, which can possibly form a new strategy for the prevention of viral infection. However, alphaherpesvirus including PRV have received little to no investigation in the context of trained immunity. Here, we found that BG pretreatment improved the survival rate, weight loss outcomes, alleviated histological injury and decreased PRV copy number of tissues in PRV-infected mice. Type I interferons (IFNs) including IFN-α/β levels in serum were significantly increased by BG. However, these effects were abrogated in the presence of Dectin-1 antagonist. Dectin-1-mediated effect of BG was also confirmed in porcine and murine macrophages. These results suggested that BG have effects on type I IFNs with antiviral property involved in Dectin-1. In piglets, oral or injected immunization with BG and PRV vaccine could significantly elevated the level of PRV-specific IgG and type I IFNs. And it also increased the antibody levels of porcine reproductive and respiratory syndrome virus vaccine and classical swine fever vaccine that were later immunized, indicating a broad-spectrum effect on improving vaccine immunity. On the premise that the cost was greatly reducing, the immunological effect of oral was better than injection administration. Our findings highlighted that BG induced type I IFNs related antiviral effect against PRV involved in Dectin-1 and potential application value as a feed additive to help control the spread of PRV and future emerging viruses., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier B.V.)

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (10 9 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (10 4 TCID 50 ) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3 + CD4 + T cells, but CD3 + CD8 + T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion., Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus., Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69 + T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ + IL-2 + CD4 + T cells and IFN-γ + TNF-α + CD8 + T cells, as well as PCV2-specific IFN-γ + TNF-α + CD8 + T cells., Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lu, Li, Chen, Li, Ma, Peng, Yan, Dong, Jin, Pan, Shang, Gu and Zhou.)

Although vaccination with inactivated vaccines is a popular preventive method against pseudorabies virus (PRV) infection, inactivated vaccines have poor protection efficiency because of their weak immunogenicity. The development of an effective adjuvant is urgently needed to improve the efficacy of inactivated PRV vaccines. In this study, a promising nanocomposite adjuvant named as MIL@A-SW01-C was developed by combining polyacrylic acid-coated metal-organic framework MIL-53(Al) (MIL@A) and squalene (oil)-in-water emulsion (SW01) and then mixing it with a carbomer solution. One part of the MIL@A was loaded onto the oil/water interface of SW01 emulsion via hydrophobic interaction and coordination, while another part was dispersed in the continuous water phase using carbomer. MIL@A-SW01-C showed good biocompatibility, high PRV (antigen)-loading capability, and sustained antigen release. Furthermore, the MIL@A-SW01-C adjuvanted PRV vaccine induced high specific serum antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response compared with commercial adjuvants, such as alum and biphasic 201. In the mouse challenge experiment, two- and one-shot vaccinations resulted in survival rates of 73.3 % and 86.7 %, respectively. After one-shot vaccination, the host animal pigs were also challenged with wild PRV. A protection rate of 100 % was achieved, which was much higher than that observed with commercial adjuvants. This study not only establishes the superiority of MIL@A-SW01-C composite nanoadjuvant for inactivated PRV vaccine in mice and pigs but also presents an effective method for developing promising nanoadjuvants. STATEMENT OF SIGNIFICANCE: We have developed a nanocomposite of MIL-53(Al) and oil-in-water emulsion (MIL@A-SW01-C) as a promising adjuvant for the inactivated PRV vaccines. MIL@A-SW01-C has good biocompatibility, high PRV (antigen) loading capability, and prolonged antigen release. The developed nanoadjuvant induced much higher specific IgG antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response than commercial adjuvants alum and biphasic 201. In mouse challenge experiments, survival rates of 73.3 % and 86.7 % were achieved from two-shot and one-shot vaccinations, respectively. At the same time, a protection rate of 100 % was achieved with the host animal pigs challenged with wild PRV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kong, Chen, Gong, Wang, Zhang, Guan, Yao, Kang, Lu, Zhang, Du, Sun, Zhuang, Zhao, Wan and Zhang.)

