Pseudorabies virus UL38 attenuates the cGAS-STING signaling pathway by recruiting Tollip to promote STING for autophagy degradation.

Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-ß (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-ß signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-ß 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-ß 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-ß pathway. The PRV UL38 protein may attenuate the activation of IFN-ß as a means of regulating the virus's persistence in the host.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

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.

Alginate di-aldehyde-modified metal-organic framework nanocarriers as delivery platform and adjuvant in inactivated pseudorabies vaccination.

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.

Pseudorabies Virus EP0 Antagonizes the Type I Interferon Response via Inhibiting IRF9 Transcription.

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 tegument protein UL13 recruits RNF5 to inhibit STING-mediated antiviral immunity.

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.

The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice. RESULTS: The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-γ and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4+ and CD8+ lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28 days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice. CONCLUSION: The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.

Effects of immunostimulators of microbial origin on T cells of pigs vaccinated with attenuated vaccine against Aujeszky's disease.

Aujeszky's disease (AD) is a viral infectious disease caused by Suid herpesvirus 1 (SuHV-1). Vaccination and eradication of AD in domestic pigs is possible using marker vaccines with attenuated or inactivated SuHV-1, or subunit vaccines. However, vaccines with attenuated SuHV-1 have shown to be more potent in inducing strong cell-mediated immune response. The studies have shown that Parapoxvirus ovis, as well as Propionibacterium granulosum with lipopolysacharides (LPS) of Escherichia coli have pronounced immunomodulatory effects and that in combination with the vaccines can induce stronger humoral and cellular immune responses than use of vaccines alone. In our study distribution of peripheral blood T cell subpopulations was analysed after administration of vaccine alone (attenuated SuHV-1), immunostimulators (inactivated Parapoxvirus ovis or combination of an inactivated P. granulosum and detoxified LPS of E. coli) and combinations of vaccine with each immunostimulator to the 12-week old piglets. Throughout the study no significant changes were found in the proportions of γδ and most αß T cell subpopulations analysed. However, on the seventh day of the study combination of an inactivated P. granulosum and LPS of E. coli with vaccine induced transient but significant increase of the proportions of CD4+CD8α+ and CD4-CD8α+ αß T cells, that have been strongly associated with early protection of SuHV-1 infected pigs. Our findings indicate that combination of inactivated P. granulosum and detoxified E. coli LPS could be used for enhancement of a cellular immune response induced by vaccines against AD.

The construction and immunogenicity analyses of a recombinant pseudorabies virus with porcine circovirus type 3 capsid protein co-expression.

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.

Peroxiredoxin 1 Interacts with TBK1/IKKε and Negatively Regulates Pseudorabies Virus Propagation by Promoting Innate Immunity.

Peroxiredoxin 1 (PRDX1) is a cellular antioxidant enzyme that is crucial for diverse fundamental biological processes, such as autophagy, inflammation, and carcinogenesis. However, molecular mechanisms underpinning its diverse roles are not well understood. Here, we report that PRDX1 positively regulates interferon (IFN) induction and that pseudorabies virus (PRV) targets PRDX1 to evade IFN induction. PRV UL13 encodes a serine/threonine kinase important for PRV infection, although its biological function remains obscure. We identified PRDX1 as a UL13-interacting protein. Virological and biochemical assays demonstrate that PRDX1 promotes IFN induction by interacting with TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε). Conversely, UL13 accelerates PRDX1 degradation via the ubiquitin-proteosome pathway in a kinase-dependent manner. In doing so, PRV inhibits IFN induction during productive infection, which requires PRDX1 expression. This study uncovers an essential role of PRDX1 in the innate immune response and reveals a new viral immune evasion strategy to counteract cellular defenses. IMPORTANCE PRV interacts with numerous cellular proteins during productive infection. Here, we demonstrated the interaction of viral protein UL13 with the antioxidant enzyme PRDX1, which functions in multiple signal transduction pathways. We found that PRDX1 participates in the type I IFN pathway by interacting with TBK1 and IKKε, thereby negatively regulating PRV propagation. However, UL13 ubiquitinates PRDX1, which routes PRDX1 into proteasomes for degradation and effectively reduces its expression. These results illuminate the fundamental role that PRDX1 plays in the IFN pathway, and they identify a potential target for the control of PRV infection.

Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

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.

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.

Anti-inflammatory effects of Flos Lonicerae Japonicae Water Extract are regulated by the STAT/NF-κB pathway and HO-1 expression in Virus-infected RAW264.7 cells.

This study examined the effect of the Flos Lonicerae Japonicae water extract (FLJWE), chlorogenic acid, and luteolin on pseudorabies virus (PRV)-induced inflammation in RAW264.7 cells and elucidated related molecular mechanisms. The results revealed that FLJWE and luteolin, but not chlorogenic acid, inhibited the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in PRV-infected RAW 264.7 cells. We found that the FLJWE and luteolin suppressed nuclear factor (NF)-κB activation by inhibiting the phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3, respectively). Moreover, the FLJWE significantly upregulated the expression of pNrf2 and its downstream target gene heme oxygenase-1 (HO-1). Our data indicated that FLJWE and luteolin reduced the expression of proinflammatory mediators and inflammatory cytokines, such as COX-2 and iNOS, through the suppression of the JAK/STAT1/3-dependent NF-κB pathway and the induction of HO-1 expression in PRV-infected RAW264.7 cells. The findings indicate that the FLJWE can be used as a potential antiviral agent.

A novel paper biosensor based on Fe3O4@SiO2-NH2and MWCNTs for rapid detection of pseudorabies virus.

