PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs.

Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection 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 reactive oxygen species (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.

Classical swine fever virus (CSFV) poses a major threat to the pig industry. The mechanisms that CSFV uses to evade host innate immunity are not fully understood. Acetylation of histones and non-histone proteins is involved in modulating innate immune responses. Histone deacetylase 1 (HDAC1) could be proviral or antiviral by modulating the acetylation status of histones, viral proteins or non-histone host proteins, depending on the type of viruses involved. First, we found that CSFV infection in IPEC-J2 cells resulted in reduced expression of HDAC1. By chemical inhibition, gene silencing, and overexpression, we revealed that HDAC1 acts as a negative regulator of CSFV replication in IPEC-J2 cells probably through activation of poly(I:C) and IFN-λ3-induced IFN-I/III innate immunity. Mechanistically, CSFV N ^pro downregulated HDAC1 and its transcriptional regulator specificity protein 1 (Sp1). N ^pro interacted with Sp1 to facilitate its degradation through the ubiquitin-proteasome pathway via its N-terminal domain, a region that does not have significant effect on IRF3 stability. Thus, it is clear that CSFV deploys the two domains of its N ^pro to counteract the innate immune responses, the C-terminal one targeting the IRF3 pathway as previously reported, and the N-terminal one targeting the Sp1-HDAC1 axis. IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express N ^pro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its N ^pro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving N ^pro -mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral N^pro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or N^pro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV N^pro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent N^pro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote N^pro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its N^pro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

Porcine enteric coronaviruses (CoVs), including swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine transmissible gastroenteritis virus (TGEV), are major pathogens causing porcine viral diarrhea syndrome (VDS), which brings significant economic losses to the swine industry; distinguishing between these clinically similar viruses has become a serious challenge. We developed a highly specific and interference-resistant porcine CoV multiplex digital PCR (dPCR) assay. The assay exhibited robust anti-interference capabilities, as the concentrations of the four viruses did not affect their accurate quantification. The coefficients of variation (CV%) of intra-batch and inter-batch repeatability for all target viruses were less than 11%. The limit of quantification (LoQ) of this dPCR assay reached 7.5 copies/reaction for each target, and it was one order of magnitude more sensitive than qPCR. The limits of detection (LoD) for SADS-CoV, PEDV, PDCoV, and TGEV were 2.72, 3.00, 3.56, and 3.19 copies/reaction, respectively. A total of 408 known samples were used for validation tests, and the results were highly consistent with the known conditions, showing a compliance rate of 97–100%. The diagnostic specificity (Dsp) of the method was 99–100%. In conclusion, the developed multiplex dPCR assay is highly suitable for early detection and quarantine in four porcine CoVs. The results indicate that this dPCR method is characterized by high specificity, anti-interference capabilities, repeatability, and high sensitivity. It also demonstrates a high compliance rate and diagnostic specificity in sample detection. This multiplex dPCR will contribute to the control of porcine enteric CoV-caused VDS and provide clues for subsequent research.