Adjuvants including aluminum adjuvant (Alum) and oil-water emulsion have been widely used in inactivated pseudorabies virus (PRV) vaccines to improve their performance, however, they are not sufficient to protect from PRV infection because of the weak immune response and poor Th1-type immune response. Divalent manganese ion (Mn 2+ ) has been reported to increase the cellular immune response significantly. In this work, a xanthan gum and carbomer-dispersed Mn 2+ -loaded tannic acid-polyethylene glycol (TPMnXC) nanoparticle colloid is developed and used as an adjuvant to improve the performance of the inactivated PRV vaccine. The good in vitro and in vivo biocompatibility of the developed TPMnXC colloid has been confirmed by the cell viability assay, erythrocyte hemolysis, blood routine analysis, and histological analysis of mouse organs and injection site. The TPMnXC-adjuvanted inactivated PRV vaccine (TPMnXC@PRV) significantly promotes higher and more balanced immune responses indicating with an increased specific total IgG antibody and IgG2a/IgG1 ratio, efficient splenocytes proliferation, and elevated Th1- and Th2-type cytokine secretion than those of control groups. Wild PRV challenge experiment is performed using mice as a model animal, achieving a protection rate of up to 86.67 %, which is much higher than those observed from the commercial Alum. This work not only demonstrates the high potentiality of TPMnXC in practical applications but also provides a new way to develop the Mn 2+ -loaded nanoadjuvant for veterinary vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo . Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo . In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies., Competing Interests: The authors declare no conflict of interest.

Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-β (interferon-beta) signaling pathway, being a typical example of innate immunity, plays a vital function. This study aimed to elucidate the function of pseudorabies virus (PRV) UL38 protein (unique long region 38) in suppressing the activation of the IFN-β signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-β by poly (dA: dT) (poly(deoxyadenylic-deoxythymidylic)) and 2'3'-cGAMP (2'-3'-cyclic GMP-AMP). Furthermore, UL38 exhibits spatial co-localization with STING (stimulator of interferon genes) and effectively hinders STING dimerization. Subsequently, STING was downgraded to suppress the production of IFN-β and ISGs (interferon stimulated genes). Immunoprecipitation analysis revealed that the interaction between UL38 and STING, which subsequently initiated the degradation of STING via selective autophagy mediated by TOLLIP (toll interacting protein). To summarize, this research elucidates the function of UL38 in counteracting the cGAS (cGAMP synthase)-STING-induced IFN-β pathway. The PRV UL38 protein may attenuate the activation of IFN-β as a means of regulating the virus's persistence in the host., (© 2024. The Author(s).)

Pseudorabies virus (PRV) is a highly contagious viral disease, which leads to severe financial losses in the breeding industry worldwide. Presently, PRV is mainly controlled using live attenuated and inactivated vaccines. However, these vaccines have an innate tendency to lose their structural conformation upon exposure to environmental and chemical stressors and cannot provide full protection against the emerging prevalent PRV variants. In this work, first, we synthesized aminated ZIF-7/8 nanoparticles (NPs), and then chemical bond-coated alginate dialdehyde (ADA, a type of dioxide alginate saccharide) on their surface via Schiff base reaction to obtain ZIF-7/8-ADA NPs. The as-fabricated ZIF-7/8-ADA NPs exhibited high stability, monodispersity and a high loading ratio of antigen. Furthermore, the ZIF-7/8-ADA NPs showed good biocompatibility in vitro and in vivo . Using ZIF-7/8-ADA NPs as an adjuvant and inactivated PRV as a model antigen, we constructed a PR vaccine through a simple mixture. The immunity studies indicated that ZIF-7/8-ADA induced an enhancement in the Th1/Th2 immune response, which was superior to that of the commercial ISA201, alum adjuvant and ZIF-7/8. Due to the pH-sensitive release of the antigen in lysosomes, the as-prepared PR vaccine subsequently accelerated the antigen presentation and improved the immune responses in vitro and in vivo . The results of PRV challenge using mice as the model demonstrated that ZIF-7/8-ADA achieved the same preventive effect as the commercial ISA201 and was much better than the alum adjuvant, and thus can serve as a promising delivery system and adjuvant to enhance humoral and cellular responses against PRV infection.

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 10 6.0 TCID 50 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 10 5.0 TCID 50 dose after 6 weeks PII ( p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 10 5.0 TCID 50 , the level of NA was close to that in the triple-gene deletion virus group with 10 6.0 TCID 50 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 10 6.0 TCID 50 . 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 10 5.0 TCID 50 , 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.

