Pseudorabies virus (PRV) has evolved multiple strategies to evade host antiviral responses to benefit virus replication and establish persistent infection. Recently, tripartite motif 26 (TRIM26), a TRIM family protein, has been shown to be involved in a broad range of biological processes involved in innate immunity, especially in regulating viral infection. Herein, we found that the expression of TRIM26 was significantly induced after PRV infection. Surprisingly, the overexpression of TRIM26 promoted PRV production, while the depletion of this protein inhibited virus replication, suggesting that TRIM26 could positively regulate PRV infection. Further analysis revealed that TRIM26 negatively regulates the innate immune response by targeting the RIG-I-triggered type I interferon signalling pathway. TRIM26 was physically associated with MAVS independent of viral infection and reduced MAVS expression. Mechanistically, we found that NDP52 interacted with both TRIM26 and MAVS and that TRIM26-induced MAVS degradation was almost entirely blocked in NDP52-knockdown cells, demonstrating that TRIM26 degrades MAVS through NDP52-mediated selective autophagy. Our results reveal a novel mechanism by which PRV escapes host antiviral innate immunity and provide insights into the crosstalk among virus infection, autophagy, and the innate immune response.

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.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Pseudorabies virus (PRV)-the causative agent of Aujeszky\'s disease-was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk-based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA-SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

This study was based on similar physicochemical characteristics of pseudorabies virus (PRV) and African swine fever virus (ASFV). A cellular model for evaluation of disinfectants was established with PRV as an alternative marker strain. In the present study, we evaluated the disinfection performance of commonly used commercialized disinfectants on PRV to provide a reference for the selection of good ASFV disinfectants. In addition, the disinfection (anti-virus) performances for four disinfectants were investigated based on the minimum effective concentration, onset time, action time, and operating temperature. Our results demonstrated that glutaraldehyde decamethylammonium bromide solution, peracetic acid solution, sodium dichloroisocyanurate, and povidone-iodine solution effectively inactivated PRV at concentrations 0.1, 0.5, 0.5, and 2.5 g/L on different time points 30, 5, 10, and 10 min, respectively. Specifically, peracetic acid exhibits optimized overall performance. Glutaraldehyde decamethylammonium bromide is cost effective but requires a long action time and the disinfectant activity is severely affected by low temperatures. Furthermore, povidone-iodine rapidly inactivates the virus and is not affected by environmental temperature, but its application is limited by a poor dilution ratio such as for local disinfection of the skin. This study provides a reference for the selection of disinfectants for ASFV.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, responsible for severe economic losses to the swine industry worldwide. The interferon-inducible GTPase guanylate-binding protein 1 (GBP1) exhibits antiviral immunity. Our findings show that there is a robust upregulation in the expression of porcine GBP1 during PRV infection. GBP1 knockout promotes PRV infection, while GBP1 overexpression restricts it. Importantly, we found that GBP1 impeded the normal structure of actin filaments in a GTPase-dependent manner, preventing PRV virions from reaching the nucleus. We also discovered that viral US3 protein bound GBP1 to interfere with its GTPase activity. Finally, the interaction between US3 and GBP1 requires US3 serine/threonine kinase activity sites and the GTPase domain (aa 1 to 308) of GBP1. Taken together, this study offers fresh perspectives on how PRV manipulates the host\'s antiviral immune system.

Pseudorabies virus (PRV) is an enveloped, linear double-stranded DNA herpesvirus that resulted in huge financial losses to the swine industry. In addition to vaccination, the development of antiviral molecules is also a beneficial supplement to the control of Pseudorabies (PR). Although our previous studies have shown that porcine Mx protein (poMx1/2) significantly inhibited the proliferation of RNA virus, it was unknown whether poMx1/2 could inhibit porcine DNA virus, such as PRV. In this study, it was investigated the inhibitory effect of porcine Mx1/2 protein on PRV multiplication. The results showed that both poMx1 and poMx2 had anti-PRV activities, which required GTPase ability and stable oligomerization. Interestingly, the two GTPase deficient mutants (G52Q and T148A) of poMx2 also had the antiviral ability against PRV, which was consistent with previous reports, indicating that these mutants recognized and blocked the viral targets. Mechanistically, the antiviral restriction of poMx1/2 came from their inhibition of the early gene synthesis of PRV. Our results for the first time shed light on the antiviral activities of two poMx proteins against DNA virus. The data from this study provide further insights to develop new strategies for preventing and controlling the diseases caused by PRV.

Pseudorabies (PR) is an important infectious disease of swine that causes enormous economic losses to the swine industry in China. Immunization with vaccines is a routine practice to control this disease. PRV inactivated vaccines usually require a booster vaccination to provide complete immune protection. Therefore, Astragalus saponins (AST) have been added as an immunopotentiator to improve the immune efficacy and reduce the immunization times for the PRV inactivated vaccine. The results in mice have shown that a single dose of AST-adjuvanted PRV inactivated vaccine promoted higher production of gB-specific IgG, IgG1, and IgG2a and neutralizing antibody, secretion of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines, and lymphocyte proliferation than mice immunized without AST. Compared to mice immunized without AST, a single dose of the AST-adjuvanted PRV inactivated vaccine improved the survival percentage of mice and reduced the PRV viral loads in the lungs and brains after lethal challenge. In summary, AST was an effective immunopotentiator to improve the immune efficacy of a single dose PRV inactivated vaccine.

Pseudorabies virus (PRV) has evolved various strategies to escape host antiviral immune responses. However, it remains unclear whether and how PRV-encoded proteins modulate the RIG-I-like receptor (RLR)-mediated signals for immune evasion. Here, we show that the PRV tegument protein UL13 functions as an antagonist of RLR-mediated antiviral responses via suppression of the transcription of RIG-I and MDA5, but not LGP2. UL13 overexpression significantly inhibits both the mRNA and protein levels of RIG-I and MDA5, along with RIG-I- or MDA5-mediated antiviral immune responses, whereas overexpression of RIG-I or MDA5 counteracts such UL13-induced suppression. Mechanistically, UL13 suppresses the expression of RIG-I and MDA5 by inhibiting activation of the transcription factor NF-κB. Consequently, overexpression of p65 promotes the activation of RIG-I and MDA5 promoters. Moreover, deletion of the p65-binding sites in the promoters of RIG-I or MDA5 abolishes the suppression role of UL13. As a result, mutant PRV lacking UL13 elicits stronger host antiviral immune responses than PRV-WT. Hence, our results provide a novel functional role of UL13-induced suppression of host antiviral immunity through modulating receptors\' transcription.

Pseudorabies virus (PRV), an etiological agent of pseudorabies in livestock, has negatively affected the porcine industry all over the world. Epithelial cells are reported as the first site of PRV infection. However, the role of host proteins and its related signaling pathways in PRV replication is largely unclear. In this study, we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney (PK-15) epithelial cells. Totally 5723 phosphopeptides, corresponding to 2180 proteins, were obtained, and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells (P ​< ​0.05). GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways. Further functional studies of NF-κB, transcription activator factor-2 (ATF2), MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference (RNAi) knockdown. It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number. JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV, especially during the biogenesis of viral genome DNA. Subsequently, by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2, it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication. These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.