PRRSV is a major swine pathogen, suppresses innate immunity, and causes persistent infection and coinfection with other pathogens. As a central immune mediator, MALT1 plays essential roles in regulating immunity and inflammation.

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of ROS and nucleocytoplasmic migration of HMGB1 via PERK activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei, hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical PCVAD mostly results from activation of latent PCV2 infection by confounding factors such as co-infection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals where PCV2 in infected cells might utilize the elevated ROS to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross-talk among ER stress, autophagy/mitophagy and mitochondria-sourced ROS in favor of PCV2 replication.

Background Virus-like particles (VLPs) are supramolecular structures composed of multiple protein subunits and resemble natural virus particles in structure and size, making them highly immunogenic materials for the development of next-generation subunit vaccines. The orderly and repetitive display of antigenic epitopes on particle surface allows efficient recognition and cross-link by B cell receptors (BCRs), thereby inducing higher levels of neutralizing antibodies and cellular immune responses than regular subunit vaccines. Here, we present a novel multiple antigen delivery system using SpyCatcher/Spytag strategy and self-assembled VLPs formed by porcine circovirus type 2 (PCV2) Cap, a widely used swine vaccine in solo. Results Cap-SC, recombinant Cap with a truncated SpyCatcher polypeptide at its C-terminal, self-assembled into 26-nm VLPs. Based on isopeptide bonds formed between SpyCatcher and SpyTag, classical swine fever virus (CSFV) E2, the antigen of interest, was linked to SpyTag and readily surface-displayed on SpyCatcher decorated Cap-SC via in vitro covalent conjugation. E2-conjugated Cap VLPs (Cap-E2 NPs) could be preferentially captured by antigen presenting cells (APCs) and effectively stimulate APC maturation and cytokine production. In vivo studies confirmed that Cap-E2 NPs elicited an enhanced E2 specific IgG response, which was significantly higher than soluble E2, or the admixture of Cap VLPs and E2. Moreover, E2 displayed on the surface did not mask the immunodominant epitopes of Cap-SC VLPs, and Cap-E2 NPs induced Cap-specific antibody levels and neutralizing antibody levels comparable to native Cap VLPs. Conclusion These results demonstrate that this modularly assembled Cap-E2 NPs retains the immune potential of Cap VLP backbone, while the surface-displayed antigen significantly elevated E2-induced immune potency. This immune strategy provides distinctly improved efficacy than conventional vaccine combination. It can be further applied to the development of dual or multiple nanoparticle vaccines to prevent co-infection of PCV2 and other swine pathogens.

Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated diseases, and it causes substantial economic losses in the swine industry each year. It is crucial to develop an effective vaccine against the circulating strain PCV2d, which is prone to substantial degrees of mutation. In this study, a truncated form of flagellin (tFlic: 85-111 aa) was inserted into the C-terminal sequence of 2dCap, and Western blotting results showed that recombinant Cap-tFlic VLPs were successfully expressed. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) data indicated that purified recombinant Cap-tFlic fusion proteins existed in the form of polymers and that tFlic could not affect the formation and internalization of VLPs. Integrated Cap-tFlic VLPs induced the expression of antigen presentation-related factors (MHC-II and CD86) by bone marrow-derived dendritic cells (BM-DCs), and the expression of TLR5-related factors (TNF-α) was dramatically elevated. Mice intramuscularly immunized with Cap-tFlic VLPs exhibited significantly higher levels of Cap-specific antibodies and neutralizing antibodies than mice immunized with wild-type Cap VLPs. The data obtained in the current study indicate that Cap-tFlic may be a candidate for a subunit vaccine against PCV2 in the future.