Pseudorabies virus (PRV) is a member of the genus Varicellovirus, family Herpesviridae and causes Aujeszky's disease to lead to huge economic losses in the global pig industry. The Non-POU domain-containing octamer-binding protein (NONO), as a Drosophila behavior/human splicing (DBHS) protein, plays a key role in multiple biological functions in cells, including transcriptional regulation, RNA splicing, DNA repair and so on. However, whether swine NONO (sNONO) inhibits PRV infection is less understood. In this study, we showed that sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. After PRV infection, sNONO enhanced the activation of IFN-β promoter and IFN-β expression. Furthermore, knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. Taken together, we have first elucidated the anti-PRV function and mechanism of sNONO, which may provide a new strategy for preventing DNA virus infection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.

The alphaherpesvirus pseudorabies virus (PRV) is the etiologic agent of swine Aujeszky's disease, which can cause huge economic losses to the pig industry. PRV can overcome a type I interferon (IFN)-induced antiviral state in host cells through its encoded EP0 protein. However, the exact role of EP0 in this process is poorly defined. Here, we report that EP0 transcriptionally represses IFN regulatory factor 9 (IRF9), a critical component in the IFN signaling pathway, thereby reducing the cellular levels of IRF9 and inhibiting IFN-induced gene transcription. This activity of EP0 is mediated by its C-terminal region independently of the RING domain. Moreover, compared with EP0 wild-type PRV, EP0-deficient PRV loses the ability to efficiently decrease cellular IRF9, while reintroducing the C-terminal region of EP0 back into the EP0-deficient virus restores the activity. Together, these results suggest that EP0 can transcriptionally modulate IRF9-mediated antiviral pathways through its C-terminal region, contributing to PRV innate immune evasion. IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that alphaherpesvirus can utilize its encoded early protein EP0 to inhibit the IFN-induced upregulation of antiviral proteins by reducing the basal expression levels of IRF9 through repressing its transcription. Our findings reveal a mechanism employed by alphaherpesvirus to evade the immune response and indicate that EP0 is an important viral protein in pathogenesis and a potential target for antiviral drug development.

Pseudorabies virus (PRV) has evolved various immune evasion mechanisms that target host antiviral immune responses. However, it is unclear whether and how PRV encoded proteins modulate the cGAS-STING axis for immune evasion. Here, we show that PRV tegument protein UL13 inhibits STING-mediated antiviral signaling via regulation of STING stability. Mechanistically, UL13 interacts with the CDN domain of STING and recruits the E3 ligase RING-finger protein 5 (RNF5) to promote K27-/K29-linked ubiquitination and degradation of STING. Consequently, deficiency of RNF5 enhances host antiviral immune responses triggered by PRV infection. In addition, mutant PRV lacking UL13 impaired in antagonism of STING-mediated production of type I IFNs and shows attenuated pathogenicity in mice. Our findings suggest that PRV UL13 functions as an antagonist of IFN signaling via a novel mechanism by targeting STING to persistently evade host antiviral responses., Competing Interests: The authors have declared that no competing interests exist.

Porcine circovirus-associated diseases (PCVADs) and pseudorabies (PR) are highly contagious and economically significant diseases of swine in China. Porcine circovirus type 3 (PCV3) is an emerging swine pathogen of PCVAD. Currently, no PCV3 vaccine is commercially available, and the epidemic caused by it is still spreading worldwide. In this study, we used the PRV variant strain HNX as the parental virus to construct recombinant PRV with TK/gE gene deletion and capsid (Cap) protein co-expression, named HNX-ΔTK/ΔgE-ORF2. The results revealed that PCV3 Cap protein can be detected in HNX-ΔTK/ΔgE-ORF2-infected PK-15 cells by both western blotting and immunofluorescence assays. Vaccination with HNX-ΔTK/ΔgE-ORF2 did not cause pruritus, ruffled fur, systemic infection, or inflammation (without high expression of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) in plasma). Furthermore, HNX-ΔTK/ΔgE-ORF2 immunization induced an anti-Cap specific antibody, activated a PRV-specific cellular immune response, and provided 100 % protection to mice against the challenge of the virulent HNX strain. Thus, HNX-ΔTK/ΔgE-ORF2 appears to be a promising vaccine candidate against PRV and PCV3 for the control of the PRV variant and PCV3., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC., Competing Interests: The authors have declared that no competing interests exist.

