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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. Author summary: The activation of inflammasome and pyroptosis is associated with the rapid death of mice infected with PRV. However, the specific molecular mechanism by which PRV promotes inflammasome overactivation remains elusive. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas viral UL4 deficiency reduced ASC-dependent inflammasome activation and pyroptosis. Mechanistically, the 132–145 aa of UL4 protein permitted its translocation to interact with ASC in the cytoplasm. During this process, UL4 promoted the phosphorylation levels of JNK and SYK, which enhanced the ASC phosphorylation, leading to increased ASC oligomerization, and ultimately enhanced the ASC-dependent inflammasome activation and subsequent pyropKtosis. In vivo, compared with PRV-WT infection, the PRV UL4 gene (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) not only delayed the death time of pigs and mice but also alleviated the inflammatory infiltration and pathological damage in the lung and brain tissues. Altogether, our findings unravel UL4 as an important regulator of host immune response by manipulating the inflammasome signaling, which may be regarded as a novel live gene-deleted PRV vaccine candidate. [ABSTRACT FROM AUTHOR]

Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge. [ABSTRACT FROM AUTHOR]

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication. Author summary: All phases of the viral life cycle are heavily dependent on host metabolism, thus virtually all viruses actively reprogram host metabolic networks to support their replication and spread. However, in the case of PoRVA and PEDV coinfection, the impact of host cell metabolic reprogramming induced by PoRVA infection on PEDV replication has not been reported. Herein, we demonstrated that PoRVA infection promotes PEDV replication by targeting glutamine metabolism in porcine intestinal epithelial cells. PoRVA infection induces increased expression levels of SLC1A5, GLS1, and GLUD1, which enhance glutamine uptake, TCA cycle flux, and ATP production. Notably, we found that PoRVA G9P[23] infection more effectively promoted PEDV replication than PoRVA G5P[7] infection by significantly increasing glutamine uptake and anaplerosis into the TCA cycle. [ABSTRACT FROM AUTHOR]