Porcine precision-cut lung slices (PCLS) were used to analyze the effect of the ciliary activity on infection of airway epithelial cells by influenza viruses. Treatment of slices with 2% NaCl for 30 min resulted in reversible ciliostasis. When PCLS were infected by a swine influenza virus of the H3N2 subtype under ciliostatic conditions, the viral yield was about twofold or threefold higher at 24 or 48 h post-infection, respectively, as compared to slices with ciliary activity. Therefore, the cilia beating not only transports the mucus out of the airways, it also impedes virus infection.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo . This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

Respiratory coronaviruses (RCoVs) significantly threaten human health, necessitating the development of an ex vivo respiratory culture system for investigating RCoVs infection. Here, we successfully generated a porcine precision-cut lung slices (PCLSs) culture system, containing all resident lung cell types in their natural arrangement. Next, this culture system was inoculated with a porcine respiratory coronavirus (PRCV), exhibiting clinical features akin to humans who were infected by SARS-CoV-2. The results demonstrated that PRCV efficiently infected and replicated within PCLSs, targeting ciliated cells in the bronchioles, terminal bronchioles, respiratory bronchioles, and pulmonary alveoli. Additionally, through RNA-Seq analysis of the innate immune response in PCLSs following PRCV infection, expression levels of interferons, inflammatory cytokines and IFN stimulated genes were significantly upregulated. This ex vivo model may not only offer new insights into PRCV infection in the porcine respiratory tract but also serve as a valuable tool for studying human respiratory CoVs infection.

African swine fever virus (ASFV) infection causes a severe hemorrhagic disease in pigs, characterized by excessive inflammatory responses and tissue damage, posing substantial threats to the pig industry worldwide. Given the lack of vaccines and effective antiviral treatments, as well as the largely unknown functions of most ASFV-encoded proteins, it’s urgent to study the proteins that are crucial in triggering inflammatory responses and how they do so. This study demonstrated that ASFV exploited the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome to induce pyroptosis and inflammatory responses, effectively replacing the non-functional porcine AIM2 pseudogene. Screening over 150 proteins encoded by the ASFV genome, EP364R was identified as the viral factor responsible for driving NLRP3-mediated pyroptosis and high-level cytokine production. Ectopic expression of EP364R in mice elicited significant upregulation of serum pro-inflammatory cytokines and splenomegaly, while its expression in bone marrow-derived macrophages (BMDMs) from NLRP3-knockout mice abrogated pyroptosis and related effects. Mechanistic investigation revealed that the helicase DDX3X acted as a molecular bridge, enabling EP364R to interact with NLRP3 to promote the aggregation and activation of inflammasomes. Depletion of DDX3X abolished EP364R’s ability to induce NLRP3-dependent pyroptosis and pro-inflammatory cytokine production. We found that the NACHT domain of porcine NLRP3 interacted with DDX3X, and EP364R established a connection with the NACHT and LRR domains of NLRP3 through DDX3X. However, EP364R bound to all the domains of DDX3X. Molecular docking analysis revealed that DDX3X interacted with EP364R through a spatially defined interface, thereby exerting its function. Furthermore, a natural compound library was employed to screen functional compounds targeting EP364R, and HAMNO was identified as an inhibitor that bound to E256, K259, and D260 of EP364R, consequently suppressing ASFV replication. Our findings explain how ASFV triggers pyroptosis and excessive cytokine release, and identify a potent small-molecule inhibitor of ASFV, aiding the development of vaccines and therapies to prevent and control African swine fever (ASF) caused by ASFV infection.