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 IC 50  = 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, 316 QPAEPFP 322 , 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pseudorabies virus (PRV) is an enveloped double-stranded DNA virus that is the etiological agent of Aujeszky's disease in pigs. Vaccination is currently available to prevent PRV infection, but there is still an urgent need for new strategies to control this infectious disease. Histone deacetylases (HDACs) are epigenetic regulators that regulate the histone tail, chromatin conformation, protein-DNA interaction and even transcription. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and HDACs that remodel chromatin and regulate gene expression. We reported here that genetic and pharmacological inhibition of HDAC1 significantly influenced PRV replication. Moreover, we demonstrated that inhibition of HDAC1 induced a DNA damage response and antiviral innate immunity. Mechanistically, the HDAC1 inhibition-induced DNA damage response resulted in the release of double-strand DNA into the cytosol to activate cyclic GMP-AMP synthase and the downstream STING/TBK1/IRF3 innate immune signaling pathway. Our results demonstrate that an HDAC1 inhibitor may be used as a new strategy to prevent Aujeszky's disease in pigs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pseudorabies virus (PRV) is the causative pathogen of Aujeszky's disease in pigs. Although vaccination is currently applied to prevent the morbidity of PRV infection, new applications are urgently needed to control this infectious disease. Poly(ADP-ribose) polymerase 1 (PARP1) functions in DNA damage repair. We report here that pharmacological and genetic inhibition of PARP1 significantly influenced PRV replication. Moreover, we demonstrate that inhibition of PARP1 induced DNA damage response and antiviral innate immunity. Mechanistically, PARP1 inhibition-induced DNA damage response resulted in the release of double-stranded DNA (dsDNA) into the cytosol, where dsDNA interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS subsequently catalyzed cGAMP production to activate the STING/TBK1/IRF3 innate immune signaling pathway. Furthermore, challenge of mice with PARP1 inhibitor stimulated antiviral innate immunity and protected mice from PRV infection in vivo . Our results demonstrate that PARP1 inhibitors may be used as a new strategy to prevent Aujeszky's disease in pigs. IMPORTANCE Aujeszky's disease is a notifiable infectious disease of pigs and causes economic losses worldwide in the pig industry. The causative pathogen is PRV, which is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV has a wide range of hosts, such as ruminants, carnivores, and rodents. More seriously, recent reports suggest that PRV can cause human endophthalmitis and encephalitis, which indicates that PRV may be a potential zoonotic pathogen. Although vaccination is currently the major strategy used to control the disease, new applications are also urgently needed for the pig industry and public health. We report here that inhibition of PARP1 induces DNA damage-induced antiviral innate immunity through the cGAS-STING signaling pathway. Therefore, PARP1 is a therapeutic target for PRV infection as well as alphaherpesvirus infection.

Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease., (Copyright © 2021. Published by Elsevier B.V.)

Background: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry., Results: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 10 5 TCID 50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013., Conclusions: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Background: Pseudorabies (PR), caused by the pseudorabies virus (PRV), is an endemic disease in some regions of China. Although there are many reports on epidemiological investigations into pseudorabies, information on PRV gI antibody dynamics in one pig farm is sparse., Objectives: To diagnose PR and analyze the course of PR eradication in one pig farm., Methods: Ten brains and 1,513 serum samples from different groups of pigs in a pig farm were collected to detect PRV gE gene and PRV gI antibody presence using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively., Results: The July 2015 results indicated that almost all brain samples were PRV gE gene positive, but PRV gI antibody results in the serum samples of the same piglets were all negative. In the boar herd, from October 2015 to July 2018 three positive individuals were culled in October 2015, and the negative status of the remaining boars was maintained in the following tests. In the sow herd, the PRV gI antibody positive rate was always more than 70% from October 2015 to October 2017; however, it decreased to 27% in January 2018 but increased to 40% and 52% in April and July 2018, respectively. The PRV gI antibody positive rate in 100-day pigs markedly decreased in October 2016 and was maintained at less than 30% in the following tests. For 150-day pigs, the PRV gI antibody positive rate decreased notably to 10% in April 2017 and maintained a negative status from July 2017. The positive trend of PRV gI antibody with an increase in pig age remarkably decreased in three tests in 2018., Conclusions: The results indicate that serological testing is not sensitive in the early stage of a PRV infection and that gilt introduction is a risk factor for a PRV-negative pig farm. The data on PRV gI antibody dynamics can provide reference information for pig farms wanting to eradicate PR., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