In this study, a novel paper biosensor based on Fe3O4@SiO2-NH2magnetic polymer microspheres and multi walled carbon nanotubes (MWCNTs) for rapid detection of pseudorabies virus (PRV) was first developed. Fe3O4@SiO2-NH2were functionalized with PRV antibody and doped in cellulose nitrate paper to fabricate the magnetic paper biosensor with good magnetic response and biocompatibility. Using MWCNTs to build conductive network of sensors, PRV antigen binds specifically to the immunomagnetic microspheres on the sensor, and the resulting immune complex changes the magnetic domain structure of the sensor and the structural gap of MWCNTs, causing the magnetic property and impedance change. TEM and EDS characterization proved that the biosensor was successfully doped with Fe3O4@SiO2-NH2and effectively recognized PRV. Under optimized conditions, the impedance variation was found to be linearly related to the logarithm value of PRV concentrations in the range of 10-1 mg ml-1, with the detection limit of 10 ng ml-1. This paper biosensor demonstrated advantages of portability, high sensitivity and specificity, providing a valuable method for early control of PRV.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

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 105 TCID50 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.

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018-2019.

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.

Pseudorabies Virus Infection Causes Downregulation of Ligands for the Activating NK Cell Receptor NKG2D.

Herpesviruses display a complex and carefully balanced interaction with important players in the antiviral immune response of immunocompetent natural hosts, including natural killer (NK) cells. With regard to NK cells, this delicate balance is illustrated on the one hand by severe herpesvirus disease reported in individuals with NK cell deficiencies and on the other hand by several NK cell evasion strategies described for herpesviruses. In the current study, we report that porcine cells infected with the porcine alphaherpesvirus pseudorabies virus (PRV) display a rapid and progressive downregulation of ligands for the major activating NK cell receptor NKG2D. This downregulation consists both of a downregulation of NKG2D ligands that are already expressed on the cell surface of an infected cell and an inhibition of cell surface expression of newly expressed NKG2D ligands. Flow cytometry and RT-qPCR assays showed that PRV infection results in downregulation of the porcine NKG2D ligand pULBP1 from the cell surface and a very substantial suppression of mRNA expression of pULBP1 and of another potential NKG2D ligand, pMIC2. Furthermore, PRV-induced NKG2D ligand downregulation was found to be independent of late viral gene expression. In conclusion, we report that PRV infection of host cells results in a very pronounced downregulation of ligands for the activating NK cell receptor NKG2D, representing an additional NK evasion strategy of PRV.

THE PRESENCE OF ANTIBODIES AGAINST PSEUDORABIES VIRUS IN WILD BOARS (SUS SCROFA) IN KOREA.

The control and elimination of pseudorabies (PR) is one of the most important goals in the pig industry. After the first PR outbreak in Korea in 1986, all pigs infected with PR virus (PRV) were removed, and a vaccination program for pigs was implemented. No PR has occurred in Korea since 2010, and vaccination was discontinued after 2013. Information on the seroprevalence of PRV in pigs, including wild boars (Sus scrofa), is important for evaluating the PR status in a country. In this study, 2.65% (28/1057) of the wild boars tested had antibodies against PRV in 2018, indicating that PRV has been circulating continuously in the wild boar population in Korea. Effective means should be implemented to prevent the transmission of PRV between wild and domestic pigs, because the wild boar is a potential reservoir host for PRV.

Pseudorabies virus glycoprotein gE suppresses interferon-ß production via CREB-binding protein degradation.

Cyclic GMP-AMP synthase (cGAS) is a main sensor used to detect microbial DNA in the cytoplasm, which subsequently induces the production of interferon (IFN) via the cGAS/STING/IRF3 signaling pathway, leading to an antiviral response. However, some viruses have evolved multiple strategies to escape this process. Pseudorabies virus (PRV) is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, which can cause serious damage to the porcine industry. Many herpesvirus components have been reported to counteract IFN production, whereas little is known of PRV. In the present study, we found that PRV glycoprotein E (gE) was involved in counteracting cGAS/STING-mediated IFN production. Ectopic expression of gE decreased cGAS/STING-mediated IFN-ß promoter activity and the level of mRNA expression. Moreover, gE targeted at or downstream of IRF3 was found to inhibit IFN-ß production. However, gE did not affect the phosphorylation, dimerization and nuclear translocation of IRF3. Furthermore, gE is located on the nuclear membrane and could subsequently degrade CREB-binding protein (CBP). MG132, a proteasome inhibitor, decreased CBP degradation and restored the IFN-ß production induced by gE. Finally, gE-deleted PRV induced a higher level of IFN-ß production and reduced CBP degradation compared to wild-type PRV. Together, these results demonstrate that PRV gE can inhibit cGAS/STING-mediated IFN-ß production by degrading CBP to interrupt the enhanced assembly of IRF3 and CBP.

Immunogenicity and protective efficacy induced by an mRNA vaccine encoding gD antigen against pseudorabies virus infection.

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.

Host antiviral protein IFITM2 restricts pseudorabies virus replication.

Pseudorabies virus (PRV) is one of the most destructive swine pathogens and leads to huge economic losses to the global pig industry. Type I interferons (IFNs) plays a pivotal role in the innate immune response to virus infection via induction of a series of interferon-stimulated genes (ISGs) expression. IFN-induced transmembrane (IFITM) proteins, a group of ISGs, are important host self-restriction factors, possessing a broad spectrum of antiviral effects. They are known confer resistance to a variety of RNA and DNA viruses. However, little is known about the role of IFITMs in PRV infection. In this study, we show that IFITM is crucial for controlling PRV infection and that IFITM proteins can interfere with PRV cell binding and entry. Furthermore, we showed that IFITM2-mediated inhibition of PRV entry requires the cholesterol pathway. Collectively, these results provide insight into the anti-PRV role of IFITM proteins and this inhibition possible associated with the change of cholesterol in the endosome, further underlying the importance of cholesterol in virus infection.