Background: Pseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear., Objectives: Our study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection., Methods: Kunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi., Results: At 10⁵-10⁶ TCID 50 PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID 50 of PRV produced a significant inflammatory mediator increase., Conclusions: An inflammatory model induced by PRV infection was established in mice, and 10² TCID 50 PRV was considered as the best concentration for the establishment of the model., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

Messenger RNA-based vaccines represent new tools with prophylactic and therapeutic potential characterized by high flexibility of application for infectious diseases. Pseudorabies virus (PRV) is one of the major viruses affecting the pig industry. PRV has serious effects in piglets, sows, and growing-fattening pigs and can lead to huge economic losses. In this study, an envelope glycoprotein D (gD) gene-based specific mRNA vaccine was generated, and a mouse model was used to investigate the protective efficacy of the vaccine. The gD mRNA vaccine and the recombinant plasmid pVAX-gD were transfected into BHK21 cells, and the antigenicity of the expressed proteins was detected by Western blot analysis. Groups of mice were vaccinated with the gD mRNA vaccine, pVAX-gD, and PBS. T cell immune responses were measured by flow cytometry or ELISA and serum neutralization tests every two weeks. The challenge with the PRV-XJ strain was performed eight weeks after the primary immunization, and the response was monitored for 15 days. The levels of specific and neutralizing antibodies in the gD mRNA vaccine group were significantly increased in 8 weeks compared to those in the control group, and cytokine levels, including that of IFN-γ/IL-2, were considerably higher than those in the control animal. Additionally, the proportion of CD4 + /CD8 + cells in peripheral lymphocytes was remarkably increased. Our data demonstrate that mRNA is a promising and effective tool for the development of vaccines. The PRV-gD-based mRNA vaccine can elicit an efficient neutralizing antibody response and induce effective protection in mice in defense against PRV infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE - /gI - /TK - ) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE - /gI - /TK - strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 10 6.0 TCID 50 of rPRV NY-gE - /gI - /TK - and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE - /gI - /TK - were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3 + , CD4 + and CD8 + in rPRV NY-gE - /gI - /TK - -immunized mice was higher than that in DMEM-injected mice. After challenge with 10 6.0 TCID 50 PRV NY at 42 dpi, all rPRV NY-gE - /gI - /TK - -immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE - /gI - /TK - could be a promising next-generation vaccine to control now epidemic PR in China., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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.

Pseudorabies virus (PRV), a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine, was recently reported to infect human and led to endophthalmitis and encephalitis. A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%, 14.25%, and 6.52% in 2012, 2013, and 2017, respectively. The virus neutralizing antibody titers of positive samples correlated well with ELISA results. The pseudorabies virus antibody positive rate of patients with encephalitis were higher than that of healthy people in 2017. The above results suggest that some undefined human encephalitis cases may be caused by PRV infection., (Copyright © 2020 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

A novel time-resolved fluorescence blocking lateral flow immunoassay (TRF-BLFIA) was developed for on-site differential diagnosis of pseudorabies virus (PRV)-infected and vaccinated pigs using europium nanoparticles (EuNPs)-labeled virion antigens and high titer PRV gE monoclonal antibodies (PRV gE-mAb). Upon application of a positive serum sample, the specific epitopes of gE protein on the EuNPs-PRV probe were blocked, inhibiting binding to the PRV gE-mAb on the T line, resulting in low or negligible fluorescence signal, whereas when a negative sample was applied, EuNPs-PRV probes would be able to bind the antibody at the T line, leading to high fluorescence signal. Under optimized conditions, TRF-BLFIA provided excellent sensitivity and selectivity. When testing swine clinical samples (n = 356), there was 96.1% agreement between this method and a most widely used commercial gE-ELISA kit. Moreover, our method was rapid (15 min), cost-efficient and easy to operate with simple training, allowing for on-site detection. Thus, TRF-BLFIA could be a practical tool to differentially diagnose PRV-infected and vaccinated pigs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Differentiating infected from vaccinated animals (DIVA) strategies have been central enabling techniques in several successful viral disease elimination programs. However, owing to their long and uncertain development process, no DIVA-compatible vaccines are available for many important diseases. We report herein a new DIVA strategy based on hybrid protein-peptide microarrays which can theoretically work with any vaccine. Leading from our findings from peste des petits ruminants (PPR) virus, we found 4 epitope-containing short peptides (ECSPs) which have distinct IgG serodynamics: anti-ECSP IgGs only exist for 10 to 60 days postvaccination (dpv), while anti-protein IgGs remained at high levels for >1,000 dpv. These data enabled the design of a DIVA diagnostic microarray containing 4 ECSPs and 3 proteins, which, unlike competitive enzyme-linked immunosorbent assay (cELISA) and virus neutralization tests (VNTs), enables ongoing monitoring of serological differences between vaccinated individuals and individuals exposed to the pathogen. For 25 goats after 60 dpv, 13 were detected with positive anti-ECSP IgGs, indicating recent infections in vaccinated goat herds. These DIVA diagnostic microarrays will almost certainly facilitate eradication programs for (re)emerging pathogens and zoonoses. IMPORTANCE Outbreaks of infectious diseases caused by viruses, such as pseudorabies (PR), foot-and-mouth disease (FMD), and PPR viruses, led to economic losses reaching billions of dollars. Both PR and FMD were eliminated in several countries via large-scale vaccination programs using DIVA-compatible vaccines, which lack the gE protein and nonstructural proteins, respectively. However, there are still extensive challenges facing the development and deployment of DIVA-compatible vaccines because they are time-consuming and full of uncertainty. Further, the negative marker strategy used for DIVA-compatible vaccines is no longer functional for live-attenuated vaccines. To avoid these disadvantageous scenarios, a new strategy is desired. Here, we made the exciting discovery that different IgG serodynamics can be monitored when using protein-based assays versus arrays comprising ECSPs. This DIVA microarray strategy should, in theory, work for any vaccine., (Copyright © 2020 Xue et al.)

Background: Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV., Methods: The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals., Results: A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10)., Conclusions: The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

Newborn humans and animals are highly susceptible to viral infections. The Aujeszky´s disease virus (ADV) is a porcine herpes virus 1 which infects the respiratory tract and is lethal during the first weeks of life. Current intramuscular vaccines, applied at weaning, induce poor mucosal immunity and frequently fail to prevent and control the disease. Additionally, early vaccination has not been studied thoroughly. Therefore, we studied a systemic/mucosal route of immunization using an inactivated ADV vaccine in two-and fourteen-day-old groups of unweaned SPF miniature Vietnamese pigs, measuring the anti ADV antibody (ELISA) and cytokine (qPCR) responses in systemic and mucosal samples. The results showed that the serum ADV-specific IgG response was higher in the 14-day groups. However, the nasal IgA responses were similar in immunized groups, although the response in saliva was higher in the 2-day old group. Moreover, in vitro ADV stimulated peripheral blood mononuclear cells and lung cells from immunized pigs showed higher IFN-γ mRNA production in the 14-day old group than in younger animals and similar levels of IL-4 and IL-10 transcripts. Our data suggest that early mucosal immunization induce humoral and cellular systemic and mucosal immune responses against ADV in young pigs and younger animals may have compensatory mechanisms to overcome early immaturity and maternal-driven immune interference. Therefore, early protection in susceptible animals could be induced using this immunization protocol, opening the possibility for its application against other viral pathogens of pigs and for traslational studies in humans., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRV delUS7/US8/UL23 -infected cells formed cell-cell fusion, but the rPRV delUS7/US8/UL23/US3 -infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Since 2011, there have been outbreaks of pseudorabies (PR) in several pig farms despite vaccination coverage, which causes substantial economic loss to the swine industry in China. The emergence of a pseudorabies virusvariant strain with high virulence and antigenic variation (e.g., PRV ZJ01), is considered to be the primary cause. In this study, truncated gB, gC, and gE of PRV ZJ01 was expressed and used to generate seven monoclonal antibodies (mAbs) against gB, gC, or gE. An indirect immunofluorescence assay (IFA) revealed that these mAbs were specific against PRV. Subsequently we identified the B cell epitopes recognized by these mAbs by Western blot. The mAbs 5A2 and 6G5 against gB recognized the same B cell linear epitope at 576 SAVATAA 582 , the mAb 5D10 against gC recognized the B cell linear epitope at 134 GETFE 138 , mAb 7C5 against gC recognized the B cell linear epitope at 143 RRGRFRSPDAD 153 , and mAbs 3E1, 3H8, and 4D2 against gE recognized the same B cell linear epitope at 151 IGDYL 155 of gE. Biological information analysis showed that these B cell linear epitopes are highly conserved among different PRV isolates and the epitope 143 RRGRFRSPDAD 153 with a high antigenic index and high hydrophilicity, fully exposed on the surface of the gC, is likely to be an important B cell epitope. These mAbs and their defined epitopes may provide useful tools for the study of the structure and function of the PRV protein, analysis of antigenic epitope characteristics, and establishment of antibody detection methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Pseudorabies caused by pseudorabies virus (PRV) mainly infects the swine and seriously threatens the biosafety of the other animals, including humans. Since 2011, the outbreaks of PRV mutants have caused enormous economic losses in the swine industry, and traditional vaccines cannot offer enough protection. PRV can transmit by direct contact, aerosol transmission and pollutants. PRV mainly transmit through the nasal mucosa. After infecting the nasal epithelial cells, PRV can quickly infect the olfactory nerve and establish a potential infection of sensory neurons. Therefore, nasal immunity can effectively prevent viral colonization infection. Recombinant Bacillus subtilis has been widely used to deliver antigen and achieve adequate protective immune responses., Results: The present study successfully constructed recombinant Bacillus subtilis (B. subtilis) expressing the dominant antigen regions of PRV gC and gD proteins (named B. subtilis-gCa and B. subtilis-gDa). Furtherly, we evaluated the immunogenicity of the two recombinant B. subtilis in mice. The mice intranasal administration with B. subtilis-gCa and B. subtilis-gDa effectively stimulated IgA and IgG immune responses, further regulated specific T lymphocytes proliferative response by IFN-γ and IL-10, and ultimately produced high titers of neutralizing antibodies against PRV infection. In particular, B. subtilis-gDa possessed more excellent immune effect than B. subtilis-gCa in mice., Conclusions: These results suggested that B. subtilis-gCa and B. subtilis-gDa could trigger high levels of mucosal and systemic immune responses and would be potential candidates for developing PRV vaccines.

Natural killer (NK) cells are components of the innate immunity and are key players in the defense against virus-infected and malignant cells. NK cells are particularly important in the innate defense against herpesviruses, including alphaherpesviruses. Aggravated and life-threatening alphaherpesvirus-induced disease has been reported in patients with NK cell deficiencies. NK cells are regulated by a diversity of activating and inhibitory cell surface receptors that recognize specific ligands on the plasma membrane of virus-infected or malignant target cells. Although alphaherpesviruses have developed several evasion strategies against NK cell-mediated attack, alphaherpesvirus-infected cells are still readily recognized and killed by NK cells. However, the (viral) factors that trigger NK cell activation against alphaherpesvirus-infected cells are largely unknown. In this study, we show that expression of the gB glycoprotein of the alphaherpesvirus pseudorabies virus (PRV) triggers NK cell-mediated cytotoxicity, both in PRV-infected and in gB-transfected cells. In addition, we report that, like their human and murine counterpart, porcine NK cells express the activating receptor paired immunoglobulin-like type 2 receptor beta (PILRβ), and we show that gB expression triggers increased binding of recombinant porcine PILRβ to the surfaces of PRV-infected cells and gB-transfected cells. IMPORTANCE Natural killer (NK) cells display a prominent cytolytic activity against virus-infected cells and are indispensable in the innate antiviral response, particularly against herpesviruses. Despite their importance in the control of alphaherpesvirus infections, relatively little is known about the mechanisms that trigger NK cell cytotoxicity against alphaherpesvirus-infected cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we found that the conserved alphaherpesvirus glycoprotein gB triggers NK cell-mediated cytotoxicity, both in virus-infected and in gB-transfected cells. In addition, we report that gB expression results in increased cell surface binding of porcine paired immunoglobulin-like type 2 receptor beta (PILRβ), an activating NK cell receptor. The interaction between PILRβ and viral gB may have consequences that stretch beyond the interaction with NK cells, including virus entry into host cells. The identification of gB as an NK cell-activating viral protein may be of importance in the construction of future vaccines and therapeutics requiring optimized interactions of alphaherpesviruses with NK cells., (Copyright © 2019 American Society for Microbiology.)

As a key cellular transcription factor that plays a central role in cellular responses to a broad range of stress factors, p53 has generally been considered as a host cell restriction factor for various viral infections. However, the defined roles of p53 in pseudorabies virus (PRV) replication, pathogenesis, and host responses remain unclear. In the present study, we initially constructed a p53 overexpressing a porcine kidney epithelial cell line (PK-15) to detect the effect of p53 on PRV replication in vitro. The results show that viral glycoprotein B (gB) gene copies and the titers of virus were significantly higher in p53 overexpressing PK-15 cells than in PK-15 and p53 inhibitor treated p53 overexpressing PK-15 cells. A similar result was also found in the p53 inhibitor PFT-α-treated PK-15 cells. We then examined the effects of p53 on PRV infection in vivo by using p53-knockout (p53 -/- ) mice. The results show that p53 knockout not only led to significantly reduced rates of mortality but also to reduced viral replication and development of viral encephalitis in the brains of mice following intracranial inoculation. Furthermore, we examined the effect of p53 knockout on the expression of the reported host cell regulators of PRV replication in the brains of mice by using RNA sequencing. The results show that p53 knockout downregulated the interferon (IFN) regulator genes, chemokine genes, and antiviral genes after PRV infection. This finding suggests that p53 positively regulates viral replication and pathogenesis both in vitro and in vivo. These findings offer novel targets of intrinsic host cell immunity for PRV infection.

Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-γ, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8 + and CD4 + CD8 + PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies.

Background: Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gC S ) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gC S was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions., Results: The results showed that a BAC clone of Bartha-K61 and a B-gD&gC S clone were successfully generated. The growth kinetics of PRV B-gD&gC S strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gC S strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gC S and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gC S strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group., Conclusions: Our results suggest that PRV B-gD&gC S strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.

Pseudorabies virus (PRV) is one of the most vital pathogens of swine, leading to huge economic losses to the pig industry. Functioning in innate immunity, type-I interferon (IFN) plays a vital role in initial stage of viral infection. ISG15, an IFN-stimulated ubiquitin-like protein, is highly increased during virus infection and participates in the IFN-mediated antiviral immune response. However, limited attention has been paid to the functional role of porcine ISG15 (pISG15) in PRV infection. In this study, we generated a PK15 inducible cell line stably expressing the pISG15 gene and investigated the potential anti-PRV response of pISG15. We demonstrated that pISG15 was upregulated in an early stage of PRV infection, and pISG15 overexpression efficiently inhibited PRV replication by reducing the viral titers and mRNA levels of PRV, and also increased expression of IFN-β and activation of the ISRE promoter. However, knockdown of pISG15 by siRNA did not affect PRV replication, and potentiated IFN-I-mediated signaling, resulting in an increase in antiviral response in the process of PRV infection. The results showed that pISG15 has a potential immunodulatory role in cellular antiviral response against PRV., (Copyright © 2018. Published by Elsevier B.V.)

Pseudorabies (PR) has been prevalent in Chinese swine breeding farms since the outbreak at the end of 2011. For investigating current prevalence of PR, a nationwide surveillance has been performed in this study. The swine serum samples were collected from 93, 100, 92, and 91 swine farms in China during 2013-2016, respectively. Since the extensive use of gE-deleted pseudorabies virus (PRV) vaccine, we could apply the PRV-gE antibody for determining wild-type virus infection and the PRV-gB antibody for evaluating vaccine immunization. The results were concluded as follows: (1) Nationally, the positive rate of PRV-gB was maintained at a high level (> 90%), while the positive rate of PRV-gE continued to decrease (from 22.17 to 13.14%). (2) The positive rates of PRV-gE were greatly varied in different geographical regions and swine farms (0~100%), while the positive rate of PRV-gB was generally high (> 90%). (3) The number of imported PRV attenuated vaccines were about twice that of domestic PRV attenuated vaccines, while the positive rate of PRV-gB was not significantly different (P > 0.05). (4) The performance of PR eradication developing or developed farms was better than the performance of common farms, with higher positive rate of PRV-gB (> 90%) and much lower positive rate of PRV-gE (nearly 0%).